Investigation of space charge effects and ion trapping capacity on direct introduction ultra‐high‐resolution mass spectrometry workflows for metabolomics

Ultra‐high‐resolution mass spectrometry, in the absence of chromatography, is finding its place for direct analyses of highly complex mixtures, such as those encountered during untargeted metabolomics screening. Advances, however, have been tempered by difficulties such as uneven signal suppression experienced during electrospray ionization. Moreover, ultra‐high‐resolution mass spectrometers that use Orbitrap and ICR analyzers both suffer from limited ion trapping capacities, owing principally to space‐charge effects. This study has evaluated and contrasted the above two types of Fourier transform mass spectrometers for their abilities to detect and identify by accurate mass measurement, small molecule metabolites present in complex mixtures. For these direct introduction studies, the Orbitrap Fusion showed a major advantage in terms of speed of analysis, enabling detection of 218 of 440 molecules (<2 ppm error, 500 000 resolution at m/z 200) present in a complex mixture in 5 min. This approach is the most viable for high‐throughput workflows, such as those used in investigations involving very large cohorts of metabolomics samples. From the same mixture, 183 unique molecules were observed by FT‐ICR in the broadband mode, but this number was raised to 235 when “selected ion monitoring‐stitching” (SIM‐stitching) was employed (<0.1 ppm error, 7 T magnet with dynamic harmonization cell, 1.8 million resolution at m/z 200, both cases). SIM‐stitching FT‐ICR thus offered the most complete detection, which may be of paramount importance in situations where it is essential to obtain the most complete metabolic profile possible. This added completeness, however, came at the cost of a more lengthy analysis time (120 min including manual treatment). Compared to the data presented here, future automation of processing, plus the use of absorption mode detection, segmented ion detection (stepwise detection of smaller width m/z sections), and higher magnetic field strengths, can substantially reduce FT‐ICR acquisition times.     

Commercial formaldehyde standard for mass calibration in mass spectrometry

Common calibration standards for mass spectrometry can be a source of many problems including instrument contamination, ionization suppression and formation of unidentified ions during subsequent analysis. In this article, we present a new approach for the calibration of mass analyzers such as a quadrupole–time‐of‐flight mass spectrometry using a diluted solution of commercial formaldehyde. Formaldehyde is an inexpensive and commonly used solvent, and its intrinsic polymerization leads to the formation of polyoxymethylene (POM) oligomers, which are excellent multiple calibration standards for a low‐mass spectral region (up to m/z 400) in the positive and negative mode of electrospray ionization. We explore the nature and origin of these polymeric species and attributed them to chemical reactions of formaldehyde and stabilizing agents in commercial formaldehyde solutions and during electrospray ionization. In contrast to other calibrants, POM oligomers do not contaminate the instrument and can easily be removed from the sample delivery system. Using tandem mass spectrometry, we elucidate the structures of the detected POM oligomers and report their reference masses, which are tightly spaced by 30 mass units. In our calibration method, mass errors of <5 ppm can be obtained from m/z 20–400 using external calibration with a simple one‐point zero‐order correction of spectral data and without the need for operation of a dual spray or internal calibrants. Our approach will be particularly useful for those interested in the analysis of fragile ions with low m/z values and can function at instrumental conditions required for analysis of the most labile metabolites and environmental contaminants. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of new metabolites from in vivo biotransformation of 2‐amino‐ 3‐methylimidazo[4,5‐f]quinoline in mouse by mass spectrometry

In studying the metabolic pathways underlying the mechanism of carcinogenesis of the heterocyclic amine of 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ), we recently found a new metabolite which gave an [M + H]⁺ ion of m/z 217 when subjected to electrospray ionization (ESI) in positive‐ion mode. Following ip injection of this metabolite of m/z 217 (designated as m/z 217) to beta‐naphthoflavone‐treated mice, 57% of the total radioactivity was recovered in a 24‐h mouse urine sample. HPLC separation followed by MS analysis indicates that the urine sample contained m/z 217 (36 ± 3% of total recovered radioactivity) and two other peaks that gave rise to the [M + H]⁺ ions of m/z 393 (31 ± 4%, designated as m/z 393) and m/z 233 (14 ± 1%, designated as m/z 233). Beta‐glucuronidase treatment of m/z 393 resulted in a radioactive peak corresponding to m/z 217. ESI in combination with various mass spectrometry techniques, including multiple‐stage mass spectrometry, exact mass measurements and H/D exchange followed by tandem mass spectrometry, was used for structural characterization. The urinary metabolites of m/z 217, 393 and 233 were identified as 1,2‐dihydro‐2‐amino‐5‐hydroxy‐3‐methylimidazo[4,5‐f]quinoline, 1,2‐dihydro‐2‐amino‐5‐O‐glucuronide‐3‐methylimidazo[4,5‐f]quinoline and 1,2‐dihydro‐2‐amino‐5,7‐dihydroxy‐3‐methylimidazo[4,5‐f]quinoline, respectively. Our results demonstrated that m/z 217 is biotransformed in vivo to m/z 393 by O‐glucuronidation and to m/z 233 by oxidation. The observation of these more polar metabolites relative to IQ suggests that they may arise from a previously undescribed detoxicification pathway. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of xylazine metabolism in rat liver microsomes using liquid chromatography–hybrid triple quadrupole–linear ion trap–mass spectrometry

ABSTRACT: Xylazine is an α₂‐adrenoceptor agonist and it is widely used in veterinary anesthesia in combination with ketamine. There is limited information on the metabolism of xylazine. A quantitative method for the determination of xylazine by HPLC‐ESI/MS/MS was developed. The method consisted of a protein precipitation extraction followed by analysis using liquid chromatography electrospray tandem mass spectrometry. The chromatographic separation was achieved using a Thermo Betasil Phenyl 100 × 2 mm column combined with an isocratic mobile phase composed of acetonitrile, methanol, water and formic acid (60:20:20:0.4) at a flow rate of 300 μL/min. The mass spectrometer was operating in selected reaction monitoring mode and the analytical range was set at 0.05–50 μm . The precision (%CV) and accuracy (%NOM) observed were 2.3–7.2 and 88.2–96.4%. In vitro metabolism studies were performed in rat liver microsomes and results showed moderate cytochrome P450 affinity (K_m = 10.1 μm ) and a low metabolic stability of xylazine with a half‐life of 4.1 min in rat liver microsomes. Five phase 1 metabolites were observed. The main metabolite observed was an oxidation of the thiazine moiety at m/z 235 and, to a lesser extent, we observed the formation of N‐(2,6‐dimethylphenyl)thiourea at m/z 181 and three distinctive hydroxylated metabolites at m/z 237. Further experiments with ketamine and ketoconazole strongly supported that the metabolism of xylazine to its main metabolite is mediated by CYP3A in rat liver microsomes. Copyright © 2013 John Wiley & Sons, Ltd.     

Shotgun lipidomics on a LTQ Orbitrap mass spectrometer by successive switching between acquisition polarity modes

Top–down shotgun lipidomics relies on direct infusion of total lipid extracts into a high‐resolution tandem mass spectrometer and implies that individual lipids are recognized by their accurately determined m/z. Lipid ionization efficiency and detection specificity strongly depend on the acquisition polarity, and therefore it is beneficial to analyze lipid mixtures in both positive and negative modes. Hybrid LTQ Orbitrap mass spectrometers are widely applied in top–down lipidomics; however, rapid polarity switching was previously unfeasible because of the severe and immediate degradation of mass accuracy. Here, we report on a method to rapidly acquire high‐resolution spectra in both polarity modes with sub‐ppm mass accuracy and demonstrate that it not only simplifies and accelerates shotgun lipidomics analyses but also improves the lipidome coverage because more lipid classes and more individual species within each class are recognized. In this way, shotgun analysis of total lipid extracts of human blood plasma enabled to quantify 222 species from 15 major lipid classes within 7 min acquisition cycle. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparison between a high‐resolution single‐stage Orbitrap and a triple quadrupole mass spectrometer for quantitative analyses of drugs

The capabilities of a high‐resolution (HR), accurate mass spectrometer (Exactive‐MS) operating in full scan MS mode was investigated for the quantitative LC/MS analysis of drugs in patients' plasma samples. A mass resolution of 50 000 (FWHM) at m/z 200 and a mass extracted window of 5 ppm around the theoretical m/z of each analyte were used to construct chromatograms for quantitation. The quantitative performance of the Exactive‐MS was compared with that of a triple quadrupole mass spectrometer (TQ‐MS), TSQ Quantum Discovery or Quantum Ultra, operating in the conventional selected reaction monitoring (SRM) mode. The study consisted of 17 therapeutic drugs including 8 antifungal agents (anidulafungin, caspofungin, fluconazole, itraconazole, hydroxyitraconazole posaconazole, voriconazole and voriconazole‐N‐oxide), 4 immunosuppressants (ciclosporine, everolimus, sirolimus and tacrolimus) and 5 protein kinase inhibitors (dasatinib, imatinib, nilotinib, sorafenib and sunitinib). The quantitative results obtained with HR‐MS acquisition show comparable detection specificity, assay precision, accuracy, linearity and sensitivity to SRM acquisition. Importantly, HR‐MS offers several benefits over TQ‐MS technology: absence of SRM optimization, time saving when changing the analysis from one MS to another, more complete information of what is in the samples and easier troubleshooting. Our work demonstrates that U/HPLC coupled to Exactive HR‐MS delivers comparable results to TQ‐MS in routine quantitative drug analyses. Considering the advantages of HR‐MS, these results suggest that, in the near future, there should be a shift in how routine quantitative analyses of small molecules, particularly for therapeutic drugs, are performed. Copyright © 2012 John Wiley & Sons, Ltd.     

Study of tamoxifen urinary metabolites in rat by ultra‐high‐performance liquid chromatography time‐of‐flight mass spectrometry

Tamoxifen (TMX) is a nonsteroidal estrogen antagonist drug used for the treatment of breast cancer. It is also included in the list of banned substances of the World Anti Doping Agency (WADA) prohibited in and out of competition. In this work, the excretion of urinary metabolites of TMX after a single therapeutic dose administration in rats has been studied using ultra‐high‐performance liquid chromatography electrospray time‐of‐flight mass spectrometry (UHPLC‐TOFMS). A systematic strategy based on the search of typical biotransformations that a xenobiotic can undergo in living organisms, based on their corresponding molecular formula modification and accurate mass shifts, was applied for the identification of TMX metabolites. Prior to UHPLC‐TOFMS analyses, a solid‐phase extraction step with polymeric cartridges was applied to urine samples. Up to 38 TMX metabolites were detected. Additional collision induced dissociation (CID) MS/MS fragmentation was performed using UHPLC‐QTOFMS. Compared with recent previous studies in human urine and plasma, new metabolites have been reported for the first time in urine. Metabolites identified in rat urine include the oxygen addition, owing to different possibilities for the hydroxylation of the rings in different positions (m/z 388.2271), the incorporation of two oxygen atoms (m/z 404.2220) (including dihydroxylated derivatives or alternatives such as epoxidation plus hydroxylation or N‐oxidation and hydroxylation), epoxide formation or hydroxylation and dehydrogenation [m/z 386.2114 (+O –H₂)], hydroxylation of the ring accompanied by N‐desmethylation (m/z 374.2115), combined hydroxylation and methoxylation (m/z 418.2377), desaturated TMX derivate (m/z 370.2165) and its N‐desmethylated derivate (m/z 356.2009), the two latter modifications not previously being reported in urine. These findings confirm the usefulness of the proposed approach based on UHPLC‐TOFMS. Copyright © 2015 John Wiley & Sons, Ltd.     

Optimization of a quadrupole ion storage trap as a source for time‐of‐flight mass spectrometry

Designs of a quadrupole ion trap (QIT) as a source for time‐of‐flight (TOF) mass spectrometry are evaluated for mass resolution, ion trapping, and laser activation of trapped ions. Comparisons are made with the standard hyperbolic electrode ion trap geometry for TOF mass analysis in both linear and reflectron modes. A parallel‐plate design for the QIT is found to give significantly improved TOF mass spectrometer performance. Effects of ion temperature, trapped ion cloud size, mass, and extraction field on mass resolution are investigated in detail by simulation of the TOF peak profiles. Mass resolution (m/Δm) values of several thousand are predicted even at room temperature with moderate extraction fields for the optimized design. The optimized design also allows larger radial ion collection size compared with the hyperbolic ion trap, without compromising the mass resolution. The proposed design of the QIT also improves the ion–laser interaction volume and photon collection efficiency for fluorescence measurements on trapped ions. Copyright © 2011 John Wiley & Sons, Ltd.     

Chemometrics comparison of gas chromatography with mass spectrometry and comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry Daphnia magna metabolic profiles exposed to salinity

The performances of gas chromatography with mass spectrometry and of comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry are examined through the comparison of Daphnia magna metabolic profiles. Gas chromatography with mass spectrometry and comprehensive two‐dimensional gas chromatography with mass spectrometry were used to compare the concentration changes of metabolites under saline conditions. In this regard, a chemometric strategy based on wavelet compression and multivariate curve resolution–alternating least squares is used to compare the performances of gas chromatography with mass spectrometry and comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry for the untargeted metabolic profiling of Daphnia magna in control and salinity‐exposed samples. Examination of the results confirmed the outperformance of comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry over gas chromatography with mass spectrometry for the detection of metabolites in D. magna samples. The peak areas of multivariate curve resolution–alternating least squares resolved elution profiles in every sample analyzed by comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry were arranged in a new data matrix that was then modeled by partial least squares discriminant analysis. The control and salt‐exposed daphnids samples were discriminated and the most relevant metabolites were estimated using variable importance in projection and selectivity ratio values. Salinity de‐regulated 18 metabolites from metabolic pathways involved in protein translation, transmembrane cell transport, carbon metabolism, secondary metabolism, glycolysis, and osmoregulation.     

Food contaminant analysis at ultra‐high mass resolution: application of hybrid linear ion trap – orbitrap mass spectrometry for the determination of the polyether toxins,azaspiracids, in shellfish

The biotoxins, azaspiracids (AZAs), from marine phytoplankton accumulate in shellfish and affect human health by causing severe gastrointestinal disturbance, diarrhea, nausea and vomiting. Specific and sensitive methods have been developed and validated for the determination of the most commonly occurring azaspiracid analogs. An LTQ Orbitrap mass spectrometer is a hybrid instrument that combines linear ion trap (LIT) mass spectrometry (MS) with high‐resolution Fourier transform (FT) MS and this was exploited to perform simultaneous ultra‐high‐resolution full‐scan MS analysis and collision‐induced dissociation (CID) tandem mass spectrometry (MS/MS). Using the highest mass resolution setting (100 000 FWHM) in full‐scan mode, the methodology was validated for the determination of six AZAs in mussel (Mytilus galloprovincialis) tissue extracts. Ultra‐high mass resolution, together with a narrow mass tolerance window of ±2 mDa, dramatically improved detection sensitivity. In addition to employing chromatographic resolution to distinguish between the isomeric azaspiracid analogs, AZA1/AZA6 and AZA4/AZA5, higher energy collisionally induced dissociation (HCD) fragmentation on selected precursor ions were performed in parallel with full‐scan FTMS. Using HCD MS/MS, most precursor and product ion masses were determined within 1 ppm of the theoretical m/z values throughout the mass spectral range and this enhanced the reliability of analyte identity. For the analysis of mussels (M. galloprovincialis), the method limit of quantitation (LOQ) was 0.010 µg/g using full‐scan FTMS and this was comparable with the LOQ (0.007 µg/g) using CID MS/MS. The repeatability data were; intra‐day RSD% (1.8–4.4%; n = 6) and inter‐day RSD% (4.7–8.6%; n = 3). Application of these methods to the analysis of mussels (M. edulis) that were naturally contaminated with azaspiracids, using high‐resolution full‐scan Orbitrap MS and low‐resolution CID MS/MS, produced equivalent quantitative data. Copyright © 2010 John Wiley & Sons, Ltd.     

Ion trap mass analysis at high pressure: an experimental characterization

In recent years, it has become increasingly interesting to understand the performance of mass spectrometers at pressures much higher than those employed with conventional operating conditions. This interest has been driven by several influences, including demand for the development of reduced‐power miniature mass spectrometers, desire for improved ion transfer into and through mass spectrometers, enhanced‐yield preparative mass separations, and mass filtering at the atmospheric pressure interface. In this study, an instrument was configured to allow for the performance characterization of a rectilinear ion trap (RIT) at pressures up to 50 mtorr with air used as the buffer gas. The mass analysis efficiency, mass resolution, isolation efficiency, and collision‐induced dissociation (CID) efficiency were evaluated at pressures ranging from 1 to 50 mtorr. The extent of degradation of mass resolution, isolation efficiency and ion stability as functions of pressure were characterized. Also, the optimal resonance ejection conditions were obtained at various pressures. Operations at 50 mtorr demonstrated improved CID efficiency in addition to peak widths of 2 and 5 m/z units (full width at half‐maximum, FWHM) for protonated caffeine (m/z 195) and Ultramark (m/z 1521) respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Liquid chromatography–tandem mass spectrometry of porphyrins and porphyrinogens in biological materials: separation and identification of interfering poly(ethylene) glycol by travelling wave ion mobility spectrometry/tandem mass spectrometry

Biological and clinical samples for porphyrin and porphyrinogen analyses by liquid chromatography–tandem mass spectrometry (LC‐MS/MS) are often contaminated with poly(ethylene)glycol (PEG), which complicates the interpretation of mass spectra and characterisation of new porphyrin metabolites. Two contaminating PEG molecules (m/z 833 and m/z 835) were completely separated from uroporphyrin I (m/z 831) by travelling wave ion mobility spectrometry and characterised by tandem mass spectrometry. One of the PEG species (m/z 835) also co‐eluted with uroporphyrinogen I (m/z 837) and was unresolvable by travelling wave ion mobility spectrometry/MS, therefore contaminating the MS/MS mass spectra owing to isotope distribution. These PEG species, with the [M + H]⁺ ions at m/z at 833 and/or m/z 835, co‐eluted with uroporphyrin I and uroporphyrinogen I by LC‐MS/MS and could be wrongly identified as uroporphomethenes. Copyright © 2013 John Wiley & Sons, Ltd.     

Dialkyl phosphates determination by gas chromatography: Evaluation of a microwave‐assisted derivatization†

Dialkyl phosphates are organophosphate insecticide metabolites and their urinary analysis is useful for assessing human exposure to these compounds. This study presents a sample preparation method with microwave‐assisted derivatization for two dialkyl phosphates to make the process faster before gas chromatographic analysis. The optimized conditions for derivatization procedure were: 250 μL of 2,3,4,5,6‐pentafluorobenzyl bromide 3% in acetonitrile for derivative; microwave for 5 min with intensity of 160 W. The electron ionization mass spectrometric analysis was performed using a gas chromatography with mass spectrometry QP‐2010 from the Shimadzu^® equipped with RTx^®‐5MS capillary column. Ions were monitored at selected‐ion monitoring mode at m/z 350 for diethyl thiophosphate and m/z 366 for diethyl dithiophosphate. The developed method was linear for both metabolites. The intra‐assay precision was the values ranged between 1.1 and 9.1%, for diethyl thiophosphate, and between 4.06 and 6.9%, for diethyl dithiophosphate. The interassay precision showed relative standard deviation between 10.3 and 15.1%, for diethyl thiophosphate and between 4.9 and 11.9%, for diethyl dithiophosphate. The results obtained suggests that derivatization assisted by microwave, before gas chromatography with mass spectrometry analysis, can be applied to monitoring of exposure to organophosphates once is fast, sensible, and precise method to determinate dialkyl phosphates.     

Development and optimization of a supercritical fluid chromatography tandem mass spectrometry method for the high‐throughput determination of nimodipine in beagle plasma

A simple, sensitive, and efficient supercritical fluid chromatography with tandem mass spectrometry method was established for the determination of nimodipine in beagle plasma. One‐step protein precipitation with acetone was used to extract the analytes from the plasma. Nitrendipine was used as the internal standard. The chromatographic separation was achieved on an ACQUITY UPC²? BEH 2‐EP column, and a gradient elution program was applied at a flow rate of 1.5 mL/min. The detection was carried out on a triple quadrupole tandem mass spectrometer with an electrospray ionization source operating in positive ion mode. Quantification was performed using multiple reaction monitoring of the transitions of m/z 419.3→301.3 for nimodipine and m/z 361.4→315.2 for nitrendipine. A satisfactory linearity was obtained over the concentration range of 0.5–800 ng/mL (r > 0.996). The intra‐ and interday precision and accuracy results were <9.1% across the quality control levels. The peak concentration and area under concentration‐time curve (0–720 min) values of the test and reference formulations were 279.28 ± 211.46 and 265.13 ± 149.26 ng/mL, 25608.00 ± 17553.65 and 28553.67 ± 20207.92 ng·min/mL, respectively. The validated method was successfully applied to reveal the pharmacokinetic profiles of nimodipine in beagle dogs after oral administration. Moreover, the analytical method could be used for further bioequivalence studies.     

Advantages of high-resolution and high-mass range magnetic-sector mass spectrometry for electrospray ionization

Electrospray ionization (ESI) mass spectra have been measured on a magnetic-sector double-focusing mass spectrometer for a number of proteins and peptides. It is pointed out how in theory raising the mass resolution of a mass spectrometer from 800–1000 to 2400–3000 significantly increases the precision with which the envelope of isotopic peaks of a protein ion (or other organic ion) can be defined, particularly at higher masses. Better definition of the isotopic envelope ought to lead to higher precision in the experimental determination of molecular mass, which has been demonstrated. It is shown how ESI mass spectra of high-mass molecules are significantly less congested at higher m/z values, so that for these molecules (RMM > 40 000) there is an advantage in being able to record peaks at higher m/z values (m/z > 2000) representing ions with fewer charges. Fragmentation of a small peptide in the ESI source has been found to provide sequence information.     

In vitro metabolism study of Strychnos alkaloids using high‐performance liquid chromatography combined with hybrid ion trap/time‐of‐flight mass spectrometry

In this report, the in vitro metabolism of Strychnos alkaloids was investigated using liquid chromatography/high‐resolution mass spectrometry for the first time. Strychnine and brucine were selected as model compounds to determine the universal biotransformations of the Strychnos alkaloids in rat liver microsomes. The incubation mixtures were separated by a bidentate‐C₁₈ column, and then analyzed by on‐line ion trap/time‐of‐flight mass spectrometry. With the assistance of mass defect filtering technique, full‐scan accurate mass datasets were processed for the discovery of the related metabolites. The structural elucidations of these metabolites were achieved by comparing the changes in accurate molecular masses, calculating chemical component using Formula Predictor software and defining sites of biotransformation based upon accurate MSⁿ spectral information. As a result, 31 metabolites were identified, of which 26 metabolites were reported for the first time. These biotransformations included hydroxylation, N‐oxidation, epoxidation, methylation, dehydrogenation, de‐methoxylation, O‐demethylation, as well as hydrolysis reactions. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous quantification of tizoxanide and tizoxanide glucuronide in mouse plasma by liquid chromatography–tandem mass spectrometry

Nitazoxanide (NTZ) is a broad‐spectrum antimicrobial agent. Tizoxanide (T) and tizoxanide glucuronide (TG) are the major circulating metabolites after oral administration of NTZ. A rapid and specific LC–MS/MS method for the simultaneous quantification of T and TG in mouse plasma was developed and validated. A simple acetonitrile‐induced protein precipitation method was employed to extract two analytes and the internal standard glipizide from 50 μL of mouse plasma. The purified samples were resolved using a C₁₈ column with a mobile phase consisting of acetonitrile and 5 mm ammonium formate buffer (containing 0.05% formic acid) following a gradient elution. An API 3000 triple quadrupole mass spectrometer was operated under multiple reaction‐monitoring mode with electrospray ionization. The precursor‐to‐product ion transitions m/z 264 → m/z 217 for T and m/z 440 → m/z 264 for TG were used for quantification. The developed method was linear in the concentration ranges of 1.0–500.0 ng/mL for T and 5.0–1000.0 ng/mL for TG. The intra‐ and inter‐day precision and accuracy of the quality control samples at low, medium and high concentrations exhibited an RSD of <13.2% and the accuracy values ranged from ?9.6 to 9.3%. We used this validated method to study the pharmacokinetics of T and TG in mice following oral administration of NTZ. Copyright © 2016 John Wiley & Sons, Ltd.     

Structural elucidation of new urinary tamoxifen metabolites by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In this study, tamoxifen metabolic profiles were investigated carefully. Tamoxifen was administered to two healthy male volunteers and one female patient suffering from breast cancer. Urinary extracts were analyzed by liquid chromatography quadruple time‐of‐flight mass spectrometry using full scan and targeted MS/MS techniques with accurate mass measurement. Chromatographic peaks for potential metabolites were selected by using the theoretical [M + H]⁺ as precursor ion in full‐scan experiment and m/z 72, 58 or 44 as characteristic product ions for N,N‐dimethyl, N‐desmethyl and N,N‐didesmethyl metabolites in targeted MS/MS experiment, respectively. Tamoxifen and 37 metabolites were detected in extraction study samples. Chemical structures of seven unreported metabolites were elucidated particularly on the basis of fragmentation patterns observed for these metabolites. Several metabolic pathways containing mono‐ and di‐hydroxylation, methoxylation, N‐desmethylation, N,N‐didesmethylation, oxidation and combinations were suggested. All the metabolites were detected in the urine samples up to 1 week. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantitation of small molecules using high‐resolution accurate mass spectrometers – a different approach for analysis of biological samples

The quantitative capabilities of a linear ion trap high‐resolution mass spectrometer (LTQ‐Orbitrap™) were investigated using full scan mode bracketing the m/z range of the ions of interest and utilizing a mass resolution (mass/FWHM) of 15000. Extracted ion chromatograms using a mass window of ±5–10 mmu centering on the theoretical m/z of each analyte were generated and used for quantitation. The quantitative performance of the LTQ‐Orbitrap™ was compared with that of a triple quadrupole (API 4000) operating using selected reaction monitoring (SRM) detection. Comparable assay precision, accuracy, linearity and sensitivity were observed for both approaches. The concentrations of actual study samples from 15 Merck drug candidates reported by the two methods were statistically equivalent. Unlike SRM being a tandem mass spectrometric (MS/MS)‐based detection method, a high resolution mass spectrometer operated in full scan does not need MS/MS optimization. This approach not only provides quantitative results for compounds of interest, but also will afford data on other analytes present in the sample. An example of the identification of a major circulating metabolite for a preclinical development study is demonstrated. Copyright © 2009 John Wiley & Sons, Ltd.