Selective and Validated Spectrophotometric Assay for ...

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T. Anil Kumar, B. M. Gurupadayya*, M.B. Rahul Reddy. Department of Pharmaceutical Analysis, JSS College of Pharmacy, JSS University, Mysore, India.
Journal of Applied Chemical Research, 20, 1, 14-27 (2012)

Journal of Applied Chemical Research w w w. j a c r. k i a u . a c . i r

Selective and Validated Spectrophotometric Assay for Microgram Determination of Ganciclovir with 1-fluoro-2, 4-dinitrobenzene and N-Bromosuccinimide Reagents T. Anil Kumar, B. M. Gurupadayya*, M.B. Rahul Reddy Department of Pharmaceutical Analysis, JSS College of Pharmacy, JSS University, Mysore, India (Received 05 June 2011; Final version received 15 November 2011) Abstract Two simple, sensitive, selective, accurate, precise and economical methods (method A and B) have been developed for the quantitative estimation of ganciclovir in bulk drug and its pharmaceutical formulations. In method A, an aqueous solution of ganciclovir reacts with 1-fluoro-2, 4-dinitrobenzene (Sanger’s reagent) at borate buffer pH 9 and forms a yellow color complex and absorbance was measured at 354 nm. In method B, N-bromosuccinimide (NBS) used as an oxidimetric reagent and dye methyl orange was used as a spectrophotometric reagent. The method involves adding a measured excess of NBS to ganciclovir in acid medium followed by determination of residual NBS by reacting with a fixed amount of methyl orange and measuring the absorbance at 508 nm. The Beer’s law was obeyed in the concentration range of 0.2-0.6 µg/ml, 1-5μg/ml for method A and B respectively. The accuracy and reliability of the methods were further ascertained by performing recovery tests via standard-addition method. The recoveries of ganciclovir tablets are in the range 99.24, 99.16 respectively. The proposed method is simple, rapid, precise and convenient for the assay of ganciclovir in commercial tablet preparations. Keywords: Ganciclovir (95.3%), 1-fluoro-2, 4-dinitrobenzene (Sanger’s reagent), N-Bromosuccinimide (NBS)

Introduction

used in treatment of cytomegalovirus (CMV)

Ganciclovir (GCV) is chemically 2-amino- infection in AIDS patients [1]. Ganciclovir 1,9-[{2-hydroxyl-1-(hydroxymethyl)ethoxy} methyl]-6H-purine-6H-one.

Ganciclovir

exhibit antiviral activity against herpes simplex

is virus (HSV) and cytomegalovirus (CMV) at

an acyclic guanosine analog that requires relatively low inhibitory concentrations. It is triphosphorylation for activation prior to official in Martindale [2] Merck Index [3] and inhibiting the viral DNA polymerase. It is USP [4]. Literature survey reveals that few * Corresponding auther: Dr. BM Gurupadayya, Department of Pharmaceutical Analysis, JSS College of Pharmacy, JSS University, Shivarathreashwara Nagar, Mysore-570 015, Karnataka, India, Fax Number: +91-821-2548359, Mobile Number: +919242886136, E mail: [email protected]

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012)

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methods like liquid chromatography using The present study describes the evaluation of pulsed amperometric detection in plasma [5], N-Bromo succinamide and Sanger’s reagent high performance liquid chromatography used as reagents for the development of simple (HPLC) with pre column fluorescence deviation

and rapid spectrophotometric method for the

using phenyl glyoxal in serum [6].

determination ganciclovir in its pharmaceutical

Several

methods

which

include dosage forms.

radioimmunoassay (RIA) [7] and enzymelinked immunosorbent assay [8, 9] have been Method & Materials initially reported for the quantification of this

Instruments

GCV in biological fluids. Some combination

A

method

for

simultaneous

double-beam

Shimadzu

1700

UV

determination spectrophotometer, connected to computer and

of GCV, and acyclovir by flow-injection loaded with UV solution software was used. chemiluminescence method [10], acyclovir For an intermediate precision study and for and penciclovir in human plasma using

ruggedness, a different Shimadzu 1800 UV

fluorescence detection [11], teicoplanin in spectrophotometer connected to computer with plasma [12] and acyclovir and guanine [13]

UV-PC software was used. Both instruments

were developed. Only few spectrophotometric have an automatic wavelength accuracy of methods [14,15] are reported for the estimation

0.1 nm and matched quartz cells of 10 mm

of ganciclovir using spectrophotometry in (1.0 cm) cell path length. The absorbance of bulk drug or its formulations.

ganciclovir in the selected medium at respective

Sanger’s reagent has been utilized as a chromogen wavelength was determined and the apparent for the spectrophotometric estimation of many molar absorptivity was calculated. compounds of pharmaceutical interest such as desloratadine [16], enalapril [17], lisinopril [18] 1-Flouro-2-4-dinitro and gabapentin [19]. A great number of organic reagent) 0.5 %( w/v)

benzene

(Sanger’s

compounds have been spectrophotometrically 0.5 g of Sanger’s reagent was accurately determined using excess NBS as oxidant in the weighed transferred into a 100 ml calibrated presence of celestine blue for propranolol and flask, dissolved in methanol, and make up the tetracycline hydrochlorides [20], omeprazole volume up to the mark to obtain a solution [21] azathioprine and astemizole [22]. However, of 0.5% (w/v). Reagent should be protected the reactions of N-Bromo succinamide from light during use and should be handled with ganciclovir and Sanger’s reagent with carefully since it is a skin irritant. It is stored ganciclovir have not been investigated so far. in a refrigerator and it was stable for 4 months.

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T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012)

Borate buffer pH 9

A 1 ml quantity of 0.5% Sanger’s reagent and

Place 50ml 0.2M boric acid, 50 ml 0.2M 0.2 ml of borate buffer were added to test tube potassium chloride into 200ml volumetric containing 3.5 ml of ganciclovir and subjected flask, then add 20 ml of 0.2M NaOH and finally for heating at 900 C for 10 minutes and cool make up the volume with distilled water.

the solution, made up to the mark with distilled water. The absorption spectrums of the complex

N-Bromosuccinimide (NBS) 0.02 %( w/v)

were determined against blank solution and the

0.02 g of N-Bromosuccinimide was accurately wavelengths of maximum absorption (λmax) of weighed transferred into a 100 ml calibrated the products of the reactions were noted. flask and make up the volume up to the mark with distilled water. The solution was freshly Method B prepared and protected from light during the use. A 1 ml quantity of 0.02% NBS solution, 1.0 ml of 1M HCl were transferred into test tube Methyl orange 0.01%

and 3.5 ml of ganciclovir stock solution were

0.01 g of methyl orange is accurately weighed

added and kept it aside for 20 minutes. Then

and transferred into a 100.0ml volumetric flask add 1 ml of 0.01% methyl orange dye which and dissolved it with small quantity of water,

results in the formation of pink color complex.

then made up to the mark with distilled water.

The solutions were made up to 10ml with water. The absorption spectrum of the complex was

Hydrochloric acid 1M

determined against blank solution prepared

8.5 ml of concentrated HCl is accurately measured without drug. The wavelength of maximum and transferred into a 100.0 ml volumetric flask absorption (λmax) of the product was recorded. and made up to the mark with distilled water. Optimization Studies Preparation of standard solution

Effect of the Sanger’s reagent concentration

Accurately weighed 100 mg of ganciclovir In the study of Sanger’s reagent, it revealed was dissolved in small quantity of distilled that the reaction was dependent on Sanger’s water and the solution was further diluted reagent concentration. The absorbance of the with distilled water to mark to obtain a final

reaction solution increased as the Sanger’s

concentration of 100 µg/ml.

reagent concentration increased, and the highest absorption intensity was attained at

Selection of Analytical Wavelengths for ganciclovir concentration of 0.5 % (w/v). Higher Sanger’s Method A

reagent concentrations up to 1.0% had no effect

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012)

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on the absorption values. Further experiments results obtained were showed in figure 1. were carried out using 0.5 % of the reagent and

Figure 1: Effect of Sanger’s reagent (%) on formation of colour product.

Effect of NBS Concentration and hydrochloric concentration of 0.02 % (w/v). Higher NBS acid

concentrations up to 0.05 % had no effect on

In the study of Sanger’s reagent of NBS reagent,

the absorption values Further experiments were

revealed that the reaction was dependent on carried out using 0.02 % and results obtained NBS reagent concentration. The absorbance were showed in figure 2. It is also observed that of the reaction solution was increased as the 1 ml of 1M hydrochloric acid is necessary for NBS concentration increased, and the highest absorbance and further more addition has no absorption intensity was attained at NBS effect on the absorption (figure 2).

Figure 2: Effect of NBS and HCl on formation of color product

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012)

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Procedure for calibration curve

at 90o C for 10 minutes and then solution was

Method A

cooled. The contents were diluted up to 10ml

Aliquates of 0.2 ml, 0.4 ml, --- 0.6 ml of 10 µg/ with distilled water. The absorbance of each ml ganciclovir were transferred into different solution was measured at 353 nm against the 10 ml volumetric flasks, to these solutions 1.0

reagent blank prepared in the same manner,

ml of Sanger’s reagent and 0.2 ml of borate without the analyte. The absorption spectra buffer pH 9 were added. The mixture was

and calibration curve are represented in the

then gently shaken and subjected for heating figure 3 and 4 respectively.

Figure 3: Absorption spectra of ganciclovir with Sanger’s reagent and NBS reagent. 0.9 0.8 0.7 Absorbance

0.6 0.5 0.4 0.3 0.2 0.1 0 0

0.2

0.4 0.6 Concentration (µg/ml)

0.8

Figure 4: Linear graph of ganciclovir with Sanger’s reagent

Method B

of 0.02% NBS, 1.0 ml of 1M HCl and kept

Aliquates of 2 ml, 3 ml, --- 5 ml of 100 µg/ml it aside for 20 minutes and then add 1 ml of ganciclovir were transferred into different 10 methyl orange indicator. The mixture was then ml volumetric flasks, to these solutions 1.0 ml

gently shaken until the appearance of color

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012)

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chromogen. The contents were diluted up to without the analyte and the absorption spectra 10 ml with distilled water. The absorbance of and calibration curve of ganciclovir is shown each solution was measured at 508 nm against in figure 3 and 5 respectively. the reagent blank prepared in the same manner,

1 0.8 Absorbance

0.6 0.4 0.2 0 0

1

2

3 4 Concentration (µg/ml)

5

6

Figure 5: Linear graph of ganciclovir with Sanger’s reagent.

Analysis

of

commercial

pharmaceutical Quantification

preparations

The limits of the Beer’ law, molar absorptivity

Twenty capsules of ganciclovir (Natclovir and Sandell’s sensitivity values were evaluated and Ganguard) were weighed accurately and and are given in Table 1. Regression analyses ground into a fine powder. An amount of the

of the Beer law plots at their respective λmax

powder equivalent to 100 mg of ganciclovir values revealed a good correlation. Graphs of was weighed into a 100 ml volumetric flask, 60

absorbance versus concentration showed zero

ml of water added and shaken thoroughly for

intercept, and are described by the regression

about 20 min. Then, the volume was made up

equation, Y = bX + c (where Y is the absorbance

to the mark with water, mixed well and filtered

of a 1 cm layer, b is the slope, c is the intercept

through Whatmann filter paper No. 41. First

and X is the concentration of the drug in μg/

10 ml portion of the filtrate was rejected and

ml) obtained by the least-squares method. The

2.5 ml of the tablet extract was subjected to

results are summarized in Table.

analysis using the procedure described above.

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T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012) Table 1. Optical characteristics of spectrophotometric method. Parameter Color λmax(nm) Beer’s law range (μg.ml-1) Molar absorptivity(L.mol-1.cm-1) Sandell’s Sensitivity (µg.cm-2) Limit of detection (µg.ml-1) Limit of quantification (µg.ml-1) Correlation coefficient, R Slope b Intercept a Standard deviation of slope Standard deviation of intercept Percentage recovery

Method A Yellow 353nm 0.2-0.6 0.1×103 0.00068966 0.05 0.1619 0.9998 1.4204 0.0051 0.03884 0.002715 99.24

Method B Pink 508 nm 1-5 0.32×103 0.00624 0.1045 0.446 0.9997 0.789 0.0262 0.004203 0.0007805 99.16

Validation of the method

scanned from 800 to 200 nm at a speed of

The validity of the method for the assay of

200 nm min−1 and checked for change in the

ganciclovir was examined by determining the absorbance at respective wavelengths. In a precision and accuracy. This was determined by separate study, drug concentration of 0.2 μg/ml analyzing six replicates of the drug within the for method A and 2 μg/ml for method B was Beer’s law limits. The low values of the relative prepared independently from pure drug stock standard deviation (R.S.D.) indicate good solution in selected media and analyzed paired t precision of the methods. To study the accuracy test at 95 % level of significance was performed of the methods, recovery studies were carried out to compare the means of absorbance. by the standard calibration curve method. For this, known quantities of pure ganciclovir were Linearity mixed with definite amounts of pre-analyzed To establish linearity of the proposed methods, formulations and the mixtures were analyzed a separate series of solutions of ganciclovir as before. The total amount of the drug was for method A (0.1-0.6 µg/ml) and for method then determined and the amount of the added B (1-5 µg/ml) were prepared from the stock drug was calculated by difference. The average solutions and analyzed. Least square regression present recoveries obtained were quantitative analysis was performed on the obtained data. indicating good accuracy of the methods. Precision Specificity and selectivity

This good level of precision was suitable for

Ganciclovir solutions were prepared in the quality control analysis of the investigated selected media with and without common drug in their pharmaceutical dosage forms. exicipients separately. All solutions were The precision of the proposed methods was

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012)

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ascertained by actual determination of six method, recovery studies were carried out replicates of fixed concentration of the drug by adding different known amounts of bulk within the Beer’s range and finding out the samples of ganciclovir within the linearity absorbance by the proposed method in all the range were taken and added to the pre-analyzed three drugs. The results are given in Table 2.

formulation of concentrations 0.2 µg/ml and 2µg/ml for method A and B respectively. The

Accuracy

results are given in Table 3.

To determine the accuracy of the proposed Table 2. Accuracy and method precision data for the developed method. Drug

S.No

Label Claim (mg)

Amount found*

% Purity*

Average (%)

S.D

R.S.Da

RSDb

1 248.42 99.36 2 247.68 99.10 3 250.04 100.02 Method 250 99.30 0.019 0.0716 0.0710 4 249.98 99.90 A 5 250.68 100.27 6 248.98 99.59 1 250.56 100.22 2 249.12 99.65 3 250.62 100.25 Method 250 99.56 0.022 0.040 0.0396 4 247.32 98.93 B 5 246.40 98.56 6 248.46 99.35 SD. Standard deviation; SEM. Standard error of mean; RSD. Relative standard Deviation;

S.E.M

0.013

0.013

a

intra-day precision, binter-day precision. Table 3. Standard addition of ganciclovir for accuracy. formulation

Amount -1

Amount

Total found

Recovery (%)

Studied

taken(µg.ml )

added

(µg.ml-1)

Method A

0.2

0.4

0.59

98.46

Method B

2

2

0.38

99.42

Robustness and Ruggedness

robustness and the ruggedness were checked

To evaluate the robustness of the methods, at three different drug levels. The intermediate reaction time and reagent concentrations were

precision, expressed as percent RSD, which is

slightly altered with reference to optimum a measure of robustness and ruggedness was values in spectrophotometry. To check the within the acceptable limits as shown in the ruggedness, analysis was performed by four

Table. 2.

different analysts; and on three different spectrophotometers by the same analyst. The Limit of detection (LOD) and limit of

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T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012)

quantitation (LOQ)

1. The method obeys Beer’s law is obeyed

The LOD and LOQ for method A and method B in the range of 0.2-0.6 μg/ml and standard by the proposed method were determined using

deviation of slope and intercept were found

calibration standards. LOD and LOQ were

to be 0.03884 and 0.002715. Optimization

calculated as 3.3 σ/S and 10 σ/S, respectively,

of the spectrophotometric conditions was

Where Sis the slope of the calibration curve intended to take into account the various goals and σ is the standard deviation of y-intercept of method development. Analytical conditions of regression equation.

were optimized via a number of preliminary experiments. The effect of Sanger’s reagent

Results and Discussion

concentration was studied and found that

In method A Sanger’s reagent forms a yellow 0.5% gave good absorbance values so further color complex with ganciclovir in alkaline experiments were carried out using 0.5 % medium and their absorbances were measured

Sanger’s reagent and effect of heating time on

at 353nm. Therefore, the present study

formation of color product was studied and

was devoted to explore Sanger’s reagent as different buffer solutions in the pH range of derivatizing reagents for the determination 8.0-10 were tested for reaction of Sanger’s of ganciclovir in pure and pharmaceutical reagent with ganciclovir. Best results were dosage forms. The reaction mechanism of obtained in case of borate buffer pH 9. drug Sanger’s reagent shown in the Scheme

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Scheme 1: Mechanism of reaction of ganciclovir with Sanger’s. HO F NO2

O H N

H2N

OH NO2

N

Sanger's reagent N

N O

Ganciclovir

HO

O F

H N

NH

OH

N

O2N N

N O

NO2

Intermediate HO

O H N

NH

O2N N

NO2

OH

N

N O

Yellow colored complex

H+

F+

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Method B is based on the oxidation reaction a known excess of NBS in acid medium, and between ganciclovir and NBS in acidic medium. the unreacted oxidant is determined by reacting The reaction mechanism of drug Sanger’s with a fixed amount of methyl orange and reagent shown in the Scheme 2. The Beer’s law measuring the absorbance at 508 nm. In this is obeyed in the range of 1-5 μg/ml and standard method, the absorbance increased linearly with deviation of slope and intercept were found increasing concentration of drug. Ganciclovir to be 0.004203 and 0.0007805. The method when added in increasing amounts to a fixed is indirect and is based on the determination amount of NBS consumes the latter and there of residual NBS after having allowed the

will be a concomitant fall in its concentration.

oxidation reaction to go to completion under When a fixed amount of dye is added to the specified experimental conditions. The decreasing amounts of NBS, a concomitant amount of NBS reacted corresponds to the increase in the concentration of dye results. drug content in the method. The ability of This is observed as a proportional increase in NBS to oxidize ganciclovir and bleach the the absorbance at the respective wavelengths colors of methyl orange dye has been used for of maximum absorption with increasing the indirect spectrophotometric assay of the concentration of ganciclovir as indicated by the drug. In this method, the drug is reacted with

correlation coefficients of 0.9997 respectively.

Scheme 2: Mechanism of reaction of ganciclovir with NBS. O

OH

O H N

H2N

O HO

N

4 NBS N

H N

H2N

O

N

Oxidation N

N O

N O

Oxidized ganciclovir

Ganciclovir

O

H3C

4 NBS

N

N

N

S

H3C

O

O-

Methyl Orange Bromination

Br

Br O

H3C N

N

N

S

H3C

OBr

Tetra Bromo Methyl Orange

Br

O

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Preliminary experiments were performed to For a quantitative reaction between ganciclovir determine the maximum concentrations of the and NBS, a contact time of 20 min was found dye spectrophotometrically, and these were necessary and constant absorbance readings found to be 0.01%. 1M Hydrochloric acid (1

were obtained when contact times were extended

ml) was the ideal medium for the oxidation upto 20 minutes. A standing time of 5 min was of ganciclovir by NBS as well as the latter’s necessary for the bleaching of the dye color by determination employing methyl orange dye. the residual NBS. The measured color was found The reaction between ganciclovir and NBS was to be stable for several hours in the presence of unaffected when 1.0 – 2.5 ml of 1 M hydrochloric the reaction product (figure 6). Based on various acid in a total volume of about 7 ml was used.

optical and validation parameters method A is

Hence, 1.0 ml of 1 M hydrochloric acid is more sensitive and reliable method compared to used for both steps in the assay procedures.

method B.

Figure 6: Effect of reaction time with absorbance.

The results were in agreement with the labeled

for method A and B respectively (Table 4).

amounts. For comparison, a conventional UV This indicated similar accuracy and precision spectrophotometric method developed in our

in the analysis of the investigated compounds

laboratory was used for parallel comparison.

in their pharmaceutical dosage forms.

The recovery percentages for 99.24 and 99.16 Table 4. Results of determination of ganciclovir in formulations and statistical comparison with the reference method. Amount found by Recovery of Recovery of Pharmaceutical Labelled proposed methods Reference proposed methods (%) dosage form Amount (mg) method 99.38 Method A 250 mg 248.46 98.73 t=0.566 (Natclovir) f=1.084 99.65 Method B 250 mg 249.12 97.50 t=0.594 (Ganguard) f=1.182

T. Anil Kumar et al., J. Appl. Chem. Res., 20, 1, 14-27 (2012)

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Conclusion

(2007).

The reagents utilized in the proposed methods

[5] Satoshi Kishino, J Chromatography B.,

are cheap, readily available and the procedures

780, 289 (2002).

do not involve any critical reaction conditions

[6] T. Suchie Masahiko, Hara Shuuji, Kimura

or tedious sample preparation. Moreover,

Masahiko, Fujii Megumi, Ono Nobufumi

the methods are free from interference by

and Kai Masaaki, Analytical Sciences., 17,

common additives and excipients. The wide

811(2001).

applicability of the new procedures for routine

[7] C. Nerenberg and S. McClung, J. Martin,

quality control was well established by the

Pharm. Res., 3, 112 (1986).

assay of ganciclovir in pure form and in

[8] S. M. Tadepalli, R. P. Quinn and D. R.

pharmaceutical preparations.

Averett, Antimicrob. Agents Chemother., 29, 93 (1986).

Acknowledgements

[9] K. Henry, H. Cantrill and C. Fletcher, Am.

The authors express their sincere thanks to

J. Ophthalmol., 103, 17 (1987).

Strides Arco Lab Limited, Bangalore, India

[10] M. Abudukeremu, W. Nannan, T. Yuhai,

for supplying the gift sample of ganciclovir

J. Xi’an Jiaotong Univ., 28, 707 (2007).

(purity-95.3%). Thanks to the Principal, JSS

[11] Y. J. Dao, Z. Jiao and M. K. Zhong, J.

College of Pharmacy, Mysore, for providing

Chromatogr. B: Anal. Technol. Biomed. Life

the necessary facilities.

Sci., 867, 270 (2008). [12] M. Cociglio, H. Peyrière and D. Hillaire-

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