Yan Zhengyin, Caldwell Gary W, Maher Noureddine
Division of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, LLC, Spring House, Pennsylvania 19477, USA.
Anal Chem. 2008 Aug 15;80(16):6410-22. doi: 10.1021/ac800887h. Epub 2008 Jul 19.
Constant neutral loss (CNL) and precursor ion (PI) scan have been widely used for the in vitro screening of glutathione conjugates derived from reactive metabolites, but these two methods are only applicable to triple quadrupole or hybrid triple quadrupole mass spectrometers. Additionally, the success of CNL and PI scanning largely depends on structure and CID fragmentation pathways of GSH conjugates. In the present study, a highly efficient methodology has been developed as an alternative approach for high-throughput screening and structural characterization of reactive metabolites using the linear ion trap mass spectrometer. In microsomal incubations, a mixture of glutathione [GSH, gamma-glutamyl-cystein-glycin] and the stable-isotope labeled compound [GSX, gamma-glutamyl-cystein-glycin-(13)C2-(15)N] was used to trap reactive metabolites, resulting in formation of both labeled and unlabeled conjugates at a given isotopic ratio. A mass difference of 3.0 Da between the natural and labeled GSH conjugate (mass tag) at a fixed isotopic ratio constitutes a unique mass pattern that can selectively trigger the data-dependent MS(2) scan of both isotopic partner ions, respectively. In order to eliminate the response bias of GSH adducts in the positive and negative mode, a polarity switch is executed between the mass tag-triggered data dependent MS(2) scan, and thus ESI- and ESI+ MS(2) spectra of both labeled and nonlabeled GSH conjugates are obtained in a single LC-MS run. Unambiguous identification of glutathione adducts was readily achieved with great confidence by MS(2) spectra of both labeled and unlabeled conjugates. Reliability of this method was vigorously validated using several model compounds that are known to form reactive metabolites. This approach is not based on the appearance of a particular product ion such as MH(+) - 129 and anion at m/z 272, whose formation can be structure-dependent and sensitive to the collision energy level; therefore, the present method can be suitable for unbiased screening of any reactive metabolites, regardless of their CID fragmentation pathways. Additionally, this methodology can potentially be applied to triple quadrupole or hybrid triple quadrupole mass spectrometers.
恒定中性丢失（CNL）和前体离子（PI）扫描已被广泛用于体外筛选由活性代谢物衍生的谷胱甘肽缀合物，但这两种方法仅适用于三重四极杆或混合三重四极杆质谱仪。此外，CNL和PI扫描的成功很大程度上取决于谷胱甘肽缀合物的结构和碰撞诱导解离（CID）碎裂途径。在本研究中，已开发出一种高效方法，作为使用线性离子阱质谱仪对活性代谢物进行高通量筛选和结构表征的替代方法。在微粒体孵育中，使用谷胱甘肽[GSH，γ-谷氨酰-半胱氨酰-甘氨酸]和稳定同位素标记化合物[GSX，γ-谷氨酰-半胱氨酰-甘氨酸-(13)C2-(15)N]的混合物捕获活性代谢物，以给定的同位素比率形成标记和未标记的缀合物。在固定同位素比率下，天然和标记的谷胱甘肽缀合物（质量标签）之间3.0 Da的质量差构成独特的质量模式，可分别选择性触发两个同位素伙伴离子的数据依赖型MS(2)扫描。为了消除谷胱甘肽加合物在正模式和负模式下的响应偏差，在质量标签触发的数据依赖型MS(2)扫描之间执行极性切换，从而在一次液相色谱-质谱（LC-MS）运行中获得标记和未标记谷胱甘肽缀合物的ESI-和ESI+ MS(2)光谱。通过标记和未标记缀合物的MS(2)光谱，很容易以高置信度明确鉴定谷胱甘肽加合物。使用几种已知会形成活性代谢物的模型化合物对该方法的可靠性进行了有力验证。该方法不基于特定产物离子的出现，如m/z 272处的MH(+) - 129和阴离子，其形成可能取决于结构且对碰撞能量水平敏感；因此，本方法可适用于对任何活性代谢物进行无偏筛选，无论其CID碎裂途径如何。此外，该方法可能适用于三重四极杆或混合三重四极杆质谱仪。
