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Determination of the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human hair by solid-phase extraction and gas chromatography-mass spectrometry

Department of Chemistry, University of Oslo, Kristiania (historical), Oslo, Norway
Chromatographia (Impact Factor: 1.37). 04/2012; 52(7):499-504. DOI: 10.1007/BF02535726

ABSTRACT A method for the determination of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human hair has been developed and validated. Hair samples (200 mg) were dissolved in NaOH (1 M) and PhIP
was isolated by successive solid-phase extraction on a polystyrene-divinylbenzene column and on a silica-based mixed-mode
column with C8 and-SO3
− functional groups. Quantification was performed by gas chromatography-electron-impact ionization high-resolution mass spectrometry
in selected-ion-monitoring mode. The method was validated for determination of PhIP in the concentration range 0.5–25 ng g−1 hair with [2H3]PhIP as internal standard. The limit of quantification was 0.26 ng g−1 hair. Within-day and between-day precision were in the ranges 1–27% and 2–15% relative standard deviation, respectively.
The hair sample used for method validation was found to contain 0.26 ng PhIP g−1 hair.

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    • "- PhIP, 50 ng, 1 ng/ml) was added to aliquots of 3–35 ml, depending on dose level, of the solubilized fur samples, corresponding to 19.8– 231 mg fur. The samples were further purified by solid phase extraction (Hegstad et al. 2000b) on an ENVπ column, followed by a HCX column (International Sorbent Technology, Hengoed, UK). The ENVπ column (6 ml, 500 mg) was preconditioned successively with 5 ml ethyl acetate-2% ammonia in methanol (1:1, v/v), 5 ml methanol and 5 ml water. "
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    ABSTRACT: The purpose of this work was to correlate the amount of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) determined in mouse fur with the number of intestinal tumours induced by PhIP, to further evaluate incorporation of PhIP into hair as a putative exposure biomarker for food mutagens. We have previously shown that PhIP increases intestinal tumourigenesis in C57BL/6J-Min/+ (Multiple Intestinal Neoplasia) mice. Fur was sampled from mice exposed according to various PhIP protocols, and the amount of PhIP in the fur was quantitated by high performance liquid chromatography - mass spectrometry (HPLC-MS). A quantitative incorporation of PhIP in the fur was demonstrated after a single subcutaneous injection of PhIP, and between one and eight PhIP exposures either via direct subcutaneous injections or through breast milk from PhIP-injected dams. However, after higher exposures, the amount of PhIP in the fur appeared to reach saturation. After low exposures to PhIP, the number of intestinal tumours correlated with the amount of PhIP in the fur of individual mice, whereas after higher exposures, the number of tumours was relatively higher than the amounts of incorporated PhIP in the fur. Other factors, e.g. amounts of reactive PhIP metabolites formed, are also determining the number of intestinal tumours. The demonstrated quantitative incorporation of PhIP into mice fur and the correlation with number of intestinal tumours at the lower exposures, indicate that determination of PhIP in human hair may be a suitable biomarker for exposure to dietary PhIP, which is found in human hair in 10-3 lower amounts than in these mice.
    Pharmacology &amp Toxicology 04/2003; 92(3):131-6.
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    • "- PhIP, 50 ng, 1 ng/ml) was added to aliquots of 3–35 ml, depending on dose level, of the solubilized fur samples, corresponding to 19.8– 231 mg fur. The samples were further purified by solid phase extraction (Hegstad et al. 2000b) on an ENVπ column, followed by a HCX column (International Sorbent Technology, Hengoed, UK). The ENVπ column (6 ml, 500 mg) was preconditioned successively with 5 ml ethyl acetate-2% ammonia in methanol (1:1, v/v), 5 ml methanol and 5 ml water. "
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    ABSTRACT: : The purpose of this work was to correlate the amount of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) determined in mouse fur with the number of intestinal tumours induced by PhIP, to further evaluate incorporation of PhIP into hair as a putative exposure biomarker for food mutagens. We have previously shown that PhIP increases intestinal tumourigenesis in C57BL/6J-Min/+ (Multiple Intestinal Neoplasia) mice. Fur was sampled from mice exposed according to various PhIP protocols, and the amount of PhIP in the fur was quantitated by high performance liquid chromatography – mass spectrometry (HPLC-MS). A quantitative incorporation of PhIP in the fur was demonstrated after a single subcutaneous injection of PhIP, and between one and eight PhIP exposures either via direct subcutaneous injections or through breast milk from PhIP-injected dams. However, after higher exposures, the amount of PhIP in the fur appeared to reach saturation. After low exposures to PhIP, the number of intestinal tumours correlated with the amount of PhIP in the fur of individual mice, whereas after higher exposures, the number of tumours was relatively higher than the amounts of incorporated PhIP in the fur. Other factors, e.g. amounts of reactive PhIP metabolites formed, are also determining the number of intestinal tumours. The demonstrated quantitative incorporation of PhIP into mice fur and the correlation with number of intestinal tumours at the lower exposures, indicate that determination of PhIP in human hair may be a suitable biomarker for exposure to dietary PhIP, which is found in human hair in 10−3 lower amounts than in these mice.
    Pharmacology &amp Toxicology 03/2003; 92(3):131-136. DOI:10.1034/j.1600-0773.2003.920305.x
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    • "PhIP (5 pg/ml: 100 ml) was added to aliquots of 2–40 ml of the solubilised hair sample from adult mice (corresponding to 20–250 mg hair) and further treated by solid phase extraction (Hegstad et al. 2000b "
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    ABSTRACT: Mice with different hair pigmentation were studied to evaluate the role of melanin in the incorporation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) into hair. Mice C57BL/6J-c2j/+ (white), C57BL/6J-Ay (yellow), C57L/J (grey), C57BR/cdJ (brown) and C57BL/6J (black) were dosed with PhIP: 7–9 days old (total amount: 0.006 or 0.58 mg/kg b.wt., for 4 days) and adults (total amount 50 mg/kg b.wt. during 8 weeks). Hair was collected either 30 days after the last PhIP administration (new-born mice) or 8 weeks after the first administration (adult mice). PhIP was incorporated into black hair to a greater extent than into brown, grey, yellow and non-pigmented hair. The concentration of PhIP in the hair of new-born mice exposed to 0.58 mg PhIP/kg b.wt. were (mean±S.D.): 328±135 (black), 134±41 (brown), 9.1±1.2 (yellow) and 5.2±1.4 (white) ng/g hair. The PhIP concentrations in the hair of adult mice exposed to 50 mg/kg b.wt. were: 4750±1449 (black), 810±235 (brown), 541±119 (grey), 35.5±4.6 (yellow) and 21.6±8.8 (white) ng/g, and the eumelanin hair concentration in the same animals decreased in a similar pattern. A linear relationship (r2=1.00, P<0.0001) between the relative PhIP incorporation and the eumelanin concentration in hair was found.
    Pharmacology &amp Toxicology 05/2002; 90(6):333 - 337. DOI:10.1034/j.1600-0773.2002.900607.x
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