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

Summary

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+NH₄−H₂O]⁺ ions were observed from these amines). All heterocyclic amines were well separated with a Supelcosil LC-CN column except for AαC 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 μL of sample in 100 mM Tris-HCl (pH 8.5) at the flow the flow rate of 100 μL min⁻¹ with Omegawax 250 capillary column. The heterocyclic amines extracted by the capillary column were easily desorbed by aspiration of 30μL of methanol prior to injection, and carryover of heterocyclic amine was not observed. The calibration curves obtained from the ratios of heterocyclic 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.