Biomonitoring of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and its carcinogenic metabolites in urine

Abstract

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine that is produced in cooked meats. The simultaneous analysis of PhIP and its metabolites in human urine is a challenge, because these biomarkers only occur in urine at parts per billion or lower concentrations and must be selectively purifed from thousands of other urinary constituents. We have developed a facile solid-phase extraction method, employing a mixed-mode reverse-phase cation exchange resin, to simultaneously isolate PhIP, its glucuronide conjugates, and the glucuronide conjugates of the genotoxic metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine from the urine of meat eaters. PhIP and its metabolites were quantified by liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS), using a triple stage quadrupole mass spectrometer in the selected reaction monitoring scan mode. The lower limit of quantification (LOQ) of PhIP is 5 parts per trillion (ppt), and the LOQ values for the glucuronide conjugates are 50 ppt, when 25 microL of urine is employed for assay. The extraction scheme is versatile and has been employed to isolate other ring-hydroxylated and glucuronidated metabolites of PhIP, for characterization by LC-ESI/MS/MS.

Figure 1

Major pathways of metabolism of PhIP in experimental laboratory animals and humans.

Figure 2

LC-ESI/MS/MS chromatograms of PhIP metabolites produced with (A) Rat liver microsomes and (B) Human liver microsomes. Both microsomal samples were fortified with UDPGA and cofactors for P450 catalysis (respective t_R: PhIP-N3-Gl 13.6 min; PhIP-N²-Gl 15.5 min; HON-PhIP-N²-Gl 14.9 min; HON-PhIP-N3-Gl 18.6 min). The ion intensity and area counts × 10⁻³ are reported.

Figure 3

LC-ESI/MS/MS product ion spectra of PhIP and its metabolites. (A) PhIP, 4′-HO-PhIP and 5-HO-PhIP @ a CID of 32 eV, and HONH-PhIP and 1-[²H₃C]-HONH-PhIP @ a CID of 20 eV. (B) Isomeric PhIP-N-Gl and HONH-PhIP-N-Gl conjugates and 1-[²H₃C]-HONH-PhIP-N3-Gl @ 32 eV. The ion intensity and area counts × 10⁻³ are reported.

Figure 3

LC-ESI/MS/MS product ion spectra of PhIP and its metabolites. (A) PhIP, 4′-HO-PhIP and 5-HO-PhIP @ a CID of 32 eV, and HONH-PhIP and 1-[²H₃C]-HONH-PhIP @ a CID of 20 eV. (B) Isomeric PhIP-N-Gl and HONH-PhIP-N-Gl conjugates and 1-[²H₃C]-HONH-PhIP-N3-Gl @ 32 eV. The ion intensity and area counts × 10⁻³ are reported.

Figure 4

SRM traces of PhIP and its glucuronide metabolites in human urine before and after consumption of cooked beef. Note, 0.01% HCO₂H replaced the 0.1% HCO₂H that was employed in the analysis of microsomal metabolites presented in Figure 2. The change in acid concentration resulted in shifts of t_R values of metabolites: PhIP-N²-Gl 15.2 min; HON-PhIP-N²-Gl 16.7 min; and HON-PhIP-N3-Gl 20.1 min). The ion intensity and area counts × 10⁻³ are reported.

Figure 5

SRM traces of novel glucuronides of hydroxylated PhIP metabolites in human urine before and after consumption of cooked beef. The novel glucuronide metabolites elute at t_R 13.9 – 14.6 min, the t_R of HON-PhIP-N²-Gl is 16.7 min; and t_R of HON-PhIP-N3-Gl is 20.1 min.

Figure 6

Calibration curves for PhIP, PhIP-N²-Gl, HONH-PhIP-N²-Gl, and HONH-PhIP-N3-Gl.

Scheme 1

Proposed pathways of fragmentation of HOHN-PhIP [M+H]⁺ at m/z 241.1, To form the product ions at m/z 223.1 and 224.1.

Scheme 2

Proposed pathways of fragmentation of PhIP-N3-Gl To produce the fragment ions at m/z 267.2 and 237.2.

Scheme 3

Proposed pathways of fragmentation of HON-PhIP-N3-PhIP [M+H]⁺ at m/z 417.1 To produce the product ions at m/z 241.1 and 225.1, and secondary fragmentations to form the product ions at m/z 223.1 and 224.1 (from HONH-PhIP) and m/z 210.1 (from PhIP).