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

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 chromatographytandem 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, longterm 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.

Do dried blood spots (DBS) have the potential to support result management processes in routine sports drug testing?

“…This information combined with the reported viability of quantifying cocaine and its main metabolites ( e.g. benzoyl ecgonine) in DBS 23–27 could allow adapting a strategy by routine doping controls that utilizes both routine urine sports drug testing samples and DBS. If a urine sample is found to contain cocaine and/or its major metabolite(s) such as e.g.…”

Blood and Plasma Volumetric Absorptive Microsampling (VAMS) Coupled to LC-MS/MS for the Forensic Assessment of Cocaine Consumption

“…Although COC stability in biosamples is usually reported to be low, even in dried microsamples such as DBS [37], several recent studies have found that the drying step significantly increases stability [33][34][35][36]. It should be noted that the different nature of the VAMS support as opposed to the DBS support (synthetic polymer vs. natural cellulose) could have some effect on analyte stability and extraction as well.…”

“…Previous studies performed on DBS observed a lower correlation between capillary blood sampled by VAMS and venous blood. This could be due to the higher overall result variability obtained on DBS, which in turn may be due to several factors, including volume of blood spotted onto the card, blood hematocrit and lack of spot homogeneity [37,51].…”

“…Other papers describe COC, BEG, EME [32], and CET [33,34] determination in capillary or venous whole blood. Some papers deal with the possibility of microsampling for COC and metabolites determination in capillary whole blood, and all of them use some form of DBS for this purpose [33][34][35][36][37]. For obvious reasons, these methods suffer from the general drawbacks of DBS sampling, including the dependence of sampling volume on hematocrit.…”

Detection of cocaine and its metabolites in whole blood and plasma following a single dose, controlled administration of intranasal cocaine

“…By contrast, whole blood is often the only specimen available in forensic investigations, including confirmatory analyses associated with drugs‐driving offences . In a parallel study to that reported here, a liquid chromatography−tandem mass spectrometry‐based method was developed for quantifying cocaine and its metabolites in dried blood spots (DBSs) . In this study, concentrations in DBS were compared to whole blood and plasma where agreement between paired samples was poor, with an overestimation of DBS concentrations.…”