Determination of 12 commonly found compounds in DUID cases in whole blood using fully automated supported liquid extraction and UHPLC-MS/MS.

A high-throughput UHPLC-MS/MS method for the most frequently found compounds; tetrahydrocannabinol (THC), amphetamine, methamphetamine, MDMA, clonazepam, diazepam, nordiazepam, oxazepam, alprazolam, nitrazepam, morphine, and codeine, in driving under the influence of drugs (DUID) cases in whole blood, is presented. Automated sample preparation by 96-well supported liquid extraction (SLE) plates with ethyl acetate + heptane (80 + 20, v/v) as organic solvent was carried out on a Freedom Evo 200 platform from Tecan. An aliquot of 100 µL whole blood was used. Sample preparation time for 96 samples was 1.5 h. Compounds were separated with gradient elution on a C18 column (50 × 2.1 mm, 1.7 µm) with a mobile phase consisting of 5 mM pH 10.2 ammonium formate and methanol. The run time was 4.5 min and 1 µL was injected on an Acquity UPLC I-Class system with a Xevo TQS tandem-quadrupole mass spectrometer in multiple-reaction monitoring mode (MRM) from Waters. Isotope labelled, 13C, internal standards (ISs) were used for all compounds except for alprazolam and morphine, which had deuterated analogs. Quantification was carried out with calibrators without whole blood matrix. Full validation was carried out according to international guidelines, and a new approach for evaluation of process efficiency (PE) has been presented. Linear or quadratic weighted (1/x) calibration curves were used with R2 ≥ 0.999. The method showed satisfactory deviations ±16% when compared to the existing methods, and satisfactory agreement with proficiency testing control samples (z-score -1.6 to 1.8, n = 16 samples). The precision, estimated as the relative standard deviation (RSD) of the concentration difference between results from two independent analyses of authentic whole blood samples, was ≤7.2% in antemortem and ≤9.3% in postmortem samples. Recovery was ≥85% for all the compounds, except morphine ≥62% and THC ≥ 50%. PE was satisfactory for all the compounds with low variation in IS response, RSD ≤ 16% (THC 27%) in antemortem samples and ≤34% (THC 66%) in postmortem samples. To the best of our knowledge, this is the first automated 96-well SLE UHPLC-MS/MS method developed for the simultaneous determination of these 12 compounds in whole blood covering the concentration ranges found in forensic samples. The method has been used in routine work during the last ten months, analysing about 9900 antemortem and 1000 postmortem whole blood samples, and has proven to be robust and reliable.

Determination of benzodiazepines in ante-mortem and post-mortem whole blood by solid-supported liquid-liquid extraction and UPLC-MS/MS.

A solid-supported liquid-liquid extraction ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of benzodiazepines commonly found in Norway, for use in cases with suspected driving impairment and autopsy cases by analysis of human whole blood samples. The following compounds were included: alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, lorazepam, midazolam, nitrazepam, nordiazepam (metabolite of diazepam), oxazepam and phenazepam. Aliquots of 500 µL whole blood were added 500 µL of borate buffer pH 11 and extracted by solid-supported liquid-liquid extraction on ChemElut(®) columns using three times 2.5 mL of methyl tert-butyl ether. Deuterated analogues were used as internal standards (IS) for all analytes, except for midazolam, phenazepam and bromazepam which had no commercially available deuterated analogues at the time the method was developed, and therefore used diazepam-d(5), flunitrazepam-d(7) and nitrazepam-d(5), respectively. The analytes were separated using UPLC with a 2.1×100 mm BEH C(18)-column, 1.7 µm particle size, and quantified by MS/MS using multiple reaction monitoring (MRM) in positive mode. Two transitions were used for the analytes and one transition for the IS. The run time of the method was 8 min including equilibration time. The concentrations of the benzodiazepines in the method span a broad range varying from the lowest concentration of 0.005 µM for flunitrazepam to the highest of 20 µM for oxazepam. The calibration curves of extracted whole blood standards were fitted by second-order calibration curves weighted 1/x, with R(2) values ranging from 0.9981 to 0.9998. The intermediate precision had a CV (%) ranging between 2 and 19%. Recoveries of the analytes were from 71 to 96%. The LLOQs for the analytes varied from 0.0006 to 0.075 µM and the LODs from 0.005 to 3.0 nM. Matrix effects were studied by post extraction addition and found to be between 95 and 104% when calculated against an internal standard. A comparison with two other LC-MS methods was performed during method validation. Good correlation was seen for all analytes. The method has been running on a routine basis for several years, and has proven to be very robust and reliable with good results for external quality samples. The method also meets the requirements of the legislative limits for driving under the influence of non-alcohol drugs to be introduced in the Norwegian legislative system from 2012.