Development of in-tube solid-phase microextraction/liquid chromatography/electrospray ionization mass spectrometry for the analysis of mutagenic heterocyclic amines

In-tube solid-phase microextraction (in-tube SPME) coupled with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) was developed for the analysis of mutagenic heterocyclic amines. LC/MS analyses of heterocyclic amines were initially performed by liquid injection onto a LC column. All heterocyclic amines tested in this study gave very simple ESI mass spectra and strong signals corresponding to [M + H](+) were observed for all heterocyclic amines except for Glu-P-1 and Glu-P-2 ([M + NH(4) - H(2)O](+) ions were observed from these amines). All heterocyclic amines were well separated with a Supelcosil LC-CN column except for A alpha C and Glu-P-2 which consistently co-eluted. In order to optimize the extraction of heterocyclic amines, several in-tube SPME parameters were examined using five representative compounds. The optimum extraction conditions were as follows: 10 aspirate/dispense steps of 30 mu L of sample in 100 mM Tris-HCl (pH 8.5) at the flow rate of 100 mu L min(-1) with Omegawax 250 capillary column. The heterocyclic amines extracted by the capillary column were easily desorbed by aspiration of 30 mu L of methanol prior to injection, and carryover of heterocyclic amine was not observed. The calibration curves obtained from the ratios of heterosyclic amine area counts against that of 4,7,8-TriMeIQx as an internal standard were linear in the range of 5 to 200 ng mL(-1), with correlation coefficients above 0.9976 (n = 18). The detection limits (S/N = 3)were 0.2-3.1 ng mL(-1). This method was successfully applied to the analysis of food samples.