Quantitative Determination of HIV-Antiviral Agent Using Complexing Reagent in Bulk Material and Formulations

Thuttagunta Manikya Sastry¹*, Karripeddi Ramakrishna² Kotturu Venkata Lavanya³ and Kallempudi Siva Iva Prasad⁴

¹Department of Chemistry, G V P College of Engineering (Autonomous), Madhurwada, Visakhapatnam-530048, A.P, India ²Department of Chemistry, GITAM University, Rushikonda, Visakhapatnam-530045, A.P, India

³Department of Humanities and Basic Sciences, Chaitanya Engineering College, Kommadi Junction, Visakhapatnam -530048, A.P, India

⁴Department of Basic Sciences and Humanities, Vignan’s Institute of Information Technology, Duvvada, Visakhapatnam - 530 049, India.

DOI : http://dx.doi.org/10.13005/ojc/300144

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Article Published : 05 Mar 2014

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ABSTRACT:

A sensitive visible spectrophotometric method was developed for quantitative determination of Antiviral [Abacavir sulphate (AVS)] agent against HIV in bulk material and dosage forms. An extraction spectrophotometric method for the assay of abacavir sulphate is based on the formation of colored co-ordination complex with cobalt thiocyanate (CTC) which can be extractable in nitrobenzene (l_max=620 nm) is described. Beer's law limits and Molar absorptivity values were found to be 10 – 50 mg mL^-1 and 9.58 x 10³ L mol^-1cm^-1 respectively. The limit of detection and limit of quantification values are found to be 3.07 x 10^-1 µg mL^-1 and 9.3 x 10^-1 µg mL^-1 respectively. Precision and accuracy of the developed method was evaluated. The results are validated statistically and compared with reported methods.

KEYWORDS:

Spectrophotometry; Abacavir sulphate; Ziagen Nitrobenzene; Cobalt thiocyanate (CTC) and dosage forms

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Copy the following to cite this article: Sastry M. T, Ramakrishna K, Lavanya K. V, Prasad K.S.I . Quantitative Determination of HIV-Antiviral Agent Using Complexing Reagent in Bulk Material and Formulations. Orient J Chem 2014;30(1)

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Introduction

Abacavir is an antiviral agent which is carbocyclic synthetic nucleoside analogue with inhibitory activity against HIV. The chemical name of Abacavir sulphate is: [(1S, 4R) – 4 – [2 – Amino – 6 – (cyclopropylamino) – 9H – purin – 9 – yl] – 2 -cyclopentene-1-methanol sulphate (salt) (2:1)] (Fig. 1). In literature, a number of analytical methods have been described for estimation of AVS including Electrochemical determination¹, HPLC ^2,3, LC^4,5 and LC-MS^6,7, and UV ^8,9  methods. But, relatively little attention has been paid in developing visible spectrophotometric methods^10-13.The analytically important functional groups in AVS have not been exploited properly so far and hence there is a need to develop sensitive and flexible visible spectrophotometric methods. So authors have made some attempts in this direction and succeeded in developing more accurate, reliable and economical analytical method using cobalt thocyanate (CTC) for quantitative determination of AVS in bulk material and formulations.

EXPERIMENTAL PROCEDURE

Instrumentation
 The measurements were made on a SL-177 model (Elico, India) visible spectrophotometer with 1 cm glass cells and on a UNICAM UV 500 spectrophotometer (Thermo Electron Corporation, UK). All pH measurements were made using a LI 120 digital pH meter (Elico, India).

Reagents and materials

All the reagents were of analytical grade and all solutions were prepared in double distilled water. Aqueous solutions of CTC (BDH; 2.5 x 10^-1M), buffer solution (P^H=2.0). and Nitrobenzene (Qualigens) solvent are used. The bulk drug Abacavir sulphate (Ranbaxy laboratories Ltd., India) was selected for the study. Formulation, Ziagen (Glaxo- welcome Inc., India) containing Abacavir sulphate was purchased from local commercial sources. Tablets equivalent to 300 mg of different batches were selected for this study.

Standard drug solution

The free base solution (mg mL^-1) was prepared by mixing 100 mg of bulk drug with 10.0 ml of 10% Na₂CO₃ solution.  The resulting solution was taken into a 150 ml separating funnel and extracted with 3 x 25 mL portions of chloroform and combined chloroform layer was brought up to 100 mL with the same solvent. A portion of the above free base stock (mg mL^-1) solution was further diluted stepwise with the same solvent to obtain working standard solution of 100 mg mL^-1

Analytical procedure

Aliquots of standard drug (free base form) solution (1.0 – 5.0 mL; 100 mg mL^-1), were delivered into series of calibrated tubes and solvent was completely removed by gentle heating on a boiling water bath. To the residue in each tube, 2.0 ml of buffer (pH 2.0) and 5.0 mL of 2.5 x 10^-1M CTC solutions were added.  The total volume of aqueous phase in each separating funnel was adjusted to 15.0 mL with distilled water.  These solutions in the test tubes were transferred to 125 mL separating funnels. To each separating funnel 10.0 mL of nitrobenzene was added and the contents were shaken for 2 min.  The two phases were allowed to separate and the absorbances of the separated nitrobenzene layer were measured at 620nm against a similar reagent blank after 10 min. The amount of drug (AVS) was deduced from its calibration curve. (Figure 2)

Fig.1:  Structure of Abacavir Sulphate Click here to View Figure

Pharmaceutical formulations

Since only one formulation is available (Tablets), four different batches of this formulation were collected and analyzed as 4 sets to verify the validity of proposed methods. Accurately weighed quantity of tablet powder equivalent to 100 mg of AVS was extracted with methanol (3 x 25.0 mL portions) and filtered.  The volume of combined extract was evaporated to dryness. The residue was used to prepare free base stock solution (mg mL^-1) and working standard solution of concentration 100 mg mL^-1 as described in the standard drug solution. UV spectrophotometric method which was suggested for the identification of drug has been moulded for its assay and chosen as the reference method for ascertaining the accuracy of the proposed method.

RESULTS AND DISCUSSION

Optimization of reaction conditions

Optimum conditions for the proposed method were established by varying one parameter at a time and keeping the others fixed and observing the effect produced on the absorbance of the colored species. The effects of various parameters such as buffer solution, concentration of the CTC, organic solvent used for extraction, ratio of organic phase to aqueous phase during extraction, stability period and intensity of colored species were studied.The optimum conditions were as follows: 4.5-6.5 mL (1.1 – 1.6 × 10 ^-1 mol L^-1) of CTC solution, 1.5-2.5 mL (1.5 -2.5 × 10 ^-2 mol L^-1) of buffer solution (P^H2.0), 1-4 min. of shaking time ,the ratio of aqueous to organic phase on extraction is 3:2  were found to be optimal. In the procedure, 5.0 mL of (1.3 × 10 ^-3 mol L^-1) CTC, 2.0 mL (2.0 × 10 ^-2 mol L^-1)   buffer solution and 2 min. shaking period required for maximum color development were found to be optimum conditions. Nitrobenzene was preferred for its selective extraction of the complex from the aqueous phase. The ratio of aqueous to organic phase on extraction was taken as 3:2. The colored species were stable for 40 min. The λ_max (nm) and ε_max(Lmol^-1cm^-1) values were found to be 620 and  9.589 ´ 10³ .

Fig. 2: Beer’s law plot of AVS-CTC method  Click here to View Figure

Absorption spectrum

For the selection of analytical wavelength, 100 mg.ml^-1 solution of abacavir sulphate was prepared by appropriate dilution of standard stock solution and scanned in the spectrum mode from 800nm – 400 nm. The spectrum of the colored species produced by the suggested procedure is shown to possess maximum absorbance at 620nm which was selected for the analysis (Fig.2). The calibration curve was prepared in the concentration range of 10-50 mg mL^-1 at 620nm. By using the calibration curve (Fig.3), the concentration of the sample solution can be determined. The linearity was found in the concentration range of 10-50 mg mL^-1 as shown in the Figure 2

Fig. 3: Absorption spectrum of AVS – CTC method Click here to View Figure

Mechanism of Reaction

The coloured species formed can be regarded as a co-ordinate complex of the drug (electron donor) and the central metal atom of cobalt thiocyanate (electron acceptor) which is extractable into nitrobenzene from aqueous solution Formation of the blue coloured complex when AVS is treated with CTC due to the presence of aliphatic secondary group is the basis in the present investigation. It was observed that the drug (AVS), Cobalt and thiocyanate were in the ratio of 2:1:4 in the complex. The probable sequence of reactions based on analogy in previous work^{14, 15} is presented in Scheme.

Scheme- Extraction of co-ordination complex in nitrobenzene for abacavir sulphate  Click here to View Scheme

Method of validation

The developed method was validated as per ICH guidelines¹⁶ for its linearity, precision, and accuracy, limit of detection and limit of quantification. Regression analysis using the method of least square was made to evaluate the slope (b), intercept (a), and correlation coefficient(R) obtained from different concentrations of drug. The result of slope (1.43 x 10^-2) and intercept (1.2 x 10^-3) of drug by the proposed method was given in Table 2.

 Table-2 Regression parameters of proposed method

Linearity

Linearity was found in the concentration range 10–50 µg mL^–1. Beer’s law plots (n = 6) were linear with a correlation coefficient of 0.9999 (Table 2)

Limit of detection (LOD) and limit of quantification (LOQ)

Limit of detection (LOD) and limit of quantification (LOQ) were established according to ICH guidelines and determined by using the formula LOD = K.SDa / b where K= 3.3 for LOD and and 10 for LOQ. SDa is the standard deviation of the intercept and b is the slope of the calibration line.LOD and LOQ were found to be as low as 3.07 x 10^-1 µg mL^-1 and 9.3 x 10^-1 µg mL^-1 respectively. The results are presented in

Table-1 Optical characteristics of the proposed method 

Precision        

The repeatability of the proposed method was studied by repeating the method six times (n = 6).To study intra-day precision, the method was repeated six times a day. Similarly, the method was repeated on six consecutive days to determine inter-day precision. The results are summarized in Table 3.

Table-3 Evaluation of precision and accuracy of proposed method

Accuracy

The accuracy of the method was determined in terms of % recovery of AVS standard. Recovery studies were carried out by addition of standard drug solution at three different levels (8, 10, 12 µg mL^-1) to previously analyzed sample (tablet) solution. Values of recovery ± SD were found to be in the range of 99. 7 + 0.7 -100.2 + 1.2 (n=3).The results are given in Table 4.

Table-4 Rrecovery studies by standard addition method

Selectivity studies

The extents of interference by various excipients that often accompany pharmaceutical formulations are tabulated in Table-6. To study the interference, 100 mg mL^-1 of AVS was taken and a known amount of the interfering substance was added and the reaction was carried out as described under general procedure. The interference studies were carried out by repeating the method five times. The results showed the same absorbance as that of pure AVS. The high percentage of recovery showed that excipients did not interfere with the proposed method and the results are presented in Table 5.

Table-5 Assay of AVS in the presence of excipients ^a

Application of the proposed method

The application of the proposed method for the assay of pharmaceutical formulations was examined for tablets and the results were statistically compared with those obtained by UV reference method. The results obtained by the proposed and UV reference method for the

formulations were compared by means of Student’s  t-test and F- test and it was found that the proposed method do not to differ significantly in precision and accuracy. The results are summarized in Table 6.

Table -6 Assay of AVS in pharmaceutical formulations

The results obtained by the proposed method are compared with reported methods¹² and found to be more sensitive in the range of 10-50 mg mL^-1 with ε_max value 9.58× 10³ L mol^-1cm^-1.      .Other analytical parameters like limit of detection (LOD) and limit of quantification (LOQ) were also be evaluated for proposed method but not available in reported methods. The advantage of proposed method is that it requires less expensive equipment, low cost reagents and also free from stringent conditional procedures. The results are given in Table 7.

Table-7 Comparison of proposed method with reported methods for Abacavir sulphate

CONCLUTIONS

In the present study abacavir sulphate was determined successfully as pure compound as well as in formulations by exploiting specific functional group present. The proposed method is sensitive, accurate and precise enough to be successfully adopted as an alternative method of GLC or HPLC technique for routine analysis and also in quality control laboratories for quantitative determination of drugs both in bulk material and dosage forms without interference from excipients and additives.

ACKNOWLEDGEMENTS

One of the authors (TMS) is thankful to the Management of Gayatri Vidya Parishad College of Engineering (Autonomous), Visakhapatnam for providing facilities

REFERENCES

1. Dogan B, Uslu B, Ozkan SA &  Zuman P,  Anal Chem, 80, 209(2008).
2. Shanta Kumari A, Prakash K, Nagoji KEV & Rao MEB , Asian J Chem, 19, 3412(2007)
3. Seshachalam U, Haribabu B& Chandrasekhar KB,   J of Separation Sci, 30(1), 28 (2007).
4. Mistri HN,  Shrivastav P, Jangid  AG & Sanyal M,  Anal. Lett, 40(6), 1147(2007)
5. Sudha T, Ravikumar VR & Hemalatha PV,   Int. J of Pharm and Biomed Res., 1(4), 108 (2010).
6. Raviolo MA, Breva IC & Esteve-Romero J, J o f Chromatography A, 1216(16) ,3546 (2009)  .
7. Rao RN,  Vali RM, Ramachandra B & Raju SS, J of Pharm and Biomed Anal, 54(2), 279 (2011)
8. Surya Rao S, Imran K, Srujana Divya G, Manikantha Kumar J, Thejonadh K & Kishore G, Archives of Applied Sci. Res, 2 (3),
   23(2010)  .
9. Sudha T, Saminathan J, Anusha K, Keerthi M, Bhargavi Y& Ganesan V,  J Chem Pharm Res, 2(5), 45 (2010).
10. Appala Raju N, Venkateswara Rao J, Vanitha Prakash K & Mukkanti K,  E-J of Chemistry, 5(3) 511 (2008).
11. Venkatamahesh R & Dhachinamoorthi D,  Int J of Pharm Tech Res, l.3 (1), 356 (2011).
12. Gowrisankar D, Prakash  S &  Raju S A, Int  J of Chem  Sci, 5(2), 2379 (2007)  .
13. Prasada Rao CH, Seshagiri Rao JVLN, Dhachinamoorthi D, Lakshmi Aswini G & Ashok K, Res J of Pharm and Tech,
    4(2) ,234(2011).
14. Sastry C S P, Lingeswara Rao J S V M & Rama Rao K, Talanta, 42, 199 (1979).
15. Adelusi S A & Oneyekweli A O, Pak. J. Sci. Ind. Res., 39 22 (1996).
16. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonized Tripartite Guideline, Text and Methodology on   Validation of Analytical Procedures, Q2 (R1) 2005.

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