INTRODUCTION:

Lopinavir chemically (2S)-N-[(2S,4S,5S)-5-[2- (2,6dimethylphenoxy) acetamido]-4-hydroxy-1,6-phenylhexan-2-yl]-3-methyl-2-(2-oxo-1,3-diazinan-1-yl)butanamide and its empirical formula is C₃₇H₄₈N₄O₅ with a molecular weight of 628.80 (figure 1 A) ^[1-3]and Ritonavir (5s, 8s, 10s,11s)-10-hydroxy-2-methyl-5-(1-methylethyl)-1-[2-(1-methylethyl) -4-thiazolyl]-3,6-dioxo-8,11-is (phenylmethyl)-2,4,7,12-etraazatridecan-13-oic acid 5-thiazolyl methyl ester of molecular formula C₃₇H₄₈N₆O₅S₂ and its molecular weight is 720.95(figure 1 B)^[1-3].These are antiretroviral drugs from protease inhibitor class. The drugs have been proved to be effective in anti-HIV treatment.

A: Lopinavir

B: Ritonavir

Figure 1: Structures of Lopinavir and Ritonavir

Lopinavir with Ritonavir combination patients who have not responds to other regimens for treatment of HIV. In combination ritonavir act as a booster by inhibiting lopinavir metabolism by CYP-3A4 and increases plasma concentration of lopinavir.

Ritonavir is the most potent protease inhibitor in its ability to inhibit CYP-450 and efflux pump-P-glycoprotein as a result the potential for severe drug interaction is quite great because of strong CYP-450 inhibiting effect of Ritonavir. The drug has found value when used in fixed dosage form combination with other PIs to block their metabolism and acts as a booster for these drugs. In these cases Ritonavir is used in a sub therapeutic dose but boosts the effectiveness of co administered drug. ^[4-7]

Many RP-HPLC methods have been reported for the simultaneous determination of Lopinavir and Ritonavir in tablet dosage form and in suspension. But in all cases P^H adjustment with buffer solution were required. None of the method is available without P^H adjustment. ^[8-10]

Therefore it was the purpose of this research to develop a rapid, simple sensitive, reliable and validated analytical method for measurement of both drugs, which will be first for their simultaneous analysis of API and tablet dosage form. The present RP-HPLC method was validated following the ICH guidelines. ^[11-19]

EXPERIMENTAL:

Chemical and reagents used:

The reference standard of lopinavir and ritonavir were obtained as gift samples from Emcure pharmaceuticals from Bhosari, Pune. All chemicals used such as Acetonitrile, Methanol were of HPLC grade of Merck Ltd. Tablet dosage form used for estimation in dosage form was Lopimune manufactured by Lupin pharmaceutical. Each tablet contains 200mg of Lopinavir and 50 mg of Ritonavir.

Instrumentation:

HPLC of CYBER Lab. including JASCO PU-2080 Plus pump equipped with universal injector Rheodyne with injection volume 20µl, Jasco UV-2075 plus UV Visible detector, Analytical balance of Shimadzu (d=0.1mg) were used for weighing the drugs ,Ultrasonicator used.

Chromatographic condition:

The separation was achieved on octadecyl column (C18) and with mobile phase containing mixture of acetonitrile: water: Methanol (55:30:15v/v/v) at a flow rate 1ml/min, UV detection was carried out at 260 nm. The retention time of Lopinavir was found to be 10.82±1min and for Ritonavir 8.94±1min. The injection volume of standard and sample solution was 20µl.

Preparation of standard and sample solution:

Weigh accurately and transfer about 10 mg of Ritonavir in100ml volumetric flask and 25mg of Lopinavir in 25ml of volumetric flask separately. Dissolve and dilute to volume with mobile phase (Lopinavir-1000µl/ml and Ritonavir 100µl/ml) from this stock solution working standard solution of both the drugs were prepared using mobile phase.

Preparation of Calibration curve:

Different binary mixture solution of Lopinavir and Ritonavir were chromatographed in 15 minutes time period and peak area of these solution were measured at 10.82 ±1 and 8.94±1 minute corresponding to RT of Lopinavir and Ritonavir respectively. Calibration curves for both the drugs were plotted separately. The method shows good linearity in the range of 50-250 µg/ml for Lopinavir and 10-50µg/ml for Ritonavir. The results are given in figure 2 a and 2 b

Figure 2: (a) Calibration curve of Lopinavir

Figure 2: (b) Calibration curve of Ritonavir

RESULT AND DISCUSSION:

Optimization of analytical condition:

With the help of C18 column different mobile phases were tried. Individual drug solution was injected into column, both elution pattern and resolution parameters studied as a function of mobile phase component and their ratio. To develop a suitable LC method for estimation of Lopinavir and Ritonavir in formulation, different mobile phases were employed to achieve the best separation. The selected and optimized mobile phase was Acetonitrile: water: methanol (55:30:15v/v/v) at a flow rate 1 ml/min. UV detection was carried out at 260 nm (Fig.3: Overlain spectra of lopinavir and ritonavir showing 260 nm wavelength selected) The retention time of was found to be for lopinavir as 10.82±1min and for ritonavir 8.94±1min respectively. Run time was 15 minutes. Here the peaks were separated and showed better resolution, theoretical plate count and asymmetry were also found good for Lopinavir and Ritonavir. The typical chromatogram of drugs assayed is shown in figure 4. Overlain chromatograph of lopinavir and ritonavir calibration is shown in figure 5.

Fig No.3.: Overlain spectra of lopinavir and Ritonavir showing 260 nm wavelength selected

Figure 4: Typical chromatogram of two drugs assayed: retention time 8.95 for Ritonavir and 10.84 for Lopinavir.

Figure 5: Overlain chromatograph of lopinavir and Ritonavir

Method Validation:

System suitability:

The system suitability of the method was studied to determine the reproducibility of chromatographic system and column performance was acceptable for the intended analytical application. Four parameters i.e., precision of peak area of five replicate injections, retention time of eluted drugs, numbers of theoretical pates, asymmetry factor and resolution between two peak of the analytes were evaluated. The results are shown in table 1

Table 1: Results of system suitability:

System suitability parameter	Proposed Method
System suitability parameter	Lopinavir	Ritonavir
Retention time (minute)	10.82±1	8.84±1
Relative Retention time (minute)	1.98±1
Tailing Factor	1.25	1.086
Theoretical plate (N)	12171.66	10911.13
Resolution (Rs)	3.94

Linearity:

The linearity of analytical method is its ability to obtain test results which are directly proportional to the concentration of the analyte in the assay method, was established by injecting test sample in the range of 10–50 µg/ml for Ritonavir and 50-250µg/ml for Lopinavir. Each solution was injected twice into HPLC and the average area at each concentration was calculated. The regression analysis was carried out from graph of peak area Vs concentration; correlation coefficient and Y intercept of plot was also evaluated. The test results are shown in table 2. Linear regression equation and correlation coefficient was found as

Lopinavir: y=441.9x-318.7 R²=0.997

Ritonavir: y=572.1x+550.2 R²=0.996

Where, ‘y’ is area of peak and ‘x’ is the concentration of drug solution respectively.

Table 2: Results of Linearity study:

Lopinavir		Ritonavir
Concentration (µg/ml)	Area	Concentration (µg/ml)	Area
50	3946.6	10	6800.96
100	7878.9	20	11783.63
150	133303.4	30	17988.1
200	17584.83	40	24154.43
250	21670.97	50	28398.27

Accuracy:

The accuracy of the test method demonstrated by preparing recovery samples at the level of 80%, 100%, and 120% of target concentration. The recovery samples were prepared in triplicate at each level. The above samples were injected and the percentage recovery at each level was determined by computing the relative standard deviation of triplicate recovery results. The result for accuracy is as shown in table-3 indicating good accuracy of the method for simultaneous determination of two drugs

Precision:

Precision was determined by two ways; by system precision and intermediate precision. System precision was demonstrated by making five replicate injections of standard solution. The peak area of the analyte for replicate injections was recorded. The %RSD for the analyte peak area of the replicate injections was evaluated. The intermediate precision of test method was demonstrated by carrying out precision study at three different concentrations. Intermediate precision study includes intraday and inter day analysis. The result summary of intermediate precision as shown in table 4 and 5.

Limit of detection and Limit of Quantitation:

Limit of detection and Limit of Quantitation was established based on the residual standard deviation method. LOD and LOQ was found to be 1.28µg/ml and 3.88µg/ml for lopinavir and 0.299µg/ml and 0.909µg/ml for Ritonavir, respectively.

Analysis of marketed formulation:

Twenty tablets were weighed and crushed to fine powder. A quantity of powder equivalent to 20mg/ml of Lopinavir and 5mg of Ritonavir were weighed and transferred to volumetric flask 25 ml mobile phase was added and sonicated for 20 min. allow the solution to cool, and then make up the volume with mobile phase. The solution was filtered through Whatmann filter paper No. 41. The filtrate of 10ml was taken and diluted with mobile phase to get 200µg/ml of Lopinavir and 50µg/ml of Ritonavir. The results of analysis of marketed formulation are given in table 6.

CONCLUSION:

The data demonstrate that the RP HPLC method showed acceptable linearity, specificity, accuracy precision and robustness in concentration range 50-250µg/ml for Lopinavir and 10-50µg/ml for Ritonavir as per the requirement ICH guidelines. The method described is rapid since chromatographic run time is 15 min. and retention time of Lopinavir was 10.82±1min and for Ritonavir 8.94±1min. The propose method is precise, accurate and robust and does not suffer from any interference from other excipients. In conclusion the proposed method could be routinely used for analysis of lopinavir and ritonavir in pharmaceutical dosage form.

ACKNOWLEDGEMENT:

The authors are grateful to Emcure Pharmaceutical Ltd., Bhosari, Pune, for providing the gift samples. We are also thankful to University of Pune for giving grant for our project. We are also thankful to Principal, Modern college of pharmacy [for ladies], Moshi, Pune for providing required facilities.

Table 3: Results of Accuracy Study

Sample Conc. (µg/ml)		Amount of standard added(µg/ml)		Amount Recovered (µg/ml)		% Recovery ±SD
Lopinavir	Ritonavir	Lopinavir	Ritonavir	Lopinavir	Ritonavir	Lopinavir	Ritonavir
150	37.5	120	30	259.60	65.01	96.18 (±0.0945)	96.31 (±0.4722)
150	37.5	150	37.5	308.41	79.10	102.80 (±0.7247)	105.47 (±0.5485)
150	37.5	180	45	347.45	85.11	105.28 (±0.7834)	103.17 (±0.4994)

Table 4: Results of Intermediate Precision for Lopinavir

Conc (µg/ml)	Intraday (n=3 mean peak area ± SD)		RSD	Inter day (n=3 mean peak area ± SD)		RSD
50	3946.6	±34.33235	0.0086	3943.8	±52.100	0.01321
100	7878.9	±132.2736	0.0167	7829.7	±150.2784	0.019193
150	13303.4	±221.8032	0.0166	13648.8	±262.5344	0.019235

Table 5: Results of Intermediate Precision for Ritonavir

Conc (µg/ml)	Intra day (n=3 mean peak area ± SD)		RSD	Inter day (n=3 mean peak area ± SD)		RSD
10	6800.967	±52.00522	0.007647	6866.833	±57.71103	0.008404
20	11783.63	± 235.13	0.019954	11704.3	±190.7747	0.0163
30	17988.1	±125.5879	0.006982	17839.2	±176.8157	0.009912

Table 6: Results of marketed Formulation

Formulations	Label claim(mg)		Amount taken(µg/ml)		Amount Found(µg/ml)		*%Assay±SD**
Lopimune	Lopinavir	Ritonavir	Lopinavir	Ritonavir	Lopinavir	Ritonavir	Lopinavir	Ritonavir
Lopimune	200	50	200	50	203.6	50.86	101.72±0.4956	103.8±0.1514

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Received on 22.05.2013 Modified on 12.06.2013

Asian J. Research Chem. 6(6): June 2013; Page 555-559