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

Abstract

Reliable, feasible analytical methods are needed for forensic and anti-doping testing of cocaine and its most important metabolites, benzoylecgonine, ecgonine methyl ester, and cocaethylene (the active metabolite formed in the presence of ethanol). An innovative workflow is presented here, using minute amounts of dried blood or plasma obtained by volumetric absorptive microsampling (VAMS), followed by miniaturized pretreatment by dispersive pipette extraction (DPX) and LC-MS/MS analysis. After sampling 20 µL of blood or plasma with a VAMS device, the sample was dried, extracted, and loaded onto a DPX tip. The DPX pretreatment lasted less than one minute and after elution with methanol the sample was directly injected into the LC-MS/MS system. The chromatographic analysis was carried out on a C8 column, using a mobile phase containing aqueous formic acid and acetonitrile. Good extraction yield (> 85%), precision (relative standard deviation, RSD < 6.0%) and matrix effect (< 12%) values were obtained. Analyte stability was outstanding (recovery > 85% after 2 months at room temperature). The method was successfully applied to real blood and plasma VAMS, with results in very good agreement with those of fluid samples. The method seems suitable for the monitoring of concomitant cocaine and ethanol use by means of plasma or blood VAMS testing.

Keywords: LC-MS/MS; VAMS; benzoylecgonine; cocaethylene; cocaine; ecgonine methyl ester; microsampling; plasma; whole blood.

Figure 1

Chemical structures of (a) cocaine (COC); (b) benzoylecgonine (BEG); (c) ecgonine methyl ester (EME); (d) cocaethylene (CET).

Figure 2

LC-MS/MS chromatograms of (a) a blank whole blood VAMS sample; (b) a blank plasma VAMS sample spiked with the analytes and the ISs at the concentration of 50 ng/mL; (c) a blank plasma VAMS sample spiked with the analytes at the limit of quantitation (LOQ) values: 1—EME; 2—EME-D₃; 3—BEG; 4—BEG-D₃; 5—COC; 6—COC-D₃; 7—CET; 8—CET-D₃.

Figure 2

LC-MS/MS chromatograms of (a) a blank whole blood VAMS sample; (b) a blank plasma VAMS sample spiked with the analytes and the ISs at the concentration of 50 ng/mL; (c) a blank plasma VAMS sample spiked with the analytes at the limit of quantitation (LOQ) values: 1—EME; 2—EME-D₃; 3—BEG; 4—BEG-D₃; 5—COC; 6—COC-D₃; 7—CET; 8—CET-D₃.

Figure 3

LC-MS/MS chromatogram of a plasma VAMS sample from a COC and ethanol user: 1—EME (12 ng/mL); 2—EME-D₃ (IS, 50 ng/mL); 3—BEG (196 ng/mL); 4—BEG-D₃ (IS, 50 ng/mL); 5—COC (18 ng/mL); 6—COC-D₃ (IS, 50 ng/mL); 7—CET (40 ng/mL); 8—CET-D₃ (IS, 50 ng/mL).

Figure 4

Analyte level comparison between dried micromatrices and fluid plasma.