Ultra-fast, accurate and simultaneous quantification of ritonavir and lopinavir in human plasma

Sensitive and robust results using the Echo® MS System

Rahul Baghla, Rolf Kern
SCIEX, USA

Abstract

Lopinavir and ritonavir are two protease inhibitors (class of anti-viral drugs) often used in a fixed-dose combination, along with other medications, for prevention and treatment of HIV/AIDS. Monitoring of lopinavir and ritonavir in human plasma is needed to ensure proper therapeutic levels are maintained. A very high throughput bioanalytical method (3 sec/sample) for the simultaneous analysis of ritonavir and lopinavir in human plasma is described using the Echo MS platform. Using a simple liquid-liquid extraction (LLE) for sample preparation, the assay provided very sensitive, accurate and reproducible results.

RUO-MKT-2-11695 Echo

Introduction

Protease inhibitors (PIs) are a class of anti-viral drugs that prevent viral replication by selectively binding to viral proteases and inhibiting their function. The development of PI-based therapies has been of enormous benefit to people infected with HIV. Used in combination with other drugs, PIs have dramatically reduced the number of people who become ill from HIV-related opportunistic infections or who die from AIDS.

Unfortunately, the effectiveness of protease inhibitors can fade over time. Mutations during viral replication can result in viruses that produce new, different proteases that are not targeted by current PI therapies.1 The best way to avoid this drug resistance is to reduce or stop HIV replication. With less HIV replication, there is less of a chance of a new strain that is resistant to anti-HIV drugs. To keep HIV levels as low as possible, PIs are typically taken in combination with at least two other anti-HIV drugs. Such combination therapies are referred to as highly active antiretroviral therapy (HAART).2

Lopinavir and ritonavir are two protease inhibitors that are often used as part of a fixed-dose combination, and serve as the model compounds in this study.

Acoustic Ejection Mass Spectrometry (AEMS), as implemented in the Echo® MS System with a SCIEX Triple Quad™ 6500+ LC-MS/MS system, offers clear benefits for quantification of lopinavir and ritonavir in human plasma. Requiring minimal sample preparation and no chromatographic separation, it provides high sample throughput without sacrificing robustness or reproducibility. The Echo® MS System combines Acoustic Droplet Ejection technology with an Open Port Interface (OPI) to deliver nanoliter sample volumes with minimal carryover. The small sample size reduces matrix suppression and facilitates easy sample preparation.

 

Figure 1. High reproducibility in quantification. 6 replicates at each concentration level of lopinavir and ritonavir in extracted human plasma, along with 6 replicates at quality control levels, showed excellent %CVs, below 15% at all levels.

Key features of the Echo® MS System for high-throughput bioanalysis quantification

  • Ultra-fast 3 sec/sample analyses provide high throughput for quantifying large number of samples
  • Liquid-liquid extraction (LLE) sample preparation ensures excellent quantitative accuracy
  • Exceptional sensitivity of the Echo® MS System yields high quality data at all concentration levels