METHOD DEVELOPMENT AND VALIDATION OFBTIPIVACAINE IN HT]MAN PLASMA AND THE

FEASABILITY TO BE APPLIED ON PREGNANT WOMENDURING LABOR

ByEnass Ali Alkhader

SupervisorProf. Tawfiq Arafat

Co-SupervisorDr. Wael Abu Dawih

A Thesis Submitted in Partial Fulfillment of the Requirements for theDegree of Master of Science in Pharmaceutical Sciences at

Universitv of Petra

Faculty of Pharmacy and Medical Sciences

Amman-Jordan

June 2013

Method Development and Validation of Bupivacaine inHuman Plasma and the Feasibility to be Applied on Pregnant

Women During Labor

By

Enass Ali Al-khader

A Thesis Submitted in Partial Fulfillment of the Requirements for the

Degree of Master of Science in Pharmaceutical Sciences at University of

Petra

Faculty of Pharmacy and Medical Sciences

Amman-Jordan

June 2013

Supervisor

Prof. Tawfiq Arafat

Co-Supervisor

Dr, Wael Abu Dayyih

Examination Committee

Name

1. Dr, Eyad Mallah

2. Dr, Faisal Al Akayleh

3. Dr. Kamal Sweidan

Signature

Signature

Signature

DEDICATIONS

First and foremost, I dedicate my work to Allah, the Almighty, lor he empowered

me the strength to persist all the way long, and granted me the blessing to finish

this thesis the wav it is.

To you my father; making you proud has always been my first aim, you have always

been my source of encouragement, support and inspiration throughout my whole life,

thank you for teaching me the impossible is nothing, with you by my side, I can reach

the stars.

To my beloved mother; you lend me a hand to stand up when everything seemed

like falling apart, without your full heart prayers I would have never achieved any

ofthis.

To my dear husband, Emad, thank you for your understanding and encouragement in

my moments ofcrisis, I would happily consider rny success as yours.

My brothers, sisters and friends deserve my wholehearted thanks as well.

lll

AKNOWLEDGMENTS

First of all, I would like to express my sincere appreciation to my supervisor Prol

Tawfiq Arafat for the continuous support in all stages of this thesis. His knowledge,

expertise, patience and guidance helped me to keep moving ahead with my work.

From the formative stages ofthis thesis, to the final draft, I owe an immense debt of

gratitude to my co-supervisor Dr. Wael Abu Dayyih, his assistance, advice and

spiritual and technical support enabled me to complete my work successfully and Ican't thank him enough.

I would like also to express my thanks and respect to Dr. Eyad Al-Mallah, Dr. Faisal

Al-Akayleh and Dr. Kamal Sweidan for being part of my committee and giving this

thesis a chance of their valuable time and scientific experience.

Last but not Least, I would like to thank the staff in Jordan Center for Pharmaceutical

Research (JCPR) and all of the staff in Faculty of Pharmacy and Medical Sciences at

University ofPetra for offering all the possible help.

IY

Table of Contents

No. Subject Page

Committee Decision ll

Dedication rii

Acknowledgments lv

Table of Contents

Abbreviations X1

List of Tables xlll

List ofFigures xvl

Abstract XVIl

Chapter 1: Introduction I1 Introduction 2

1.1 History 2

1.2 Chemical Properties 3

l.J Physical Properties 4

1^ Clinical Pharmacology 5

1.4.1 Indications 6

1.4.2 Mechanism of Action 8

1.4.3 Adverse Effects 9

1.4.4 Toxicity/ Overdose 1l

1.5 Pharmacokinetics 12

1.5.I Absorption 12

1.s.2 Distribution IJ

1.5.3 Metabolism and Excretion IJ

1.5.4 Heptic Impairment 1A

1.5.5 Contraindications 15

1.6 Labor and Delivery l5

1.7 Precautions t6

1.8 Interactions 1',7

1.9 Epidural 17

1,9.1 What is Epidural Delivery 1'7

1.9.2 Epidural Side Effects and Management 18

1.9.3 Bupivacaine Epidural 18

l.l0 Determination of Bupivacaine in Biological Plasma 20

1.1 I Method Validation 22

1.11.1 Standard Calibration Curve and Linearity !)

Ll1.2 Lower Limit of Quantification (LLOQ) z+

i. I 1.3 Accuracy and Precision 25

1.11.4 Recovery 25

l1l5 Specifity and Selectivity 25

1.11.5.1 Specifity 25

t .11 5.2 Selectivity lo

1.11.6 Dilution Integrity 27

1.11.7 srability 28

1. i 1.7.1 Short Term Stability 28

1.1r.7 .2 Long Term Stability 29

111.7.3 Stock Solution Stability 29

1.r2 Internal Standard 29

113 Previous Analytical Studies for Bupivacaine Determinationin Bioloeical Fluid

30

t.14 The Obj ectives JJ

Chapter 2: Expiremental Jt

2 Expiremental J]

2.1 Chemicals and Reaeents l5

2.2 Instrumentation 35

z.J Method Develorrment JO

2.3.1 Selection Methods for Analysis JO

2.3.2 Chromatographic Conditions 38

.A Preparation of Stock Solution and Serial Dilution 39

2.4.1 Preparation of Stock Solution ofBupivacaine 39

a ^., Preparation of Stock Solution ofBisoprolol (InternalStandard)

39

Preparation of Internal Standard Working Solution

(Bisoprolol)

39

2.4.4 Preparation of Bupivacaine in Plasma 39

2.4.4.1 Preparation of Standard Calibration Curve 39

2.4.4.2 Preparation of QC Samples 41

2.5 Sample Preparation and Method of Extraction Development 43

?qr Solvent 43

2 5.2 Buffer +J

z.) 3 Vortex Mixins Duration 43

2.5.4 Back Extraction 43

2.6 Method Validation 44

2.6.r Recovery 44

LO.Z Standard Calibration Curve and Linearity 44

2.6.2.1 Linearity 44

Standard Calibration Curve Response 45

2.6.3 Sensitivity 45

z.o.+ Accuracy and Precision 45

2.6 4.1 Accuracy 46

2.6.4.2 Precision 46

2.6.5 Inter and Intra-day Accuracy and Precision 46

?.6.5.1 Intra-day Accuracy and Precisron 46

2.6.s.2 Inter-day Accuracv and Precision 46

2.6.6 Recovery 47

2.6'1 Specifity and Selectivity 47

2.6 7.r Specifity 47

267.2 Selectivity 47

2.6.8 Dilution Integrity 4'.7

2.6.9 Stability 48

2.6.9.r Short Term Stability 48

2.6.9.1.1 Room Temperature (Bench top) Stability 48

2.6.9.1.2 Post-operative Stability (Auto-sampler) Stability 48

2.6.9.1.3 Freeze and Thaw Stability 49

2.6.9.2 Long Term Stability 49

2.6.9 3 Stock Solution Stability 50

Chapter 3: Clinical Study Design ll

.' Clinical Study Design J2

JI Ethics 52

3.1.1 Independent Ethics Committee (IEC) 52

).t z Declaration of Helsinki 52

3.1.3 Ethical, Legal and Administrative Aspects )J

3. 1.4 Subject Information and Consent Form fJ

J -Z Overall Study Design and Plan Description ff

3.2.1 Benefit Risk Evaluation 55

3.2.2 Choice of Preenant Woman ll

Choice ofthe Dosaee 55

Choice of Sampling 56

3.2.s Selection of Patient 56

3 .2.5.1 Justification of Inclusion and Exclusion criteria 56

3 2.5.2 Inclusion Criteria )/3.2.5.3 Exclusion Criteria .)/

J.Z.O Other Conditions 58

3.2.7 Patient's Obligation 58

3 2.8 Drug Handling and Accountability 58

1?R r Dosage and Duration of Treatment 59

).2.6.2 Labeling of Empty Ampoule 59

Compliance 59

3.2 10 Concomitant Medication 60

3.2 11 Rescue Medication 60

3.2.12 Emergency Procedure 6l

J.2.l) Follow Up ofPatient Experiencing Adverse Events 61

3.2.14 Safety Assessment 61

3.3 Study Procedure bl

J.J I General Procedure 6l

).J.Z Special Procedure 62

3.3.3 Daily Activities in Each Period of the Trial 62

3 3 3.r Entry Examination 62

).J J.Z Day 1 63

3.3 .4 Drue Administration 64

3.3.5 Blood Sampling for Drug Analysis 64

J.J. O Analvsis of Bupivacaine 64

3.4 Study Documentation 65

3.4.1 Investigator's File ol

Case Report Form (CRF) oo

J.) Pharmacokinetic Evaluation o/

3.6 Biometrics and Statistical Aspects o/

J O. l Data Presentation ot

J. I Good Clinical Practice 68

3.7 .1 Legal Requirements 68

3.7.2 Adherence to the Protocol 68

3.7 .3 Data Handline Procedures 69

Quality Assurance Aspects 70

3 7.5 Monitoring 70

3.7.6 Confidentiality 71

Chapter 4: Results and Discussion 72

4 Results and Discussion

4.1 Internal Standard

1n Description of Method 73

4.2.r Sample Preparation and Method ofExtraction Procedure

^an Separation and Chromatography 74

4.3 Validation Results 78

4.3.1 Validation Day One, Two and Three, linearity, Accuracy and

Precision Results

78

4.4 Recovery 105

4.5 Sensitivity 109

4.6 Specifity and Selectivity 111

4.6.1 Specifity 1il+.o.2 Selectivity 1r4

4.7 Dilution Integrity 115

4.8 Stability 116

4.8.1 Short Term Stability 116

4,8 1, I Room Temoerature Stabilitv 116

4.8.r.2 Autosampler Stability 118

4,8. 1.3 Freeze and Thaw Stability 119

4.8.2 Long Term Stability of Plasma Samples 120

4.8.3 Stock Solution Stability 123

5 Conclusion 125

6 References t26

7 APPENDLX: Chromatosrams and UV Scan 139

8 APPENDIX: Study Protocol 148

APPENDD(: lnformed Consent Form 1',76

10 APPENDIX: Case Repoft Form 182

1l Abstract (In Arabic) 1.97

Abbreviations

AUC Area Under the Curve

B.N, Batch Number

BP Blood Pressure

C,,,,'u,, Maximum Plasma Concentration

Conc, Concentration

CRF Case Report Form

CV Coeffi cient of Variation

DW Dry Weight

ED Expiry Date

FDA Food and Drus Administration

5 Gram

FIPLC High Performance Liquid Chromatography

h Heart Rate

Hr Hour

ICH Intemational Conference on Harmonization

I,S Internal Standard

INR International Normalized Value

TV Intravenous Infusion

JCPR Jordan Center for Pharmaceutical Research

LC Liquid Chromatography

LLOQ Lower Limit Of Quantification

MP Mobile Phase

No. Number

QC Quality Control

QC Levels Low, mid and high

QC Samples Low, mid and high

R Corelation Coefficient

R' Coefficient of D etermination

RP Reversed Phase

RPA Relative Peak Area

RSD Relative Standard Deviation

RSQ Regression squared or coeffi cient of Determination

RT Room Temperature

RPA Relative Peak Area

SD Standard Deviation

STD Standard Calibration Curve

TCA Trichloroacetic Acid

TEA Triethylamine

T-u" Median Time to Maximum Plasma Concentration

TILOQ Upper Limit of Quantification

USFDA United States Food and Drus Administration

WS Working Standard

Zero-sample Blank with internal standard

List of Tables

Table No, Caption Page No.

Table I Bupivacaine routes of Administration and Indicated

Concentrations

7

Table 2 Chromatographic Conditions for Method (1) 36

Table 3 Chromatographic Conditions for Method (2) 36

Table 4 Chromatographic Conditions for Method (3)

Table 5 Chromatographic Conditions for Method (4) JI

Table 6 Chromatographic Conditions for Method (5) JI

Table 7 Chromatographic Conditions for Method (6) 38

Table 8 Chromatographic conditions of Bupivacaine analysis 38

Table 9 Standard calibration curve preparation procedure 40

Table 10 Quality Control Samples preparation procedure 42

Table 1 1 Results of each developed method 74

Table 12 Standard calibration curve ofvalidation day one,

Intraday acclfiacy data derived from standard

calibration curve back

80

Table 13 Raw Data of the Standard Curve with Regards to

Correlation, Slope, R2 and intercept for Day L

80

Table 14 Standard calibration curve ofvalidation day two,

intraday accuracy data derived from standard

calibration curve back calculation.

82

Table 15 Raw Data of the Standard Curve with Regards to

Correlation, Slope, R'? and intercept for Day II82

Table 16 Standard calibration curve ofvalidation day three,

Intraday accuracy data derived from standard

calibration curve back calculation.

84

Table 17 Raw Data of the Standard Curve with Regards to

Correlation, Slope, R'z and intercept for Day III84

Table 18 Standard calibration curve number 4, Intraday accuracy

data derived from standard calibration curve back

calculation.

86

Table 19 Raw Data of the Standard Curve number 4 with

Regards to Conelation, Slope. R2 and intercept

86

Table 20 Standard calibration curve number 5, Intraday accuracy

data derived from standard calibration curve back

calculation.

88

Table 21 Raw Data of the Standard Curve number 5 with

Regards to Correlation, Slope, R2 and intercept.

88

Table22 Linearity and linear working range ofBupivacaine data

based on back calculated area ratio derived from

standard calibration curve,

90

Table 23 Linearity and linear working range ofBupivacaine data

based on normalized concentration derived from

standard calibration curves

91

Tahle 24 Linearity and linear working range ofBupivacaine data

based on calculated concentration derived from

standard calibration curves

o?

Table 25 Raw Data for five calibration Curves with Regards to

Correlation, Slope, R'? and intercept

93

Table26 Intra-day precision and accuracy data for QC samples

derived from standard calibration curve ofday one

validation.

95

Table 27 Intra-day precision and accuracy data for QC samples

derived from standard calibration curve ofday two

validation

98

Table 28 Intra-day precision and accuracy data for QC samples

derived from standard calibration curve of day three

validation

101

Table 29 Inter-day accuraoy and Precision for the quality control

Samples ofBupivacaine in the three days ofvalidation

104

Table 30 Data of Bupivacaine in solution for the quality control

samples

106

Table 3 1 Data of Bupivacaine in plasma for the quality control

samples

107

Table 32 Recovery o% for Bupivacaine 108

Table 33 Recovery % for Bisoprolol (I. S) 108

Table 34 Precision and Accuracy for Bupivacaine LLOQ (10

nglml)

110

Table 35 Specifity ofblank for Bupivacaine r12

Table 36 Specifity of Zero of Bupivacaine 113

Table 37 Selectivitv for Buoivacaine rl4Table 3 8 Dilution Integrity for Bupivacaine 115

Table 39 Room Temperature stability of short term for three

levels of QC samples (low, mid and high) for

Buoivacaine.

117

Table 40 Auto-sampler stability of short term for three levels of

QC samples (low, mid and high) for Bupivacaine

118

Table 41 Freeze and thaw stability of short term for three levels

of QC samples (low, mid and high) for Bupivacaine

119

Table 42 Long term stability for four weeks ofthree levels of

QC samples (low, mid and high) for Bupivacaine

121

Table 43 Stock solution stability (500 pg/ ml) diluted to 3750

ng/ml for Bupivacaine

123

Table 44 Stock solution stability (1000 pgl ml) diluted to2.5 ytg/

ml for Bisoprolol (I.S)

124

List of Figures

Figure No. Caption Page No.

Figure 1 Buoivacaine Chemical Structure 3

Figure 2 Steps in HPLC method development 20

Figure 3 Bisoprolol Chemical Structure 39

Figure 4 Labeling of Empty Ampoule 59

Figure 5 HPLC chromatogram of plasma blank 76

Figure 6 HPLC chromatogram ofLLOQ ofBupivacaine (10 ng/ml)

76

Figure 7 HPLC chromatogram of Zero BupivacaineConcentration

77

Figure 8 HPLC Chromatogram ofBupivacaine LILOQ (4000 ng/ml)

71

Figure 9 The plot ofcalibration curve levels against their

analytical response and regression linear equation in

validation Day I

81

Figure 10 The plot of calibration curve levels against their

analytical response and regression linear equation in

validation Day II

83

Figure 1 1 The plot ofcalibration curve levels against their

analytical response and regression linear equation in

validation Day III

85

Figure 12 The plot ofcalibration curve number 4 levels against

their analltical response and regression linear equation

87

Figure 13 The plot ofcalibration curve number 5 levels against

their analytical response and regression linear equation

89

Figure 14 Plot of linearity offive calibration curves levels against

their analytical response and regression linear equation

93

Method Development and Validation of Bupivacaine in Human Plasma and the

Feasibility to be Applied on Pregnant Women During Labor

ByEnass Ali Al-khader

SupervisorProf. Tawfiq Arafat

Co-SupervisorDr, Wael Abu Dayyih

University of Petra, 2013

Abstract

A highly accurate and simple HPLC method using C18 BDS column for the

determination of Bupivacaine in plasma was successfully developed and validated.

Bisoprolol was used as an internal standard. A constant mobile phase was composed

of water and acetonitrile (66.5:33.5) v/vo/o in a combination with 0.1% triethylamine

buffer of pI{=2.5. The flow rate was 1,0 mllmin with respect to retention time and

peak resolution, UV detector was used and l, max was 2O2 nm. The effect of different

chromatographic parameters (composition of mobile phase, pH and column

temperature) have been tested during the development and validation of the method.

The method was intended to be implemented in determination of Bupivacaine in

mother and placenta during labor, the method is validated, protocol and consent form

is ready and approved by ethics committee for future direct use.

xvll

Chapter I

Introduction

I Introduction

Bupivacaine is a local anesthetic which has various uses like infiltration, nerve block,

epidural, and intrathecal anesthesia. Bupivaoaine often is administered by epidural

injection before total hip arttroplasty (Joun A.C. et al., 2002; Rosenblatt M.A. et al.,

2006; Weinberg G.L. et al.,2006). Also, it is commonly injected to surgical wound sites

to reduce pain for up to 20 hours after the surgery. Bupivacaine is also used as a local

anesthetic by epidural injection during labor. Sometimes, Bupivacaine is co-administered

with epinephrine to prolong the duration of its action, fentanyl for epidural analgesia, or

glucose (Gaurav K. and Geeta C.,2011).

1.1 History

In 1 850 the Austrian Von Scherzer brought a suffioient quantum of cooa leaves to Europe

to permit the isolation of cocaine (Oeppen R.S., 2003 and RuestschY.A. et a1.,2001).

As suggested by his friend Sigmund Freud, descriptions of the properties of the coca

prompted by the Austrian Koller to perform in 1884 the first clinical operation under

local anesthesia, by admrnistration ofcocaine on the eye. And then, the use ofcocaine for

local and regional anesthesia rapidly spread throughout Europe and America (Barash

P.G., 2009; Calatayd I. and Gonzalez A., 2003 and Miller RD. and Pardo M, 2011).

Unfortunately, the toxic effects of cocaine caused many deaths among patients and

addicted medical staff Local anesthesia was in a profound crisis until the development of

modern organio chemistry which led to the synthesis of pure cocaine in 1891. New amino

ester local anesthetics were synthesized between 1891 and 1930, such as tropocaing

eucaine, holocaine, orthoform, benzocaine, and tetracaine, In addition, amino amide local

anesthetics were prepared between i898 and 1972 including nirvaquine, procaine,

chloroprocaine, cinchocaine, lidocaine, mepivacaine, prilocaine, efocaine, Bupivacaine,

etidocaine, and articaine. All of these drugs were ostensibly less toxic than cocaine, but

they had the problem of differing amounts of central nervous system (CNS) and

cardiovascular (CV) toxicity (Avery P. et al, 1984 and Groban L,, 2003).

Bupivacaine is ofspecial interest because it has the advantages oflong duration ofaction

and history of clinioal application. Synthesized in 1957, and introduced to the markets in

1965 but was paralleled with the progressive and cumulative reports of CNS and CV

toxicity which lead to the restriction of its use and the identification of a special therapy-

resistant CV toxicity. Numerous experimental studies were conducted to identify the fine

cellular mechanism of this toxicity, which refines our understanding of the action of local

anesthetics (Malamed S.F." 2006 and Sureka C. and Radha M.K.,2012).

1.2 Chemical PropeIties

Bupivacaine hydrochloride is chemically designated as 2- piperidinecarboxamide, l-

butyl-N-(2,6- dimethylphenyl)-, monohydrochloride, monohydrate (Smith G., 2001 and

Reddy A, 2012) with a molecular mass of 342.9 and has the structure is shown in frgure

I (Weiner D,, et al., 2002) ,

. l-{Cl n HrO

X'igure l: Bupivacaine Chemical Structure.

cl-tF{cl-lu}Fsl-{!

(J-*o*'*

*to

--< { '!1'|,{

ffH*

Each I mL of Bupivacaine vial contains 7.5 mg Bupiracaine hydrochloride anhydrous

and 82.5 mg dextrose anhydrous (Chambers W.A. et al., 1982; Gessel E.F.er al, 1991,

and Logan M.R et al.,1986).

The specific gravity of Bupivacaine Spinal is between 1.030 and 1.035 at 25'C and 1,03

at 37oC (Abouleish E.I, 1987; Hideyuki H et a|,2004, and Horlocker T.T. and Wedel

D.J., 1993). pKa value of Bupivacaine is 8.1, Lipid solubility (octanoywater partition

coefficient) is 346 and protein binding of96 (Strichartz G.R, 1990 and The International

pharmacopeia, 1979).

Sulphated Ash. : Not more than 1.0 mg/g (Dodds L.J, 2004).

Loss on drying: Dry to constant weight at 105'C; it loses not less than 45 mg/ g and not

more tian 60 mg/g (Mantanari L. et ol, 2003 and Irngston M. et al,2O0O).

13 Physical Properties

Bupivacaine hydrochloride monohydrate is a white crystalline odorless powder that is

fteely soluble in 957o ethanol, soluble in water, and slightly soluble in chloroform ot

ether (Dodds L.,2004, Reddy A,, 2072 andTrngY . et a1.,2009).

1.4 Clinical Pharmacolory

Bupivacaine slows the propagation ofthe nerve impulse and reduce the rate ofrise ofthe

action potential which leads to the increase ofthe tlreshold for electrical excitation in the

nerve which finally blocks the generation and the conduction of nerve impulses and that's

how the Bupivacaine works (Coutney K.R, 1980; Jong R.H., 1977; Kevin V et a(,2006

and l\,Ialamed S.F., 2004),

In general, the progression of anesthesia is related to the diameter, myelination, and

conduction velocity of affected nerve fibers. @iebuyck J. and Raymond B., 1989 and

Raymond S.A., 1992).

Systemic absorption ofBupivacaine produces effeots on the cardiovascular and

central nervous system (CNS). At blood concentrations achieved with normal therapeutic

doses, changes in cardiac conduction, excitability, refractoriness, contractility, and

peripheral lascular resistance are minimal. However, recent clinical reports and animal

research suggest that cardiovascular changes like depressed cardiac conduction and

excitability which finally leads to cardiac arrest are more likely to occur after unintended

direct intravascular injection of Bupivacaine. Thereforg when epidural anesthesia with

Bupivacaine is considered, incremental dosing is necessary (Brian R.L. et al, 1988;

Marcel P.V. et a|,2000 and Spencer L. et al, 1995).

Following systemic absorptiorq Bupivacaine will produce central nervous system

stimulation, depression or both. Apparent central stimulation manifestations are

restlessness, tremors, shivering, progressing to conrulsions, followed by depression and

coma progressing ultimately to resp atory arrest. On the other hand, the local anesthetics

have a primary depressant effect on the medulla and on higher centers. The depressed

stage may occur without a prior excited stage (Thomas RD. et a|,1986).

Adding sodium bicarbonate on the dose of 1-2 ml to the Bupivacaine solution can

accelerate the Bupivacaine onset of action. This acts by maximizing the amount of

Bupivacaine in the more lipid-soluble (unionized) form (Nathen N. er al, 1996 and

Weignberg G.L. et al,2006). However, this is not available in the local Jordanian market.

Bupivacaine is marketed as hydroohloride salts to maximize aqueous solubility. After

injection, the salts are buffered in the tissue to physiologio pH, thereby providing

sufficient free base ooncentration for diffusion through the axonal membrane (Rom S.A..

1989), However, repeated injections ofBupivacaine can deplete the buffering oapacity of

the local tissue (Fawcett J,P. et al., 2002).

The ensuing acidosis increases the extracellular cationic form, which diffuses poorly and

results in taohyphylaxis. Tachyphylaxis to Bupivacaine is comrnon in areas wrth a limited

buffer capacity (Renck H., 1980 and Skarda RT. and Tranquilli W.J., 2011).

1.4.1 Indications

Bupivacaine Hydrochloride is indicated for the production of local or regional anesthesia

or analgesia for surgery, dental surgery procedures, oral surgery procedures, diagnostic

procedures, therapeutic procedures, and for obstetrical procedures (Butterworth J.F. el a/.,

1993 and Reiz S. et al.,1989). Only the 0.25o/s ar.d 0.5%o concentratiors are indicated for

obstarical anesthesia (Hayman M. and Shinder S., 1971).

Experience with non-obstetrical surgical procedures in pregnant patients is not sufficient

to recommend use of 0.75% concentration of Bupivaoaine Hydrochloride in these

patients (Albright G.,1979; Reynols F. and Taylor G., 1970 and Seltzer J. et al., 1987).

1,4,2 Mechanism of Action

Bupivacaine reduce sodium permeability and increase action potential threshold which

leads to the prevention of nerve impulses generation (Covino B., 1981 and Scholz A. er

al, 1997).

As the primary MOA of Bupivacaine, it blocks voltage-gated sodium channels.

Excitable membrane of nerve axons, just like the membrane of cardiac muscle and

neuronal cell bodies, maintains a resting transmembrane potential of -90 to -60 mV

(Tetzlaff J., 2000).

When excitation occurs, the sodium channels open, and a fast inward of sodium current

quickly depolarizes the membrane toward the sodium equilibrium potential which is

+40mV. Due to this depolarization process, the sodium channels close (inactivate) and

potassium channels open. The outward flow of potassium repolarizes the membrane

toward the potassium equilibrium potential (about -95 mV); repolarization retums the

sodium channels to the rested state with a characteristic recovery time that determines the

refractory period. The transmembrane ionic gradients axe maintained by the sodium

pump. Bupivaoaine has the above effects similarly in both nerves and heart muscles

(Lipkind G. and Fozzard H,, 2005 and Valenzuela C. et al., 1995).

As the Bupivacaine concentration increases progressively and applied to a nerve fiber,

the threshold for exoitation inoreases, impulse conduction slows, the rate of rise of the

action potential declines, the action potential amplitude decreases, and, finally, the ability

to generate an action potential is completely abolished. These progressive effects result

from binding of Bupivacaine to more and more sodium channels. Ifthe sodium current is

blocked over a critical length of the nerve, propagation across the blocked area is no

longer possible. In myelinated nerves, the critical length is two to tluee nodes of Ranvier

at the minimum dose required to block propagation, the resting potential in not

significantly altered (Lee-Son el a 1., 1,992).

The recovery from Bupivacaine block is 10 to 100 times slower than normal block

recovery of channels from normal inactivation As a result, the refractory period is

lengthened and the nerve conducts fewer electrical impulses (Moller R and Covino G.,

1988).

1,4,3 Adveme Effects

Reactions to Bupivacaine are characteristic of those associated with other amide-type

local anesthetics (Berahord C. et a1.,2002 and Fuzier R et al., 2002).

The two main acute adverse experiences which demand immediate countermeasures

following the administration of spinal anesthesia is hypotension due to loss of

sympathetic tone and respiratory paralysis or under ventilation due to cephalic extension

ofthe motor level ofanesthesia. Ifnot treated these adverse effects may lead to a cardiac

arrest (Dong P. et al.,2OOO).

Hypotersion is more commonly observed in patients with shrunken blood volumg

shrunken interstitial fluid volume, cephalad spread of the local anesthetic, and/or

mechanical obstruction of venous return Nausea and vomiting are frequently associated

with hypotensive episodes following the administration of spinal anesthesia (Harrock N.

et al., 1993 and Scull T. and Carli F., 1996).

Cardiovascular System: Beside the adverse effect of hypotension due to the loss of

sympathetic tone. Also, High doses, or inadvertent intravascular injectioq may lead to

high plasma levels and related depression ofthe myocardium, decreased cardiac output,

bradycardia, heart block, ventricular arrhythmias, and, possibly, cardiac arrest (Horten J.

et a1.,2004 and Rolf N. et 41, 2000).

Respiratory System: As previously mentioned the upward extersion ofthe level of spinal

anesthesia may lead to respiratory paralysis or under ventilation. Other causes which may

contribute to under-ventilation are pre-anesthetic medicatioq intra-operative analgesics

and sedatives, as well as surgical manipulation, may contribute to under-ventilation. This

will usually be noted within minutes of the injection of spinal anesthetic solutiorl but

because of differing surgical maximal onset times, differing intercurrent drug usage and

differing manipulation, it may occur at any time during surgery or the immediate

reoovery period (ones J. et al., l99O and Penlnnt J.,2001).

Central Nervous Slstem: Respiratory paralysis or underventilation secondary to cephalad

spread of the level of spinal anesthesia and hypotension for the same reason are the two

most commonly encountered central nervous system-related adverse observations which

demand immediate countermeasures (Crreenhalgh C., 1996 and Wittpenn J. et al.,1986).

Sensory loss may include numbness ofthe legs and tongue and shivering (Popescu A. el

al.,2073).

Neurologic: Neurologic effeots following spinal anesthesia may include loss of perineal

sensation and sexual function, persistent anesthesia, paresthesia, weakness and paralysis

of the lower extremities, and loss of sphincter control all of which may have slow,

incomplete, or no recovery, hypotensioq high or total spinal block, urinary retention,

headache, backache, septic meningitis, meningismus, arachnoiditis, slowing of labor,

increased incidence offorceps delivery, shivering, cranial nerve palsies due to traction on

l0

nerves from loss of cerebrospinal fluid, fecal and urinary incontinence (Atalay C. et al.,

2010; Fuzier R et al., 2009; Ioshi K. et al.,2012 ard Sarotti D. et al.,20ll).

Allergic: Allergio-type reactions are rare and rftry occur if the patient has sensitivity to

Bupivacaine, These reactions are charaatertzed by signs such as urticaria, erlthema,

pruritus, angioneurotic edema (including laryngeal edema), tachyoardia, nausea,

vomiting, sneezing, dizziness, syncope, elevated temperature, excessive srveating, and,

rarely, anaphylactoid-like symptorrratology (including severe hypotension). Cross

sensitivity among members of the amidetype local anesthetio group has been reported

and there is no definite advantage has been reported for Bupivacaine sensitivity screening

before administration. (Sambrook etal.,20ll1, Speoa S. eta1.,2010 and Thyssen J. e/al,

2008).

Urinary retention; as a result of the sacral level block there is a reduced ability for the

urinary sphinoher to relax (Huang Y. et al., 1998 and Olofsson C. et a|.,1,997).

Other adverse like nausea and vomiting may occur during spinal anesthesia (Watcha M.

and Whire P., 1992).

Many of the above mentioned ADRs may be related to Bupivacaine administratron

techniques, with or without a contribution from it (Knudsen K., et al., 1997).

1.4,4 Toxicity/ Overdose

Toxicity is the degree to which a substance can harm humans or animals and is studied to

avoid it in the first place and to be prepared for the required management if happened

(Rozmon K., 1998).

High plasma levels encountered during therapeutic use or to underventilation (and

perhaps apnea) secondary to upward extension of spinal anesthesia requires acute

ll

emergency. Hypotension is commonly encountered during the conduct of spinal

anesthesia due to relaxation of sympathetio tone, and sometimes, contributory mechanical

obstruction of venous return (Rashma K., 2012 and Mukherji S., 2008).

High doses or inadvertent intravascular injection may lead to high plasma levels and

related central nervous system toxicity characterized by excitement and/or depression.

(Weinberg G. et al.,2OO3)

Restlessness, anxiety, drzzrness, tinnitus, sleepiness, light-headedness, auditory

disturbances, blurred vision or tremors firay occur, possibly proceeding to conulsions.

However, excitement may be transient or absent, with depression being the first

manifestation of an adyerse rcaotion. This may quickly be followed by drowsiness meting

into unconsciousness and respiratory arrest (Mccloskey J. et al., 1992 and Spence A.,

2007),

1.5 Pharmacokinetics

Pharmacokinetics is the action of drugs in the body over a period of time; including the

processes of absorption, distributioq localization in tissues, biotransformation, and

excretion and its primary goal is to enhance efficacy and decrease toxicity ofa patient.

Bupivacaine pharmacokinaics have not been extensively studied owing to their rapid

breakdown in plasma <1 min (Veng-Pedeson P., i989),

Absorption and distribution are not important in controlling the onset of effect because

they are administered epidurally (Martinez M. and Amindon G.,20OZ).

1.5.1 Absorption

Absorption is the movement of drug into a blood stream and it's a primary focus in drug

development and medicinal chemistry since the drug must be absorbed before any

12

medicinal effects can take place. Moreover, the drug's pharmacokinetic profile can be

easily and significantly changed by adjusting factors that affect absorption (Egan W, et

a|,,2002).

Factors affecting systemic absorption of Bupivacaine are: dosage, drug-tissue binding,

local tissue blood flow and use ofvasoconstrictor (Tucker G. and Mather L., 1979).

Adding vasoconstrictors lowers the blood levels of Bupivacaine up to 30%, localized

neuronal uptake is enhanced because of higher local tissue concentrations and the risks of

systemic toxic effects are reduced (Eisenach J. etal.,1987).

1.5.2 Distribution

Drug distribution includes studying the pattern of distribution of drug molecules by

various tissues after the chemical enters the circulatory system. Because of differences in

pH, lipid content, cell membrane functions, and other individual tissue factors, most

drugs are not distributed equally in all parts of the body and it's a major parameter in

drug development and medicinal chemistry (William P. and Riviere E., 1989).

Bupivacaine is widely distributed after IV administration with 28 minutes half-time of

distribution. The initial rapid distribution phase leads to uptake into highly perfused

organs such as braiq liver, kidney, and heart, followed by a slower distribution phase

occurs with uptake into moderately well-perfused tissues, such as the muscle and

gastrointestinal tract (DeRossi R. et al.,2012).

1.5.3 Metabolism and Excretion

Drug metabolism is the chemical alteration of drug or substance in the presence of

various enzymes that acts as a catalyst and themselves not consumed in a chemical

reaction and thereby may activate or deactivate the drug while excretion is the process,

l3

act or function of discharging or ejecting waste product of metabolism and their studies

are highly important in drug delivay process as they play a major role and determine the

fate of the prospective drugs (Wilkinson G., 2005),

Since Bupivacaine in the uncharged form diffuse readily through lipid membranes, liule

or no urinary excretion of the neutral form occurs. Acidification of the urine promotes

ionization of the tertiary amine base to the more water-soluble charged form, leading to

more rapid elimination (Bianconi M., 1998 and Burm A. and Van Kleef J., 1984).

Bupivaoaine is converted in the liver to more water-soluble metabolites, which are

excreted in the urine; the amide linkage is hydrolyzed by liver microsomal Cytochrome

P450 isozymes with elimination triz of 3.5 hr. As a result, Bupivacaine toxicity is more

likely to occur in patients with hepatic disease. Many other drugs used in anesthesia are

metabolized by the same P450 isozymes, and ooncomitant administration of these

competing drugs may slow the hepatic metabolism of Bupivacaine (Sjostrand U. and

Wildman 8., 1973).

1.5,4 Hepatic Impairment

As Bupivaoaine is dealkylated in the liver, it is evident that defective liver function,

whether due to primary cellular rnsufficiency or to how liver blood flow in cardiac

failure or due to B-adrenoreceptor block, may both delay elimination and allow higher

peak plasma concentrations of both types of Bupivacaine. This is likely to be signifioant

only with large or repeated doses or infusions. So the rate of elimination will be higher

than distribution (Murphy E , 2005 and Padnit J., 201 1).

t4

1.5.5 Contraindications

According to the USP Bupivacaine is contraindicated in patients with a known

hypersensitivity to it or to any local anesthetic agent ofthe amide-type.

The following conditions preclude the use ofspinal anesthesia:

* Severe hemorrhage, severe hypotension or shook and arrhy'thmias, such as complete

heart block, which severely restrict cardiac output.

* Local infection at the site of proposed lumbar puncture.

* Septicemia (Hidebrand K. et a1.,2001and Reiz S. and Nath S., 1986)

1,6 Labor and Delivery

Spinal anesthesia has a recognized use during labor and delivery. Bupivacaine

hydrochloride, when administered properly, via the epidural route in doses 10 to l2 times

the amount used in spinal anesthesia has been used for obstetrical analgesia and

anesthesia (Owen M. et al.,1998).

Maternal hypotension has resulted from regional anesthesia. Bupivacaine produces

vasodilation by blocking sympathetic nerves. As a prophylaxis Physician can elevate the

patient's legs and position her on her left side which will help in preventing blood

pressure decreasing. The fetal heart rate also should be monitored continuously and

electronic fetal monitoring is highly advisable (Gaisar R. et a|.,1991).

It is extremely important to avoid aortocaval compression by the gravid uterus during

administration ofregional block to parturients. To do this, the patient must be maintained

in the left lateral decubitus position or a blanket roll or sandbag may be placed beneath

the right hip and the gravid uterus displaoed to the left (BelfrageP. et al.,1975; D'Angelo

K et al., 1994 and Loftus J. et al.,1995).

15

Spinal anesthesia may alter the forces of parturition through changes in uterine

contractility or maternal expulsive efforts. Spinal anesthesia has also been reported to

prolong the second stage of labor by removing the parturient's reflex urge to bear down

or by interfering with motor function. The use of obstetrical anesthesia may increase the

need for forceps assistance (GaisarP.. et al., 1997).

Bupivacaine has not reported diminished musole strength and tone for the flust day or two

of life as some local anesthetic drug products during labor and delivery may be followed

with iChestnut D. et al., 1988).

There have been reports of cardiac arrest during use of Bupivacaine hydrochloride

0.75% solution for epidural anesthesia in obstetrical patients. The package insert for

Bupivacaine hydrochloride for epidural, nerve block, e1c. has a more oomplete discussion

of preparation for, and management of this problem. These cases are compatible with

systemic toxicity following unintended intravascular injection of the much larger dose

recommended for epidural anesthesia and have not occurred within the dose range of

Bupivacaine hydrochloride 035V0 recommended for spinal anesthesia in obstetrics. The

0.75yo concentration of Bupivacaine hydrochloride is therefore not recommended for

obstetrical epidural anesthesia. Bupivaoaine Spinal (Bupivacaine in dextrose injection,

USP) is recommended for spinal anesthesia in obstetrics (Mclure H. and Rubin A., 2005

and Owen M. et a1.,1998).

1.7 Precautions

The USP have set precautions in order to assure a functioning IV pathway that the patient

should have LV, fluids running via an indwelling catheter, The lowest dosage of local

anesthetic that results in effective anesthesia should be used. Asoiration for blood should

It)

be performed before injection and injection should be made slowly. Tolerance varies with

the status of the patient. Debilitated, elderly patients and acutely ill patients may require

reduced doses. Reduced doses may also be indicated in patients with increased intra-

abdominal pressure (including obstetrical patients), if otlerwise suitable for spinal

anesthesia (Covino B. and Wildsmith J., 1998).

If hypotension occurs, it may be oontrolled by vasooonstrictors in dosages depending on

the severity of hypotension and response of treatrnent. The level ofanesthesia should be

carefully monitored because it is not always controllable in spinal techniques (Dong P. er

al. , 2OOO and Rolf N. eta|.,2000).

Because amide-type local anesthetics such as Bupivacaine are metabolized by the liver,

especially repeat doses which should be used cautiously in patients with hepatic disexe.

Patients with severe hepatic disease, because of their inability to metabolize local

anesthetics normally, dre at a greater risk of developing toxic plasma concentrations

(Murphy E., 2005 and Sjostrand U. and Wildman 8.,1973).

1.E Interactions

Bupivacarne levels may elevate while taking azole antifungal agents (Fluconazole,

ketoconazole, Itraconazole) as the metabolism ofBupivacaine by CYP3A4 is inhibited by

Azoles, this interaction should be considered (Tatro D., 2010)

1.9 Epidural

1,9,1 What is Ipidural Delivery

Epidural anesthesia is regional anesthesia that blocks pain in a particular region of the

body. The goal of an epidural is to provide analgesia, or pain relief, rather than anesthesia

which leads to total lack of feeling. Epidurals block the nerve impulses from the lower

l7

spinal segments. This results in decreased sensation in the lower half of the body

(Rosequist R and Rosenberg J.,2009).

1.9,2 Epidural Side Effects and Management

Side effects can include urine retentioq hypotensiorl sensory loss, and (rarely) pruritus,

respiratory depression and nausea (Block B. et a1.,2003).

Management of epidural side effeots could be by close monitoring of the patient, and

adhering to a standardized system and the use of and the use of color-coded, dedicated

epidural lines. Serious drug side-effeots with epidurals are usually the result ofaccidental

lV administration instead of epidural administration. There is also a small risk of

paralysis during the insertion of epidural catheter (Auroy T., et al., 1997 andLorriL. et

al.,2004).

As a result of media attention about epidurals, patients are often very concerned about

having an epidural inserted and this need to be discussed and informed patient consent

obtained. Patients also need to understand that as a result of epidural administration they

may become unaware of the need to urinate; a sensory awareness of the bladder can be

lost. Depending on local practice, patients will either be routinely oatheterized on

initiation of therapy or monitored for this adverse event and catheterized if needed (Jung

S. et a1.,2010 and Kane R, 1981).

1,9.3 Bupivacaine Epidural

Bupivaceine is used alone; a review of the literature suggests that Bupivacaine dose is

0,125-0.15% results in effective pain reliel Some literatures advice adding fentanyl

2pglml which maximize the Bupivacaine duration of effect @loeckinge B. et al., 1,995).

l8

Literature mentioned that a number of pharmacologic adjuncts, including bicarbonate,

epinephrine and rapid-acting local anesthetics have been added to solutions of

Bupivacaine 1o speed analgesic omet, reduce dose requirements, and lower risks of

toxicity. However, none of these are added in the local Jordanian markets. (MaGee D. er

al., 1983 and Raymond S. et a1.,7991).

Few Jordanian hospitals use Bupivacaine combined with Fentanyl, most of them use

single dose of Bupivacaine HCI (Macraine @) in a dose of 1.0-1.25 mg/ml.

lq

1,10 Determination of Bupivacaine in Biological Plasma

Chromatography techniques including LC, GC and FIPLC have been widely used in the

analysis of drugs; analysts often face the need to develop separation methods on daily

basis,

In order to develop the chnomatographic separation method a series of steps should be

made, these steps are summarized in figure 2. Good methods develop strategy should

require as many experimental runs as necessary to achieve the desired final results

(Snyder L. et a1.,20Q6)

2. Need for special HPLC Drocodure.

4. Choose LC method; preliminary

run; estimate bgst soparahon

6. Ch€ck for problems or requiromentfor special procedure.

8. Validate methcxl for release to

routine laboratory.

Figure 2: Steps in HPLC method development

Before developing a new method we need to review all the required information about

the sample.

Sample's information whioh will be important to be known:

1. Number of compounds.

2. Chemical structure of compounds,

3. Molecular weight of compounds.

4. pKa values ofcompounds.

5. Samples solubility.

6. Concentration range of compounds of samples in interest (llussein 4.,2012).

The chemical composition ofthe sample can provide clues for the best choice of initial

conditions for a HPLC separation, depending on the use made ofthis sample information,

two somewhat different approaches to HPLC method development are possible, some

chromatographers try to match the chemistry of the sample to a best choice of initial

HPLC conditions when they rely heavily on ther past experience (i.e. separation of

compounds with similar structure), other workers proceed to an initial chromatographic

separation paying little attention to the nature of the sample. These two kinds of HPLC

method development might be characterzed theoretical vs. empirical (Snyder er al and

Dolan G., 2006).

Once an initial separation has been carried out the choice of ensuing experiment can be

rnade on the basis of similar considerations (theoretical vs. empirical). Best strategy in

chromatography is based on blend ofthese two procedures, it should also be kept in mind

that the composition of many samples is not fully known at the beginning of method

21

development (e.g. samples containing impurities, degradation products, metabolites, etc.)

in these cases an empirical approach maybe the only option (Snyder el a/. and Dolan G,

2006).

1,11 Method Validation

According to the United States for Food and Drug Administration (USFDA) guidelines;

validation is the process of determining the suitability of a measurement system for

providing useful analytical data and it applies to all aspects of the measurement,

validation of the analytical method is a ralue of judgment in whioh the performance

parameters of the method are compared with the requirements for the analytical data

(FDA,2001).

lntraday validation means within the day and interday means between days (FDA" 2001

and Viswanathan er. al., 2007).

QC sample is a spiked plasma used to monitor the performance ofa biological analytical

method and to assess the integrity and validity of the results of the unknown samples

analyzed in an analytical batch (FD,\ 2001 and Viswanattan et. a1.,2007).

Standard blank is a sample of a biological matrix to which no analle has been added

that is used to assess the specifity of a bioanalytical method (FDA, 2001 and

Viswanathan e/. al., 2007).

Quantification range is the range of concentration, including ULOQ and LLOQ that can

be reliably and reproducibly quantified with acouracy and precision through the use ofa

concentration response relationship (FDA, 2001 and Viswanathan et. al.,2OO7).

22

To guarantee reliability and reproducibility ofthe assay for quantification of Bupivacaine

in human plasma the method was validated in ooncordance with the USFDA guideline

requirements (FDA 2001).

Validation of this procedure was performed in order to evaluate the method in terms of

accul.aay, preoisioq recovery, linearity of response, sensitivity, stability and specifity

(FDA, 2001 and Viswanathan et. al., 2007).

1.11.1 Standard Calibration Curye and Linearity

A calibration (standard) otnve is the relationship between instrument response and known

concentrations of the anallte.

A calibration curve is constructed using blank sample, zero-sample (blank with internal

standard) and 7 non-zero raterial standard samples, starting from lower limit of

quantification (LLOQ sample and ending with upper limit of quantification (ULOQ)

sample @DA, 2001 and Viswanatbarr et. al., ?007).

The following conditions should be met in developing a calibration curve (FDA, 2001

and Viswanathan er. al., ?0O7)

- Consist of at least six out of seven non-zero standards including the LLOQ and the

calibration standard at the highesr concentration.

- 15% deviation from nominal concentration for each concentration except the IIOQ

concentration 20% deviation is acceptable. At least ?5%o out of the non-zero standards

should meet the above criteria.

- The coe{Iicient of determination (rt-21 shoutd be equal or more than 0.98-

- Rejected points must not be the LLOQ or ULLOQ in the standard curve.

LJ

- During validation; the calibrant levels on regression line were shown to be linear. Best

fit calibration lines of ckomatographic response versus concentration were determined

by weighted le€st squares regression analysis.

- The correlation coeffrcient (R) must be consistently greater than 0.99 during the course

ofthe validation.

The linearity of an analytical method is its ability ivvithin a given range) to obtain test results

which are directly proportional to the concentration ofanalyte in the sample, Linearity is

usually expressed in terms of variance around the slope of the regression line calculated

according to an established mathematical relationship from the results obtained (FDA,

2001 and Viswanathan et. al.,2OO7).

1,11.2 Lower Limit of Quantification (LLOQ)

The lowest point on the standard calibration curve is accepted as the lower limit of

quantltation (LLOQ if the following criteria's should be met:

1. The interferences peak area in blank must be less ttan 2oo/o of analyte peak area in

LLOQ at the retention time ofthe analltes.

2. Analyte peak is identifiable, discrete and with an accuracy between 80% -

120%o in relation to the nominal standard concentration.

3. Precise on CV % of 200k and accuracy of 2ffl0, calculated among duplicate standards

when both are approved if a sample has an estimated concentration that falls below the

lower Limit of Quantification (LLOQ) its value was not extrapolated and, therefore,

reported as zero or BLQ @elow the Limit of Quantification) (FDA, 2001 and

Viswanathan et. al., 2007).

aA

1.11,3 Accurary and Precision

Accuracy of the assay was defined as the absolute value ofthe ratio ofthe baok calculated

amount mean values of the unknown samples and ther specified values expressed as a

percentage. For the LLOQ aocuracy should be in the range of 80 - 120/o to be accepted

while a range of85 - 115% is acceptable for other concentration range. Inter-day and intra-

day accuracy was estimated for the standard curve (FDA, 2001 and Viswanathan et. al.,

2007).

Precision of an analytical method reflects and describes the closeness of individual

measures ofan analltes when the procedure is applied repeatedly to multiple aliquots ofa

single homogeneous volume of plasma matrix. The precision of the assay was measured

by the intra-day and inter-day percent coefficient of variation 15% of the ooefficient of

variation (CV) is the maximum accepted determined CV at each concentration level

except for the LLOQ, where it should not exceed CVo% of 20% (FDA, 2001 and

Viswanathan &. al., 2007).

1.11.4 Recovery

Recovery refers to the extraction effioiency of an analytical method. Within the limits of

variability; recovery experiment should be made to all analyte subject to quantification

(TDA, 2001 and Viswanathan et. al.,2007).

f.11.5 Specifity and Selectivity

1.11.5.1 Speci{ity

Specifrty of the method should be examined by analyzing blank and standard zero

samples of the appropriate biological matrix (Saliva, plasma, Urine or any other matrix)

and it should be obtained from at least six sources, Each ofthe blank and zero standard

25

sample should be tested for interference, LLOQ is used to insure specifity. Batch

Specifity Test should include the following:

l- Calibration standards I quantification range must be obtained in calibration curve.

2- Six standard blank samples of the matrix from different sources extracted using the

proposed extraction procedure.

3- Six standard zero samples extraoted using the proposed extraction procedure (FDA,

2001 and Viswanathan et. al., 2007).

1.11.5,2 Selectivity

The extraction or precipitation technique of drug from plasma and chromatographic

conditions used shall provide an assay which is free of interfering endogenous plasma

components.

Plasma samples chnomatograms should be interferurg peaks free and that is how the

selectivity is evident.

Common Drug should be injected to check that there are no interferences at drug or

internal standard retention time. For example; Caffiene, Salicylic acid, Ibuprofeq

niootine, acetaminophen, metronidazole and sulfamethoxazole. If the method is intended

to quantit/ more than one analyte, eaoh analyte should be tested to ensure lack of

interference.

Acceptance criteria for both specifity and selectivity:

L In standard zero samples the area ratio (or height ratio) of interfering peak at the

anallte retention time (if found) should be less than 20Vo of the peak area (or herght)

ration of the anahe in the LLOQ standard used to calculate the calibration function.

.1,O

2. The response peak (or height) for the interfering peak at the intemal standard retention

time (if found) in "standard blank samples'' should be < 5%o of the mean IS peak area or

height) in the calibration standards used to calculate the calibration function.

3. The first group of twelve samples (which are 6 blank samples and 6 zero standard

samples) should meet the mentioned criteria.

4. If more than 1 sample out of each 6 samples (more than 2U/o) did not meet the

required mentioned criteria, the second group (which are for the same type of the first

group, 6 blank samples and 6 zero standard samples but of different source) should be

tested.

5. If more than 1 sample out of each 6 samples of the seoond group (more tl:u;n 20o/oi) did

not also meet the mentioned criteria, the method should be modified in order to eliminate

interference (FDA, 2001 and Viswanathan et. a1.,2007).

1.11.6 Dilution Integrity

The ability to dilute samples of concentrations above the upper limit of the linear

standard curve is demonstrated by accuracy and precision parameters. Liner dilution

should result in a linear analytioal response. For dilution integrity evaluation 3 QC

samples which are two times greater than QC-high and other 3 QC samples which are

four times greater than QC-high should be prepared, diluted into the calibration range

with the same biological matrix of the sample and tested, so the first dilution was

prepared by adding 125 pl ofQC sample with a concentration of6000 ngi ml and diluted

with 125 pl of blank plasma, and the second dilution was prepared by adding 62.5 itl of

QC sample with a concentration of 12000 ngl ml and diluted with 187,5 pl blank plasma,

after testing these samples the back measured concentrations were multiplied by the

27

dilution factor which is 2 for the fust dilution and 4 for the second one, 15% deviation of

the nominal peak of the QC-high is the maximum deviation accepted for the resulting

peak are (or heigh| (FDA, 2001 and Viswanatftn:n et. al.,2007\.

1.11.7 Stability

The stability results from long-term accelerated and stress which should be completed

according to ICH guidelines demonstrated adequate stability of the active substance.

It should be confirmed that the active substance is very stable when exposed to a variety

ofstressed conditions such as acid, base, oxidation, thermal, humidity and light exposure.

Drug stability in biological fluid is a function of the storage conditions, the chemical

properties ofthe drug in the matrix; stability procedures should evaluate the stability of

the analyte (FDA, 2001 and Viswanathan et. a1.,2007):

1. Short term stability which includes (bench top) storage, after going through freeze and

thaw cycles and Autosampler stabilrty.

2. After long-term (frozen at the intended storage temperature).

3. Stability ofanalyte and I.S in the master solution (stock solution stability) (FDA 2001

and Viswanathan et. al.,2007).

1,11.7.1 Short Term Stability

Short term stability refers to the stability status during the needed time for Bupivacaine

analysis in the plasma samples, including plasma thawing, sample preparation and

analysis of last sample of the anallical batch on HPLC (FDA 2001 and Viswanathan et.

al.. 2007).

28

1.11.7,2 Long "Ierm Stability

Long term refers to the stability status of Bupivacaine from the date of the flrst sample

collection to the date of last sample analysis, the volume of samples should be sufficient

for analysis on three separate occasions at least (FDA 2001 and Viswanathan et. al.,

2007).

1 l1.7.3 Stock Solution Stability

Stock solution stability refers to the stability examination of analytes stock solutions at

different conditions such as: room temperature, refrigerator or deep freezer (FDA5 2001

and Viswanathan et. al., 2007).

l.l2 Internal Standard:

An internal standard is a known amount of a compound which is different from the

analyte and is added to the unknown. Signal from analyte is compared with signal from

the internal standard and then we can find the analyte's concentration. (Song T. er al,

19e8).

The internal standards advantages are especially for analyses in which the quantity of

sample analyzed or the instrument response varies slightly from run to run for reasons

that are difhcult to control. For example, liquid flow rates that vary by a few percent in a

HPLC experiment could change the detector response. A calibration curve is only

accurate for the one set of conditions under which is obtained. However. the relative

response ofthe detector to the analyte and standard could increase because of a change in

solvent flow rate as well as the signal from the analyte. As long as the concentration of

standard is known, the correct concentration of analyte can be derived. Internal standards

are widely used in HPLC because the small quantity of sample solution injected into the

29

HPLC is not very reproducible in some experiments (Wal P et al., ?QIO).

Internal standards are also desirable when samples loss can occur during sample

preparation steps prior to analysis. If a known quantity of standard is added to the

unknown prior to any manipulation, the ratio of standard to analyte remains constant

because the same fraction of each is lost in any operation as they both will face the same

analytical steps in the analysis procedure (Wal P. et al,, 2010).

The required features of an internal standard that it should be chemically inert, similar

chemical structure from the analyte, the internal standard should elute out faster. It needs

to add the same amount of same compound to all calibration and samples. The internal

standard must have resolution and detectability and mimics analytes in pretreatment steps

NrralP. et al.,2OlO).

1.13 Prwious analytical studies for Bupivacaine Determination in Biological Fluid

There are few reported methods for the determination of Bupivacaine levels in human

plasma, examples are:

A method for determination of Bupivacaine using HPLC, and the possibility of

pharmacological interferences produced by seven commonly used drugs administered

before, during and after surgery: diazepanl midazolam, epinephnine, naloxone,

flumacenil, atropine and ephedrine was developed. The method has an average recovery

of 102.8 + 5.4yo. The detection limit is 0.125 pg/ml The within-day coefficient of

variation is 5.88% and the between-day coefiicient of variation is 159/0. No drug

interferences at generally encountered serum concentrations were found (Ruzafa A. et al.,

1991), Ruzafa A. etal. method t:u;s the disadvantage of low sensitivity.

30

Huy-Riem and others developed a method in 1984 for the determination of Bupivacaine

and was performed on a HPLC equipped with a variable wavelength UV detector set at

210 nm, the IIPLC column was packed by forcing 1 gm of micro-bondapak C18-10

micron in 30 ml of toluene:cyclohexanol (l:2) aI 60"C into a stainless steel column (15

cm; internal diameter 0.32 cm) with methanol at flow rate 5 mV min. The mobile phase

consisted ofacetonitrile, 0.05 M NazIIPOa, solution was adjusted at pH 5.80 (25:7 5 wlv).

The flow rate was 0.9 mV min, the data showed little day to day rariability in slopes and

intercepts with good linearity (r!0 9996) over the conoentration ranges studied. Within-

day and between days reproducible measurements are between 0.2-40%. Recovery was

more than 9370 and retention time of 8,7 min (Huy-Riem H. etal., 1984).

Huy-Riem H. et al. method has the disadvantages of: low recovery, low sensitivity and

long retention time.

Another coupled-column liquid chromatographic method for determining the {iee

concentration of ropivacaine and Bupivacaine in blood plasma was developed. Following

adjustment ofthe temperature and pH, the plasma samples were uhrafiltrated.

Ropivacaine or Bupivacaine in the ultrafiltrate was determined by direct injection into a

coupled-oolumn HPLC systenq consisting of one reversed-phase and one ion-exchange

column.

The system was highly seleotive. Ropivacaine and Bupivacaine were detected by UV at

210 nm. The limit of determination was 10 nM and the inter-assay precision at a

concentration level of about 100 nM was 6% (RS.D., n : 30) for ropivacaine and 70%

(RS.D., r = 30) for Bupivacaine (Arcidsson T. and Eklund E., 1995).

Arcidsson T. and Eklund E. method has the disadvantage oflong retention time.

JI

In 1993 a reversed- phase HPLC method has been developed and validated for the

quantification of Bupivacaine in human plasma. Bupivacaine and the internal standard,

lidocaing are extracted from alkalinized plasma using n-hexane before chromatographing

on a reversed-phase system. The mobile phase comprises 62 parts of 0.05 M phosphate

buffer and 38 parts of methanol at pH 5.9 and it is pumped at 1.0 mVmin, The retention

times of Bupivacaine and lidocaine were observed to be 3.8 and 5.9 min respectively.

Ultraviolet deteotion at 254 nm enabled a limit of detection of 25 ng/ml to be aohieved.

The reproducibility of the method was good at 1000 nglml (C. Y .=4.3%, n=6). The

method is linear from 50 to 3200 nglml (Morello 1. et a|.,1993).

Morello I. et al. method has the disadvantages of low sensitivrty and using phosphate

buffer which has negative effects on HPLC instrument on the long run.

As shown there are few analytical methods reported for the determination ofBupivacaine

in human plasma and this work will concentrate on developing a new HPLC-IIV method

that is rapid, sensitive, selective, and reproducible to overcome the disadvantages found

in the previous methods.

1,14 The Objectives

Objectives ofthe current study include:

1- To develop a sensitive TIPLC-W method for quantitative determination of

Bupivacaine in human plasma.

2- To validate the method in terms of linearity, sensitivity, accuracy, precision, recovery,

specifity, selectivity, dilution integrity and stability according to the United States food

and drug administration.

3- To study the feasibility of applying this method for the determination of Bupivacaine

in pregnant women during labor-

*Note: This thesis was intended to be practically applied on pregnant women. protocol

and consent forms were ready and approved by the ethics committee. We waited for more

than a year but unfortunately for no justified reason none ofthe Jordanian hospitals gave

the approval to take cases from

JJ

Chapter 2

Expiremental

2 Expiremental

2.1 Chemicals and Reagents

- Bupivacaine Hydrochloride was obtained from JCPR; B.N.: 25, Re-stand ardization date. 6l

9/2013.

- Bisoprolol as intemal standa.rd was obtained from Taboukl B.N: RD-WS304, ED:

28/12/2012.

- The blank plasma was collected from Blood Bank (harvested from donors), Islamic

Hospital (Ammaq Jordan), plasma was obtained by centrifugation of blood treated with

sodium heparin.

- Methanol HPLC grade (Fischer, USA); B.N.: 1 156250, ED: tl 91 201,3.

- Aoetonitrile HPLC grade (Fischer, USA); B.N: 1155826,ED:158/2013.

- Phosphoric Acid 85%. (Fischer, USA); B,N: 08E050526, ED: Il 5/ ZOt3.

- Quality deionized wateq HPLC grade; B.N: 120?702, ED: t 5/ l)l 2013.

- Triethylamine(TEA) (ACROS, Geel - Belgium); B.N: 402345634,ED:21 I0l 2013.

2.2 Instrumentation

- A Dionex HPLC auto-sampler system composed of a constant solvent delivery system

@580), a 100 pL frxed volume injeotor (Rheodyne 7125), W Detector (t\D340S), I:

202 nn1 Autosampler (ASI-100), Computer System, MicroSoftWindows 2000, Hypersil

Thermo Electron Corporatiorq BDS C-18 Column ( 150 mm x 4.6 mm, 5pm).

- Balance: Sartorious, model M82355-

- Centrifuges: Eppendorf centrifuge model 5417 C and 57O2k

- Vortex Mixer: EKA" manual.

35

2.3 Method Development

2.3.1 Selection Methods for Analysis

The effect of different chromatographic conditions on the separation of Bupivacaine were

studied; pI! ion pair concentratiorq composition of mobile phase and column were studied

to find the most proper method for the determination ofBupivacaine in plasma.

Method ofanalysis was developed as indicated in the tables 2-7 the resultant chromatograms

are attached in the appendices:

Table 2: Chromatographic conditions for method (1)

Table 3: Chromatographic conditions for method (2)

Column BDS Hlpersil Crr Colunn (150mm x 4.6mm, 5pn1 ThermoElectron Corp.

Solvent system{Mobile phase)

25.Oo/o Acetonrttile, 75.0/o water, ad.justed at pll:3.6 usingH:PO+

Detection UV Detector l" : 220 nm.

Injection Volume 75 micro litters

Flow Rate 1.0 mVmin.

Oven Temperature 2s"c

Column BDS Hlpersil Crs Column (150mm x 4.6mn! 5prq ThermoElectron Com.

Solvent system(Mobile phase)

30.0% Acetonitrile, 70,0Vo water,adjusted at pH:2.5 usine H3POr

I ml/L Triethylamine,

Detection UV Detector ),: 220 nm.

Iniection Volume 75 micro lrtters

Flow Rate 1.0 mVmin.

Oven Temperaturc 25"C

-to

Table 4l Chromatographic conditions for method (3)

Column BDS Hypersil Crs Column (150mm x 4.6mrr1 5pn1 ThermoElectron Com,

Solvent system(Mobile phase)

30.0% Acetonitrile, Tj.U/o water, 1 myl triethylamine, adjustedat pH:2,5 using H:PO,I

Detection UV Detector ?',: 220 nm.

Iniection Volume 75 micro litters

Flow Rate 1.0 mVmin,

Oven Temperafure 40'c

Table 5: Chromatographic conditions for method (4)

Table 6: Chromatographic conditions for method (5)

Column BDS Hypersil Crs Column (150mm x 4.6mnu 5prq ThermoElectron Corp.

Solvent system(Mobile phase)

30.ff/o Acetonitrile, 70.U/o water, 1 ml4l, triethylaming adjustedat pH=2.5 using l{:POr

Detection W Detector l" = 220 nm.

Injection Volume 75 micro litters

Flow Rate 1.0 mVmin.

Oven Temperature 50"c

Column BDS Hypersil Cqs Colurnn (150mm x 4.6mm, 5pnr, ThermoElectron Coro.

Solvent system(Mobile phase)

30.0% Acetonitrile, 70.0To water, I ml./L triethylamine, adjustedat pH=2.5 usrng H:PO+

Detection UV Detector l, = 210 nm.

Injection Volume 75 micro litters

Flow Rate 1.0 mVmin.

Oven Temperature 40'c

Table 7: Chromatographic conditions for method (6)

Column BDS Hypersil Crs Column (150mm x 4.6mm, 5pnr, ThermoElectron Corp.

Solvent system(Mobile phase)

33.5% Acetonitrile, 66.5 % water, 1 ml/L triethylaming adjusted

^t pH1.5 using H:PO+

Detection UV Detector l: 210 nm.

Iniection Volume 50 micro litters

Flow Rate 1.0 mVmin.

Oven Temperature 25"C

2.3.2 Chromatographic Conditions

Cluomatographic conditions in this experiment are illustrated in table 8:

Table 8l chrcmatographic conditions of Bupivacaine analysis

Column BDS HypersilElectron Corp.

Cre Column (l50mm x 4.6mnL 5pnt, Thermo

Solvent system

fMobile phase)66.5 9'o water, 33.5 % Acetonitrile, I ml,il- Triethylamine, pH :2.5 adiust with H:PO+

Detection UV Detector i.: 202 nm.

Iniection Volume 50 micro litters

Retention Times*BisoprololBupivacaine

3.1 - 3.3 minutes Qnternal Standard)3.5 - 3.7 minutes

Flow Rate 1.0 mVmin,

Oven Temperature .10"C

Retention times dependent upon HPLC conditions.

2,4 Preparation of Stock Solution and Serial dilution

2,4.1 Prepamtion of Stock Solution of Bupivacaine

10 mg of Bupivacaine was dissolved in 20 ml of methanol IIPLC to get 500

solution ofBupivacaine and labeled as Bupivacaine solution A

2.4.2 Prepatation of Stock Solution of Bisoprolol (Internal standard)

10 mg of Bisoprolol (Figure 3) was dissolved in 10 ml of Methanol HPLC

pglml stock solution A of Bisoprolol:

pg/ml stock

to get 1000

t-fuA[^o"Y

\r-.,--....-Orrr'-lFigure 3: Bisoprolol Chemical Structure

2.4.3 Preparation of Internal Standard Working Solution @isoprolol)

0.05 ml from solution (A) withdrawn and diluted in 50 ml of Methanol to give solution (B)

that contains 10 pglml.

2.4.4 Preparation of Bupivacaine in Plasma

2.4.4.1 Preparation of Standard Calibration Curve

Spiked plasma samples were prepared by using Bupivacaine solution A (stock solution)

which contains 500 pg/ml of Bupivacaine.

Table 9, describes the dilution procedure to prepare serial working solution (W.S), and

sprking of 500,0 pglml from W.S in a subsequent step into 10 ml of plasma to get 7

levels of standard calibration curve.

39

.\^

EA!N 8 8

C.l

-E-EFEFa,=

F€re N c.l o.l c-l c.l a.l N

U (J atO (J d

O (J \oU

F.(J

o,

+€ d E

;;,58 -i c{N

ol 8c.l

.E3EF a.=

= 6 .,:r

=& 2^s-€!=a - v) .= clo O

N $

'':itrcoI(D*3 8 o O

z

(t) a Na (A!+a a (A F-a

{l

q,

I

:t

a

F

Samples of the standard curve in plasma were prepared using seven conc€ntrations, not

including zero and these concentrations are:

10, 50,25q 500, i000, 2000 and 4000 ng/ml.

Each concerrration of the plasma samples was divided to 0,25 ml in an eppendorf tube and

kept at -40 "C, standard samples were given daily together with the quality control samples.

2.4'4.2 Preparation of QC Samples

Spiked plasma samples were prepared using Bupivacaine solution A (stock solution) which

contains 500 pglml ofBupivacaine (stock A).

Table l0 shows data of preparation of Quality Control samples in plasma, quality control

samples were kept at - 40 0C until required for subsequent analysis.

4l

-!a ^vtrtr5n O

_E3=-a-

trrE9a^sEi3 O 8

QF.]O

zO

b,)

O

.H: e+'! d E

f;58 t.r

-E.E3E

EF-EJ ?^^e -cs lJ

=a-(h-=RO

.,ll troon

ah .= 8

z

(t) aNa cn

q,

tr

L

|-

F

t

2.5 Sample Preparation and Method of Extraction Development

2.5.1 Solvent

Plasma sample cant be directly injected to FIPLC system. It should be in solution and that is

why solvent should be used. Three Drfferert solvents were tested for sample prepamtion and

method of extraction; diethyl ether, /a/r-butyl-methyl-ether and diethylether:

dichloromathane (70: 30).

2.5,2 Buffer

Buffer s used to enhance the baseline, buffers 1.0 M NaOH and 1.0 M Na2CO3 were

studied and compared with water.

2.5.3 Vortex Mixing Duration

In the extraction procedure two vortex mixing is used. Flrst vortex mixing was studied at 2,4

and 6 minutes and the second vortex mixing was studied for 1,2,4 and 6 minutes.

2.5.4 Back Extraction

Back extraction was used to avoid reconstitution, formic acid with concentrations of (0.1%,

0.5Yo and 1%) and perchloric acid with concentration of (1, 2.5o/o and 5%) were studied.

43

2,6 Method Validation

The validation was performed on three separate days, with seven standard calibration

level lines (not including zero) on each day. Each day of validation included plasma

samples representing blank, Zero, standard calibration curve, l0 replicates of Quality

Control (QC) samples (Q.C. I-ow, Q.C. Md, Q.C. High) The validation parameters have

not to exceed the limits by the FDA Guidance for Industry.

2.6,1 Recovery

The percent recovery was determined by measuring the absolute peak area of

Bupivacaine and IS fiom a plasma sample prepared according to the method ofanalysis.

Plasma samples containing concentration of QC Low 30 nglml, QC Mid 1500 nglml and

QC Htgh 3000 nglml analytes were prepared in triplicate. The absolute peak areas

obtained from the injections of the prepared plasma standards were compared to the

absolute peak areas of an equivalent aqueous standard, which was prepared to contain a

concentration of analytes standard assuring 100% recovery, and same treatment of the

LS. The recovery edent of analytes and IS should be consistent, precise, and

reproduoible.

2.6.2 Standard Calibration Curwe and Linearity

2.6.2.1 Linearity

The linearity was measured by plotting the peak area ratio (analytes pak areal I.S peak

area) versus specified concentrations. The data was fitted to a straight line with a linear

best-fit line was used to measure the concentrations ofall samples throughout the batch.

Five calibration curves consisting ofa blank, zero and seven non-zero standards prepared

in human plasma were analvzed.

44

The concentrations of calibration standards cover the range from LLOQ to the highest

expected concentration.

The calibration standards were prepared as described in the methodology and the linearity

was evaluated by calculating the linear regression (conelation ooefficient, n$, ana Uy

evaluating the back calculated concentrations ofthe calibration standards,

2.6,2.2 Standard Calibration Curve Response

The linearity of the method was illustrated by weighted squares regression analysis of

standard plots associated with a seven point validation, graphical plot of analysis

response was made by plotting peak area ration of standards versus concentration for

each sample set.

2.6.3 Sensitivity

The LLOQ in the calibration plot should provide that its chnomatographic response

(analytical signal to noise ratio in the calibration curve) must be > 5 times that of blank

responsg with accuracy 80-l2oyo and precision s20%. No attempts were made to obtain

measurements below this limit. Ten replicates were tested at LLOe samples from

different plasma sources to assess sensitivity. Sensitivity samples were prepared along

with the calibration curve at the same time (FDA,, 2001 and Viswanathan et. a1.,2007).

2,6.4 Accuracy and Precision

The reliability of the method in terms of accuracy and precision was evaluated from

chrornatograms obtained from pooled plasma samples spiked with a fixed concentration

ofthe IS and different volumes ofquality control low, mid and high levels ofBupivacaine.

45

2.6.4,1 Accuracy

The accuracy was measured using ten replicates for each of QC levels; low, mid and high

for Bupivacaing peak areas of replicates were analyzed daily and the concenfations were

back-calculated by employing the regression equation established on the same day.

2,6.4.2 Precision

The precision of the assay was measured by the intra-day and inter-day percent

coeffioient of variation. 15o/o of the coeffioient of variation (CV) is the maximum

accepted determined CV at each concentration level except for the LLOQ, where it

should not exceed CVolo of 20%. Precision was estimated from QC samples and standard

ourve levels (FDA, 2001 and Viswanathan et. a|.,2O07).

2,6.5 Inter and Intra-day Accuracy and Precision

2,6.5,1 Intra-day Accuracy and Precision

For the assay, Intra-day accuracy and preoision was measured by aralyzing ten replicates

for each of QC level (Low, Mid and High) in the batch for Buprvacarne.

2.6.5.2 Inter-day Accuracy and Precision

Ten replioates for each of QC levels (Low, Mid and high) in the batch of Bupivacaine

were analyzed to measure the inter-day accuracy and precision in the batch for

Bupivacaine, the above was applied for 3 successive days. Peak areas ofall replicates of

each concentration were measured and concentrations were back-caloulated by

employing the regression equation established on the corresponding day.

46

2,6.6 Recovery

Recovery \ras tested by preparing triplicates from each QC level of plasma samples and

triplicates from each QC level prepared in the mobile phase (out of extraction procedure)

FDA,, 2001 and Viswanathan et. a1.,2007).

2,6.7 Specifity and Selectivity

2.6.7,1 Specifity

Specifity of the method was examined by analyzing blank and standard zero samples of

the biological matrix (plasma) and it was obtained from six different sources, Each ofthe

blank and zero standards were tested for interference.

2.6,7,2 Selectivity

Selectivity test was done by the analysis of six different most common drugs that most

people might take and it was done to make sure there is no sisnificant interference at

Bupivacaine and Bisoprolol retention time.

For selectivity common drugs were injeoted to check interferences at drug or internal

standard retention time e.g (caffiene, acetylsalicylic acid, acetaminophen, ibuprofeq

nicotin, mefanimic acid and sulfamethoxazole).

2.6.8 Dilution Integrity

For the evaluation of dilution integrity three QC samples whioh are two times greater

than QC-high and other 3 QC samples which are four times greater than QC-high were

prepared, diluted into the calibration range with the same biological matrix of the sample

and teste4 15Vo deviation of the nominal peak of the Qc-high is the maximum deviation

accepted for the resulting peak area (or herght) (FDA, 2OOl and Viswanatha n et. al.,

2007).

47

2,6,9 Stability

Bupivacaine is a stable analyte based on its physicaVchemical (physiooohemical)

properties it's breakdown is not expected to occur under the normal conditions of storage

on analysis (Gaurav K. and Geeta C., 2011).

Plasma samples containing QC Low, QC Mid and QC High concentrations were prepared

in order to determine the analyte's stability under the analytical conditions as indicated.

The analytical results for the stored samples were aru,lyzd against a freshly prepared

standard solution. The stabilily ofanalyte was determined as follows.

2,6.9.1 Short Term Stability

2,6,9.1.1 Room Temperature (Bench top) Stability

Tkee QC samples (triplicate) of each level (Low, Mid and High) were thawed unassisted

at room temperature (RT) for 8 hours based on the time expected for the samples to be

maintained in the temperature during handling. Another tluee eC samples of each level

(Low, Mid and High) were prepared and thawed at the right time (0,0 hour samples)

unassisted at RT.

After that all of the samples (total of 18 samples) were processed using the processed

extraction prooedure and quantified against a calibration curve.

The mean concentration of the target samples were compared to the one obtained from

the 0.0 hour samples to oalculate the stability %o of each cono. Level (FDA, 2001 and

Viswanathan et. al., 2007 ).

2,6.9.1.2 Post-Operative Stability (Auto-Sampler Stability)

Post-preparative stability means auto sampler stability. The stabitity of processed

samples, including the required resident time in the auto sampler, was determined. The

48

stabilrty of Bupivacaine and IS were assessed over the anticipated run time for the batch

long in validation samples by determining concentrations on the basis of original

calibration standard. Triplicate for each level of QC samples (low, medium and high)

were prepared as mentioned in the sample preparation procedure together and kept at the

auto sampler for 0.0 and 24.0 hours. The accuracy determined at each concentration level

should not exceed 15% (FDA,, 2001 and Viswanattan et. al.,2007).

2,6,9.1.3 X'reeze and Thaw Stability

Freeze and thaw stability was investigated over thLree cycles in which analytes stability

were determined after each cycle of the three freeze and thaw cycles. Triplicate for each

level of QC samples (low, medium and high) were stored at - 40 "C for 24 hours and

thawed unassisted at room temperature, when completely thawed, the samples were

frozen again for 24 hours under the same conditions. The freeze-thaw cycle should be

repeated two more times, and then analyzed after each cycle and oompared together on a

freshly prepared standard curve in order to determine stabilrtf/0. Ifanalytes are unstable

at the intended storage temperature, the stability sample should be frozen this time at -

70'C during the three freeze and thaw cycles (FDA, 2001 and Viswanathan eL al.,2007).

2.6.9,2 Long T erm Stability

Long-term stability should be determined by storing tkee samples from each QC level

(Loq QC Mid and QC High) at - 40"C. Each concentration should be analyzed daily

from day 1 until the end of the study and then analyzed at the end of the unknown

volunteer plasma sample (and since I could not achieve volunteer plasma samples it was

analyzed at the end ofthe method preparation and validation).

49

The conoentrations of all the stability samples were compared to the mean of back

calculated values for the standards from the first day of long-term stability testing.

There should be not more than 15% decrease in the analytical results (FDA, 2001 and

Viswanathan eL al., 2007).

2.6.9,3 Stock Solution Stability

The stability of stock solution ofBupivaoaine and IS were evaluated at room temperature

for at least 24 hours. Samples then refrigerated and the stability ofthe refrigerated stock

solutions was determined after 14 days, then the stability were tested by comparing the

instrument response with that of freshly prepared solutions with acceptance criteria 85-

115%, the samples from Bupivacaine and bisoprolol were analyzed to cover the period of

analysis,

50

Chapter 3

Clinical Study Design

3 Clinical Study Design

This work was planned to have two parts; analyical part which is the method

development and validation and clinical part in which 50 cases each of 5 different

blood samples were supposed to be taken (details described later), case report form,

Informed Consent Form and Protocol were all prepared and approved by the ethics

committee (they are attached in the appendices). Unfortunately the clinical part was

not done and we were not able to take even one sample for many obstacles we faced.

We waited for more than one year with daily follow up with no positive reply. We

will keep fighting for the whole idea to be completed as planned for the sake of the

future generations.

3.1 Ethics

3.1.1 Independent Ethics Committee (IEC)

Copies of study design, a sample of a case report form (CRF), consent form, and

information to subjects, were submitted to the IEC at Farah Hospital.

3.1,2 Declaration of Helsinki

The study will be performed in accordance with the relevant articles of the

Declaration of Helsinki (1964) as revised in Tokyo (1975), Venice (1983), Hong

Kong (1989), and Somerset West, RSA (1996) and Edinburgh (2000). the "Note of

Clarification on Paragraph 29" added by the WMA General Assembly, Washington

(2002) and the "Note of Clarifrcation on Paragraph 30" added by the WMA General

Assembly, Tokyo (2004) ( Note of Clarification on Paragraph 30 added), and rhe 59'h

WMA general Assembly, Seoul, October 2008.

52

3.1.3 Ethical, Legal and Administrative Aspects

Prior to the initiation ofthe study, the protocol, the patient information leaflet and the

informed consent form will be submitted to the Ethics committee (IRB/IEC)

responsible for the review and approval.

In case of approval the study can be started immediately after a copy of the ethics

approval / favorable opinion has been sent to the Sponsor'

If significant protocol changes are necessary which require the preparation of an

amendment, this amendment has to be submitted to the IRB/IEC for approval'

The study will only be performed when full approval has been obtained from the

IRB/IEC and copy ofthe certification has been received, including a list ofthe actual

members of the IRBAEC.

3,1.4 Subject Information and Consent Form

Before being admitted to the clinical study, each subject should be given her consent

to participate by signing the informed consent form, enough time and explanation of

the nature, purpose and likely duration ofthe study and should be advised about any

discomfort and possible adverse effects on her health or well being that the

supervising doctor believes may result.

Following points were covered in the informed consent form and the information for

subj ects:

* The objectives ofthe study.

* The fact that the subject will not directly receive health benefits from the study and

all of the given information should be fully true and subject should fully comply with

the directions given.

* Drug product information, indications and adverse reactions ofthe drug'

* The freedom to ask for further information at any time.

JJ

* Study procedure; the starting time of the study, information required, samples that

will be collected (type, volume and samples' time) and follow-up examination

measurements.

* Subject's qualifications that should be met in order to be included in the study.

* The subject's right to withdraw from the study at any time without giving

reasons (except for AEs) and without jeopardizing the further course of

treatment,

* The confidentiality ofthe personal inlormation given in the study

Both documents, the informed consent form and the information for subjects are

presented in Jordan Center for Pharmaceutical Research (JCPR) Amman-Jordan, in

English and Arabic version. Personal information will be treated as strictly

confidential and will not be made publicly available,

The confirmation of the subject's consent will be certified by the signatures both of

the subject and ofthe investigator as well as an independent witness.

The investigator will not to undertake any investigations specifically required only for

the clinical study until valid consent is obtained.

To ensure medical confidentiality and data protection, the signed informed consent

forms and the source data of laboratory and other examinations will be remained with

the investigator. They will further be kept there for at least 15 years after the

completion of the trial. The investigator allows the documents to be inspected on

request and affirmed - by signing and dating - in the CRF's that informed consent had

been obtained.

The signed informed consent forms were checked by the monitor in the course of

onsite monitorinq.

54

3.2.4 Choice of Sampling

The duration of sampling (4 hours) was chosen taking into account the terminal

elimination half-life of the compounds Bupivacaine is approximately 3 5 hr (Martinez

M. and Amindon G., 2002).

One sample will be taken before the drug delivery to be considered as zero point,

another sample will be taken half an hour after drug administration and immediately

post delivery for Bupivacaine corrcentration checking.

One sample will be taken post delivery from the placenta to check if the drug crosses

the placenta (as it should not), the sample was chosen to be taken from the placenta

and not directly from the baby to avoid harming the baby.

3.2.5 Selection of Patient

50 pregnant women, who fulfrll the inclusion criteria (Section f .2.5.2), do not present

any of the exclusion criteria (Section 3.2.5.3), and who have given wdtten patient

informed consent (Section 3.I 2), will be entered into the study.

Drug assays and statistical analysis of the data will be performed on all plasma

samples ofpatients who complete the study according to the protocol, Patients will be

selected and recruited from a group ofpregnant women.

3.2.5.1 Justification oflnclusion and Exclusion Criteria

The criteria are set to en$ure a homogeneous pregnant women population without

accompanying diseases interfering with the conduct and scientific evaluation of the

study. Additionatly, the criteria have been selected to minimize risk to the patients'

well-beins.

56

3.2.5.2 Inclusion Criteria

Only pregnant women presenting all of the following criteria should be enrolled in

the present fial:

* Pregnant female.

* Age between 18 and 45 years.

* Physically and mentally healthy as judged by means of a medical and standard

laboratory examination.

* No allergic history to Bupivacaine, or any ofthe excepients.

* No alcohol consumption or a history of alcohol of alcoholism or drug/chemical

abuse.

* Informed consent given in written form according to section 3.1.2 of the study

protocol.

3.2.5,3 Exclusion Criteria

Pregnant women presenting any ofthe following will not be included in the trial:

* Allergic diathesis or any clinically significant allergic disease

* History ofallergic response to Bupivacaine.

* Presence or history of heart failure, liver diseases, muscular edema, cardiovascular

disease or cardiovascular risk factors.

* Severe renal or hepatic insufficiency.

* Participation in another clinical trial within the last 2 months.

* Donation of blood or plasma within the last 2 months.

* Clinically relevant abnormalities at physical examination or laboratory tests.

* Any chronic disease which might interfere with absorption, distribution,

metabolism or excretion ofthe drug.

* Intake any other anesthetic, narcotic, or any drug during the delivery.

57

* Knowledge to have hepatitis B infection or HIV, or carrier of the respective

antigens Evidence of an uncooperative attitude.

* Legal incapacity and/or other circumstances rendering the patient unable to

understand the nature, scope and possible consequences ofthe study.

3,2,6 Other Conditions

Any concomitant disorder not interfering with the exclusion criteria listed in section

3.2.5.3, the relevant medication and any changes in the concomitant disorders and/ or

medication have to be documented appropriately in the Case Report Forms.

Additional illnesses present at entry into the study are regarded as concomitant

illnesses and generally as an exclusion criterion. Illnesses occurring during the study

period (Inter-current illnesses) are to be regarded as adverse events and will each be

documented on a separate adverse event form.

3.2.7 Patient's Obligation

Patients must adhere to the protocol instructions as presented in the Informed Consent

Form, section 3.1 .2. The pregnant women must notify the clinical investigator of all

adverse events experienced or ifthey are unable to follow the study procedures.

The clinical investigator must be informed by the patient, or any other responsible

persoq of the occurrence of any serious adverse event, or if the patient requires

additional medication of any kind, irrespective of the time ofday or night.

3.2.8 Drug Handling and Accountability

This is a controlled drug and it's not available at JCPII it is only available at the

hospital and will be given under medical supervision ofthe gynecologist.

Before this medication given to pregnant women by gynecologist it will be checked

by the investigator to be sure that the patient given the right medication and

documented that by clinical investigator and a second medical professional.

)6

All empty ampoules must be collected in labeled plastic bag as in section 3.2 8 2 and

accounted for at the end ofthe study. A1l that will documented in proper way.

3,2.8,1 Dosage and Duration of Treatment

Pre-delivery time within one to two hours the medication will be given to the patients,

all patients will receive a single dose of 1.00-1.25 mg/ ml Bupivacaine @CL)/ 2 00-

5.00 pg as an epidural injection.

3.2.8,2 Labeling of Empty AmPoule

Labeling of empty ampoules will be as shown in figure 4:

LABELING OF EMPTY AMPOULEStudy No. : Bttp-07 -2012Patient No.:DrugName:manufactured by.......,..............Dose: 1.00-1.25 mgl ml Bupivacaine(HCl).epidural injection, single dose

BatchNo.:...Expiry Date:Storage: keep in closed locker. [n hospital.

Figure 4: Labeling of Empty AmPoule

3.2.9 Compliance

Administration of the study medication will be performed by the clinical investigatof

and supervised by a second medical professional to ensure the drug intake and in

order to verifu the compliance of the patients. The administration of the study

medication is to be documented in the CRF and certified by the Investigator.

fv

3.2,10 Concornitant Medication

Administration of any other medication than the study drug is defined as concomitant

medication and will be carefully documented in the CRF as previous and/or

concomitant medication, concomitant medication is generally not allowed for the

duration of the trial. If this is considered to be necessary for the patient's welfare it

may be given at the discretion of the Investigator. The patients have to inform the

investigator about any intake ofother drugs in the course ofthe trial.

It will be the clinical Investigator's decision whether the patient is withdrawn or if

she is allowed to continue the study. This decision depends on the type of illness and

the kind of drug to be used.

Ifthe clinical investigator decided that the patient must be withdrawn from the study,

the intake ofconcomitant medication has to be documented in the adverse event form

and "drop-out sheet" in the case report form specifying the substance, dose, time and

reason for use of concomitant medication and is to be regarded as an exclusion

criterion.

3,2.11 Rescue Medication

Since respiratory arrest may result either through direct depression ofthe respiratory

centre or as the result of hypoxi4 primary attention should be given to the

establishment of adequate respiratory exchange through provision of a patent airway

and institution of assisted or controlled ventilation.

The opioid antagonist, naloxone, is a specific antidote Naloxone (see package

information for full information) should be administered intravenously,

simultaneously with respiratory resuscitation. As the duration of effect of naloxone

60

may be considerably shorter than that ofBupivacaing repeated administration may be

necessary.

3.2,12 Em ergency Procedures

Emergency equipment and drugs will be available within the clinical unit. In case

emergency :reatment will be necessary, the treatment and the drugs used during the

emergency should be documented.

32.13 Follow Up of Patient Experiencing Adverse Events

Any adverse event, which occurs in the course ofthe study, should be monitored and

followed up until it has resolved according to the judgment ofthe investigator.

3.2.14 Safety Assessment

All pregnant women involved in the study will be included in the listing of safety

data. Reason for withdrawal and date of any withdrawal will be reported. All adverse

experiences will be listed and tabulated by severity, treatment and relationship to the

therapy.

3.3 Study Procedure

33.1 General Procedure

Pregnant women within the age limits as defined in section 3.2.2 will be questioned

with reference to the inclusion and exclusion criteria. Patients who seem suitable for

inclusion will be asked for informed consent as described in section 3.1.2. Patients

will then sign the Informed Consent Form and their code will be recorded in the

Screening List.

A copy of all the patient laboratory tests will be taken before the trial in the last visit

to her doctor.

61

A list ofthe normal ranges and units of measurement ofthe laboratory parameters to

be determined during the study will be provided by the Investigator and documented

in each CRF.

The laboratory printouts have to be inserted into the CRF as original or authorized

copy. Any values out of range have to be assessed as "not clinically relevant" (NCR)

or "clinically relevant" (CR) on the laborato4' printout.

If in the course of enlry screening any clinical relevant abnormal value is observed,

this finding will be regarded as an exclusion criterion. Single laboratory values

outside the normal range will generally not be regarded as an exclusion criterion

provided that:

1. They are not accompanied by clinical symptoms,

2. The context of related laboratory values gives no indication of a pathological

process and,

3. The investigator regards them as "not clinically rclevant O\ICR).

The investigator will check on each Patient's well being prior to their discharge from

the clinic.

33.2 Special Procedures

No further special procedures are planned in the present trial.

3.3.3 Daily Activities in Each Period of the Trial

3.3.3.1 Entry Examination

The entry examination will be carried out at the last visiting to her doctor, before the

beginning ofthe trial.

Following parameters will be documented and /or investigated:

* Thorough explanation ofthe study medication from the Investigator.

* Date of inclusion (date of signing the informed consent).

62

* Date of examination.

* Inclusion criteria (according to protocol),

* Exclusion criteria (according to protocol).

* Informed consent (if inclusion criteria fulfilled).

* Demographic data: date of birth, ethnic group, sex, height, weight, BMI.

* Additional information concerning: illness within the last 4 weeks prior start ofthe

trial, last participation in any clinical trial, last donation of blood or plasma, last

administration of any medication (including OTC or topical medication), last

administration of drugs known to alter the major organs or systems as well as

specification ofthe drug name, dosage, start and end oftreatment.

* Vital signs: registration of body temperature, measurements of supine heart rate

(HR) and blood pressure @P) after 5 minutes of supine rest.

* Physical examination.

3.3.3.2 Day I

The following procedures will be performed and documented in CRF on this day:

* Check of exclusion criteria, adverse events and concomitant medication

* A venous cannula is inserted and 2 ml blood sample is drawn pre-drug

administration.

* Blood sampling will continue at the following times: immediately after labor from

mother, half and 4 hours post drug administration.

x A blood sample of 2 ml will be withdrawn from the placenta after child birth.

* Registration of vital signs at 0, 1,2,3, 4 hours post dose.

* Questioning for and registration of adverse events: 0, 1,2,3, 4 hours post dose.

* Discharge from the hospital should be recorded.

63

33.4 Drug Administration

The precise instructions for drug administration are given to patients by the

investigator.

All patients will receive in the period of the trial a single dose of 1.00-1.25 mg/ ml

Bupivacaine(HCl) as epidural injection.

3.3.5 Blood Sampling for Drug Analysis

Blood sample for drug analysis will be taken pre-drug administration and at the

following times after drug administration: immediately after labor, half and 4 hours

post drug administration from the mother and 2 ml from the placenta immediately

post the delivery.

The blood samples 2 ml will be collected into tubes using heparin as anticoagulating

agent, After sampling the tubes will be immediately centrifuged (4000 rpm, room

temperature, for 5.0 min), the separated plasma will be transferred into polypropylene

tubes immediately frozen and stored at a temperature of -100 C or less until analysis.

The samples will be transferred frozen from the hospitals to the laboratories ofJordan

Center for Pharmaceutical Reseaxch (JCPR) for analysis.

3.3,6 Analysis of Bupivacaine

The determination of Bupivacaine plasma concentrations will be performed by means

ofa suitable chromatographic assay method, at the laboratories of Jordan Center for

Pharmaceutical Research (JCPR) A validated method as worked out in due time

before the start ofthe study.

The method will be validated for linearity and selectivity and be able to determine

Bupivacaine concentrations with sufficient selectivity and accuracy.

Standard samples and quality control samples will be distributed through each

analysis day.

64

3.4 Study Documentation

3,4.1 Investigator's File

The investigator will be having a study file where the following documents will be

archived:

L lnvestigator Brochure and its updates.

2. Study Protocol, with all its appendixes and their amendments if any, with the

relevant communications during the study.

3. IRB/IEC composition and dated approval / favorable opinion ofthe Study Protocol

with the relevant appendixes.

4. Curriculum Vitae and /or other relevant documents evidencing qualifications of

investigator(s) and sub- investigator(s).

5. Normal value(s)and range(s) for medical / laboratory /technical procedure(s) and /

or test(s) and there updated values.

6. Medical / laboratory / technical procedures /tests (certification, accreditation,

established quality control and or extemal quality assessment or other validation).

7. Sample of label(s) attached to investigational product containers.

8. Signed Informed Consent Forms.

9. Source Documents.

10. Signed, dated, and completed Case Report Form (CRF) and their corrections after

initial data were recorded.

I l. Patient identification code.

12. Investigational products accountability at the sit€ to ensure that they have been

used according to the Protocol, to document the final accounting of investigational

product(s) received at the site dispensed to patients, returned by the patient and

returned to Sponsor or destructed by the investigator.

b)

13. Signature Sheet to document signature and initials of all persons authorized to

make entries and / or corrections on CRF.

14. Publication and Literature Survey.

15. Ethical and legal information on clinical trials (e.g. ICH Guideline or Good

Clinical Practice, Declaration of Helsinki).

3,4.2 Case Report Form (CRF)

Standardized CRFs will be used to document the patients' data during the course of

the study.

The investigator will assure that all data are entered promptly, legibly, completely,

and accurately and confirm to the CRF relevant attached source documents. This also

applies to the data for those patients who -after having consented to participate -underwent baseline examinations required especially for inclusion into the study but

whom- because a criterion of exclusion was met or for other reasons - were not

included into the study.

To ensure legibility the case report forms should be filled out with a blue ballpoint

pen (not pencil, felt-tip or fountain pen). Any corrections to the case report forms

must be carried out by the investigator. A single strike must be drawn through the

original entry. The reason for the correction has to be given, ifnecessary and it has to

be dated and initialed. Incorrect entries must not be covered with correcting fluid, or

obliterated, or made illegible in any way, Even if there are no changes from a

previous examination, in the interests of completeness of data acquisition the

questions which are repeated in each section of the case report forms should be

answered in full. A reasonable explanation must be given by the investigator for all

missins data.

66

All medical records upon which the case report forms are based must be kept for at

least 5 years after completion ofthe study.

3.5 Pharmacokinetic Evaluation

For each Pregnant women, the concentration of Bupivacaine in the plasma will be

determined as well as the concentration of Bupivacaine in the new bom infants (by

measuring the concentration of drug in placenta immediately after labor).

3.6 Biometrics and Statistical Aspects

The biostatistical evaluation will be

management unit) at JCPR, by means

Innaphase Corporation, France.

3,6.1 Data Presentation

carried out by Dr. Nasir Idkaidek (data

of the software Kinetica 2000 version 4.1,

The concentration vs. Time profiles after administration of Bupivacaine epidural

injection will be represented in the form of tables and graphics for each pregnant

women and the concentration of Bupivacaine in the new bom infants will be

measured and tabulated.

All safety data will be presented in tabular form as listings of data by patient. For the

laboratory data tables ofout-of - range values will be also presented.

The Study Report will be prepared as per ICH Topic E3, Structure and Content of

Clinical Studv Reports.

ol

3.7 Good Clinical Practice

3.7.1 Legal Requirements

This study will be conducted in accordance with the following:

* Commission of the European Communities, EC Note for Guidance: Good Clinical

Practice for trials on medicinal products in the European Community, July 1991. ( as

well as with respective laws and regulations in the countries where clinical centers are

recruited).

* ICH Topic E3. Structure and Content of Clinical Study Reports, Consensus

Guideline from 17 .07 .1996 (6IFR37320).

* ICH Topic E 6. Note for Guidance on Good Clinical Practice, step 5. Consolidated

Guideline form 01.05, 1996

* Arrangements for archiving of documentation according to the Commission

Directive of 19.07, I 991 (91 /597 IEEC)

* Guidance for Industry, Bioanalltical Method Validation. May 2001

x Note for Guidance on the Investigation of Bioequivalence (CPMP/

QWP,EWP/l40 l/98 Rev. l) (2010)

* Declaration ofHelsinki (Seoul Revision, 2008)

* Jordan's Drug Law

* JCPR standard operating procedures

3.7.2 Adherence to the Protocol

Protocol violations are any deviations from the procedures outlined in this document,

missed evaluations, incorrect timing of evaluations, non-compliance with study

medications and intake of prohibited medications.

After a patient has been enrolled, it is the investigator's responsibility to make a

reasonable effort to avoid any protocol violations in order not to exclude the patient

68

form the study. Protocol violations do not themselves constitute a justification for

withdrawal of a patient from the study.

All protocol violations will be reported immediately to the Sponsor during the course

ofthe study. The nature ofthese violations will be defined in the monitoring protocol.

A11 protocol deviations will be listed and the availability of the patients concerned

will be discussed with the Sponsor prior to the statistical analysis.

3.7.3 Data Handling Procedures

The results from screening and the data collected during the study will be recorded in

the patient's Case Report Form (CRF) which will be designed and printed by the

principal investigator. Each case report form will be signed and dated by the

investigator.

All corrections in the CRFs are to be made legible and signed by the investigator-

Each patient receives a code number. His personal identification remains in a separate

confidential file that can be used only together with the investigator. A print out ofthe

free texl information is compared by a second person with the CRFs to check its

accuracy. The results ofthis check are documented. During the input of data a list of

values is made, that seem to be not plausible in respect to the normal ranges given

form the investigator. All these values are to be discussed with the investigator.

Ifthe reason for a not plausible value cannot be clarified consulting the investigator,

this value is defined as a "missing value" in a written form in order to maintain

patient confidentiality- All data recorded during the course ofthe study will only be

identified by patient codes and patient study number. However, the investigator

agrees to record the complete patient identification on the Screening List, This list

will be treated with strict adherence to confidentialitv and will be filed in the

investisator's file.

69

t

For the purpose ofthis study, the following will be considered as source data:

* Data from sxternal sources such as Laboratory investigation result that will be

attached with the CRF.

* Patient Enrollment Log Sheet which will be kept in the JCPR documentation room,

any records filled by hand and then retyped by computer.

3.7.4 Quality Assurance Aspects

The study will be conducted in accordance to the Quality Assurance System

implemented by JCPR as well as all other requirements mentioned in the Protocol.

All aspects ofthe Study will be subjected to internal audit perlormed by the Quality

Assurance Unit. The audit records and the Quality Assurance Statement will be

included in the final report.

3.7.5 Monitoring

It is the responsibility of the investigator to assure that the study is conducted in

accordance with the protocol and that valid data are entered into the CRF.

Data verification is legally required and will be done by direct comparison with

source documents in case of patient's respective consent or by cross-checking with

source documents in the presence ofthe investigator- always giving due consideration

to data protection and medical confidentiality

The investigator will permit a representative from the Sponsor part to monitor the

study as frequently as necessary to determine that data recording and protocol

adherence are satisfactory. The CRFs and related documents will be reviewed in

detail in accordance with the Good Clinical Practice regulations.

It is the investigator's obligation to assure documentation of all relevant data in the

patient's file, such as medical history / concomitant diseases, date ofstudy enrolment,

visit dates, results of examinations, administrations of medication and adverse events.

70

Throughout the study, all data will only be identified by patient number and patient

codes. The data will be blinded correspondingly in all data analyses.

All obtained data will be checked for plausibility and completeness by the in-house

Quality Control Monitor.

3,7.6 Confidentiality

All information obtained during the conduct ofthe study with respect to the patient's

state ofhealth will be regarded as confidential.

Patients will be informed that all study findings will be stored on computer and

handled strictly confidential. Patients will be identified throughout documentation and

evaluation by the individual patient number and patient code only, whereas names

will be kept secret by the investigator

All information concerning study medication, all study material and study drugs shall

remain the property ofthe sponsor and the investigator, where he obliged to keep all

data and out coming information ofthe study confidential and to use those data only

in context with the soonsor.

71

Chapter 4

Results and Discussion

4 Results and Discussion

4.1 Internal Standard

Internal standard chosen should match the chromatographic properties, recovery and

ionization properties ofthe analyte (Singh and Sharma, 2005).

Bisoprolol and propanolol were found to match these criteria, upon running them after

applying the extraction procedure both suggested I.S showed no interfering but

Propanolol retention time was 4.5 min while bisoprolol retention time was in the

ralge of 3.1-3.3 min so bisoprolol was found to match the required criteria and also

serve our purpose of method development, therefore it was chosen as an IS.

Bisoprolol did not alter or deteriorate the performance ofthe proposed method.

4.2 Description of Method

4,2,1 Sample Preparation and Method of Extraction procedure

According to the sample preparation and method of extraction development

mentioned in section (2 5.1) 5.0 ml of tert-butyl-methyl-ether was the chosen solvent

as it has the best intensity, 1,0 M sodium carbonate was chosen as buffer as it has the

best baseline, for the vortex mixing duration all the tested durations have the same

AUC so the shortest vortex mixing duration was used which is 2 minutes for the first

mixing and I minute for the second one, back extraction was used with 200 pt of

formic acid,

The chosen sample preparation and method of extraction in details is: A 50 pl of

Bisoprolol (10.0 pglml) was added to 0.25 ml of plasma in test tubg then 50 prl of 1.0 M

sodium carbonate was added, the mixture was vortex-mixed, the next step is adding 5.0

ml of lerl-butyl-methyl-ether, vortex for 2.0 minutes followed by centrifugation for 6.0

minutes at 4400 rpr4 The organic layer was quantitatively transferred to another test

tube and a volume of 200 pl of l% formic acid was added, and vortex_mixed for 1.0

Method Result

Method I Good intensity, bad chromatogram

Method 2 Interference at same retention time

Method 3 There is still interferins

Method 4 Interference in blank, Bupivacaine and LS peaks become so closeto each other

Method 5 Better chromatogram and no interference in blank brrt *lerejsinterference in the running time 4 min. needs to be overcome

Method 6 Better chromatogram but still needs enhancement

minute. After 6 min of centrifugation at 4400 rprn, the lower layer was carefully

transfened to 350 pl flat bottom insert and 50 pl ofthis sample was injected on BDS

Hypersil Crs column, Bupivacaine and the internal standard were separated from

endogenous substances.

4.2.2 Separation and Chromatography

The chromatographic conditions were optimized through several test and error trials

to achieve symmetric peaks shapes for the analyte and I.S, as well as shortest run

time.

Seven methods were evaluated to optimize the chromatographic conditions, the result

ofeach method is illustrated in table I 1:

Table 11: Results ofeach developed method

The good results were obtained with a mobile phase consists of water/ acetonitrile

(66.5/33.5o/o v/v), 0.1% triethylamine and pH of 2.5 adjusted with phosphoric acid

and oven temperature set at 40 oc. The development of the current method was

focused on the short run time to assure high throughput, with good peak shapes and

74

high sensitivity. The retention times of Bupivacaine and IS were 3.1-3.3 and 3.5-3.7,

respectively.

Figure 5 shows plasma blank chromatogram, it is obviously clean indicated by y-axis

which represents the intensity and no endogenous interfering peaks with comparison

to LLOQ chromatogram, which is represented in figure 6 with a concentration of l0

ng/ml. Zerc concentration of plasma sample showing in its chromatogram in figure 7

also a clean area in the place of Bupivacaine and I.S peaks in this chromatogram we

could see the I.s peak. A good chromatographic separation can be indicated by upper

Limit of Quantitation (uLoQ) level of standard calibration curve that appears in

f,tgure 8, its specified amount is 4000 nglml for Bupivacaine, theoretical plates in

HPLC chromatogram for ULOQ of Bupivacaine is 8948, asymmetry value is 1.04 and

resolution value is 2.84.

75

Figure 5: HPLC Chromatogram of plasma Blanlc

f igure 6: IfPLC Chromatogram of LLOe of Bupivacaine (10 ngl mt).

Figure 7: HPLC Chromatogram ofZero Bupivacaine Concentration.

Figure 8: HPLC Chromatogram of Bupivacaine ULOe (a000 ngl ml)

4.3 Validation Results

FDA guidelines were taken in consideration to guarantee reliability and

reproducibility ofthe assay for quantitation ofBupivacaine in human plasma matrix,

the method was validated in a concordalce with the FDA regulation requirements.

Validation parts were successfully tested in terms of recovery, linearity of response,

accuracy, precision, sensitivity, stability and specificity, (FDA, 2001; Viswanathan et.

al., 2007). The result of each validation section is arranged together after describing

all validation sections:

4,3.1 Validation Day One, Two and Three, linearity, Accuracy and Precision

Data

Inter and intraday accuracy, precision and linear response for standard calibration

curve and QC samples are represented from their analytical sequences which are

arraigned in the following tables:

Table 12 represents validation ofday one for staldard calibration curve.

Intra-day accuracy data derived from standard calibration curve back calculation, and

Figure 9 shows the plot of calibration curve levels against their analyical response

and regression linear equation.

Day 1 validation results showed an accuracy range of 98 6l-102.11% while the

accepted criteria according to USFDA is 85.00-115.00% except for the LLOQ which

is 80 00-120 00%, this work's day 1 validation results passed the required criteria in

terms of accuracy.

The coeffrcient of determination (R'?) should be equal or more than 0.98 to be within

the accepted criteria while in figure 9 we could find it equals 0.999930 which

represents the strength of the corelation, the correlation coefficient of standard

78

calibration curve was consistently greater than 0.99 during the course of the

validation.

79

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Table 14 represents validation ofday two for standard calibration curve.

Intra-day accuracy data derived from standard calibration curve back calculation, and

Figure l0 shows the plot of calibration curve levels against their analytical response

and regression linear equation.

Day 2 validation results showed an accuracy range of 96.85-103.37% while the

accepted criteria according to USFDA is 85.00-115.00% except for the LLOe which

is 80.00-120.000%, this work's day 2 validation results passed the required criteria in

terms of accuracy.

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Table 16 represents validation of day three for standard calibration curve. Intra-day

accuracy data derived from standard calibration curve back calculation, and figure 11

shows the plot of calibration curve levels against their analytical response and

regression linear equation.

Day 3 validation results showed an accuracy range of 92.00-104.71% while the

accepted criteria according to USFDA is 85.00-115.00% except for the LLOQ which

is 80.00-120.00%, this work's day 3 validation results passed the required criteria in

terms of accuracy.

The coefficient of determination 1R2) should be equal or more than 0.98 to be within

the accepted criteria while in figure l1 we could find it equals 0.998942 which

represents the strength of the corelation, the correlation coeffrcient of standard

calibration curve was consistently greater than 0-99 during the course of the

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Table l8 represents calibration curve Number 4. Intra-day accuracy data derived from

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equation.

calibration curve No. 4 results showed an accuracy range of9g.27-101.3g% while the

accepted criteria according to USFDA is 85.00-115.00% except for the LLoe which

is 80.00-120.00%, this work's calibration curve No. 4 results passed the required

criteria in terms of accuracy.

The coefficient of determination (R2) should be equal or more than 0.9g to be within

the accepted criteria while in figure 12 we could find it equals 0.999936 which

represents the strength of the correlation, the correlation coefficient of standard

calibration curve was consistently greater than 0.99 during the course of the

validation.

coY= 0.000588X+0.000150Gf= o.eese42 )

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Figure 12: The plot of calibration curve number 4 levels against their analytical

response and regression linear equation.

Table 20 represent Calibration curve Number 5. Intra-day accuracy data derived from

standard calibration curve back calculation, and figure 13 shows the plot of

calibration curve levels against their analytical response and regression linear

equation.

Calibration curve No. 5 results showed an accuracy range of96.38-103.78% while the

accepted criteria according to USFDA is 85.00-115.00% except for the LLOQ which

is 80.00-120.0070, this work's calibration curve No. 5 results passed the required

criteria in terms of accuracy.

The coeffrcient of determination (R'z.) should be equal or more than 0.98 to be within

the accepted criteria while in figure 13 we could find it equals 0.999378 which

represents the strength of the correlation, the conelation coeffrcient of standard

calibration curve was consistently greater than 0.99 during the course of the

validation.

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Table 22 shows linearity and linear working range ofBupivacaine data based on back

calculated area ratio derived from standard calibration curve.

Table 23 shows linearity and linear working range of Bupivacaine data based on

normalized concentration derived from standard calibration curves.

Table 24 shows linearity and linear working range of Bupivacaine data based on

calculated concentration derived from standard calibration curves while figure 14

shows the plot of linearity of five calibration curves levels against their analltical

response and regression linear equation.

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Tables 26 represents intra-day precision and accuracy data for QC samples derived from the

standard calibration curve of day one validation, accuracy for the 10 replicates of QC low

was in the range of 95.82-10'7.53Yo and precisionoZ for QC low samples was 4.581%.

QC mid 10 replicates of day one validation accuracy was in the range of 97 .92-100.22Vo and

precision% of 0.801, finally QC high 10 replicates accuracy was in the range of 95.09-

97 .93%;o and precision% of 1.157.

Comparing the above mentioned accuracy ranges for QC low, mid and high for validation

day 1 with the accepted criteria which is 85.00-115,00% except for LLOQ the range should

be 80.00-120.0004 we could find them within the required range while the CV% didn't

exceed 20.00% for the LLOQ and 15.00% for the rest of the concentrations which reflects

the closeness ofthe individual measures when the procedure is applied repeatedly to multiple

aliquots of a single homogenous volume of the plasma matrix.

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Tables 27 represents intra-day precision and accuracy data for ec samples derived from

standard calibration curve of day two validation, accuracy for the 10 replicates of ec low

was in the range of 98.90-108.64Yo and precisiono/o for QC low samples was 3.37'l%.

QC mid 10 replicates of day one validation accuracy was in the range of 95.22-102,50% and

precision% of 2.647, finally QC high l0 replicates accuracy was in the range of 93.g2-

l01.2lyo and precisiono% of 2.605.

comparing the above mentioned accuracy ranges for ec low, mid and high for validation

day 2 with the accepted criteria which is 85.00-115.00% except for LLoe the range should

be 80,00-120.000% we could find them within the required range while the cv% didn't

exceed 20.00% for the LLoQ and 15.00% for the rest ofthe concentrations which reflects

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aliquots of a single homogenous volume of the plasma matrix.

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QC mid l0 replicates of day one validation acouracy was in the range of 101.25-

106.090 and precision% of 1.630, finally QC high 10 replicates accuracy was in the

range of 101.34-102.88% and precision% of 0.625.

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Bupivacaine in the three davs ofvalidation.

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4.4 Recovery

The recovery of Bupivacaine from its biological matrix in this bioanalytical method

shows a high value and good enough to be used to determine Bupivacaine in human

plasma, and it was acceptable at the studied concentration range.

Table 30 shows data ofBupivacaine in the mobile phase for the triplicates of quality

control samples while table 31 shows data of Bupivacaine in plasma for the triplicates

of quality control samples.

Absolute recoveries for Bupivacaine and IS were calculared by dividing average peak

area of triplicate from each QC level of plasma samples (Table 31) over same set of

QC samples but prepared in mobile phase (out of extraction procedure)(Table 30)

multiplied by 100%, namely (mean of drug in Plasma./mean of drug in mobile phase) *

t00%.

Table 32 shows Recovery % of Bupivacaine while table 33 shows the recovery o/o of

Bisoprolol (LS) a high recovery oZ is shown and hcceptable at the studied

concentratlon.

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Precision and accuracy data for Bupivacaine LLOQ (10 nglml) are shown in table 33.

The accuracy data ofthe 10 replicates ofthe LLOQ showed a range of 97.40-115.53yo

while the precision result is 4.866, both accuracy and precision are within the

acceptance criteria mentioned in the USFDA which reflects that this method is

sensitive at this concentration.

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4.6 Specifity and Selectivity

4.6.1 Specifity

Six different human plasma samples were used to prepare 6 samples of blank and 6

samples of zero, the samples were then acquiesced with the 7 standard calibration

curve points.

The data in tables 34 and 35 show the specifity ofblank and zero samples respectively.

Specifity of blank samples were measured by dividing (AUC of the LS in the blank

over the AUC of the LS of the standard calibration curve (STD) points)* 100%, the

acceptance criteria are less than 5%.

Specifity of zero samples was measured by dividing (AUC of Bupivacaine in zero

over the AUC ofBupivacaine in LLOQ)* 100% or (the calculated amount in zero over

the calculated amount in the LLOQ)* 100%, the acceptance criteria is less than 20%.

No significant interfering peaks from the plasma samples were found at the retention

time of Bupivacaine or for the L S.

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4,6.2 Selectivity

Selectivity test ofthe validation procedure was done by analyzing six different most

common drugs that most people might take in their daily life, this test is made to

insure that there is no significant interferences at the retention time ofBupivacaine and

I.S, Table 37 shows that there were no peaks at the specified retention time of

Bupivacaine and I. S.

Table 37: Selectivity for Bupivacaine

No. Drug Name Peak at retention time 2.5-4 minutes

t Salicylic Acid No Peak

2 Paracetamol No Peak

3 Ibuprofen No Peak4 Mefenamic Acid No Peak

5 CaIIiene No Peak

6 Nicotinamide No Peak

l.t 4

4,7 Dilution Integrity

Table 38 shows the data for dilution integrity indicated by QC high prepared in the

tiplicate concentration for Bupivacaine after dilution procedure.

Acceptance criteria for the dilution integrity is 85.00-115.00% of accuracy , so these

data lie with the accepted range.

Table 38: Dilution integrity for Bupivacaine

Concentration Measured Conc. Accuracy %o

3000.000nglml

3056 695 3056.214 101.890 101 ,870

2817.366 2819.133 93.910 93.970

2858,348 2853.324 95.280 95.1 10

D16000.000ng/ml(Two times)

5704.639 5703.750 95.080 95.060

5709.388 5712.006 95.160 95.200

6046.510 6046.868 100.780 100.780

D2r2000.000ng/ml(Four times)

1rr79.7 53 1t207.'169 93.160 93.400

1 1619.108 t1769.st2 96.830 98.080

11325.846 I 1991.665 94.380 99.930

115

4.8 Stability

4.8.1 Short Term Stability

4.8,1.1 Room Temperature Stability

Freshly prepared 0 hour triplicate QC's of each level were taken as a reference upon

calculating stability of Bupivacaine at room temperature.

Stability9'o : mean of measured concentration at 0 hrl mean of measured concentration

at 8 hr" 100%

Table 39 shows data for short term stability indicated by three levels of QC samples

(low, mid and high) lor Bupivacaine respectively at room temperature.

From the table's data we find the room temperature stability test is passed according to

the acceptance criteria which is in the range 85 00-115.00%.

116

Table 391 Room temperature stability of short term for three levels of QC

samples (low, mid and high) for Bupivacaine,

QC Low ( 30.000 ng/ ml )

TimeAUCDrug AUC IS Ratios

MeasuredConc.

MeanMeasured

Accuracyo/o Stability

0Hour

0. 132 7.816 0.017 28.780

29.085

95.930

100.0000.126 7.633 0.016 28.14s 93.820

0.139 7.811 0.018 ?o Itq 101.100

8Hours

0.1 t2 6.'199 0.016 28.045

28.574

93.480

98 2400 125 7.270 0.017 29.201 97.340

0.1 14 6.828 0.017 28.475 94.920

QC Mid ( 1500.000 nglml

0Hour

7.t69 7.811 0.918 1476.0171477.628

98.400100.0007 232 '7.842 0.922 1483.030 98.870

7.754 7.806 0.917 1473 836 98.260

8Hours

6.544 6.888 0.950 152'.7.841

1496. 181

101.860

101,2606.384 7.163 0.891 t433.4'.t7 9s.5706.52r 6.866 0.950 t527.2?4 101.810

QC Hish ( 3000.000 nelml )

0Hour

t4 049 7.794 1.803 2897.0402904.096

96.570100.00014.314 7.834 r.827 2936.686 97.890

14.009 7.822 t.791 2878.563 95.950

8Hours

13.089 7.227 1.811 2910 781

2899.81097.O30

99 85012 952 7.232 1.791 2878.436 95,95013 079 114^ 1.811 2910.213 97.010

4,8. 1.2 Autosampler Stability

Table 40 shows data for shoft term stability indicated by three levels of QC samples

(low, mid and high) for Bupivacaine after preparation procedure (Auto-sampler

stability), T=20'C.

From the table's data we find the autosampler stability test is passed according to the

acceptance criteria where the accvrac5f/o doesn't exceed l5ol0.

Table 40: Auto-sampler stability of short term for three levels of QC samples

(low, mid and high) for Bupivacaine

QC Low ( 30.000 ngl ml )

Time AUCDrug AUC IS Ratios

MeasuredConc.

MeanMeasured

Accuracyo Stabiliry

0

Hour

0.t32 7.816 0.017 28.780

29.085

95.930

100.0000.126 7.633 0,016 28.145 93.820

0. 139 7.811 0.018 30.329 101. 100

24Hours

0.112 6.799 0,016 28.045

31.380

r04,350

107.8900.125 7.270 0.017 29.201 r04 910

0.1 14 6.828 0.017 28.475 104.530

QC Mid ( rs00.000 nglml )

0

Hour

7.169 7.81 1 0 918 1476.O17

1477.62898.400

100,0007.232 7.842 0.922 1483 030 98.8707.154 7.806 0.917 14'.73.836 98.260

24Hours

8.535 8.963 0.952 153 I .307

153 7.458102.090

104.5008.343 8.669 0.962 1547.598 103.170

8.439 8.850 0.954 1533.469 102.230

QC High ( 3000.000 nglml )

0Hour

14.049 7.794 1.801 2897.040

2904.09696.570

100.00014.314 7.834 1.827 2936.686 97.890

14.009 7.822 1.791 2878.563 95.950

24Eours

16320 8.789 1.857 2984.3142998.698

99.480103.26016.417 8.777 1.870 3006.296 100.210

76.023 8.569 1.870 3005.42s 100.180

118

4.8.1,3 Freeze and Thaw Stability

Table 41 shows data of three cycles of freeze and thaw for short term stability

indicated by three levels ofQC samples (low, mid and high) for Bupivacaine over 72

hours,

From the table's data we find the freeze and thaw stability test is passed according to

the acceptance criteria.

Table 4l: Freeze and thaw stability of short term for three levels of QC samples

(low, mid and high) for Bupivacaine

QC Low ( 30.000 ngl ml )

Time AUCDrug AUC IS Ratios

MeasuredConc.

MeanMeasured

AccuracyVo

Stability

0Hour

0.132 7.816 0.ofi 28.780

29.085

95.930

100.0000.t26 7.633 0.016 28.145 93.820

0.139 7.811 0.018 30.329 101.100

17

Hours

0.t25 7.083 0.018 29.9'79

30.53 5

99.930

704.9900.135 7.388 0.018 31.007 103.360

0.128 7.085 0.018 30.618 102.060

QC Mid ( 1500.000 nglmt

0

Hour

7.169 7.811 0.918 t476.O171477.628

98.400100.0007.232 7.842 0.922 1483.030 98.870

7.154 7.806 0.917 1473.836 98.260

72Houn

6.646 7.501 0.886 1424.750

13'76.620

94.98093.1605.837 7.232 0.807 1298.095 86.540

6.594 7.538 0.875 1407.015 93.800

QC High ( 3000.000 nglml ,

0Hour

14.o49 '7.794 1.803 2897.0402904.096

96.570100.00014.314 7.834 1.827 2936.686 97.890

14.oo9 7.822 1.791 2878.563 95.950

1''Hours

14.284 8.287 1.724 2770.4402783.5',74

92.35095.85014.509 8,356 t.736 2790.908 93,030

73.299 7.663 t.736 2789.374 92.980

119

4.8.2 Long Term Stabilify of Plasma Samples

Table 42 shows a two times examining tests have taken over 4 weeks as specified time

in tables for long term stability to proof stability ofthree levels ofQC samples (low,

mid and high) for Bupivacaine respectively.

From the table's data we find that the long term stability of plasma samples test is

passed according to the acceptance criteria where the decrease in the analytical results

is not more than I5oZ.

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Tables 43-44 are examining tests for stock solutions stability which have taken over

two weeks as specified time in tables to proof stability of the mid level of QC samples

of Bupivacaine and Bisoprolol respectively.

From the table's data we find the stock solution stability test is passed according to the

acceptalce criteria.

Table 43: Stock solution stability (500 pg/ ml) diluted to 3750 ng/ml for

Bupivacaine

Time AUC Drug Mean Accuracy

0hr14.721 14.644

14.631

96.30

14.664 14.605

14.620 14.534

Room temp.(24)hr

14.r93 1.4.063

14.09114.1 l8 14.015

14.126 14.031

Refrigerator 'CTwo weeks

13.779 13.696

13.745 93.9413.790 13.718

13.774 t3.713

tz)

Table 44: Stock solution stability (1000 pgl ml) diluted to 2,5 pg ml for Bisoprolol

(r.s)

Time AUC Internal standard Mean Accuracy

0.0 hr7.639 7.645

7.642

97.91

7.654 7.646

7.638 | .o3L

Room temp,(24)hr

7 494 7.474

7.4837.488 7.459

7.5r0 7.470

Refrigerato r oC

Two weeks

7.330 7.270'7.321 95.80t.J)) I,JLJ

7.352 7.318

5. Conclusion

This method was developed to overcome the disadvantages ofthe previous analytical

methods which were achieved. This method is simple, reproducible, selective, has a

high resolution and sensitive with a LLOQ of 10 nglml which is from our knowledge

the lowest validated detection limit, a short total run time of 4 minutes with a retention

time of 3.1-3.3 min. for the internal standard arrd 3.5-3.7 min. for Bupivacaine

compared to the range of 3.8-4.0 min. for the methods of low sensitivity and 13.0 min.

for the methods of medium sensitivity. An excellent recovery was achieved in this

method which isMin.97.07Yo compared to a range of 93% of previous methods.

The method was fully validated with all of the data within the acceptance criteria in

concordance to USFDA and the clinical part is agreed by the ethics committee and

could be directly used to determine Bupivacaine in biological plasma once available.

1?5

f

6. References

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Appendix (1):

Chromatograms and UV Scan

2 10 ug/ml_2s% ACN pH=3.6

Sample Name:Vial NumberSample Type:

Control Program:Quantif. Method:

10 ug/mL2s% ACN pH=3.6RA2

unknown3dEsomeprazole9125t201213:34

lnjection Volume:Channel:Wavelength:Bandwidth:Dilution Factor:

7 5.0uv_vts_12201

1.000011.39Time: Run l1me

Operator:analyst Timebase:HPLC Sequence:25.09.2012 Page 2-16t3t2013 12.43 PM

Chromeleon (c) Dionex 199e2001versron b.JU uu,lo c,/b

139

No. Ret.Time Peak Name Height nre? -net.nrea nrnountmin mAU mAu*min %1 O.Ag eupiv.

Total: 48.239 11.852 100.00 0.000

Retention Time Name Value Channel0.000 min l\4inimum Area 0.01 ISionall'min All Channels0.000 min Inhibit Inteqration On All Channels0.488 min Inhibit Inteoration off All Channels0.753 min Inhibit Inteqration On All Channels4.026 min lnhibit Inteoration off All Channels4 340 min lnhibit lnteoration On All Channels

bupivacaine./lnlegratjon

Operator:analyst Timebase:HPLC Sequence:25.09.2012 Page 31-16t312013 1:O2 PM

31 0.1% FA_30% ACN pH=2,5

Sample Name:Vial Number:Sample Type:Control Program:Quantif. Method:

0.1% FA_30% ACN pH=2.5GA5unknown3dEsomeprazole912612012'l3i1a

lnjection Volume:Channel:

Wavelength:Bandwidth:Dilution Fadar:

75.0uv_vts_1n,a.1

r.00005.63Time: Run Time

No. Ret.Time Peak Name Height Area Rel.Area Amountmin mAU mAu'min Yo

4.32 n.a. 47.249 5.430 100.00 n.a.

Total: 47 .249 5.430 100.00 0.000

ReJention Time Name Value Channel0.000 min Minimum Area 0.01 [Sionall'min All Channels0.000 min nhibit Inteqration On All Channels0.488 min nhibit lnteoration off All Channels0.753 min nhibit Integration On All Channels4.026 min nhibit Inteqration off All Channels4 731 min nhibit lnteoration (rn All Channels

chromeleon (c) Dionex '1996-2001

Version 6.30 Build 576bu pivaca ine/l ntegration

140

51 Bf k 0.1% F.A_30o/o ACN pH=2.5 0.5TEA

Sample Name:Vial NumberSample Type:

Control Program:Quantif. Method:

Blk 0.1% F.A_30% ACN pH=2.5 0.5TEAlnjection Volume: 75.0UV-VIS-.I2101

'1.0000

5.50

GA8unknownbupivacaineEsomeprazole9127120129i11

Channel:Wavelength:Bandwidth:Dilution Factor:

TIme: Run Time

Operator:analyst Timebase:HPLC Sequence:25.09.2012 Page 51-16/32013 1:09 PlVl

Chromeleon (c) Dionex 1996-2001Version 6.30 Build 576

No, Ret.Time Peak Name Height Are,a Rel.Afea Amountmin mAU mAU*min olo

1 4.11 n.a. Q.257 0.040 39.57 n.a.

J0.3650.201

4.274.45

0.024 23.880.037 36.55

Total: o.824 0.102 100.00 0.000

Retention Time Name Value Channel0.000 min lVinimum Area 0.0'l ISiqnall.min All Channels0.000 min Inhibit Inteoration On All Channels0.488 min Inhibit Inteoration otf All Channels0.753 min Inhibit Inteoration On All Channels4.026 min lnhibit Integration off All Channels4 731 min lnhibit lnteorafioD On All Channels

bupivacaine/lntegration

r4l

Operator:analyst Timebase:HPLC Sequence:25.09.2012 Page 7 4-16/3/2013 1:15 PM

74 Blk 1.0 % F.A_30% ACN pH=2.5 1.OTEA 40C

Sample Name:Vial NumberSample Type:

Control Program:

Quantit Method:

Blk 1.O oh F.A_30o6 ACN pH=2.5 1.0f El hjection Volume: 75.Ouv_vts_tn.a.1

1.00005.00

GA9unknown?,{

Esomeprazole91271201211i59

Channel:Wavelength:Bandwidth:Dilution Factor:

Time: Run Time

No. Ret.Time Peak Name Height Area Rel.Area Amountmin mAU mAu*min

1 4.04 n.a. 0.161 0.014 100.00 n.a.

Total: 0.161 0.014 100.00 0.000

ReJentialn Timc Name Value Channel0.000 min l\4inimum Area 0.01 [Siqnall"min All Channels0.000 min lnhibit lnteoration On All Channels0.488 min Inhibit Inteqration off All Channels0.753 min Inhibit Inteoration UN All Channels4.026 min lnhibit lnteoration off Al, Channels4 731 min lnhibit lnfedretion (Jn All Chennels

chromeleon (c) Dionex 1996-2001Version 6.30 Build 576bupivacaine/lnteg ration

142

Operator:analyst Timebase:HPLC Sequence:27.09.2012 Page 24-16/3n013 1.21 PM

Chromeleon (c) Dionex 1996-2001Version 6.30 Build 576

Sample Name:Vial NumberSampb Type:Control Program:Quantif. Method:

mixG85unKnown3dbupivacaine9127m1214:20

lnjection Volume;Channel:

Wavelength:Bandwidth:Dilution Factor:

75.0uv_vls_1n.a.1

1.00005.10

No. Ret.Time Peak Name Height Area Ret.area Amountmin mAU mAU'min

1

23.533.92

t.sBupiv.

146.68429.491

15.176 83.303.043 16.70

Total: 176.175 18.219 100.00 0.000

Retention Time Name Valtre Channel0.000 min Minimum Area 0.01 ISionall.min All Channels0.000 min Inhibit Integration On All Channels3.454 min Inhibit Inteqration All Channels3.987 min Inhibit Inteoration On All Channels4.042 min Inhibit lnteoration .)ff All Channels4.498 min lnhibit lnteoration (Jh All Channels

bupivacaine/lntegration

l+J

17 Blk 0.1 % F.A_30% ACN pH=2.5 1.0TEA 40C

Sample Name:Vial Number:Sample Type:

Qontrol Prcgram:Qumtif. Method;

Blk 0.1 % F.A_30% ACN pH=2.5 1.OTE| lnjection Votume: 75.0uv_vts_12'to1

1.0000

5.10

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Channel:Wavelength:Bandwidth:Dilution Factor

Time: Run Time

Operator:analyst Timebase:HPLC Sequence:27.09.2012 Page 17-161312013 1:28 PM

Chromeleon {c) Dionex 19%-2001Version 6.30 Build 576

No. Ret.Time Peak Name Height area net.area Emour*-min mAU mAU,min %Bupiv. 0.180 0.018 100.00 n.a

Total: 0. 180 0.018 100.00 0.000

Retention Time Name Valtle Channel0.000 min Minimum Area 0.01 lsiqnall-min All Channels0.000 min Inhibit lnteoration UN All Channels3.454 min Inhibit Inteqration Ali Channels3.987 min Inhibit Inteoration 9n All Channels4.042 min Inhibit Inteoration All Channels4.498 min lnhibit ,nteoralion UD All Channels

bupivacaindlntegration

144

12 1O nglml

Samp/e NameiVial Number:Sample Type:

Control Program:

Quantif . Method:

10 ng/mlGA5

Injection Volume:Channel:

Wavelength:Bandwidth:Dilution Factor:

50.0UV-VIS-1210

Time:

unknownbupivacainebupivacaine10l1nO129t4'l Run Time

,|

1.00004.75

Operator:analyst Timebase:HPLC Sequence:01.10.2012 Page 12-16i 312013 1:38 PIV

Chromeleon {c) Dionex 1996-2001Version 6.30 Build 576

No. Ret.Time Peak Name Height Area Rel.Area Amountmin mAU mAu,min %

1

)3.794.36

t.sBupiv.

77 .1320.523

8.1170.061

99 250.75

Tota,: 77 .654 8.178 100.00 0.000

Retention Time Name Value Channel0.000 min Minimum Area 0.01 lsionalfm,n Al, Channels0.000 min Inhibit lnteoration On All Channels2.953 min Inhibit Inteqration All Channels3.326 min Inhibit lnteqration On All Channels3.386 min nhibit Inteqration All Channels3.778 min nhibit lnteorelion On All Channels

bu pivacaine/lnteg ration

l4s

Operator:analyst Timebase:HPLC Sequence:25.09.2012 Page 31-161312013 1:54 PM

31 0.1% FA_30% ACN pH=2.5

Sample Name:Vial NumberSample Type:

Control Program:Quantit. Method:Recording Time:Run Time (min):

0.1% FA_30o6 ACN pH=2.5GA5unknown1A

Esomeprazole91261201213i'lg5.50

lnjection Volume:Channel:

Wavelength:Bandwidth:Dilution FaLtor:Sample Weight:Sample Amount:

75.Ouv_vts_12101

1.0000

1.00001.0000

No. Ret.Time Peak Name Area Height Lambda Max # 1 tamUOa wlax *t Zmin mAu*min mAU nm nm4.32 Bupiv. 10.540 92.510 399.5 399.s

Total 10.flo 92.510

Chromeleon {c) Dionex 199G2001verston b.3u bu o 5/bCalibration/Purity (Curr.Peak)

146

Appendix (2):

Study Protocol

Jordan Center for -@JCPR

Pharmaceufical Research

Protocol No.:Buo-07-2012

Confidential

A STUDY TO DBTERMINE THE CONCENTRATION OFBUPIVACAINE EPIDURAL INJECTION DURING LABOR

FOR MOTHBR AND NEWBORN INFANT

Sponsor:

TEST MEDICATION:Bapivacaine Ampoule

Jordan Center for Phurmaceutical ResearchP.O. Box 950435 Amman i 1195 JordanTel.: 00962-6-5814953Fax: 00962-6-5814952E-mai I : Lalaf_a.l@i-c.p_r:jp,,.qpm

Prof. Dn TawJiq Arafatlordan Center for Pharmuceutical ResearchP.O. Box 950435 Amman, 11195 JordanTel.: 00962-6-5814953Fax: 00962-6-5814952E-mail: t. arafat@,icpr-jp. com

Pri ncip al I nv estigolo r :

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A STTJDY TO DETERMINE THE CONCENTRATION OFBUPIVACAINE EPIDURAL INJECTION DURING LABOR

FOR MOTHER AND NEWBOR}I INFANT

REFERENCE PRODUCT:Bapivacuine Ampoule

I, the undersigned below hereby certify that I revised and approved

This final version of the protocol

Tawfiq Arafat, Ph.D.Principal Investigator

Dr. Basem HalasehConsultant in Anesthesia and Intensive Care

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Table of Contents

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Title Page

I. RESPONSIBILITIES AND ADDRESSES J2. LIST OF ABBREVIATIONS AND DEFIMTION OF TER}IS 63. SUMMARY 74. FLOW CH,{.RT 7

5. INTRODUCTION 8

6. OBJECTIVE OF THE STUDY 1l7. BENEFIT - RISK EVALUATION ll8, DESIGN l19. SELECTION OF PATIENT t2

9.1 Justification for Inclusion and Exclusion Criteria t29.2 Inclusion Criteria 1')

9.3 Exclusion Criteria 12

9,4 Other Conditions t-t9,5 Patient s' Oblieations IJ

10. MEDICATION 13l0.l Studv Medication 13

i 0. l. 1 Test drug 13

10. 1,2 Blinding IJ10.2 Drug t{andling and Accountability IJ

10.2.1 Dosage and Duration of Treatment IJ10.2.2 Label of empty ampouls T4

10.3 Compliance 1410.4 Concomitant Medication l4I0.5 Rescue Medication t410.6 Emergency Procedures l410.7 Follow up of Patients Experiencing Adverse Evenrs 15

10.8 Safety Assessment l511. STUDY PROCEDURE 15

11.I General Procedure l5I 1.2 Special Procedures 15

1 I . 3 Daily Activities in Each Period Of The Trial 15

I L4 Drug Administratron loI 1,5 Blood Sampling for Drug Analysis 16

I L6 Analysis to12. ADVERSE EVENTS l7

12.I Definition of Adverse Events 1.7

I 2.2 Relationship to the Test Drus t712.3 Classification ofAdverse Events and Adverse Drue Reactions l8

12.4 Reporting and Documentation ofAdverse Events l813. STUDY DOCUMENTATION l9

13. I Investigator's File 19

13.2 Case Report Form (CI{F) l914. PHARMACOKINETIC EVALUATION 2015. BIOMETRICS AND STATISTICAL ASPECTS 20

15.l Data Prosentation 20

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Title PageI6. ETHICAL CONSIDERATIONS 20

16. I Declaration of Helsinki 2016.2 Ethical , Leeal and Administrative Aspects 2016.3 Patient Information and Informed Consent ZI

17. GOOD CLINICAL PRACTICE 2l17. I Legal Requirements 21

1 7. 2 lnvestigator's Obligations 2217.3 Adherence to the Protocol 22

17.4 Data Handline Procedures17.5 Quaiiw Assurance Aspects LJ

17.6 Monitoring z)17.7 Confidentiality 2417.8 Qualification of the Principal Investigator 24

18, PROTOCOL AMENDMENTS 2419 REPORTS 24

l9.l Archiving 2520. COMMUNICATION OF STUDY RESULTS 2621, REI'ERENCES 2722. APPENDICES t1

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1. RESPONSIBILITIES AND ADDRESSES

1,1 Sponsor:

1.2 Principal Investigator* :

1,3 Clinical Investigators* :

1.4 Principal Investigatorassistant:

1.5 Biostatistician:

1.6 Quality Assurance:

1.7 Clinical QualityAssurance Supervisor:

fordan Center for Pharmaceutical ResmrchP.O. Box 950435 Amman, 11195 JordanTel.: 00962-6-5814953Fax 0Q962-6-581.4952E-mai I : t.ars&t@i9pl-jp, _c..-oln

Prof, Dr. Tawfiq Arafat, Ph.D, Pharm, AnalysisJordan Center for Pharmaceutical ResearchP.O.Box 950435 Amman, 11195 JordanTel.: 00962-6-5814953Fax: 00962-6-5814952E-mail : : t. arafat@jcpr-jp.com

Dr. Basem HalasehConsultant in Anesthesia and Intensive Care

Dnass Al-khader

Data Management UnitNasir M Idkaideli, Ph.D. PharmaceuticsE-mail ; nidkaidek@uop.ecl4ieJordan Center for Pharmaceutical Research

Mr, Munther Melhim (M.Sc), Chem, Eng.Jordan Center for Pharmaceutical Research

Farah Abu AFhial (B.Sc), PharmacyJordan Center for Pharmaceutical Research

1.8 Clinical Qualiry Control Ayah Emad Al-Ubaidi (B.Sc), PharmacySupervisor: Jordan Center for Pharmaceutical Research

1.9 Analysts: Mr. Ahmed Abu Awad, {B.Sc) Chemist- In tie absence of L'rof. Dr. TatrfLq Arafal, the Co ifes''igator ot the clinixal inwstgator teil! .tct as principat inj'es..igator for ttud!-relaad dpaisions.

C.r's for the inrettigators arc bcated itr Aryerulix (01)

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2. LIST OF ABBREVIATIONS AND DEFINITION OF'TERMS

ADR Adverse Drus ReactionAE Adverse EventANOVA Analvsis of VarianceAUC Area Under Plasma Concentration-Time Curve

AUC O-tArea Under The Plasma Concentration-Time Curve FromZero(0) Hours to Time (T)

AUC 0-o Area Under The Plasma Concentration-Time Curve FromZero(0) Hours to Time (o)

BA BioavailabilityBMI Body Mass IndexCLn Renal Clearancec.""'. Maximal Plasma Drue ConcentrationCp Concentration PointCRF Case Report FormCV Curriculum VitaeFDA Food and Drug AdministrationGCP Good Clinical PracticeGMP Good Manufacturins PracticeHPLC High Performance Liquid ChromatographyIEC Independent Ethics CommitteeI.M, Intra MuscularIRB Institutional Review BoardI.V. IntravenousJCPR Jordan Center for Pharmaceutical ResearchK"t Elimination Rate ConstantLCMS Liquid Chromatography Mass SpectroscopySAE Serious Adverse EventsoP Standard Operating ProcedureT Timet,, Terminal Elimination Half -LifeTID Tres In Dies, Three Times DailyT.* Time of Maximum Plasma Drus Concentration

u MicrowHo World Health Oreanization

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3. SUMMARY

NAME OFCOMPANY:Jordan Center forPharmaceutical Research

NAME OF FINISHED PRODUCT:Bupir acaine(HCl) L0- l.25 mg/m.,amooules

NAME OF ACTI\E INGREDIENTS:Bupivacaine HCL

TITLE OF STUDYA STUDY TO DETERMINE THE CONCENTRATION OF EPIDI.,IRALINJECTION DURING LABOR FOR MOTFIER AND NEWBORN INFANT

PIIASE Clinical study

OBJECTIYETo assess the amount ofBupivacaine in the mother and nerv bom infant afterlabor

STUDY DESIGN An open, single dose, one period, one treatment.PLANNED SAMPLE SIZE n : 12 pregnaft wornen to complete the studv.PATIENT SELECTIONCRITERIA Pregnant women under labor aged between 18-45 years.

TEST DRUG Bupivacaine(HCl) 1.0-1.25 me/ml ampoules for epidural iniection-ROUTE OF ADMINISTMTIOI\ Single dose of 1.0-1.25 mg/ml Bupivacaine(HCl)DURATION OF TREATMENT 5 min pre-drug admrnistration until 4 hours post drug administrahon.PRIMARY ENDPOINTS Concentration in the blood for the mother and the nerv bom infant

SAFETY ENDPOINTS Adverse events and vital signs questioningTotal blood withdra\\'n during the studv is 10 ml.

PROCEDURE

Each patient will receive in an open design a single dose of 1.0- 1.25 mglmlBupivacaine(HCl)epidural injection on one occasion, 2 ml blood samples rvill betaken pre-drug administration, immediately post delivery two samples will betaken one from mother and the other from the placenta, post halfhour of drugadministration and the last sample will be taken post 4 hours post drugadministration.

ANALYSIS OF BUPIVACAINE Plasma samples will be analyzed by using suitable chromatographic methodSTATISTICAL ANALYSIS The statistical aralyses ofconcentration of Bupivacaine in the plasma

4, f,.LOW CHART

Typ€ of Assessment planned Entry examination Trail Day

Study day:In last visit to Doctor pre{elivery

not more than one week Delivery dayInformed consent

DemographyMedical and zurgical history

Lif€ style, habitsAdditional infomation

Vital signs at:0. 1. 2. 3. 4 hours Dost drus adninistratlon

Blood sampling timePre- drug administratiorl inmediat€ly post delivery from mother

and from plac€lta , halfend 4 hous post druq administationAdverse events questioning

0, l, 2, 3,4 hows Dost drus administrationPhysical examination

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s. INlRo,Uwrerl'uo'Bupivacaine injections contain bupivacaine HCI which is chemically designated as 2-piperidinecarboxamide, I -butyl-N-(2,6- dimethylphenyl)-, monohydrochloride, monohydrate and has

the followins structure:

ct-{e {0H?}i,cH:rI

".N,f'

'l - noNtl

LJ

INDICATIONSBupivacaine is indicated for the production oflocal or regional anesthesia or analgesia for surgery, fororal surgery procedures, for diagnostic and therapeutic procedures, and for obstetrical procedures. Onlythe 0.25%o and 0.5Yo concentrations are indicated for obstetrical anesthesia.Experience with non-obstetrical surgical procedures in pregnant patients is not sufficient to recommenduse ofthe 0.7 5%6 concentration in these patients. Bupivacaine hcl injections is not recommended forintravenous regional anesthesia (Bier Block).

Use in Epidural AnesthesiaDuring epidural administration of(bupivacaine hci injections) , 0.5% and 0.75% solutions should beadministered in incremental doses of 3 mL to 5 mL with sufficient time between doses to detect toxicmanifestations ofunintentional intravascular or intrathecal injection. In obstetrics, only the 0.5%;o and0.25V0 concentrations should be used; incremental doses of3 mL to 5 mL ofthe 0.5% solution notexceeding 50 mg to 100 mg at any dosing interval are recommended. Repeat doses should be precededby a test dose containing epinephrine if not contraindicated. Use only the single dose ampules andsingle dose vials for caudal or epidural anesthesia; the multiple dose vials contain a preservative andtherefore should not be used for these orocedures.

CONTRAINDICATIONS:Bupivacaine is contraindicated in patients with a known hypersensitivity to it orto any local anestheticagent ofthe amide type or to other components ofbupivacaine solutions.

PrecautionsBupivacaine During epidural administration ofBupivacaine hcl injections, concentrated solutions (0.5-0.75%) should be administered in incremental doses of 3 to 5 mL with sufficient time between doses todetect toxic manifestations ofunintentional intravascular or intrathecal injection. Syringe aspirationsshould also be performed before and during each supplemental injection in continuous (intermittent)catheter techniques. An intravascular injection is still possible even if aspirations for blood are negative.During the administration ofepidural anesthesia, it is recommended that a test dose be administeredinitially and the effects monitored before the full dose is given. When using a "continuous" cathetertechnique, test doses should be given prior to both the original and all reinforcing doses, because plastictubing in the epidural space can migrate into a blood vessel or through the dura. When clinical

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/.u

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conditions permit, the test dose should contain epinephrine (10 to 15 mg have been suggested) to serveas a warning ofunintentional intravascular injection. Ifinjected into a blood vessel, this amount ofepinephrine ls likely to produce a transient "epinephrine response" within 45 seconds, consisting ofanincrease in heart rate and systolic blood pressure, circumoral pallor, palpitations and nervousness in theunsedated patient. The sedated patient may exhibit only a pulse rate increase of20 or more beats perminute for l5 or more seconds. Therefore, following the test dose, the heart rate should be monitoredfor a heart rate increase. Patients on beta-blockers may not manifest changes in heart rate, but bloodpressure monitoring can detect an evanescent rise in systolic blood pressure. The test dose should alsocontain l0 to l5 mg ofBupivacaine or an equivalent dose ofa short-acting amide anesthetic such as 30to 40 mg oflidocaine, to detect an unintentional intrathecal administration. This will be manilestedwithin a few minutes by signs ofspinal block (eg, decreased sensation ofthe buttocks, paresis ofthelegs, or, in the sedated patient, absent kneejerk). An intravascular or subarachnoid injection is stillpossible even if results ofthe test dose are negative. The test dose itself may produce a systemic toxicreaction, high spinal or epinephrine-induced cardiovascular effects.

Injection ofrepeated doses oflocal anesthetics may cause significant increases in plasma levels witheach repeated dose due to slow accumulation ofthe drug or its metabolites or to slow metabolicdegradation. Tolerance to elevated blood levels varies with the physical condition ofthe patient.Debilitated, elderly patients, acutely ill patients and children should be given reduced dosescommensurate with their age and physical condition. Local anesthetics should also be used with cautionin patients with hypotension or heart block.

Careful and constant monitoring of cardiovascular and respiratory vital signs (adequacy ofventilation)and the patient's state ofconsciousness should be performed after each local anesthetic injection. Itshould be kept in mind at such times that restlessness, anxiety, incoherent speech, light-headedness,numbness and tingling of the mouth and lips, metallic taste, tinnitus, dizziness, blurred vision, tremors,twitching, depressiorq or drowsiness may be early warning signs of central nervous system toxicity.

Labor and DeliveryBupivacaine regarding obstetrical use in 0.75% concentrationLocal anesthetics rapidly cross the placenta, and when used for epidural, caudal or pudendal blockanesthesia, can cause varying degrees of maternal, fetal and neonatal toxicity. (The incidence anddegree oftoxicity depend upon the procedure performed, the tlpe and amount of drug used, and thetechnique of drug administration. Adverse reactions in the parturient, fetus and neonate involvealterations of the central nervous system, peripheral vascular tone and cardiac function.Maternal hypotension has resulted from regional anesthesia. Local anesthetics produce vasodilation byblocking sympathetic nerves. Elevating the patient's legs and positioning her on her left side will helpprevent decreases in blood pressure. The fetal heart rate also should be monitored continuously, andelectronic fetal monitoring is highly advisable.

Epidural, caudal, or pudendal anesthesia may alter the forces of parturition through changes in uterinecontractility or maternal expulsive efforts. Epidural anesthesia has been reported to prolong the secondstage oflabor by removing the parturient's reflex urge to bear down or by interlering with motorfunction. The use of obstetrical anesthesia may increase the need for forceps assistance. The use of

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some local anesthetic drug products during labor and delivery may be followed by diminished musclestrengh and tone for the first day or two of life. This has not been reported with Bupivacaine .

It is extremely important to avoid aortocaval compression by the gravid uterus during administration ofregional block to parturients. To do this, the patient must be maintained in the left lateral decubitusposition or a blanket roll or sandbag may be placed beneath the right hip and the gravid uterus displacedto the left.Side effects:Bupivacaine

. weak or shallow breathing;o fast heart rate, gasping, feeling unusually hot;o slow heart rate, weak pulse;o feeling restless or anxious, ringing in the ears, metallic taste, speech problems, numbness or

tingling around your mouth, tremors, feeling light-headed, or fainting; oro problems with urination.

Less serious side effects include:. nausea, vomiting;o headache, back pain;o dizziness;o or problems with sexual function.

Pharmacokinetics:BupivacaineThe rate of systemic absorption oflocal anesthetics is dependent upon the total dose and concentrationof drug administered, the route of administration, the vascularity ofthe administration site, and thepresence or absence of epinephrine in the anesthetic solution. A dilute concentration ofepinephrine(l:200,000 or 5 mglml-) usually reduces the rate ofabsorption and peak plasma concentration ofbupivacaine, permitting the use of moderately larger total doses and sometimes prolonging the durationofaction.The onset of action with bupivacaine is rapid and anesthesia is long-lasting. The duration ofanesthesiais significantly longer with bupivacaine than with any other commonly used local anesthetic. It has alsobeen noted that there is a period ofanalgesia that persists after the retum of sensation, during whichtime the need for potent analgesics is reduced,Local anesthetics are bound to plasma proteins in varying degrees. Generally, the lower the plasma

concentration ofdrug, the higher the percentage of drug bound to plasma proteins. Local anestheticsappear to cross the placenta by passive diffi.rsion. The rate and degree of diffusion is governed by: (1)the degree of plasma protein binding, (2) the degree of ionization, and (3) the degree of lipid solubility,FetaVmatemal ratios oflocal anesthetics appear to be inversely related to the degree of plasma proteinbinding, because only the free, unbound drug is available for placental transfer. Bupivacaine, with a

high protein binding capacity (95%), has a low fetaVmaternal ratio (0.2-0.4). The extent ofplacentaltransfer is also determined by the degree ofionization and lipid solubility ofthe drug. Lipid soluble,nonionized drugs readily enter the fetal blood from the maternal circulation.

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Depending upon the route of administration, local anesthetics are distributed to some extent to all bodytissues, with high concentrations found in highly perfused organs such as the liver, lungs, heart, and

brain.Pharmacokinetic studies on the plasma profile ofbupivacaine after direct intravenous injection suggest

a three-compartment open model. The first compartment is represented by the rapid intravasculardistribution ofthe drug. The second compartment represents the equilibration ofthe drug throughoutthe highly perfused organs such as the brain, myocardium, lungs, kidneys, and liver. The thirdcompartment represents an equilibration of the drug with poorly perfused tissues, such as muscle and

fat. The elimination of drug from tissue depends largely upon the ability ofbinding sites in thecirculation to carry it to the liver where it is metabolized.After injection of Sensorcaine (bupivacaine hci injections) (bupivacaine HCI) for caudal, epidural or

peripheral nerve block in man, peak levels ofbupivacaine in the blood are reached in 30 to 45 minutes,

followed by a decline to insignificant levels during the next 3 to 6 hours.Various pharmacokinetic parameters ofthe local anesthetics can be significantly altered by the presence

ofhepatic or renal disease, addition ofepinephrine, factors affecting urinary pH, renal blood flow, theroute of drug administratioq and the age ofthe patient. The halflife ofbupivacaine in adults is 2,7hours and in neonates 8.1 hours.

In clinical studies, elderly patients reached the maximal spread ofanalgesia and maximal motorblockade more rapidly than younger patients. Elderly patients also exhibited higher peak plasma

concentrations following administration of this product. The total plasma clearance was decreased inthese patients.Amidetype local anesthetics such as bupivacaine are metabolized primarily in the liver via conjugationwith glucuronic acid. Patients with hepatic disease, especially those with severe hepatic disease, may be

more susceptible to the potential toxicities ofthe amide+ype local anesthetics. The major metabolite ofbupivacaine is 2,6-pipecoloxylidine.The kidney is the main excretory orgal for most local anesthetics and their metabolites. Urinaryexcretion is affected by renal perfusion and factors affecting urinary pH. Only 5%o ofbupivacaine is

excreted unchanged in the urine.When administered in recommended doses and concentrations, Sensorcaine (bupivacaine hci injections)(bupivacaine HCI) does not ordinarily produce irritation or tissue damage and does not cause

methemoglobinemia.

6. OBJECTIVEOF'THESTUDYThe objective ofthe present study is to assess the concentration ofBupivacaine in the blood ofthe mother

and her new born infant (by measure the concentration in placenta) after having a single epiduralinjection dose of 1.0-1.25 mglml Bupivacaine(HCl).

7. BDNEF'IT - RISK EVALUATIONThe planned procedures within the present trial represent no special risk to the pregnant women, AllPregnant women will be subjected to safety examinations. The screening examination will be carried outnot more than one week before the beginning of the trial. Blood samples of 2 ml for drug analysis will be

taken at the following times: pre-drug administration, immediately post delivery two samples will be

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taken one from mother and the other from the placenta, post halfhours of drug administration, then lastsample will be taken post 4 hours post drug administration.

8. DESIGNThe study will be conducted according to open, single dose, one period, one treatments, in pregnant

women under labor. A total number of 50 pregnant women under labor have to complete the trial.

Choice of pregnant women: The objective of this study is based on the selection ofpregnant womanwho already intended to take this anesthetic medication, from the all ages between 18-45.

Choice ofdosage: The dosage ofthe test product epidural injection of 1.0-1.25 mg/mlBupivacaine(HCl). which is the recommended dose given to women during labor in Jordan.

9. SELECTIONOFPATIENT100 pregnant women, who fulfill the inclusion criteria (Section 9.2), do not present any ofthe exclusioncriteria (Section 9.3), and who have given written patient informed consent (Section 16.3), will beentered into the study.Drug assays and statistical analysis of the data will be performed on all plasma samples of patients whocomplete the study according to the protocol. Patients will be selected and recruited from a group ofpregnant women.

9.1 Justification for Inclusion and Exclusion CriteriaThe criteria are set to ensure a homogeneous pregnart women population without accompanyingdiseases interfering with the conduct and scientific evaluation ofthe study. Additionally, the criteriahave been selected to minimize risk to the oatients' well-beine,

9.2 Inclusion CriteriaOnly pregnant women presenting all ofthe following criteria should be enrolled in the present trial:o Pregnant female.o Age between 18 and 45 years.. Physically and mentally healthy as judged by means ofa medical and standard laboratory examination.o No allergic history to Bupivacaine, or any ofthe excepients.. No alcohol consumtion or a history of alcohol of alcoholism or drug/chemical abuse.r Informed consent given in written form according to section 16.3 ofthe study protocol.

9.3 Exclusion CriteriaPregnant women presenting any ofthe following will not be included in the trial:

o Allergic diathesis or any clinically significant allergic disease. History of allergic response to Bupivacaine .

o Presence or history of heart failure, liver diseases, muscular edema, cardiovascular disease orcardiovascular risk factors.

o Severe renal or hepatic insufficiency.ion in another clinical trial within the last 2 months.

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o Donation of blood or plasma within the last 2 months,r Clinically relevant abnormalities at physical examination or laboratory tests.o Any ckonic disease which might interfere with absorption, distribution, metabolism or excretion

of the drug.o Intake any other anesthetic, narcotic, or any drug during the delivery.o Knowledge to have hepatitis B infection or HIV, or carrier ofthe respective antigens Evidence of

an uncooperative attitude.. Legal incapacity and/or other circumstances rendering the patient unable to understand the

nature, scope and possible consequences ofthe study,

9.4 Other ConditionsAny concomitant disorder not interfering with the exclusion criteria listed in section 9.3, the relevantmedication and any changes in the concomitant disorders and/ or medication have to be documentedappropriately in the Case Report Forms.Additional illnesses present at entry into the study are regarded as concomitant illnesses and generallyas an exclusion criterion. Illnesses occurring during the study period (Inter-cunent illnesses) are to beregarded as adverse events and will each be documented on a separate adverse event form.9,5 Patients' ObligationPatients must adhere to the protocol instructions as presented in the Informed Consent Form, Appendix(02). The pregnant women must notify the clinical investigator of all adverse events experienced or ifthey are unable to follow the study procedures.The clinical investigator must be informed by the patient, or any other responsible person, oftheoccurrence ofany serious adverse event, or ifthe patient requires additional medication ofany kind,irrespective ofthe time ofday or night.

10, MEDICATION10.1 Study MedicationThe investigational drug will be present in hospitals (that have IEC to do clinical study) and thepregnant women will take the medication in hospital at pre-labor time.

10,1.1 Test drugName BupivacaineEach patient will take injection containing as active ingredient 1,0- 1.25 mg/ml Bupivacaine(HCl).Mode of administration epidural injection.

10.1.2 BlindingNo blinding is planned.

10.2 Drug handling and accountabilityThis is a controlled drug and it's not available at JCPR's pharmacy, it is only available at the hospitaland will be given under medical supervision ofthe gynecologist.Before this medication given to pregnant women by gynecologist it will be checked by one ofJCPRstaffto be sure that the patienl given the right medication and documented that by clinical investigatorand a second medical orofessional.

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All empty ampoules must be collected in labeled plastic bag as in section 10 2.2 and accounted for at

the end ofthe study. All that will documented in proper way.

10,2.1 Dosage and Duration of TreatmentPre-delivery time within one to two hours the medication will be given to the patients, all patients willreceive a single dose of i.0- i.25 mg/ml Bupivacaine(HCl)/ 2-5pg as epidural injection .

10.2.2LABELING OF EMPTY AMPOULE

Study No.: Bup-07-2012Patient No.:Drug Name:manufactured by..Dose: L 0- l. 25 mg/ml Bupivacaine(HCl). epiduralinjection, single doseBatch - No.:Expiry date:Storage: keep in closed locker. In hospital.

The instructions for dosage ofthe study medication are given in section 11.4 and separately as a sheet,Patient Information Leaflet, Appendix (03), to be kept in the room where administration takes place

10.3 ComplianceAdministration ofthe study medication will be performed by the clinical investigator and supervised bya second medical professional to ensure the drug intake and in order to verify the compliance ofthepatients. The administration ofthe study medication is to be documented in the CRF and certified bythe Investigator.

10.4 Concomitant MedicationAdministration of any other medication than the study drug is defined as concomitant medication and

will be carefully documented in the CRF as previous and/or concomitant medication.Concomitant medication is generally not allowed for the duration ofthe trial. If this is considered to benecessary for the patient's welfare it may be given at the discretion ofthe Investigator. The patientshave to inform the Investigator about any intake ofother drugs in the course ofthe trial.It will be the Clinical Investigator's decision whether the patient is withdrawn or if she is allowed tocontinue the study. This decision depends on the type of illness and the kind of drug to be used.

Ifthe clinical investigator decided that the patient must be withdrawn from the study, the intake ofconcomitant medication has to be documented in the adverse event form and "drop-out sheet" in thecase report form specifying the substance, dose, time and reason for use ofconcomitant medication andis to be regarded as an exclusion criterion.

10,5 Rescue MedicationSince respiratory arrest may result either through direct depression ofthe respiratory centre or as theresult ofhypoxia, primary attention should be given to the establishment of adequate respiratoryexchange through provision ofa patent airway and institution ofassisted or controlled ventilation.

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The opioid antagonist, naloxone, is a specific antidote. Naloxone (see package information for fullinformation) should be administered intravenously, simultaneously with respiratory resuscitation. Asthe duration of effect ofnaloxone may be considerably shorter than that ofBupivacaine , repeated

administration may be necessary.

10.6 Emergency ProceduresEmergency equipment and drugs will be available within the clinical unit. In case emergency treatmentwill be necessary, the treatment and the drugs used during the emergency should be documented.

10.7 Follow Up ofPatient Experiencing Adverse EventsAny adverse event, which occurs in the course ofthe study, should be monitored and followed up untilit has resolved according to the judgment of the investigator

10.8 Safety AssessmentA1l pregnant women involved in the study will be included in the listing of safety data, Reason forwithdrawal and date of any withdrawal will be reported. All adverse experiences will be listed and

tabulated by severity, treatment and relationship to the therapy.

11. STUDYPROCEDURE11.1 General ProcedurePregnant women within the age limits as defined in section 9.2 will be questioned with reference to theinclusion and exclusion criteria. Patients who seem suitable for inclusion will be asked for informedconsent as described in section 16.3. Patients will then sign the Informed Consent Fornr, Appendix (02)and their code will be recorded in the Screening List.A copy ofall the patient laboratory tests will be taken before the trial in the last visit to her doctor.a list ofthe normal ranges and units of measurement ofthe laboratory parameters to be determinedduring the study will be provided by the Investigator and documented in each CRF.

The laboratory printouts have to be inserted into the CRF as original or authorized copy. Any values outofrange have to be assessed as "not clinically relevant" {NCR) or "clinically relevant" (CR) on thelaboratory printout.

If in the course ofentry screening any clinical relevant abnormal value is observed, this finding will be

regarded as an exclusion criterion. Single laboratory values outside the normal range will generally notbe regarded as an exclusion criterion provided that:1. They are not accompanied by clinical symptoms,2 . The context of related laboratory values gives no indication of a pathological process and,3. The investigator regards them as "not clinically relevant QIICR).

The investigator will check on each Patient's well being prior to their discharge from the clinic.

11.2 Special ProceduresNo further special procedures are planned in the present trial.

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11.3 Daily Activities in Each Period of the TrialEntrv examination:The entry examination will be carried out at the last visiting to her doctor, before the beginning ofthetrial.Following parameters will be documented and /or investigated. Thorough explanation ofthe study medication from the Investigators. Date ofinclusion (date ofsigning the informed consent),. Date of examination,. Inclusion criteria (according to protocol),. Exclusion criteria (according to protocol).. Informed consent (if inclusion criteria fulfilled).. Demographic data: date of birth, ethnic group, sex, height, weight, BML. Additional information concerning: illness within the last 4 weeks prior start ofthe trial, last

participation in any clinical trial, last donation ofblood or plasma, last administration ofanymedication (including OTC or topical medication), last administration of drugs known to alter themajor organs or systems as well as specification ofthe drug name, dosage, start and end oftreatment,

. Vital signs: registration ofbody temperature, measurements ofsupine heart ratc (FIR) and bloodpressure (RR) after 5 minutes supine rest,

. Physicalexamination.

Day 1:The following procedures will be performed and documented in CRF on this day:

o Check ofexclusion criteria, adverse events and concomitant medicationr A venous cannula is inserted and 2 ml blood sample is drawn pre-drug administration.. Blood sampling will continue at the following times: immediately after labor from mother, half

and 4 hours post drug administration.o A blood sample of 2 ml will be withdrawn from the placenta after child birth.. Registration ofvital signs at: 0,7,2,3, 4 hours post dose.o Questioning for and registration ofadverse events: 0, 1,2,3,4 hours post dose.

o Discharge from the hospital should be recorded,

11.4 Drug AdministrationThe precise instructions for drug administration are given to patients by the investigators.A11 patients will receive in the period of the trial a single dose of 1.0-1.25 mglml Bupivacaine(HCl) as

epidural injection

11,5 Blood Sampling for Drug AnalysisBlood sample for drug analysis will be taken pre-drug administration and at the following times afterdrug administration: immediately after labor, half and 4 hours post drug administration from the mother

and 2 ml from the placenta immediately post the delivery.The blood samples 2 ml will be collected into tubes using heparin as anticoagulating agent. After samplingthe tubes will be immediately centrifuged (4000 rpm, room temperature, for 5.0 min), the separated plasma

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will be transferred into polypropylene tubes immediately frozen and stored at a temperature of -10" C orless until analysis.

The samples will be transferred frozen from the hospitals to the laboratories ofJordan Center forPharmaceutical Research (JCPR) for analysis.

I1.6 AnalysisThe determination of Bupivacaine plasma concentrations will be performed by means of a suitablechromatographic assay method, at the laboratories ofJordan Center for Pharmaceutical Research (JCPR)

A validated method as worked out in due time before the start ofthe study.

The method will be validated for linearity and selectivity and be able to determine Bupivacaineconcentrations with sufficient selectivity and accutacy.Standard samples and quality control samples will be distributed through each analysis day.

12. ADVERSE EVENTSl2.l Definition of Adverse EventsAn adverse Event (AE) is any untoward medical occurrence in a pregnant women or clinicalinvestigation patient administered a pharmaceutical product and which does not necessarily have a

causal relationship with this treatment. An adverse event can therefore be any unfavorable andunintended sign (including any abnormal laboratory finding), symptom or disease temporally associatedwith the use ofa medicinal (investigational) product according to ICH Guide line for Good ClinicalPractice 1996.

All adverse events including intercurrent illness must be reported and documented as described below.All patients experiencing adverse events - whether considered associated with the use ofthe studymedication or not - must be monitored until symptoms subside and any abnormal laboratory values

have returned to baseline. Or until there is a satisfactory explanation for the changes observed. A1l

findings must be recorded on an adverse event form and (if appropriate) in the patients medical records.

The data required are: the type of adverse event, an estimate of seriousness, severity, date and time ofonset, date and time ofcessation, actions required, course and outcome ofthe event and an assessment

ofrelationship to administration of the study drug (certain, probable / likely, possible, unlikely,conditional / unclassified and un-assessable / unclassified).

L2,2 Relationship to the Test DrugThe investigator or his delegate will judge whether or not, in his opinion, the adverse event was

connected with the trial medication accoiding to the classification given below (Venulet and Ham)(axs).Each adverse event has to be reported, even ifthe investigator feels that it is not connected with the trialmedication.o Certain: A clinical event, including laboratory test abnormality, occurring in a plausible time

relationship to drug administration, and which cannot be explained by a concurrent disease orother drugs or chemical. The response to withdrawal ofthe drug(dechallenge) should be clinically plausible. The event must be definitive pharmacologicallyor phenomenological, using a satisfactory rechallenges procedure if necessary.

o Probable / likely: A clinical event, including laboratory test abnormality, with a reasonabletime sequence to administration ofthe drug, unlikely to be attributed to concurrent disease or

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other drugs or chemicals, and which follows a clinically reasonable response on withdrawal(dechallenge). Rechallenge information is not required to fulfill this criterion.Possible: A clinical event, including laboratory test abnormality, with a reasonable timesequence to administration ofthe drug, but which could also be explained by concurrent disease orother drugs or chemicals, or where information on drug withdrawal may be lacking or unclear.

Unlikely: A clinical event, including laboratory test abnormality, with a temporal relation ship todrug administration which makes a causal relationship improbable, and in which other drugschemicals or underlying disease provide plausible explanation.

Conditional / unclassified: A clinical event including laboratory test abnormality, reported as an

adverse reaction about which more data is essential for a proper assessment or the additional data are

under examination.Un-assessable / unclassified: A report suggestion on adverse reaction which cannot be judged

because information is insufficient or contradictory, and which cannot be supplemented or verified.

12,3 Classification of Adverse Events (and Adverse Drug Reactions) (6)

In the pre-approval phase ofa product all noxious and unintended responses to a medicinal productrelated to any dose should be considered to be Adverse Drug Reactions.

Regarding marketed medicinal products an Adverse Drug Reaction is a reaction to a drug which isnoxious and unintended and which occurs at doses normally used in man for prophylaxis, diagnosis, ortherapy ofdiseases or for modification ofphysiological function according to ICH guideline for goodclinical practice 1996. An adverse reaction, the nature or severity ofwhich is not consistent with the

applicable product information (e.g, investigators Brochure for an unproved investigational product orpackage insert / summary ofproduct characteristics for an approved product is an Unexpected ADR(from ICH guideline for Good Clinical practice 1996).

Adverse Events and adverse Drug Reactions are divided into the categories serious and non- serious.

This determines the procedure that must be used to report and document the adverse eventA Serious Adverse Event (SAE) or Serious Adverse Drug Reaction (Serious ADR) is any untowardmedical occurrence that at any dose

. result in deatho is life - threateningr Requires inpatient hospitalization or prolongation of existing hospitalization.o Results in persistent or significant disability / incapacity.. Or is a congenital anomaly/ birth defect.

Regardless ofthe classification ofan AE and ADR as serious, non- serious and / or unexpected (see

above) its severity must be assessed as mild, moderate or severe according to medical criteria exclusively.. Mild: awareness of symptoms but does not interfere with routine activities.o Moderate: discomfort enough to interfere with routine activities.r Severe: impossible to perform routine activities.

It should be noted that a severe adverse event needs not to be serious in nature and that a serious

adverse event needs not, by definition, to be severe. Regardless of severity all serious adverse eventsmust be reported as described below.

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12.4 Reporting and Documentation ofAdverse EventsIfan adverse event occurs, it must be recorded on the adverse event form

Serious Adverse Events (SAEs): All SAEs should be reported immediately to the Sponsor except for

those SAEs that the protocol or other document, (e.g. Investigators Brochure), identifies as not needing

immediate reporting. And should be documented in SAE formThe immediaie and follow - up reports should be followed promptly by detailed, r'r'itten reports The

immediate and follow-up reports ihould identify patients by unique code numbers assigned to the trial

patients rather than by the patient's names, personal identification numbers, and /or addresses.

ihe investigator should also comply with the applicable regulatory authority (ies) and the IRB/IEC (s)

where required, and to the regulaiory authority (ies) of all adverse drug reactions that are both serious

and unexpected. Such expecied reports should comply with the applicable regulatory requirement (s)

and withihe ICH Guideline for Clinical Safety Data management: Definitions and Standards for

Expedited ReportingAdverse events andT or laboratory abnormalities defined in the protocol as critical to safety evaluations

should be reported to the Sponsoi according to the reporting requirements and within 24 hours after

information become available. For reported deaths, the Investigator should supply the Sponsor and the

IRB/ IEC with any additional requested information (e,g., autopsy reports and terminal medical

reports).Non- serious Adverse Events: These ars to be documented on an AE- page in the case report forms.

There are no special reporting procedures to be followed.Occurrence ofuny se.ious ad-verse event (including death, irrespective ofthe reason) has to be notified

immediately (within 24 hours), at latest the next working day, to the Principle investigator to the

corresponding addresses (see below):

Principal Investigator Prof, Dr, Tawfiq ArafatJordan Center for Pharmaceutical Research

P.O. Box 950435 Amman, 11195 Jordan

Tel.: 00962-6-5814953Fax Q0962-6-5814952

13. STUDYDOCUMENTATIONf3.1 Investigator'sFileThe investigat& will be having a study file where the following documents will be archived :

1. Investigator Brochure and its updates.2. Study Protocol, with all its appendices and there amendments ifany, with the relevant

communications during the study.3. IRB/IEC composition and dated approval / favorable opinion ofthe Study Protocol with the relevant

appendices.4.

-Curriculum Vitae and /or other relevant documents evidencing qualifications of investigator(s) and

sub- investigator(s).5. Normal value(s) range(s) for medical / laboratory /technical procedure(s) and / or test(s) and there

updated values,e. l,tedical / laboratory / technical procedures /tests (certihcation, accreditation, established quality

control and or extemal quality assessment or other validation).

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7. Sample oflabel(s) attached to investigational product containers.8. Signed Informed Consent Forms-9. Source Documents.10, Signed, dated, and completed Case Report Form (CRF) and their corrections after initial data were

recorded,I L Patient identification code.12. Investigational products accountability at the site to ensure that they have been used according to

the Protocol, to document the final accounting of investigational product(s) received at the site

dispensed to patients, retumed by the patient and retumed to Sponsor or destructed by the investigator.

13. Signature Sheet to document signature and initials of all persons authorized to make entries and / or

corrections on CRF.14. Publication and Literature Survey.15. Ethical and legal information on clinical trials (e.g. ICH Guideline or Good Clinical Practice,

Declaration of Helsinki).16. GCP- statement ofthe Principal Investigator.

13.2 Case Report Form (CRF)Standardized CRFs will be used to document the patients' data during the course ofthe study.

The investigator will assure that all data are entered promptly, legibly, completely, and accurately and

Confirm to the CRF relevant attached source documents. This also applies to the data for those patients

who -after having consented to participate - underwent baseline examinations required especially forinclusion into the study but whom- because a criterion ofexclusion was met or for other reasons - were

not included into the study,To ensure legibility the case report forms should be filled out with a blue ballpoint pen (not pencil, felrtip or fountain pen). Any corrections to the case report forms must be carried out by the investigator orhis designate. A single strike must be drawn through the original entry. The reason for the correction

has to be given, if necessary, and it has to be dated and initialed. Incorrect entries must not be covered

with correcting fluid, or obliterated, or made illegible in any way. Even if there are no changes from a

previous examinatior; in the interests of completeness ofdata acquisition the questions which are

repeated in each section ofthe case report forms should be answered in full. A reasonable explanation

must be given by the investigator for all missing data.A.ll medical records upon which the case report forms are based must be kept for at least 5 years after

completion of the study.

14. PHARMACOKINETICEVALUATIONFor each Pregnant women, the concentration ofBupivacaine in the plasma will be determined as well as

the concentration ofBupivacaine in the new born infants (by measure the concentration of drug inplacenta immediately after labor)15. BIOMETRICS AND STATISTICAL ASPECTS

The biostatistical evaluation will be carried out by Dr. Nasir ldkaidek (data management unit) at JCPR'

by means ofthe software Kinetica 2000 version 4.1, Innaphase Corporation, France.

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The concentration vs. Time profiles after administration ofBupivacaine epidural injection will berepresented in the form oftables and graphics for each pregnant women and the concentration ofBupivacaine in the new born infants will be measured ard tabulated.All safety data will be presented in tabular form as listings ofdata by patient. For the laboratory data

tables ofout-of- range values will be also presented.The Study Report will be prepared as per ICH Topic 83, Structure and Content of Clinical Study Reports.

16.

The study will be performed in accordance with the relevant articles ofthe Declaration ofHelsinki(1964) as revised in Tokyo (1975), Venice (1983), Hong Kong (1989), and Somerset West, RSA (1996)

and Edinburgh (2000). the "Note of Clarification on Paragraph 29" added by the WMA GeneralAssembly, Washington (2002)h and the "Note of Clarification on Paragraph 30" added by the WMAGeneral Assembly, ToLao (2004) ( Note of Clarification onParugraph 30 added), and the 59* WMAgeneral Assembly, Seoul, October 2008.

16.2 Ethical, Legal and Administrative AspectsPrior to the initiation ofthe study, the protocol, the patient information leaflet and the informed consentform will be submitted to the Ethics Committee (IRB[EC) responsible for the review and approval.In case of approval the study can be started immediately after a copy ofthe ethics approval / favorableopinion has been sent to the Sponsor.

If significant protocol changes are necessary which require the preparation ofan amendment, thisamendment has to be submitted to the IRB/IEC for approvalThe study will only be performed when full approval has been obtained from the IRB/IEC and copy ofthe certification has been received, including a list ofthe actual members ofthe IRB/IEC.

16.3 Patient Information and Informed ConsentBefore being admitted to the clinical study, the patient must have given his consent to participate inresponse to a complete written and verbal explanation ofthe nature, scope and possible consequencesofthe clinical study by a physician in a form understandable to him as presented in Appendix (02)The pregnant women must be able to understand the full implications of their decision.

The Patient Information Leaflet, Appendix (03), will be prepared by the principal investigator" and willbe translated into local language whereby the translated documents will be used for confirmation ofthepregnant women consent by the signature ofthe investigator and the pregnant women.It will explain the nature ofthe study, its objectives and potential risks. In addition, the following

points must also be covered:o A description ofthe aims of the study and howitwill be organizedo The type of treatment and the way in which the pregnant women will be allocated to the

treatment.o The positive effects which can be expected ofthe study treatmentso Any negative effects possibly attributable to the study treatmentso The freedom to ask for fuither information at any time

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o The patient's right to withdraw from the clinical study at any time without givingreasons and withoutjeopardizing the further course oftreatment

Personal information will be treated as strictly confidential and not be publicly available the informedconsent forms will be used for confirmation ofthe patient's consent by the signature ofthe investigatorand the patient.

The study specific information for pregnant women and informed consent will be handled to thepatients and enough time will be given to read these documents. The signed and dated informed consentform remains with the investigator as part ofthe confidential data and will be kept there for at least 5

years after the study has been completed- The other personally signed and dated original shall bereceived by the pregnant women,To ensure medical conftdentialtty and data protectioq the patient identification remains with theinvestigator and must be kept there for at least 5 years after the study has been completed. The patientfiles and other source data shall be kept for the maximum period of time permitted by the hospital,institution or private practice.The investigator will allow these documents to be inspected on request and will affirm - by signing anddating - in the case report forms that informed consent has been obtained.

17. GOOD CLINICAL PRACTICE (6)'(A

17,l LegalRequirementsThis study will be conducted in accordance with the following:

e Commission of the European Communities, EC Note for Guidance: Good ClinicalPractice for trials on medicinal products in the European Community, July 1991.( as well as with respective laws and regulations in the countries where clinicalcenters are recruited).

o ICH Topic E3. Structure and Content of Clinical Study Reports, ConsensusGuideline from 17,07.1996 (6IFR37320).

e ICH Topic E 6. Note for Guidance on Good Clinical Practice, Step 5. ConsolidatedGuideline form 01.05, 1996

. Arrangements for archiving of documentation according to the CommissionDirective of 19.07. I 99 I (9 1 | 597 /EEC)

o Guidance for Industry, Bioanalytical Method Validation. May 2001o Note for Guidance on the Investigation of Bioequivalence (CPMP/ QWPEWP/14O1/98

Rev. l) (2010)o Declaration ofHelsinki (Seoul Revision, 2008)r Jordan Drug Lawr JCPR standard operating proceduresa

17.2 Investigator's ObligationsPrior to initiation ofthis study, the investigator will approve this protocol by signing the signature page.

This signature confirms that the study will be performed in compliance with the protocol.

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The investigator must ensure that the Sponsor provides adequate documents (i.e. Investigator'sBrochure or Product Information) giving information about the pharmacological and toxicologicalproperties ofthe test product,

The Investigator or his medically educated representative will review the CRFs for completeness andaccuracy and will sign and date the CRFs and any changes in the CRF.The signatures serve to attest that the information contained on the CRFs is true and have not beenfalsified, in case ofa conection the reason for it shall also be given. It is the Investigator'sresponsibility to assure completion and to review and approve all CRFs. At all times the investigatorhas final responsibility for the accuracy and authenticity ofall clinical and laboratory data entered in theCRFs (s. section 13.2).The Investigator may delegate some ofhis work to certain responsible persons.

17.3 Adherence to the ProtocolProtocol violations are any deviations from the procedures outlined in this document, missedevaluations, incorrect timing of evaluations, non-compliance with study medications and intake ofprohibited medications.

After a palient has been enrolled, it is the investigator's responsibility to make a reasonable effort toavoid any protocol violations in order not to exclude the patient form the study, Protocol violations donot themselves constitute a justification for withdrawal of a patient from the study.

All protocol violations will be reported immediately to the Sponsor during the course ofthe study. Thenature of these violations will be defined in the monitoring protocol. All protocol deviations will belisted and the availability ofthe patiens concerned will be discussed with the Sponsor prior to thestatistical analysis.

17.4 Data Handling ProceduresThe results from screening and the data collected during the study will be recorded in the patient's Case

Report Form (CRF) which will be designed and printed by the principal investigator. Each case reportform will be signed and dated by the investigator.All corrections in the CRFs are to be made legible and signed by the investigator. Each patientReceives a code number. His personal identification remains in a separate confidential file that can beused only together with the investigator. A print out ofthe free text information is compared by asecond person with the CRFs to check its accuracy- The results ofthis check are documented. Duringthe input ofdata a list ofvalues is made, that seem to be not plausible in respect to the normal rangesgiven form the investigator. All these values are to be discussed with the investigator.

Ifthe reason for a not plausible value cannot be clarified consulting the investigator, this value isdefined as a "missing value" in a written form. In order to maintain patient confidentiality. AII datarecorded during the course ofthe study will only be identified by patient codes and patient studynumber. However, the investigator agrees to record the complgte patient identification on the ScreeningList,This list will be treated with strict adherence to confidentiality and will be filed in the investigator'sfile.For the purpose ofthis study, the following will be considered as source data:

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o Data from external sources such as Laboratory investigation result that will be attachedwith the CRF,

. Patient Enrollment Log Sheet which will be kept in the JCPR documentation room,Any records filled by hand and then retyped by computer

17.5 Quality Assurance AspectsThe study will be conducted in accordance to the Quality Assurance System implemented by JCPR as

well as all other requirements mentioned in this Protocol. A1l aspects ofthe Study will be subjected tointernal audit performed by the Quality Assurance Unit. The audit records and the Quality Assurance

Statement will be included in the final report.

17,6 MonitoringIt is the responsibility ofthe investigator to assure that the study is conducted in accordance with theprotocol and that valid data are entered into the CI{F.

Data verification is legally required and will be done by direct comparison with source documents incase ofpatient's respective consent or by cross-checking with source documents in the presence oftheinvestigator- always giving due consideration to data protection and medical confidentialityThe investigator will permit a representative from the Sponsor part to monitor the study as frequently as

necessary to determine that data recording and protocol adherence are satisfactory, The CRFs and

related documents will be reviewed in detail in accordance with the Good Clinical Practice regulations.It is the investigator's obligation to assure documentation of all relevant data in the patient's file, such

as medical history / concomitant diseases, date ofstudy enrolment, visit dates, results of examinations,administrations of medication and adverse events.

Throughout the study, all data will only be identified by patient number and patient codes. The data willbe blinded correspondingly in all data analyses.All obtained data will be checked for plausibility and completeness by the in-house Quality ControlMonitor.17.7 ConfrdentialityAll information obtained during the conduct ofthe study with respect to the patient's state of health willbe regarded as confidential.

Patients will be informed that all study findings will be stored on computer and handled strictlyconfidential. Patients will be identified throughout documentation and evaluation by the individualpatient number and patient code only, whereas names will be kept secret by the investigatorAll information concerning study medication, all study material and study drugs shall remain theproperty ofthe sponsor and the investigatoq where he obliged to keep all data and out cominginformation ofthe study confidential and to use those data only in context with the sponsor.

17.8 Qualification ofthePrincipallnvestigatorBy this signature ofthe study protocol, the principal investigator certifies that he has more than 15

years experience in conducting clinical trials.

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18. PROTOCOLAMENDMENTSAmendments to the conduct ofthe clinical trial may arise from changes to the protocol or from newinformation relating to the scientific documents in support ofthe trial.All amendments have to be discussed and signed by the sponsor and the principal investigator prior toimplementation. Any amendment must be set out in writing, at the same time giving the reasons, andsigned by all parties concerned, The amendment then becomes part ofthe Study Protocol.Amendments which might have an impact on the well - being of the patient such as the use ofadditional invasive examination methods, require a new approvaV favorable opinion by the IRB/IECand a further informed consent form that is to be siened bv all oatients enrolled in the trial who are

affected by the amendment.

Other changes will only be submitted to the IRB/IEC in writing. The investigator may implement a

deviation form, or a change ofthe protocol to eliminate an immediate hazard(s) to trial patients withoutprior IRB/IEC approval. As soon as possible, the implemented deviation or change, the reason for it,and if appropriate, the proposed protocol amendment(s) should be submitted to:a) the IRB/IEC for review and approval opinionb) the Sponsor for agreement.

The investigator should not implement any deviation from or changes ofthe protocol withoutagreement by the sponsor and prior review and documented approval from IRB/IEC ofan amendment,except where necessary to eliminate an immediate hazard(s) to trial patients, or when the change(s)involves only logistical or administrative aspects ofthe trial.The implemented amendments on the Protocol or any of its appendices shall be subjected to JCPR

Quality Assurance System and recorded on Document Revision Record,

19. REPORTSThe Principal Investigator will prepare a final report and submit that to the Sponsor.The final report will contain the following information:o The name and address ofthe investigator and the dates on which the study was initiated and completed.o The objectives study design and procedures as stated in the approved protocol and amendments.r The identity ofthe test and reference drugs. Information on batch numbers, expiry dates and storage

conditions.o Dose levels, dosage regimen and route of administration.. Summary of demographic information, laboratory and clinical safety data, information on adverse

events and withdrawals.o Description ofthe target parameters and results form the statistical comparison including a

description of any calculations and transformations performed.. The signature ofthe Principal investigator and other scientists involved in the study as

authentication of the report.r The Ethics Committee approval.o The analltical report with validation data. The location of storase of all raw data and the final reoort.

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. Report will be written as per ICH Topic E3 Structure and Content of Clinical Study Reports, 17*July 1996. Archiving of essential documents will be performed according to the CommissionDirective of 19.07. I 99 1, (9 | / 59'l /EEC).

19.1 ArchivingThe company responsible for placing the medicinal product on the market shall make

arrangements for archiving of documentation according to the Commission Directive of1 9.07. 1 99 1 (9 1/597 IEEC).

o The investigator shall arrange for the retention of the patient identification codes for at least 5

years after the completion or discontinuation ofthe trial.. Patient files and other source data shall b e kept for the maximum period of time permitted by the

institution or private practice. All raw data generated in connection with this study, together withthe original copy ofthe final report, will be retained in the scientific Archives ofthe Jordan Centerfor Pharmaceutical Research for 5 years and after that the sponsor will be contacted for the fate ofthe documents.

. The sponsor or other owner of the data shall retain all other documentation pertaining to the trialas long as the product is authorized .These procedures shall include:

. The protocol including the rational, obj ectives and statistical design and methodology of the trial,with conditions under which it is performed and managed, and details ofthe investigationalproduct, the reference medicinal product and / or the placebo used.

. Standard operating procedures

. All written opinions on the protocol and procedures,r The investigator's brochureo The final reporto The Sponsor or subsequent owner shall retain the final report, for five years after the product is no

longer authorized.

Any change ofownership ofthe data shall be documented. All data shall be made available ifrequested by relevant authorities.

20. COMMUNICATION OF STUDY RSSULTSIt is envisaged that the findings ofthe study will, in due time and by mutual agreement, be published ina scientific joumal and / or presented at a scientific meeting. It is generally preferable that the results ofthis study will be published as a whole,By signing this protocol the Investigator agrees that the results ofthis study may be used for submissionto national and /or intemational supervising authorities.Any publication or presentation requires the consent ofthe Sponsor.

:26 of 27 Final Version Date: 17 .0'7 .12

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2I. REFDRENCES

1. Available at: www, drugs.com

2. Available at: www.rxlist.com

3. Declaration ofHelsinki, fifty ninth amendment World Medical Assembly. available at :

http://www.wma. net

4. Shah \ry, Midha KK, Dighe S, McGilveray, Skelly JP, Yacobi A, Layloff T, Viswanathan CT,

Cook CE, McDowall RD, Pittman KA Spector S: Analyical Methods Validation: Bioavailability,Bioequivalence, and Pharmacokinetic Studies J. ofPharmaceut. Sci. 1992;81,3: 3Q9-312.

5. Venulet J, Ham MT: Methods for monitoring and documenting adverse drug reactions. Int. J. Clin.Pharmacol. Ther. 1996; 34:112-129 .

6. Guidance for Industry : FDA Bioanalytical Method validation Guidelines US dept. of Health andHuman Services, Food and Drug Administration (FDA), Center for Drug Evaluation and Research

(CDER), March 2003. Available at; http://www.fcla.gov/cder/guidance/index.html

7. Guidance on General Consideration for Clinical Trials (CPMP/ICW29|/95) The European Agencyfor the Evaluation of Medical Products (EMEA), March 1998. available at :

httn://www.ema.eurooa. eu

22, APPENDICES

Appendix (01): Case Report Form, Form # protocol-0lAppendix (02): Inforrned Consent Form, Form # protocol-O3 & protocol-O4Appendix (03): patient lnformation Leaflet, Forms # protocol-os & protocol-06Appendix (04): Investigators Curriculum Vitae

e:27 of 27 Final Version Date: 17.07 .\2

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Appendix (3):

Informed Consent Form

Jordan Center forw

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INFORMED CONSENT FORM

Confidentialstudv Name: A STLIDY To DETERMTND Tr{E CONCENTRATTON oF BtrprvAcArND DURrNLABOR FOR MOTHER AND NEWBORN rtlFANTr

Patient Name: ..,....... (Nam€ of patient ln caDital letters)

Test Medication Bupivacaine(HCL

I)ose 1.0-1.25 mg/ml ampoule forepidural injection

trarulated into Arabio &!

I.the above signed, voluntarily agree to take part in the mentioned clinical study and I have been given enoughtime and explanation ofthe nature, purpose and like$ duration ofthe study and I have been advised about anydiscomfort and possible adverse effects on my health or well being that the supervising doctor believes mayresult, I have also read the volunteer information document relating to the study,I have also read all the information in patient information leaflet (Form # protocor-05),

I have been given the oppoftunity to ask questions about all aspects ofthe study and have understood theadvice and information given as a result.

I have given all information about my previous and present illnesses, together with any medicatron or drugs Ihave taken in the last 3 moths ofmy pregnancy, and about any consultation that I have had wlth any doctor inthe last six months and any medications I am planning to take.

1. Studv's ObiectivesThe aim ofthe present trial is to assess the amount of 1.0-1.25 mglml Bupivacaine in the mother and new borninfant after labor . The efticacy and safety ofBupivacaure drug which has been proved in clinical trials.

2, BenefitsI amaware that, although this study msy help the advance of medical knowledge, I will not dlectly receivehealth benefrts from it, and ifl have not been truthfirl in any ofthe informationl have supplied, and ifl do notfully comp$ with the directions given to me, I may harm myself by participation in ttre siuOy. I confirm thatwhile participation; I will not knowingly do any thing that could reasonably be assumed to ;ffect the results ofthe clinical study.

3, Drug Prcduct InformationBupivacaine is indicated for the production oflocal or regional anesthesia or analgesia for surgery, for oralsurgery procedures, for diagnostic and therapeutic procedures, and for obstetrical procedures.

Study No. : Buo-O'1-2O12 Patient No: Patient Initral:

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Pharmaceutical Research@

-ffiJCPRJordan Center for

INFORMED CONSENT FORM

Confidential

Study No.: Buo-07-2012 Patient No: Patient Initial:

4. Adverse Drus ReactionsBupivacainer weak or shallow breathing;. fast heart rate, gasping, feeling unusually hot;o slow hean rate, weak pulse.. feeltrg restless or anxious, ringing in the ears, metallic taste, speech problems, numbness or tingling around

your mouth, tremors, feeling light-headed, or fainting; orr problems with urination.

Irss serious side effects include:. nausEr, vomiting;. headachg back pain;o dzziness;. or problems wlth sexual function.

5. Studv ProceduresThe study will start at delivery day; the patient will receive one treatment drugl.0-1.25 mg ampoule forepidural injection.

In last doctor visit:Study procedures will be read to you, you have to listen carefully and make sure that you understand it.Copy ofyour laboratory examination that you did it before the delivery will be taken. Also the standardclinical soreening includes medical history, a complete clinical examination, a determination of weight, height,body temperature, measurements of blood pressure and heart rate after 5 minutes supine rest. You will beasked about your medical history and lifestyle, all that information will be collected in a screening form.

Admission:On your delivery day two pharmacists from our staff will be available in that time to collect the data and timefor your delivery.

Durine Studv Conduction:The delivery procedure will be conducted as usually except that before drug administration 2 ml blood samplewill be collected by a short intravenous catheter (Cannula) already present in your vein. The catheter maycause redness or a bruise in its place, just notit/ the clinical investigator if it happen.

Then you will administrate Bupivacaine drug as your doctor recommendation 1.0-l.25 mg ampoule epiduralinteotion

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INFORMED CONSENT FORM

Studv No.: Buo-07-2O12 Patient No: Patient Initial:lrlto tuabic s! D€r Fonn #

ConfidentialOther two blood sample will be oollected immediately post delivery 2ml from mother and 2 ml form theplacenta, half hour post drug administration form mother, and the last sample will be collected post 4 hour ofdrug administration,

- A series of5 x 2 ml blood samples are collected over all the clinical study. Blood samples will be collectedprior to drug administration, immediately post delivery from mother and from placenta, post half and post 4hours of drug administration.

- Total blood volume per patient to be collected during the study will not exceed l0 r (About 8 ml frommother and 2 ml form placenta). Blood samples will be used only for the purposes listed in the study protocol.

- Vital signs will be measured prior to drug administratiorl and post drug administration at the following timel, ?,3 and' 4 hours. Adverse events will be questioned prior to drug administration and 1,2,3and 4 hour postdrug administration.

At follow- un examinatio n:Standard clinical evaluation includes measurements of blood pressure, heart rate, body temperature andphysical examination will be done for all patients.

6. Oualifications1, Pregnant female.2, Age between 18 and 45 years.3. No allergic history to Bupivacaine, or any ofthe excepients.4. Physically and mentally healthy as judged by means of a medical and standard laboratory examination.5. No alcohol consumtion or a history ofalcohol ofalcoholism or drug/chemical abuse.

I have also been informed that:

2.

1. I have been asked about my medical history and I have been rnformed that in case that I am not honest Imight harm myself.I agree to comply with any instruction given during the study and to cooperate r /ith the supervisingdoctors and to tell the doctor immedlately if I suffer fiom any deterioration in my health or well - beingor any unexpected or unusual symptoms whenever they have arisen, IfI fail to oomplete the studybecause my participation is terminated by supervising doctor for failure to comply with instructionsgiven to mg I will not be entitled to any payment.

7, Voluntarv Pafticioation and the Risht to Withdraw

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INFORMBD CONSENT FORMo tra lated inlo A.rabio as D€r Form F

ConfidentialYou are free to withdraw from the clinical study at any time without giving reasons and without jeopardizingthe further course of treatment. Your signature under the informed consent form does by no means oblige youto complete the study.

I further understand that any information that become available during the oourse ofthe study that may affectmy willingness to take part in the study will be disolosed to me as soon as praoticable.

9. ConfidentialitvPersonal information will be treated as strictly confidential and not be publicly, only Jordan center forpharmaceutical research's staff, the sponsors of the study, Jordan FDA, Ministry of health or any othercompetent authority provided with this study, all the files relating to my identity and a socialist in the studyconfidential as pernitted by law

10. Information related to the riqhts of the patients /study related iniuries

For more information about your rights as a volunteer, or In case ofany emergency within or after thestudy conduct don't hesitate to call Dr. Basem lfulaseh (Teh 0096-2-

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INFORMED CONSENT FORMinlo Arabic as D€r Form #

Confidentialll, Siqnatures

I confirm that I have received a copy ofthis consent form and ofthe volunteer information sheet relating to thestudy.

As investigator who performed the informed consent discussion. I am satisfied that the volunteer hasunderstood the explanation and having read the relevant volunteer information, (also I checked the volunteerability to read by selecting certain paragraphs fiom this consent form and I asked him to read them), has freelyconsented to participate in the study.

Name Signature Date

Principal Investigator

name signaturc date

Patient Name

ID#

Impaltial WitnessName

ID#

Page 5 of 5

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Appendix (4):

Case Report Form

Jordan Center forw

-r*JCPRPharmaceutical Research

Case Re rt Form

A STUDY TO DETERMINE THE CONCENTRATION OFBUPryACAINE EPIDURAL INJECTION DURINGLABOR FOR MOTHER AND NEWBORN INFANT

TESTMEDICATION:Bupivacaine Ampoule

Patient No.:

lnitials of Patient:

This Patient isl

Only screened:

Study completer:

Drop - out:

Withdrawal

trtroE Reason:

Study No.: Bup-07-2012 Patient No: Patient Initial:Confidential

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Renort Form

ConfidentialNo.: Buo-07-2012 Patient No: Patient Initial:

General Instructions

Avoid writing outside the space provided

Always use a blue ballpoint pen

Sign and date forms

Infomation must be immediatelv written in the CRF

. Enorc:

Cross out the error with a single horizontal line and write any correction next to it

Do not use correction fluid

Make sure that the error, although crossed out, remains legible

Sign the oorrection

Types of errors:

For writing errors (W.E)

Typing errors (T.E)

Date error (D.E)

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Case Report FormNo.: Bup-07-2012 Patient No: Patient Initial:

Confidential

Flow Chart

Type of Assessment planned Entry examination Trail Day

Study day:In last visit to Doctorpre-delilrry not more

than one weekDelivery day

Informed consentDemogr4\

Medical and surgical historyLife style, habits

Additional informationVital signs at:

0, 1,2,3,4 hours post drug administratiotl

Blood sempling timePre- drug adminishation, immediately post delivery from mother

and from nlacenta . halfand 4 hours post drug administration

Adverse events questioningO. 1.2.3.4 houls post drug administation

Phyiical examination

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Patient No: Patient Initial:Confidential

Motherts Demoqranhic DataDate of birth:

Height:

Blood type:

Race: ECaucasian tr

Medical Historv:

Disabilities:

Allergy:

Hyper - / H5,potension:

Diabetic:

Major surgery:

Other diseases:

Micturition:

Defecation:

Sleep:

Alcohol:

Nicotine:

Caffeine:

Entry ExaminationQ'{ot more than 7 days pre-delivery day)

Date of examination

(DD/ MN,f/ YY)

tDtN#YY+Y)

Weight kg BMI=weight (kg)

Herght (m)'

Black DRed EOriental DOthers. specifv

D yes, please speciry . . . . . , . . . . . . . . .

I yes, pl"ur" .p"cify ... . .. ...

E yes, pl"u.e .p"ciry ... ... ...

E yes, plea." rpori$, ... . .. . . .

E yes, pl*." "p"ciry

. . . . . . . . .

Eyes,pl"u.".p""'".....'..............'.'

E abnormal, please speciJy

E abnormal, please specifu

0 abnormal, please specifu

E yes, please specr& ........ ......Units / weeksflJnit equals 0.5 I beer, 500 ml wine or 50ml spirits )

O yes, please specrry ... . . . . . . . .. .Cigarette s or equivalents /day.

E yes, please specii, ... ... ... ... Cups / day

trnoAnotrnotrnoDnotrno

O normal

E normal

D normal

trno

trnotrno

185

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Renort Form

Confidential

Study No.r Blo-07-2O12 Patient No: Patient Initial:

(DD /MM /YY)

Last administration of anymedication

Last administration of anyinvestigational drug *

l.ast participation in anyclinical trial **

* Inyestigational drug means Bupivacainc** Clinical trial

iig"",r"" "i

i"t"rrigu,o"Entry Examination

flot more than 7 days pre-delivery day)

Presnancy historv:

How many times did you get pregnant? , , ,

Did you take this medication before? . . . ... ...

Ifyes, how did you respond?

Was there any side effect?

Ifyes, please specifr ... , , .

For how long you have been pregnant? ....,,......

Are there any complications in your pregnancy? . . . . . . . . . . . .

Ifyes, please specfy ... ... . . .

Date

t::"t"*:-

or I more than 12 months ago or El none

or E more than 12 months ago or E none

Signature of investigator

(DD A4N,I/YY )

(MM/ YY

ttl(MM / YY )

(DD/MM/YY)

186

Jordan Center for Pharmaceutical Research

Case Renort FormStudv No.: Buo-07-2O12 Patient Nol Patient Initial:

ConfidentialEntry Examination

Clinical Oxamination(Not more thaa 7 days pre-delivery day)

Informed Consent formDid the patient receive a signed copy from the Informed Consent Form? -.. . . ,

Ifyes. mention the date signed: | | | |

t-DDlffiiE)Phvsical examination:

General condition: ......,,....

Pathological findings at physical examination

If yes, please specry . .

tr yes Ono

Body temperature

Heart rate and blood pressure after 5 min supine rest:

HR........ ..... /mm

Clinical findings: tr trnormal abnormal

lf abnormal, please specifuIfabnormal findings present, are these clinically relevant? E yes

Date lrtl(DD/MM/Yg Signature of investigator

Eno

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Report FormNo.: Bun-07-2012 Patient No: Patient Initiat:

ConfidentialEntry Examinationmore than 7

'

Please check all following criteria(Ifone criterion is answered with "no " the patient is not eligible for this study)

yes No

o Pregnant female J trr Age between 18 and 45 years J o

Physically and mentally healthy asjudged by means ofa medical and standard laboratoryexamination. J n

. No allergic history to Bupivacaine. J trr lnformed consent given in written form 3 D

Please check all following criteria(Ifone criterion is answered with "yes" thepatient is not eligible for this studv)

yes Noo Allergio diathesis or any olinically significant allergic disease J Do History of allergic response to Bupivaceine. D Do Presence or history of heart failure, liver diseases, muscular edema, cardiovascular

disease or cardiovascular risk fictors. D D

o Severe renal or hepatic insuffciency. D Do Clinically relevant abnormalities at phlsiqal examination or laboratory tests. o oo Any chronic disease which might interfere with absorption, distribution, metabolism or

excretion ofthe drug. D oo Intake of phenytoirL rifampicin, or phenobarbitone or mono-amino oxidase inhibitors

within 2 weeks prior to start of the study. 3 tro Intake any other anesthetic drug during the delivery. D Do Knowledge to have hepatitis B infection or HIV, or carrier of the respective antigens

Evidence of an uncooperative attitude. T 3. Legal inoapacity and/or other circumstances rendering the patient unable to understand

the naturg scope and possible consequences ofthe study.D D

(DD/MM / YY)Date

Signature of Investigator

188

Jordan Center for -=fr*JCPR

Pharmaceutical Research

Patient No: Patient Initial:Confidential

Laboratory InvestigationClinical Ilaemato

*all this laborstory test arc recorded acaording to patient laboratoryie.sult, iTsGEN.A: not applicable.NCR = Outside normal range, no clinical relevance.CR = Outside normal range, clinically relevantTh,e_piysician responsible for drug safety as per the Combined Delegation and Signature List determines clinical relevance.

a - Values outside normal range with clinical relevance at entry examination are an exclusion criterion.b - A control investigation should be performed if any value out of range with clinical relevance is forurd ar final examination. Allsuch values should be regarded as adverse events.

Date

bdm-/Yd Signature ofPhysician

189

Jordan Center for@

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No,: Buo-07-2012 Patient No: Patient Initial:Confidential

Laboratory Investigation (to be continued)

N.A: not applicable.NCR = Outside normal rangg no clinical relevance.CR = Outside normal range, climcally relevantThe physician responsible for drug safety as per the Combined Delegation and Signature List deterrmnes clinical

relevance.

a - Values outside normal range with clinical relevance at entry examination are an exclusion criierion .

b - A control investigation should be performed if any value out ofrange with clinical relevance is found at finalexamination. All such values should be regarded as adverse events,

If abnormal findings present, are these clinically relevant?Ifyes, this is to be regarded as an exclusion criterion

trNo trYes tr N.A

Subject suitable for the trial:

Dateltll

pYes

*all this laboratory t€st are recorded according to patient labomtory result, ifshe has.

No

190

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Pharmaceutical Research

No.: Bup-07-2012 Patient No: patient Initial:

Date

(DD7VM7YY)

Case Renort Form

ConfidentialSignature of Physician

talisation and Blood

dnffi+Td Signature of Investigator

191

Jordan Center for Pharmaceutical Research

Reoort FormNo.: Buo-07-2012 Patient Nor Patient Initial:

exclusion criteria met at the t visitadministration

adverse events occurred until or the visitr Post t hours of administration. Post 2 hours of administration. Post 3 hours of administrationo Post 4 hours of administration

ConfidentialExclusion Criteria and Adverse Events during the Trial

DateSignature of Investigator

Please note: Concomitant diseases after beginning of the trial are regarded as adverse events and exclusion

criteria. Concomitant medication after beginning ofthe trial are regarded as exclusion criteria. In this case, €fplease fill in the'drop-out sheet'and if needed, 'adverse events form'A final examination has to be performed at the time of withdrawal if possible.

11of 15 Form # nrotocol-0l rev.a

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Case Renort FormNo,: Bup-07-2012 Patient Nor Patient Initial:

General condition:

Occurrence ofadverse events E trno yes

ifyes , please fill in the Adverse Event form

DatettlIDD-7t"tiaITD

ConfidentialFinal Examination

Qrlot more than 2 days after the administration of the test drug)

Date of examination

rttl/MM/YY

Clinical ExaminationPhysical examination

8P....../.. ( mmHg )

oabnormal

Pathologioal findings at physical examination

Bodytemperature .......... C'

Heart rate and blood pressure after 5 min supine rest:HR.,,..,. (/ rnin)

Clinical findings:tr

normalif abnormal , please specify:

if abnormal findings presentthese clinicaUy relevant ? D E

trno

tryes

Signature of investigator

193

Jordan Center forwry

JCPRPharmaceutical Research

No.: Buo-07-2012 Patient No: Patient InitialtConfidential

Adverse Events(olease lill in onlv one adverre event per form)

FirstAdverse Event

Definition: any undesirable experience occurring to a subject, during a clinical trial,*hether or not considered related to the invesigatic,nal product(s).

r- | Please. write legibly and lill in the appropriate numbers. Documenr completely each ad\erse evenL

Nalure of adverse event

OnsetLIJ | | ttt tt | |

DD MM YY hr:min

Cessation| | lt I tl I ll | |

DD MM YY hr:min

Last dose before onsetrrrr r|LLJ ltl

DD MM YY hr:minCause Severity Course/

DAnemSeriousness Relationship

To DrusAction talen Outcome to

date1=Concomitsnldbease2= Int€rcurent

3= oth€r

o

Ifl-3, pleale ffI Inb€lol:

LI

l=Mll'l (nointerfer€nce widrroutin€ ootivitiei)2=Mod€nte(int€rf€reno€ withroutine activities)3=S€v€re

n

l=Iiolsr€d

2=Intermitt€nt

3=Condnuolr!

n

2=Sertoushoepitalisatii:n orprolonged hospita-lisatioq signifiolrt orp€rsirt€nt disability , lifelhr*tening d€ath,

congenital anonaly6(irthdefeat)

{e

if2, pl€o3e tnformThe peruon3 below!

aLI

l=Cerlain,- probabte/likelyF po$lble.l= UnllkelyS Condltloreyuncl63sltied6=Un'sse!sable/unclalsilied

( for d€6nitions seeprotoool , page 21 )

T1l_l

l=Not r€qulred2=Sperific

3=Dos. rcductlon,t=Subject$lthdrntlals-O{her

+o

if2,3pr 5Pleale ffll inbelow!

1__l

1= Full

2=Improred3=Unalrered4=Aggravat€d5=D€rrh6= UnknowD

rOrq

if applicable concomitant disease, medications or other causes related to adverse event:

ea Sponsor: Jordan Center for Pharmaceutical Research, Tel: 00962.6-5814953Principaf Investigator: Dr. Tawfiq Arafat, Tel: 00962-79-5653403

Ob. Action taken:.

Date tttl(DD/MM /YY) Signature of Investigator

194

Jordan Center forwff

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No,: Bun-07-2012 Patient No: Patient Initial:Confidential

Adverse Events(please fill in only one adverse event per form)

SecondAdverse Event

Definition: any undesirable experience occuning to a subject, during a clinical trial,whether or not considered related to the inrestigational product(s).

(a' I Please, urite legibly and hll in the appropfiate numbers. Docrxnent completely each adverse event

Nattrre of adverse evmt

OnsetLIJ I I lll ll t I

DD MM YY hr:min

Cessation| | lt | | | I ll | |

DD MM YY hr:min

Last dose before onsetllll lll tlll I

DD MM YY hr:minCause Severity Course/

DatternSeriousness Relationsbip

To DrusAction taken Outcome to

date

2= Intcrcurrtddt!eBe3= other

tlo

If I J, pl€'s€ fill inbelor!

T-lt_l

r=M d (nointerf€ienoe withroutine acrivities)2=Moderat(interferE roe withrou.ine sotivities)J=Severe

tr

l= Isolsied

2=Intermlttent

3=Continuoul

4=Unlnorrll

n

1=Not s€riou!2=Seriou!hcrpitalilation or prolongedhc6pita-lis.tion, signficantor pesistEnr disability, lifethreatening d€ath,congenital anornaly/b(irrhdefect)

tlo

if2, pleri€ informTh€ persom belowl

a

n

l=Certsin2= probrble/likeb3= po33tble4= Unlikely5= Condtfionayuncla!stfied6=Una!!e!!able

( lor definitions seeprotoaol , pas€ 21 )

r-t_l

1=Not rcquircd2=Spe3ilic

3=Dosereductlon{=Subjectwithdraqial5=Oth€r

+o

if2J,or sPlece lill in

l= Full

2=Impmved3=Unsft€r€d

d5=D€ath6= Unknown

nob.

if applicable concomitant disease, medications or other caus€s rolated to adt'erso event:

EE Sponsor: Jordan Center for Pharmaceutical Research, Tel: 00962-65814953Principal Investigator: Dr. Tawfiq Arafat, Tel: 00962-79-56$443

gts. Action taken:

Date(DD/MM /YY) Signaturc of Investigator

14 of 15 Form # Drrtocol-Ol rev.a

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Case Report FormNo.: Bun-07-2012 Patient Nol patient Initial:

Date

Statement of the principal Investigator

I hereby confirm that the clinical trial was conducted in accordance with the principlesof the Declaration of Helsinki and ICH GCp - guidelines and the data in this case

Report Form is to the best of my knowledge a truthtrl and correct representation ofthe source data and results of the trial.

Signature of Investigator(DD /MM / YY)

196

l5 of 15 Form # protocol-0l rev.a

4u,i{ll ujDl+lt+ OS$rr_..lu+Jt a*,,'Jid 4JJE eir;..;E &EAt J rJJ!:irYjJl rE3i ., &lJ.ll ,;e l.d#Ll 4. u.r+Jl r

rle!j!.:i:t sJe (J/lrl

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