Complete automation of solid-phase extraction with subsequent liquid chromatography-tandem mass spectrometry for the quantification of benzoylecgonine, m-hydroxybenzoylecgonine, p-hydroxybenzoylecgonine, and norbenzoylecgonine in urine--application to a high-throughput urine analysis laboratory.

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.