Development and validation of a methodology for the analysis of ritonavir and lopinavir in combined dosage form by high-performance liquid chromatography

Introduction. Qualitative and quantitative analysis of pharmaceutical substances using reverse-phase high-performance liquid chromatography based on domestic hardware is an important stage in the approval of medicines. Drug «Kalidavir» is widely used in the treatment of human immunodeficiency virus. The developed methodology assumes significant advantages in comparison with foreign methods involving the use of imported equipment and standards from the United States recommended by regulatory documentation.

The aim of the study was to develop and conduct validation procedures for the quantitative analysis of ritonavir and lopinavir in the combined drug «Kalidavir».

Material and methods. The optimized parameters of chromatographic operations included the column size (75´2 mm), the choice of the polymer sorbent ProntoSil 120-5C AQ, as well as a binary mobile phase consisting of a 0.1% solution of trifluoroacetic acid in an aqueous medium and methanol, respectively, with a flow rate setting of 150 µl per minute and a column temperature of 35^oC. An isocratic elution regime was applied, ensuring a continuous supply of eluent and the correctness of the analytical conclusion. Additionally, the injection volume of 2 µl and the determination of absorption at four wavelengths from the range from 200 to 240 nm were established.

Results. Retention times of ritonavir and lopinavir were 7.2 min and 11.9 min, respectively. Linearity was observed in the range from 0.025 mg/ml for ritonavir and 0.1 mg/ml for lopinavir and reached 0.5 mg/ml for both substances, with a good correlation coefficient (> 0.999). Relative errors according to the developed quantitative determination methods did not exceed 0.85% for lopinavir and 0.96% for ritonavir.

Conclusions. The methods of qualitative and quantitative determination of lopinavir and ritonavir in the combined tablet dosage form "Kalidavir" by high-performance liquid chromatography using domestic equipment have been developed and approved, which allowed to obtain sufficiently reliable and reproducible results.