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.

Figure 1. Ritonavir

Figure 2. 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

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

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