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Quantification of cocaine and cocaine metabolites in dried blood spots from a controlled administration study using liquid chromatography–tandem mass spectrometry

Research output: Contribution to journalArticle

Lars Ambach, Eleanor Menzies, Mark C. Parkin, Andrew Kicman, John R.H. Archer, David M. Wood, Paul I. Dargan, Christophe Stove

Original languageEnglish
Pages (from-to)709-720
Number of pages12
JournalDrug Testing And Analysis
Volume11
Issue number5
DOIs
PublishedMay 2019

King's Authors

Abstract

Cocaine is a common illicit stimulant and is mainly metabolized by hydrolysis to benzoylecgonine (BE) and ecgonine methyl ester (EME), but also to minor metabolites like norcocaine, or hydroxy-BE. When ethanol is present, cocaethylene is formed. Dried blood spot (DBS) sampling is a minimally invasive microsampling technique with possible advantages for analyte stability and ease of storage, making it an attractive matrix in forensic and clinical settings. We developed a liquid chromatography–tandem mass spectrometry-based (LC–MS/MS) method for quantifying cocaine, BE, EME, norcocaine, hydroxy-BE, and cocaethylene in DBS. Six-mm punches were extracted with aqueous buffer followed by protein precipitation, evaporation and reconstitution in mobile phase. Separation was achieved on a Polar-RP column (Phenomenex) in a 6-minute gradient including baseline-separation of norcocaine and BE. For MS detection, a QTRAP 5500 (Sciex) was used in positive electrospray ionization (ESI) multiple reaction monitoring (MRM) mode. The method was validated for selectivity, sensitivity [lower limited of quantification (LLOQ) 1.0–5.0 ng/mL], imprecision (≤13.4%, ≤19.6% at LLOQ), accuracy (≤ ± 14.9%), matrix effects, extraction efficiency (≥20.9%), hematocrit effect, volume spotted, punch location, long-term and autosampler stability. Concentrations in DBS from a controlled cocaine administration study in healthy volunteers were compared to whole blood and plasma. Although concentrations correlated moderately to strongly (Spearman's ρ 0.603–0.958), agreement between paired samples was poor, with overestimation of DBS concentrations and wide confidence intervals in Bland–Altman analysis. A possible cause are differences in capillary and venous blood concentrations, with the underlying mechanism requiring further research before DBS analysis for cocaine and its metabolites can be considered equivalent to whole blood or plasma analysis.

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