P53 Automated sample clean-up for a comprehensive forensic toxicology urine screening LC-MS/MS method

Abstract

(For research use only, not for use in diagnostic procedures). Here we present a novel beta glucuronidase hydrolysis that hydrolyses urine samples in 30 minutes. This in combination with rapid cleanup of samples using disposable pipette extraction (DPX) automated tips using the Tecan Freedom EVO® Liquid Handling Workstation, allows for high throughput analysis. Spiked Urine with 157 drugs was hydrolysed with IMCS β-Glucuronidase hydrolysis performed in 30 minutes. All DPXWAX- S automated tip preparations were performed using a Tecan Freedom EVO® Liquid Handling Workstation. The LC-MS/MS system consisted of an Eksigent ekspert™ ultraLC 100 interfaced to an AB SCIEX QTRAP® 4500 LC/MS/MS System. LC separation was performed on a Phenomenex Kinetex 2,6 μm C18, 100 Å, 50 × 3 mm column at 40 °C; 15 minute gradient of water and methanol with ammonium formate buffer; 0.4 mL/min flow rate 10 μL injection volume. The data acquisition, processing, and reporting was performed using the Cliquid® software, which enabled the direct import of batch lists from the Freedom Evo® liquid handling system. Upon completion of data acquisition, the software automatically performed quantitation for the 157 analytes included in the method, and automatically generated and printed out reports. The reproducibility of the automated protocol was assessed by preparing and analyzing replicates of each calibration standard, and the measured CVs were less than 15 % over the entire concentration range covered by the assay. The method displayed good linearity for all analytes, with R>0.99. Recoveries for over 60 % of the analytes were greater than 80 %. Not only does method automation eliminate human error, thus increasing reproducibility, it also eliminates subjectivity during data processing, and furthermore has the potential to save a great deal of time. A rapid urine hydrolysis in 30 minutes followed by automated DPX cleanup using the Tecan Freedom EVO® Liquid Handling Workstation has been investigated and has been shown to provide a viable alternative approach to provide automated sample clean-up for a comprehensive forensic toxicology urine screening LC-MS/MS method.