Development and validation of a liquid chromatography–tandem mass spectrometry assay for the analysis of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and its metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) in plasma, urine, bile, intestinal contents, faeces and eight selected tissues from mice
Introduction
Heterocyclic amines are a large group of carcinogenic compounds found in proteinaceous foods such as cooked meats and fish, coffee, alcohol beverages, cigarette smoke, air, river and rainwater [1]. The most abundant of these heterocyclic amines, 2-amino-1-methyl-6-phenylimidazo[4-5-b]pyridine (PhIP), has been shown to specifically induce tumors of the colon, breast and prostate in rodents [2], [3], [4]. PhIP is formed as a by-product of the Maillard reaction during cooking or frying of protein-rich foods at high temperatures [5]. Shioya et al. suggested phenylalanine, creatinine and glucose to be the most probable precursors of PhIP [6]. For PhIP to exert its carcinogenic effect, a multiple metabolism pathway has been suggested [7], [8], [9], starting with N-hydroxylation at the amine group by CYP1A1 and CYP1A2 [10], [11], [12] resulting in 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP).
The hydroxyl group can be sulphated by SULT1A1 enzymes [13], [14], [15], [16], [17] or acetylated by NAT1 or NAT2 enzymes [13], [17], [18]. Heterolytic cleavage of this sulphate or acetate group results in a reactive radical cation which can form adducts with DNA purines [19], [20]. PhIP and N-OH-PhIP are precursors for this multiple metabolism pathway which stresses the importance of the ability to quantify PhIP and N-OH-PhIP in biological samples in order to understand the distribution of PhIP and the formation and distribution of its carcinogenic metabolite N-OH-PhIP.
The enzymes that are mainly responsible for the metabolism of PhIP are found in different organs and therefore biotransformation of PhIP is not restricted to specific tissues such as the liver [10], [21], [22], [23], [24], [25], [26]. As it has been shown that PhIP induces tumors in numerous organs such as the colon, breast and prostate, the distribution profile of PhIP may be very relevant. This could provide further insight into the carcinogenic potential of PhIP.
A partially validated assay for the analysis of PhIP and N-OH-PhIP in human liver was published in 2001 by Prabhu et al. [27]. A fully validated bioanalytical liquid chromatography–tandem mass spectrometry method for the quantification of PhIP and its metabolite N-OH-PhIP in plasma, urine, bile, faeces, intestinal contents and eight selected tissues from mice has, to the best of our knowledge, not been published hitherto. We designed such an assay with the purpose to support preclinical toxicokinetic studies.
Section snippets
Chemicals
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its deuterated internal standard 2-amino-1-(trideuteromethyl)-6-phenylimidazo[4,5-b]pyridine (PhIP-D3) were purchased from Toronto Research Chemicals (North York, Ontario, Canada). 2-Hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine was purchased from the National Cancer Institute Chemical Carcinogen Reference Standard Repository at the Midwest Research Institute (Kansas City, USA). 2-Amino-1-methyl-6-phenylimidazo[4,5-b]-5-hydroxypyridine
Linearity
Eight non-zero calibration standards in plasma, urine and 4% (m/v) BSA were prepared in duplicate for each run and analyzed in three independent runs. Calibration curves (area ratio with the internal standard versus nominal concentration) were fitted by least-squares linear regression using the reciprocal of the squared concentration (1/x2) as a weighting factor. To assess linearity, deviations from the mean calculated concentrations over three runs should be within ±15% of nominal
HPLC-MS/MS
To obtain high sensitivity, a triple quadrupole mass spectrometer was chosen as a detector. An analytical column was selected with a stationary phase endcapped with polar groups to provide retention of both hydrophobic as well as hydrophilic compounds.
The protonated molecular ions ([M+H]+) were used as precursor ions to generate product-ion spectra (Fig. 1A and B). The most intense product ions were optimized and used as MRM transitions to ensure high sensitivity and selectivity. Ionization and
Toxicokinetic study
The applicability of the assay was demonstrated by i.v. administration of 1 mg/kg PhIP into the tail vein of a male mouse. After 0.5 h plasma, urine, intestinal contents and tissues were collected and analyzed. Bile was obtained in a separate experiment where gall bladder cannulation in a male mouse was performed. After cannulation, 1 mg/kg PhIP was administered i.v. and bile was collected in 15 min fractions. Faeces were collected for 24 h in a metabolic cage after i.v. administration of 1 mg/kg
Conclusion
We have developed an assay for the sensitive analysis of PhIP and N-OH-PhIP in murine plasma, urine, bile, intestinal contents, faeces and tissue homogenates. The influence of matrix effects on the quantification of analytes is described. Two LC-gradients were developed: a step gradient and a linear gradient.
For the analysis of PhIP, a stable isotope labelled internal standard was available to compensate for matrix effects. Both gradients can be used for the quantitative analysis of this
Acknowledgement
Abadi Gebretensae is acknowledged for his excellent technical assistance.
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