Gaudreau Éric, Bérubé René, Bienvenu Jean-François, Fleury Normand
Laboratoire du Centre de Toxicologie (CTQ), Institut national de santé publique du Québec (INSPQ), 945, avenue Wolfe, Québec, QC, G1V 5B3, Canada.
Anal Bioanal Chem. 2016 Jun;408(15):4021-33. doi: 10.1007/s00216-016-9491-2. Epub 2016 Apr 20.
Data on the stability of monohydroxy polycyclic aromatic hydrocarbons (OH-PAHs; metabolites of PAHs) in urine are needed in order to effectively study the effects of PAHs in the body, but the relevant data are not available in the literature. Therefore, in this work, we investigated the stability of OH-PAHs in urine. For each OH-PAH studied, the free form (as opposed to the conjugated form) comprised <10 % of the total OH-PAH in urine samples obtained from a normal population, except for 9-OH-phenanthrene (where the free form represented 22.2 % of the total 9-OH-phenanthrene). 1-Naphthol and 9-OH-phenanthrene were found to be less stable in their free forms in urine than in their conjugated forms when the urine samples were stored at 4 °C or room temperature. Free 3-OH-fluoranthene was also very unstable at 4 °C or room temperature. The conjugated forms of the OH-PAHs were more stable than their corresponding free forms. However, the free and conjugated forms of all the OH-PAHs were stable in urine at -20 °C and -80 °C. A freeze and thaw assay also revealed that freezing and thawing had minimal impact on the stability of the OH-PAHs in urine. For the derivatized extracts, storing the samples under an argon atmosphere at 4 °C was found to maintain sample integrity. In order to measure the stabilities of 19 hydroxylated metabolites of PAHs in urine, we developed a method with sensitivity in the low pg/mL range using nine labeled internal standards. This method combined enzymatic deconjugation with liquid-liquid extraction, derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), and gas chromatography/tandem mass spectrometry (GC-MS/MS). Graphical abstract Stability of the conjugated forms of the OH-PAHs versus free forms (e.g. 1-naphthol).
为了有效研究多环芳烃（PAHs）在体内的作用，需要尿液中单羟基多环芳烃（OH-PAHs；PAHs的代谢产物）稳定性的数据，但文献中尚无相关数据。因此，在本研究中，我们调查了尿液中OH-PAHs的稳定性。对于所研究的每种OH-PAH，除了9-羟基菲（其游离形式占总9-羟基菲的22.2%）外，在从正常人群获得的尿液样本中，游离形式（与结合形式相对）占总OH-PAH的比例小于10%。当尿液样本在4℃或室温下储存时，发现1-萘酚和9-羟基菲的游离形式在尿液中的稳定性低于其结合形式。游离的3-羟基荧蒽在4℃或室温下也非常不稳定。OH-PAHs的结合形式比其相应的游离形式更稳定。然而，所有OH-PAHs的游离和结合形式在-20℃和-80℃的尿液中都是稳定的。冻融试验还表明，冻融对尿液中OH-PAHs的稳定性影响最小。对于衍生化提取物，发现在4℃的氩气气氛下储存样品可保持样品完整性。为了测量尿液中19种PAHs羟基化代谢产物的稳定性，我们开发了一种使用9种标记内标的低pg/mL范围内具有灵敏度的方法。该方法将酶解结合液液萃取、用N-甲基-N-（三甲基硅基）三氟乙酰胺（MSTFA）衍生化以及气相色谱/串联质谱（GC-MS/MS）。图形摘要OH-PAHs结合形式与游离形式（如1-萘酚）的稳定性对比。
