High-performance liquid chromatography–tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites

Abstract

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)(2003)(2004)(2005)(2006)(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.

Identification of the absorbed components and metabolites in rat plasma after oral administration of Rhizoma Chuanxiong decoction by HPLC–ESI-MS/MS

“…For example, glucuronidation, sulfation and conjugation with l-glutamine cause 176, 80, 129 shifts of exact mass, respectively. Based on above metabolism rule of drugs [27] and MWs of the absorbed components, the MWs of probable metabolites were firstly postulated. Then EICs of the protonated molecular ion of probable metabolites were obtained from dosed rat plasma and blank rat plasma, and compared successively.…”

Editorial: Drug Metabolism and Toxicology

“…Liquid chromatography-mass spectrometry (LC-MS) is the most popular and versatile analytical technique which has been widely used for the identification of trace levels of drugs and their metabolites in various biological matrices due to its high sensitivity [9][10][11][12]. Several strategies, such as MS/MS, MS n , on-line hydrogen/ deuterium exchange experiments, accurate mass measurements, softwares dedicated to the metabolite prediction (in silico tools), chemical derivatization and radio-labeling of parent drugs were used for structural characterization of metabolites [9][10][11][12][13][14][15]. …”

Analytical Methods for Quantification of Drug Metabolites in Biological Samples

“…Glutathione conjugation +C10H15O6N3S +305 Table 1. The nominal mass changes in biotransformation of drugs by common metabolic reactions [28,34] …”

“…Twin peaks of metabolites with known mass difference [14] In case of reactive metabolite studies there are typical approaches to identify glutathione conjugates: increased mass shift 305 Da according to the parent, constant neutral loss of pyroglutamic acid (m/z 129) in the positive ionization mode and/or precursor ion of m/z 272 in the negative ionization mode [34,38]. Recently, an in vitro bioactivation study using these identification approaches has confirmed that bazedoxifene does not show the formation of glutathione conjugates compared to raloxifene what offers an improved safety profile of this third generation drug relative to other available SERMs [39].…”

“…Firstly, in general metabolites are much more hydrophilic than parent drug, especially glucuronides [34]. That fact has represented a hindrance for direct metabolite determination because chromatographic separation between these polar analytes and interfering matrix components could not be achieved in many cases.…”