Hakala Kati S, Kostiainen Risto, Ketola Raimo A
Drug Discovery and Development Technology Center (DDTC), Faculty of Pharmacy, P. O. Box 56, FI-00014 University of Helsinki, Finland.
Rapid Commun Mass Spectrom. 2006;20(14):2081-90. doi: 10.1002/rcm.2562.
The purpose of the study was to determine the advantages of different mass spectrometric instruments and commercially available metabolite identification programs for metabolite profiling. Metabolism of tramadol hydrochloride and the excretion of it and its metabolites into human urine were used as a test case because the metabolism of tramadol is extensive and well known. Accurate mass measurements were carried out with a quadrupole time-of-flight mass spectrometer (Q-TOF) equipped with a LockSpray dual-electrospray ionization source. A triple quadrupole mass spectrometer (QqQ) was applied for full scan, product ion scan, precursor ion scan and neutral loss scan measurements and an ion trap instrument for full scan and product ion measurements. The performance of two metabolite identification programs was tested. The results showed that metabolite programs are time-saving tools but not yet capable of fully automated metabolite profiling. Detection of non-expected metabolites, especially at low concentrations in a complex matrix, is still almost impossible. With low-resolution instruments urine samples proved to be challenging even in a search for expected metabolites. Many false-positive hits were obtained with the automated searching and manual evaluation of the resulting data was required. False positives were avoided by using the higher mass accuracy Q-TOF. Automated programs were useful for constructing product ion methods, but the time-consuming interpretation of mass spectra was done manually. High-quality MS/MS spectra acquired on the QqQ instrument were used for confirmation of the tramadol metabolites. Although the ion trap instrument is of undisputable benefit in MS(n), the low mass cutoff of the ion trap made the identification of tramadol metabolites difficult. Some previously unreported metabolites of tramadol were found in the tramadol urine sample, and their identification was based solely on LC/MS and LC/MS/MS measurements.
本研究的目的是确定不同质谱仪器和市售代谢物鉴定程序在代谢物谱分析方面的优势。将盐酸曲马多的代谢及其在人尿中的排泄作为测试案例，因为曲马多的代谢广泛且为人熟知。使用配备LockSpray双电喷雾电离源的四极杆飞行时间质谱仪（Q-TOF）进行精确质量测量。应用三重四极杆质谱仪（QqQ）进行全扫描、产物离子扫描、前体离子扫描和中性丢失扫描测量，并使用离子阱仪器进行全扫描和产物离子测量。测试了两个代谢物鉴定程序的性能。结果表明，代谢物程序是节省时间的工具，但尚未能够实现完全自动化的代谢物谱分析。检测意外代谢物，尤其是在复杂基质中低浓度的代谢物，仍然几乎不可能。对于低分辨率仪器，即使在寻找预期代谢物时，尿液样本也被证明具有挑战性。通过自动搜索获得了许多假阳性结果，因此需要对所得数据进行人工评估。使用更高质量精度的Q-TOF避免了假阳性。自动化程序对于构建产物离子方法很有用，但质谱图的耗时解释是人工完成的。在QqQ仪器上获得的高质量MS/MS谱图用于确认曲马多代谢物。尽管离子阱仪器在MS(n)方面具有无可争议的优势，但离子阱的低质量截止使得曲马多代谢物的鉴定变得困难。在曲马多尿液样本中发现了一些先前未报道的曲马多代谢物，它们的鉴定仅基于LC/MS和LC/MS/MS测量。
