Ultra-fast forensic toxicological screening and quantitation under 3 minutes with a QTOF HPLC-MS/MS system

Objectives HPLC-MS/MS technology combines separation (HPLC) and detection (MS, and MS/MS) and is widely used for screening applications. Mostly, the duration of HPLC runtime in these studies vary from 5 to 20 min. For high-throughput laboratories, a fast screening method is desired. There are mainly two approaches to acquire fragmentation information for screening purpose of compounds by HPLC-MS/MS: Information-Dependent-Acquisition (IDA) or Data-Dependent-Acquisition (DDA) and MS/MS All . For IDA-MS/MS, a survey scan is performed to collect the information on the precursor ions, followed by multiple dependent MS/MS scans for a number of most abundant precursor/candidate ions. MS/MS All with SWATH ® acquisition is a novel MS/MS All technique. In every data cycle, the instrument acquires TOF-MS information first, and then it sequentially acquires MSMS information of all precursor ions across a specified mass range in pre-divided mass windows. Both of these two data acquisition approaches are compatible with retrospective data interrogation. In this study, we aimed to develop an ultra-fast screening method in a forensic toxicological setting using the SCIEX X500R QTOF system with SCIEX OS software 1.0. We also aim to compare IDA-MS/MS and MS/MS All with SWATH ® acquisition for this fast screening method. Methods Blank human urine samples were spiked with multiple drugs commonly found in forensics setting at different concentration levels. Typically, samples were diluted 10-fold in 10% methanol and centrifuged. The clear supernatants were transferred to auto-sampler vials and 10 μL samples were injected. HPLC separation was on a 20 × 2 mm cartridge-type column with cartridge holder. Mobile phase A was 10 mm ammonium formatted in water and mobile phase B was 0.1% formic acid in methanol. HPLC runtime was 2.5 min Data was acquired, processed and reported in SCIEX OS software 1.0. Results Good separation for all the analytes tested and good retention for polar compounds. Sample set 1 was blank urine samples spiked with more than 85 compounds at different levels (CO1 means cutoff number for Sample set 1). Despite a very short HPLC runtime (less than 3 min), the method showed excellent sensitivity. The calibration curve for one of the analytes (JWH 210 5-OH metabolite) is showed. Sample set 2 were also urine samples with both calibrators (spiked with a different group of 84 compounds, CO 2 means cutoff number for sample set 2) and unknown samples. It showed true positive rate in the calibrators for set 2. No false positive was identified. It showed the true positive rate with different HPLC methods and MS/MS acquisition methods. Conclusion In this study, we have developed a super-fast screening/quantitation method in forensic setting under 3 minutes using the SCIEX X500R QTOF system. Two non-targeted data acquisition methods: IDA-MS/MS and MS/MS All with SWATH ® acquisition were both tested and compared. Depending on the specific requirement of the screening method, both IDA-MS/MS and SWATH® acquisition have their advantage and disadvantage in certain areas and the user should be flexible to adopt either approach with appropriate method settings. For Research Use Only. Not for use in diagnostic procedures.