Development of automated in-tube SPME/ LC / MS method for drug analysis

The technique of automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) was developed for the determination of stimulants and β-blockers in biological samples. In-tube SPME is an extraction technique for organic compounds in aqueous samples, in which analytes are extracted from the sample directly into an open tubular capillary column by repeated draw/eject cycles of sample solution. LC/MS analyses of stimulants and β-blockers were initially performed by liquid injection onto a LC column. Five stimulants and nine β-blockers tested in this study gave very simple ESI mass spectra, and strong signals corresponding to [M + H]⁺ were observed for all drugs. The stimulants were separated with a Supelcosil LC-CN column using acetonitrile/50 mM ammonium acetate (15:85) as a mobile phase. The β-blockers were separated with a Hypersil BDS C₁₈ column using acetonitrile/methanol/water/acetic acid (15:15:70:1) as a mobile phase. In order to optimize the extraction of these drugs, several in-tube SPME parameters were examined. The optimum extraction conditions were 15 draw/eject cycles of 30-35 μL of sample in 50-100 mM Tris-HCl (pH 8.5) at a flow rate of 100 μL/min using Omegawax 250 capillary column (Supelco, Bellefonte, PA). The drugs extracted on the capillary column were easily desorbed by mobile phase flow, and carryover of drugs was not observed. Using in-tube SPME/LC/ESI-MS with selected ion monitoring, the calibration curves of drugs were linear in the range of 2-100 ng/mL with correlation coefficients above 0.9982 (n = 18), and detection limits (S/N = 3) of 0.1-1.2 ng/mL. This technique was successfully applied to the analysis of biological samples, such as urine and serum, without interference peaks. The recoveries of drugs spiked into human urine and serum samples were above 81 and 71%, respectively.