Determination of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in hemoglobin using on-line coupling of restricted access material to liquid chromatography–mass spectrometry

A new method for the analysis of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in hemoglobin has been developed. The method is based on a protein hydrolysis with hydrochloric acid to release the bound PhIP followed by purification and preconcentration steps. The sample clean-up consisted of an ultrafiltration of the hydrolyzed protein and preconcentration of PhIP by freeze-drying. An ultimate cleaning step of the sample using restricted access material (RAM) coupled to liquid chromatography performs extraction and enrichment of the analyte from the sample matrix. Three different RAM columns were tested, LiChrospher®ADS C4, C8 and C18. Two mass spectrometers (ion trap and triple quadrupole) operating in different modes were also evaluated for the determination of PhIP released from hemoglobin adducts. Quality parameters were established and good precision (relative standard deviation (R.S.D.) < 15%), suitable accuracy (>95%) and low detection limits were reached, up to 0.03 fmol PhIP/mg hemoglobin, when using the triple quadrupole.