79

2. VISIBLE SPECTROPHOTOMETRIC DETERMINATION OF

IRINOTECAN HYDROCHLORIDE

2.1: INTRODUCTION

Irinotecan is an antineoplastic agent that is primarily used in the

treatment of metastatic colorectal cancer.The drug is official in I.P80

and also in Martindale, The Extra Pharmacopoeia 81.

Drug profile of Irinotecan HCl is shown in the following table 2.1:

80

TABLE 2.1: DRUG PROFILE OF IRINOTECAN HCL

1 Official Name Irinotecan Hydrochloride

2 Chemical

Name

(S)-10-[4-(-piperidino) piperidinocarbonyl

oxoyl]-4,7-diethyl-4-hydroxy-1H-

pyrano[3,4:6,7]indolizino[1,2-b]diethyl-

3,14[4H,12H]-dione

3 Chemical

Structure

Fig 2.1

4 Analytically

useful

functional

group

Secondary amino group

5 Molecular

Formula

C33H38N4O6

6 Molecular

Weight

586.678

7 Color Yellow to pale yellow crystalline powder

8 Solubility Water

9 Therapeutic

Use

Antineoplastic agent that is primarily used in

the treatment of metastatic colorectal cancer.

10 Mechanism of It is a semisynthetic, water-soluble derivative of

81

Action camptothecin, a cytotoxic alkaloid extracted

from plants such as Camptotheca acuminata.

Irinotecan and its active metabolite, SN-38,

inhibit the action of topoisomerase I, an

enzyme that produces reversible single-strand

breaks in DNA during DNA replication. These

single-strand breaks relieve torsional strain

and allow DNA replication to proceed.

Irinotecan and SN-38 bind to the

topoisomerase I-DNA complex and prevent

religation of the DNA strand, resulting in

double-strand DNA breakage and cell death.

Irinotecan is cell cycle phase-specific (S-phase)

81.

Aim of the Work

Few HPLC methods for quantitative determination of irinotecan were

reported in the literature82-87 and are mainly useful for therapeutic

monitoring of the drug in biological fluids (Table 2.2). No visible

spectrophotometric method for quantitative determination of

irinotecan in bulk drug samples and injections was reported. The aim

of the present work is to develop and validate rapid, economical and

sensitive visible spectrophotometric method for quantitative

determination of irinotecan in bulk drug samples and injectable

preparations. In the present investigation, two new visible

spectrophotometric methods were developed and validated for

82

irinotecan with two chromogenic reagents namely Bromo Cresol Green

(BCG) and Bromo Phenol Blue (BPB).

TABLE 2.2: LIST OF REPORTED HPLC METHODS FOR IRINOTECAN HCl

Sample Internal

standard

Column Mobile Phase Detector Reference

Injectio

ns

Reference

solution

(a):0.04% w/v

of Irinotecan

HCL with

mobile phase

Reference

solution (b):

Dilute 1 ml of

this to 100 ml

with mobile

phase.

ODS (25

cm X 4.6

cm,5 µm)

Disodium

hydrogen

phosphate and

1-heptane

sulphonic acid

in water.pH

adjusted to 3

with

orthophosphoric

acid and

Acetonitrile(72:2

8) and Flow rate

1 ml/min

UV at 254

nm

80

Rat

plasma

samples

Camptothecin Hypersil

C18

0.1M potassium

dihydrogen

phosphate

containing 0.01

M TBAHS (pH

6.4)-acetonitrile

(75:25, v/v)

Florescence 82

Human

plasma

Camptothecin Xterra

RP18

The sample

pretreatment

Florescence 83

83

and

saliva

involved protein

precipitation

with methanol-

acetonitrile

(50:50 by

volume) followed

by acidification

with

hydrochloric

acid to convert

the lactone ring-

opened form into

its lactone form

Human

whole

blood

and in

red

blood

cells

Hypersil

ODS

Sample

pretreatment

involved

deproteinization

of whole blood or

plasma-diluted

RBCs isolated by

MESED

instruments,

with a mixture

of aqueous

perchloric acid

and methanol

(1:1, v/v)

Florescence 84

Human

plasma

Zorbax

SB-C18

sample

pretreatment

Florescence 85

84

consisted of a

simple protein

precipitation

with acetonitrile-

methanol (1:1,

v/v), after which

CPT-11 and SN-

38 were

quantitatively

converted to

their carboxylate

form by adding

0.01 mol/L

sodium

tetraborate

(pH,9)

Human

plasma

Camptothecin

(III)

C18

reversed-

phase

Acetonitrile,phos

phate buffer and

heptanesulphoni

c acid

Florescence 86

2.2: EXPERIMENTAL

Instruments:

ELICO-SL-164 Ultraviolet-Visible spectrophotometer (double beam)

was used for all spectral measurements.

85

Digisun model DI-707 pH meter was used for all the pH

measurements.

Chemicals and reagents and their preparation:

All the chemicals used were of analytical grade.

BCG (0.1% w/v), BPB (0.1% w/v), phthalate buffer of pH 2.2 and

chloroform.

BCG(0.1% w/v): was prepared by dissolving 100 mg of BCG in 0.72

ml of 0.1N NaOH and 20 ml of methanol. After solution was affected,

sufficient distilled water was added to produce 100 ml.

BPB(0.1% w/v): was prepared by dissolving 100 mg of BPB in 1.5 ml

of 0.1N NaOH and 20 ml of methanol. The solution was then diluted

with distilled water to make up the volume to 100 ml. This solution

was treated with methanol to remove methanol soluble impurities.

Phthalate buffer: of pH 2.2 was prepared as per I.P.

All the reagents and solvents were of analytical grade and were used

as they are purchased without any further purification.

Procedure:

Preparation of standard drug solution:

About 50 mg of irinotecan was accurately weighed and dissolved in 50

ml of distilled water in a standard volumetric flask to obtain a stock

solution of 1 mg/ml. A volume of 5 ml of this solution was further

86

diluted with distilled water to 50 ml to get 100 µg/ml of working

standard solution.

Preparation of sample solution:

Volume of injection equivalent to 25 mg of the drug was diluted to 50

ml with distilled water to get the required concentration as given in

the preparation of standard drug solution.

For Bulk Drug Samples

Method IRT1 (BCG Method):

Aliquots of standard drug solution (0.3-3.75 ml) were added to 5 ml of

phthalate buffer of pH 2.2 contained in a separating funnel followed

by 0.5 ml of 0.1% w/v BCG solution.

The solution was extracted with chloroform and collected chloroform

layer was dried over anhydrous sodium sulfate. Volume was made up

to 10 ml with water. A linear graph was obtained at 420 nm after the

waiting period of 15 min, against reagent blank prepared

simultaneously.

Method IRT2 (BPB Method):

Aliquots of standard drug solution (0.1-1.25 ml) were added to 3 ml of

phthalate buffer of pH 2.2 contained in a separating funnel followed

by 1.0 ml of 0.1% w/v BPB solution.

The solution was extracted with chloroform and collected chloroform

layer was dried over anhydrous sodium sulfate. Volume was made up

87

to 10 ml with water. A linear graph was obtained at 380 nm against

reagent blank prepared simultaneously.

Analysis of Formulations:

Method IRT1 (BCG Method):

Volume of injection equivalent to 25 mg of the drug was diluted to 50

ml and further diluted with distilled water to get the required

concentration. To this solution were added 3 ml of phthalate buffer of

pH 2.2 contained in a separating funnel followed by 0.5 ml of 0.1%

w/v BCG solution. The solution was extracted with chloroform and

collected chloroform layer was dried over anhydrous sodium sulfate.

Volume was made up to 10 ml with water. Absorbance of this solution

was measured at 380 nm against reagent blank prepared

simultaneously.

Method IRT2 (BPB Method):

Volume of injection equivalent to 25 mg of the drug was diluted to 50

ml and further diluted with distilled water to get the required

concentration. To this solution were added 3 ml of phthalate buffer of

pH 2.2 contained in a separating funnel followed by 1.0 ml of 0.1%

w/v BPB solution. The solution was extracted with chloroform and

collected chloroform layer was dried over anhydrous sodium sulfate.

Volume was made up to 10 ml with water. Absorbance of this solution

88

was measured at 380 nm against reagent blank prepared

simultaneously.

2.3: RESULTS AND DISCUSSION

Analytical Data and Method Validation:

Optimization of parameters for Methods IRT1 and IRT2

The optimum conditions were established by changing one parameter

while fixing the other parameters and noting the effect on absorbance

of chromogen.

The different parameters such as volume of phthalate buffer, strength

and volume of dye solution, time gap between the addition of

reagents, order of addition of reagents and stability of colored species

were studied and presented in table 2.3.

TABLE 2.3: OPTIMUM CONDITIONS ESTABLISHED FOR

METHODS IRT1 AND IRT2

Parameter Optimum

Range

Conditions In

The Methods

Remarks

Volume of 0.1%

w/v dye

solution

required for

complex

formation

1-2 ml 1 ml Addition of less than

1ml results in low

absorbance

particularly with high

concentrations of

Beer‟s law limits.

Addition of more than

89

1 ml results in high

blank value.

Volume of

phthalate buffer

7-10 ml 3 ml To maintain pH at 2.2

which is necessary for

maximum color

development

Time and

temperature

required for

complex

formation

5-15 min at

temp 28± 50

C

15 min at room

temp(290 C)

Change of time and

temperature beyond

optimum limits

produced erratic

results.

Stability after

complete color

development

40 min >40 min Complex formed is

stable for sufficient

period of time.

90

The optimum pH required for complexation and efficiency of the

solvent to extract the ion pair were studied with respect to maximum

sensitivity, color stability, adherence to Beer‟s law and other optimum

conditions are incorporated in the procedure.

Optical Characteristics

Absorption Maximum

Absorption spectra of irinotecan for methods IRT1 and IRT2 were

shown in figures 2.2 and 2.3.

91

Absorption Spectrum of Irinotecan with BCG

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

300 350 400 450 500

Wavelength (nm)

Ab

so

rba

nc

eChromogen Vs BCG,BCG

0.1% W/V in Methanol,IRT-

12.5 μg/mlReagent Blank vs Distilled

Water

Fig 2.2 Absorption spectrum of Irinotecan with BCG

Absorption Spectrum of Irinotecan with BPB

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

300 350 400 450 500

Wavelength (nm)

Ab

so

rba

nc

e

Chromogen Vs BPB, BPB

0.1% W/V in Methanol,

IRT- 5 µg/mlReagent blank vs

Distilled water

Fig 2.3 Absorption spectrum of Irinotecan with BPB

92

Linearity

By using the method of least squares regression analysis was

performed to evaluate the slope (m), intercept (b) and correlation

coefficient (r) was computed from various concentrations and the

results are presented in Tables 2.4 and 2.5. The graph showed

negligible intercept as described by the regression equation y = mx + b

where y is the absorbance and x is the concentration in µg/ml.

Calibration curves for methods IRT1 and IRT2 were shown in figures

2.4 and 2.5

The optical characteristics such as molar absorptivity, Beer‟s law

limits, absorption maxima and Sandell‟s sensitivity are presented in

Table 2.6.

93

Table 2.4 Linearity Data of Irinotecan HCl with BCG

Conc µg/ml Absorbance

3 0.073

6.25 0.145

12.5 0.276

25 0.573

37.5 0.834

Linearity Of Irinotecan with BCG y = 0.0222x + 0.0055

R2 = 0.9995

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 5 10 15 20 25 30 35 40

Concentration in µg/ml

Ab

so

rban

ce

Fig 2.4 Calibration Curve of Irinotecan with BCG

94

Table 2.5 Linearity Data of Irinotecan HCl with BPB

Conc µg/ml Absorbance

1 0.116

2.5 0.182

5 0.354

7.5 0.525

10 0.698

12.5 0.882

Linearity of Irinotecan HCl with BPBy = 0.0073x + 0.0272

R2 = 0.9978

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 2 4 6 8 10 12 14

Concentration in µg/ml

Ab

so

rban

ce

Fig 2.5 Calibration Curve of Irinotecan with BPB

95

TABLE 2.6: OPTICAL CHARACTERISTICS AND PRECISION OF

THE METHODS

S.

NO Parameter IRT1 IRT2

01 λ max (nm) 420 380

02 Beer‟s law range

(µg/ml)

3.0-37.5 1.0-12.5

03 Molar extinction

coefficient(1.mole-1

cm-1)

1.4 x 104

5.8 x 107

04 Sandell‟s sensitivity

(µg/cm2/0.001)

0.066 0.094

05 Regression equation

(y = mx + b) *

Slope (m)

Intercept (b)

0.0222

0.0055

0.0073

0.0272

06 Correlation

coefficient (r)

0.9998 0.9989

07 Precision (%Relative

Standard Deviation)

0.274 0.847

08 Standard Error of

Mean

0.0086 0.0184

* y = mx + b, where y is the absorbance unit and x is the

concentration in µg/ml.

96

Accuracy and Recovery

Commercially available injections of irinotecan (Table 2.7) were

analyzed by the proposed methods and as an additional check on the

accuracy of the method, recovery experiments were also conducted by

spiking known amounts of pure drug in preanalysed injection and the

recovery was calculated in each of the case using the regression line

equation developed under the linearity experiment. The average

recovery across the concentration range studied was 99.86 % with a

relative standard deviation of 0.52% for BCG and 100.05% with a

relative standard deviation of 0.21% for BPB. The correlation

coefficient between the amount added to amount found was

calculated as 0.9999 in both the methods, indicating a strong

correlation between the amount added and amount found.

Assay results of the proposed methods were compared with that of

reference method and statistically evaluated using one-way ANOVA

with post-test followed by Dunnett multiple comparison test. The

means of the proposed methods are not significantly different from

that of reference method (p>0.05). The assay and accuracy results

were presented in Table 2.8. The proposed methods are accurate, fast,

precise, sensitive, and efficient and can be used in quality control

laboratories.

97

The interference studies indicated the common additives and

excipients present in formulations did not interfere with the proposed

methods.

98

TABLE 2.7: COMMERCIAL FORMULATIONS OF IRINOTECAN HCl

Generic Name

Proprietary Name Dosage Form

Content

Irinotecan

HCl

Irinotel inj, Dabur, India Injection 40 mg and

100 mg

Irinogen 100, BGSS

Pharma, Argentina Injection 100 mg

Irnocam,Dr.Reddy‟s,India Injection 40 mg and

100 mg

TABLE 2.8: EVALUATION OF IRINOTECAN HCl IN

PHARMACEUTICAL DOSAGE FORMS (n=6)

Samplea Labelled

Amount (mg)

Amount obtained (mg)b Percentage Recoveryb,c

Proposed method d I.P

Reference method 80

IRT1

IRT2

IRT1 IRT2 HPLC

I1 100 100.05±0.63 100.05±0.23 99.96±0.64 99.86±0.01 100.05±0.01

I2 100 99.84±0.74 99.84±0.70 99.51±0.82 99.88±0.03 99.98±0.02

a - I1 and I2 are the injections from different batches (Irinogen 100, BGSS Pharma, Argentina).

b – Mean ± SD of 6 determinations.

c – 50 mg of pure drug was added and recovered.

For both the samples I1 and I2 One-way ANOVA with post-test

followed by Dunnett multiple comparison test was performed. The results showed that p > 0.05 and the means of the proposed methods

are not significantly different from that of reference method.

99

2.4: Chemistry of the colored species formed

Irinotecan is an indolizino derivative, having an amino group in the

molecular structure making it possible to form the ion-pair complexes

with acidic dyes namely bromocresol green (BCG) and bromophenol

blue (BPB) 88-94.The ion-association complex or adduct (commonly

known as ion pair, if two ions are involved) is a special form of

molecular complex resulting from two components extractable into

organic solvents from aqueous phase at suitable pH, one component

is a chromogen (dye or metal complex) possessing charge (cationic or

anionic in nature) and so it is insoluble in organic solvents. The

second component is colourless, possessing opposite charge (anionic

or cationic) to that of chromogen. Reaction mechanisms are

represented in proposed scheme 2.1 for BCG and proposed scheme

2.2 for BPB.

+Irinotecan HCl Ion-Association complex

BCG (Dye) (Drug)

Proposed Scheme 2.1: Reaction mechanism of Irinotecan HCl

with BCG

100

+ Irinotecan HCl Ion-association complex

BPB (Dye) (Drug)

Proposed Scheme 2.2: Reaction mechanism of Irinotecan HCl

with BPB

2.5 Conclusion

The proposed visible spectrophotometric method enables quantitative

determination of irinotecan in bulk drug samples and injections.

Efficient visible spectrophotometric detection at the respective

absorption maxima enabled determination with no interference from

the excipients or solvents of injectable solution. The calibration curves

were linear over a concentration range from 3.0-37.5 µg/ml for BCG

and 1.0-12.5 µg/ml for BPB. The relative standard deviation‟s (R.S.D.)

were less than 1% and average recovery was above 99.86%. Assay

results of the proposed methods were compared with that of reference

method and statistically evaluated using one-way ANOVA with post-

test followed by Dunnett multiple comparison test. The means of the

proposed methods are not significantly different from that of reference

method (p>0.05). The proposed method is fast, sensitive, precise,

accurate, and efficient and can be used in for analysis in quality

control laboratories.

101

3. VISIBLE SPECTROPHOTOMETRIC DETERMINATION OF

BICALUTAMIDE

3.1 INTRODUCTION

Bicalutamide is an oral non-steroidal anti-androgen for prostate

cancer. It was first launched in 1995 as a combination treatment

(with surgical or medical castration) for advanced prostate cancer and

subsequently launched as monotherapy for the treatment of earlier

stages of the disease. The drug is official in Martindale, The Extra

Pharmacopoeia 95 and also in Merck Index 96.

Drug profile of Bicalutamide is shown in the following table 3.1:

TABLE 3.1: DRUG PROFILE OF BICALUTAMIDE

1 Official Name Bicalutamide

2 Chemical Name N-[4-cyano-3-(trifluoromethyl)phenyl]-3-(4-

fluorophenyl)sulfonyl-2-hydroxy-2-methyl-

propanamide

3 Chemical

Structure

Fig 3.1

4 Analytically

useful functional

group

Secondary amino group

5 Molecular C18H14F4N2O4S

102

Formula

6 Molecular Weight 430.374

7 Color White powder

8 Solubility Methanol

9 Therapeutic Use Used for the treatment of stage D2

metastatic prostate cancer in combination

with a luteinizing hormone-releasing

hormone analogue or as a monotherapy.

Aim of the Work

Only few HPLC methods for quantitative determination of

bicalutamide were reported in the literature and these methods were

applied in the determination of bicalutamide and it‟s metabolites in

biological fluids 97-100 and are mainly useful for therapeutic

monitoring of the drug (Table 3.2).No visible spectrophotometric

method for quantitative determination of bicalutamide in bulk drug

samples and formulations was reported. The aim of the present work

is to develop and validate rapid, economical and sensitive visible

spectrophotometric method for quantitative determination of

bicalutamide in bulk drug samples and formulations. In the present

investigation,two new visible spectrophotometric methods were

developed for bicalutamide with two chromogenic reagents namely

MBTH and NQS.

103

TABLE 3.2: LIST OF REPORTED METHODS FOR BICALUTAMIDE

Sample Internal standard

Column Mobile Phase Detector Reference

Rat blood plasma

Letrozole C18 column

mobile phase consisting of water:

acetonitrile (adjusted to pH 3.0 with

20% o-phosphoric

acid) (60:40), at a flow rate of 1.0 ml/min

UV 97

Bulk drug and

pharmaceutical formulatio

ns.

Symmetry C(18)

(4.6 mm x 250 mm;

particle size 5

µm) column , isocratic

mode

Mobile phase was 0.01 M

KH(2)PO(4) (pH 3.0):acetonitri

le (50:50 v/v)

PDA detector

set at 215 nm

98

Tablets

(UV Method)

Twenty tablets of bicalutamide were weighed and

powered in glass mortar. Amount equivalent to 5 mg was transferred to 50 ml volumetric flask, dissolved in 5 ml of DMF and made up the

volume with 1% SLS to obtain a concentration of 100 µg/ml. The solution was filtered through

Whatman filter paper No. 41 and filtrate was diluted to obtain concentration in between linearity range. The absorbance of sample

solution was measured and amount of bicalutamide was determined by referring to the calibration curve (λmax was found to be 272 nm).

101