Aqueous phase hexylchloroformate derivatization and solid phase microextraction: Determination of benzoylecgonine in urine by gas chromatography-quadrupole ion trap mass spectrometry

A derivatization/solid phase microextraction (SPME) method for the determination of benzoylecgonine in urine was developed. The derivatization is conducted directly in 1 mL of urine while sonicating for 3 min with 12 μL of hexyl chloroformate and 70 μL of a mixture containing acetonitrile:water:hexanol:2-dimethylaminopyridine (5:2:2:1 v/v), yielding benzoylecgonine hexyl ester (BHE) as the product. After the 3 min period, an aliquot of 250 μL is transferred to a vial for SPME. After the desired extraction time the 100 μm polydimethylsiloxane SPME fiber was transferred to the GC-MS for separation and analysis with a quadrupole ion trap mass spectrometer. The hexyl chloroformate derivatization and SPME procedures were optimized for compatibility and sensitivity. The method was found linear for 0.10 to 20.0 μg/mL (r² = 0.999) of benzoylecgonine in urine using benzoylecgonine-d₃ as an internal standard (1.5 μg/mL). Intra-day precisions were 8.8 and 6.8% RSD for 0.30 μg/mL and 17 μg/mL benzoylecgonine standards in urine (n = 6), respectively. Inter-day precision (n = 3) were ≤3.3% RSD, indicating good reproducibility. A detection limit of 0.03 μg/mL (S/N = 3) was achieved, thus making the SPME method a simplified alternative to SPE for GC-MS confirmation after EMIT tests for benzoylecgonine which have a cutoff of 0.30 μg/mL. Quantitative results by SPME and SPE of two clinical urine specimens known positive for cocaine by EMIT were in excellent agreement. Benzoylecgonine was detected by the derivatization/SPME method in 22 out of 22 other urine specimens known positive for cocaine.