Efficient forensic toxicological screening and quantitation workflow using QTOF LC–MS/MS system

Abstract

Quadrupole time-of-flight (QTOF) mass spectrometry is becoming the desired technology for sensitive and selective screening workflows in a forensic toxicological setting. The technology overcomes many challenges faced when using traditional techniques and more significantly captures all information about the sample in one injection to allow for retrospectively mining the data. Here we describe a new benchtop QTOF system with revolutionary N-optic designed flight path and new, intuitive software for easy adoption of accurate mass technology to forensic testing. Blank human urine was spiked with more than 50 common drugs found in forensics setting at different concentration levels. HPLC separation was performed using the SCIEX ExionLC AC system on a reverse-phase column. Data was collected on a SCIEX X500R QTOF mass spectrometer with SCIEX OS software: TOF – MS survey scan with Information Dependent Acquisition (IDA) – triggering of up to 16 product ion scans; or SWATH® acquisition. For SWATH® acquisition, the precursor isolation window width was varied for each MS/MS experiment. Data processing was also performed in SCIEX OS software with simultaneous identification and quantification being accomplished in single software. Screening: multiple confidence criteria including mass accuracy, retention time, isotope, and most importantly, library matching were used. The availability of MS/MS information for identification was required in this study through library searching the acquired spectra against a SCIEX high resolution forensic library containing MS/MS spectra of 1700 compounds. Excellent true positive rates were accomplished for both acquisition modes (89% with IDA and 98% with SWATH® acquisition). This result is consistent with the expectation that the IDA-MS/MS data acquisition has the risk of occasionally missing acquiring MS/MS. The acquisition of MS/MS data in the screening approach allowed the confident identification of structural isomers that have similar retention times through unique fragment ions and their ratios. Simultaneous Quantification Linearity on the new QTOF system for the majority of compounds tested was four orders linear dynamic range covering 0.1 to 1000 ng/mL. SWATH® acquisition ensures that all data, including MS/MS, is acquired at all times. Due to the fast scanning speed (up to 100 Hz at 35 K resolution) in MS/MS mode, a fast cycletime (0.5 sec) was used for the 6.5-min LC method that allowed for one TOF-MS scan and 16 MS/MS scans that covered 120 to 500 m/z range. A sensitive and selective workflow was developed for toxicological compound screening in a forensic setting using the new X500R QTOF system. The newly designed software that accompanies the new benchtop QTOF instrument allows for simultaneous compound identification and quantification in one step all in one place. SCIEX OS software allows the easy adoption of accurate mass technology for routine forensic workflows.