International Journal of Drug Development and Research

Keywords

Lopinavir; Ritonavir; Antiretrovirals; HPLC-UV

INTRODUCTION

Lopinavir and ritonavir is a human immunodeficiency virus (HIV) protease inhibitor [1] and is chemically designated as [1S-[1R*, (R*), 3R*, 4R*]]-N-[4 [[(2,6dimethyl-phenoxy) acetyl] amino]- 3-hydroxy-5-phenyl 1(phenyl methyl) pentyl] tetrahydro-alpha-(1-methylethyl)-2-oxo-1(2H) pyrimidine acetamide and 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*) respectively. The Chemical structure of lopinavir and ritonavir were given in (Fig.1). This class of drugs inhibits the HIV protease preventing cleavage of the gag-pol polyprotein, reducing the probability of viral particles reaching a mature infectious state. Administered alone, lopinavir has insufficient bioavailability. However, like several HIV protease inhibitors, its blood levels are greatly increased by low doses of ritonavir, a potent inhibitor of cytochrome P450 3A4 [2, 3] and therefore lopinavir is co-administered with sub-therapeutic doses of ritonavir by oral route of administration. Several HPLC methods have been described in the literature for the determination of lopinavir [4–20]. These are determination of lopinavir alone and simultaneously with other antiretrovirals in human plasma by RP-HPLC [4–18].

Donato et al. have reported a HPLC method for the assay of lopinavir and ritonavir in soft-gel capsules [19]. Seshachalam et al. have reported an isocratic RP-HPLC method for the lopinavir assay and determination of its process and degradation impurities in bulk drug and pharmaceutical formulation [20]. Sreenivasa et al. have reported a validated method for impurity profile of lopinavir [21]. However, there is no method reported regarding the quantitation of lopinavir and ritonavir

Hence, the aim of this study was to develop and validate a simple HPLC method, using UV detection to quantify lopinavir and ritonavir in pure form and pharmaceutical formulations. Due to the low molar absorptivity of lopinavir and ritonavir, the UV region was found to be at 254nm. The validated method was applied to the analysis of tablets containing lopinavir-ritonavir (200+50 mg).

Experimental

Reagents and materialsq

Lopinavir and ritonavir reference standards were purchased from Glenmark Laboratories, Mumbai. Tablets were purchased from local pharmacies which was manufactures by Emcure Pharmaceuticals Emletra®. Ultra-pure water was obtained from a Millipore system (Bedford, MA, USA). Acetonitrile, methanol (HPLC grade) was obtained from E-Merck (India) Ltd - Mumbai - India. All other chemicals used in the analysis were AR grade.

Instrumental and analytical conditions

The HPLC analyses were carried out on Thermo scientific (Spectra serious P4000) system with UV detector, Hypersil (ODS) C8 (250 × 4.6mm, 5μm) column from Thermo, USA. UV detection was performed at 254nm. UV spectra from 190 to 400nm were online recorded for peak identification. The injection volume of sample was 20 μL. An isocratic mobile phase containing acetonitrile, methanol and 0.01M potassium dihydrogen orthophosphate buffer at pH 3.0 (30:20:50, v/v/v) was carried out with the flow rate of 1mL/min. The separation of lopinavir and ritonavir was evaluated in different proportions of these solvents and, for each condition, retention factor (k) and resolution (R) was calculated.

Preparation of standard solution

About 100 mg of lopinavir and 25 mg of ritonavir reference standards was accurately weighed and transferred to a 100 mL volumetric flask, 10 mL of acetonitrile was added to ensure the complete solubilisation and the volume was adjusted with the mobile phase. Further dilutions were made to get the final concentration of 100 μg mL-1 of lopinavir and 50 μg mL-1 of ritonavir. The solution was filtered through 0.45μ membrane filter.

Analysis of fixed dose tablets

Three different batches of Emletra® were analyzed using the validated method. Lopinavir and ritonavir standard was added to the samples, with the aim of increasing the peak area of lopinavir and ritonavir in the chromatograms and thereby improving the detection of this compound. For the analysis, six replicates of each batch were assayed. The tablets were weighed and finely powdered. An accurately weighed portion of the powder, equivalent to about 100 mg of lopinavir and 25 mg of ritonavir was transferred to a 100 mL volumetric flask followed by the addition of 25 mL of acetonitrile. The solution was sonicated for 3 minutes and diluted with mobile phase to volume. Further dilutions were made to get the final concentration equivalent to 100 μg mL-1 of lopinavir and 50 μg mL-1 of ritonavir.

Validation [22-23]

Linearity

Standard solutions containing 0.1 mg mL-1 of lopinavir and 0.05 mg mL-1 of ritonavir was prepared, in triplicate. Aliquots of these solutions were diluted in mobile phase to five different concentrations, corresponding to 80, 90, 100, 110, 120 μg mL-1 of lopinavir and 40, 45,50,55,60 μg mL-1 of ritonavir. The calibration curves for concentration versus peak area were plotted for lopinavir and ritonavir; obtained data were subjected to regression analysis using the least squares method with a weighting factor of 1/x.

Precision

The intra-day precision was evaluated by analyzing six sample solutions (n = 6), at the final concentration of analyses (100 μg mL-1) of lopinavir and (50 μg mL-1) of ritonavir. Similarly the inter-day precision was evaluated in three consecutive days (n=18). The lopinavir and ritonavir concentrations were determined and the relative standard deviation (RSD) was calculated.

Accuracy

Accuracy of the method was demonstrated at three different concentration levels (80?120%) by spiking a known quantity of standard drugs of Lopinavir and ritonavir into the pre-analyzed sample in triplicate. Specificity

Spectral purities of lopinavir and ritonavir chromatographic peaks were evaluated using the UV spectra recorded by a UV detector. In addition, a solution containing a mixture of the tablets excipients was prepared using the sample preparation procedure and injected on to the chromatograph, to evaluate possible interfering peaks.

Robustness

Six sample solutions were prepared and analyzed under the established conditions and by variation of the following analytical parameters: flow rate of the mobile phase (0.8 and 1.1 mL/min), acetonitrile, methanol and buffer as mobile phase (28.5:19:52.5, 31.5:21:47.5), mobile phase pH (2.8 and 3.2) and column temperature (23, 27oC).The lopinavir and ritonavir contents was determined for each condition and the obtained data was submitted for statistical analysis (ANOVA test).

Detection and quantitation limits

Limit of detection LOD (signal-to- noise ratio of 3) and limit of quantification LOQ (signal-to- noise ratio of 10) were measured based on the signal-tonoise ratio. Determination of the signal-to-noise is performed by comparing measured signals samples with known low concentration of analyte with those of blank samples establishing the minimum concentration at which the analyte can be reliably detected and quantified

Results and discussion

The chromatographic parameters were initially evaluated using a Thermo Hypersil (ODS) C8 (250 × 4.6mm, 5μm) column and a mobile phase composed of acetonitrile, methanol and 0.01M potassium dihydrogen orthophosphate buffer at pH 3.0 (30:20:50,v/v/v). Using this column, different proportions of mobile phase solvents were evaluated, to obtain a good peak (Table 1). Under these conditions the retention factor obtained for lopinavir and ritonavir were 4.66, 3.44 and a short run time (6 min), and so, this condition was adopted in subsequent analysis (Fig. 2). After the evaluation of the lopinavir and ritonavir UV spectrum in the range of 200-400 nm, the wavelength of 254 nm was selected for detection, due to the adequate molar absorptivity of lopinavir and ritonavir in this region and the higher selectivity of this wavelength regarding possible interfering compounds or solvents in the sample

Validation

Linearity

A linear correlation was found between the peak areas and the concentrations of lopinavir and ritonavir in the assayed range. The regression analysis data were presented in (Table 2). The regression coefficients (r2) obtained was higher than 0.999 for the both compounds (Fig.3), which attest the linearity of the method.

Precision

Mean contents of lopinavir and ritonavir in the intraday precision analysis (n = 6) was 100 μg mL-1 (RSD = 0.21%) and 50 μg mL-1 (RSD=0.66%) respectively. For the intra-day precision (n=18) the mean contents obtained were 100.3 μg mL-1 (RSD = 0.25%) and 50 μg mL-1 (RSD =0.36%) for lopinavir and ritonavir respectively. The RSD values lower than 2.0% assure the precision of the method.

Analysis of fixed dose combination tablets

Samples of fixed dose combination tablets Emletra® containing 200 mg of lopinavir and 50 mg of ritonavir were analyzed using the validated method. The results obtained were presented in (Table 3). All the analyzed batches presented lopinavir and ritonavir were very close to the labeled amount. The lopinavir content in the tablets samples varied from 99.87 to 100.52%, while ritonavir content varied from 99.61 to 100.57%.

Accuracy

It was investigated by means of addition of lopinavir and ritonavir reference standards to a mixture of the tablets excipients. Lopinavir mean recovery (n =9) was 101.51% (RSD = 0.21 %) and ritonavir mean recovery was 100.29% (RSD = 0.36 %) demonstrating the accuracy of the method. The results of accuracy (Table 4) revealed that the method was more accurate.

Specificity

Peak purities higher than 99.0% were obtained for lopinavir and ritonavir in the chromatograms of sample solutions, demonstrating that other compounds did not co-elute with the main peaks. The chromatogram obtained with the mixture of the tablets excipients showed no interfering peaks in the same retention time of lopinavir and ritonavir.

Robustness

Statistical analysis showed no significant difference between results obtained employing the analytical conditions established for the method and those obtained in the experiments in which variations of some parameters were introduced. Thus, the method showed to be robust for changes in mobile phase flow rate from (0.8, 1.1 mL/min), acetonitrile, methanol and buffer proportion from (28.5:19:52.5, 31.5:21:47.5), mobile phase pH (2.8, 3.2) and column temperature (23, 27oC). The analysis data were presented in (Table 5).

3.1.7. Detection and quantitation limits

According to the determined signal-to-noise ratio, lopinavir and ritonavir presented limits of detection of 0.70-0.15μg mL-1 and limits of quantitation of 1.63-0.63 μg mL-1 respectively where the compounds proportion found in the sample solutions injected onto the chromatograph. However, the objective of the method is the quantitation of lopinavir and ritonavir, so that the values obtained for lopinavir and ritonavir should be considered as the limit of method sensitivity.

The development of simple and reliable method is essential to assure the identification and quantitative determination of antiretrovirals drugs, since the problem of counterfeit or substandard antiretrovirals is well established all over the world. The quality control of the antiretrovirals pharmaceutical preparations marketed nowadays may help to assure the treatment efficacy and avoid the development of resistance to antiretrovirals drugs.

Conclusion

This study proposed is for the development and validation of lopinavir and ritonavir in pure form and pharmaceutical formulations. It could precisely and accurately quantity of lopinavir and ritonavir in the presence of other tablet excipients. The developed method showed to be a simple, sensitive and suitable technique to quantify the antiretrovirals and might be employed for quality control analysis, as well as in further studies in other matrices, such as plasma. The lopinavir and ritonavir tablets analyzed by the validated method showed adequate quality and drug contents in concordance with the labeled amount.

Tables at a glance

Table 1 Table 2 Table 3 Table 4 Table 5

Figures at a glance

Figure 1 Figure 2 Figure 3

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