Differentiating crack cocaine from regular cocaine in whole blood samples in drugs and driving cases

Abstract

In this study, the procedure for analyzing cocainic drugs and metabolites (anhydroecgnonine, anhydroecgnonine methyl ester, benzoylecgonine, cocaine, cocaethylene, ecgnonine ethyl ester, and ecgnonine methyl ester) in whole blood samples obtained from drugs and driving cases using a mixed mode solid phase extraction (SPE) C8/SCX cartridge (Clean Screen DAU206), is described. This extraction and analysis procedure allows forensic analysts to differentiate between drivers who have used “crack cocaine” against those using regular cocaine. Samples of whole blood (containing deuterated internal standards) were diluted with an aqueous phosphate buffer (pH 6). Each sample was applied to a conditioned SPE column. The filtrate was collected and adjusted to pH 2. The sorbent was rinsed with deionized (DI) water, aqueous hydrochloric acid and methanol. After drying, cocaine, cocathylene, benzoylecgnonine, and anhydroecgnonine methyl ester were eluted from the SPE column with 3 mL of an elution solvent consisting of methylene chloride–isopropanol–ammonium hydroxide. The original filtrate was applied to a second conditioned SPE column and washed with DI water, aqueous hydrochloric acid and methanol. After drying the SPE sorbent, anhydroecgnonine was eluted with methanol containing 4% ammonium hydroxide (3 mL). The eluates were combined and evaporated to dryness, and the residue was dissolved in the mobile phase for analysis by LC-MS/MS in positive multiple reaction monitoring mode (MRM). Chromatography was performed in gradient mode employing a C₁₈ column and a mobile phase consisting of acetonitrile and 0.1% aqueous formic acid. The total run time for each analysis was within five minutes. The limits of quantitation/detection for this method were determined to be 0.5 ng mL⁻¹ and 1.0 ng mL⁻¹ respectively. The method was found to be linear from 1.0 ng mL⁻¹ to 100.0 ng mL⁻¹ (r² > 0.995). The recoveries of the noted cocaine type drugs were found to be greater than 90%. This method was applied to twenty positive cocaine completed drugs and driving cases, it was found that anhydroecgnonine concentrations ranged from 0 ng mL⁻¹ to 23 ng mL⁻¹, anhydroecgonine methyl ester concentrations ranged from 0 ng mL⁻¹ to 66 ng mL⁻¹, while cocaine was found to range from 25 ng mL⁻¹ to 250 ng mL⁻¹, cocaethylene ranged from 0 ng mL⁻¹ to 80 ng mL⁻¹, benzoylecgnonine concentrations were found to be in the range 90 ng mL⁻¹ to 790 ng mL⁻¹, ecgonine methyl ester concentrations ranged from 40 ng mL⁻¹ to 500 ng mL⁻¹ and ecgonine ethyl ester concentrations were found to be in the range 0 ng mL⁻¹ to 180 ng mL⁻¹.