A liquid chromatography–tandem mass spectrometry method for the determination of cocaine and metabolites in blood and in dried blood spots collected from postmortem samples and evaluation of the stability over a 3‐month period

Abstract

The aims of this study were (1) to identify and quantify cocaine (COC), benzoylecgonine (BE), ecgonine methyl ester (EME), and cocaethylene (CE) in DBS; (2) to compare dried blood spots (DBSs) analytical results with the routine blood analyses; (3) to monitor analytes stability on DBS within a 3-month period. Eighty-five μL of blood from postmortem cases were put on a card for DBS analysis and kept in the dark, at room temperature. Samples were extracted through solid-phase extraction (SPE) cartridges and injected in the liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The analytical procedure is simple, sensitive, and specific. Limits of detection (LODs) and quantification (LOQs) were calculated at 1.0 and 5.0 ng/mL(g) for COC and CE, and at 0.5 and 2 ng/mL for EME and BE, respectively. Validation parameters fulfilled all the acceptance criteria. Fifty-five postmortem cases were evaluated. Eighteen cases were positive for COC (44-2456 ng/mL) and BE (228-4700 ng/mL), 12 for EME (92-1500 ng/mL), and 11 cases for CE (11-273 ng/mL). Stability was evaluated on 8 cases collected in the period January 2017-January 2018. For each case, 5 DBSs were collected at T0. Four DBSs were analyzed within the 4 following weeks and 1 sample was analyzed after 3 months. The concentrations on DBSs, stored at room temperature, always matched the ones obtained on blood samples kept at -20°C (<20% variation, both at T0 and after 3 months). BE and COC concentrations remained stable after a 3-month storage, EME concentrations slightly increased after 3 weeks in the 2 analyzed samples, while CE provided a less homogeneous stability depending on the sample.

Determination of benzodiazepines in blood and in dried blood spots collected from post‐mortem samples and evaluation of the stability over a three‐month period

“…Though a different SPE procedure could improve the efficiency, we decided not to change it, in order to achieve the best compromise and extract as many compounds as possible with the same procedure. Indeed, thanks to the developed procedure, it is possible to extract many other analytes of forensic interest, as already published in previous papers . A significant matrix effect was observed for desalkylflurazepam.…”

“…As described previously, we decided to analyze the whole blood spot deposed on the paper substrate after deposing an exact amount of blood on the paper in order to minimize the variability of factors such as different hematocrit levels, which could lead to a different diffusion on the filter …”

“…Indeed, thanks to the developed procedure, it is possible to extract many other analytes of forensic interest, as already published in previous papers. 15,28,29 The method allowed the evaluation of 27 different compounds.…”

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

“…If a defined volume of blood is brought onto specimen collection paper, samples with a high HCT will consequently spread out less and form smaller spots whereas low HCT samples will form bigger spots. 23,26,[31][32][33][34][35][36][37][38][39][40] However, these methods include only a few of the metabolites, such as BE 23,38 ; BE and nor-COC 31 ; BE and COC-Et 36 ; BE, EME, and COC-Et 8,26,39 ; or only BE without COC determination. Although in principle full spot analysis can cope with this, this requires volumetric disposition of a blood drop, which is impractical and/or requires the use of specialized devices.…”

“…Although in principle full spot analysis can cope with this, this requires volumetric disposition of a blood drop, which is impractical and/or requires the use of specialized devices. 17,32,39 With the exception of the initial study by Henderson et al, which used a modified radioimmunoassay, 32 all published methods used high performance liquid chromatography (HPLC) coupled to a variety of detectors, such as spectrofluorimetry (Fluo), 36 tandem mass spectrometry (MS/MS), 8,17,26,33,35,39 or high resolution mass spectrometry (HRMS). HCT bias can be circumvented by spotting-defined volumes of blood and analyzing the entire DBS or by using volumetric absorptive microsampling (VAMS).…”