Quantitative Analysis of HIV-1 Protease Inhibitors in Cell Lysates Using MALDI-FTICR Mass Spectrometry

Abstract

In this report we explore the use of MALDI-FTICR mass spectrometry for the quantitative analysis of five HIV-1 protease inhibitors in cell lysates. 2,5-Dihydroxybenzoic acid (DHB) was used as the matrix. From a quantitative perspective, DHB is usually a poor matrix due to its poor shot-to-shot and poor spot-to-spot reproducibilities. We found that the quantitative precisions improved significantly when DMSO (dimethylsulfoxide) was added to the matrix solution. For lopinavir and ritonavir, currently the most frequently prescribed HIV-1 protease inhibitors, the signal-to-noise ratios improved significantly when potassium iodide was added to the matrix solution. The mean quantitative precisions, expressed as % relative standard deviation, were 6.4% for saquinavir, 7.3% for lopinavir, 8.5% for ritonavir, 11.1% for indinavir, and 7.2% for nelfinavir. The mean quantitative accuracies, expressed as % deviation, were 4.5% for saquinavir, 6.0% for lopinavir, 5.9% for ritonavir, 6.6% for indinavir, and 8.0% for nelfinavir. The concentrations measured for the individual quality control samples were all within 85-117% of the theoretical concentrations. The lower limits of quantification in cell lysates were 4 fmol/microL for saquinavir, 16 fmol/microL for lopinavir, 31 fmol/microL for ritonavir, and 100 fmol/microL for indinavir and nelfinavir. The mean mass accuracies for the protease inhibitors were 0.28 ppm using external calibration. Our results show that MALDI-FTICR mass spectrometry can be successfully used for precise, accurate, and selective quantitative analyses of HIV-1 protease inhibitors in cell lysates. In addition, the lower limits of quantification obtained allow clinical applications of the technique.

Biomedical application of MALDI mass spectrometry for small‐molecule analysis

“…leads to an observed optimum affinity for K þ (van Kampen et al, 2008aKampen et al, , 2009a. The optimum affinity for K þ is indeed the case when 2,5-DHB is used as the matrix.…”

“…Dwell time of the ions does not contribute significantly to the analysis time, although the detection time of ions can take second(s) per scan in Fourier-transform ion cyclotron resonance (FTICR) MS and Orbitrap MS. With a high repetition rate laser that fires at 1,000-2,000 Hz, an averaged mass spectrum of high quality can be obtained in a few seconds with MALDI-time-of-flight (TOF) MS or MALDI-triple quadrupole MS (Hatsis et al, 2003;McLean, Russell, & Russell, 2003;Moskovets et al, 2006;Rathore et al, 2008). For quantitative analysis of, for instance, HIV protease inhibitors, analysis times for a single spot on the target plate have been reported of $3 min for MALDI-FTICR MS (20 Hz laser), $30 sec for MALDI-TOF MS (50 Hz laser), and $5 sec for MALDI-triple quadrupole MS (1,000 Hz laser; van Kampen et al, 2006Kampen et al, , 2008aKampen et al, ,b, 2009b. Recently, an analysis time of 1.75 min for an entire 384-well target plate was reported for an assay to screen for small-molecule inhibitors of enzymes with MALDI-triple quadrupole MS (Rathore et al, 2008).…”

“…When MALDI-FTICR is used for small-molecule analysis, a LMW matrix might be preferred over a high MW matrix (van Kampen et al, 2008a). Attempts to use the high MW matrix F20TPP for MALDI-FTICR analysis showed extensive fragmentation of F20TPP, which resulted in matrix-derived peaks in the low mass range.…”

“…We have shown an improved precision for the analysis of HIV-1 protease inhibitors with 2,5-DHB as matrix by adding a small amount of dimethylsulfoxide to the sample/matrix mixture (van Kampen et al, 2008a). Although the deposited sample/ matrix solutions dry at a much slower rate, homogeneous sample surfaces are obtained.…”

“…The correlation coefficients for the calibration curves also improved when ILM were used. Other properties of ILM compared to their solid counterparts are that slightly increased laser fluences are needed for ionization, and that ILM favor formation of sodium and potassium adducts (Mank, Stahl, & Boehm, 2004;ZabetMoghaddam, Heinzle, & Tholey, 2004); those properties can be exploited for analytes with poor protonation (Gouw et al, 2002;van Kampen et al, 2006van Kampen et al, , 2008avan Kampen et al, , 2009b. For further information on ILM, see the review of .…”