bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒05‒31
28 papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

  1. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 May 19. pii: S1570-0232(20)30223-3. [Epub ahead of print]1149 122157
      Since cannabis and amphetamine-type stimulants (ATS) are drugs of abuse used alone and concurrently worldwide, it is important to analyze them simultaneously. However, there are no reports of analytical method for the simultaneous extraction of these compounds and metabolites in hair samples of suspected drug abusers, due to the different chemical properties of acidic and lipophilic cannabinoids, and basic and hydrophilic ATS. We developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for the quantification of cannabidiol (CBD), cannabinol (CBN), (-)-trans-Δ9-tetrahydrocannabinol (THC), THC metabolites such as 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) and THC-COOH-glucuronide (THC-COOH-glu), and six ATS of amphetamine, methamphetamine, phentermine, methylenedioxyamphetamine, methylenedioxymethamphetamine, and methylenedioxyethylamphetamine in the least amounts of human hair samples. The pulverized hair samples (10 mg) were extracted with 1 mL of 0.5% formic acid in methanol three times, and the supernatants were evaporated in a stream of nitrogen gas. The residue was dissolved and the aliquot was analyzed by LC-HRMS using positive electrospray ionization and the parallel reaction monitoring mode. The lower limits of quantitation were 0.1 pg mg-1 for THC-COOH and THC-COOH-glu, 4 pg mg-1 for CBD, CBN, and THC, and 10 pg mg-1 for the six ATS. Linearity, accuracy, precision, matrix effect, recovery, and post-preparation stability for the 11 analytes were fully validated. The present method was successfully applied to the determination of 11 analytes in hair samples of 10 suspected drug abusers.
    Keywords:  (−)-trans-Δ(9)-tetrahydrocannabinol and metabolites; Amphetamine-type stimulants; Cannabinoids; Human hair; Liquid chromatography-high resolution mass spectrometry
  2. J Chromatogr A. 2020 Jul 05. pii: S0021-9673(20)30400-3. [Epub ahead of print]1622 461160
      The glutathione (GSH) trapping assay is commonly utilized for the screening and characterization of reactive metabolites produced by drug metabolism. This study describes a fluorous derivatization method for a more sensitive and selective analysis of reactive metabolites trapped by GSH using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, the GSH-trapped reactive metabolites, which were obtained after incubation of the test compounds with human liver microsome (HLM) in the presence of GSH and NADPH, were derivatized using the perfluoroalkylamine reagent through oxazolone chemistry. Since this reaction enabled the selective modification of the α-carboxyl group in GSH, the structural compositions of the metabolites were not affected by the derivatization. Furthermore, the selective analysis of the resulting derivatives could be performed using perfluoroalkyl-modified stationary phase LC separation via the interaction between the perfluoroalkyl-containing compounds, such as fluorous affinity, followed by detection with the precursor ion and/or enhanced product ion scan modes in MS/MS. Finally, we demonstrated the applicability of this method by analyzing perfluoroalkyl derivatives of some drug metabolites trapped by GSH in HLM incubation.
    Keywords:  Fluorous derivatization; Glutathione; LC–MS/MS; Oxazolone chemistry; Reactive metabolite
  3. Molecules. 2020 May 27. pii: E2483. [Epub ahead of print]25(11):
      Background: Determination of psychotropic drugs in clinical study is significant, and the establishment of methodologies for these drugs in biological matrices is essential for patients' safety. The search for new methods for their detection is one of the most important challenges of modern scientific research. The methods for analyzing of psychotropic drugs and their metabolites in different biological samples should be based on combining a very efficient separation technique including high-performance liquid chromatography (HPLC), with a sensitive detection method and effectively sample preparation methods. Objective: Retention, peaks symmetry and system efficiency of vortioxetine on Hydro RP, Polar RP, HILIC A (with silica stationary phase), HILIC-B (with aminopropyl stationary phase), and ACE HILIC-N (with polyhydroxy stationary phase and SCX columns were investigated. Various mobile phases containing methanol or acetonitrile as organic modifiers and different additives were also applied to obtained optimal retention, peaks shape, and systems efficiency. The best chromatographic procedure was used for simultaneous analysis of vortioxetine and its metabolites in human serum, urine and saliva samples. Methods: Analysis of vortioxetine was performed in various chromatographic systems: Reversed phase (RP) systems on alkylbonded or phenyl stationary phases, hydrophilic interaction liquid chromatography (HILIC), and ion-exchange chromatography (IEC). Based on the dependence of log k vs the concentration of the organic modifier, log kw values for vortioxetine in various chromatographic systems were determined and compared with calculated log P values. Solid phase extraction (SPE) method was applied for sample pre-treatment before HPLC analysis. HPLC-QTOF-MS method was applied for confirmation of presence of vortioxetine and some its metabolites in biological samples collected from psychiatric patient. Conclusions: Differences were observed in retention parameters with a change of the applied chromatographic system. The various properties of stationary phases resulted in differences in vortioxetine retention, systems' efficiency, and peaks' shape. Lipophilicity parameters were also determined using different HPLC conditions. The most optimal systems were chosen for the analysis of vortioxetine in biological samples. Both serum and urine or saliva samples collected from patients treated with vortioxetine can be used for the drug determination. For the first time, vortioxetine was detected in patient's saliva. Obtained results indicate on possibility of application of saliva samples, which collection are non-invasive and painless, for determination and therapeutic drug monitoring in patients.
    Keywords:  HPLC-DAD; HPLC-QTOF-MS; SPE; lipophilicity; saliva; serum; urine; vortioxetine
  4. Rapid Commun Mass Spectrom. 2020 May 26. e8844
      RATIONALE: In recent environmental research, multi-methods using high-performance liquid chromatography-mass spectrometry (HPLC/MS) have become more and more important for the analysis of organic micropollutants in environmental matrices. As the targeted compounds usually have quite different physico-chemical properties, the optimization of these methods is challenging.METHODS: The pH in the electrospray of the mass spectrometer ion source was modified independently from the pH used for the HPLC separation, using a post-column HPLC flow in order to make the spray acidic or alkaline. The method development was conducted manually in a systematic multistep way.
    RESULTS: The method used for the analysis of organic biocides (e.g. terbutryn, propiconazole, isothiazolinones) was optimized. It was shown that the use of a neutral as well as an acidified gradient could not be optimized for all target compounds, while a neutral gradient with post-column acidification was optimum for all target compounds.
    CONCLUSIONS: Acidic or alkaline post-column infusion allows pH optimization for ionization conditions, independently of the pH optimization for chromatographic separation. The introduction of an additional modifier might also allow minimization of matrix effects or directed formation of sodium adducts, without affecting the chromatographic separation.
  5. ACS Nano. 2020 May 28.
      Mass spectrometry imaging (MSI) based on matrix-assisted laser desorption ionization (MALDI) is widely used in proteomics. However, matrix-free technologies are gaining popularity for detecting low molecular mass compounds. Small molecules were analyzed with nanostructured materials as ionization promoters, which produce low-to-no background signal, and facilitate enhanced specificity and sensitivity through functionalization. We investigated the fabrication and the use of black silicon and gold-coated black silicon substrates for surface-assisted laser desorption/ionization mass spectrometry imaging (SALDI-MSI) of animal tissues and human fingerprints. Black silicon was created using dry etching while gold nanoparticles were deposited by sputtering. Both methods are safe for the user. Physicochemical characterization and MSI measurements revealed the optimal properties of the substrates for SALDI applications. The gold-coated black silicon worked considerably better than black silicon as SALDI-MSI substrate. The substrate was also compatible with imprinting, a sample-simplification method that allows the efficient transference of metabolites from the tissues to the substrate surface, without compound delocalization. Moreover, by modifying the surface with hydrophilic and hydrophobic groups, specific interactions were stimulated between surface and sample, leading to a selective analysis of molecules. Thus, our substrate facilitates targeted and/or untargeted in situ metabolomics studies for various fields such as clinical, environmental, forensics and pharmaceutical research.
  6. Rapid Commun Mass Spectrom. 2020 May 29. e8845
      RATIONALE: High throughput LC-MS is an increasing topic in analytical chemistry. Especially the idle time of a mass spectrometer should be reduced for an efficient and cost saving use. Therefore, a fast-switching dual ion source was developed, which uses the most important ionization techniques at atmospheric pressure, ESI and APCI, with one or more LC systems.METHODS: The performance of the developed ion source is shown by infusion experiments and chromatographic analyses of different standard substances. A high throughput method is demonstrated by coupling two UHPLC systems to the dual ion source with a triple quadrupole mass spectrometer.
    RESULTS: No decrease of the ion abundance and a stable performance of the MS is presented while using the dual ion source. Instrumental limits of detection are 30 ng L-1 for testosterone using ESI and 1 μg L-1 for vitamin D3 using APCI. A fast switching between two UHPLCs and the dual ion source is leading to a high sample throughput of 50 samples in 75 min with relative standard deviations for testosterone and vitamin D3 of 1.5 % and 3.8 %, respectively.
    CONCLUSION: This work presents the development of a dual ESI and APCI ion source operating simultaneously or in switched mode. The results show sensitive and reliable performance as well as the hyphenation to one or more HPLC systems.
  7. Turk J Pharm Sci. 2019 Jun;16(2): 227-233
      Objectives: The fixed dose combination of saxagliptin and dapagliflozin is a recently approved antidiabetic medication. It is marketed under the brand name Qtern. The aim of this study was to develop a simple, rapid, sensitive, and validated isocratic reversed phase-high performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of saxagliptin and dapagliflozin in human plasma using linagliptin as internal standard as per US-Food and Drug Administration guidelines.Materials and Methods: The method was performed on a Waters 2695 HPLC equipped with a quaternary pump. The analyte separation was achieved using an Eclipse XDB C18 (150 × 4.6 mm × 5 µm) column with a mobile phase consisting of 0.1% ortho phosphoric acid and acetonitrile (50:50) with pH adjusted to 5.0 at 1 mL/min flow rate.
    Results: The analyte was detected at 254 nm. The retention time of the internal standard, saxagliptin, and dapagliflozin was 2.746, 5.173, and 7.218 min, respectively. The peaks were found to be free of interference. The method was validated over a dynamic linear range of 0.01 to 0.5 μg/mL and 0.05 to 2 μg/mL for saxagliptin and dapagliflozin, respectively, with a correlation coefficient of 0.998. The precision and accuracy of samples of six replicate measurements at lower limits of quantification level were within the limits. The analytes were found to be stable in human plasma at -28°C for 37 days.
    Conclusion: The stability, sensitivity, specificity, and reproducibility of this method make it appropriate for the determination of saxagliptin and dapagliflozin in human plasma.
    Keywords:  Saxagliptin; dapagliflozin; human plasma; isocratic; linagliptin
  8. Biomed Chromatogr. 2020 May 27. e4907
      Creatinine is an important diagnostic marker and is also used as a standardization tool for the quantitative evaluation of exogenous/endogenous substances in urine. An aim of this study was to evaluate and compare three analytical approaches, based on hyphenations of different separation (2D capillary isotachophoresis, CITP-CITP; capillary zone electrophoresis, CZE; ultra-high performance liquid chromatography, UHPLC) and detection (conductivity, CD; ultraviolet, UV; tandem mass spectrometry, MS/MS) techniques, for their ability to provide reliable clinical data along with their suitability for the routine clinical use (cost, simplicity, sample throughput). The developed UHPLC-MS/MS, CITP-CITP-CD, and CZE-UV methods were characterized by favorable performance parameters, such as linearity (r˃0.99), precision (RSD 0.22-2.97% for the creatinine position in analytical profiles), and recovery (87.1-115.1%). Clinical data, obtained from the analysis of 24 human urine samples by a reference enzymatic method, were comparable with those obtained by the tested methods (Passing-Bablok regression, Bland-Altman analysis), approving their usefulness for the routine clinical use. In this context, the UHPLC-MS/MS method provides benefits of enhanced orthogonality/accuracy and high sample throughput (3-fold shorter total analysis times than the CE methods) while advantages of the CE methods for routine labs are simplicity and low cost of both the instrumentation as well as measurements.
    Keywords:  Capillary isotachophoresis; Capillary zone electrophoresis; Clinical analysis; Creatinine; Mass spectrometry; Ultra high-performance liquid chromatography
  9. Anal Chem. 2020 May 29.
      We demonstrate a method for facile differentiation of acidic, isomeric metabolites by attaching high proton affinity, piperidine-based chemical tags to each carboxylic acid group. These tags attach with high efficiency to the analytes, increase signal, and result in formation of multiply positively charged analyte ions. We illustrate the present approach with citrate and isocitrate which are isomeric metabolites each containing three carboxylic acid groups. We observe a 20-fold increase in signal-to-noise for citrate, and an 8-fold increase for isocitrate as compared to detection of the untagged analytes in negative mode. Collision-induced dissociation of the triply tagged, triply charged analytes results in distinct tandem mass spectra. The phenylene spacer groups limit proton mobility and enable access to structurally informative C-C bond cleavage reactions. Modeling of the gas-phase structures and dissociation chemistry of these triply charged analyte ions highlights the importance of hydroxyl proton mobilization in this low proton mobility environment. Tandem mass spectrometric analyses of deuterated congeners and MS3 spectra are consistent with the proposed fragment ion structures and mechanisms of formation. Direct evidence that these chemistries are more generally applicable is provided by subsequent analyses of doubly tagged, doubly charged malate ions. Future work will focus on applying these methods to identify new metabolites and development of general rules for structural determination of tagged metabolites with multiple charges.
  10. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 May 15. pii: S1570-0232(19)31823-9. [Epub ahead of print]1149 122155
      Homocysteine thiolactone (HTL) is a chemically reactive thioester that has been implicated in cardiovascular disease. So far, its presence has been documented in human and mouse plasma and urine. Here, using a new method, we show that HTL is present in human saliva. The assay involves chloroform-methanol extraction of HTL, lyophilization, and derivatization with N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS). The method is based on a gas chromatography coupled with mass spectrometry (GC-MS) and quantifies HTL in a linear range from 0.05 to 1 µmol L-1 saliva and urine. The limit of quantification (LOQ) was 0.05 µmol L-1. With respect to saliva specimen, the accuracy was 98.7-112.6%, and 90.2-100.5%, while the precision was 7.1-13.5% and 12.5-15.0% for the intra- and inter-day variation, respectively. In relation to urine samples, the accuracy was 91.9-110.9% and 91.2-103.3%, while the precision varied from 2.2% to 14.5% and 7.4% to 14.3% for intra- and inter-day measurements, respectively. Using this method, we show that in apparently healthy individuals (n = 18), HTL levels in saliva are not positively correlated with urinary HTL levels. Undoubtedly, larger population should be investigated to get more meaningful results.
    Keywords:  Gas chromatography – mass spectrometry; Homocysteine thiolactone; Human saliva; Human urine; Liquid-liquid extraction; N-trimethylsilyl-N-methyl trifluoroacetamide
  11. Biomed Chromatogr. 2020 May 25. e4905
      A simple and specific LC-MS/MS method was developed and validated for the determination of ethyl ester of eicosapentaenoic acid (EPAEE) and ethyl ester of docosahexaenoic acid (DHAEE). After de-protein with acetonitrile, the plasma samples were separated on a C18 column using a gradient elution system consisted of methanol and 1.0 mM ammonium acetate in water. The detection used an APCI ion source in positive mode with multiple reaction monitoring for the quantitation of EPAEE and DHAEE. The acceptable linearity was achieved over the concentration ranges of 1.00~1000 ng/mL for EPAEE and 2.50~2500 ng/mL for DHAEE, respectively. The method was successfully applied to a pharmacokinetic study of EPAEE and DHAEE in healthy Chinese subjects after oral administration of 4 g Omega-3-acid ethyl esters 90 soft capsule. The pharmacokinetic profiles of EPAEE and DHAEE were observed for the first time in Chinese volunteers, which reached maximum concentration of 499 ± 243 ng/mL and 1596 ± 476 ng/mL for EPAEE and DHAEE, respectively. The areas under the plasma concentration-time curve were 1290 ± 765 ng/mL·h for EPAEE and 4369 ± 1680 ng/mL·h for DHAEE, respectively.
    Keywords:  ethyl ester of docosahexaenoic acid; ethyl ester of eicosapentaenoic acid; liquid chromatography/tandem mass spectrometry; pharmacokinetics
  12. Bioanalysis. 2020 May 29.
      Aim: FLZ, a novel promising dopamine neuroprotective agent, is designed to treat Parkinson's disease. F7G and F21G are FLZ major active Phase II metabolites whose exposure are nearly 100-times higher than FLZ, may chiefly produce effectiveness in human. Measurement of F7G and F21G in plasma samples is critical for investigating its pharmacokinetics in clinical studies. Methodology & results: Plasma samples were extracted by SPE method and then analyzed by a newly established ultra-UHPLC-MS/MS method. Conclusion: For the first time, a reliable and robust bioanalytical method for F7G and F21G detection was successfully applied in a first-in-human study.
    Keywords:  Parkinson's disease; UHPLC–MS/MS; human plasma; metabolites
  13. J Chromatogr Sci. 2020 May 25. pii: bmaa020. [Epub ahead of print]
      Gemcitabine is a deoxycytidine analog that has been used for a broad spectrum of tumor, such as nonsmall-cell lung cancer, bladder cancer and pancreatic cancer. Because gemcitabine is hydrophilic, hydrophilic interaction liquid chromatography (HILIC), where analytes are retained on a polar column according to their hydrophilicity, should be adequate for separation analysis of gemcitabine. In the present study, we proposed a hydrophilic interaction chromatography with ultraviolet (HILIC-UV) method with liquid-liquid extraction and adding tetrahydrouridine to plasma samples for gemcitabine analysis of clinical samples with respect to daily and wide usage. The method successfully determined gemcitabine in 56 plasma samples of 30 unique patients. Mean plasma concentration of gemcitabine was 15.0 ± 6.0 μg/mL (mean ± standard deviation). The concentration range is consistent with the data from previous literatures. Our proposed HILIC-UV method is simple and easy handling, and is widely and clinically usable for determination of gemcitabine in human plasma.
  14. Curr Protoc Toxicol. 2020 Jun;84(1): e93
      Bone marrow transplantation is used to treat particular types of cancers such as lymphoma, leukemia, and multiple myeloma. Appropriate dosing of busulfan during the preparative phase is critical for a successful allograft; if blood concentrations get too high significant liver toxicity can occur, if blood concentrations are too low, then graft-versus-host disease (GVHD) can develop. Busulfan monitoring in blood allows hospitals with the opportunity to provide individualized medicine to patients and improve overall patient outcome. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) is an important analytical method for quantification of busulfan in plasma in order to optimize the dose. © 2020 Wiley Periodicals LLC. Basic Protocol: Analysis of busulfan by liquid chromatography/mass spectrometry.
    Keywords:  bone marrow transplantation; busulfan; chromatography; tandem mass spectrometry; therapeutic drug monitoring
  15. Anal Chem. 2020 May 29.
      We developed a new method for monitoring the distribution of administrated fatty acids in the body by combination of stable isotope-labeling technique and imaging mass spectrometry (IMS). The developed stable isotope-labeling technique is very simple and able to adapt to all the fatty acid species. In this study, we synthesized stable isotope-labeled arachidonic acid (AA) and docosahexaenoic acid (DHA) and they were simultaneously administrated to mice to examine their migrations and distributions in the brain. The administrated AA and DHA have two more molecular weight compared to original those and were apparently distinguished from originally accumulated AA and DHA in the brain using IMS. As the results, we could reveal that the administered AA and DHA firstly accumulated in the hippocampus and cerebellar cortex in the brain. This technique does not use radio isotope and would be able to apply to elucidate the role of all the kinds of fatty acid species in the body.
  16. Metabolomics. 2020 May 25. 16(6): 68
      BACKGROUND: Metabolomics provides measurement of numerous metabolites in human samples, which can be a useful tool in clinical research. Blood and urine are regarded as preferred subjects of study because of their minimally invasive collection and simple preprocessing methods. Adhering to standard operating procedures is an essential factor in ensuring excellent sample quality and reliable results.AIM OF REVIEW: In this review, we summarize the studies about the impacts of various preprocessing factors on metabolomics studies involving clinical blood and urine samples in order to provide guidance for sample collection and preprocessing.
    KEY SCIENTIFIC CONCEPTS OF REVIEW: Clinical information is important for sample grouping and data analysis which deserves attention before sample collection. Plasma and serum as well as urine samples are appropriate for metabolomics analysis. Collection tubes, hemolysis, delay at room temperature, and freeze-thaw cycles may affect metabolic profiles of blood samples. Collection time, time between sampling and examination, contamination, normalization strategies, and storage conditions may alter analysis results of urine samples. Taking these collection and preprocessing factors into account, this review provides suggestions of standard sample preprocessing.
    Keywords:  Metabolomics; Plasma; Preprocessing; Serum; Urine
  17. J Vis Exp. 2020 May 09.
      The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines. Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode. This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method generates reliable results and can be further applied to other in vitro cellular models.
  18. Anal Chem. 2020 May 28.
      Trifluoroacetic acid (TFA) is a commonly used mobile phase additive in liquid chromatography-mass spectrometry (LC-MS) based biopharmaceutical characterization to enhance reversed-phase chromatographic performance of peptide separation, however it leads to significant mass spectrometry signal suppression during electrospray ionization. Previous studies have shown that introducing large amounts of carboxylic acids or ammonium hydroxide to LC eluents post-column can improve MS sensitivity. In this study we discovered glycine as a simple additive for TFA mobile phases which mitigates ion suppression through a similar mechanism but in a more effective way than weak acid or weak base and boosts mass spectrometry responses (signal-to-noise ratio) of peptides by more than one order of magnitude on average after directly adding a small amount (e.g., 2 mM) into TFA mobile phases without compromising the chromatographic performance of peptide separation. Other small molecule additives in TFA mobile phases, such as amino acids containing extended side chains with different chemical properties and glycine-based variants, were also evaluated. The results demonstrated that glycine offered the best response boosting on peptides. The discovery of this glycine additive in TFA mobile phases provides a simple and conventional approach to achieve greater mass spectrometry detection sensitivity than TFA mobile phases for LC-MS based characterization of biopharmaceuticals. Keywords: Trifluoroacetic acid (TFA), Liquid chromatography-mass spectrometry (LC-MS), Biopharmaceutical characterization, Glycine additive, Increased signal-to-noise ratio.
  19. Prostaglandins Other Lipid Mediat. 2020 May 22. pii: S1098-8823(20)30056-3. [Epub ahead of print] 106463
      Low basal endogenous concentrations (< 20 pg/mL) of the 5-lipoxygenase (5-LO) pathway biomarker leukotriene E4 (LTE4) in human plasma present a significant analytical challenge. Analytical methods including liquid chromatography-mass spectrometry and enzyme linked immunosorbent assays have been used to quantify plasma LTE4 in the past but have not provided consistent data in the lower pg/mL-range. With our new method, a detection limit (<1 pg/mL plasma) significantly below basal levels of LTE4 was achieved by combining large volume sample purification and enrichment by anion-exchange mixed mode solid phase extraction (SPE) with large volume injection followed by chromatographic separation by ultra performance liquid chromatography (UPLC) and quantification by highly sensitive negative-ion electrospray tandem mass spectrometry (MS/MS). The method was reproducible, accurate and linear between 1-120 pg/mL plasma LTE4. The method was used to perform an analysis of plasma samples collected from healthy volunteers in a Phase 1 study with the FLAP (5-lipoxygenase activating protein) inhibitor AZD5718. Basal endogenous LTE4 levels of 5.1 ± 2.7 pg/mL were observed in healthy volunteers (n = 34). In subjects that had been administered a single oral dose of AZD5718, significant suppression (>80%) of plasma LTE4 level was observed, providing pharmacological evidence that endogenous 5-LO pathway activity could be assessed.
    Keywords:  5-lipoxygenase; AZD5718; FLAP; LTE4; Leukotriene E4; Liquid Chromatography; cardiovascular disease; mass spectrometry
  20. J Pharm Biomed Anal. 2020 May 24. pii: S0731-7085(20)30227-2. [Epub ahead of print]187 113358
      A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of sorafenib (SORA), its N-oxide active metabolite and of regorafenib (REGO) and its two active metabolites regorafenib N-oxide and N-desmethyl regorafenib N-oxide in hepatocellular carcinoma patients' plasma. A proper analytes' separation was obtained with Synergi Fusion RP column (4 μm, 80 Å, 50 × 2.0 mm) using a gradient elution of 10 mM ammonium acetate with 0.1% formic acid (mobile phase A) and methanol:isopropanol (90:10, v/v, mobile phase B) containing 0.1% formic acid. The analysis was then performed by electrospray ionization in negative mode coupled with a triple quadrupole mass spectrometry, API 4000QT, monitoring two transitions for each analyte, one for the quantification and the other for confirmation. The method could be easily applied to the clinical practice thanks to the short run (7 min), the low amount of patient plasma necessary for the analysis (5 μL) and the fast sample processing based on protein precipitation. The method was therefore fully validated according to FDA and EMA guidelines. The linearity was assessed (R2≥0.998) over the concentration ranges of 50-8000 ng/mL for SORA and REGO, and 30-4000 ng/mL for their metabolites, that appropriately cover the therapeutic plasma concentrations. The presented method also showed adequate results in terms of intra- and inter-day accuracy and precision (CV ≤ 7.2% and accuracy between 89.4% and 108.8%), recovery (≥85.5%), sensitivity, analytes stability under various conditions and the absence of the matrix effect. Once the validation was successfully completed, the method was applied to perform the Cmin quantification of SORA, REGO and their metabolites in 54 plasma samples collected from patients enrolled in a clinical study ongoing at the National Cancer Institute of Aviano.
    Keywords:  Hepatocellular carcinoma; LC–MS/MS; Regorafenib; Sorafenib; Therapeutic drug monitoring
  21. ACS Omega. 2020 May 19. 5(19): 10919-10926
      Oxidative stress (OS) plays a major role in the pathogenesis of various diseases in humans. OS is a result of an imbalance between reactive oxygen species (ROS) and the biologically available antioxidants that prevent or repair damage that ROS inflict on the host cells. ROS are naturally generated during normal mitochondrial respiration and by oxidative burst during the immune response. Many factors may influence OS, including genetics, diet, exercise, and exposure to environmental toxicants (e.g., tobacco smoke). A nonenzymatic peroxidation product of arachidonic acid (AA), 8-iso-PGF2α (8-isoprostane), is a validated biomarker of OS that is present in urine as both glucuronide conjugate and free acid. Previous studies report that the conjugated forms of 8-isoprostane can vary between 30 and 80% of the total 8-isoprostane levels. By hydrolyzing the conjugated forms, it is possible to obtain a total (free + conjugated) measurement of 8-isoprostane in urine samples. Here, we describe a robust, automated, and high-throughput method for measuring total urinary 8-isoprostane using a polymeric weak anion-exchange solid-phase extraction (SPE) and isotope-dilution ultrahigh performance liquid chromatography electrospray ionization-tandem mass spectrometry (UHPLC-MS/MS). This method, using a 96-well plate platform, showed good sensitivity (8.8 pg/mL LOD) and used only 400 μL of the sample volume with a cycle time of 11 min. The inter- and intraday precision, calculated from 20 repeated measurements of two quality control pools, varied from 4 to 10%. Accuracy, calculated from the recovery percentage at three spiking levels, ranged from 92.7 to 106.7%. We modified this method to allow for the exclusive measurement of free 8-isoprostane by removing the hydrolysis step. We measured both free and total 8-isoprostane in urine collected from 30 cigarette smokers (free: 460 ± 78.8 pg/mL; total: 704 ± 108 pg/mL) and 30 nonusers of tobacco products (free: 110 ± 24.2 pg/mL; total: 161 ± 38.7 pg/mL). This method is robust, accurate, and easily adaptable for large population studies.
  22. Talanta. 2020 Aug 15. pii: S0039-9140(20)30102-8. [Epub ahead of print]216 120811
      Herein we propose, for the first time, a rapid method based on flow injection analysis, electrospray ionization-tandem mass spectrometry (FIA-ESI-MS/MS) and multivariate calibration for the determination of l-leucine, l-isoleucine and L-allo-isoleucine in saliva. As far as we know, multivariate calibration has never been applied to the data from this non-separative approach. The possibilities of its use were explored and the results obtained were compared with the corresponding ones when using univariate calibration. Partial least square regression (PLS1) multivariate calibration models were built for each analyte by analyzing different saliva samples, and were subsequently applied to the analysis of another set of samples which had not been used in any calibration step. For Leu, the model worked satisfactorily with root mean square errors in the prediction step of 17%. This error can be considered acceptable and is common in methodologies that do not include a separation step. Results were compared with those obtained when univariate calibration was used, using the m/z transition 132.1 → 43.0 as the quantitation variable. In this case, the obtained results were not acceptable, with RMSEP of 236%, due to the fact that saliva samples contained another compound, different to the target analytes, which also shared the same transition. Ile and aIle have the same fragmentation patterns, so quantification of the sum of both compounds was performed, with RMSEP of 14% using a PLS1 model. Similar results were obtained when a univariate calibration model using the m/z transition 132.1 → 69.0 was employed. However, the use of this transition should be carefully examined when other compounds present in the matrix contribute to the analytical signal. The method increases sample throughput more than one order of magnitude compared to the corresponding LC-ESI-MS/MS method and is especially suitable as screening. When abnormally high or low concentrations of the analytes studied are obtained, the use of the method that includes separation is recommended to confirm the results.
    Keywords:  Electrospray ionization tandem mass spectrometry; Leucines; Multivariate calibration; Non-separative method; Saliva
  23. Anal Bioanal Chem. 2020 May 26.
      N-containing heterocycles (NCHs) are largely used as precursors for pharmaceuticals and can enter the environment. Some NCHs have been shown to be toxic, persistent, and very mobile in the environment. Thus, they have received increasing attention in the past years. However, the analysis of these polar compounds in environmental samples is still a challenge for liquid chromatography. This paper investigates the use of mixed-mode liquid chromatography (MMLC), which has reversed-phase and ion exchange characteristics for measurements of NCHs in water. NCHs with low pKa (i.e., < 2.5) display mainly reversed-phase interactions (neutral species) with the stationary phase and those with higher pKa (i.e., > 5) interact by a mixture of reversed-phase/ion exchange/HILIC mechanism. It was also shown that the presented method performs well in the quantification of the majority of the selected NCHs in surface water with MDLs between 3 and 6 μg/L, a low matrix effect and recoveries in the range of 77-96% except for pyridazine exhibiting 32% were achieved. The method was successfully employed to follow the degradation of NCHs in ozonation.
    Keywords:  Cation exchange; Liquid chromatography; Mixed-mode chromatography; Nitrogen-containing heterocycles; Reversed-phase
  24. Anal Bioanal Chem. 2020 May 28.
      The new ultra-high performance liquid chromatography method with tandem mass spectrometry detection (UHPLC-MS/MS) has been optimized to allow fast, selective, and high-throughput analysis of two Candida albicans quorum sensing molecules (QSM), farnesol and tyrosol. The problem of the presence of the interference in the samples and system was successfully solved by careful optimization of chromatographic conditions. Charged hybrid stationary phase modified with pentafluorophenyl group and optimized gradient elution provided adequate separation selectivity and peak shapes. The impurity was identified as dibutyl phthalate and had the same m/z ions as farnesol leading to an important interference on selected reaction monitoring channel. Two different types of biological matrices originating from vaginal fluid, supernatant and sediment, were analysed. Micro-solid phase extraction in pipette tips was optimized for the selective isolation of QSM from the supernatant. The insufficient retention of farnesol on the extraction sorbent was improved when 1% of organic solvent was added prior to extraction, while the retention of tyrosol was only possible when using combined C8 and polymer sorbent type. Strong retention of farnesol had to be solved by increasing elution solvent strength and volume up to 600 μL. However, this approach did not allow the pretreatment of sediment samples due to the sorbent clogging. Therefore, our previously developed protein precipitation method was modified and validated to analyse the sediments. New developed UHPLC-MS/MS method provided suitable accuracy and precision for the determination of QSM in vaginal fluid while using only 50 μL sample volume and two different sample preparation methods.
    Keywords:  Candida albicans; Farnesol; Microextraction; Quorum sensing; Tyrosol; UHPLC-MS/MS
  25. J Food Sci. 2020 May 25.
      Short-chain fatty acids (SCFAs) are the main metabolites of the intestinal flora and play an important role in the interaction between the intestinal flora and host metabolism. Therefore, reliable methods are needed to accurately measure SCFAs concentrations. SCFAs are commonly analyzed by gas chromatography-mass spectrometry (GC-MS), which requires lengthy sample treatments and a long run time. This study aimed to develop a fast GC method with formic acid pretreatment for SCFAs quantification in the plasma of rat. Baseline chromatographic resolution was achieved for three SCFAs (acetic, propionic, and butyric) within an analysis time of 10.5 min. The method exhibited good recovery for a wide range of concentrations with a low limit of detection for each compound. The relative standard deviations (RSDs) of all targeted compounds showed good intra- and interday precision (<10%). We used our method to measure SCFAs levels in plasma samples from rats fed with a high fructose diet (HFD) to test the accuracy of the developed method. It was shown that SCFAs are indeed affected negatively by a HFD (60% fructose). This method was successfully employed to accurately determine SCFAs in the rat plasma with minimum sample preparation. Results showed potential damage of HFD, which produced lower SCFAs. PRACTICAL APPLICATION: Increasingly, microbiota and gut health research are being conducted by many food scientists to elucidate the relationships among the factors of food components, particularly the nondigestible carbohydrates, food processing conditions, and potential health impact. This research provides a useful, rapid, and accurate method that can save time in the analysis of short-chain fatty acids, which are commonly analyzed in gut health research.
    Keywords:  gas chromatography; rat plasma; short-chain fatty acid
  26. Anal Chem. 2020 May 29.
      Blood metabolomics has been widely used for discovering potential metabolite biomarkers of various diseases. In this study, we report our investigation of the effects of freeze-thaw cycles (FTCs) of human serum samples on quantitative metabolomics using a differential chemical isotope labeling (CIL) LC-MS method. A total of 99 serum samples collected from healthy individuals (47 females and 52 males) were subjected to 5 FTCs, followed by 12C-/13C-dansylation labeling LC-MS analysis. A total of 2790 peak pairs or metabolites were relatively quantified among the 495 comparative samples, including 150 positively identified metabolites, 235 high-confident putatively identified metabolites and 1949 mass-matched metabolites from database searches. Multivariate analysis of the metabolome data showed a clustering of the 3rd-5th FTC samples in contrast to the separation of the 1st and 2nd FTC samples, indicating that the extent of FTC-induced metabolome changes became smaller after the third cycle. The changing patterns among the FTC-effected metabolites were found to be complex. Using sex as a biological factor for grouping, we observed a clear separation of males and females when the samples were subjected to the same number of FTCs. However, when the male- and female-samples with different numbers of FTCs were compared, the number of significant metabolites found in male-female comparison increased dramatically, indicating that FTC effects could lead to a large number of false positives in biomarker discovery. Finally, we proposed a method of detecting the FTC effects by re-analyzing the original samples after subjecting them an additional FTC.
  27. Crit Rev Anal Chem. 2020 May 24. 1-33
      Microfluidic capillary electrophoresis (MCE) is the novel technique resulted from the CE mininaturization as planar separation and analysis device. This review presents and discusses various application fields of this advanced technology published in the period 2017 till mid-2019 in eight different sections including clinical, biological, single cell analysis, environmental, pharmaceuticals, food analysis, forensic and ion analysis. The need for miniaturization of CE and the consequence advantages achieved are also discussed including high-throughput, miniaturized detection, effective separation, portability and the need for micro- or even nano-volume of samples. Comprehensive tables for the MCE applications in the different studied fields are provided. Also, figure comparing the number of the published papers applying MCE in the eight discussed fields within the studied period is included. The future investigation should put into consideration the possibility of replacing conventional CE with the MCE after proper validation. Suitable validation parameters with their suitable accepted ranges should be tailored for analysis methods utilizing such unique technique (MCE).
    Keywords:  Microfluidic capillary electrophoresis; diverse applications; future prospective; recent advancements
  28. Biomed Chromatogr. 2020 May 27. e4908
      In this study, a simplified, sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established and validated for the quantification of ulipristal acetate (UPA) in human plasma and for the investigation of pharmacokinetic profile of UPA following single oral administration of ella® (UPA 30 mg tablet) in healthy Chinese volunteers. Plasma samples were analyzed after being processed by protein precipitation with methanol. Chromatographic separation was performed on a Kinetex® EVO C18 column (2.1 × 50 mm, 2.6 μm) using gradient elution with a mobile phase composed of methanol and water containing 2 mM ammonium acetate and 0.3% formic acid at a flow rate of 0.3 mL/min. The chromatographic running time was 4.0 min per sample. The MS detection was operated in the positive ion electrospray ionization (ESI) by multiple reaction monitoring (MRM) mode using the transition of m/z 476.2→134.1 for UPA and m/z 479.3→416.2 for UPA-d3 (internal standard, IS), respectively. UPA and IS were monitored without severe interference from the biological matrices. The method was linear over the wide concentration range of 0.300-300 ng/mL. The intra- and inter-day precision and accuracy were well within the limits required for bioanalytical assays. The method was firstly used to describe the pharmacokinetic characteristic of UPA after single oral administration of Ella® in healthy Chinese subjects. Based upon between-study comparison, there were statistically significant differences (P < 0.05) between Chinese and Caucasian subjects for the systemic exposure of UPA, suggesting that race seems to significantly impact the systemic exposure of UPA.
    Keywords:  Human plasma; LC-MS/MS; Pharmacokinetics; Ulipristal acetate