A comparison of an optimized automated sample preparation of PEth in blood pretreated by freezing versus manual preparation in whole blood, analyzing by UHPLC – MS/MS

Abstract

A robust and simple high-throughput automatic sample preparation in a 96-well plate format for quantification of PEth16:0/18:1 with UHPLC-MS/MS in pre-treated frozen blood samples has been simplified as well as optimised regarding liquid handling parameters. To keep the air cushion in the pipetting step as small as possible, 300 µl pipette tips were used instead of 1000 µl tips for pipetting the minimum volume of 2-propanol required for the sample preparation. The 96-well plate was mixed vigorously to acquire a good reproducibility of the PEth results. A 96-wellplate with a well volume of 2.0 ml was used to eliminate cross contamination between the wells, and the calibration curve covered a concentration range from 0.03 to 5 µmol/l. The method was sensitive, and the limit of quantification was 0.005 µmol/l for PEth. The matrix effect in whole blood was small and corrected to 106% by using an isotope-labelled internal standard. There was no carry-over observed after the highest standard 5 µmol/l. The intermediate precision and accuracy were, respectively, less than 9% and 101% when using both the automated and manual sample preparation method. 19 external controls were analysed from an Equalis proficiency testing program for a period of 16 months, and our results were always within 79% of the assigned value. 114 samples were analysed by manual and automatic sample preparation with UHPLC-MS/MS in respectively whole blood and pre-treated frozen blood samples. The mean percentage difference of these measurements between the two methods was calculated in accordance with (Bland & Altman) to be (−4.8 ± 5.6) %.