Lopinavir and Ritonavir Tablets Assay Method Following United States Pharmacopeia Monograph Guidelines Using an Ascentis C8 Column and UV Detection

Introduction

This application note illustrates how it is possible to set-up an assay method for Lopinavir and Ritonavir tablets. The given experimental conditions follow the current USP43-NF38 monograph; the chromatographic assay method (HPLC-UV) is isocratic in nature and calls for an L7 column (octyl silane; C8 modification). The method has been validated following the guidelines in USP General Chapters <621>, <1225>, and <1226>.

Lopinavir and Ritonavir were baseline resolved (Rs > 5) within 40 minutes using an Ascentis C8 HPLC column (150 x 4.6 mm, 5 µm) with a mixture of 4.1 g/L monobasic potassium phosphate solution, acetonitrile, methanol, and tetrahydrofuran as the mobile phase. Under the applied conditions ritonavir elute prior to lopinavir, and all system suitability requirements are met. The method demonstrates good selectivity, reproducibility, sensitivity and accuracy.

Ritonavir

Lopinavir

 

Experimental Conditions
Column Ascentis C8 (5µm) 150 x 4.6 mm (L7) Injection volume 50 µL
Detection UV @ 215 nm Flow rate 1.5 mL/min
Buffer 4.1 g/L of monobasic potassium phosphate in water Temperature Column: 40° C : Autosampler: 10° C
Mobile phase Acetonitrile, Methanol, Tetrahydrofuran, Buffer (175:100:100: 625) Pressure drop 156 bar (2262 psi)
Diluent Acetonitrile and Buffer  (50:50)    
System suitability solution 6.25 µg/mL of USP Ritonavir RS and 25 µg/mL of USP Lopinavir RS in Solution A
Standard solution 6.25 µg/mL of USP Ritonavir RS and 25 µg/mL of USP Lopinavir RS in Solution A

System Suitability Solution

System Suitability Solution

 

Chromatographic Data (System Suitability Solution)
 

Peaks Compound Retention Time (min) Retention Time %RSD Resolution Tailing Factor Theoretical Plates
1 Ritonavir 29.0 0.07 - 1.09 7065
2 Lopinavir 37.0 0.04 5.3 1.05 -

 

Ritonavir Linearity

Ritonavir  
Concentration (μg/mL) Mean Area
12.5 487
20 818
40 1657
50 2160
   

 

Standard Repeatability (6.25 μg/mL)
STD 1 242.38
STD 2 241.40
STD 3 243.41
 
Mean 242.40
Standard Deviation 1.0
RSD (%) 0.4

Ritonavir Linearity

Lopinavir Linearity

Lopinavir  
Concentration (μg/mL) Mean Area
12.5 671
20 1023
40 2068
50 2633
Standard Repeatability (25 μg/mL)
STD 1 1340.92
STD 2 1342.53
STD 3 1341.86
 
Mean 1341.77
Standard Deviation 0.8
RSD (%) <0.1

Lopinavir Linearity

Materials