New analytical strategies in studying drug metabolism

Identification and elucidation of the structures of metabolites play major roles in drug discovery and in the development of pharmaceutical compounds. These studies are also important in toxicology or doping control with either pharmaceuticals or illicit drugs. This review focuses on: new analytical strategies used to identify potential metabolites in biological matrices with and without radiolabeled drugs; use of software for metabolite profiling; interpretation of product spectra; profiling of reactive metabolites; development of new approaches for generation of metabolites; and detection of metabolites with increased sensitivity and simplicity. Most of the new strategies involve mass spectrometry (MS) combined with liquid chromatography (LC

The Life Sciences Mass Spectrometry Research Unit

The Life Sciences Mass Spectrometry (LSMS) research unit focuses on the development of novel analytical workflows based on innovative mass spectrometric and software tools for the analysis of low molecular weight compounds, peptides and proteins in complex biological matrices. The present article summarizes some of the recent work of the unit: i) the application of matrix-assisted laser desorption/ionization (MALDI) for mass spectrometry imaging (MSI) of drug of abuse in hair, ii) the use of high resolution mass spectrometry for simultaneous qualitative/quantitative analysis in drug metabolism and metabolomics, and iii) the absolute quantitation of proteins by mass spectrometry using the selected reaction monitoring mode

Mass Spectrometric QUAL/QUAN Approaches for Drug Metabolism and Metabolomics

A liquid chromatography–high-resolution mass spectrometry platform was used for simultaneous qualitative and quantitative (QUAL/QUAN) acquisition, enabling drug metabolism and metabolomics investi- gations. Plasma study samples were monitored for three different groups of patients at a single time-point (1 h after drug administration): one group received acetaminophen (APAP), one group received both APAP and ketorolac and one group was a control group. The quantification of APAP and two of its metabolites (APAP-glucuronide and APAP-cysteine) was performed on a fast acquisition quadrupole-Time-Of-Flight (50–100 ms duty cycle, resolving power of 30,000) compatible with UHPLC time constraints. High-resolution Selected Reaction Monitoring was used for quantification of APAP and its metabolites from 50–10,000 ng/mL using a 50 ?L plasma aliquot. Average measured concentrations were for APAP 6,650 ng/mL vs 6,160 ng/mL, APAP-CYS concentrations were 154.2 ng/mL vs 140.6 ng/mL and APAP-GLU concentrations 8,750 ng/mL vs 8,430 ng/mL between the group that received only APAP (n = 11) and the group that received APAP in combination with ketorolac (n = 11). No major differences were observed between the two groups of patients, as it would be expected due to the differing metabolism pathway for both substances. For the qualitative aspect, a metabolomics data processing platform with biological QC samples was applied to the study samples to search for unanticipated metabolites and biomarkers related to APAP and ketorolac metabolism. Multivariate analysis (i.e. Principle Component Analysis), variables grouping tools (i.e. PCVG) and high-resolution MS(/MS) spectra from the MSALL acquisition strategy enabled the profiling and characterization of circulating metabolites of APAP in plasma such as APAP-sulfate, APAP-mercapturate as well as ketorolac

High-resolution mass spectrometry for integrated qualitative and quantitative analysis of pharmaceuticals in biological matrices

Quantitative and qualitative high-resolution (HR) dependent and independent acquisition schemes on a QqTOF MS (with resolving power 20,000-40,000) were investigated for the analysis of pharmaceutical compounds in biological fluids. High-resolution selected reaction monitoring (HR-SRM) was found to be linear over three orders of magnitude for quantitative analysis of paracetamol in human plasma, offering a real alternative to triple quadrupole LC-SRM/MS. Metabolic stability of talinolol in microsomes was characterized by use of three different acquisition schemes: (i) information-dependent acquisition (IDA) with a TOF MS experiment as survey scan and product-ion scan as dependent scan; (ii) MSALL by collecting TOF mass spectra with and without fragmentation by alternating the collision energy of the collision cell between a low (i.e., 10eV) and high setting (i.e., 40eV); and (iii) a novel independent acquisition mode referred to as "sequential window acquisition of all theoretical fragment-ion spectra” (SWATH) or "global precursor ions scan mode” (GPS) in which sequential precursor ions windows (typically 20 u) are used to collect the same spectrum precursor and fragment ions using a collision energy range. SWATH or GPS was found to be superior to IDA or MSALL in combination with UHPLC for qualitative analysis but requires a rapidly acquiring mass spectrometer. Finally, the GPS concept was used for QUAL/QUAN analysis (i.e. integration of qualitative and quantitative analysis) of bosentan and its metabolites in urine over a concentration range from 5 to 2,500ngmL−

Real-time 2D separation by LC × differential ion mobility hyphenated to mass spectrometry

The liquid chromatography-mass spectrometry (LC-MS) analysis of complex samples such as biological fluid extracts is widespread when searching for new biomarkers as in metabolomics. The success of this hyphenation resides in the orthogonality of both separation techniques. However, there are frequent cases where compounds are co-eluting and the resolving power of mass spectrometry (MS) is not sufficient (e.g., isobaric compounds and interfering isotopic clusters). Different strategies are discussed to solve these cases and a mixture of eight compounds (i.e., bromazepam, chlorprothixene, clonapzepam, fendiline, flusilazol, oxfendazole, oxycodone, and pamaquine) with identical nominal mass (i.e., m/z 316) is taken to illustrate them. Among the different approaches, high-resolution mass spectrometry or liquid chromatography (i.e., UHPLC) can easily separate these compounds. Another technique, mostly used with low resolving power MS analyzers, is differential ion mobility spectrometry (DMS), where analytes are gas-phase separated according to their size-to-charge ratio. Detailed investigations of the addition of different polar modifiers (i.e., methanol, ethanol, and isopropanol) into the transport gas (nitrogen) to enhance the peak capacity of the technique were carried out. Finally, a complex urine sample fortified with 36 compounds of various chemical properties was analyzed by real-time 2D separation LC×DMS-MS(/MS). The addition of this orthogonal gas-phase separation technique in the LC-MS(/MS) hyphenation greatly improved data quality by resolving composite MS/MS spectra, which is mandatory in metabolomics when performing database generation and searc

The Application of Liquid Chromatography and Capillary Zone Electrophoresis Combined with Atmospheric Pressure Ionisation Mass Spectrometry for the Analysis of Pharmaceutical Compounds in Biological Fluids

HPLC coupled to atmospheric pressure ionisation mass spectrometry has almost replaced HPLC assays with UV, fluorescence, or electrochemical detection, due to its enhanced speed, sensitivity, and selectivity, especially when tandem-MS is used. To increase the speed and sensitivity of the drug assays further, high-speed HPLC, multi-component analysis, and µHPLC are used on a routine basis. Sample preparation is recognized as an important issue in bioanalytics. The use of a 96-well plate format with automated liquid-handling systems, off-line and on-line solid-phase extraction or automated liquid-liquid extraction allows to cope with the high sample throughput enabled by LC-MS. Although LC-MS/MS represents the highest standard with respect to sensitivity and selectivity, LC-MS, as a less expensive alternative, is useful in many stages of drug discovery and development. CE-MS and CEC-MS appear to be an attractive alternative to HPLC-MS with respect to separation power, but both are a challenge in application and only seldomly used for the quantification of new drug candidates in biological fluids