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  • Fast targeted analysis of 132 acidic and neutral drugs and poisons in whole blood using LC-MS/MS.

Fast targeted analysis of 132 acidic and neutral drugs and poisons in whole blood using LC-MS/MS.

Forensic science international (2014-05-06)
Matthew Di Rago, Eva Saar, Luke N Rodda, Sophie Turfus, Alex Kotsos, Dimitri Gerostamoulos, Olaf H Drummer
ABSTRACT

The aim of this study was to develop an LC-MS/MS based screening technique that covers a broad range of acidic and neutral drugs and poisons by combining a small sample volume and efficient extraction technique with simple automated data processing. After protein precipitation of 100μL of whole blood, 132 common acidic and neutral drugs and poisons including non-steroidal anti-inflammatory drugs, barbiturates, anticonvulsants, antidiabetics, muscle relaxants, diuretics and superwarfarin rodenticides (47 quantitated, 85 reported as detected) were separated using a Shimadzu Prominence HPLC system with a C18 separation column (Kinetex XB-C18, 4.6mm×150mm, 5μm), using gradient elution with a mobile phase of 25mM ammonium acetate buffer (pH 7.5)/acetonitrile. The drugs were detected using an ABSciex(®) API 2000 LC-MS/MS system (ESI+ and -, MRM mode, two transitions per analyte). The method was fully validated in accordance with international guidelines. Quantification data obtained using one-point calibration compared favorably to that using multiple calibrants. The presented LC-MS/MS assay has proven to be applicable for determination of the analytes in blood. The fast and reliable extraction method combined with automated processing gives the opportunity for high throughput and fast turnaround times for forensic and clinical toxicology.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Metronidazole, BioXtra
Sigma-Aldrich
Trimethoprim, ≥98.5%
Sigma-Aldrich
Sodium hydroxide solution, 1.0 N, BioReagent, suitable for cell culture
Sigma-Aldrich
Phenylbutazone
Sigma-Aldrich
Chlorothiazide, thiazide diuretic
Sigma-Aldrich
Cyclobenzaprine hydrochloride
Sigma-Aldrich
Ipratropium bromide monohydrate, ≥98% (TLC), powder
Supelco
Metronidazole, analytical standard
Supelco
Prednisolone, VETRANAL®, analytical standard
Sigma-Aldrich
Salicylic acid, meets analytical specification of Ph. Eur., BP, USP, 99.5-100.5% (calc. to the dried substance)
Sigma-Aldrich
Ammonia, puriss., anhydrous, ≥99.95%
Sigma-Aldrich
Salicylic acid, ≥99%, FG
Sigma-Aldrich
Trimethoprim, ≥99.0% (HPLC)
Supelco
Trimethoprim, VETRANAL®, analytical standard
Supelco
Metronidazole, VETRANAL®, analytical standard
Sigma-Aldrich
Ammonia-14N, 99.99 atom % 14N
Supelco
Salicylic acid, 1.0 mg/mL in acetonitrile, ampule of 1 mL, certified reference material, Cerilliant®
Salicylic acid, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Methanol, HPLC Plus, ≥99.9%
Sigma-Aldrich
Methanol, suitable for HPLC, gradient grade, suitable as ACS-grade LC reagent, ≥99.9%
USP
Chlorothiazide, United States Pharmacopeia (USP) Reference Standard
Valproic acid for system suitability, European Pharmacopoeia (EP) Reference Standard
Metronidazole, European Pharmacopoeia (EP) Reference Standard
Prednisolone, British Pharmacopoeia (BP) Assay Standard
Sigma-Aldrich
Sodium hydroxide, reagent grade, 97%, powder
Sigma-Aldrich
Methanol, ACS reagent, ≥99.8%
Sigma-Aldrich
Methanol, Absolute - Acetone free
Cyclobenzaprine hydrochloride, European Pharmacopoeia (EP) Reference Standard
USP
Trimethoprim, United States Pharmacopeia (USP) Reference Standard
Supelco
Chlorothiazide, Pharmaceutical Secondary Standard; Certified Reference Material