bims-metlip 2020-03-01 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2020–03–01
sixteen papers selected by
Sofia Costa, Cold Spring Harbor Laboratory

Absolute Quantification of the Central Carbon Metabolome in Eight Commonly Applied Prokaryotic and Eukaryotic Model Systems.
Simultaneous quantification of candesartan and irbesartan in rabbit eye tissues by liquid chromatography-tandem mass spectrometry.
rMSIproc: an R package for mass spectrometry imaging data processing.
Determination of Eight Coccidiostats in Eggs by Liquid-Liquid Extraction-Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry.
What's in a whisker? High-throughput analysis of twenty-eight C19 and C21 steroids in mammalian whiskers by ultra-performance convergence chromatography-tandem mass spectrometry.
Simultaneous quantitation of glycyrrhetic acid and puerarin in plasma by UFLC-MS/MS, and application in a pharmacokinetics study in healthy and alcoholic liver injury rats.
Validation of an ultra-sensitive detection method for steroid esters in plasma for doping analysis using positive chemical ionization GC-MS/MS.
Improved oxytocin analysis from human serum and urine by orbitrap ESI-LC-HRAM-MS.
Structure elucidation of urinary metabolites of fentanyl and five fentanyl analogues using LC-QTOF-MS, hepatocyte incubations and synthesized reference standards.
The determination of common anabolic steroid and stimulants in nutritional supplements by HPLC-DAD and LC-MS.
Mass spectral fragmentation analyses of isotopically labelled hydroxy steroids using low resolution gas chromatography - mass spectrometry (GC/EI-MS): a practical approach.
Screening procedure for 38 fentanyl analogues and five other new opioids in whole blood by liquid chromatography-tandem mass spectrometry.
A New Validated HPLC-MS/MS Method for Quantification and Pharmacokinetic Evaluation of Dovitinib, a Multi-Kinase Inhibitor, in Mouse Plasma.
Quantification of endogenous steroid sulfates and glucuronides in human urine after intramuscular administration of testosterone esters.
Detection of Chemical Weapon Nerve Agents in Bone by Liquid Chromatography-Mass Spectrometry.
Evaluation of oxysterol levels of patients with silicosis by LC-MS/MS method.

Metabolites. 2020 Feb 19. pii: E74. [Epub ahead of print]10(2):

Absolute Quantification of the Central Carbon Metabolome in Eight Commonly Applied Prokaryotic and Eukaryotic Model Systems.

Lisa M Røst, Lilja Brekke Thorfinnsdottir, Kanhaiya Kumar, Katsuya Fuchino, Ida Eide Langørgen, Zdenka Bartosova, Kåre Andre Kristiansen, Per Bruheim.

  Absolute quantification of intracellular metabolite pools is a prerequisite for modeling and in-depth biological interpretation of metabolomics data. It is the final step of an elaborate metabolomics workflow, with challenges associated with all steps-from sampling to quantifying the physicochemically diverse metabolite pool. Chromatographic separation combined with mass spectrometric (MS) detection is the superior platform for high coverage, selective, and sensitive detection of metabolites. Herein, we apply our quantitative MS-metabolomics workflow to measure and present the central carbon metabolome of a panel of commonly applied biological model systems. The workflow includes three chromatographic methods combined with isotope dilution tandem mass spectrometry to allow for absolute quantification of 68 metabolites of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and the amino acid and (deoxy) nucleoside pools. The biological model systems; Bacillus subtilis, Saccharomyces cerevisiae, two microalgal species, and four human cell lines were all cultured in commonly applied culture media and sampled in exponential growth phase. Both literature and databases are scarce with comprehensive metabolite datasets, and existing entries range over several orders of magnitude. The workflow and metabolite panel presented herein can be employed to expand the list of reference metabolomes, as encouraged by the metabolomics community, in a continued effort to develop and refine high-quality quantitative metabolomics workflows.

Keywords:  B. subtilis; S. cerevisiae; absolute quantification; central carbon metabolism; human cell lines; intracellular metabolite pools; metabolome database; microalgae; tandem mass spectrometry; targeted metabolite profiling

DOI:  https://doi.org/10.3390/metabo10020074
Biomed Chromatogr. 2020 Feb 25. e4808

Simultaneous quantification of candesartan and irbesartan in rabbit eye tissues by liquid chromatography-tandem mass spectrometry.

Aimin Tan, Xuan Gui, Molly Wong, Hui Deng, Guifen Gu, Constantine Fanaras, John C Fanaras.

  Diabetic retinopathy is a major cause of vision loss in adults. Novel eye-drop formulations of candesartan and irbesartan are being developed for its cure or treatment. To support a preclinical trial in rabbits, it was critical to develop and validate a new LC-MS/MS method for simultaneous quantification of candesartan and irbesartan in rabbit eye tissues (cornea, aqueous humor, vitreous body and retina/choroid). Eye tissue samples were first homogenized in H2 O-diluted rabbit plasma. The candesartan and irbesartan in the supernatants together with their respective internal standards (candesartan-d4 and irbesartan-d4 ) were extracted by solid-phase extraction. The extracted samples were injected onto a C18 column for gradient separation. The MS detection was in the positive electrospray ionization mode using the multiple reaction monitoring transitions of m/z 441 → 263, 445 → 267, 429 → 207, and 433 → 211 for candesartan, candesartan-d4 , irbesartan and irbesartan-d4 , respectively. For the validated concentration ranges (2-2000 and 5-5000 ng/g for candesartan and irbesartan, respectively), the within-run and between-run accuracies (% bias) were within the range of -8.0-10.0. The percentage CV ranged from 0.6 to 7.3. There was no significant matrix interference nor matrix effect from different eye tissues and different rabbits. The validated method was successfully used in the Good Laboratory Practice (GLP) study of rabbits.

Keywords:  LC-MS; candesartan; eye tissue; irbesartan; rabbit

DOI:  https://doi.org/10.1002/bmc.4808
Bioinformatics. 2020 Feb 28. pii: btaa142. [Epub ahead of print]

rMSIproc: an R package for mass spectrometry imaging data processing.

Pere Ràfols, Bram Heijs, Esteban Del Castillo, Oscar Yanes, Liam A McDonnell, Jesús Brezmes, Iara Pérez-Taboada, Mario Vallejo, María García-Altares, Xavier Correig.

SUMMARY: Mass spectrometry imaging (MSI) can reveal biochemical information directly from a tissue section. MSI generates a large quantity of complex spectral data which is still challenging to translate into relevant biochemical information. Here we present rMSIproc, an open-source R package that implements a full data processing workflow for MSI experiments performed using TOF or FT-based mass spectrometers. The package provides a novel strategy for spectral alignment and recalibration, which allows to process multiple datasets simultaneously. This enables to perform a confident statistical analysis with multiple datasets from one or several experiments. rMSIproc is designed to work with files larger than the computer memory capacity and the algorithms are implemented using a multi-threading strategy. rMSIproc is a powerful tool able to take full advantage of modern computer systems to completely develop the whole MSI potential.
AVAILABILITY AND IMPLEMENTATION: rMSIproc is freely available at https://github.com/prafols/rMSIproc.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btaa142
Molecules. 2020 Feb 22. pii: E987. [Epub ahead of print]25(4):

Determination of Eight Coccidiostats in Eggs by Liquid-Liquid Extraction-Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry.

Bo Wang, Jianyu Liu, Xia Zhao, Kaizhou Xie, Zhixiang Diao, Genxi Zhang, Tao Zhang, Guojun Dai.

  A method for the simultaneous determination of robenidine, halofuginone, lasalocid, monensin, nigericin, salinomycin, narasin, and maduramicin residues in eggs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The sample preparation method used a combination of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) technology to extract and purify these target compounds from eggs. The target compounds were separated by gradient elution using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC). Tandem mass spectrometry was used to quantitatively and qualitatively analyze the target compounds via electrospray ionization (ESI+) and multiple reaction monitoring mode. The HPLC-MS/MS and UPLC-MS/MS methods were validated according to the requirements defined by the European Union and the Food and Drug Administration. The limits of detection and limits of quantification of the eight coccidiostats in eggs were 0.23-0.52 µg/kg and 0.82-1.73 µg/kg for HPLC-MS/MS, and 0.16-0.42 µg/kg and 0.81-1.25 µg/kg for UPLC-MS/MS, respectively. The eggs were spiked with four concentrations of the eight coccidiostats, and the HPLC-MS/MS and UPLC-MS/MS average recoveries were all higher than 71.69% and 72.26%, respectively. Compared with the HPLC-MS/MS method, utilizing UPLC-MS/MS had the advantages of low reagent consumption, a short detection time, and high recovery and precision. Finally, the HPLC-MS/MS and UPLC-MS/MS methods were successfully applied to detect eight coccidiostats in 40 eggs.

Keywords:  HPLC–MS/MS; LLE; SPE; UPLC–MS/MS; coccidiostats; eggs

DOI:  https://doi.org/10.3390/molecules25040987
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Feb 25. pii: S1570-0232(19)31664-2. [Epub ahead of print]1141 122028

What's in a whisker? High-throughput analysis of twenty-eight C19 and C21 steroids in mammalian whiskers by ultra-performance convergence chromatography-tandem mass spectrometry.

Nico Lübcker, Liezl M Bloem, Therina du Toit, Pieter Swart, P J Nico de Bruyn, Amanda C Swart, Robert P Millar.

  Obtaining longitudinal endocrinological data from free-ranging animals remains challenging. Steroid hormones can be extracted sequentially from non-invasively sampled biologically inert keratinous tissues, such as feathers, nails, hair and whiskers. However, uncertainty regarding the type and levels of steroids incorporated into such tissues complicates their utility in wildlife studies. Here, we developed a novel, comprehensive method to analyze fourteen C19 and fourteen C21 steroids deposited chronologically along the length of seal whiskers in a single, 6-minute chromatographic step, using ultra-performance convergence chromatography-tandem mass spectrometry. The limits of detection and quantification ranged from 0.01 to 2 ng/mL and from 0.1 to 10 ng/mL, respectively. The accuracy and precision were within acceptable limits for steroids at concentrations ≥2 ng/mL. The recovery (mean = 107.5% at 200 ng/mL), matrix effect and process efficiency of steroids evaluated, using blanked whisker matrix samples, were acceptable. The method was applied to the analysis of steroid hormone levels in adult female whisker segments obtained from southern elephant seals (Mirounga leonina), n = 10, and two fur seal species, Antarctic fur seals (Arctocephalus gazella; n = 5) and subantarctic fur seals (Arctocephalus tropicalis; n = 5), sampled between 2012 and 2017. In the whisker subsamples analyzed (n = 71), the median concentration of steroid hormones detected above the LOQ ranged from 2.0 to 273.7 pg/mg. This was the first extraction of multiple C19 and C21 steroids, including their C11-oxy metabolites, from the whiskers of mammals. Measuring hormones sequentially along the whisker lengths can contribute to our understanding of the impact of stress associated with environmental/climate changes that affect the health, survival of organisms, as well as to delineate the reproductive cycles of free-living mammals with cryptic life stages.

Keywords:  Androgens; Glucocorticoids; Keratin and feathers; Marine mammal endocrinology; Metabolites; Progestogens; Southern elephant seal; UPC(2)-MS/MS; Vibrissae; Whiskers

DOI:  https://doi.org/10.1016/j.jchromb.2020.122028
Biomed Chromatogr. 2020 Feb 28. e4818

Simultaneous quantitation of glycyrrhetic acid and puerarin in plasma by UFLC-MS/MS, and application in a pharmacokinetics study in healthy and alcoholic liver injury rats.

Chenxiao Shan, Qi Yuan, Xiaobing Cui, Chuan Chai, Sheng Yu, Hongmei Wen, Xi Huang.

  A rapid, sensitive, and accurate UFLC-MS/MS method was developed and validated for simultaneous quantitation of glycyrrhetic acid and puerarin in plasma derived from healthy and alcoholic liver injury rats. Plasma samples from healthy and model rats were deproteinated with methanol using liquiritin as an internal standard. Chromatography separation was performed by a Waters BEH C18 column (2.1 × 50 mm; 1.7 μm) using a gradient elution from acetonitrile and water (containing 0.1% formic acid) and the flow rate that was set at 0.4 mL/min. Quantitation was performed on a Triple Quad 4500 tandem mass spectrometer coupled with an electrospray ionization source in negative multiple reaction monitoring mode. Full method validation, including specificity, carryover, dilution integrity, recovery, linearity, precision and accuracy, matrix effect, and stability were within acceptable limits. The newly established method was successfully applied in a pharmacokinetics study to investigate glycyrrhetic acid and puerarin in healthy and alcoholic liver injury rats.

Keywords:  UFLC-MS/MS; alcoholic liver injury rat; glycyrrhetic acid; pharmacokinetics; puerarin

DOI:  https://doi.org/10.1002/bmc.4818
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Feb 19. pii: S1570-0232(19)31575-2. [Epub ahead of print]1141 122026

Validation of an ultra-sensitive detection method for steroid esters in plasma for doping analysis using positive chemical ionization GC-MS/MS.

Pieter Van Renterghem, Wouter Viaene, Wim Van Gansbeke, Juliana Barrabin, Michele Iannone, Michael Polet, Guy T'Sjoen, Koen Deventer, Peter Van Eenoo.

  The standard approach to detect misuse with testosterone in sport is based on the determination and evaluation of the urinary steroid profile followed by the confirmation of atypical profiles using isotope ratio mass spectrometry. The detection capacity of these methods can be attenuated by confounding factors or testosterone preparations with endogenous isotopic fingerprints. An alternative detection method for misuse of an endogenous steroid in sports is the direct detection of the administered steroid ester present in most preparations. Thus unambiguous proof for doping misuse can be delivered. In this work, the sensitivity of gas chromatography coupled to a triple quadrupole with chemical ionization (GC-CI-MS/MS) is applied to detect trace levels of 10 testosterone and 2 nandrolone esters in plasma for in human doping analysis. The detection method was developed employing a liquid-liquid extraction and HPLC cleanup step before analysis on the GC-CI-MS/MS. The quantitative method was validated in a linear range of 100-2000 pg/ml and proved to be selective, reproducible and very sensitive with limits of detection as low as to 10 pg/ml. A clinical study with the administration of testosterone undecanoate in 3 volunteers was carried out and the compound was detectable up to 86 days after administration.

Keywords:  Chemical ionisation; Direct detection; Doping control; GC-MS/MS; Steroid esters; Testosterone

DOI:  https://doi.org/10.1016/j.jchromb.2020.122026
Drug Test Anal. 2020 Feb 25.

Improved oxytocin analysis from human serum and urine by orbitrap ESI-LC-HRAM-MS.

Adrian A Franke, Xingnan Li, Chester Dabalos, Jennifer F Lai.

  Native circulating oxytocin (OT) levels in non-pregnant/non-lactating/non-medicated humans are very low (≤8 pg/mL). The lower limit of detection (LLOD) of our previous liquid chromatography mass spectrometry (LC-MS) method (10-25 pg/mL) precluded their quantification in serum and urine. Thus, we sought to improve the LC-MS sensitivity of OT measurements in these matrices by hydrophobic tagging and solid phase extraction (SPE). In the former approach, OT was reduced then alkylated with N-alkyl acetamide (C12, C14, C16, and C18) tags or derivatized using sulfonyl chloride-based reagents. In the latter approach, native OT in serum and urine was concentrated by offline SPE using gradient acetonitrile washings after first crashing with acetonitrile. Peak urinary eluate fractions were further concentrated online then analyzed by orbitrap-based LC-MS with electrospray ionization. All hydrophobic OT derivatives had lower sensitivity than native OT. Washing with a water-acetonitrile gradient during SPE improved the LLOD of OT in spiked serum to 2.5 pg/mL while adding a subsequent online-concentration step improved the LLOD in spiked urine to 1-5 pg/mL and allowed us to detect OT in urine from lactating women. We were unable to improve the sensitivity of OT measurements by hydrophobic tagging or by derivatization using sulfonyl chloride-based reagents. However, we were successful in improving the sensitivity of native OT measurements in serum and urine 2- and 5-fold, respectively, from our previous orbitrap-based LC-MS method. Offline SPE was mandatory for both matrices and a subsequent online-concentration step was required for urine.

Keywords:  SPE; orbitrap LC-MS; oxytocin; serum; urine

DOI:  https://doi.org/10.1002/dta.2783
J Anal Toxicol. 2020 Feb 27. pii: bkaa021. [Epub ahead of print]

Structure elucidation of urinary metabolites of fentanyl and five fentanyl analogues using LC-QTOF-MS, hepatocyte incubations and synthesized reference standards.

Jakob Wallgren, Svante Vikingsson, Tobias Rautio, Enas Nasr, Anna Åstrand, Shimpei Watanabe, Robert Kronstrand, Henrik Gréen, Johan Dahlén, Xiongyu Wu, Peter Konradsson.

  Fentanyl analogues constitute a particularly dangerous group of new psychoactive compounds responsible for many deaths around the world. Little is known about their metabolism and studies utilizing LC-QTOF-MS analysis of hepatocyte incubations and/or authentic urine samples does not allow for determination of the exact metabolite structures, especially when it comes to hydroxylated metabolites. In this study seven motifs (2-, 3-, 4- and β-OH as well as 3,4-diOH, 4-OH-3-OMe and 3-OH-4-OMe) of fentanyl and five fentanyl analogues, acetylfentanyl, acrylfentanyl, cyclopropylfentanyl, isobutyrylfentanyl and 4F-isobutyrylfentanyl were synthesized. The reference standards were analyzed by LC-QTOF-MS, which enabled identification of the major metabolites formed in hepatocyte incubations of the studied fentanyls. By comparison with our previous data sets, major urinary metabolites could tentatively be identified. For all analogues, β-OH, 4-OH and 4-OH-3-OMe were identified after hepatocyte incubation. β-OH was the major hydroxylated metabolite for all studied fentanyls, except for acetylfentanyl where 4-OH was more abundant. However, the ratio 4-OH/β-OH was higher in urine samples than in hepatocyte incubations for all studied fentanyls. Also, 3-OH-4-OMe was not detected in any hepatocyte samples, indicating a clear preference for the 4-OH-3-OMe, which was also found to be more abundant in urine compared to hepatocytes. The patterns appear to be consistent across all studied fentanyls and could serve as a starting point in the development of methods and synthesis of reference standards of novel fentanyl analogues where nothing is known about the metabolism.

Keywords:  Fentanyl analogue; human hepatocytes; metabolism; new psychoactive substances; reference standards

DOI:  https://doi.org/10.1093/jat/bkaa021
J Chromatogr Sci. 2020 Feb 26. pii: bmz121. [Epub ahead of print]

The determination of common anabolic steroid and stimulants in nutritional supplements by HPLC-DAD and LC-MS.

Riyadh Salih Al-Khadhra.

  A high-performance liquid chromatography method employing a diode-array detector and mass spectrometry detector was developed, validated and implemented for determining Synephrine, Caffeine, Clenbuterol, Nandrolone, Testosterone and Methylhexaneamine in Nutritional supplements. The use of Nutritional supplements is widespread. Hazards relating to concentration, composition, individual contaminants, supplements interactions as well as positive doping results among athletes present increasing concerns regarding nutritional supplement consuming. The proposed method was validated according to the International Conference on the Harmonization of the Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) standards. The proposed method observed to be accurate, linear, precise, sensitive, required minimal sample preparation and uncomplicated mobile phase. The implementation of the proposed method on nine commercial supplements shows that inaccurate labeling for some supplements regarding the concentration of the ingredients.

Keywords:  Caffeine; Clenbuterol; Doping; HPLC-DAD; LC-MS; Methylhexaneamine; Nandrolone; Nutritional supplements contamination; Synephrine; Testosterone

DOI:  https://doi.org/10.1093/chromsci/bmz121
Rapid Commun Mass Spectrom. 2020 Feb 27. e8769

Mass spectral fragmentation analyses of isotopically labelled hydroxy steroids using low resolution gas chromatography - mass spectrometry (GC/EI-MS): a practical approach.

Annette Sophie Kollmeier, Maria Kristina Parr.

RATIONALE: Gas chromatography coupled to electron ionization mass spectrometry (GC/EI-MS) is used for routine screening of anabolic steroids in many laboratories after the conversion of polar groups to trimethylsilyl (TMS) derivatives. The aim of this work is to elucidate the origin and formation of common and subclass specific fragments in the mass spectra of TMS-derivatized steroids. Especially in the context of metabolite identification or analysis of designer drugs, isotopic labelling is helpful to better understand fragment ion generation, identify unknown compounds and update established screening methods.
METHODS: Stable isotope labelling procedures for the introduction of [2 H9 ]-TMS or 18 O were established to generate perdeuterotrimethylsilylated, mixed deuterated and 18 O-labelled derivatives for 13 different hydroxysteroids. Fragmentation proposals were substantiated by comparison of the abundances of isotopic labelled and unlabelled fragment ions in unit mass resolution GC/MS. Specific fragmentations were also investigated by high resolution MS (GC/quadrupole time-of-flight MS, GC/QTOFMS).
RESULTS: Methyl radical cleavage occurs primarily from the TMS-groups in saturated androstanes and from the steroid nucleus in case of enol-TMS of oxo or a,b-unsaturated steroid ketones. Loss of trimethylsilanol (TMSOH) is dependent on steric factors, degree of saturation of the steroid backbone and availability of hydrogen and TMSO-group in the 1,3-diaxial position. For the formation of the [M - 105]+ fragment ion, methyl radical cleavage predominates from the angular methyl groups in position C-18 or C-19 and is independent of the site of TMSOH loss. The common [M - 15 - 76]+ fragment ion was found in low abundance and identified as [M - CH3 - (CH3 )2 SiH-OH]+ . For the different steroid subclasses further diagnostic fragment ions were discussed and structure proposals postulated.
CONCLUSION: Stable isotope labelling of oxo groups as well as derivatization with deuterated TMS groups enables detection of structure related fragment ion generation in unit mass resolution GC/EI-MS. This may in turn allow us to propose isomeric assignments that are otherwise almost impossible using MS only.

DOI: https://doi.org/10.1002/rcm.8769
J Appl Toxicol. 2020 Feb 26.

Screening procedure for 38 fentanyl analogues and five other new opioids in whole blood by liquid chromatography-tandem mass spectrometry.

Piotr Adamowicz, Zhanetta Bakhmut, Anna Mikolajczyk.

  In recent years, many new opioids, particularly fentanyl analogues, have appeared on the drug market. The extreme potency of even low doses of these compounds leads to numerous fatal poisonings. This also results in the fact that only sophisticated techniques are capable of detecting fentanyl analogues at concentrations that can be expected in blood. In this context, the purpose of this study was to develop a fast liquid chromatography-tandem mass spectrometry screening method for the detection of fentanyl analogues, and other new synthetic opioid receptor agonists in whole blood. Blood samples were extracted with ethyl acetate under basic conditions. The separation was achieved with the gradient of the mobile phase composition and the gradient of the flow rate in 13 minutes. The detection of all compounds was based on dynamic multiple reaction monitoring. Most of the compounds were well differentiated by their retention times and/or transitions; however, separation of some isomers has not been achieved. The validation was performed for 21 compounds. The limits of detection were in the range 0.01-0.20 ng/mL. The developed procedure enables simultaneous qualitative screening, detection and identification of 38 fentanyl analogues and five other new opioids. The method was implemented to analyze authentic samples (positive; n = 3) demonstrating its suitability for this application. The procedure can be easily expanded to include new emerging opioids, which is an indispensable advantage in the dynamically developing drug market. The developed protocol can be adopted for routine work in both forensic and clinical analytical laboratories worldwide.

Keywords:  LC-MS/MS; new fentanyls; new opioids; new psychoactive substances; screening analysis

DOI:  https://doi.org/10.1002/jat.3962
Drug Des Devel Ther. 2020 ;14 407-415

A New Validated HPLC-MS/MS Method for Quantification and Pharmacokinetic Evaluation of Dovitinib, a Multi-Kinase Inhibitor, in Mouse Plasma.

Haitham AlRabiah, Adnan A Kadi, Haya I Aljohar, Mohamed W Attwa, Nasser S Al-Shakliah, Sabry M Attia, Gamal Ae Mostafa.

   Background: Dovitinib (TKI 258) is a small-molecule multi-kinase inhibitor for the treatment of different types of cancer. There is currently no validated method for its quantitative determination; therefore, we aimed to develop a reliable method to assay dovitinib.
Method and Results: An electrospray ionization tandem mass spectrometry (ESI-MS/MS) method was used to separate dovitinib using an analytical C18 column (50 × 2.1 mm, 1.8 μm) at 25°C. Bosutinib was used as the internal standard (IS). Dovitinib was extracted from mouse plasma using a precipitation procedure. The mobile phase consisted of 10 mM ammonium formate: acetonitrile (68:32, v/v, pH 4.3) run at a rate of 0.3 mL min-1. MS detection was performed in the positive ion mode. Multiple reaction monitoring transitions were 393→337 and 393→309 for dovitinib, and 530→141 and 530→113 for bosutinib. The investigated method was validated as a bio-analytical method based on FDA guidelines. The linearity of the developed method was over the range of 5-500 ng mL,-1 coefficient of determination (r2= 0.9998). The average intra-day recovery and relative standard deviation (RSD) of the quality control (QC) sample were 97.24% and 1.32%, whereas the overall inter-day accuracy and precision were 97.99% and 0.54%, respectively. Dovitinib was stable during sample storage and handling conditions. Furthermore, the dilution integrity of the method was demonstrated by good recovery (97-99%) and RSD values (0.5-0.7%).
Conclusion: This method was selectively sensitive and exhibited no matrix effect, with an acceptable accuracy and precision according to the FDA guidelines. The developed method could be efficiently used for pharmacokinetic studies of dovitinib.

Keywords:  HPLC; MS detection; dovitinib; mouse plasma; pharmacokinetics

DOI:  https://doi.org/10.2147/DDDT.S223573
Steroids. 2020 Feb 22. pii: S0039-128X(20)30039-8. [Epub ahead of print] 108614

Quantification of endogenous steroid sulfates and glucuronides in human urine after intramuscular administration of testosterone esters.

Guro Forsdahl, Katharina Zanitzer, Damir Erceg, Günter Gmeiner.

  For an effective detection of doping with pseudo-endogenous anabolic steroids, the urinary steroid profile is of high value. In this work, the aim was to investigate steroid metabolism disruption after exogenous intramuscular administration of different testosterone esters. The investigation focused on both sulfo - and glucoro conjugated androgens. A single intramuscular injection of either 1000 mg testosterone undecanoate (Nebido®) or a mixture of 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone isocaproate, and 100 mg testosterone decanoate (Sustanone®), was given to six healthy volunteers. Urine was collected throughout a testing period of 60 days. A LC-MS method was developed and validated for the analysis of eight conjugated steroids in their intact form. The results show that urinary changes in both sulfo - and glucuro conjugated steroid levels are prominent after the injection of testosterone esters. A promising potential marker for the intake of exogenous testosterone is the combined ratio of epitestosterone sulfate/epitestosterone glucuronide to testosterone sulfate/testosterone glucuronide ((ES/EG)/(TS/TG)) as a complementary biomarker for testosterone abuse. This represents a new piece of evidence to detect testosterone doping, representing a new approach and being independent from the metabolic connections of the markers in the steroid passport.

Keywords:  Doping analysis; Phase II metabolites; Steroid glucuronides; Steroid sulfates; Urinary steroid profile

DOI:  https://doi.org/10.1016/j.steroids.2020.108614
J Anal Toxicol. 2020 Feb 25. pii: bkz118. [Epub ahead of print]

Detection of Chemical Weapon Nerve Agents in Bone by Liquid Chromatography-Mass Spectrometry.

Katie M Rubin, Bruce A Goldberger, Timothy J Garrett.

A recently proposed model for the incorporation of xenobiotics of forensic interest into the human skeleton suggests nerve agent metabolites may incorporate into bone at relatively elevated concentrations based on their unique chemical properties. To test the hypothesis that nerve agent metabolites interact with bone, methods for the extraction, isolation and semi-quantitative detection of nerve agent metabolites (MPA, EMPA, IMPA, iBuMPA, CMPA and PMPA, corresponding to the nerve agents VX, Russian VX, sarin, cyclosarin and soman, respectively) from osseous tissue were developed using liquid chromatography-mass spectrometry with both quadrupole time-of-flight and triple quadrupole (QqQ) instruments. The optimized methods were validated on the QqQ instrument. Despite high ion suppression, the achieved limits of detection (5-20 pg/g for four analytes; 350 pg/g for the fifth analyte) were lower than many of those published for the same analytes in other biomatrices, including serum and urine. These methods were tested on the skeletal remains of minipigs exposed to the chemical weapon VX in vivo. The VX metabolite was detected in multiple minipig bone samples; to the authors' knowledge, this is the first time in vivo nerve agent exposure has been detected from bone. Further, detected concentrations and diaphyseal-to-epiphyseal area count ratios reflect animal exposure history. Although the results are limited, they are promising, indicating that nerve agent metabolites may interact with bone as a pharmacokinetic compartment and can be extracted from bone postmortem. Additional studies, assessing the effects of different agents, exposure pathways and taphonomic variables, are needed; however, these results suggest the method may be used with human bone to detect use of chemical weapons from postmortem biomatrices even well after a suspected attack. More general implications for both nerve agent toxicology and skeletal toxicology are also discussed.

DOI: https://doi.org/10.1093/jat/bkz118
Mol Cell Biochem. 2020 Feb 27.

Evaluation of oxysterol levels of patients with silicosis by LC-MS/MS method.

Neslihan Aksu, Afshin Samadi, Ahmet Yalçınkaya, Tuğçe Çetin, Burcu Eser, İncilay Lay, Türkan Nadir Öziş, Yeşim Öztaş, Suna Sabuncuoğlu.

  Silicosis is one of the prolonged and irreversible occupational diseases. Crystalline silica dust, which has been linked with silicosis, occurs in different industrial areas such as constructions, ceramic, quarry, and pottery. There are significant numbers of newly diagnosed cases every year in Turkey. Patients with silicosis suffer from inflammatory respiratory disorders and silicosis-related complications such as rheumatoid arthritis, systemic sclerosis, and vasculitis. Oxysterols are defined as 27-carbon intermediates or end products of cholesterol. They are also implicated in the etiology of disease states such as atherosclerosis, neurodegenerative, and inflammatory diseases. The aim of the study is to evaluate cholesterol oxidation products in the patients with silicosis and determination of sphingosine-1-phosphate (S1P) levels which is a sphingolipid metabolite. In addition to these parameters, it is aimed to determine the possible lipid peroxidation by different parameters. For this purpose, blood samples and urine were collected from 47 patients and 30 healthy individual with their consents. In order to evaluate oxysterols, 7-ketocholesterol and cholestan 3β,5α,6β-triol levels were measured by LC-MS/MS method. The measured levels of 7-KC were 0.101 ± 0.005 µmol/l in patient and 0.050 ± 0.003 µmol/l in control plasma samples. Triol levels were measured as 0.038 ± 0.005 µmol/l in patient group and 0.033 ± 0.004 µmol/l in control group (p < 0.001). In addition, lipid peroxidation products were measured by human-8-isoprostane, human-4-hydroxynonenal (4-HNE), and human malondialdehyde (MDA) ELISA kits. The measured levels of HNE in the patient and control groups were 735.14 ± 288.80 pg/ml and 595.72 ± 108.62 pg/ml in plasma and 606.02 + 118.23 pg/ml and 531.84 + 107.18 pg/ml in urine, respectively (p < 0.05). F2-iP results of patients and controls were 450.0 + 101.40 pg/dl and 386.9 + 112.7 pg/ml for urine and 432.7 ± 188,8 pg/dl and 321.9 ± 69.4 pg/dl for plasma, respectively (p < 0.05). MDA levels of plasma were measured as 44.1 ± 14.6 nmol/ml in the patient and 31.9 ± 10.5 nmol/ml in the control (p < 0.05). Levels of MDA for urine samples were 30.15 + 5.06 nmol/ml and 25.15 + 6.07 nmol/ml in patients and controls, respectively (p < 0.05). S1P levels were decreased in patients compared to control group (49.05 ± 10.87 and 67.57 ± 16.25, p < 0.001). The results not only indicate a correlation between cholesterol oxidation, lipid peroxidation, and silicosis, but also provide better understanding of the role of the lipids in the mechanism of this inflammatory disease.

Keywords:  Cholesterol; Lipid peroxidation; Oxysterols; S1P; Silicosis

DOI:  https://doi.org/10.1007/s11010-020-03706-w