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

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 (lmax=620 nm) is described. Beer’s law limits and molar absorptivity values were found to be 10 – 50 μg mL-1 and 9.58 x 103 L mol-1 cm-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 × 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.


EXPERIMENTAL 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 -1 M), 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% Na 2 CO 3 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 µg mL -1

Analytical procedure
Aliquots of standard drug (free base form) solution (1.0 -5.0 mL; 100 µg 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 -1 M 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 3)

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 µg 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.

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 H 2.0), 1-4 min. of shaking time ,the ratio of aqueous to organic phase on extraction is 3:2.In this 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.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 l max (nm) and µ max (Lmol - 1 cm -1 ) values were found to be 620 and 9.589 × 10 3 respectively.

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.

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 [16][17] is presented in Scheme.

Method of validation
The developed method was validated as   per ICH guidelines 18 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 × 10 -2 ) and intercept (1.2 × 10 -3 ) of drug by the proposed method was given in Table 2.

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 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 I.

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 interday precision.The results are summarized in Table 3.

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.

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.

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.
The results obtained by the proposed method are compared with reported methods 12 and found to be more sensitive in the range of 10-50 µg mL -1 with µ max value 9.58× 10 3 L mol -1 cm -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.

CONCLUSIONS
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.

Table 2 : Regression parameters of proposed method
Y = a + bC where C is the concentration of analyte in mg/ml and Y is the absorbance unit.

Table 3 : Evaluation of precision and accuracy of proposed method Precession and accuracy parameter CTC Method
a: Average of six determinations (n = 6) b = AVS concentration: 100 µg mL-1

Table 4 : Rrecovery studies by standard addition method
a: Average value of 3 determinations

Table 5 : Assay of AVS in the presence of excipients a Excipient Concentration (mg mL -1 ) Recovery (%) a,b
a: Concentration of drug 100 mg ml -1 ,b: Mean ± SD, n = 5.