Holland Ricky D, Taylor Jason, Schoenbachler Laura, Jones Richard C, Freeman James P, Miller Dwight W, Lake Brian G, Gooderham Nigel J, Turesky Robert J
Division of Chemistry, National Center for Toxicological Research, 3900 NCTR Road, Jefferson, Arkansas 72079, USA.
Chem Res Toxicol. 2004 Aug;17(8):1121-36. doi: 10.1021/tx049910a.
A rapid and facile tandem solvent solid phase extraction method was established to isolate the heterocyclic aromatic amines (HAAs) 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and 2-amino-9H-pyrido[2,3-b]indole from urine. The HAAs were separated by reversed phase liquid chromatography and quantified by electrospray ionization tandem mass spectrometry (ESI/MS/MS) using selected reaction monitoring. The limits of detection and quantitation of these HAAs approached 1-3 and 2-8 pg/mL, respectively, using only 0.3 mL of urine for analysis. Full product ion spectra were acquired to corroborate analyte identities. The pretreatment of urine from human volunteers that had consumed a grilled beef meal with acid or base at 70 degrees C increased the concentration of HAAs by as much as 6-fold, indicating the presence of phase II conjugates of the parent compounds. HAAs containing an N-methylimidazole moiety undergo facile cleavage of the N-methyl group under collision-induced dissociation conditions, and MS/MS analysis in the constant neutral loss scan mode monitoring the transition M + H --> M + H - CH(3)(*) revealed the presence of two other HAAs. 2-Amino-3-methylimidazo[4,5-f]quinoxaline (IQx) was identified by coelution of the analyte with synthetic IQx and by acquisition of the product ion spectrum. The second HAA was present in a relatively high abundance in urine. The molecule had the same nominal mass as 8-MeIQx (MH(+) at m/z 214), and the product ion spectrum was similar to that of 8-MeIQx. This novel HAA was also found in the grilled meat consumed by the volunteers at a concentration of 8 parts per billion. The accurate mass measurement and product ion spectrum of this molecule by ESI quadrupole time-of-flight mass spectrometry revealed that it was an isomer of 8-MeIQx. This tandem solvent solid phase extraction LC/ESI/MS/MS procedure may be used to rapidly assess the daily exposure to a variety of HAAs in urine.
建立了一种快速简便的串联溶剂固相萃取方法，用于从尿液中分离杂环芳香胺（HAA）2-氨基-3,8-二甲基咪唑并[4,5-f]喹喔啉（8-MeIQx）、2-氨基-3,4,8-三甲基咪唑并[4,5-f]喹喔啉、2-氨基-1-甲基-6-苯基咪唑并[4,5-b]吡啶和2-氨基-9H-吡啶并[2,3-b]吲哚。通过反相液相色谱分离HAA，并使用选择反应监测通过电喷雾电离串联质谱（ESI/MS/MS）进行定量。仅使用0.3 mL尿液进行分析时，这些HAA的检测限和定量限分别接近1-3 pg/mL和2-8 pg/mL。采集全产物离子光谱以确证分析物的身份。对食用烤牛肉餐的人类志愿者的尿液在70℃下用酸或碱进行预处理，可使HAA浓度提高多达6倍，表明存在母体化合物的II相缀合物。含有N-甲基咪唑部分的HAA在碰撞诱导解离条件下容易发生N-甲基基团的裂解，在恒定中性损失扫描模式下进行的MS/MS分析监测跃迁M + H --> M + H - CH(3)(*)，揭示了另外两种HAA的存在。通过分析物与合成IQx的共洗脱以及产物离子光谱的采集，鉴定出2-氨基-3-甲基咪唑并[4,5-f]喹喔啉（IQx）。第二种HAA在尿液中的丰度相对较高。该分子的标称质量与8-MeIQx相同（m/z 214处的MH(+)），产物离子光谱与8-MeIQx相似。在志愿者食用的烤肉中也发现了这种新型HAA，浓度为十亿分之八。通过ESI四极杆飞行时间质谱对该分子进行精确质量测量和产物离子光谱分析，表明它是8-MeIQx的异构体。这种串联溶剂固相萃取LC/ESI/MS/MS方法可用于快速评估尿液中多种HAA的每日暴露量。
