High-throughput quantitation of large molecules using multiplexed chromatography and high-resolution/accurate mass LC-MS.

BACKGROUND: High-throughput screening with LC-MS has been routinely implemented to various degrees throughout the entire drug-discovery process. One of the major advantages of utilizing LC-MS earlier at the lead discovery stage is reducing the cost of sample analysis while increasing assay selectivity. Avoiding labeling agents and other non-native species in an assay environment can reduce costly sample preparation, while chromatographic separation of the analyte of interest from interferences in the sample matrix has been shown to increase selectivity and sensitivity. METHOD: In this paper, we utilize high-resolution MS-LC multiplexing to analyze phosphorylated peptides as part of a screening assay. Commonly used enzyme buffers were used to prepare phosphorylated peptide standards of varying concentrations and these were plated into a 96-well plate format for LC-MS analysis. The overall cycle time for analysis from sample to sample, LLOQ, Z' and coefficient of variance were determined. CONCLUSION: High-resolution MS coupled with LC multiplexing provides high-quality sample analysis at sampling rates of up to 18 s per sample. Samples analyzed in both simple and complex sample matrixes demonstrated an LOQ of 5 nM with linear response across the working range of the assay. Overall statistical analysis of the large samples produced Z' = 0.85 for sample sets in sodium citrate solution and Z' = 0.66 for sample sets in HEPES solution indicating a robust analytical method.

Application of a linear ion trap/orbitrap mass spectrometer in metabolite characterization studies: examination of the human liver microsomal metabolism of the non-tricyclic anti-depressant nefazodone using data-dependent accurate mass measurements.

We report herein, facile metabolite identification workflow on the anti-depressant nefazodone, which is derived from accurate mass measurements based on a single run/experimental analysis. A hybrid LTQ/orbitrap mass spectrometer was used to obtain accurate mass full scan MS and MS/MS in a data-dependent fashion to eliminate the reliance on a parent mass list. Initial screening utilized a high mass tolerance ( approximately 10 ppm) to filter the full scan MS data for previously reported nefazodone metabolites. The tight mass tolerance reduces or eliminates background chemical noise, dramatically increasing sensitivity for confirming or eliminating the presence of metabolites as well as isobaric forms. The full scan accurate mass analysis of suspected metabolites can be confirmed or refuted using three primary tools: (1) predictive chemical formula and corresponding mass error analysis, (2) rings-plus-double bonds, and (3) accurate mass product ion spectra of parent and suspected metabolites. Accurate mass characterization of the parent ion structure provided the basis for assessing structural assignment for metabolites. Metabolites were also characterized using parent product ion m/z values to filter all tandem mass spectra for identification of precursor ions yielding similar product ions. Identified metabolite parent masses were subjected to chemical formula calculator based on accurate mass as well as bond saturation. Further analysis of potential nefazodone metabolites was executed using accurate mass product ion spectra. Reported mass measurement errors for all full scan MS and MS/MS spectra was <3 ppm, regardless of relative ion abundance, which enabled the use of predictive software in determining product ion structure. The ability to conduct biotransformation profiling via tandem mass spectrometry coupled with accurate mass measurements, all in a single experimental run, is clearly one of the most attractive features of this methodology.

Automated screening with confirmation of mechanism-based inactivation of CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP1A2 in pooled human liver microsomes.

A strategy is proposed to profile compounds for mechanism-based inactivation of CYP3A4, CYP2C19, CYP2C9, CYP2D6, and CYP1A2 based on an apparent partition ratio screen. Potent positives from the screen are confirmed by time- and concentration-dependent inactivation assays. Quasi-irreversible inhibitions are then differentiated from irreversible inactivations by oxidation with potassium ferricyanide and/or dialysis. The three-step screening procedure has been validated with acceptable accuracy and precision for detection and confirmation of mechanism-based inactivators in drug discovery. We report here the apparent partition ratios for 19 mechanism-based inactivators and four quasi-irreversible inhibitors obtained under the same experimental conditions. The apparent partition ratio screen was automated to provide throughput for determining structure-mechanism-based inactivation relationships. Information about reversibility can be used to assess potential toxicity mediated by covalent adducts, as well as the potential for pharmacokinetic drug-drug interactions. Direct comparison of known mechanism-based inactivators and quasi-irreversible inhibitors, based on our screening of apparent partition ratios, has identified ritonavir, mibefradil, and azamulin as highly effective mechanism-based inactivators; e.g., 1 mol of CYP3A4 was inactivated on turnover of about 2 mol of compound. Other mechanism-based inactivators we identified include bergamottin (CYP1A2 besides previously reported CYP3A4), troglitazone (CYP3A4), rosiglitazone (CYP3A4), and pioglitazone (CYP3A4). Comparison of the apparent partition ratios and inactivation clearance data for the three glitazones suggests that the chromane moiety on troglitazone contributes to its greater potency for mechanism-based inactivation.