Determination of tinidazole by potentiometry, spectrophotometry and ...

Indian Journal of Chemical Technology Vol. 12, May 2005, pp. 273-280

Determination of tinidazole by potentiometry, spectrophotometry and high performance liquid chromatography K Basavaiah*, P Nagegowda & U Chandrashekar Department of Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India Received 24 May 2004; revised received 20 December 2004; accepted 8 February 2005 Three assay procedures based on potentiometry, spectrophotometry and high performance liquid chromatography (HPLC) have been developed for the determination of tinidazole in tablets and injections. In the potentiometric titration method, the drug in glacial acetic acid is titrated with acetous perchloric acid with potentiometric end point detection. Spectrophotometry involves the addition of different amounts of tinidazole to a fixed amount of perchloric acid-malachite green mixture followed by measurement of absorbance at 615 nm. The HPLC determination was carried out on a reversed phase C18 column using a mobile phase consisting of acetonitrile-0.1 % phosphoric acid (70:30) at a flow rate of 1.0 mL min-1 with UV-detection at 316 nm. Potentiometric tiration is applicable over 1-10 mg range of tinidazole and in spectrophotometry, the calibration graph is linear from 15-180 μg mL-1 with a molar absorptivity of 9.02 × 102 L moL-1 cm-1 and a Sandell sensitivity of 385.2 ng cm-2. The limits of detection and quantification are calculated to be 3.05 and 10.15 μg mL-1, respectively. In HPLC method, a rectilinear relationship was observed between 6.25 and 250 μg mL-1 tinidazole with a detection limit of 0.625 μg mL-1 and a quantification limit of 1.875 μg mL-1. The analysis time was less than 5 min. The statistical evaluation of the methods was examined by determining intra-day and inter-day precision. The methods when applied to the determination of tinidazole in tablets and injections gave satisfactory results. The accuracy and reliability of the proposed methods were further ascertained by parallel determination by the reference methods and by recovery studies using standard-addition technique. Keywords: Tinidazole, potentiometry, spectrophotometry, HPLC, pharmaceuticals IPC Code: G01J3/00; B01D15/00; A61K

Tinidazole (TNZ) is 1-[2-(ethyl sulphonyl) ethyl]-2methyl-5-nitroimidazole1 (Fig. 1). It is an antiprotozoal and is used in the treatment of susceptible protozoal infection and in the treatment of prophylactics of anaerobic bacterial infection. TNZ is also used in the treatment of amoebiasis. The therapeutic importance of this compound justifies research to establish analytical methods for its determination in pharmaceutical preparations and biological samples. Several methods are available for determining TNZ in its dosage forms. The pharmacopoeical method1 involves measurement of absorbance of methanolic solution of the drug at 310 nm. Methods proposed for the determination of TNZ include gas liquid chromatography2, packed column supercritical fluid chromatography3, high performance liquid chromatography(HPLC)4-9, voltammetry10,11, ___________________ HPLC work was presented at “The National Conference on Emerging Trends in Chemical Sciences in The New Millenium” Feb 5-7, 2004, Amaravathi, India. * For correspondence (E-mail: [email protected]; Fax: 0091-0821-2421263)

polarography12, infrared spectrophotometry13 and UVspectrophotometry14. For the visible spectrophotometric determination, either TNZ itself15-17 or the amino compound obtained after reduction18-23 by Zn/HCl has been reacted with several reagents. Methods based on oxidative coupling15,18, Schiff base formation19,20, complex formation16,17,23, charge-transfer complexation21, and condensation22 reactions are found in the literature for the determination of this drug in formulations. Other methods involve the use of chromogenic agents24,25 that react with TNZ to form species that absorb in the visible region.The proposed methods suffer from one or the other disadvantages (Tables 1 & 2).

Fig. 1 ⎯ Structure of tinidazole

INDIAN J CHEM. TECHNOL., MAY 2005

274

Table 1⎯Comparison of the existing HPLC methods with the proposed method Sl No.

Column & mobile phase used

Linear range, μg ml-1

Detection method

Remarks

Ref

1

ODS, aq. 25 % MeOH

uv- detection, 380 nm

Uses an internal standard

4

2

Nucleosil SGE C18, 0.02M KH2PO4/MeOH (7:3)

0.2 – 1.0


Uses an internal standard, narrow range of response

5

3

Cosmosil 5C18 – AR, 0.01 M phosphate buffer pH 5.5 – acetonitrile (17:3)

20 – 200


Uses an internal standard

6

4

Lichrosorb RP-8, MeOH – 0.01M KH2PO4 (3:7)

2 – 10

uv-detection, 313 nm

Uses an internal standard, narrow range of linear response

7

5

Micro Bondapak C18, MeOH – 0.05M H3PO4 (pH 3.0) (8:3)

0.2 – 8.0


Uses an internal standard, narrow range of linear response

8

6

Micro Bondapak C18, 22 % acetonitrile – acetate buffer (pH 4.7)

0.5 (LOD)


Uses internal standard

9

7

Hypersil ODS C18, acetonitrile – 0.1 % H3PO4 (pH 3.0) (70:30)

6.25 – 250.0

uv-detection, 316 nm

No internal standard, long and dynamic linear range of response

Present method

Table 2⎯Comparison of the existing visible spectrophotometric methods with the proposed method Sl No.

Reagent/s uesd

Linear range, μg ml-1

λmax, nm

Remarks

Ref

1

CAT - MBTH*

2.5 –20.0

540

Uses an expensive reagent (MBTH); contact time 30 min

15

2

CAT – NNDPD*

4.0 – 36.0

540

Involves prereduction of analyte; contact time 30 min.

18

3

Vanillin

10 - 50

412

Involves prereduction and boiling for 2 min

19

4

DMAB*

10 – 100

550

Involves prereduction step

20

5

NH2OH – SNP*

2.5 – 30

660


16

6

Tetramethyl ammonium hydroxide

4 – 32

365

Measurement made at shorter wavelength where the interference is more, uses DMF as a diluent

17

7

Salicyaldehyde

20 – 70

380

Measurement made at shorter wavelength

23

8

K2Cr2O7 - metol

3 – 30

530

Involves prereduction step, colour is stable for only 10 min

21

9

9 - chloroacridine

1 – 10

---


22

10

HClO4 – crystal violet

15 – 180

615

No pre reduction step, colour is stable for several days, measurement is made at a longer wavelength where the interference from the excipients is generally less

Present method

* CAT: Chloramine-T MBTH: 3-Methyl-2-benzothioazolinone-hydrazone hydrochloride NNDPD: N,N-dimethyl-p-phenylnediamine dihydrochloride DMAB.: Dimethylaminobenzaldehyde SNP: Sodium nitroprusside

The versatility of non-aqueous titrimetry and HPLC in pharmaceutical analysis particularly in industrial quality control is well known26. Visible spectrophotometry because of its simplicity,

sensitivity, speed, reliability and accuracy continues to be a widely used technique in drug quality control27. In this paper, three methods are proposed for the determination of TNZ both in pure form and in

BASAVAIAH et al.: DETERMINATION OF TINIDAZOLE BY POTENTIOMETRY, SPECTROPHOTOMETRY AND HPLC275

tablets and injections. In the titrimetric procedure, the drug solution in acetic acid is titrated with perchloric acid, the end point being determined potentiometrically. Spectrophotometry involves treating a fixed amount of perchloric acid-malachite green mixture with varying amounts of TNZ, and measuring the change in absorbance of the dye colour at 615 nm. The increase in absorbance is related to drug concentration. The HPLC analysis was carried out by injecting the drug solution onto a Hypersil ODS C18 column with the elution being effected by a mobile phase consisting of acetonitrite-0.1 % orthophosphoric acid (70:30) and UV detection at 316 nm. Experimental Procedure Reagents and Materials

All chemicals used were of analytical reagent grade. A 0.1 M perchloric acid was prepared by adding 4.5 mL of 70 % perchloric acid (s d Fine Chem, Mumbai) to 150 mL of glacial acetic acid (s d Fine Chem, Mumbai) and mixed well. To the mixture was added 10.5 mL of acetic anhydride and the solution was allowed to cool for 30 min; finally diluted to 500 mL with glacial acetic acid and allowed to stand overnight. This perchloric acid (~ 0.1 M) was diluted to get 0.01 M with glacial acetic acid and standardized with pure potassium biphthalate and crystal violet indicator. Acid dye mixture (10 mM HClO4-0.5 mM malachite green) was prepared by mixing 10 mL of 0.1 M HClO4 and 40 mL of 1000 μg mL-1 dye solution (prepared in glacial acetic acid) and diluting to 100 mL in a calibrated flask with glacial acetic acid. HPLC grade acetonitrile (RANKEM, India) acetic acid (s d Fine Chem, Mumbai), AR grade H3PO4 (Qualigens Fine Chem, India) and distilled water filtered through a 0.45 μM filter (Millipore) were used. A 0.1 % H3PO4 solution was prepared by diluting 1 mL of the acid to 1 L with distilled water and filtered through a 0.45 μM filter. The diluent solution was prepared by mixing acetonitrile and water in the ratio 60:40. The mobile phase used for chromatography consisted of acetonitrile-0.1% H3PO4 (70:30). Pharmaceutical grade TNZ was received as gift from Cipla India Ltd., Mumbai, and was used as such. A 1 mg mL-1 drug solution for use in titrimetry was prepared by dissolving 250 mg of pure TNZ in glacial acetic acid and diluting to 250 mL in calibrated flask. This solution (1000 μg mL-1) was diluted appropriately to get 300 μg mL-1 with glacial acetic

acid for use in spectrophotometric work. For chromatographic assay, a stock standard solution of TNZ (250 μg mL-1) was prepared in diluent. Apparatus

Potentiometric titration was performed with an Elico Model L1-120 digital pH meter provided with a combined glass-SCE system. The KCl of the salt bridge was replaced with 0.1 M lithium perchlorate solution in glacial acetic acid. All absorbance measurements were made with a Systronics Model 106 digital spectrophotometer provided with 1 cm matched quartz cells. The chromatographic system consisted of an Agilant 1100 series chromatograph equipped with an inbuilt solvent degasser, quartenary pump, photodiode array detector with variable injector and auto sampler and reversed phase 5 μm column (Hypersil ODS C18, 25 cm long and 4.6 mm id Thermosil). Potentiometric titration (Method A)

A 10 mL aliquot of standard drug solution equivalent to 1-10 mg of TNZ was pipetted out into a clean and dry beaker and the solution was diluted to about 30 mL by adding glacial acetic acid. A combined glass-SCE system was dipped in the solution. The content was stirred magnetically and the titrant (0.01 M HClO4) was added from a microburette. Near the equivalence point, the titrant was added in 0.1 mL increments. After each addition of titrant, the solution was stirred magnetically for 30 s and the steady potential was noted. The addition of titrant was continued until there was no significant change in potential on further addition of titrant. The equivalence point was determined by graphical Gran’s plot method. The amount of drug in the measured aliquot was calculated from: Amount(mg) =

VMwR 1.5

where V = volume of perchloric acid added, mL, MW = relative molecular mass of drug, R= strength of prechloric acid, moL L-1. Spectrophometric method (Method B)

Different aliquots (0.5-6.0 mL) of 300 μg mL-1 TNZ solution (standard) were accurately transferred into a series of 10 mL calibrated flasks each containing 1 mL of perchloric acid - malachite green (10 – 0.5 mM) mixture, and the volume was diluted to the mark with glacial acetic acid. The contents were

276


mixed well and absorbance was measured at 615 nm against a reagent blank. The increasing values of absorbance at 615 nm were plotted against the concentration of TNZ to obtain the calibration graph. The concentration of the unknown was read from the calibration graph or computed from the regression equation derived from Beer’s law data. Chromatographic assay (Method C) Chromatographic conditions

Chromatographic separation was achieved at ambient temperature on a reversed phase Hypersil ODS 5 μm C18 column using a mobile phase consisting of acetonitrile – 0.1 % phosphoric acid (pH 3.0) (70:30) at a flow rate of 1.0 mL min-1. The detector wavelength was set at 316 nm with a sensitivity of 0.2 a.u.f.s. Calibration graph

Working standard solutions containing 6.25 – 250.0 μg mL-1 TNZ were prepared by appropriate dilution of the stock solution with the diluent solution. Twenty μL aliquot of each solution was injected automatically onto the column in duplicate and the chromatograms were recorded. Calibration graph was constructed by plotting the mean peak area as a function of TNZ concentration. Analysis of formulations

Ten tablets were weighed and finely powdered. The contents of ten ampoules were emptied into a beaker and mixed. Methods A & B

An accurately weighed amount of the tablet powder equivalent to 100 mg of TNZ was transferred into a clean and dry 100 mL calibrated flask, 60 mL of glacial acetic acid was added and shaken thoroughly for about 20 min. Then, the volume was made up to the mark with glacial acetic acid, mixed well and filtered using Whatman No.42 filter paper. A suitable aliquot of the filtrate was subjected to analysis by titrimetry. The filtrate (1000 μg mL-1) was appropriately diluted to obtain 300 μg mL-1 solution with glacial acetic acid and a convenient aliquot was analyzed by spectrophotometry. Fifty mL of injection solution equivalent to 100 mg of TNZ was quantitatively transferred into a 250 mL separating funnel and extracted with three 20 mL portions of chloroform. The organic extract was dried over anhydrous sodium sulphate and the solvent from the pooled extract was evaporated over a water bath.

The residue was dissolved in glacial acetic acid and diluted to the mark in a 100 mL calibrated flask with the same solvent. The solution was then analyzed by titrimetric and spectrophotometric methods as described for tablets. Method C

An amount of the tablet powder equivalent to 25 mg of TNZ was accurately transferred into a 100 mL calibrated flask, 60 mL of diluent solution added and shaken thoroughly for about 20 min; the volume was diluted to the mark and mixed well. A small portion of this solution (10 mL) was withdrawn and filtered through a 0.2 μm filter to ensure the absence of particulate matter. The filtered solution was appropriately diluted to get the final solution. In the case of injectable product, 12.5 mL of injection solution was accurately transferred into a 50 mL calibrated flask and diluted to the mark with the diluent solution and filtered through a 0.45 μm filter. The filtered solution was then analyzed after suitable dilution. Results and Discussion Titrimetry and spectrophotometry

The methods make use of the basic property of the TNZ molecule and are based on the fact that substances which are too weakly basic in aqueous solution exhibit enhanced basic property in nonaqueous solvents thus allowing their easy determination. In the present titrimetric method, the weakly basic character of TNZ was enhanced due to the non-levelling effect of glacial acetic acid, and the drug was titrated with acetous perchloric acid with potentiometric end point detection. A steep rise in the potential (96 mV per 0.1 mL titrant) was observed at the equivalent point (Fig. 2). The Gran’s plot method

Fig. 2 - Potentiometric titration curve & Gran’s plot


was used to ascertain the equivalence point. A reaction stoichiometry of 1:1.5 (drug : titrant) was obtained which served as the basis for quantification. The relationship between the drug amount and titration end point was examined. The linearity between the two is apparent from the correlation coefficient of 0.9964 obtained by the method of least squares. From this it is implied that the reaction between TNZ and perchloric acid proceeds stoichiometrically in the ratio 1:1.5 in the range studied (1-10 mg). Malachite green (C.I. 42000) is a dye exhibiting blue colour in the base form and yellow colour in the acid form. The spectrophotometric method is based on the facts that the colour of the dye is dependent on the pH of the solution and that the colour change is not sudden but occurs continuously as the pH changes over a definite range. When different amounts of TNZ are added to a fixed amount of perchloric acid malachite green mixture where the dye is in the acid form (yellow colour) the colour changes from yellow to blue, and the absorbance of the solution increases at 615 nm. This is caused by the progressive increase in the pH of the solution because of the neutralization of the acid (HClO4) of the mixture by the added TNZ (base) in increasing amounts. This is shown by the proportional increase in the absorbance of the solution at 615 nm (Fig. 3) which is corroborated by the correlation coefficient of 0.9872. In a preliminary study, 40 μg mL-1 malachite green in the base form was found to show a convenient maximum absorbance at 615 nm. In the presence of 1 mL of 10 mM perchloric acid and in a total volume of 10 mL, this absorbance decreased to a constant minimum. Hence, different amounts of TNZ were treated with a fixed amount of acid dye mixture i.e., 1 mL of 10 mM HClO4 – 0.5 mM malachite green (400 μg mL-1 w.r.t. malachite green), to determine the concentration range over which the method is applicable. The dye colour was found to be stable for several hours in the presence of drug, and the order of addition of reactants was not critical. The increasing absorbance values at 615 nm were plotted against concentration of TNZ to obtain the calibration graph. Beer’s law is obeyed over the concentration range 15-180 μg mL-1, the equation of the line being A = 1 × 10-3 + 2.6 × 10-3 C, where A is the absorbance and C concentration in μg mL-1. The correlation coefficient of the calibration

Fig. 3 – Beer’s law Curve

plot was calculated to be 0.9872 (n=7) confirming a linear increase in absorbance with increasing concentration of TNZ. The calculated molar absorptivity was 9.02 × 102 L moL-1 cm-1 and the Sandell sensitivity was 385.2 ng cm-2. The limits of detection and quantification were calculated from the standard deviation of the absorbance measurements obtained from a series of seven blank solutions. The limits of detection and quantification established according to IUPAC definitions28 were 3.05 and 10.15 μg mL-1, respectively. HPLC

TNZ was also determined by HPLC. A solution of TNZ was injected onto a reversed phase ODS column at 316 nm. The composition and the pH of the mobile phase were varied to optimize the chromatographic conditions. A pH 3 mobile phase consisting of acetonitrile – 0.1 % H3PO4 (70:30) was used. Acetonitrile and phosphoric acid increase the solubility of TNZ and prevent its adherence to the packing material in the column. At a flow rate of 1.0 mL min-1, the retention time for TNZ was 2.8 min (Fig. 4). Under the described experimental conditions the analyte peaks were well defined and free from tailing. TNZ was determined by measuring the peak area. A plot of peak area against concentration gave a linear relationship (r=0.9999) over the concentration range 6.25 – 250.0 μg mL-1. Using regression analysis the linear equation, Y = 24.31+ 40.02 X was obtained, where Y is mean peak area and X concentration in μg mL-1. The limit of detection and the limit of quantification were 0.625 and 1.875 μg mL-1, respectively.

278


Precision of the methods

The intra-day precision of the methods was determined by replicate analysis of the standard solution containing TNZ at three different levels, and the results for methods A and B are presented in Table 3. For HPLC method, the relative standard deviation (RSD) values for both peak area and retention time were calculated and are given in Table 4. The inter-day precision was established by performing analyses over a five day period on solutions prepared freshly each day. The RSD values were no more than 2.8 and 3.2 % for methods A and B, respectively. The peak area based and retention time based inter-day RSD values did not exceed 5.6 % for HPLC method.

Fig. 4 - Chromatogram of 100 μg mL-1 tinidazole under optimum conditions

Accuracy of the methods

The accuracy of the developed methods was evaluated by carrying out recovery experiments on known amounts of standard TNZ solution. The % relative error, which is a measure of accuracy compiled in Tables 3 and 4, indicates the excellent accuracy of the methods. Application

The proposed methods were successfully applied for the determining TNZ in tablets and injections, and the results are summarized in Table 5. The same batch tablets and injections were analyzed by reference methods1,17 for comparison. The results obtained by the proposed methods agreed well with those of the reference methods and with the label claim. The results were statistically compared using Student’s tand F-tests. As shown in Table 5, the calculated t- and F-values were less than the theoretical values, indicating that the proposed methods have the same accuracy and precision. Accuracy and reliability of the methods were further established by performing recovery experiments. The pre-analyzed tablets and injections were spiked with pure drug at three different levels and the total was found by the proposed methods. Each determination was repeated three times. The recoveries of the pure drug added were in the range 95.48 – 106.26 % indicating that commonly added excipients, additives and diluents in formulations did not interfere in the determination. Conclusion Tinidazole has been determined in bulk drug and in pharmaceutical products by employing three different

Table 3⎯Accuracy and precision data of titrimetric and spectrophotometric methods Titrimetry Drug taken, mg

Drug found, mg

Relative error, %

RSD % (n=3)

Range of error, %

2.0

2.03

1.50

1.81

± 4.49

3.0

3.07

2.33

1.03

± 2.56

7.0

6.82

2.57

1.22

± 3.03

Spectrophotometry Drug taken, μg mL-1

Drug found, μg mL-1

Relative error, %

RSD % (n=7)

Range of error, %

30.00

29.45

1.83

1.36

± 1.19

90.00

91.06

1.18

1.43

± 1.26

150.00

148.23

1.18

0.93

± 0.82

RSD-Relative standard deviation Table 4 ___ Accuracy and precision data of HPLC method Drug taken, μg mL-1

Drug found, μg mL-1

Relative error, %

RSD*% (n=7)

RSD** % (n=7)

25.00

24.28

2.88

0.78

1.23

100.00

96.54

3.46

1.26

0.85

170.00

168.72

0.75

0.86

2.12

* Based on peak area ** Based on retention time


Table 5⎯Results of assay of formulations by the proposed methods Formulation and brand name* Tina a

Method A 300

500

1000

Tinipidib Injecttions

Foundψ(% recovery of nominal amount ± SD)

Label claim mg/tablet or /mL

2

Method B

Method C

101.18 ± 1.58

99.36 ± 0.65

101.08 ± 1.46

t=1.42 F=1.31

t=1.12 F=4.50

t=2.71 F=1.19

99.68 ± 1.04

100.34 ± 0.58

102.38± 1.03

t=2.27 F=1.82

t=2.06 F=2.19

t=2.11 F=1.74

100.86 ± 0.72

101.12 ± 0.54

99.48 ± 0.68

t=1.15 F=2.51

t=1.95 F=4.46

t=1.04 F=2.81

100.63 ± 0.92

101.13 ± 1.06

101.14 ± 1.78

t=1.10 F=0.00

t=1.97 F=1.23

t=1.84 F=1.78

Reference**method 98. 64 ± 1.38

101.18 ± 0.77

100.08 ± 1.14

99.89 ± 0.92

ψ

Mean value of three determinations in method A & five determinations in methods B &C ∗ Marketed by (a) Bombay Tablets Mfg. Co., India, (b) Parenteral Drugs, India **Tablets were analysed by the method cited in Ref. 1 and injections were analysed by the method cited in Ref.17.

techniques. The methods offer the advantages of simplicity, speed and convenience since they do not require special working conditions unlike many other reported methods. The titrimetric and spectrophotometric methods are characterized by the absence of experimental variables that normally affect the results and this is aptly demonstrated by the high accuracy and precision of the results. The titrimetric method is applicable over a micro-scale (1-10 mg) and the spectrophotometric method is superior to many methods reported earlier in terms of simplicity and specificity. The reaction employed in the spectrophotometric method is instantaneous and results in a stable coloured besides using an inexpensive reagent. The determination can be performed at room temperature. The absorbance measurement is made at 615 nm where the interference from the associated inactive ingredients is generally far less at longer wavelength than at shorter wavelengths used in most of the reported methods including the pharmacopoeial method. The HPLC method does not require extensive sample treatment and involves a HPLC system employing an inexpensive mobile phase. The assay procedure is applicable over a wide dynamic range compared to many proposed earlier and is more sensitive than several existing HPLC procedures. There was no interference from matrix sources.

Acknowledgement The authors are thankful to the Quality Control Manager, Cipla India Ltd, Mumbai, for the supply of pure tinidazole as gift. The authors (PN & UC) thank the authorities of the University of Mysore, Mysore, for facilities. References 1 The India Pharmacopoeia, Controller of Publications, Ministry of Health and Family Welfare, Govt. of India, New Delhi, 1996, 764, 765. 2 Sadana G S & Gaonkar M V, Indian Drugs, 25 (1987) 121. 3 Patel Y, Dhorda U J, Sundaresan M & Bhagawat H M, Anal Chim Acta, 362 (1998) 271. 4 Fenz M X, Gao H & Yu F Y, Yaowu Fenxi Zazhi, 17 (1997) 247. 5 Nadakarni D R, Merehan R N, Sundaresan & Bhagwat A M, Indian Drugs, 34 (1997) 393. 6 Ku Y R, Tsai M J & Wen K C, Yaowu Shipin Fenxi, 4 (1996) 141. 7 Talwar N, Karajgi J S & Jain N K, Indian Drugs, 29 (1991) 55. 8 Ray S, East Pharm, 32(38) (1989) 125. 9 Menouer M, Guermouche S & Guermouche M H, J Pham Belg, 42 (1987) 243. 10 Warowna M, Fijalek Z, Dziekanska A & Koorzaniewska A, Acta Pol Pharm, 48 (1991) 17. 11 Ozkan S A, Analysis, 25 (1997) 130. 12 Joshi D M & Joshi A P, Indian Drugs, 33 (1996) 338. 13 Sanghavi N M, Joshi N G & Dubal K S, Indian Drugs, 18 (1981) 354. 14 Lopez Martinez L, Luna Vazquez F J & Lopez de Alba P L, Anal Chim Acta, 340 (1997) 241.

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