Column-switching high-performance liquid chromatography–electrospray ionization mass spectrometry for identification of heroin metabolites in human urine

In order to prove heroin (DAM) use, a simple, rapid and sensitive analytical method has been established by combining semi-microcolumn HPLC, a column switching technique and electrospray ionization mass spectrometry (ESI-MS). Urine samples were directly introduced to the system, and endogenous urinary constituents were removed by using on-line column switching solid-phase extraction with a strong cation-exchange (SCX) cartridge column (2.0 mm I.D.×10 mm). Heroin and its metabolites enriched on the top of the column were then successfully analyzed with excellent separation by use of a SCX semi-microcolumn (1.5 mm I.D.×150 mm), accompanied by ESI mass spectral detection. The proposed conditions are as follows: mobile phase, 10 mM ammonium acetate (pH 6.0)–acetonitrile (30:70, v/v) (for main separation) and 30 mM ammonium acetate (for trapping); flow-rates, 120 μl/min (for main separation) and 200 μl/min (for trapping); capillary voltage, +4.5 kV; cone voltage, 50 V. Linear calibration curves were obtained in the selected ion monitoring (SIM) mode using protonated molecular ions (m/z 370 for DAM, m/z 328 for MAM and m/z 286 for MOR) over the concentration ranges from 10 to 1000 ng/ml for morphine (MOR) and 1–100 ng/ml for DAM and 6-acetylmorphine (MAM). The detection limits were 0.1–3 ng/ml. Upon applying the scan mode, 2–30 ng/ml were the detection limits. The present HPLC–ESI-MS method was successfully applied to the determination of opiates in users’ urine samples.