Ultra-fast searching assists in evaluating sub-ppm mass accuracy enhancement in U-HPLC/Orbitrap MS data

A strategy, detailed methodology description and software are given with which the mass accuracy of U-HPLC-Orbitrap data (resolving power 50,000 FWHM) can be enhanced by an order of magnitude to sub-ppm levels. After mass accuracy enhancement all 211 reference masses have mass errors within 0.5 ppm; only 14 of these are outside the 0.2 ppm error margin. Further demonstration of mass accuracy enhancement is shown on a pre-concentrated urine sample in which evidence for 89 (342 ions) potential hydroxylated and glucuronated DHEA-metabolites is found. Although most DHEA metabolites have low-intensity mass signals, only 11 out of 342 are outside the ±1 ppm error envelop; 272 mass signals have errors below 0.5 ppm (142 below 0.2 ppm). The methodology consists of: (a) a multiple internal lock correction (here ten masses; no identity of internal lock masses is required) to avoid suppression problems of a single internal lock mass as well as to increase lock precision, (b) a multiple external mass correction (here 211 masses) to correct for calibration errors, (c) intensity dependant mass correction, (d) file averaging. The strategy is supported by ultra-fast file searching of baseline corrected, noise-reduced metAlign output. The output and efficiency of ultra-fast searching is essential in obtaining the required information to visualize the distribution of mass errors and isotope ratio deviations as a function of mass and intensity

Advances in structure elucidation of small molecules using mass spectrometry

The structural elucidation of small molecules using mass spectrometry plays an important role in modern life sciences and bioanalytical approaches. This review covers different soft and hard ionization techniques and figures of merit for modern mass spectrometers, such as mass resolving power, mass accuracy, isotopic abundance accuracy, accurate mass multiple-stage MS(n) capability, as well as hybrid mass spectrometric and orthogonal chromatographic approaches. The latter part discusses mass spectral data handling strategies, which includes background and noise subtraction, adduct formation and detection, charge state determination, accurate mass measurements, elemental composition determinations, and complex data-dependent setups with ion maps and ion trees. The importance of mass spectral library search algorithms for tandem mass spectra and multiple-stage MS(n) mass spectra as well as mass spectral tree libraries that combine multiple-stage mass spectra are outlined. The successive chapter discusses mass spectral fragmentation pathways, biotransformation reactions and drug metabolism studies, the mass spectral simulation and generation of in silico mass spectra, expert systems for mass spectral interpretation, and the use of computational chemistry to explain gas-phase phenomena. A single chapter discusses data handling for hyphenated approaches including mass spectral deconvolution for clean mass spectra, cheminformatics approaches and structure retention relationships, and retention index predictions for gas and liquid chromatography. The last section reviews the current state of electronic data sharing of mass spectra and discusses the importance of software development for the advancement of structure elucidation of small molecules

Bioactivation of sitaxentan in liver microsomes, hepatocytes, and expressed human P450s with characterization of the glutathione conjugate by liquid chromatography tandem mass spectrometry

Sitaxentan is a selective endothelin-A receptor antagonist that was marketed as Thelin in several European countries and Canada for pulmonary arterial hypertension. Sitaxentan was undergoing further clinical trials in the United States but due to four deaths and one case of liver transplantation from severe liver toxicity that appeared to be idiosyncratic in nature, it was withdrawn worldwide in December, 2010. Sitaxentan contains a 1,3-benzodioxole ring that undergoes enzymatic demethyleneation to an ortho-catechol metabolite that can further oxidize to a reactive ortho-quinone metabolite. Here, we report the detection and mass spectral characterization of a glutathione conjugate of this sitaxentan quinone reactive metabolite that was trapped in vitro using mouse, rat, dog, and human liver microsomes supplemented with NADPH and glutathione and that was also observed in rat and human hepatocytes. Using human liver microsomes, we also demonstrated that P450 3A4 undergoes time-dependent inhibition. Density functional calculations on the catechol metabolite of sitaxentan indicated that the reaction leading to the quinone was thermodynamically favorable with an enthalpy change of -6.3 kcal/mol. Using density functional methodology, we modeled the attack of glutathione on the quinone with an S-methyl thiolate anion which allowed us to predict, based on the difference in transition state energies, that the 2-position on the phenyl ring was more likely than the 5-position as the site of glutathione conjugation. Overall, our results demonstrated that sitaxentan is capable of facile formation of a reactive ortho-quinone metabolite capable of reacting with glutathione and may rationalize the idiosyncratic nature of the hepatotoxicity that led to its withdrawal. 2013 American Chemical Societ