Holcapek M, Kolárová L, Nobilis M
Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Nám. Cs. Legií 565, 53210, Pardubice, Czech Republic.
Anal Bioanal Chem. 2008 May;391(1):59-78. doi: 10.1007/s00216-008-1962-7. Epub 2008 Mar 15.
Applications of tandem mass spectrometry (MS/MS) techniques coupled with high-performance liquid chromatography (HPLC) in the identification and determination of phase I and phase II drug metabolites are reviewed with an emphasis on recent papers published predominantly within the last 6 years (2002-2007) reporting the employment of atmospheric pressure ionization techniques as the most promising approach for a sensitive detection, positive identification and quantitation of metabolites in complex biological matrices. This review is devoted to in vitro and in vivo drug biotransformation in humans and animals. The first step preceding an HPLC-MS bioanalysis consists in the choice of suitable sample preparation procedures (biomatrix sampling, homogenization, internal standard addition, deproteination, centrifugation, extraction). The subsequent step is the right optimization of chromatographic conditions providing the required separation selectivity, analysis time and also good compatibility with the MS detection. This is usually not accessible without the employment of the parent drug and synthesized or isolated chemical standards of expected phase I and sometimes also phase II metabolites. The incorporation of additional detectors (photodiode-array UV, fluorescence, polarimetric and others) between the HPLC and MS instruments can result in valuable analytical information supplementing MS results. The relation among the structural changes caused by metabolic reactions and corresponding shifts in the retention behavior in reversed-phase systems is discussed as supporting information for identification of the metabolite. The first and basic step in the interpretation of mass spectra is always the molecular weight (MW) determination based on the presence of protonated molecules M+H and sometimes adducts with ammonium or alkali-metal ions, observed in the positive-ion full-scan mass spectra. The MW determination can be confirmed by the M-H ion for metabolites providing a signal in negative-ion mass spectra. MS/MS is a worthy tool for further structural characterization because of the occurrence of characteristic fragment ions, either MS( n ) analysis for studying the fragmentation patterns using trap-based analyzers or high mass accuracy measurements for elemental composition determination using time of flight based or Fourier transform mass analyzers. The correlation between typical functional groups found in phase I and phase II drug metabolites and corresponding neutral losses is generalized and illustrated for selected examples. The choice of a suitable ionization technique and polarity mode in relation to the metabolite structure is discussed as well.
综述了串联质谱（MS/MS）技术与高效液相色谱（HPLC）联用在I相和II相药物代谢产物鉴定与测定中的应用，重点关注主要在过去6年（2002 - 2007年）发表的近期论文，这些论文报道了采用大气压电离技术作为在复杂生物基质中灵敏检测、准确鉴定和定量代谢产物的最有前景的方法。本综述致力于人类和动物体内外的药物生物转化。HPLC - MS生物分析的第一步在于选择合适的样品制备程序（生物基质采样、匀浆、添加内标、去蛋白、离心、萃取）。后续步骤是正确优化色谱条件，以提供所需的分离选择性、分析时间，并与MS检测具有良好的兼容性。如果不使用母体药物以及预期的I相有时还有II相代谢产物的合成或分离化学标准品，通常无法实现这一点。在HPLC和MS仪器之间加入额外的检测器（光电二极管阵列紫外、荧光、偏振光等）可产生有价值的分析信息，补充MS结果。讨论了代谢反应引起的结构变化与反相系统中保留行为相应变化之间的关系，作为代谢产物鉴定的辅助信息。质谱解释的第一步也是基本步骤始终是基于在正离子全扫描质谱中观察到的质子化分子M + H以及有时与铵或碱金属离子的加合物来确定分子量（MW）。对于在负离子质谱中产生信号的代谢产物，MW测定可通过M - H离子来确认。由于特征碎片离子的出现，MS/MS是进一步进行结构表征的有价值工具，对于使用基于阱的分析仪研究裂解模式的MS(n)分析，或对于使用基于飞行时间或傅里叶变换质谱仪进行元素组成测定的高质量精度测量。还概括并举例说明了I相和II相药物代谢产物中发现的典型官能团与相应中性丢失之间的相关性。还讨论了根据代谢产物结构选择合适的电离技术和极性模式。
