Determination of 12 commonly found compounds in DUID cases in whole blood using fully automated supported liquid extraction and UHPLC-MS/MS.
J Chromatogr B Analyt Technol Biomed Life Sci
; 1093-1094: 8-23, 2018 Sep 01.
Article
in En
| MEDLINE
| ID: mdl-29980102
ABSTRACT
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 acetateâ¯+â¯heptane (80â¯+â¯20, v/v) as organic solvent was carried out on a Freedom Evo 200 platform from Tecan. An aliquot of 100⯵L whole blood was used. Sample preparation time for 96 samples was 1.5â¯h. Compounds were separated with gradient elution on a C18 column (50â¯×â¯2.1â¯mm, 1.7⯵m) with a mobile phase consisting of 5â¯mM pHâ¯10.2 ammonium formate and methanol. The run time was 4.5â¯min and 1⯵L 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 R2â¯≥â¯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, nâ¯=â¯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 THCâ¯≥â¯50%. PE was satisfactory for all the compounds with low variation in IS response, RSDâ¯≤â¯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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Dronabinol
/
Opiate Alkaloids
/
Automation, Laboratory
/
Driving Under the Influence
/
Amphetamines
Type of study:
Diagnostic_studies
/
Prognostic_studies
Limits:
Humans
Language:
En
Journal:
J Chromatogr B Analyt Technol Biomed Life Sci
Journal subject:
ENGENHARIA BIOMEDICA
Year:
2018
Document type:
Article