contribution for the analysis of certain drugs which treat cerebrovascular insufficiency

إال )) تعبدوا أال ربك قضى إال و تعبدوا أال ربك قضى وإما إحسانا وبالوالدين إما إياه إحسانا وبالوالدين إياه

أو أحدهما الكبر عندك أو يبلغن أحدهما الكبر عندك يبلغنوال أف لهما تقل فال وال كالهما أف لهما تقل فال كالهما

+ قوال لهما وقل + تنهرهما قوال لهما وقل تنهرهماجناح* لهما واخفض جناح* كريما لهما واخفض كريمارب وقل الرحمة من رب الذل وقل الرحمة من الذل

+ صغيرا ربيانى كما + ارحمهما صغيرا ربيانى كما (*(*ارحمهما

Contribution for the Contribution for the analysis of certain drugs analysis of certain drugs

which treat which treat cerebrovascular cerebrovascular

insufficiencyinsufficiency

This thesis consists of six partsThis thesis consists of six parts Part I: General introductionPart I: General introduction Part II: New spectrophotometric method for Part II: New spectrophotometric method for

simultaneous determination of binary simultaneous determination of binary mixtures of nicergoline and cinnarizine and mixtures of nicergoline and cinnarizine and stability indicating for vincamine.stability indicating for vincamine.

Part III: Simultaneous determination of Part III: Simultaneous determination of nicergoline and cinnarizine. nicergoline and cinnarizine.

Part IV: Stability indicating methods for the Part IV: Stability indicating methods for the determination of meclophenoxate determination of meclophenoxate hydrochloride.hydrochloride.

Part V: Stability indicating methods for the Part V: Stability indicating methods for the determination of vinpocetine.determination of vinpocetine.

Part VI: Stability indicating methods for the Part VI: Stability indicating methods for the determination of Pyritinol dihydrochloride.determination of Pyritinol dihydrochloride.

Part IPart I

General introductionGeneral introduction

Types of cerebrovascular diseaseTypes of cerebrovascular disease Cerebrovascular insufficiency Etiology Cerebrovascular insufficiency Etiology

and Pathophysiologyand Pathophysiology Complications of cerebrovascular Complications of cerebrovascular

insufficiencyinsufficiency Mechanism of action of the selected Mechanism of action of the selected

drugsdrugs

Part IIPart IINew spectrophotometric New spectrophotometric method for simultaneous method for simultaneous determination of binary determination of binary mixtures of nicergoline mixtures of nicergoline

and cinnarizine and and cinnarizine and stability indicating for stability indicating for

vincaminevincamine

This part includes a general introduction This part includes a general introduction about the chemistry of nicergoline, about the chemistry of nicergoline, cinnarizine and vincamine.cinnarizine and vincamine.

Review article on the reported methods Review article on the reported methods used for their quantitative used for their quantitative determination. determination.

This part is subdivided into two sections:This part is subdivided into two sections: Section(A):Section(A): Determination of vincamine Determination of vincamine

in presence of its acid degradation in presence of its acid degradation product by the ratio subtraction methodproduct by the ratio subtraction method

Section(B):Section(B): Determination of nicergoline Determination of nicergoline and cinnarizine by the ratio subtraction and cinnarizine by the ratio subtraction and isosbestic point methodsand isosbestic point methods

Section(A)Section(A)Determination of Determination of

vincamine in presence of vincamine in presence of its acid degradation its acid degradation product by the ratio product by the ratio subtraction methodsubtraction method

Structure of Vincamine:Structure of Vincamine:

NN

O

HO

H3CO

H

CH3

Theory of ratio subtraction method:Theory of ratio subtraction method:The method depends on that, if you have a The method depends on that, if you have a mixture of two drugs (X) and (Y) with mixture of two drugs (X) and (Y) with overlapping spectra and the spectrum of (Y) is overlapping spectra and the spectrum of (Y) is extended than (X), the determination of (X) extended than (X), the determination of (X) can be done by dividing the spectrum of the can be done by dividing the spectrum of the mixture by a certain concentration of (Y) as a mixture by a certain concentration of (Y) as a devisor (Y'). The division will give a new curve devisor (Y'). The division will give a new curve that represents .If we subtract this that represents .If we subtract this constant, then multiply the new curve obtained constant, then multiply the new curve obtained after subtraction by (Y') (the devisor), therefore after subtraction by (Y') (the devisor), therefore we can obtain the original curve of (X). we can obtain the original curve of (X). This can This can be summarized as follows:be summarized as follows: tcons

Y

X

Y

Y

Y

X

Y

YXtan

''''

'tantan

' Y

Xtconstcons

Y

X

XYxY

X'

'

The constant can be determined directly from the curve

by the straight line which is parallel to the wavelength axis in the region where (Y) is extended.

tconsY

Xtan

'

tconsY

Xtan

'

Figure (2): Absorption spectra of vincamine 20 µg ml-1 (———) degradation product 20 µg ml-1 (---------- )

and deg.product 16 µg ml-1 (devisor) (———) using 0.1N hydrochloric acid as a solvent.

268nm

Figure (5): Division spectra of laboratory prepared mixtures of vincamine (X) and its degradation product (Y) using 16 µg ml-1 of degradation product (Y') as a divisor and 0.1 N HCl as a solvent.

A (X+Y/Y’)

Figure (6): Division spectra of laboratory prepared mixtures of vincamine (X) and its degradation product (Y) using 16 µg ml-1 of degradation product (Y') as a divisor and 0.1 N HCl as a solvent after subtraction of the constant.

A (X/Y')

Figure (7): The obtained absorption spectra of vincamine in lab.mixtures 8-32 g.ml-1

A (X/Y’*Y’ = X)

Figure (7): The original absorption spectra of vincamine in cal.curve from 8-40 g.ml-1

268nm268nm

Figure (4): Linearity of the absorbance of the zero order curve at 268.2 nm to the corresponding concentration of vincamine.

y = 0.0239x - 0.0017

R2 = 0.9998

00.20.40.60.8

11.2

0 10 20 30 40 50

Concentration ug.ml-1

Peak

abso

rbanc

e Absorbance

Table (I): Determination of vincamine in Table (I): Determination of vincamine in laboratory prepared mixtures by the laboratory prepared mixtures by the proposed methodproposed method..

Concentration (µg.ml-1) Percentage % Ratio subtraction

method

VincamineDegradation

productVincamine

Degradation product

Recovery %

Vincamine

8 32 20 80 98.60

12 28 30 70 98.91

16 24 40 60 98.24

20 20 50 50 99.51

24 16 60 40 100.36

28 12 70 30 99.32

32 8 80 20 99.85

Mean 99.25

S.D. 0.732

Table (II): Determination of vincamine Table (II): Determination of vincamine in oxybral capsules by the proposed in oxybral capsules by the proposed methodmethod..

Oxybral capsules claimed to

contain 30 mg vincamine

Batch number

Ratio subtraction method

Reported method * Company method **

Found % ± S.D.** Found % ± S.D.** Found % ± S.D.**

052831 A 99.01 ± 0.762 99.07 ± 0.466 99.32 ± 0.956

0012261 A(expired 5/04)

84.63 ± 1.025 84.11 ± 0.501 97.56 ± 0.857

* Stability indicating spectrophotometric method.** Spectrophotometric method

Table (III): Application of standard Table (III): Application of standard addition for the determination of addition for the determination of vincamine by the proposed methodvincamine by the proposed method..

Batch number

Vincamine Ratio subtraction method

Standard added in mg Recovery % of added

052831 A 2537.550

98.5699.0199.99

Mean ± S.D.* 99.98 ± 0.731

Table (IV): Statistical comparison for Table (IV): Statistical comparison for the results obtained by the proposed the results obtained by the proposed method and the reported method for method and the reported method for the analysis of vincamine in pure the analysis of vincamine in pure powder formpowder form

* Stability indicating spectrophotometric method.

Item Ratio subtraction method Reported method *

Mean 99.72 99.90

S.D. 0.917 1.041

Variance 0.841 1.084

n 9 6

F test 1.288 (3.69)

Student’s t test 0.353 (2.160)

The figures in parenthesis are the corresponding tabulated values at P=0.05

Section [B]Section [B]Determination of Determination of

nicergoline and cinnarizine nicergoline and cinnarizine by the ratio subtraction by the ratio subtraction

and isosbestic point and isosbestic point methodsmethods

Structure of Structure of nicergoline:nicergoline:

Structure of Structure of cinnarizine:cinnarizine:

N

N

N

O

OBr

H3C

CH3H

H3CO

N

N

H

H

Figure (9): Absorption spectra of Nicergoline 20 µg ml-1 (———) Cinnarizine 20 µg ml-1 (------------- ) and mixture of 10 µg ml-1 of each drug (……….) using methanol as a solvent.

235.8 nm

270.2 nm

Figure (10): Zero order absorption spectra of nicergoline 6- 36 μg ml-1

y = 0.0119x - 0.0001

R2 = 0.9999

0

0.1

0.2

0.3

0.4

0.5

0 10 20 30 40


Peak

abs

orba

nce

y = 0.041x + 0.0349

R2 = 0.9998

00.20.40.60.8

11.21.41.6

0 10 20 30 40


Peak

abs

orba

nce

Figure (12): Linearity of the absorbance of the zero order curve at 270.2 nm to the corresponding concentration of nicergoline.

Figure (13): Linearity of the absorbance of the zero order curve at 235.8 nm to the corresponding concentration of nicergoline

Absorbance Absorbance

A (X+Y/Y’)

Figure (15): Division spectra of laboratory prepared mixtures of cinnarizine (X) and nicergoilne (Y) using 6 µg ml-1 of nicergoline (Y') as a divisor and methanol as a solvent. (Scale x 0.1)

Figure (16): Division spectra of cinnarizine (X) and nicergoilne (Y) using 6 µg ml-1 of nicergoline (Y') as a divisor and methanol as a solvent after subtraction of the constant.(scale x 0.1)

A (X/Y')

Figure (17): The obtained absorption spectra of cinnarizine in lab.mixtures.

A (X/Y‘*Y’ = X)

Figure (17): The original absorption spectra of cinnarizine in cal.curve.

252nm

y = 0.0578x + 0.0193

R2 = 0.9997

0

0.5

1

1.5

0 5 10 15 20 25


Peak

abs

orba

nce

Figure (14): Linearity of the absorbance of the zero order curve at 252.0 nm to the corresponding concentration of cinnarizine.

Absorbance

Table (V): Determination of nicergoline Table (V): Determination of nicergoline and cinnarizine in laboratory prepared and cinnarizine in laboratory prepared mixtures by the proposed methodsmixtures by the proposed methods

Concentration (µg ml-1) Ratio Isosbestic point method

Ratio subtraction

method

Nicergoline cinnarizine Nicergoline : Cinnarizine

Recovery % Recovery %

Nicergoline Cinnarizine

270 nm 235 nm

9.0 13.5 2 : 3 100.51 100.88 100.97

6.0 10.5 2 : 3.5 101.05 101.47 101.45

7.0 14.0 2 : 4 99.54 99.77 100.43

6.0 13.5 2 : 4.5 98.96 99.17 101.07

6.0 15.0 2 : 5 98.68 99.07 99.43

6.0 16.5 2 : 5.5 99.75 100.31 100.00

6.0 18.0 2 : 6 99.31 99.27 100.14

6.0 19.5 2 : 6.5 99.03 98.66 99.96

6.0 21.0 2: 7 100.25 100.00 100.48

Mean S.D. 99.67 0.790

99.84 0.918

100.43 0.635

Table (VI): Determination nicergoline Table (VI): Determination nicergoline and cinnarizine in cinibral tablets by and cinnarizine in cinibral tablets by the proposed methodthe proposed method..

Cinibral tablets Cinibral tablets claimed to contain claimed to contain 10 mg nicergoline 10 mg nicergoline

& 25 mg & 25 mg cinnarizinecinnarizine

Batch numberBatch number

Isosbestic Isosbestic point methodpoint method

Ratio Ratio subtraction subtraction

methodmethod

Reported methodReported method* *

Found % ± Found % ± S.D.S.D.**for for

nicergolinenicergoline

Found % ± Found % ± S.D.S.D.**for for

cinnarizinecinnarizine

Found % ± Found % ± S.DS.D..**

NicergolineNicergoline

Found % ± Found % ± S.DS.D..**

cinnarizinecinnarizine

λ1λ1 λ2λ2

3912339123 99.2499.24 ± ±

0.8770.877

98.6198.61 ± ±

0.8740.874

99.1599.15 ±±

0.2120.212

99.3299.32± ±

0.9560.956

98.3798.37 ± ±

0.8920.892

4008240082 100.8100.866 ± ±

0.6190.619

99.8699.86 ± ±

0.7330.733

99.5899.58± ±

0.3010.301

98.5698.56 ±±

0.8570.857

98.9998.99 ± ±

0.8250.825

* HPLC method.

Table (VII): Application of standard Table (VII): Application of standard addition for the determination of addition for the determination of nicergoline and cinnarizine by the nicergoline and cinnarizine by the proposed methodproposed method..

Batch number Standard added (mg) Isosbestic point method

Ratio subtraction method

nicergoline cinnarizine Recovery % of added

nicergoline

Recovery % of added

cinnarizine

λ1 λ2

39123 10.0015.0020.00

25.0037.5050.00

99.26100.03100.32

99.66100.83101.05

98.1898.9398.06

Mean S.D.* 99.87 0.54

7

100.51

0.747

98.39

0.471

* Average of four determinations.

Table (VIII): Statistical comparison for Table (VIII): Statistical comparison for the results obtained by the proposed the results obtained by the proposed method and the reported method for method and the reported method for the analysis of nicergoline and the analysis of nicergoline and cinnarizine in pure powder formcinnarizine in pure powder form

* HPLC method.

Item Isosbestic point method Ratio subtraction method

Reported method*

nicergoline cinnarizine nicergoline Cinnarizine

270 nm 235 nm

Mean 99.58 99.83 99.91 99.64 99.27

S.D. 0.847 1.039 0.703 0.978 0.714

Variance 0.717 1.079 0.494 0.956 0.509

n 9 9 9 6 6

F test 1.333(3.69)

1.128(4.82)

1.030(3.69)

Student's t test 0.126(2.160)

0.354(2.160)

1.717(2.160)


Table IX: Assay parameters and method Table IX: Assay parameters and method

validationvalidation Parameter

Ratio subtraction method Isosbestic point method Ratio subtraction method

Vincamine nicergoline Cinnarizine

λ=270nm λ=235nm

Range (µgml-1) 8.0-40.0 6- 36 6- 36 6.0-22.0

Slope 0.0239 0.0119 0.041 0.0578

Intercept 0.0017 -0.0001 0.00349 0.0193

Mean 99.72 99.58 99.83 99.91

S.D. 0.917 0.847 1.039 0.703

Variance 0.840 0.717 1.079 0.494

Correlation coefficient (r)

0.9998 0.9999 0.9998 0.9997

* RSD%a 0.788, 0.903 0.620, 0.805 0.942, 0.641 0.732, 0.897

*RSD%b 0.936, 0.984 0.881, 1.173 0.892, 1.274 0.841, 0.875

RSD %a, RSD %b the intra-day, inter-day respectively (n=5) relative standard deviation of concentrations (28, 32 µg/ml) for vincamine, (6, 8 µg/ml) for nicergoline and (14, 16 µg/ml) for cinnarizine..

Part IIIPart IIISimultaneous Simultaneous

determination of determination of nicergoline and cinnarizine nicergoline and cinnarizine

in their binary mixturein their binary mixture

This part is subdivided into four sections: This part is subdivided into four sections: Section [A]:Simultaneous determination Section [A]:Simultaneous determination

of nicergoline and cinnarizine by the of nicergoline and cinnarizine by the derivative spectrophotometryderivative spectrophotometry

Section [B]: Section [B]: SimultaneousSimultaneous determination determination of nicergoline and cinnarizine by of nicergoline and cinnarizine by densitometric methodsdensitometric methods

Section [C]: Section [C]: Simultaneous determination Simultaneous determination of nicergoline and cinnarizine by high-of nicergoline and cinnarizine by high-performance liquid chromatographyperformance liquid chromatography

Section [D]: Simultaneous determination Section [D]: Simultaneous determination of nicergoline and cinnarizine by of nicergoline and cinnarizine by chemometric methodschemometric methods

Section [A]Section [A]Simultaneous Simultaneous


by the derivative by the derivative spectrophotometryspectrophotometry

Figure (67): Absorption spectra of Nicergoline 22 µg ml-1 (———) and Cinnarizine 12 µg ml-1 (---------- ) using methanol as a solvent.

dA/dλ

Figure (68): First order spectra of Nicergoline 22 μg ml-1 (______) Cinnarizine 12 μg ml-1 (_ _ _ _ _ _) using methanol as a solvent.

307nm

dA/dλ

Figure (69): First – derivative absorption spectra of 6 - 38 µg ml-1 nicergoline.

307nm

Figure (70): Linearity of the peak amplitude of the first derivative at 307.6 nm to the corresponding concentration of nicergoline.

y = 0.0238x + 0.0072

R2 = 0.9998

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 10 20 30 40


Pea

k am

plitu

de

A (cinnarizine/nicergoline)

Figure (71): Zero order of ratio spectra of cinnarizine 6- 22 μg ml-1 using 10 µg ml-1 of nicergoline as a divisor.

dA(cinnarizine/nicergoline)/dλ

Figure (72): First order of ratio spectra of cinnarizine 6- 22 μg ml-1 using 10 µg ml-1 of nicergoline as a divisor.

244nm

y = 0.2706x + 0.0854

R2 = 0.9996

0

2

4

6

8

0 5 10 15 20 25


Peak

ampli

tude

Figure (73): Linearity of the peak amplitude of the first derivative of the ratio spectra at 244.6 nm to the corresponding concentration of cinnarizine.

Table (LXIII): Simultaneous Table (LXIII): Simultaneous determination of nicergoline and determination of nicergoline and cinnarizinee in laboratory prepared cinnarizinee in laboratory prepared mixtures by the proposed methodsmixtures by the proposed methods..

Concentration (µg ml-1) Ratio First derivative method

Derivative ratio method

Nicergoline cinnarizine Nicergoline : Cinnarizine

Recovery % Recovery %

Nicergoline Cinnarizine

9.0 13.5 2 : 3 98.88 100.74

6.0 10.5 2 : 3.5 99.25 101.62

7.0 14.0 2 : 4 98.24 100.89

6.0 13.5 2 : 4.5 99.35 101.11

8.0 20.0 2 : 5 99.81 99.81

6.0 16.5 2 : 5.5 99.81 100.09

7.0 21.0 2 : 6 99.35 99.90

6.0 19.5 2 : 6.5 99.81 100.06

6.0 21.0 2: 7 101.75 100.63

Mean S.D. 99.58 0.961 100.530.616

Table (LXIV): Determination of Table (LXIV): Determination of nicergoline and cinnarizine in cinibral nicergoline and cinnarizine in cinibral

tablets by the proposed methods.tablets by the proposed methods.

Cinibral tablets claimed to contain 10 mg

nicergoline & 25 mg cinnarizine

Batch number

First derivative method


Reported method *

Found % ± S.D.*for

nicergoline

Found % ± S.D.*for

cinnarizine

Found % ± S.D.*for

nicergoline

Found % ± S.D.*for

cinnarizine

39123 99.54 ± 0.869 100.25 ±0.396 99.32 ± 0.956 98.37 ± 0.892

40082 98.86 ± 0.639 99.46 ± 0.589 98.56 ± 0.857 98.99 ± 0.825

* HPLC method.

Table (LXV): Application of standard Table (LXV): Application of standard addition for the determination of addition for the determination of nicergoline and cinnarizine by the nicergoline and cinnarizine by the

proposed methodproposed method.. Batch number Standard added (mg) First derivative

methodDerivative ratio

method

nicergoline cinnarizine Recovery % of added

nicergoline

Recovery % of added

cinnarizine

39123 10.0015.0020.00

25.0037.5050.00

99.6298.5498.14

98.1898.9398.06

Mean S.D.* 98.76 0.765 98.39 0.471


Table (LXVI): Statistical comparison for the Table (LXVI): Statistical comparison for the results obtained by the proposed method and results obtained by the proposed method and the reported method for the analysis of the reported method for the analysis of nicergoline andnicergoline and

cinnarizine in pure powder formcinnarizine in pure powder form

* HPLC method.

Item First derivative

method


Reported method*

nicergoline cinnarizine nicergoline Cinnarizine

Mean 99.77 99.95 99.64 99.27

S.D. 0.767 0.752 0.978 0.714

Variance 0.588 0.565 0.956 0.509

n 9 9 6 6

F test 1.625 (3.69) 1.110 (4.82)

Student's t test 0.288 (2.160) 1.750 (2.160)


Section [B]Section [B]SimultaneousSimultaneous

determination of determination of nicergoline and cinnarizine nicergoline and cinnarizine by densitometric methodsby densitometric methods

Figure (74): TLC chromatogram of nicergoline and cinnarizine A= nicergoline, Rf= 0.505. B= cinnarizine, Rf = 0.807. M= mixture of both drugs Developing system, chloroform : methanol : ethyl acetate(5: 3 :2 v/v/v)

Figure (75): Scanning profile of the TLC chromatogram of nicergoline at 287 nm.

Figure (76): Scanning profile of the TLC chromatogram of cinnarizine at 252 nm.

Distance (mm) Distance (mm)

Reflectance

Reflectance

y = 0.2195x - 0.1541

R2 = 0.9997

0

2

4

6

8

0 10 20 30 40

Concentration in ug.spot-1

Peak

area

(x

10

-4)

Figure (77): Linearity of the area under the peak to the corresponding concentration of nicergoline

Figure (78): Linearity of the area under the peak to the corresponding concentration of cinnarizine

y = 0.6517x + 0.1104

R2 = 0.9995

0123456

0 2 4 6 8 10

Concentration in ug.ml-1

Peak

area

(x

10-4

)

spot-1

Table (LXVII): Determination of Table (LXVII): Determination of nicergoline and cinnarizine in nicergoline and cinnarizine in laboratory prepared mixtures by the laboratory prepared mixtures by the

proposed methodproposed method.. Volume taken from stock solns (ml)

Concentrationg.spot-1

Ratio nicergo

line : cinnari

zine

Densitometric method

nicergoline CinnarizineB

nicergoline Cinnarizine Recovery % for

nicergoline

Recovery % for

Cinnarizine

2 3 8 12 3: 2 98.05

2 4 8 16 4: 2 98.56

2 5 8 20 5: 2 98.01

2 6 8 24 6: 2 98.99

2 7 8 28 7:2 99.38

0.5 0.75 2 3 3: 2 100.02

0.5 1 2 4 4: 2 98.71

0.5 1.25 2 5 5: 2 98.50

0.5 1.5 2 6 6: 2 98.64

0.5 1.75 2 7 7: 2 98.17

Mean S.D. 98.59

0.594 98.80

0.708

Table (LXVIII): Determination of Table (LXVIII): Determination of nicergoline and cinnarizine in cinibral nicergoline and cinnarizine in cinibral

tablets by the proposed methodtablets by the proposed method..

Cinibral tablets claimed to contain 5 mgBatch number

Densitometric method Reported method *

% Found for nicergoline ± S.D.*

% Found for cinnarizine ± S.D.*

% Found for nicergoline ±

S.D.*

% Found for cinnarizine ±

S.D.*

39123 100.19 ± 0.605 99.74 ± 0.927 99.32 ± 0.956 98.37 ± 0.892

40082 98.90 ± 1.125 99.01 ± 1.401 98.56 ± 0.857 98.99 ± 0.825

* HPLC method.

Table (LXIX): Application of standard Table (LXIX): Application of standard addition for the determination of addition for the determination of nicergoline and cinnarizine by the nicergoline and cinnarizine by the

proposed methodproposed method.. Batch number Standard added (mg) Densitometric method

nicergoline cinnarizine Recovery % of added nicergoline

Recovery % of added cinnarizine

39123 20.0030.0040.00

50.0075.00

100.00

98.65100.6297.87

99.0499.60

101.63

Mean S.D. 99.04 1.417 100.09 1.362


Table (LXX): Statistical comparison for the Table (LXX): Statistical comparison for the results obtained by the proposed method and results obtained by the proposed method and the reported method for the analysis of the reported method for the analysis of nicergoline and cinnarizine in pure powder nicergoline and cinnarizine in pure powder

formform

* HPLC method.

Item Densitometric method Reported method*

nicergoline cinnarizine nicergoline cinnarizine

Mean 99.93 99.95 99.64 99.27

S.D. 1.128 1.418 0.978 0.714

Variance 1.272 2.010 0.956 0.509

n 7 7 6 6

F test 1.330 (4.95) 3.949 (4.95)

Student's t test 0.490 (2.201) 1.060 (2.201)


Section [C]Section [C]Simultaneous Simultaneous


by high-performance by high-performance liquid chromatographyliquid chromatography

Figure (79): Liquid chromatographic separation of nicergoline and cinnarizine using final assay conditions: Column: RP18 Mobile phase: methanol : acetonitrile : water (4: 4: 2 v/v/v). Flow rate: 1.5 ml min-1. Detection: 287 nm for nicergoline and 252 nm for cinnarizine. Retention time nicergoline: 2.15 min. Retention time cinnarizine: 4.72 min.

Time (min)

Detector response

y = 0.0504x - 0.0031

R2 = 0.9998

0

0.5

1

1.5

2

2.5

0 10 20 30 40 50


Relat

ive pe

ak ar

ea Figure (80): Linearity of the relative peak area to the corresponding concentration of nicergoline.

Figure (81): Linearity of the relative peak area to the corresponding concentration of cinnarizine.

y = 0.0524x - 0.0401

R2 = 0.9997

00.5

11.5

22.5

33.5

44.5

5

0 20 40 60 80 100


Rela

tive

peak

are

a

Table (LXXI): Determination of Table (LXXI): Determination of nicergoline and cinnarizine in nicergoline and cinnarizine in laboratory prepared mixtures by the laboratory prepared mixtures by the

proposed methodproposed method.. Volume taken from stock

solns (ml)Concentration

g.ml-1

Rationicergoline: cinnarizine

HPLC method

nicergoline Cinnarizine nicergoline Cinnarizine nicergoline cinnarizine

2.0 3.0 20 30 3: 2 99.03 101.06

2.0 4.0 20 40 4: 2 98.74 100.78

1.0 2.5 10 25 5: 2 97.96 100.19

1.0 3.0 10 30 6: 2 100.01 101.67

1.0 3.5 10 35 7:2 98.43 101.99

Mean S.D. 98.83 0.767

101.13 0.714

Table (LXXII): Parameters required for Table (LXXII): Parameters required for system suitability test of HPLC methodsystem suitability test of HPLC method

Parameter Obtained value Reference value

Resolution (R) 1.959 R > 0.8

T ( tailing factor) nicergoline 1.208 T = 1 for a typical symmetric peakcinnarizine 1.187

(relative retention time) 2.512 > 1

K’ (column capacity) nicergoline 3.782 1- 10 acceptable

cinnarizine 9.504

N (no.of theoretical platesno.of theoretical plates) nicergoline 463.11 Increases with efficiency of the separationcinnarizine 427.95

HETP nicergoline 0.05398 The smaller the value, the higher the column efficiencycinnarizine 0.0584

Table (LXXIII): Determination of Table (LXXIII): Determination of nicergoline and cinnarizine in cinibral nicergoline and cinnarizine in cinibral


Cinibral tablets claimed to contain

5 mgBatch number

HPLC method Reported method *

% Found for nicergoline ± S.D.*


S.D.*

% Found for nicergoline ±

S.D.*


S.D.*

39123 98.04 ± 0.683 99.51± 1.014 99.32 ± 0.956 98.37 ± 0.892

40082 99.65 ± 0.715 99.17 ± 0.961 98.56 ± 0.857 98.99 ± 0.825

* HPLC method.

Table (LXXIV): Application of standard Table (LXXIV): Application of standard addition for the determination of addition for the determination of nicergoline and cinnarizine by the nicergoline and cinnarizine by the

proposed methodproposed method.. Batch number Standard added (mg) HPLC method

nicergoline cinnarizine Recovery % of added nicergoline

Recovery % of added cinnarizine

39123 10.0015.0020.00

25.0037.5050.00

97.6398.0298.93

99.84100.45100.36

Mean S.D.* 98.19 0.667 100.21 0329


Table (LXX): Statistical comparison for the Table (LXX): Statistical comparison for the results obtained by the proposed method and results obtained by the proposed method and the reported method for the analysis of the reported method for the analysis of nicergoline and cinnarizine in pure powder nicergoline and cinnarizine in pure powder

formform

* HPLC method.The figures in parenthesis are the corresponding tabulated values at P=0.05

Item HPLC method Reported method*

nicergoline cinnarizine nicergoline cinnarizine

Mean 100.32 99.97 99.64 99.27

S.D. 1.664 0.550 0.978 0.714

Variance 2.768 0.302 0.956 0.509

n 9 8 6 6

F test 2.896 (4.82) 1.685 (3.97)

Student's t test 1.384 (2.160) 2.080 (2.179)

Table (LXXVI): Assay parameters and Table (LXXVI): Assay parameters and method validation for nicergoline and method validation for nicergoline and cinnarizinecinnarizine

Parameter

Derivative ratio spectrophotometric

method

Densitometricmethod

HPLCmethod

nicergoline cinnarizine nicergoline cinnarizine nicergoline cinnarizine

Range (µg/ml)

6- 38 6- 22 8- 32 (µg/spot)

2- 8 (µg/spot)

10- 90 10- 45

Slope 0.0238 0.2706 0.2195 0.6517 0.0524 0.0504

Intercept 0.0072 0.0854 0.1541 0.1104 -0.0401 -0.0031

Mean 99.77 99.95 99.93 99.95 100.32 99.97

S.D. 0.767 0.752 1.128 1.418 1.664 0.550

Variance 0.588 0.565 1.272 2.010 2.768 0.302

Coff. Of variation

0.767 0.752 1.128 1.418 1.658 0.550

Correl. Coef.(r)

0.9998 0.9996 0.9997 0.9995 0.9995 0.9998

* RSD%a 0.901, 0.875 0.481, 0.503 0.758, 0.951 0.989, 1.401 1.005, 1.124 0.329, 0.684

*RSD %b 1.307, 1.105 0.843, 0.742 0.894, 1.197 1.554, 1.216 1.110, 1.354 0.689, 0.921* RSD%a, RSD%b: the intra-day, inter-day respectively (n=5) relative standard deviation of concentrations (20 and 30 µg/ml nicergoline and 10 and 14 µg/ml cinnarizine) for derivative ratio, (16 and 20 µg/spot nicergoline and 4 and 6 µg/spot cinnarizine) for densitometric method and (20 and 30 µg/ml from both nicergoline and cinnarizine) for HPLC method.

Section [D]Section [D]Simultaneous Simultaneous

determination of determination of nicergoline and cinnarizine nicergoline and cinnarizine by chemometric methodsby chemometric methods

Chemometrics is the application of Chemometrics is the application of mathematical and statistical methods to mathematical and statistical methods to provide maximum chemical information provide maximum chemical information through analysis of chemical data.through analysis of chemical data.

In this section, three chemometric In this section, three chemometric techniques were applied for stechniques were applied for simultaneous imultaneous determination of nicergoline and cinnarizine determination of nicergoline and cinnarizine

Classical Least Squares (CLS) with non Classical Least Squares (CLS) with non zero intercept, zero intercept,

principal component regression (PCR) principal component regression (PCR) partial least squares (PLS). partial least squares (PLS).

Table (LXXVII): The concentration of Table (LXXVII): The concentration of different mixtures of nicergoline and different mixtures of nicergoline and

cinnarizine used in the training setcinnarizine used in the training set Sample number Nicergoline

(μg.ml –1)Cinnarizine

(μg.ml –1)

1 7.2 15.0

2 7.2 13.5

3 6.6 18.0

4 6.6 13.5

5 6.0 18.0

6 6.0 16.5

7 6.0 12.0

8 5.4 16.5

9 4.8 16.5

10 4.8 15.0

0

0.2

0.4

0.6

0.8

1

0 2 4 6 8

Principle component

RMSE

C

0

0.2

0.4

0.6

0.8

1

0 2 4 6 8

Principle component

RMSE

C

Figure (82): RMSEC plot of the cross validation results of the training set as a function of the number of principal components used to construct the PCR calibration for nicergoline.

Figure (83): RMSEC plot of the cross validation results of the training set as a function of the number of principal components used to construct the PLS calibration for nicergoline.

0

0.2

0.4

0.6

0.8

1

0 2 4 6 8

Principle component

RMSE

C

0

0.2

0.4

0.6

0.8

1

0 2 4 6 8

Principle component

RMSE

C

Figure (35): RMSEC plot of the cross validation results of the training set

as a function of the number of principal components used to

construct the PCR calibration for cinnarizine.

Figure (36): RMSEC plot of the cross validation results of the training set

as a function of the number of principal components

used to construct the PLS calibration for cinnarizine.

Table (LXXVIII): Results of the analysis Table (LXXVIII): Results of the analysis of the mixtures of the validation set of of the mixtures of the validation set of nicergoline & cinnarizine by the nicergoline & cinnarizine by the proposed methodsproposed methods..

Sample no.

Concentration g.ml-1 Nicergoline Recovery % Cinnarizine Recovery %

Nicergoline Cinnarizine CLS non zero

PCR PLS CLS non zero

PCR PLS

1 7.2 18.0 100.56 100.10 100.10 100.71 100.56 100.57

2 7.2 12.0 99.53 99.72 99.72 100.38 100.47 100.47

3 6.6 16.5 100.41 100.29 100.29 99.97 99.93 99.93

4 6.6 15.0 100.29 100.46 100.46 98.84 98.90 98.90

5 6.0 15.0 100.91 100.60 100.60 100.74 100.64 100.64

6 6.0 13.5 100.70 100.55 100.55 100.94 100.90 100.90

7 5.4 18.0 98.52 98.62 98.62 99.45 99.47 99.47

8 4.8 18.0 99.08 99.42 99.42 99.44 99.50 99.50

Mean 100.00 99.97 99.97 100.06 100.05 100.05

S.D 0.854 0.686 0.685 0.757 0.706 0.707

y = 1.0262x - 0.1596

R2 = 0.9975

0

2

4

6

8

0 2 4 6 8

Actual concentration (ug.ml-1)

Pre

dic

ted

co

nce

ntr

atio

n

(ug

.ml-1

)

y = 0.984x + 0.2573

R2 = 0.9975

0

5

10

15

20

0 5 10 15 20


Pre

dic

ted

co

nce

ntr

atio

n

(ug

.ml-1

)

Figure (84): Predicted concentration versus actual concentration of nicergoline in the validation set using CLS method

Figure (85): Predicted concentration versus actual concentration of cinnarizine in the validation set using CLS method

y = 1.0182x - 0.1129

R2 = 0.9983

0

2

4

6

8

0 2 4 6 8

Actual concentration (u.g.ml-1)

Pred

icte

d co

ncen

trat

ion

(u.g

.ml-1

)

y = 0.9825x + 0.2779

R2 = 0.998

0

5

10

15

20

0 5 10 15 20


Pre

dict

ed c

once

ntra

tion

(ug

.ml-1

)

Figure (86): Predicted concentration versus actual concentration of nicergoline in the validation set using PCR method

Figure (87): Predicted concentration versus actual concentration of cinnarizine in the validation set using PCR method

y = 1.0182x - 0.1127

R2 = 0.9983

0

2

4

6

8

0 2 4 6 8


Pred

icte

d co

ncen

tratio

n (u

g.m

l-1

)

y = 0.9825x + 0.2779

R2 = 0.998

0

5

10

15

20

0 5 10 15 20


Pred

icte

d co

ncen

tratio

n (u

g.m

l-1

)

Figure (88) : Predicted concentration versus actual concentration of nicergoline in the validation set using PLS method

Figure (89) : Predicted concentration versus actual concentration of cinnarizine in the validation set using PLS method

-0.1

-0.05

0

0.05

0.1

0 2 4 6 8


Con

cent

ratio

n re

sidu

als

(u.g

.ml

-1)

-0.2

-0.1

0

0.1

0.2

0 5 10 15 20


Con

cent

ratio

n re

sidu

als

(ug

.ml

-1)

Figure (90): Concentration residuals versus actual concentration of nicergoline in the validation set using CLS method

Figure (91): Concentration residuals versus actual concentration of cinnarizine in the validation set using CLS method

-0.1

-0.05

0

0.05

0 2 4 6 8


Conc

entra

tion

resi

dual

s (u

g.m

l-1

)

-0.2

-0.1

0

0.1

0.2

0 5 10 15 20


Conc

entra

tion

resi

dual

s (u

g.m

l-1

)

Figure (92): Concentration residuals versus actual concentration of nicergoline in the validation set using PCR method

Figure (93): Concentration residuals versus actual concentration of cinnarizine in the validation set using PCR method

-0.1

-0.05

0

0.05

0 2 4 6 8


Conc

entra

tion

resi

dual

s (u

g.m

l-1

)

-0.2

-0.1

0

0.1

0.2

0 5 10 15 20


Con

cent

ratio

n re

sidu

als

(ug

.ml

-1)

Figure (94): Concentration residuals versus actual concentration of nicergolinein the validation set using PLS method

Figure (95): Concentration residuals versus actual concentration of cinnarizine in the validation set using PLS method

Table (LXXIX): RMSEP and Q2 values of Table (LXXIX): RMSEP and Q2 values of the validation set analysis of the validation set analysis of cinnarizine hydrochloride by the cinnarizine hydrochloride by the

proposed methodsproposed methods..Item CLS PCR PLS


RMSEP 0.0491 0.11027 0.0362 0.10139 0.03624 0.10143

Q2 0.9968 0.9972 0.9979 0.9976 0.9979 0.9976

Table (LXXX): Quantitative Table (LXXX): Quantitative determination of nicergoline and determination of nicergoline and cinnarizine in cinibral tablets by the cinnarizine in cinibral tablets by the

proposed methodsproposed methods Batch no.

CLS PCR PLS


Found*

Mean± S.D.

Found*

Mean± S.D.

Found*

Mean± S.D.

Found*

Mean± S.D.

Found*

Mean± S.D.

Found*

Mean± S.D.

39123

6.01 99.990.321

15.15

101.090.601

6.01 99.450.524

15.10

99.980.726

6.02 100.040.611

15.10

99.780.820

40048

6.04 99.540.710

15.08

100.230.265

6.07 100.130.522

15.13

101.020.754

6.06 100.000.540

15.14

100.580.738

* (μg.ml –1).

Table (LXXXI): Results of the standard Table (LXXXI): Results of the standard addition technique for the addition technique for the simultaneous determination of simultaneous determination of nicergoline and cinnarizine in cinibral nicergoline and cinnarizine in cinibral tablets by the proposed methodstablets by the proposed methodsBatch.

noStandard added(mg) CLS PCR PLS

nicergoline cinnarizineRecovery

% of added nicergoline

Recovery % of

added cinnarizine

Recovery % of added nicergoline

Recovery % of

added cinnarizine

Recovery % of added nicergoline

Recovery % of

added cinnarizine

39123 0.250.3750.50

0.250.3750.50

99.2199.98

100.17

98.1198.46

100.36

100.72100.99100.52

99.20100.47100.58

102.13101.69101.15

100.73100.7599.87

Mean ± S.D.

99.78 ± 0.508

98.97 ± 1.210

100.74 ± 0.235

100.08 ± 0.766

101.65 ± 0.490

100.45 ± 0.502

Table (LXXXII): Statistical comparison for the Table (LXXXII): Statistical comparison for the results obtained by the proposed methods results obtained by the proposed methods and the reported method for the analysis of and the reported method for the analysis of nicergoline and cinnarizine in pure powder nicergoline and cinnarizine in pure powder

formform..

* HPLC method.

Item CLS PCR PLS Reported method*

nic cin nic cin nic cin nic cin

Mean 100.00 99.99 100.01 99.91 100.01 99.92 99.64 99.27

S.D. 0.575 0.905 0.567 0.966 0.566 0.962 0.978 0.714

Variance 0.330 0.819 0.321 0.933 0.320 0.925 0.956 0.509

n 10 10 10 10 10 10 6 6

F test 2.896(3.48)

1.609(4.77)

2.978(3.48)

1.833(4.77)

2.987(3.48)

1.817(4.77)

Student's t test

0.936(2.145)

1.656(2.145)

0.968(2.145)

1.401(2.145)

0.968(2.145)

1.428(2.145)


Part IVPart IVStability indicating Stability indicating

methods for the methods for the determination of determination of meclophenoxate meclophenoxate

hydrochloridehydrochloride

This part includes a general introduction about This part includes a general introduction about the chemistry of meclophenoxate hydrochloride.the chemistry of meclophenoxate hydrochloride.

Review article on the reported methods used for Review article on the reported methods used for its quantitative determination. its quantitative determination.

This part is subdivided into three sections:This part is subdivided into three sections: Section(A):Section(A): High-performance liquid High-performance liquid

chromatographic determination of chromatographic determination of meclophenoxate hydrochloride in presence of its meclophenoxate hydrochloride in presence of its acid degradation productacid degradation product

Section(B):Section(B): Kinetic study on the degradation of Kinetic study on the degradation of meclophenoxate hydrochloridemeclophenoxate hydrochloride

Section (C):Section (C): Determination of meclophenoxate Determination of meclophenoxate hydrochloride in presence of its acid degradation hydrochloride in presence of its acid degradation productproduct using ion selective electrodes using ion selective electrodes

Section [A]Section [A]High-performance liquid High-performance liquid

chromatographic chromatographic determination of determination of meclophenoxate meclophenoxate

hydrochloride in presence hydrochloride in presence of its acid degradation of its acid degradation

productproduct

-Structure of meclophenoxate

OO

Cl

O

NCH3

CH3

OO

Cl

O

NCH3

CH3

OOH

Cl

O

OHN

CH3

CH3

1 N NaOH

instantaneous

+

2 N NaOH

Reflux 25 min.then neutralize

with HCl

OO

Cl

O

NCH3

CH3

OOH

Cl

O

OHN

CH3

CH3

1 N NaOH

instantaneous

+

-The proposed mechanism for preparing the degradation product:

Figure (19): Liquid chromatographic separation of meclophenoxate.HCl and its degradation product using final assay conditions: Column: RP18 Mobile phase: 0.01 M ammonium carbonate: acetonitrile (7:3 v/v). Flow rate: 1.0 ml min-1. Detection: 277 nm. Retention time meclophenoxate hydrochloride: 5.39 min. Retention time degradation product: 2.70 min.

Time (min)

Detector response

Figure (20): Linearity of the relative peak area to the corresponding concentration of meclophenoxate hydrochloride.

y = 0.0095x + 0.0229

R2 = 0.9997

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 100 200 300 400 500


Rel

ativ

e p

eak

area

Table (X): Determination of Table (X): Determination of meclophenoxate hydrochloride in meclophenoxate hydrochloride in laboratory prepared mixtures by the laboratory prepared mixtures by the proposed methodproposed method

Concentration (µg.mlConcentration (µg.ml-1-1)) PercentagePercentage% % HPLCMethodHPLCMethod

Meclophenoxate. HClMeclophenoxate. HClDegradation Degradation productproduct

MeclophenoxateMeclophenoxate..HClHCl

Degradation Degradation productproduct

RecoveryRecovery% %

Meclophenoxate. HClMeclophenoxate. HCl

350350 5050 87.587.5 12.512.5 99.1299.12

300300 100100 7575 2525 101.56101.56

250250 150150 62.562.5 37.537.5 98.7598.75

200200 200200 5050 5050 102.03102.03

150150 250250 37.537.5 62.562.5 98.5198.51

100100 300300 2525 7575 100.47100.47

5050 350350 12.512.5 87.587.5 99.0799.07

MeanMean 99.9499.94

S.DS.D.. 1.3191.319

Table (XI): Parameters required for Table (XI): Parameters required for system suitability test of HPLC methodsystem suitability test of HPLC method

ParameterParameter Obtained valueObtained value Reference valueReference value

Resolution (R)Resolution (R) 3.6763.676 R > 0.8R > 0.8

T ( tailing factor)T ( tailing factor) Meclophenoxate.HCl 1.05Meclophenoxate.HCl 1.05 T = 1 for a typical T = 1 for a typical symmetric peaksymmetric peak

Deg. product 1.0Deg. product 1.0

(relative retention time)(relative retention time) 2.2242.224 > >11

K’ (column capacity)K’ (column capacity) Meclophenoxate.HCl 9.796Meclophenoxate.HCl 9.796 11 - -1010 acceptableacceptable


N (no.of theoretical plates)N (no.of theoretical plates) Meclophenoxate.HCl 728.4Meclophenoxate.HCl 728.4 Increases with efficiency of Increases with efficiency of the separationthe separation


HETPHETP Meclophenoxate.HCl 0.0343Meclophenoxate.HCl 0.0343 The smaller the value, the The smaller the value, the higher the column higher the column

efficiencyefficiencyDeg. product 0.093Deg. product 0.093

Table (XII): Determination of Table (XII): Determination of meclophenoxate hydrochloride in meclophenoxate hydrochloride in

lucidril tablets by the proposed methodlucidril tablets by the proposed method

* Stability indicating HPLC method.

Lucidril tablets claimed to contain 250 mg

meclophenoxate.HClBatch number


Found % ± S.D.** Recovery % ± S.D.*

5GE0941 100.87 ± 1.197 100.29 ± 1.411

010156(expired 3/04) 83.91 ± 1.002 84.12 ± 1.373

Table (XIII): Application of standard Table (XIII): Application of standard addition for the determination of addition for the determination of meclophenoxate hydrochloride by the meclophenoxate hydrochloride by the proposed methodproposed method

Batch number

Standard added(mg)

HPLC method

Meclophenoxate. HClFound of added (mg) Recovery % of added

5GE0941 100.00150.00200.00

98.96152.00202.42

98.96101.33101.21

Mean ± S.D.* 100.53 ± 1.277


Table (XIV): Statistical comparison for Table (XIV): Statistical comparison for the results obtained by the proposed the results obtained by the proposed method and the reported method for method and the reported method for the analysis of meclophenoxate the analysis of meclophenoxate

hydrochloride in pure powder formhydrochloride in pure powder form


Item HPLC method Reported method *

Mean 99.94 99.39

S.D. 1.148 1.144


n 9 6

F test 1.006 (4.82)



Section [B]Section [B]

Kinetic study on Kinetic study on degradation of degradation of

meclophenoxate meclophenoxate hydrochloridehydrochloride

Kinetic study on degradation of Kinetic study on degradation of meclophenoxate hydrochloride meclophenoxate hydrochloride

includesincludes:: Study the kinetic order of the Study the kinetic order of the

reaction reaction Study the effect of sodium hydroxide Study the effect of sodium hydroxide

concentration on the reaction rateconcentration on the reaction rate Study the effect of temperature on Study the effect of temperature on

the reaction rate the reaction rate Calculate energy of activation (Ea):Calculate energy of activation (Ea):

log =log =1

2

K

K)

21

12(

303.2 TT

TT

R

Ea

y = -0.0346x + 1.9929

R2 = 0.9993

0

0.5

1

1.5

2

2.5

0 10 20 30

Time in minutes

2 +

log

(CT/

Co)

Figure (22): First order plot of the hydrolysis of meclophenoxate hydrochloride (1000 mg %) with 2 N NaOH at 80 oC

0

0.5

1

1.5

2

2.5

0 10 20 30

Time in minutes

2 +

lo

g (

CT/

Co)

90oC

80oC

70oC

60oC

Figure (23): First order plot of the hydrolysis of meclophenoxate hydrochloride (1000 mg %) with 2 N NaOH at different temperatures.

0

0.5

1

1.5

2

2.5

0 10 20 30

Time in minutes

2 +

lo

g (

CT/

Co)

90oC

80oC

70oC

60oC

Figure (24): First order plot of the hydrolysis of meclophenoxate hydrochloride (1000 mg %) with 1.5 N NaOH at different temperatures.

0

0.5

1

1.5

2

2.5

0 10 20 30

Time in minutes

2 +

lo

g (

CT/

Co)

90oC

80oC

70oC

60oC

Figure (25): First order plot of the hydrolysis of meclophenoxate hydrochloride (1000 mg %) with 1.0 N NaOH at different temperatures.

-2

-1.5

-1

-0.5

0

2.7 2.8 2.9 3 3.1

1/T x 10-3 K-1

Lo

g K

2 N NaOH

1.5 N NaOH

1 N NaOH

Figure (26): Arrhenius Plot for the hydrolysis of meclophenoxate hydrochloride (1000 mg %) with 1.0, 1.5, 2.0 N NaOH

Ea" was found to be 12.331 kilo calories mol-1

Table (XVI): Kinetic data of Table (XVI): Kinetic data of meclophenoxate hydrochloride meclophenoxate hydrochloride hydrolysishydrolysis

Normality of Normality of NaOHNaOH

TemperatureTemperature K in minK in min-1-1 tt1/21/2 in min in min..

2.02.0 N NaOHN NaOH 9090ooCC

8080ooCC

7070ooCC

6060ooCC

0.1250.1250.0790.0790.0490.0490.0280.028

5.545.548.778.77

14.1414.1424.7524.75


8080ooCC

7070ooCC

6060ooCC

0.0930.0930.0590.0590.0380.0380.0220.022

7.457.4511.7411.7418.2318.2331.5031.50


8080ooCC

7070ooCC

6060ooCC

0.0630.0630.0400.0400.0250.0250.0140.014

11.0011.0017.3217.3227.7227.7249.5049.50

Section [C]Section [C]Determination of Determination of meclophenoxate meclophenoxate

hydrochloride in presence hydrochloride in presence of its acid degradation of its acid degradation

productproduct using ion using ion selective electrodesselective electrodes

Ion selective electrodes are electrodes Ion selective electrodes are electrodes containing membranes having a selective containing membranes having a selective response for a particular ion.response for a particular ion.

Meclophenoxate hydrochloride (Cation) Meclophenoxate hydrochloride (Cation) reacted with tetraphenylborate or reineckate reacted with tetraphenylborate or reineckate (anionic ion exchangers) to form stable 1:1, (anionic ion exchangers) to form stable 1:1, water insoluble ion association complex.water insoluble ion association complex.

-CD-based sensors form inclusion complexes -CD-based sensors form inclusion complexes in the aqueous and in solid state with organic in the aqueous and in solid state with organic molecules. molecules.

Three membranes are studied in this part:Three membranes are studied in this part: a) meclo-TPBa) meclo-TPB b) meclo-RNCb) meclo-RNC c) c) -CD-RNC-CD-RNC

The electrode assemblyThe electrode assembly

Figure (27): PVC matrix membrane ion selective electrode: [1] Shielded cable. [2] Rubber sheath. [3] Quickfit cone. [4] Quickfit socket. [5] Mercury. [6] Calomel reference electrode. [7] Internal solution. [8] PVC tubing. [9] Sensor membrane.

Figure (28): Effect of pH on the response of meclo - TPB electrode

-150

-100

-50

0

50

100

0 5 10 15

pH

E (

mV

)10 -̂4

10 -̂3

-100

-50

0

50

100

150

0 5 10 15

pH

E (

mV

) 10^-4

10^-3

Figure (29): Effect of pH on the response of meclo - reineckate electrode

-100

-50

0

50

100

150

0 5 10 15

pH

E (

mV

)10 -̂4

10 -̂3

Figure (30): Effect of pH on the response of CD- reineckate electrode.

-50

0

50

100

150

200

250

0 2 4 6 8

- log concentration

E (

mV

) 20-25oC

30oC

40oC

Figure (31): Effect of temperature on the response of CD- reineckate membrane electrode.

-100

-50

0

50

100

150

200

0 5 10

- log concentration

E (

mV

) meclo-TPB

meclo-RNC

meclo-B-CD

Figure (32): Profile of the potential in mV to the –log concentration of meclophenoxate hydrochloride with meclo- TPB, meclo – reineckate and CD- reineckate.

Table (XVIII): Electrochemical response Table (XVIII): Electrochemical response characteristics of the three characteristics of the three investigated electrodesinvestigated electrodes

ParameterParameter Meclo-TPBMeclo-TPB Meclo-RNCMeclo-RNC -CD-RNC-CD-RNC

Slope (mV/decade)Slope (mV/decade)** --52.7352.73 --51.6451.64 --54.0554.05

Intercept (mV)Intercept (mV) 222.03222.03 227.84227.84 268.6268.6

Response time Response time (seconds)(seconds)

4040 4040 3030

Working pH rangeWorking pH range 44 – – 7.57.5 5.55.5 - - 77 44 – – 7.57.5

Concentration range Concentration range (M)(M)

11 x 10x 10-5-5- 1 x 10- 1 x 10-2-2 11 x 10x 10-5-5- 1 x 10- 1 x 10-2-2 11 x 10x 10-5-5- 1 x 10- 1 x 10-2-2

Stability (weeks)Stability (weeks) 33 33 33

Average recoveryAverage recovery (%)(%)

99.9299.92 99.9699.96 100.03100.03

Standard deviationStandard deviation 1.0771.077 0.5020.502 0.7630.763

Correlation Correlation coefficientcoefficient

0.99950.9995 0.99980.9998 0.99960.9996

Table (XIX) Potentiometric selectivity Table (XIX) Potentiometric selectivity coefficients (Kcoefficients (KPlotPlotprimary ion, primary ion, interferent) for the three proposed interferent) for the three proposed

electrodeselectrodes..InterferentInterferent Selectivity coefficientSelectivity coefficient

Meclo-TPBMeclo-TPB Meclo-RNCMeclo-RNC CD-RNCCD-RNC

NaClNaCl 3.753.75 x 10x 10-2-2 3.793.79 x 10x 10-2-2 5.755.75 x 10x 10-3-3

KClKCl 3.013.01 x 10x 10-2-2 3.613.61 x 10x 10-2-2 6.076.07 x 10x 10-3-3

NHNH44ClCl 2.052.05 x 10x 10-2-2 3.303.30 x 10x 10-2-2 5.125.12 x 10x 10-3-3

CaClCaCl22 2.672.67 x 10x 10-2-2 3.363.36 x 10x 10-2-2 5.325.32 x 10x 10-3-3

MgSOMgSO44 3.513.51 x 10x 10-2-2 4.524.52 x 10x 10-2-2 7.177.17 x 10x 10-3-3

glucoseglucose 3.143.14 x 10x 10-2-2 3.053.05 x 10x 10-2-2 6.346.34 x 10x 10-3-3

lactoselactose 3.153.15 x 10x 10-2-2 3.183.18 x 10x 10-2-2 6.386.38 x 10x 10-3-3

sucrosesucrose 2.922.92 x 10x 10-2-2 2.672.67 x 10x 10-2-2 5.875.87 x 10x 10-3-3

UreaUrea 2.922.92 x 10x 10-2-2 3.183.18 x 10x 10-2-2 4.764.76 x 10x 10-3-3

L-phenyl alanineL-phenyl alanine 2.882.88 x 10x 10-2-2 2.562.56 x 10x 10-2-2 4.334.33 x 10x 10-3-3

Deg.productDeg.product 2.092.09 x 10x 10-2-2 2.192.19 x 10x 10-2-2 5.415.41 x 10x 10-3-3

Table (XX): Determination of Table (XX): Determination of meclophenoxate hydrochloride in meclophenoxate hydrochloride in laboratory prepared mixtures by the laboratory prepared mixtures by the proposed methodproposed method

Concentration (M)Concentration (M) RatioRatio** Recovery % of meclophenoxate. HClRecovery % of meclophenoxate. HCl

Meclophenoxate Meclophenoxate HClHCl

Degradation Degradation productproduct

Meclo-Meclo-TPBTPB

Meclo-Meclo-RNCRNC

CD-RNCCD-RNC

11 x 10x 10-3-3 11 x 10x 10-4-4 1010 : :11 98.6398.63 97.5497.54 99.2199.21

11 x 10x 10-3-3 55 x 10x 10-4-4 22 : :11 99.0699.06 99.4199.41 99.2399.23

11 x 10x 10-3-3 11 x 10x 10-3-3 11 : :11 99.5899.58 100.06100.06 100.53100.53

11 x 10x 10-3-3 55 x 10x 10-3-3 11 : :55 100.69100.69 98.7398.73 102.87102.87

Table (XXI): Determination of Table (XXI): Determination of meclophenoxate hydrochloride in meclophenoxate hydrochloride in

lucidril tablets by the proposed methodlucidril tablets by the proposed method Lucidril tablets Lucidril tablets claimed to claimed to

contain 250 contain 250 mgmg


Meclo-TPBMeclo-TPB Meclo-RNCMeclo-RNC CD-RNCCD-RNC Reported Reported methodmethod* *

% %Found ± Found ± S.DS.D**.**.



Found % ± Found % ± S.D.**S.D.**

5GE09415GE0941 100.78100.78 0.916 0.916 99.2399.23 1.137 1.137 100.74100.74 0.6280.628 99.5499.54 ± ±1.2321.232

010156010156((expired expired 3/043/04))

83.0983.09 1.4041.404 83.1183.11 0.675 0.675 83.1283.12 0.661 0.661 99.6599.65 ± ±0.9510.951


Table (XXII): Determination of Table (XXII): Determination of meclophenoxate hydrochloride in meclophenoxate hydrochloride in spiked human plasma by the proposed spiked human plasma by the proposed electrodeselectrodes..

Concentration(M)Concentration(M) Meclo-TPBMeclo-TPB Meclo-RNCMeclo-RNC CD-RNCCD-RNC

Recovery % ± S.DRecovery % ± S.D*.*. Recovery % ± S.DRecovery % ± S.D*.*. Recovery % ± S.DRecovery % ± S.D*.*.

11 x 10x 10-3-3 101.77101.77 ± ±0.6120.612 101.58101.58 ± ±0.6630.663 101.03101.03 ± ±0.4970.497

11 x 10x 10-4-4 102.14102.14 ± ±0.5500.550 101.97101.97 ± ±0.6010.601 101.24101.24 ± ±0.4040.404

* Average of three determinations

Table (XXIII): Application of standard Table (XXIII): Application of standard addition for the determination of addition for the determination of meclophenoxate hydrochloride by the meclophenoxate hydrochloride by the proposed methodproposed methodBatch Batch

numbnumberer

Standard Standard addedadded

((mgmg))

Meclo-TPBMeclo-TPB Meclo-RNCMeclo-RNC CD-RNCCD-RNC

Meclo. Meclo. HClHCl

FoundFound RecoveryRecovery FoundFound RecoveryRecovery FoundFound RecoveryRecovery

5GE09415GE0941 14.6814.6822.0222.0229.3629.36

14.4114.4121.6721.6729.3129.31

98.1698.1698.4198.4199.8299.82

14.6514.6521.6121.6129.0029.00

99.7999.7998.1398.1398.7798.77

14.7014.7022.3422.3429.9129.91

100.13100.13101.45101.45101.87101.87

Mean ± Mean ± S.DS.D*.*.

98.7998.79 ± ±0.8950.895

98.8998.89 ± ±0.8370.837

101.15101.15 ± ±0.9070.907

* Average of three determinations

Table (XXIV): Statistical comparison for Table (XXIV): Statistical comparison for the results obtained by the proposed the results obtained by the proposed method and the reported method for method and the reported method for the analysis of meclophenoxate the analysis of meclophenoxate

hydrochloride in pure powder formhydrochloride in pure powder form


Item Meclo-TPB Meclo-RNC CD-RNC Reported method *

Mean 99.92 99.96 100.03 99.39

S.D. 1.077 0.902 0.763 1.144

Variance 1.159 0.813 0.582 1.308

n 4 4 4 6

F test 1.128 (9.01) 1.608 (9.01) 2.247 (9.01)

Student’s t test

0.733 (2.306) 0.833 (2.306) 0.974 (2.306)


Table (XLIV): Assay parameters and Table (XLIV): Assay parameters and method validation for meclophenoxate method validation for meclophenoxate

hydrochloridehydrochlorideParameter

HPLC method Suggested electrodes

Meclophenoxate. HCl. Meclo-TPB Meclo-RNC CD-RNC

Range (μg ml-1)

15-400 1 x 10-5- 1 x 10-2M 1 x 10-5- 1 x 10-2M 1 x 10-5- 1 x 10-2 M

Slope 0.0095 52.73 51.64 54.05

Intercept 0.0229 222.03 227.84 268.6

Mean 99.94 99.92 99.96 100.03

S.D. 1.148 1.077 0.902 0.763

Variance 1.317 1.159 0.813 0.582

RSD% 1.149 1.077 0.902 0.762

Correl. Coef.(r) 0.9997 0.9995 0.9998 0.9996

* RSD%a 1.110, 1.236 0.928, 0.903 0.936, 0.984 0.841, 0.875

*RSD %b 1.428, 1.418 1.132, 1.197 1.378, 1.521 1.004, 1.173

* RSD%a, RSD%b the intra-day, inter-day respectively (n=5) relative standard deviation of concentrations (200 and 300

µg ml-1) and (10-3M and 10-4M) for ion selective electrodes method.

Part VPart VStability indicating Stability indicating

methods for determination methods for determination of vinpocetineof vinpocetine

This part includes a general introduction about the This part includes a general introduction about the chemistry of vinpocetine.chemistry of vinpocetine.

Review article on the reported methods used for its Review article on the reported methods used for its quantitative determination.quantitative determination.

This part is subdivided into four sections: This part is subdivided into four sections: Section [A]: Determination of vinpocetine in Section [A]: Determination of vinpocetine in

presence of its acid degradation product by the presence of its acid degradation product by the derivative ratio spectrophotometryderivative ratio spectrophotometry

Section [B]: Section [B]: DensitometricDensitometric determination of determination of vinpocetine in presence of its acid degradation vinpocetine in presence of its acid degradation productproduct

Section [C]: Section [C]: High-performance liquid High-performance liquid chromatographic determination of vinpocetine in chromatographic determination of vinpocetine in presence of its acid degradation productpresence of its acid degradation product

Section [D]: Section [D]: ChemometricChemometric determination of determination of vinpocetine in presence of its acid degradation vinpocetine in presence of its acid degradation productproduct

Section [A]Section [A]Determination of Determination of

vinpocetine in presence of vinpocetine in presence of its acid degradation its acid degradation

product by the derivative product by the derivative ratio spectrophotometryratio spectrophotometry

Structure of vinpocetineStructure of vinpocetine

NN

O

O

H3C

H

CH3

Figure (34): Absorption spectra of vinpocetine 12 µg ml-1 (———) and degradation product 10 µg ml-1 (---------- ) using 0.1N hydrochloric acid as a solvent.

Figure (35): First order spectra of vinpocetine 12 μg ml-1 (______) degradation product 10 μg ml-1 (_ _ _ _ _ _) using 0.1N hydrochloric acid as a solvent.

dA/dλ

A (vinpocetine/deg.prod.)

Figure (36): Zero order of ratio spectra of vinpocetine 4-32 μg ml-1 using 10 µg.ml-1 of degradation product as a divisor.

Figure (37): First order of ratio spectra of vinpocetine 4-32 μg ml-1 using 10 µg ml-1 of degradation product as a divisor.

dA(vinpocetine/deg.product)/dλ

311nm

y = 0.1856x + 0.0197

R2 = 0.9994

0

1

2

3

4

5

6

7

0 10 20 30 40


Pe

ak

a

mp

litu

de

Figure (38): Linearity of the peak amplitude of the first derivative of the ratio spectra at 311 nm to the corresponding concentration

of vinpocetine.

Table (XXV): Determination of Table (XXV): Determination of vinpocetine in laboratory prepared vinpocetine in laboratory prepared

mixtures by the proposed methodmixtures by the proposed method.. Concentration (µg.mlConcentration (µg.ml-1-1)) PercentagePercentage)%( )%( Derivative ratio Derivative ratio

methodmethod

VinpocetineVinpocetineDegradation Degradation productproduct


RecoveryRecovery% %

VinpocetineVinpocetine

2424 88 7575 2525 99.5199.51

2020 1212 62.562.5 37.537.5 102.37102.37

1616 1616 5050 5050 101.08101.08

1212 2020 37.537.5 62.562.5 98.4298.42

88 2424 2525 7575 101.05101.05

44 2828 12.512.5 87.587.5 100.91100.91

MeanMean 100.55100.55

S.DS.D.. 1.3851.385

Table (XXVI): Determination of Table (XXVI): Determination of vinpocetine in vinporal tablets by the vinpocetine in vinporal tablets by the

proposed methodproposed method..

Vinporal tablets claimed to Vinporal tablets claimed to contain 5 mgcontain 5 mg


Derivative ratio methodDerivative ratio method Reported methodReported method** **

Found % ± S.D.Found % ± S.D.** Found % ± S.D.Found % ± S.D.**

710101710101 99.0299.02 ± ± 0.9330.933 99.3299.32 ± ± 0.9560.956

910101910101 98.9898.98 ± ±1.0141.014 98.5698.56 ± ±0.8570.857

* Spectrophotometric method.

Table (XXVII): Application of standard Table (XXVII): Application of standard addition for the determination of addition for the determination of vinpocetine in its pharmaceutical vinpocetine in its pharmaceutical

preparation by the proposed methodpreparation by the proposed method..


Standard addedStandard added((mgmg))

Derivative ratio methodDerivative ratio method

VinpocetineVinpocetine Found of added (mg)Found of added (mg) Recovery % of addedRecovery % of added

710101710101 25.0025.0037.5037.5050.0050.00

25.0625.0638.6238.6250.5350.53

100.24100.24102.98102.98101.06101.06

Mean ± S.DMean ± S.D*.*. 101.42101.42 ± ±1.4061.406


Table (XXVIII): Statistical comparison Table (XXVIII): Statistical comparison for the results obtained by the for the results obtained by the proposed method and the reported proposed method and the reported method for the analysis of vinpocetine method for the analysis of vinpocetine in pure powder formin pure powder form


Item Derivative ratio method Reported method *

Mean 100.05 99.73

S.D. 1.251 1.050


n 8 6

F test 1.420 (4.88)



Section [B]Section [B]DensitometricDensitometric

determination of determination of vinpocetine in presence of vinpocetine in presence of

its acid degradation its acid degradation productproduct

Figure (40): TLC chromatogram of vinpocetine and its degradation product. A= Vinpocetine Rf = 0.80. B= Degradation product Rf = 0.54 M= mixture of vinpocetine and its deg. product Developing system, methanol: chloroform: ethyl acetate (2:1:1 v/v/v).

Figure (41): Scanning profile of the TLC chromatogram of vinpocetine at 268 nm.

Distance (mm)

Reflectance

y = 0.4071x + 0.153

R2 = 0.9995

0

1

2

3

4

5

6

7

0 5 10 15 20

concentration ug.spot-1

inte

grat

ed p

eak

area

(x

10

-4)

Figure (42): Linearity of the area under the peak to the corresponding concentration of vinpocetine

Table (XXIX): Determination of Table (XXIX): Determination of vinpocetine in laboratory prepared vinpocetine in laboratory prepared

mixtures by the proposed methodmixtures by the proposed method.. Concentration (µg/spot)Concentration (µg/spot) PercentagePercentage)%( )%( DensitometricDensitometric

MethodMethod



Recovery %Recovery %


99 11 9090 1010 101.23101.23

88 22 8080 2020 98.0998.09

77 33 7070 3030 97.8297.82

66 44 6060 4040 100.68100.68

55 55 5050 5050 101.09101.09

44 66 4040 6060 100.15100.15

33 77 3030 7070 102.03102.03

MeanMean 100.15100.15

S.DS.D.. 1.6091.609

Table (XXX): Determination of Table (XXX): Determination of vinpocetne in vinporal tablets by the vinpocetne in vinporal tablets by the proposed methodproposed method



Densitometric methodDensitometric method Reported methodReported method** **

Found % ± S.D.Found % ± S.D.** Found % ± S.D.Found % ± S.D.**

710101710101 101.03101.03 ± ± 1.0271.027 99.3299.32 ± ± 0.9560.956

910101910101 100.31100.31 ± ±1.0031.003 98.5698.56 ± ±0.8570.857


Table (XXXI): Application of standard Table (XXXI): Application of standard addition for the determination of addition for the determination of vinpocetine in its pharmaceutical vinpocetine in its pharmaceutical

preparation by the proposed methodpreparation by the proposed method..



Densitometric methodDensitometric method

VinpocetineVinpocetineFound of added Found of added

(mg)(mg)Recovery % of Recovery % of

addedadded

710101710101 25.0025.0037.5037.5050.0050.00

25.6925.6938.0238.0249.1249.12

102.76102.76101.38101.3898.2498.24

Mean ± S.DMean ± S.D*.*. 100.79100.79 ± ±2.3162.316


Table (XXXII): Statistical comparison Table (XXXII): Statistical comparison for the results obtained by the for the results obtained by the proposed method and the reported proposed method and the reported method for the analysis of vinpocetine method for the analysis of vinpocetine

in pure powder formin pure powder form..


Item Densitometric method Reported method *

Mean 99.76 99.73

S.D. 1.644 1.050


n 8 6

F test 2.451 (4.88)



Section [C]Section [C]High-performance liquid High-performance liquid

chromatographic chromatographic determination of determination of

vinpocetine in presence of vinpocetine in presence of its acid degradation its acid degradation

productproduct

Figure (43): Liquid chromatographic separation of vinpocetine and its degradation product using final assay conditions: Column: RP18 Mobile phase: methanol: 0.01 M ammonium bicarbonate (60:40 v/v). Flow rate: 1.6 ml min-1. Detection: 268 nm. Retention time vinpocetine (II): 6.12 min. Retention time degradation product (I): 2.24 min.

Time (min)

Detector response

y = 0.1005x - 0.0175

R2 = 0.9996

0

0.5

1

1.5

2

0 5 10 15 20

Concentration ug ml-1

Relat

ive pe

ak ar

ea

Figure (44): Linearity of the relative peak area to the corresponding concentration of vincamine.

Table (XXXIII): Determination of Table (XXXIII): Determination of vinpocetine in laboratory prepared vinpocetine in laboratory prepared

mixtures by the proposedmixtures by the proposed methodmethod

Concentration (µg mlConcentration (µg ml-1-1)) PercentagePercentage(%) (%) HPLC HPLC methodmethod

VinpocetineVinpocetine Degradation productDegradation product VinpocetineVinpocetine Degradation productDegradation productRecovery %Recovery %


1616 44 8080 2020 100.39100.39

1414 66 7070 3030 100.07100.07

1212 88 6060 4040 99.6799.67

1010 1010 5050 5050 99.3299.32

88 1212 4040 6060 101.21101.21

66 1414 3030 7070 99.5499.54

44 1616 2020 8080 100.82100.82

22 1818 1010 9090 101.07101.07

MeanMean 100.26100.26

S.DS.D.. 0.7240.724

Table (XXXIV): Parameters required for Table (XXXIV): Parameters required for system suitability test of HPLC methodsystem suitability test of HPLC method



T ( tailing factor)T ( tailing factor) Vinpocetine 1.045Vinpocetine 1.045 T = 1 for a typical symmetric T = 1 for a typical symmetric peakpeakDeg. product 1.166Deg. product 1.166


K’ (column capacity)K’ (column capacity) Vinpocetine 9.200Vinpocetine 9.200 11 - -1010 acceptableacceptable


N (N (no.of theoretical platesno.of theoretical plates)) Vinpocetine 418.88Vinpocetine 418.88 Increases with efficiency of Increases with efficiency of the separationthe separationDeg. product 51.38Deg. product 51.38

HETPHETP Vinpocetine 0.05968Vinpocetine 0.05968 The smaller the value, the The smaller the value, the higher the column higher the column

efficiencyefficiencyDeg. product 0.4865Deg. product 0.4865

Table (XXXV): Determination of Table (XXXV): Determination of vinpocetine in vinporal tablets by the vinpocetine in vinporal tablets by the

proposed methodproposed method



HPLC methodHPLC method Reported methodReported method* *

% %FoundFound ± S.D ± S.D.. Found % ± S.D.Found % ± S.D.**

710101710101 99.2899.28 ±± 1.1241.124 99.8099.80 ± ± 0.9050.905

910101910101 100.71100.71 ± ± 1.1051.105 100.31100.31 ± ±1.0741.074


Table (XXXVI): Application of standard Table (XXXVI): Application of standard addition for the determination of addition for the determination of vinpocetine in vinporal tablets by the vinpocetine in vinporal tablets by the proposed methodproposed method


Standard addedStandard added))mgmg((

HPLC methodHPLC method

VinpocetineVinpocetine Found % of addedFound % of addedRecovery % of Recovery % of

addedadded

012261012261 AA 10.0010.0015.0015.0020.0020.00

10.1210.1214.8514.8519.9019.90

101.22101.2299.0699.0699.5199.51

Mean ± S.DMean ± S.D*.*. 99.9399.93 ± ± 1.1391.139


Table (XXXVII): Statistical comparison Table (XXXVII): Statistical comparison for the results obtained by the for the results obtained by the proposed method and the reported proposed method and the reported method for the analysis of vinpocetine method for the analysis of vinpocetine in pure powder formin pure powder form



Mean 100.00 99.73

S.D. 1.219 1.050


n 8 6

F test 1.347 (4.88)



Table (XXXVIII): Assay parameters and Table (XXXVIII): Assay parameters and method validation for vinpocetinemethod validation for vinpocetine

ParameterParameter

Derivative ratio Derivative ratio spectrophotometric methodspectrophotometric method

Densitometric Densitometric methodmethod


VinpocetineVinpocetine VinpocetineVinpocetine VinpocetineVinpocetine

Range (Range (µµg/ml)g/ml) 4-324-32 1-151-15))µg/spotµg/spot(( 2-162-16

SlopeSlope 0.18560.1856 0.40710.4071 0.10050.1005

InterceptIntercept 0.01970.0197 0.1530.153 0.01750.0175

MeanMean 100.05100.05 99.7699.76 100.00100.00

S.DS.D.. 1.2511.251 1.6441.644 1.2191.219

VarianceVariance 1.5651.565 2.7022.702 1.4851.485

Coff. Of Coff. Of variationvariation

1.2501.250 1.6471.647 1.2191.219

Correl. Coef.(r)Correl. Coef.(r) 0.99940.9994 0.99950.9995 0.99960.9996

* *RSD%RSD%aa 0.2050.205 , ,0.3180.318 0.4870.487 , ,0.3390.339 0.294,0.460.294,0.4633

**RSD %RSD %bb 0.4410.441 , ,0.4090.409 0.6290.629 , ,0.5780.578 0.665,0.540.665,0.5477

*RSD%a, RSD%b: the intra-day, inter-day respectively (n=5) relative standard deviation of concentrations (16 and 20 µg/ml) for derivative ratio, (9 and 11µg/spot) for densitometric method and (8 and 12 µg/ml) for HPLC method.

Section [D]Section [D]ChemometricChemometric

determination of determination of vinpocetine in presence of vinpocetine in presence of

its acid degradation its acid degradation productproduct

Table (XXXIX): The concentration of Table (XXXIX): The concentration of different mixtures of vinpocetine and different mixtures of vinpocetine and its degradation product used in the its degradation product used in the

training settraining set.. Sample numberSample number VinpocetineVinpocetine))μμg.ml g.ml –1–1((

Deg. productDeg. product ))μμg.ml g.ml –1–1((

11 8.08.0 10.010.0

22 8.08.0 14.014.0

33 16.016.0 6.06.0

44 16.016.0 10.010.0

55 20.020.0 6.06.0

66 20.020.0 10.010.0

77 12.012.0 10.010.0

88 12.012.0 18.018.0

99 12.012.0 0.00.0

1010 16.016.0 0.00.0

0

1

2

3

4

1 2 3 4 5 6

Principal component

RMSE

C

0

1

2

3

4

1 2 3 4 5 6

Principle component

RMSE

C

Figure (45): RMSEC plot of the cross validation results of the training set as a function of the number of principal components used to construct the PCR calibration for vinpocetine.

Figure (46): RMSEC plot of the cross validation results of the training set as a function of the number of principal components used to construct the PLS calibration for vinpocetine.

Table (XXXX): Results of the analysis of Table (XXXX): Results of the analysis of the mixtures of the validation set of the mixtures of the validation set of vinpocetine and its degradation vinpocetine and its degradation

product by the proposed methodsproduct by the proposed methods Sample noSample no.. Concentration (Concentration (μμg.ml g.ml –1–1)) Vinpocetine RecoveryVinpocetine Recovery% %

VinpocetineVinpocetine Deg. productDeg. product CLSCLS PCRPCR PLSPLS

11 4.04.0 14.014.0102.19102.19 102.16102.16 102.1671102.1671

22 8.08.0 6.06.0 101.77101.77 102.26102.26 102.26102.26

33 8.08.0 18.018.0 102.06102.06 102.14102.14 102.14102.14

44 16.016.0 14.014.0 99.7399.73 99.5299.52 99.5299.52

55 20.020.0 2.02.0 100.70100.70 100.39100.39 100.39100.39

66 20.020.0 14.014.0 99.7299.72 99.6499.64 99.6499.64

77 20.020.0 18.018.0 99.8199.81 100.03100.03 100.03100.03

88 12.012.0 6.06.0 100.29100.29 100.46100.46 100.46100.46

99 12.012.0 14.014.0 99.0999.09 99.0599.05 99.0599.05

1010 20.020.0 0.00.0 100.62100.62 100.64100.64 100.64100.64

MeanMean 100.60100.60 100.63100.63 100.63100.63

S.DS.D . . 1.0841.084 1.1751.175 1.1751.175

y = 0.9951x + 0.1144

R2 = 0.9997

0

10

20

30

0 5 10 15 20 25


Pred

icte

d co

ncen

tratio

n(u

g.m

l-1

)

Figure (47): Predicted concentration versus actual concentration of vinpocetine in the validation set using CLS method

y = 0.9934x + 0.1374

R2 = 0.9997

0

5

10

15

20

25

0 5 10 15 20 25


Pre

dict

ed

conc

entr

atio

n (u

g.m

l-1)

Figure (48): Predicted concentration versus actual concentration of vinpocetine in the validation set using PCR method

y = 0.9934x + 0.1374

R2 = 0.9997

0

5

10

15

20

25

0 5 10 15 20 25


Pre

dict

ed

conc

entr

atio

n (u

g.m

l-1)

Figure (49): Predicted concentration versus actual concentration of vinpocetine in the validation set using PLS method

-0.2

-0.1

0

0.1

0.2

0 5 10 15 20 25


Con

cent

ratio

n re

sidu

als

(ug

.ml-1

)

Figure (50): Concentration residuals versus actual concentration of vinpocetine in the validation set using CLS method

-0.2

-0.1

0

0.1

0.2

0 5 10 15 20 25


Con

cent

ratio

n re

sidu

als

(ug

.ml

-1)

Figure (51): Concentration residuals versus actual concentration of vinpocetine in the validation set using PCR method

-0.2

-0.1

0

0.1

0.2

0 10 20 30


Conc

entra

tion

resi

dual

s (u

g.m

l-1

)

Figure (52): Concentration residuals versus actual concentration of vinpocetine in the validation set using PLS method

Table (XXXXI): RMSEP and Q2 values of Table (XXXXI): RMSEP and Q2 values of the validation set analysis of the validation set analysis of vinpocetine by the proposed methodsvinpocetine by the proposed methods..

ItemItem CLSCLS PCRPCR PLSPLS

RMSEPRMSEP 0.104820.10482 0.109170.10917 0.109170.10917

QQ22 0.999660.99966 0.9996320.999632 0.9996320.999632

Table (XXXXII): Quantitative Table (XXXXII): Quantitative determination of vinpocetine in determination of vinpocetine in vinporal tablets by the proposed vinporal tablets by the proposed

methodsmethods..Batch noBatch no CLSCLS PCRPCR PLSPLS

FoundFound**))μμg.ml g.ml –1–1((

Mean ±Mean ± S.D S.D.. FoundFound**))μμg.ml g.ml –1–1((

Mean ±Mean ± S.D S.D.. FoundFound**))μμg.ml g.ml –1–1((

Mean ±Mean ± S.D S.D..

710101710101 20.1220.12 100.41 0.65±100.41 0.65±22

19.6519.65 99.41 0.80±99.41 0.80±88

19.6519.65 99.41 0.80±99.41 0.80±88

910101910101 19.3619.36 99.03 0.781±99.03 0.781± 19.5419.54 99.65 0.84±99.65 0.84±55

19.5419.54 99.65 0.84±99.65 0.84±55


Table (XXXXIII): Results of the standard Table (XXXXIII): Results of the standard addition technique for the addition technique for the determination of vinpocetine in determination of vinpocetine in vinporal tablets by the proposed vinporal tablets by the proposed methodsmethods

Batch Batch numbnumb

erer

Standard Standard addedadded

((mgmg))

CLSCLS PCRPCR PLSPLS

VinpocetineVinpocetineRecovery % of Recovery % of

addedaddedRecovery % of Recovery % of

addedaddedRecovery % of Recovery % of

addedadded

710101710101 25.0025.0037.5037.5050.0050.00

101.54101.54100.98100.98100.06100.06

100.96100.96101.20101.2099.0399.03

100.96100.96101.20101.2099.0399.03

Mean ± Mean ± S.DS.D..

100.86100.86 ± ±0.7470.747

100.39100.39 ± ±1.1891.189

100.39100.39 ± ±1.1891.189

Table (XXXXIV): Statistical comparison Table (XXXXIV): Statistical comparison for the results obtained by the for the results obtained by the proposed methods and the reported proposed methods and the reported method for the analysis of vinpocetine method for the analysis of vinpocetine in pure powder formin pure powder form..


Item Proposed methods Reported method*

CLS PCR PLS

Mean 100.05 100.21 100.21 99.73

S.D. 0.774 0.968 0.968 1.050

Variance 0.599 0.937 0.937 1.102

n 10 10 10 6

F test 1.839 (3.48) 1.176 (3.48) 1.176 (3.48)

Student’s t test 0.702(2.145) 0.931(2.145) 0.931(2.145)


Part VIPart VIStability indicating Stability indicating

methods for the methods for the determination of Pyritinol determination of Pyritinol

dihydrochloridedihydrochloride

This part includes a general introduction about This part includes a general introduction about the chemistry of pyritinol dihydrochloride.the chemistry of pyritinol dihydrochloride.

Review article on the reported methods used for Review article on the reported methods used for its quantitative determination.its quantitative determination.

This part is subdivided into three sections: This part is subdivided into three sections: Section [A]:Determination of pyritinol Section [A]:Determination of pyritinol

dihydrochloride in presence of its degradation dihydrochloride in presence of its degradation product by the derivative ratio product by the derivative ratio spectrophotometryspectrophotometry

Section [B]: High-performance liquid Section [B]: High-performance liquid chromatographic determination of pyritinol chromatographic determination of pyritinol dihydrochloride in presence of its degradation dihydrochloride in presence of its degradation productproduct

Section [C]: Section [C]: Determination of pyritinol Determination of pyritinol dihydrochloride in presence of its degradation dihydrochloride in presence of its degradation productproduct using ion selective electrodes using ion selective electrodes

Structure of Structure of pyritinol.2HClpyritinol.2HCl

The proposed The proposed scheme for its scheme for its degradationdegradation

N NCH3

HOS -S

CH3

OH

OH

2HCl.. H2O

HO

N N NCH3

HOS -S

CH3

OHHO

CH3

HO

HO

H2 O2 30% v/v

1 hr at room temp 2

SO3HOH

mol.wt 233

Section [A]Section [A]Determination of pyritinol Determination of pyritinol

dihydrochloride in dihydrochloride in presence of its presence of its

degradation product by degradation product by the derivative ratio the derivative ratio spectrophotometryspectrophotometry

Figure (54): Absorption spectra of pyritinol dihydrochloride 20 µg ml-1 (———) and degradation product 20 µg ml-1 (---------- ) using distelled water as a solvent.

Figure (55): First order spectra of pyritinol dihydrochloride 20 µg ml-1 (———) and degradation product 20 µg ml-1 (---------- ) using distelled water as a solvent.

dA/dλ

Figure (56): Zero order of ratio spectra of pyritinol dihydrochloride 10-26 μg ml-1 using 20 µg.ml-1 of degradation product as a divisor.

A (pyritinol.2HCl/deg.prod.)

dA(pyritinol.2HCl/deg.product)/dλ

Figure (57): First order of ratio spectra of pyritinol dihydrochloride 10-26 μg ml-1 using 20 µg ml-1 of degradation product as a divisor.

357nm

Figure (58): Linearity of the peak amplitude of the first derivative of the ratio spectra at 357.4 nm to the corresponding concentration of pyritinol dihydrochloride.

y = 0.1119x + 0.0235

R2 = 0.9991

0

0.5

1

1.5

2

2.5

3

3.5

0 5 10 15 20 25 30


Pea

k am

plitu

de

Table (XXXXV): Determination of Table (XXXXV): Determination of pyritinol dihydrochloride in laboratory pyritinol dihydrochloride in laboratory prepared mixtures by the proposed prepared mixtures by the proposed

methodmethod.. Concentration (µg.mlConcentration (µg.ml-1-1)) PercentagePercentage(%) (%) Derivative Derivative

ratio ratio methodmethod

Pyritinol.2HClPyritinol.2HClDegradation Degradation productproduct

Pyritinol.2HClPyritinol.2HClDegradation Degradation productproduct


Pyritinol.2HClPyritinol.2HCl

2424 22 92.3192.31 7.697.69 99.1099.10

2222 44 84.6284.62 15.3815.38 99.7599.75

1818 88 69.2469.24 30.7630.76 101.28101.28

1414 1212 53.8553.85 46.1546.15 100.15100.15

1010 1616 38.4738.47 61.5361.53 99.6299.62

MeanMean 99.9899.98

S.DS.D.. 0.8170.817

Table (XXXXVI): Determination of Table (XXXXVI): Determination of pyritinol dihydrochloride in Encephabol pyritinol dihydrochloride in Encephabol



Encephabol tablets claimed to contain 200 mg pyritinol.2HClBatch number

Derivative ratio method Reported method *

Found % ± S.D.** Found % ± S.D.**

13476 100.04 ± 0.921 99.36 ± 0.421

13168 99.42 ± 0.775 98.95 ± 0.551

Table (XXXXVII): Application of Table (XXXXVII): Application of standard addition for the determination standard addition for the determination of pyritinol dihydrochloride by the of pyritinol dihydrochloride by the

proposed methodproposed method..

Batch number

Standard added(mg)


Pyritinol dihydrochlorideFound of added

(mg)Recovery % of added

13476 25.0037.5050.00

25.38 37.0850.30

101.5298.89

100.61

Mean ± S.D.* 100.34 ±1.335


Table (XXXXVIII): Statistical Table (XXXXVIII): Statistical comparison for the results obtained by comparison for the results obtained by the proposed methods and the the proposed methods and the reported method for the analysis of reported method for the analysis of pyritinol dihydrochloride in pure pyritinol dihydrochloride in pure powder form.powder form.


Item Derivative ratio method Reported method *

Mean 99.52 99.92

S.D. 1.037 1.172


n 9 6

F test 1.279 (3.48)



Section [B]Section [B]High-performance liquid High-performance liquid

chromatographic chromatographic determination of pyritinol determination of pyritinol

dihydrochloride in dihydrochloride in presence of its oxidative presence of its oxidative

degradation productdegradation product

Figure (60): Liquid chromatographic separation of pyritinol dihydrochloride and its degradation product using final assay conditions: Column: RP 8 Mobile phase:acetonitrile: solution pH 4 (58:42 v/v). Flow rate: 1.0 ml min-1. Detection: 296 nm. Retention time pyritinol dihydrochloride: 1.62 min. Retention time degradation product: 2.97 min.

Time (min)

Detector response

y = 0.0247x + 0.0033

R2 = 0.9997

0

0.5

1

1.5

2

2.5

0 20 40 60 80 100


Rela

tive

peak

are

a

Figure (61): Linearity of the relative peak area to the corresponding concentration of pyritinol dihydrochloride.

Table (IL): Determination of pyritinol Table (IL): Determination of pyritinol dihydrochloride in laboratory prepared dihydrochloride in laboratory prepared mixtures by the proposed methodmixtures by the proposed method

Concentration (µg.mlConcentration (µg.ml-1-1)) PercentagePercentage% % HPLCHPLCMethodMethod

Pyritinol. 2HClPyritinol. 2HClDeg. Deg.

productproductPyritinol. 2HClPyritinol. 2HCl

Deg. Deg. productproduct


Pyritinol. 2HClPyritinol. 2HCl

8080 2020 8080 2020 99.0199.01

6060 4040 6060 4040 98.2598.25

4040 6060 4040 6060 101.74101.74

2020 8080 2020 8080 99.6199.61

1010 9090 1010 9090 100.85100.85

MeanMean 99.8999.89

S.DS.D.. 1.4041.404

Table (L): Parameters required for Table (L): Parameters required for system suitability test of HPLC methodsystem suitability test of HPLC method



T ( tailing factor)T ( tailing factor) Pyritinol dihydrochloride 1.23Pyritinol dihydrochloride 1.23 T = 1 for a typical T = 1 for a typical symmetric peaksymmetric peak



K’ (column capacity)K’ (column capacity) Pyritinol dihydrochloride 4.946Pyritinol dihydrochloride 4.946 11 - -1010 acceptableacceptable


N (no.of theoretical plates)N (no.of theoretical plates) Pyritinol dihydrochloride141.41Pyritinol dihydrochloride141.41 Increases with Increases with efficiency of the efficiency of the

separationseparationDeg. product 85.69Deg. product 85.69

HETPHETP Pyritinol dihydrochloride 0.176Pyritinol dihydrochloride 0.176 The smaller the value, The smaller the value, the higher the the higher the

column efficiencycolumn efficiencyDeg. product 0.291Deg. product 0.291

Table (LI): Determination of pyritinol Table (LI): Determination of pyritinol dihydrochloride in Encephabol tablets dihydrochloride in Encephabol tablets

by the proposed methodby the proposed method..

Encephabol tablets claimed to contain

200 mg pyritinol.2HClBatch number


Found % ± S.D.** Found % ± S.D.**

13476 100.19 ± 0.911 99.36 ± 0.421

13168 99.22 ± 0.912 98.95 ± 0.551


Table (LII): Application of standard Table (LII): Application of standard addition for the determination of addition for the determination of pyritinol dihydrochloride by the pyritinol dihydrochloride by the proposed methodproposed method..


Standard addedStandard added))mgmg((


Pyritinol dihydrochloridePyritinol dihydrochlorideFound of added Found of added

(mg)(mg)Recovery % of addedRecovery % of added

1347613476 10.0010.0015.0015.0020.0020.00

9.82 9.82

14.9614.9620.2220.22

98.2698.2699.7499.74

101.12101.12

Mean ± S.DMean ± S.D*.*. 99.4799.47 ±1.430±1.430


Table (LIII): Statistical comparison for Table (LIII): Statistical comparison for the results obtained by the proposed the results obtained by the proposed methods and the reported method for methods and the reported method for the analysis of pyritinol dihydrochloride the analysis of pyritinol dihydrochloride in pure powder formin pure powder form..



Mean 99.59 99.92

S.D. 0.963 1.172


n 8 6

F test 1.483 (3.97)



Section [C]Section [C]Determination of pyritinol Determination of pyritinol

dihydrochloride in dihydrochloride in presence of its presence of its

degradation productdegradation product using using ion selective electrodesion selective electrodes

Pyritinol dihydrochloride (Cation) Pyritinol dihydrochloride (Cation) reacted with tetraphenylborate or reacted with tetraphenylborate or reineckate (anionic ion exchangers) to reineckate (anionic ion exchangers) to form stable 1:2, water insoluble ion form stable 1:2, water insoluble ion association complex.association complex.

-CD-based sensors form ion seiving -CD-based sensors form ion seiving membranes. membranes.

Three membranes are studied in this Three membranes are studied in this part:part:

a) Pyrit-TPBa) Pyrit-TPB b) Pyrit-RNCb) Pyrit-RNC c) c) -CD-RNC-CD-RNC

-50

0

50

100

150

200

0 5 10 15

pH

E(m

V)

10 -̂5

10 -̂4

Figure (62): Effect of pH on the response of pyrit - TPB electrode

-50

0

50

100

150

200

0 5 10 15

pH

E(m

V) 10^-5

10^-4

Figure (63): Effect of pH on the response of pyrit - reineckate electrode

0

50

100

150

200

250

0 5 10 15

pH

E(m

V)

10 -̂5

10 -̂4

Figure (64): Effect of pH on the response of CD- reineckate electrode

0

50

100

150

200

250

0 2 4 6 8

-log concentration

E(m

V) 20-25oC

30oC

40oC

Figure (65): Effect of temperature on the response of CD- reineckate membrane electrode

0

50

100

150

200

250

0 2 4 6 8

- log concentration

E (

mV

) pyrit-TPB

pyrit-RNC

pyrit-B-CD

Figure (66): Profile of the potential in mV to the –log concentration of pyritinol dihydrochloride with pyrit- TPB, pyrit – reineckate and CD- renikate.

Table (LVI): Electrochemical response Table (LVI): Electrochemical response characteristics of the three characteristics of the three investigated electrodesinvestigated electrodes

ParameterParameter Pyrit-TPBPyrit-TPB Pyrit-Pyrit-RNCRNC -CD--CD-RNCRNC

Slope (mV/decade)Slope (mV/decade)** --30.60030.600 --31.10031.100 --32.89132.891

Intercept (mV)Intercept (mV) 266.84266.84 286.89286.89 317.07317.07

Response time Response time (seconds)(seconds)

4040 4040 4040

Working pH rangeWorking pH range 2.52.5 - -44 2.52.5 - -44 2.52.5 - -44

Concentration range Concentration range (M)(M)

3.1623.162 x 10x 10-6-6- 3.162 x - 3.162 x 1010-4-4

3.1623.162 x 10x 10-6-6- 3.162 x - 3.162 x 1010-4-4

11 x 10x 10-6-6- 3.162 x 10- 3.162 x 10-4-4

Stability (weeks)Stability (weeks) 44 44 44

Average recoveryAverage recovery (%)(%)

99.9999.99 100.00100.00 99.9999.99

Standard deviationStandard deviation 0.8270.827 0.7750.775 0.6800.680

Correlation Correlation coefficientcoefficient

0.99940.9994 0.99960.9996 0.99900.9990

Table (LVII) Potentiometric selectivity Table (LVII) Potentiometric selectivity coefficients (Kcoefficients (KPlotPlotprimary ion, primary ion, interferent) for the three proposed interferent) for the three proposed electrodeselectrodes..

InterferentInterferent Selectivity coefficientSelectivity coefficient

Pyrit-TPBPyrit-TPB Pyrit-Pyrit-RNCRNC CD-CD-RNCRNC

NaClNaCl 6.316.31 x 10x 10-3-3 2.222.22 x 10x 10-3-3 8.678.67 x 10x 10-4-4

KClKCl 5.975.97 x 10x 10-3-3 2.272.27 x 10x 10-3-3 8.888.88 x 10x 10-4-4

NHNH44ClCl 6.026.02 x 10x 10-3-3 2.842.84 x 10x 10-3-3 8.978.97 x 10x 10-4-4

CaClCaCl22 5.285.28 x 10x 10-3-3 3.363.36 x 10x 10-3-3 9.249.24 x 10x 10-4-4

MgSOMgSO44 6.076.07 x 10x 10-3-3 3.253.25 x 10x 10-3-3 9.619.61 x 10x 10-4-4

glucoseglucose 4.974.97 x 10x 10-3-3 3.773.77 x 10x 10-3-3 9.119.11 x 10x 10-4-4

lactoselactose 5.045.04 x 10x 10-3-3 3.293.29 x 10x 10-3-3 8.458.45 x 10x 10-4-4

sucrosesucrose 5.235.23 x 10x 10-3-3 3.203.20 x 10x 10-3-3 8.278.27 x 10x 10-4-4

UreaUrea 4.614.61 x 10x 10-3-3 1.991.99 x 10x 10-3-3 1.001.00 x 10x 10-3-3

L-phenyl alanineL-phenyl alanine 5.225.22 x 10x 10-3-3 2.062.06 x 10x 10-3-3 9.859.85 x 10x 10-4-4

Deg.productDeg.product 1.101.10 x 10x 10-1-1 1.021.02 x 10x 10-1-1 9.719.71 x 10x 10-2-2

Table (LVIII): Determination of pyritinol Table (LVIII): Determination of pyritinol dihydrochloride in laboratory prepared dihydrochloride in laboratory prepared mixtures by the proposed methodmixtures by the proposed method

Concentration (M)Concentration (M) RatioRatio** Found % of pyritinol Found % of pyritinol dihydrochloridedihydrochloride

Pyritinol.2HClPyritinol.2HCl Degradation Degradation productproduct

Pyrit-Pyrit-TPBTPB

Pyrit-Pyrit-RNCRNC

CD-CD-RNCRNC

11 x 10x 10-4-4 11 x 10x 10-5-5 1010 : :11 99.1299.12 100.14100.14 98.2698.26

11 x 10x 10-4-4 55 x 10x 10-5-5 22 : :11 98.6598.65 101.22101.22 100.26100.26

11 x 10x 10-4-4 11 x 10x 10-4-4 11 : :11 108.27108.27 108.00108.00 107.01107.01

11 x 10x 10-4-4 55 x 10x 10-4-4 11 : :55 139.11139.11 136.56136.56 137.81137.81

Table (LIX): Determination of Pyritinol Table (LIX): Determination of Pyritinol diydrochloride in encephabol tablets by diydrochloride in encephabol tablets by

the proposed methodthe proposed method

Encephabol tablets

claimed to contain 200

mgBatch number

Pyrit-TPB Pyrit-RNC CD-RNC Reported method *

% Found ± S.D.**

% Found ± S.D.**

% Found ± S.D.**

Found % ± S.D.**

13476 100.25 0.923 100.02 0.866 100.32 0.413 99.36 ± 0.421

13168 99.41 ± 1.361 99.57 ± 1.302 98.60 ± 0.741 98.95 ± 0.551


Table (LX): Determination of pyritinol Table (LX): Determination of pyritinol dihydrochloride in spiked human dihydrochloride in spiked human

plasma by the proposed electrodesplasma by the proposed electrodes..Concentration(M)Concentration(M) Pyrit-TPBPyrit-TPB Pyrit-RNCPyrit-RNC CD-RNCCD-RNC

Recovery % ± S.DRecovery % ± S.D*.*. Recovery % ± S.DRecovery % ± S.D*.*. Recovery % ± S.DRecovery % ± S.D*.*.

3.1623.162 x 10x 10-4-4 100.92100.92 ± ±0.6710.671 99.7499.74 0.601±0.601± 100.76100.76 ± ±0.3410.341

11 x 10x 10-4-4 101.14101.14 ± ±0.7700.770 100.41100.41 ± ±0.8260.826 100.11100.11 ± ±0.4750.475

* Average of three determinations.

Table (LXI): Application of standard Table (LXI): Application of standard addition for the determination of addition for the determination of pyritinol dihydrochloride by the pyritinol dihydrochloride by the proposed methodproposed method

Batch Batch numbnumb

erer


Pyrit-TPBPyrit-TPB Pyrit-Pyrit-RNCRNC CD-CD-RNCRNC

Pyrit.2HClPyrit.2HClFoundFound RecoveryRecovery FoundFound RecoveryRecovery FoundFound RecoveryRecovery

1347613476 23.0023.0034.5034.5046.0046.00

22.7122.7134.6234.6246.1146.11

98.7398.73100.34100.34100.23100.23

23.1123.1134.2534.2547.1947.19

100.47100.4799.2899.28

102.58102.58

22.7122.7134.1834.1846.3446.34

98.7398.7399.0799.07

100.76100.76

Mean ± Mean ± S.DS.D*.*.

99.7699.76 ± ±0.7890.789

100.77100.77 ± ±1.6711.671

99.5299.52 ± ±1.0871.087

* Average of three determinations.

Table (LXII): Statistical comparison for Table (LXII): Statistical comparison for the results obtained by the proposed the results obtained by the proposed method and the reported method for method and the reported method for the analysis of Pyritinol dihydrochloride the analysis of Pyritinol dihydrochloride

in pure powder formin pure powder form


Item Pyrit-TPB Pyrit-RNC CD-RNC Reported method

**

Mean 99.99 100.00 99.99 99.92

S.D. 0.827 0.775 0.680 1.172

Variance 0.683 0.600 0.462 1.375

n 5 5 6 6

F test 2.013 (6.26) 2.291 (6.26) 2.976 (5.05)

Student’s t test 0.111 (2.262) 0.130 (2.262) 0.126 (2.228)


Table (LXXXII): Assay parameters and method Table (LXXXII): Assay parameters and method validation for pyritinol dihydrochloridevalidation for pyritinol dihydrochloride

Parameter


HPLC method Suggested eledtrode

Pyritinol.2HCl Pyritinol.2HCl Pyrit-TPB Pyrit-RNC CD-RNC

Range (μg

ml-1)

10-26 10-80 3.162 x 10-6- 3.162 x 10-

4M

3.162 x 10-6- 3.162 x 10-4

M

1 x 10-6- 3.162 x 10-4M

Slope 0.119 0.0247 30.600 31.100 32.891

Intercept 0.0235 0.0033 266.84 286.89 317.07

Mean 99.52 99.59 99.99 100.00 99.99

S.D. 1.037 0.963 0.827 0.775 0.680

Variance 1.075 0.927 0.683 0.600 0.462

RSD% 1.04 0.966 0.827 0.600 0.680

.(r) 0.9991 0.9997 0.9994 0.9996 0.9990

* RSD%a 0.962, 0.841 1.401, 1.277 0.954, 0.778 0.821, 0.973 0.857, 0.816

*RSD %b 1.218, 1.300 1.334, 1.387 1.286, 1.301 1.312, 1.275 1.176, 1.211

* RSD%a, RSD%b the intra-day, inter-day respectively (n=5) relative standard deviation of concentrations (18 and 20 µg/ml) for derivative ratio method, (40

and 50 µg ml-1) for HPLC method and (10-4M and 10-5M) for ion selective electrodes method.

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contribution for the analysis of certain drugs which treat cerebrovascular insufficiency

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