Unbiased high-throughput screening of reactive metabolites on the linear ion trap mass spectrometer using polarity switch and mass tag triggered data-dependent acquisition.
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.
Yan
Zhengyin
Z
Division of Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, LLC, Spring House, Pennsylvania 19477, USA. zyan@prdus.jnj.com
Caldwell
Gary W
GW
Maher
Noureddine
N
D002851:Chromatography, High Pressure Liquid; D003408:Cresols / Q000378:metabolism*; D003627:Data Interpretation, Statistical; D004008:Diclofenac / Q000378:metabolism*; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006846:Hydrocarbons, Halogenated / Q000378:metabolism*; D007211:Indoles / Q000378:metabolism*; D007553:Isotope Labeling; D008862:Microsomes, Liver / Q000378:metabolism*; D000078183:Oxindoles; D021241:Spectrometry, Mass, Electrospray Ionization
D003408:Cresols; D006846:Hydrocarbons, Halogenated; D007211:Indoles; D000078183:Oxindoles; D004008:Diclofenac; C035500:3-methyloxindole; C032538:4-cresol; C030373:2,3-dimethylindole; D005978:Glutathione