Absolute Quantification of Rifampicin by MALDI Imaging Mass Spectrometry Using Multiple TOF/TOF Events in a Single Laser Shot

Abstract

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the visualization of molecular distributions within tissue sections. While providing excellent molecular specificity and spatial information, absolute quantification by MALDI IMS remains challenging. Especially in the low molecular weight region of the spectrum, analysis is complicated by matrix interferences and ionization suppression. Though tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity and improve sensitivity by eliminating chemical noise, typical MALDI MS/MS modalities only scan for a single MS/MS event per laser shot. Herein, we describe TOF/TOF instrumentation that enables multiple fragmentation events to be performed in a single laser shot, allowing the intensity of the analyte to be referenced to the intensity of the internal standard in each laser shot while maintaining the benefits of MS/MS. This approach is illustrated by the quantitative analyses of rifampicin (RIF), an antibiotic used to treat tuberculosis, in pooled human plasma using rifapentine (RPT) as an internal standard. The results show greater than 4-fold improvements in relative standard deviation as well as improved coefficients of determination (R2) and accuracy (>93% quality controls, <9% relative errors). This technology is used as an imaging modality to measure absolute RIF concentrations in liver tissue from an animal dosed in vivo. Each microspot in the quantitative image measures the local RIF concentration in the tissue section, providing absolute pixel-to-pixel quantification from different tissue microenvironments. The average concentration determined by IMS is in agreement with the concentration determined by HPLC-MS/MS, showing a percent difference of 10.6%.

Multiple Time-of-Flight/Time-of-Flight Events in a Single Laser Shot for Improved Matrix-Assisted Laser Desorption/Ionization Tandem Mass Spectrometry Quantification

“…40 This instrument is equipped with a 349 nm, diode-pumped, frequency-tripled Nd:YLF laser capable of laser repetition rates up to 5 kHz (Spectra-Physics, Santa Clara, CA). The laser energy for all experiments was ~66 μ J/pulse (as measured prior to attenuation).…”

Chemical and topographical 3D surface profiling using atmospheric pressure LDI and MALDI MS imaging

“…Over the last decade the analytical sensitivity 9 and the acquisition speed 10 for MALDI MSI instruments improved dramatically. As a consequence, new fields of application for MALDI MSI evolved in chemistry 11,12 , biology 13 , medicine 14 and life sciences 15,16 .…”

A Quantitative MALDI-MSI Study of the Movement of Molecules in Biological Systems

“…One approach could concern the normalisation strategy to adopt. Over the past year, the normalisation to a stable isotope internal standard has been shown to increase the quantitative capabilities of MSI analysis (Pirman and Yost, 2011;Prentice, Chumbley and Caprioli, 2017). In this study, in the absence of an internal standard, the MSI data were normalised to total ion current (TIC).…”

“…Multiple studies have shown how normalisation to an internal standard increases the quantitative capabilities of MSI analysis (Pirman and Yost, 2011;Prentice, Chumbley and Caprioli, 2017). The internal standard is a molecule with chemical and physical characteristics similar to analytes under study.…”

“…Over the past years, the employment of an internal standard has been demonstrated to increase the quantitative capabilities of MSI analysis (Pirman and Yost, 2011;Prentice, Chumbley and Caprioli, 2017). The choice of an appropriate internal standard represents a crucial aspect for a successful MALDI quantitative investigation (Wilkinson et al, 1997).…”