Quantification of HIV Antiretroviral Drugs in Human Plasma by Matrix Assisted Laser Desorption Ionisation Time-of-Flight Mass Spectrometry

User Benefits

- Minimal sample preparation required prior to acquisition. - Data can be rapidly acquired without the need for chromatographic separation. - The quantification assay can be developed according to guidelines.

Introduction

Matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is widely used in many fields of application thanks to its robustness, simple operation and high sensitivity. The majority of applications are almost exclusively for qualitative analysis aimed at fast and accurate confirmation of the molecular mass. Quantitative analysis with MALDI-TOF MS has traditionally been viewed as impractical due to non-uniform distribution of the analyte(s) and matrix across the spot surface, giving rise to variations in the ion intensities. For this reason, LC–MS/MS is the mainstream technique for quantifying drugs in plasma. These unavoidable limitations can be overcome by adopting the ‘internal standard (I.S.)’ approach, where a fixed amount of an exogenous compound is added to the sample and the peak heights or areas of the endogenous analytes are corrected relative to it. The I.S. serves as a mimic of the analyte(s) and should exhibit similar chemical and physical properties, so that in all the stages of the assay (from sample preparation to analysis) it behaves in a manner close/identical to the sample analyte(s). A calibration curve is usually generated containing various amounts of a single analyte of interest and a fixed amount of the internal standard. This way, the ‘corrected’ ion intensities allow calculation of absolute amounts as the constant of proportionality can be established. Here, we show a proof-of-principle example of determination and quantification of HIV protease inhibitors in human plasma using a Shimadzu benchtop MALDI-8030 EasyCare instrument. Lopinavir (LPV) was used as an example of HIV antiretroviral drug and quantified against Ritonavir (RTV) internal standard. The quantitative assay was developed according to the FDA guidelines. In this study, by employing MALDI‑TOF MS, we achieved quantitative analysis that enables both simplified sample preparation and high‑throughput measurements.

May 11, 2026 GMT