3-hydroxybenzoylecgonine and benzoylecgonine

Urinary excretion of ecgonine (EC) was compared to that of cocaine, benzoylecgonine, ecgonine methyl ester and minor metabolites, meta-hydroxybenzoylecgonine, para-hydroxybenzoylecgonine, and norbenzoylecgonine, following controlled administration of oral, intravenous, intranasal, and smoked cocaine. Urine EC concentrations peaked later than all other analytes and had longer detection times than the other minor metabolites. With a 50 ng/mL cutoff concentration and following low doses of 10 to 45 mg cocaine by multiple routes, detection times extended up to 98 h. Maximum concentrations (Cmax) were 6-14 mole % of those for benzoylecgonine, Cmax increased with dose, time to maximum concentration (Tmax) was independent of dose, and route of administration did not have a significant impact on Cmax or Tmax for metabolites. EC is an analyte to consider for identifying cocaine use due to its stability in urine and long detection times.

Topics: Adult; Biomarkers; Cocaine; Gas Chromatography-Mass Spectrometry; Humans; Male; Substance Abuse Detection

Cocaine (COC) is a potent CNS stimulant that is metabolized to benzoylecgonine (BE) and further metabolized to minor metabolites such as m-hydroxybenzoylecgonine (m-HOBE). COC is also metabolized to norcocaine (NC). Cocaethylene (CE) is formed when cocaine and ethyl alcohol are used simultaneously. Anhydroecgonine methyl ester (AEME) is a unique marker following smoked cocaine, and anhydroecgonine ethyl ester (AEEE) is found in cocaine smokers who also use ethyl alcohol. We developed a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection and quantitation of COC, BE, NC, CE, m-HOBE, AEME, and AEEE in urine. Two hundred samples previously analyzed by gas chromatography (GC) coupled with MS were extracted using solid-phase extraction. Chromatographic separation was achieved using a gradient consisting of mobile phase A [20 mM ammonium formate (pH 2.7)] and mobile phase B (methanol/acetonitrile, 50:50), an XDB-C(8) (50 x 2.1 mm, 1.8 microm) column and a flow rate of 270 microL/min. Concentrations were calculated by comparing the peak-area with the internal standard and plotted against a standard curve. The assay displayed linearity from 1.0 to 100 ng/mL. Within- and between-run coefficients of variation were < 10% throughout the linear range. A method comparison between GC-MS and LC-MS-MS showed good correlation for COC (r(2) = 0.982) and BE (r(2) = 0.955). We report here on a sensitive method to identify clinically and forensically relevant cocaine and associated analytes at concentrations as low as 1.0 ng/mL.

Topics: Chromatography, High Pressure Liquid; Cocaine; Cocaine-Related Disorders; Dopamine Uptake Inhibitors; Ethanol; Gas Chromatography-Mass Spectrometry; Humans; Reproducibility of Results; Solid Phase Extraction; Spectrometry, Mass, Electrospray Ionization; Substance Abuse Detection; Tandem Mass Spectrometry

A fully automated system utilizing a liquid handler and an online solid-phase extraction (SPE) device coupled with liquid chromatography-tandem mass spectrometry (LC-MS-MS) was designed to process, detect, and quantify benzoylecgonine (BZE), meta-hydroxybenzoylecgonine (m-OH BZE), para-hydroxybenzoylecgonine (p-OH BZE), and norbenzoylecgonine (nor-BZE) metabolites in human urine. The method was linear for BZE, m-OH BZE, and p-OH BZE from 1.2 to 10,000 ng/mL with limits of detection (LOD) and quantification (LOQ) of 1.2 ng/mL. Nor-BZE was linear from 5 to 10,000 ng/mL with an LOD and LOQ of 1.2 and 5 ng/mL, respectively. The intrarun precision measured as the coefficient of variation of 10 replicates of a 100 ng/mL control was less than 2.6%, and the interrun precision for 5 replicates of the same control across 8 batches was less than 4.8% for all analytes. No assay interference was noted from controls containing cocaine, cocaethylene, and ecgonine methyl ester. Excellent data concordance (R2 > 0.994) was found for direct comparison of the automated SPE-LC-MS-MS procedure and an existing gas chromatography-MS procedure using 94 human urine samples previously determined to be positive for BZE. The automated specimen handling and SPE procedure, when compared to the traditional extraction schema, eliminates the human factors of specimen handling, processing, extraction, and derivatization, thereby reducing labor costs and rework resulting from batch handling issues, and may reduce the number of fume hoods required in the laboratory.

Topics: Automation; Chromatography, Liquid; Cocaine; Humans; Solid Phase Extraction; Tandem Mass Spectrometry

Recently, meta-hydroxybenzoylecgonine (m-OH BE) was identified by gas chromatography-mass spectroscopy during quantitative analysis for cocaine. Identification of m-OH BE in addition to the routinely identified benzoylecgonine by gas chromatography-mass spectroscopy confirmatory assays may increase detection of cocaine-exposed infants and decrease false negative results. However, it is not known whether m-OH BE is derived directly from benzoylecgonine or from hydroxylated cocaine, or whether this metabolite is produced in the fetus or transferred across the placenta from the maternal circulation. We quantitated the recovery of cocaine, benzoylecgonine, and m-OH BE from amniotic fluid, fetal meconium, fetal intestine, and maternal urine for up to 4 days after single dose administration of either cocaine or benzoylecgonine to pregnant time-bred guinea pigs. m-OH BE was recovered from meconium after maternal injections of cocaine and benzoylecgonine. There was no significant detection of m-OH BE from amniotic fluid or intestine and minimal recovery from maternal urine after either cocaine or benzoylecgonine administration. Detection of m-OH BE in meconium increased the identification of in utero exposed guinea pigs, and the greatest yield of m-OH BE from meconium occurred later than that observed for cocaine or benzoylecgonine.

Topics: Amniotic Fluid; Animals; Cocaine; Female; Fetus; Gas Chromatography-Mass Spectrometry; Guinea Pigs; Intestinal Mucosa; Intestines; Maternal-Fetal Exchange; Meconium; Metabolic Clearance Rate; Pregnancy; Tissue Distribution

Meconium has been reported to be a more suitable specimen than maternal or neonatal urine for detecting fetal exposure to cocaine. In a study comparing various immunoassays with gas chromatography/mass spectrometry (GC/MS), several unexplained discrepancies among the assays were noted. Using methanol extracts of meconium samples, an immunoreactive spot that was more polar than benzoylecgonine was detected by thin-layer chromatography (TLC). An extract of this spot analyzed by GC/MS yielded a fragmentation pattern indicative of an aryl hydroxylated benzoylecgonine. Standards of m-hydroxybenzoylecgonine, o-hydroxybenzoylecgonine, and p-hydroxybenzoylecgonine were synthesized; it was determined that m-hydroxybenzoylecgonine had the same retention time and ion ratios as the TLC immunoreactive spot. Furthermore, m-hydroxybenzoylecgonine proved to be immunoreactive. Ten meconium samples immunoreactive for benzoylecgonine were analyzed by GC/MS. Results before and after hydrolysis with beta-glucuronidase (type IX) showed free m-hydroxybenzoylecgonine comprising 59 to 94% of the total m-hydroxybenzoylecgonine and showed total m-hydroxybenzoylecgonine values ranging from 0.2 to 6.3 times as high as benzoylecgonine. Therefore, m-hydroxybenzoylecgonine appears to be a quantitatively important cocaine metabolite in meconium, which is responsible for a significant portion of the discrepancy between benzoylecgonine concentrations in meconium extracts as measured by immunoassay and GC/MS.

Topics: Artifacts; Cocaine; Humans; Immunoassay; Meconium