INTRODUCTION:

Lopinavir (LPV) ^1-2 is chemically known as [1S-[1R*, (R*), 3R*, 4R*]]-N-[4 [[(2, 6-dimethyl-phenoxy) acetyl] amino]-3-hydroxy-5-phenyl-1(phenylmethyl) pentyl] tetrahydro-alpha-(1-methylethyl)-2-oxo-1(2H) pyrimidine acetamide. Ritonavir (RTV) ^1-2 is chemically known as 10-Hydroxy-2-methyl-5- (1-methylethyl) -1- [2-(1-methylethyl) -4-thiazolyl] -3, 6-dioxo-8, 11-bis (phenylmethyl) -2, 4,7,12 -tetraazatridecan-13-oic acid, 5-thiazolylmethyl ester, [5S-(5R*, 8R*, 10R*, 11R*)] both the drugs were used as an antiretroviral agents.

Various analytical methods have been reported for the assay of Lopinavir and Ritonavirin pure form as well as in pharmaceutical formulations. They include high performance thin layer chromatography³, reversed phase high performance liquid chromatography^4-12, liquid chromatography with tandem mass spectrometry^13,14,liquid chromatography coupled with mass spectrometry^15-18. No UV spectrophotometric study on Lopinavir and Ritonavir in combined dosage form in pharmaceutical preparations has been found in recent literature survey. In the present study,

UV spectrophotometric method has been developed for the determination of Lopinavir and Ritonavir and applied to commercial tablets; the results obtained in this method were statistically compared, because this method can be used for the drug assays in pharmaceutical sciences. Ritonavir is frequently co- formulated with Lopinavir. This paper describes simple, rapid, accurate, reproducible and economical method for the simultaneous estimation of Lopinavir and Ritonavir in tablet formulations using simultaneous equation method.

MATERIALS AND METHODS:

Apparatus:

Shimadzu UV-Visible double beam spectrophotometer model Shimadzu Pharmaspec UV-1700 was employed with spectral bandwidth of 0.5 nm and wavelength accuracy of ± 0.3 nm with automatic wavelength corrections with a pair of 1 cm UV matched quartz cells. Glassware’s used in each procedure were soaked overnight in a mixture of chromic acid and sulphuric acid rinsed thoroughly with double distilled water and dried in hot air oven.

Materials:

The standard drugs (Lopinavir and Ritonavir) were procured as a gift sample from Heterolab, Hyderabad and tablet formulation is from Emcure pharmaceuticals (Emletra) were used for the present study. Methanol, Water analytical reagent grade were purchased from E-Merck. All the solutions were protected from light and were analyzed on the day of preparations.

Selection of common solvent:

Methanol of analytical reagent grade was selected as common solvent for developing spectral characteristics of drug. The selection was made after assessing the solubility of both the drugs in different solvents.

Preparation of Standard Drug Solution:

Standard stock solutions containing Lopinavir and Ritonavir were prepared by dissolving 25 mg of Lopinavir and quantity of Ritonavir equivalent to Lopinavir base 25 mg separately in 10 ml of methanol. It was then sonicated for 10 minutes and the final volume of both the solutions were made up to 25 ml with methanol to get stock solutions containing 1000µg/ml each of Lopinavir and Ritonavir in two different 25 ml volumetric flasks.

Selection of wavelength:

Lopinavir and Ritonavir solutions 10µg/ml solution were prepared separately and λ max of both drugs was scanned individually in the range of 200-400 nm to determine the wavelength of maximum absorption for both the drugs. For estimation, the two wavelengths were selected, 259.0 nm for Lopinavir and 240.0 nm for Ritonavir in the respective solvent.

Preparation of calibration curve:

Lopinavir and Ritonavir showed linearity with absorbance in the range of 10- 50 µg/ ml and 15 - 55 µg/ ml at their respective maxima, which were validated by least square method. Coefficients of correlation were found to be 0.9995 for Lopinavir and 0.9996 for Ritonavir. For simultaneous estimation of Lopinavir and Ritonavir, a series of standard solution were prepared by diluting appropriate volume of standard stock solutions. The scanning solutions of Lopinavir and Ritonavir were carried out in the range of 200- 400 nm against methanol as blank. Absorbance and absorptivities of series of standard solutions were recorded at selected wavelengths (λ1₎ and (λ2).

Fig.1: Overlain spectra of Lopinavir (LPV) and Ritonavir (RTV) in methanol.

Analysis of Tablet Formulation:

Twenty tablets of each containing 200 mg of Lopinavir and, 50 mg of Ritonavir were weighed, and crushed into fine powder. A quantity of powder equivalent to 50 mg was dissolved in 25 ml of methanol and sonicate for 15 min. Then volume was made up to 50 ml with methanol and filtered through whatmann filter paper. The final mixed sample solution were prepared, correspond to 80 µg /ml of Lopinavir and 20 µg/ml of Ritonavir. The absorbances of resulting solutions were measured at 259 and 240 nm. The concentration of Lopinavir and Ritonavir present in the sample solution was calculated by using the equation generated from calibration curve of respective drugs. The result of analysis of the tablet formulation is presented in table no. 1.

Simultaneous equation method:

Two wavelengths selected for the method are 259 nm and 240 nm that are absorption maxima of Lopinavir and Ritonavir respectively in methanol. The absorbances were measured at the selected wavelengths and absorptivities (A 1%, 1 cm) for both the drugs at both wavelengths were determined as mean of three independent determinations. Concentrations in the sample were obtained by using following equations-

C_{x =} A₂a_y1-A₁a_y2) / (ax₂a_y1-a_x1a_y2) ……Eq. (i)

C_y = (A₁a_x2-A₂a_x1) / (ax₂a_y1-a_x1a_y2) …Eq. (ii)

Where, A₁ and A₂ are absorbance of mixture at 259 nm and 240 nm respectively, ax₁ and ax₂ are absorptivities of Lopinavir at λ₁ and λ₂ respectively and ay₁ and ay₂ are absorptivities of Ritonavir at λ₁ and λ₂ respectively. C_x and C_y are concentrations of Lopinavir and Ritonavir respectively. Figure I represents the overlain spectra of both the drugs in 1:1 ratio and the criteria for obtaining maximum precision [i.e. absorbance ratio (A₂/A₁)/ ax₂ / ax₁ and ay₂ / ay₁] by this method were calculated ⁵ and found to be outside the range of 0.1 – 2.0 which is satisfied for both the Lopinavir and Ritonavir in ratios of 1:1 and 1:2.

Recovery Studies:

The method was validated according to ICH Q2B guidelines^19-21 for validation of analytical procedures in order to determine the linearity, sensitivity, precision and accuracy for the analyte. Recovery studies by spiking different concentrations of pure drug in the preanalyzed tablet samples within the analytical concentration range of the proposed method. The added quantities of the individual drugs were estimated by above method. The results of recovery studies were found to be satisfactory and the results are presented in table no. 2.

RESULTS AND DISCUSSION:

The proposed method for simultaneous estimation of Lopinavir and Ritonavir in combined sample solutions was found to be simple, accurate and reproducible. Beer's law was obeyed in the concentration range of 10-50 µg/ml and 15-55 µg/ml for Lopinavir and Ritonavir respectively. Co-efficient of variation was found to be 0.9995 and 0.9996 for Lopinavir and Ritonavir, respectively, Regression and Optical characteristics of Lopinavir and Ritonavir are presented in table no 3. The percentage recoveries were found to be in the range of 101.41± 1.096 and 100.54 ± 1.002 for Lopinavir and Ritonavir respectively. Once the equations are determined, analysis requires only the measuring of the absorbance of the sample solution at two wavelengths selected, followed by a few simple calculations.

Table 1: Results of analysis of tablet samples

Analyte

Label claim

(mg/ tab)

% Label claim estimated* (Mean ± S.D.)

LPV

200

99.02 ± 0.0124

RTV

99.93 ± 0.0217

*Average of five determination R.S.D., Relative Standard Deviation

Table 2: Recovery study

Drug in standard mixture solution ( µg ml^-1)		% Recovery ± S. D.*
LPV	RTV	LPV	RTV
10	15	99.25 ± 1.08	99.92± 0.31
20	25	99.70 ± 0.86	97.16± 1.69
30	35	100.90 ± 0.26	100.91± 0.18

S. D.* Stands for standard deviation, the results of mean of three readings (n=3).

Table 3: Regression and Optical characteristics of Lopinavir and Ritonavir

Parameters	Value for	Value for
Parameters	LPV	RTV
Beer’s law limit ( µg ml^-1)	10-50	15-55
Correlation coefficient (r)	0.9995	0.9996
Regression equation
Slope	0.0239	0.0594
Intercept	0.0035	0.0114

CONCLUSION:

The most striking feature of this method is its simplicity and rapidity, non- requiring- consuming sample preparations such as extraction of solvents, heating, degassing which are needed for HPLC procedure. It is a new and novel method and can be employed for routine analysis in quality control studies. Analysis of authentic samples containing Lopinavir and Ritonavir showed no interference from the common additives and excipients. Hence, recommended procedure is well suited for the assay and evaluation of drugs in pharmaceutical preparations. The described method gives accurate and precise results for determination of Lopinavir and Ritonavir in tablets.

ACKNOWLEDGEMENT:

The authors are thankful to heterolab, Hyderabad for providing gift samples of Lopinavir and Ritonavir. We acknowledge to the management, Kovai Medical Center Research and Educational Trust, Coimbatore for all the facilities to do a work of this magnitude.

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Received on 07.11.2009 Modified on 01.12.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 188-191