ENCLOSURE-I

6. BRIEF RESUME OF INTENDED WORK

6.1 Need of the study

Numerous methods are available for estimation of anti-HIV drugs, For example sophisticated analytical methods like HPLC and UV which are being employed for analysis, and hence we will try to develop new methods for estimation of anti-HIV drugs.

The methods adopted will be simple, accurate, reproducible, selective and sensitive methods and can be used for routine quality control analysis of anti-HIV drugs.

These methods are less time consuming and easy to perform when compared to other methods. These methods will be tested and validated for various parameters according to

ICH guidelines. The results are reproduced so that is accurate, precise, specific and reproducible while being simple and less time consuming and hence can be suitably applied for the estimation of anti-HIV drugs in different dosage forms. Statistical analysis will be performed for reproducibility and selectivity for the estimation of anti-HIV drugs.

In the present investigation few simple, economic, reproducible spectroscopic methods were developed for the estimation of an anti-HIV drug abacavir in bulk and formulations along with statistical interpretations. ENCLOSURE- II

6.2 Review of literature

Pant et al1 developed simple spectroscopic method for routine determination of acyclovir in pharmaceutical formulations and bulk dosage forms. The method is based on the formation of colored species via reaction of the enzyme HRP

(horseradish peroxidase) with ferric chloride and 2,2ʹ-bipyridine in presence of orthophosphoric acid and finally the drug to be estimated namely acyclovir to produce a

pink colored chromogen (λmax at 400 nm). Statistical analysis of method exhibited a molar absorptivity value of 105 L/mol/cm, and Sandell’s sensitivity of 0.001912. Relative standard deviation (RSD) of the method was found to be 0.0386% indicating that the method is reproducible, based on the principle of absorption visible spectrophotometry for the determination of acyclovir in pharmaceutical formulations. The method is reliable and can be utilized for the determination of acyclovir in various pharmaceutical formulations and bulk dosage forms.

Venkata et al 2 developed a simple and reproducible spectrophotometric method for the determination of two recent antiviral drugs namely valacyclovir (drug A) and famciclovir (drug B) in bulk and pharmaceutical dosage forms. The method is based on the extraction of the drugs into organic layer of the dye tropaeolin ooo in presence of

0.1 N hydrochloric acid and the absorbances were measured at 490 nm. The method was optimized using eight parameters.

Devyani et al3 developed a novel, simple, rapid and sensitive spectrophotometric method for simultaneous estimation of lamivudine and silymarin. The method employed was formation and solving of simultaneous equation using 270.9 nm and 326.4 nm as two analytical wavelengths. Both the drugs obey Beer’s Law in the concentration ranges employed for this method. Accuracy and reproducibility of the proposed method was statistically validated by recovery studies. The method is found to be rapid, precise and accurate and can easily be employed in the laboratory for the routine estimation of drugs.

Shalini et al 4 also developed two new, simple, cost effective and sensitive spectrophotometric methods (A and B) for the determination of lamivudine in dosage and bulk forms. Lamivudine was estimated at 279.6 nm in 0.1 N HCl by method A and at

269.8 nm in 0.1 N NaOH by method B. In both the methods linearity was found to be in the range of 0-6 μg/ml for method A and 0-10 μg/ml for method B. The proposed methods were successfully applied for the determination of lamivudine in pharmaceutical formulations. The results demonstrated that the procedure is accurate, precise and reproducible (relative standard deviation <1%), while being simple, cheap and less time consuming and can be suitably applied for the estimation of lamivudine in different dosage forms.

Abd et al5 developed a simple chemometrics-assisted spectrophotometric method for the simultaneous determination of lamivudine and stavudine in pharmaceutical tablets. Beer’s law was obeyed for both drugs in the general concentration ranges of 2-12 and 3-15 μg/ml for lamivudine and stavudine respectively.

The proposed methods were successfully applied for the determination of the two drugs in laboratory prepared mixtures. Good percentage recoveries and proper statistical data obtained with both the laboratory prepared mixtures and the commercial tablets proved the suitability and efficiency of the proposed procedures for routine analysis and quality control purposes with quite satisfactory precision. The obtained F- and t-values indicate no significant differences between the results of the proposed and reported methods.

Basavaiah et al 6 developed four sensitive and rapid methods for the determination of stavudine (STV) in bulk drug and in dosage forms. In titrimetry, aqueous solution of stavudine was treated with a known excess of bromate-bromide in

HCl medium followed by estimation of unreacted bromine by iodometric back titration.

Spectrophotometric methods involve the addition of a measured excess of bromate- bromide in HCl medium and subsequent estimation of the residual bromine by reacting with a fixed amount of methyl orange, indigo carmine or thymol blue followed by measurement of absorbance at 520 nm (method A), 610 nm (method B) or 550 nm

(method C). In all the methods, the amount abrogate reacted corresponds to the amount of stavudine. Calculations in titrimetry were based on a 1:0.666 (STV: KBrO3) stoichiometry and the method was found to be applicable over 3.5-10 mg range. A linear increase in absorbance with concentration of stavudine was observed in the spectrophotometric methods, and the Beer’s law was obeyed over the concentration ranges 0.125-1.75, 1-10 and 1-9.0µg/ml stavudine for method A, method B and method

C, respectively. The methods when applied to the determination of stavudine in tablets and capsules were found to give satisfactory results.

Basavaiah et al7 also developed three new methods which described the assay of stavudine (STV) in bulk drug and in dosage forms using chloramine-T (CAT) and two dyes, methyl orange and indigo carmine, as reagents. Titrimetry involves treating

STV with a measured excess of CAT in hydrochloric acid medium, and after the oxidation of STV is judged to be complete, the unreacted oxidant is determined iodometrically. Spectrophotometric methods entail the addition of a known excess of

CAT to STV in hydrochloric acid medium followed by determination of residual oxidant by reacting with a fixed amount of either methyl orange and measuring the absorbance at

520 nm (Method A) or indigo carmine and measuring the absorbance at 610 nm (Method

B). In all the methods, the amount of CAT reacted corresponds to the amount of STV. In titrimetric method, there action follows 1:1 stoichiometry (STV: CAT), and is applicable over the range 1.5-10 mg of STV. In spectrophotometric methods, the absorbance is found to increase linearly with concentration of STV. The system obeys Beer’s law for

0.2-2.0 and 1.0-10.0 μg/ml for method A and method B, respectively. The apparent molar absorptivities are calculated to be 5.7x104 and 1.5x104 L/mol/cm for method A and method B, respectively, and the corresponding Sandell`s sensitivity values are 0.004 and

0.015 µg/cm-1. The methods were successfully applied to the assay of STV in tablet and capsule formulations and the results were compared with those of a reference method by applying Student’s t-test and F-test.

Gnana et al 8 developed two simple and sensitive spectrophotometric methods for the estimation of lamivudine in both pure and tablet dosage form. Methods A and B are based on the condensation reaction of lamivudine with carbonyl reagents such as p-dimethylaminobenzaldehyde (PDAB) and vanillin in acidic condition to form yellow colored chromogen with absorption maxima at 476 nm and 474 nm respectively. Beer’s law is valid in the concentration range of 2-10 μg/ml. This method was validated for precision, accuracy, ruggedness and robustness. Statistical analysis proves that the method is reproducible and selective for the estimation of the said drug.

Preethi et al 9 developed a simple, sensitive, rapid, accurate and precise spectrophotometric method for estimation of acyclovir in bulk and pharmaceutical dosage forms. Acyclovir shows maximum absorbance at 253nm with molar absorptivity of

1.3733×104 l/mulch Beer’s law was obeyed in the concentration range of 2-20 µg/ml.

Results of the analysis were validated statistically and by recovery studies.

Basavaiah et al 10 developed a simple, sensitive and rapid, titrimetric and two spectrophotometric methods for the assay of lamivudine in bulk drug and in tablet dosage forms using potassium iodate and two dyes, methyl orange and indigo carmine, as reagents. In titrimetry, an aqueous solution of lamivudine is titrated directly with iodate in an acidic medium, and in the presence of an excess of bromide using methyl orange as an indicator. After the decoloration of the red color of methyl orange, the residual bromine is titrated iodometrically to a starch end point. Spectrophotometric methods involve the addition of a known excess of iodate in an acidic medium and in the presence of an excess of bromide followed by the determination of residual bromine by the reaction with a fixed amount of either methyl orange and measuring the absorbance at

520 nm (method A), or indigo carmine and measuring the absorbance at 610 nm (method

B). The calculated apparent molar absorptivity values are found to be 3.3 x 104 and 9.3 x

103L/mol/cm, for method A and method B, respectively, and the corresponding Sandell`s sensitivity values are 6.94 and 24.62 ng/cm2. The methods were successfully applied to the assay of lamivudine in tablet form and the results were compared with those of a reference method by applying the Student’s t-test and F-test.

Devmurari et al 11 developed a novel, simple, rapid and sensitive spectrophotometer method for simultaneous estimation of lamivudine and abacavir. The method employs formation and solving of simultaneous equation using 280 nm and 297 nm as two analytical wavelengths. Both the drugs obey Beer’s Law in the concentration ranges employed for this method. Accuracy and reproducibility of the proposed method was statistically validated by recovery studies. The method is found to be rapid, precise and accurate and can easily be employed in the laboratory for the routine estimation of drugs.

Sudha et al 12 developed and validated simple, precise, accurate and rapid high performance thin layer chromatographic method for the simultaneous estimation of lamivudine and abacavir sulphate in combined dosage forms. The detection of spot was carried out at 265 nm. The method was validated in terms of linearity, accuracy, precision and specificity. The calibration curve was found to be linear between 500 to 3000 ng with regression coefficient of 0.9998. The proposed method can be successfully used to determine the drug content of marketed formulation.

Appala et al 13 developed two simple, accurate, rapid and sensitive methods (A and B) for the estimation of abacavir sulphate in its pharmaceutical dosage form. The method A and B are based on the formation of chloroform extractable complex of abacavir sulphate with bromophenol blue (method A) and bromocresol green (method

B), which shows absorbance maxima at 460 nm and 469 nm respectively. The absorbance-concentration plot is linear over the range of 1-10 mcg/ml for method A and

B respectively. Results of analysis for all the methods were validated statistically and by recovery studies. The proposed methods are economical and sensitive for the estimation of abacavir sulphate in bulk drug and in its tablet dosage form.

Verweij et al14 developed a reversed phase high performance liquid chromatography method for the simultaneous quantitative determination of the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine, didanosine, stavudine, zidovudine and abacavir in plasma. The method involved solid-phase extraction with

Oasis MAX cartridges from plasma, followed by high performance liquid chromatography with a Symmetry Shield RP 18 column and ultraviolet detection set at a wavelength of 260 nm. The assay was validated over the concentration range of 0.015-5 mg/l for all five NRTIs. The average accuracy for the assay were 92-102%, inter- and intra-day coefficients of variation (CV) were <2.5% and extraction recoveries were higher than 97%. This method proved to be simple, accurate and precise, and is currently in use in our laboratory for the quantitative analysis of NRTIs in plasma.

Rolf et al 15 reported combined bio-analytical assay for abacavir, a reversed transcriptase inhibitor, and mycophenolic acid (MPA), based on reversed-phase liquid chromatography and both ultraviolet (UV) absorption and fluorescence detection.

Both analytes are extracted from plasma with acetonitrile. After centrifugation, evaporation of the supernatant and reconstitution in water, the sample is injected into the chromatograph. Abacavir is detected using UV detection at 285 nm and MPA spectrofluorometrically at 345 and 430 nm for excitation and emission, respectively. The method has been validated in the 80-2000 ng/ml range for abacavir and in the 10-10,000 ng/ml range for MPA for 200 ml plasma samples. The lower limits of quantification are

80 and 10 ng/ml for abacavir and MPA, respectively. Precisions and accuracies are ≤8% in the valid concentration ranges of both analytes.

Veldkampa et al 16 described a simple and rapid high-performance liquid chromatographic method for the quantification of abacavir in human plasma. The method has been validated over the range of 20-2000 ng/ml using a volume of 300 ml of plasma.

The assay is linear over this concentration range as indicated by the F-test for lack-of-fit.

Within- and between-day precisions are less than 7.5 % for all quality control samples.

The lower limit of quantitation is 20ng/ml and the recovery of abacavir is 88.1 % (61.3

%). This validated assay is suited for use in pharmacokinetic studies with abacavir in human plasma and can readily be implemented in the setting of a hospital laboratory for the monitoring of abacavir concentrations. ENCLOSURE – III

6.3 Objective of the study

The present work is planned with the following objectives.

1. To develop methods for spectrophotometric estimation of Abacavir.

2. To estimate anti-HIV drug abacavir by UV and HPLC.

3. To validate UV and HPLC methods with statistical interpretation.

ENCLOSURE – IV

7. MATERIALS AND METHODS

7.1 Source of data

The primary data will be collected by performing various tests and investigations in the laboratory. The secondary data will be collected by referring various National and

International journals, Books, Pharmacopeia’s and Websites.

The day to day development in the area will be updated by literature survey through e-publishing, internet and current periodicals in our library and elsewhere.

All the basic facilities required for the estimation of anti-HIV drugs are available in our college laboratories. ENCLOSURE – V

7.2 Method of collection of data

The data is planned to collect from laboratory experiments which includes,

1. Instruments like UV Spectrophotometer, Colorimeter and HPLC instruments will

be used to collect above data.

2. Titrimetric methods will be used to collect above data. ENCLOSURE – VI

8. REFERENCES

1. Pant P, Saradhi SV, Felice CS, Gurung B, Divya VG, Rao NM. Spectrophotometric

determination of acyclovir through oxidative coupling of with 2, 2ʹ-bipyridine by

horsradish peroxidase (HRP). J Appl Chem Res 2009; 10: 7-12.

2. Venkata Reddy P, Sudha RB. Estimation of some antiviral drugs using tpooo as

analytical reagent. E J Chem 2006; 3: 154-8.

3. Devyani D, Vyas SP. Simultaneous Spectrophotometric Estimation of Lamivudine and

Silymarin in mixture. Int J Pharm Pharm Sci 2009; 1: 107-11.

4. Shalini S, Shanooja VP, Abdul JS, Basima, Harilal KK, Harish R et al. Application of

UV-spectrophotometric methods for estimation of lamivudine in tablets. Dig J

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5. Abd El-Maaboud I Mohamed, Workalemahu M. Determination of lamivudine and

stavudine in pharmaceutical preparations using chemometrics-assisted

spectrophotometry. Saudi Pharm J 2009; 17: 286-94.

6. Basavaiah K, Ramakrishna V, Somashekar C, Anil Kumar UR. Sensitive and rapid

titrimetric and spectrophotometric methods for the determination of stavudine in

pharmaceuticals using bromate-bromide and three dyes. Ann Braz Acad Sci 2008; 2:

253-62.

7. Basavaiah K, Ramakrishna V, Anil Kumar UR. Use of chloramine-T and two dyes in

the sensitive determination of stavudine in pharmaceuticals. Braz J Pharm Sci 2007; 43:

471-9. 8. Gnana babu CJ, Vijaya kumar G. Validated spectrophotometric estimation of

lamivudine in pure and tablet dosage form. Int J Chem Tech Res 2009; 1: 1372-5.

9. Preethi G, Nilofar M, Sudha K, Konapure NP, Kuchekar BS. Spectrophotometric

estimation of acyclovir in pharmaceutical dosage forms. Indian J Pharm Sci 2006; 68:

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spectrophotometric assay methods for the determination of lamivudine in

pharmaceuticals using iodate and two dyes. J Anal Chem 2007; 62: 542-8.

11. Devmurari VP, Patel RB. Simultaneous spectrophotometric determination of

lamivudine and abacavir in the mixture. Int J Pharm Sci Res 2010; 1: 82-6.

12. Sudha T, Ravikumar VR, Hemalatha PV. Validated HPTLC method for simultaneous

determination of lamivudine and abacavir sulphate in tablet dosage form. Int J Pharm

Sci Res 2010; 1: 107-11.

13. Appala Raju N, Venkateswara Rao J, Vanitha Prakash, Mukkanti K.

Spectrophotometric estimation of abacavir sulphate in pharmaceutical formulations. E J

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14. Verweij-van Wissena CPWGM, Aarnoutsea RE, Burgera DM. Simultaneous

determination of the HIV nucleoside analogue reverse transcriptase inhibitors

lamivudine, didanosine, stavudine, zidovudine and abacavir in human plasma by

reversed phase high performance liquid chromatography. J Chromatogr B 2005; 816:

121-9. 15. Rolf WS, Richard MWH, Jos HB. Liquid chromatographic assay for simultaneous

determination of abacavir and mycophenolic acid in human plasma using dual

spectrophotometric detection. J Chromatogr B 2001; 750: 155-61.

16. Veldkampa AI, Sparidansa RW, Hoetelmansa RMW, Beijnena JH. Quantitative

determination of abacavir (1592U89), a novel nucleoside reverse transcriptase inhibitor,

in human plasma using isocratic reversed-phase high-performance liquid

chromatography with ultraviolet detection. J Chromatogr B 1999; 736: 123-8.