bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2023‒09‒17
24 papers selected by
Sofia Costa, Matterworks

  1. J Chromatogr A. 2023 Aug 31. pii: S0021-9673(23)00567-8. [Epub ahead of print]1708 464342
      The importance of lipids seen in studies of metabolism, cancer, the recent COVID-19 pandemic and other diseases has brought the field of lipidomics to the forefront of clinical research. Quantitative and comprehensive analysis is required to understand biological interactions among lipid species. However, lipidomic analysis is often challenging due to the various compositional structures, diverse physicochemical properties, and wide dynamic range of concentrations of lipids in biological systems. To study the comprehensive lipidome, a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS)-based screening method with 1200 lipid features across 19 (sub)classes, including both nonpolar and polar lipids, has been developed. HILIC-MS/MS was selected due to its class separation property and fatty acyl chain level information. 3D models of class chromatographic retention behavior were established and evaluations of cross-class and within-class interferences were performed to avoid over-reporting these features. This targeted HILIC-MS/MS method was fully validated, with acceptable analytical parameters in terms of linearity, precision, reproducibility, and recovery. The accurate quantitation of 608 lipid species in the SRM 1950 NIST plasma was achieved using multi-internal standards per class and post-hoc correction, extending current databases by providing lipid concentrations resolved at fatty acyl chain level. The overall correlation coefficients (R2) of measured concentrations with values from literature range from 0.64 to 0.84. The applicability of the developed targeted lipidomics method was demonstrated by discovering 520 differential lipid features related to COVID-19 severity. This high coverage and targeted approach will aid in future investigations of the lipidome in various disease contexts.
    Keywords:  COVID-19; Clinical lipidomics; HILIC-MS/MS; NIST SRM 1950 plasma; Over-reporting; Quantitation
  2. J Chromatogr A. 2023 Sep 03. pii: S0021-9673(23)00574-5. [Epub ahead of print]1708 464349
      Enantioselective amino acid analysis is gaining increasing importance in pharmaceutical, biomedical and food sciences. While there are many methods available for enantiomer separation of amino acids, the simultaneous analysis of all chiral proteinogenic amino acids by a single method with one column and a single condition is still challenging. Herein, we report an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS) assay using Chiralpak QN-AX as chiral column. With 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate (AQC) as derivatization reagent, efficient enantioselective separation of D- and L-amino acids using HPLC has become possible. Thiol-containing amino acids like Cys are alkylated prior to AQC-labelling. A protocol for automated sample preparation including both derivatization step and calibrator preparation is presented. For compensating matrix effects, u-13C15N-labelled internal standards (IS) were employed. The method was validated and applied to the enantioselective analysis of amino acids in a bacterial fermentation broth.
    Keywords:  Amino acids analysis; Automation; Enantioselective separations; LC-MS/MS
  3. Talanta. 2023 Sep 06. pii: S0039-9140(23)00922-0. [Epub ahead of print]267 125171
      Purine intermediates play important roles in physiological function and participate in the kidney disorders, while a targeted quantification of the metabolic alterations in the purine metabolism in acute kidney injury (AKI) individuals has not been conducted. In the study, a novel, rapid and sensitive LC-MS method for simultaneous quantification of 16 purine metabolites was developed using hydrophilic interaction separation mode in human plasma and urine. The developed method was validated by using charcoal-stripped plasma and urine as blank matrix. The results showed that the method was good linear (R2 > 0.99) and the lower limit of quantification (LLOQ) ranged from 0.833 ng/mL to 800 ng/mL. The recovery and matrix effect were repeatable and stable. The intraday precision ranged from 0.7% to 12.7%, while the inter-day precision ranged from 1.6% to 18.5%. Most analytes were stable in the autosampler and could subject three freeze-thaw cycles. The method provided a wider coverage of purine metabolites and completed good separation of interfering compounds of nucleosides, deoxynucleosides and their corresponding nucleobases without derivatization, which was time-saving and labor-saving for the large-scale analysis. Furthermore, the method was successfully applied to plasma and urine samples of hospitalized patients without and with AKI. The results showed certain purine intermediates were up-regulated in plasma and down-regulated in urine of AKI inpatients, indicating that AKI stress may associate with inflammatory responses. The novel method can facilitate the quantitative analysis of purine metabolites in biological fluids, and exhibit great prospects in providing more information on the pathogenesis of AKI.
    Keywords:  (AKI); Acute kidney injury; HILIC; LC-MS/MS; Purine metabolites
  4. Anal Chim Acta. 2023 Oct 16. pii: S0003-2670(23)00880-2. [Epub ahead of print]1278 341659
      BACKGROUND: The kynurenine pathway (KP) generates eight tryptophan (TRP) metabolites collectively called kynurenines, which have gained enormous interest in clinical research. The importance of KP for different disease states calls for developing a low-cost and high-throughput chromatography-mass spectrometry method to evaluate the potential of different kynurenines. Simultaneous separation of TRP and its eight metabolites is challenging because they have substantial polarity differences (log P = -2.5 to +1.3).RESULTS: A low-cost, reversed-phase LC-MS/MS method based on polarity partitioning was established to simultaneously separate and quantitate all nine kynurenine pathway metabolites (KPMs) in a single run for the first time in the open literature. Based on stationary phase screening and ternary mobile phase optimization strategy, high polarity KPMs were retained while medium and low polarity KPMs were eluted in a shorter time. After method validation, we demonstrated the applicability of this LC/MS/MS method by quantitative measurement of all nine KPM in cerebrospinal fluid (CSF) and plasma among two groups of human subjects diagnosed with depression. Furthermore, we measured the differential KPMs in these two groups of low and high inflammation and correlated the results with CRP or TNF-α markers for depression.
    SIGNIFICANCE: Our proposed LC-MS/MS provides a new metabolite assay that can be easily applied in various clinical applications to simultaneously quantify multiple biomarkers in KP dysfunction.
    Keywords:  Human cerebrospinal fluid and plasma; Kynurenine pathway metabolites; Quantitation; Simultaneous separation; Solid-liquid extraction
  5. Clin Lab. 2023 Sep 01. 69(9):
      BACKGROUND: Therapeutic drug monitoring (TDM) of antifungal drugs is recommended. LC-MS/MS outperforms bioassay and high-performance liquid chromatography (HPLC) for TDM. In this study, we validated TDM for voriconazole, posaconazole, and itraconazole using HPLC-MS/MS with the multiple reaction monitoring (MRM) method.METHODS: For the validation of LC-MS/MS for antifungal TDM, accuracy, precision, linearity, carryover, lower limit of quantitation (LLOQ), ion suppression, and sample stability tests were performed according to the guidelines of the United States Food and Drug Administration (FDA) and the Clinical and Laboratory Standards Institute (CLSI).
    RESULTS: The LC-MS/MS triazole method showed that all analytes had biases less than 8.9% and coefficients of variation (CV) less than 7.7%. The linearity was validated over the ranges of 0.20 to 5.86 mg/L for voriconazole, 0.12 to 4.96 mg/L for posaconazole, 0.09 to 1.85 mg/L for itraconazole, and 0.12 to 2.38 mg/L for OH-itraconazole. Ion suppression and carryover were negligible. The lower limits of quantitation (LLOQs) for voriconazole, posaconazole, itraconazole, and OH-itraconazole were 0.114 mg/L, 0.206 mg/L, 0.118 mg/L, and 0.065 mg/L, respectively. Voriconazole, posaconazole, itraconazole, and OH-itraconazole can be stored at 4℃ for 4 - 7 days, according to sample stability. Sample preparation took < 15 minutes per batch, and analytical run time was 5 minutes per sample.
    CONCLUSIONS: We developed and validated a simple, reliable, and quick LC-MS/MS method for triazole antifungal agents TDM suitable for routine hospital practice.
  6. Se Pu. 2023 Sep;41(9): 799-806
      Carbon dioxide (CO2) absorption and capture is an effective measure to achieve the "dual carbon" goal of carbon peak and carbon neutrality in China. Organic amine compounds are widely used in the industrial separation and recovery of CO2. Thus, the establishment of analytical methods for organic amine compounds is of great significance for the research and development of carbon capture and storage (CCS) technology and carbon capture, utilization and storage (CCUS) technology. In this study, a method was developed for the determination of nine organic amine compounds in CO2 absorption liquid by hydrophilic interaction liquid chromatography (HILIC)-electrostatic field orbitrap high resolution mass spectrometry. The sample was diluted with water and filtered through a 0.22 μm nylon membrane before sampling and analysis. An Accucore HILIC column (100 mm×2.1 mm, 2.6 μm) was used for separation at 30 ℃. Gradient elution was conducted using 90% acetonitrile aqueous solution containing 5 mmol/L ammonium formate and 0.1% formic acid as mobile phase A and 10% acetonitrile aqueous solution containing 5 mmol/L ammonium formate and 0.1% formic acid as mobile phase B. Determination was performed using an electrospray ion source (ESI) in the positive ion mode. The quantitative analysis was carried out by standard addition method. The chromatographic retention performance of different chromatographic columns and the influence of different mobile phases on the separation of the organic amine compounds were compared, and the method was validated. The results showed that the linear ranges of the nine organic amine compounds were 0.04-25000 ng/mL with the linear correlation coefficients (R2) greater than 0.9910. The limits of detection (LODs) of the method were in the range of 0.0004-0.0080 ng/mL, and the limits of quantification (LOQs) of the method were in the range of 0.0035-0.0400 ng/mL. The average recoveries of the method ranged from 85.30% to 104.26% with relative standard deviations (RSDs) of 0.04%-7.95% at the spiked levels of 1, 1.5 and 3 times sample concentration. The established method was applied to detect the absorption waste liquid of a cement plant, and nine organic amine compounds could be effectively detected. The stability of the actual sample was tested, and the RSDs were 0.10%-6.35% in 48 h at 4 ℃. The method is sensitive, rapid and accurate for the determination of the nine organic amine compounds in industrial waste water. It can provide reference for the detection of organic amine compounds, and provide strong technical support for the research and industrial application of CO2 capture technology.
    Keywords:  carbon dioxide (CO2) absorbent; electrostatic field orbitrap high resolution mass spectrometry; hydrophilic interaction liquid chromatography (HILIC); organic amine compounds
  7. AAPS PharmSciTech. 2023 Sep 12. 24(7): 184
      Ketone ester ((R)-3-hydroxybutyl (R)-3-hydroxybutyrate) has gained popularity as an exogenous means to achieve ketosis. Regarding its potential as a therapeutic prodrug, it will be necessary to study its pharmacokinetic profile and its proximal metabolites (beta-hydroxybutyrate, 1,3-butanediol, and acetoacetate) in humans. Here we develop and validate two LC-MS methods for quantifying KE and its metabolites in human plasma. The first assay uses a C18 column to quantitate ketone ester, beta-hydroxybutyrate, and 1,3-butanediol, and the second assay uses a hydrophilic interaction liquid chromatography (HILIC) column for the quantitation of acetoacetate. The method was partially validated for intra- and inter-day accuracy and precision based on the ICH M10 guidelines. For both the assays, the intra- and inter-run accuracy was ±15% of the nominal concentration, and the precision (%CV) was <15% for all 4 molecules being quantified. The matrix effect for all molecules was evaluated and ranged from -62.1 to 44.4% (combined for all molecules), while the extraction recovery ranged from 65.1 to 119% (combined for all molecules). Furthermore, the metabolism of ketone ester in human plasma and human serum albumin was studied using the method. Non-saturable metabolism of ketone ester was seen in human plasma at concentrations as high as 5 mM, and human serum albumin contributed to the metabolism of ketone ester. Together, these assays can be used to track the entire kinetics of ketone ester and its proximal metabolites. The reverse-phase method was used to study the metabolic profile of KE in human plasma and the plasma protein binding of 1,3-BD.
    Keywords:  LC-MS; bioanalysis; human plasma; ketone bodies; ketone ester
  8. Anal Methods. 2023 Sep 11.
      There is a pressing need for the development of greener liquid chromatographic bioanalytical methods for antidiabetic drugs for plasma monitoring and revisiting patients' dosage regimens. Besides, analytical methods are also needed for the quality assurance of finished drug products and regulatory approval. Therefore, the present review focuses on the reported liquid chromatographic methods (LC and LC-MS/MS) that are applied for quality control, forced degradation, and pharmacokinetic studies of a newer antidiabetic agent, canagliflozin (CNG). These reported studies are summarized based on liquid chromatographic separation parameters, such as column dimensions, mobile-phase compositions, flow rate, and use of different detection systems (UV, PDA, and mass spectrometry). The sample pretreatment of biological fluids, which is important for minimizing the matrix effect, is dealt with separately. Liquid-liquid extraction was found to be the most preferred methodology adopted for sample pretreatment followed by the solid-phase extraction technique. However, miniaturized novel pretreatment methods are untraceable in the literature for the extraction of CNG. Special emphasis is paid to the assessment of the greenness profiles of the reported analytical methods for the consideration of sustainable development and green analytical chemistry. Based on the National Environmental Method Index (NEMI) assessment tool, most of the reported studies fulfilled around half of the parameters and were found to be about 50% greener. It is proposed that toxic or hazardous solvents, such as acetonitrile or methanol, should be replaced with greener and environmentally friendly solvents. Thus, there is a need to develop more robust, efficient, and greener liquid chromatographic methods for the determination of CNG in biological fluids and drug products.
  9. Clin Chim Acta. 2023 Sep 12. pii: S0009-8981(23)00358-3. [Epub ahead of print] 117556
      BACKGROUND: Reference measurement procedures for assigning values to calibration materials play a crucial role in the concept of metrological traceability in laboratory medicine. ISO standard 15193 specifies the requirements for such measurement procedures, albeit in a very general terms.MATERIALS AND METHODS: A standard structure of analysis series for value assignment by LC-MS/MS was developed and tested,this structure was complemented by a spreadsheet file for result calculation, metadata evaluation, and finally validation and confirmation of individual analysis runs and individual results, and measurement uncertainty evaluation. This framework was applied to a procedure for the quantification of ciprofloxacin in serum as an example.
    RESULTS: The approach of a detailed description of the analytical procedures of isotope dilution LC-MS/MS reference measurement methods together with a highly standardized spreadsheet-based method for data processing was found to be practical and efficient. The described measurement procedure for the quantification of ciprofloxacin in serum was found to be fit for purpose.
    CONCLUSION: A standardized, detailed procedure for the application of isotope dilution LC-MS/MS in reference measurement procedures can complement the ISO 15193 standard with respect to this particular analytical technique, which is now widely used in the context of metrological traceability in laboratory medicine.
    Keywords:  ISO 15193; Isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS); metrological traceability; reference measurement procedures; standardization
  10. Anal Chim Acta. 2023 Oct 16. pii: S0003-2670(23)00894-2. [Epub ahead of print]1278 341673
      BACKGROUND: Phospholipids (PLs) are major constituents of cell membranes, play important roles in cell proliferation and death, as well as in signal transduction, and therefore are relevant biomarkers for different pathologies. On the other hand, when the analysis of small compounds, such as therapeutics in blood is desired, then phospholipids are part of the matrix and cause serious interference during analysis. Currently, both the analysis and removal of PLs from biological samples are limited by extensive sample preparation and instrumental separation.RESULTS: A fast and simple quantitative Ti4+-modified paper spray tandem mass spectrometric (TiPS-MS/MS) method was established in urine, where the enrichment of phospholipids was achieved, as well as reduction of matrix effects (primarily caused by high salt content) that ultimately led to improved sensitivity and selectivity. The method could achieve a physiologically relevant limit of detection (0.01-0.03 μg mL-1). Also, the usefulness of the Ti4+-modified paper was investigated in the opposite mode, namely for the selective removal of phospholipids from matrices such as plasma. Clonidine is used as model compound, as the detection of this compound is known to suffer from ion suppression by phospholipids. Compared with blank paper spray tandem mass spectrometry, the limit of detection could be improved from 0.3 μg mL-1 to 0.03 μg mL-1 by employing a Ti4+-modified paper on top of the spray tip to capture phospholipids from the sample.
    SIGNIFICANCE AND NOVELTY: A novel Ti4+-modified paper was developed to allow for rapid solid-phase extraction of phospholipids from urine or selective removal from plasma, followed by direct paper spray mass spectrometric detection as a fast and convenient sample preparation and analysis combination. The paper properties are based on the Ti4+ metal ion, which can selectively bind phosphate-containing compounds under acidic conditions, and its applicability was demonstrated in relevant biological matrices.
    Keywords:  Matrix effect; Paper modification; Paper spray mass spectrometry; Phospholipids; Selective enrichment
  11. Talanta. 2023 Sep 06. pii: S0039-9140(23)00924-4. [Epub ahead of print]267 125173
      The present investigation showed that each of the three different liquid chromatography modes may be successfully used for the qualitative analysis of nusinersen metabolites in a patient's serum sample extract. However, the smallest number was detected by the hydrophilic interaction liquid chromatography. Furthermore, the response of the mass spectrometry is several times greater for ion pair chromatography compared to reversed-phase one. Various extraction methods were applied for the extraction of nusinersen metabolites from serum. Silica with bonded capture strand for hybridization was applied, as well as silica modified with amino and carboxyl groups for dispersive solid phase extraction. The hybridization allows selective extraction of nusinersen analogs, however, it fails in extraction of short metabolites. On the contrary, the efficiency of weak ion exchange-based extraction was high, even in the case of the direct extraction of nusinersen metabolites from diluted serum samples without a protein removal step. The new material is a great alternative to liquid-liquid extraction and hybridization for the isolation of nusinersen metabolites from the serum of patients with spinal muscular atrophy (SMA). It is a very simple method that uses a low concentration of organic salt and desorption occurs after changing its pH. Such complex studies were performed for the first time for nusinersen metabolites extracted from the serum of SMA patients treated with Spinraza.
    Keywords:  Dispersive solid phase extraction; Hybridization; Liquid chromatography coupled with mass spectrometry; Liquid-liquid extraction; Nusinersen; Oligonucleotides
  12. Front Pharmacol. 2023 ;14 1211383
      A fast, simple, and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established for the quantification of safinamide in rat plasma. Plasma samples were treated with acetonitrile for protein precipitation, and diazepam was used as an internal standard (IS). The analytes were separated on an Acquity UPLC C18 (2.1 mm × 50 mm, 1.7 μm) chromatographic column with gradient elution using a mobile phase (0.1% formic acid-acetonitrile). Then, the eluates were detected by electrospray ionization (ESI) in positive ion mode. The analytes were quantified by multiple reaction monitoring (MRM) using the transition m/z 303.3→215.0 of safinamide and m/z 285.0→154.0 of IS. Safinamide had good linearity in the concentration range of 1.0-2000 ng/mL, and the lower limit of quantitation (LLOQ) was 1.0 ng/mL. The intra- and inter-day precision and accuracy of safinamide were less than 7.63%, while the average recovery rate was 92.98%-100.29%. The method was validated to be stable and had low noise, short chromatographic run time, wide linear range, small sample volumes, low sample injection volumes, and high sensitivity. Therefore, it can be used in pharmacokinetics and preclinical and clinical studies.
    Keywords:  UPLC-MS/MS; pharmacokinetics; plasma; rat; safinamide
  13. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Sep 11. pii: S1570-0232(23)00296-9. [Epub ahead of print]1229 123886
      Phosphatidylethanol (PEth) is a group of phospholipids formed exclusively in the presence of ethanol on the erythrocyte membrane, making it a direct biomarker for long-term ethanol consumption for which a clinical reference interval has been established. Here, we describe an assay for quantitation for two most abundant PEth homologues, PEth 16:0/18:1 and PEth 16:0/18:2, from human whole blood, and present challenges overcome throughout the development process. Since PEth is localized within erythrocyte membranes, a reliable sample preparation technique is an important aspect of PEth analysis. Therefore, various erythrocyte lysing agents for recovery of exogenously spiked standards and controls were evaluated to identify one that performed comparably to the recovery of endogenous analytes found in authentic samples. A supported liquid extraction (SLE) technique was employed for sample cleanup and enrichment which together with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis enabled automated sample preparation, appropriate chromatographic resolution, and minimal system carryover. This resulted in a laboratory developed test with an analytical measurement range (AMR) of 10-1000 ng/mL (slope = 0.9902-1.0138, R2 = 0.9958-0.9972), that was precise (intra-day precision: 3.4-4.1%; inter-day precision: 4.4-8.2% over the AMR), accurate when compared with an available external laboratory test (slope = 0.9943-1.0206, R2 = 0.9635-0.9678, no lower decision point interpretation changes), with effective analyte recovery (77.2-83.5%), and established stability characteristics, while chromatographically separating the analytes to ensure no additive effects due to the isotopic distribution of the opposing analyte.
    Keywords:  Automated extraction; Biomarker; Erythrocytes; Ethanol; Phosphatidylethanol; Recovery
  14. J Cheminform. 2023 Sep 15. 15(1): 80
      Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI) spatially resolves the chemical composition of tissues. Lipids are of particular interest, as they influence important biological processes in health and disease. However, the identification of lipids in MALDI-MSI remains a challenge due to the lack of chromatographic separation or untargeted tandem mass spectrometry. Recent studies have proposed the use of MALDI in-source fragmentation to infer structural information and aid identification. Here we present rMSIfragment, an open-source R package that exploits known adducts and fragmentation pathways to confidently annotate lipids in MALDI-MSI. The annotations are ranked using a novel score that demonstrates an area under the curve of 0.7 in ROC analyses using HPLC-MS and Target-Decoy validations. rMSIfragment applies to multiple MALDI-MSI sample types and experimental setups. Finally, we demonstrate that overlooking in-source fragments increases the number of incorrect annotations. Annotation workflows should consider in-source fragmentation tools such as rMSIfragment to increase annotation confidence and reduce the number of false positives.
    Keywords:  Annotation; Bioinformatics; Cheminformatics; Computation; In-source decay; In-source fragmentation; Lipids; MALDI; Mass spectrometry imaging
  15. Drug Test Anal. 2023 Sep 15.
      Benzodiazepines are essential screening targets for common sleeping and sedative drugs used in forensic toxicology. Direct analysis in real-time tandem mass spectrometry was used to rapidly identify 10 benzodiazepines and related metabolites in the blood and urine. The related direct analysis in real-time tandem mass spectrometry parameters were optimized. A liquid-liquid extraction method using ethyl acetate as the extraction solvent was used for sample preparation. The established method was validated and tested on case specimens. The limits of detection of this method ranged from 0.2 to 20 ng/mL and the limits of quantification from 1 to 50 ng/mL. The recoveries ranged from 78.8% to 114%, and the matrix effects were in the range of -21.2% to 17.9%. The precision and repeatability at high and medium concentrations did not exceed 14.6%, and the limit of quantification did not exceed 18.2%, indicating a desirable linear relationship. The established method was used to determine blood and urine specimens from authentic cases, and promising results were obtained.
    Keywords:  DART-MS/MS; benzodiazepine; blood; urine
  16. J Am Soc Mass Spectrom. 2023 Sep 13.
      Lipids are structurally diverse molecules that play a pivotal role in a plethora of biological processes. However, deciphering the biological roles of the specific lipids is challenging due to the existence of numerous isomers. This high chemical complexity of the lipidome is one of the major challenges in lipidomics research, as the traditional liquid chromatography-mass spectrometry (LC-MS) based approaches are often not powerful enough to resolve these isomeric and isobaric nuances within complex samples. Thus, lipids are uniquely suited to the benefits provided by multidimensional liquid chromatography-ion mobility-mass spectrometry (LC-IM-MS) analysis. However, many forms of lipid isomerism, including double-bond positional isomers and regioisomers, are structurally similar such that their collision cross section (CCS) differences are unresolvable via conventional IM approaches. Here we evaluate the performance of a high resolution ion mobility (HRIM) system based on structures for lossless ion manipulation (SLIM) technology interfaced to a high resolution quadrupole time-of-flight (QTOF) analyzer to address the noted lipidomic isomerism challenge. SLIM implements the traveling wave ion mobility technique along an ∼13 m ion path, providing longer path lengths to enable improved separation of isomeric features. We demonstrate the power of HRIM-MS to dissect isomeric PC standards differing only in double bond (DB) and stereospecific number (SN) positions. The partial separation of protonated DB isomers is significantly enhanced when they are analyzed as metal adducts. For sodium adducts, we achieve close to baseline separation of three different PC 18:1/18:1 isomers with different cis-double bond locations. Similarly, PC 18:1/18:1 (cis-9) can be resolved from the corresponding PC 18:1/18:1 (trans-9) form. The separation capacity is further enhanced when using silver ion doping, enabling the baseline separation of regioisomers that cannot be resolved when measured as sodium adducts. The sensitivity and reproducibility of the approach were assessed, and the performance for more complex mixtures was benchmarked by identifying PC isomers in total brain and liver lipid extracts.
  17. Anal Chim Acta. 2023 Oct 16. pii: S0003-2670(23)00940-6. [Epub ahead of print]1278 341719
      Red blood cells (RBCs) are the subject of clinical attention due to their biological importance. Recently, it has been shown that certain erythrocyte pathologies could be linked to an abnormal lipid composition. In this work, we have developed a simple and fast method using online sample preparation with liquid chromatography coupled to mass spectrometry (SPE-HPLC-MS/MS), to identify a large number of sphingolipids (SL) and phospholipids (PL). The use of online sample preparation considerably reduces analysis times (15 min including extraction and separation of lipids + 2 min for system re-equilibration) and facilitates experimentation while ensuring very good extraction yields. This method was then successfully applied to the quantification of 30 sphingolipids and phospholipids in plasma and erythrocyte extracts from a cohort of individuals with Gaucher disease, treated or not by enzymotherapy. Our results for the study of this disease, led us to establish the lipid profile of the healthy red blood cells, still not very well-known to date. For this, we adopted a semi-targeted approach, based on the use of a triple-quadrupole analyzer and identified more than two hundred different lipid species. These promising results will hopefully enable us to enrich our knowledge of the normal red blood cells lipidome.
    Keywords:  Gaucher's disease; Online SPE-HPLC-MS/MS; Red blood cells lipidome; Sphingolipid and phospholipid profiling
  18. Anal Bioanal Chem. 2023 Sep 16.
      Sample preparation of complex, natural mixtures such as lignin prior to mass spectrometry analysis, however minimal, is a critical step in ensuring accurate and interference-free results. Modern shotgun-MS techniques, where samples are directly injected into a high-resolution mass spectrometer (HRMS) with no prior separation, usually still require basic sample pretreatment such as filtration and appropriate solvents for full dissolution and compatibility with atmospheric pressure ionization interfaces. In this study, sample preparation protocols have been established for a unique sample set consisting of a wide variety of degraded lignin samples from numerous sources and treatment processes. The samples were analyzed via electrospray (ESI)-HRMS in negative and positive ionization modes. The resulting information-rich HRMS datasets were then transformed into the mass defect space with custom R scripts as well as the open-source Constellation software as an effective way to visualize changes between the samples due to the sample preparation and ionization conditions as well as a starting point for comprehensive characterization of these varied sample sets. Optimized conditions for the four investigated lignins are proposed for ESI-HRMS analysis for the first time, giving an excellent starting point for future studies seeking to better characterize and understand these complex mixtures.
    Keywords:  Analytical methods; Custom software; Lignin; Mass defect; Mass spectrometry; Sample preparation
  19. Talanta. 2023 Sep 06. pii: S0039-9140(23)00919-0. [Epub ahead of print]267 125168
      The paper presents an LC-MS/MS-based approach to targeted screening of both polar and non-polar metabolites using a synthesized monolithic column which is a copolymer of styrene, divinylbenzene, and 1-vinyl-1,2,4-triazole. It was shown that this column in combination with eluents 20 mM (NH4)2CO3 + NH3 (pH = 9.8, eluent A) and ACN (eluent B) allows for separation of metabolites of different nature in two modes, HILIC and RP LC, and these methods are mutually complementary. A combination of analyses based on these two modes was proposed, allowing detection of about 400 metabolites in a total time of less than 30 min. Comparison of the developed method with those utilizing commercially available columns with sorbents of various types showed that it could provide a broader metabolite coverage. Using the developed approach, metabolomic screening of dried blood spots samples of mice exposed with X-ray was performed, and metabolites that could be considered as possible markers of irradiation exposure and organ tissue damage were detected. Analysis of marker metabolites revealed metabolic pathways that were altered by radiation exposure. Comparison of the results with literature data showed the effectiveness of the developed metabolomic screening approach.
    Keywords:  Dual retention chromatography; Liquid chromatography; Metabolomics; Monolithic column; Tandem mass spectrometry; X-ray irradiation
  20. Se Pu. 2023 Sep;41(9): 760-770
      Mycotoxins are secondary metabolites produced by toxigenic fungi under specific environmental conditions. Fruits, owing to their high moisture content, rich nutrition, and improper harvest or storage conditions, are highly susceptible to various mycotoxins, such as ochratoxin A (OTA), zearalenone (ZEN), patulin (PAT), Alternaria toxins, etc. These mycotoxins can cause acute and chronic toxic effects (teratogenicity, mutagenicity, and carcinogenicity, etc) in animals and humans. Given the high toxicity and wide prevalence of mycotoxins, establishing an efficient analytical method to detect multiple mycotoxins simultaneously in different types of fruits is of great importance. Conventional mycotoxin detection methods rely on high performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). However, fruit sample matrices contain large amounts of pigments, cellulose, and minerals, all of which dramatically impede the detection of trace mycotoxins in fruits. Therefore, the efficient enrichment and purification of multiple mycotoxins in fruit samples is crucial before instrumental analysis. In this study, a reliable method based on a QuEChERs sample preparation approach coupled with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established to determine 36 mycotoxins in fruits. In the optimal extraction method, 2.0 g of a sample was extracted with 10 mL of acetic acid-acetonitrile-water (1∶79∶20, v/v/v) in a 50 mL centrifuge tube, vortexed for 30 s, and ultrasonicated for 40 min. The mixture was then salted out with 2.0 g of anhydrous MgSO4 and 0.5 g of NaCl and centrifuged for 5 min. Next, 6 mL of the supernatant was purified using 85 mg of octadecylsilane-bonded silica gel (C18) and 15 mg of N-propylethylenediamine (PSA). After vigorous shaking and centrifugation, the supernatant was collected and dried with nitrogen at 40 ℃. Finally, the residues were redissolved in 1 mL of 5 mmol/L ammonium acetate aqueous solution-acetonitrile (50∶50, v/v) and passed through a 0.22 μm nylon filter before analysis. The mycotoxins were separated on a Waters XBridge BEH C18 column using a binary gradient mixture of ammonium acetate aqueous solution and methanol. The injection volume was 3 μL. The mycotoxins were analyzed in multiple reaction monitoring (MRM) mode under both positive and negative electrospray ionization. Quantitative analysis was performed using an external standard method with matrix-matched calibration curves. Under optimal conditions, good linear relationships were obtained in the respective linear ranges, with correlation coefficients (R2) no less than 0.990. The limits of detection (LODs) and quantification (LOQs) were 0.02-5 and 0.1-10 μg/kg, respectively. The recoveries of the 36 mycotoxins in fruits ranged from 77.0% to 118.9% at low, medium, and high spiked levels, with intra- and inter-day precisions in the range of 1.3%-14.9% and 0.2%-17.3%, respectively. The validated approach was employed to investigate mycotoxin contamination in actual fruit samples, including strawberry, grape, pear, and peach (15 samples of each type). Eleven mycotoxins, namely, altenuene (ALT), altenusin (ALS), alternariol-methyl ether (AME), tenuazonic acid (TeA), tentoxin (Ten), OTA, beauvericin (BEA), PAT, zearalanone (ZAN), T-2 toxin (T2), and mycophenolic acid (MPA), were found in the samples; three samples were contaminated with multiple mycotoxins. The incidence rates of mycotoxins in strawberry, grape, pear, and peach were 27%, 40%, 40%, and 33%, respectively. In particular, Alternaria toxins were the most frequently found mycotoxins in these fruits, with an incidence of 15%. The proposed method is simple, rapid, accurate, sensitive, reproducible, and stable; thus, it is suitable for the simultaneous detection of the 36 mycotoxins in different fruits.
    Keywords:  QuEChERS; fruits; mycotoxins; ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)
  21. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Sep 09. pii: S1570-0232(23)00294-5. [Epub ahead of print]1229 123884
      A simple, sensitive, and efficient method based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the determination of 8 coccidiostats in chicken feces and environmental water (including sewage, pond water, and lake water) surrounding the farm. Target analytes in chicken feces were extracted with 2% acetic acid in acetonitrile solution, followed by a dispersive solid-phase extraction (DSPE) cleanup step using the mixture of PSA and C18 adsorbents. Environmental water samples were pretreated using a lyophilization approach. Analysis was carried out on a UPLC-MS/MS with the combination of methanol and 0.1% formic acid aqueous solution as the mobile phase under multiple reaction monitoring in positive and negative ionization modes. Results showed that 8 coccidiostats were linear with correlation coefficients higher than 0.99. Method validation was performed using fortified samples, reaching satisfactory recoveries of 75.9%-97.8% in chicken feces and 71.9%-108.2% in environmental water. Limits of detection for 8 analytes in chicken feces and environmental water were 0.03∼2 µg/kg and 0.005∼1 µg/L, respectively. Matrix effects were calculated and strong signal suppression (>50%) for some coccidiostats was observed. The developed method was successfully applied to analyze coccidiostats in chicken feces and environmental water collected from local chicken farms.
    Keywords:  Chicken feces; Coccidiostats; Environmental water; Residues; Ultra-performance liquid chromatography-tandem mass spectrometry
  22. Clin Chim Acta. 2023 Sep 11. pii: S0009-8981(23)00356-X. [Epub ahead of print]549 117554
      BACKGROUND: Apixaban's technical sheet does not recommend its use in clinical practice for patients with chronic kidney disease undergoing haemodialysis. However, recent studies indicate that apixaban could be a safe oral anticoagulant in these kinds of patients who do not present valvular atrial fibrillation. We developed and validated ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) procedures for measuring apixaban concentrations in plasma, dialysate liquid, and urine.MATERIAL AND METHODS: Simple protein precipitation was implemented to prepare samples. Chromatographic separations were achieved on an Acquity®-UPLC®-BEHTM (2.1x100 mm id, 1.7 µm) reverse-phase C18 column using a water/acetonitrile non-linear gradient containing 0.1 % formic acid at a 0.4 mL/min flow rate. Apixaban and its internal standard (apixaban-d3) were detected by electrospray ionisation mass spectrometry in positive and multiple reaction monitoring modes, using transitions of 460.3 → 199.0/443.2 and 463.3 → 202.0, respectively.
    RESULTS: No significant interferences and carry-overs were observed. Precisions, absolute relative biases, normalised-matrix factors, and normalised recoveries were ≤ 12.2%, ≤8.0%, 94.3-105.1%, and 93.9-105.4%, respectively. Linearity was observed between 5 and 500 μg/L for plasma/dialysate liquid and 5-1000 μg/L for urine.
    CONCLUSIONS: The validated UHPLC-MS/MS procedures could help support a pharmacokinetic study in non-valvular atrial fibrillation subjects with chronic kidney disease undergoing haemodialysis and apixaban-based anticoagulant therapy.
    Keywords:  Apixaban; Dialysate liquid; Plasma; UHPLC-MS/MS; Urine
  23. Se Pu. 2023 Sep;41(9): 807-813
      Carbamates are used in broad-spectrum insecticides and herbicides, and have highly efficient, low-residue, and long-lasting characteristics. However, this type of pesticide exerts mutagenic, teratogenic, carcinogenic, and other adverse effects, and its frequent use can exceed the recommended scope and limits. Research on the determination of carbamate pesticides mainly focuses on foods of plant origin and pays less attention to foods of animal origin. The methods for carbamate determination described in the current national standards have complicated operating procedures and low efficiency. Therefore, highly efficient and accurate methods for carbamate detection in milk must be established. In this work, a rapid method based on pass-through solid-phase extraction (SPE) purification coupled with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of 10 carbamate pesticides in liquid milk. The pretreatment and instrument methods were systematically optimized. The milk sample was extracted with acetonitrile, and then purified using a Captiva EMR-Lipid filtration kit. The purified extract was separated on an ACQUITY UPLC BEH C18 column with mobile phase of methanol and 0.1% formic acid aqueous solution in gradient elution. The flow rate was 0.3 mL/min. Column temperature was 35 ℃. Quantitative analysis was performed using the external standard method with matrix matching curves. The 10 carbamate pesticides showed good linear relationships in the mass concentration range of 2-200 μg/L, with correlation coefficients greater than 0.999. The limits of detection (LODs) and quantification (LOQs) for the 10 carbamate pesticides were 0.045-0.23 and 0.15-0.77 μg/kg, respectively. Recovery tests were conducted using the blank-matrix method at three spiked levels of 15, 50, and 100 μg/kg, and good recoveries for the 10 carbamate pesticides were obtained. In particular, the recoveries for the three spiked levels of 15, 50, and 100 μg/kg were 68.7%-93.3% with relative standard deviations (RSDs) of 1.8%-8.0%. The proposed method is efficient, convenient, accurate, and suitable for the rapid detection of the 10 carbamate pesticides in liquid milk. Compared with the conventional NH2 and ENVITM-18 SPE columns used in the national standard determination method, the proposed method demonstrated better purification effects. The recoveries for aldicarb sulfoxide, aldicarb sulfone, methomyl, and carbaryl after purification using the Captiva EMR-Lipid kit increased from 60% to 80%. Thus, the proposed method is suitable for targets with strong polarity and gives measurement results with good repeatability and accuracy.
    Keywords:  carbamate pesticide; enhanced matrix removal-lipid cleaning; liquid milk; pass-through solid-phase extraction purification; ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)
  24. Anal Chim Acta. 2023 Oct 16. pii: S0003-2670(23)00962-5. [Epub ahead of print]1278 341741
      Carbohydrates play crucial regulatory roles in various physiological and pathological processes. However, the low ionization efficiency and the presence of linkage pattern, monosaccharide composition and anomeric configuration isomers make their in-depth analysis very challenging, especially for heterogeneous biological tissues. In this study, we propose a high-sensitive and isomer-specific imaging approach to visualize the spatial distributions of monosaccharide and disaccharide isomers by integrating chemical derivatization and matrix-assisted laser desorption/ionization tandem mass spectrometry imaging (MALDI-MS2I). 2-Pyridinecarbohydrazide (PYD) is developed as a novel derivatization reagent which can not only improves the MS sensitivity of carbohydrates, but also enables the identification and visualization of ketose and aldose monosaccharide isomers, as well as linkage pattern, monosaccharide composition and anomeric configuration disaccharide isomers by mass spectrometry imaging of isomer-specific MS/MS fragment ions. Moreover, we build quantitative MALDI-MS2 and MALDI-MS2I methods for disaccharide isomers based on the diagnostic fragment ions, and good linear relationships could be achieved both in solution and on glass slides. We expect that this study should provide new ideas for in-depth profiling of the spatial signatures of carbohydrates in biological tissues and lay the foundation for a deeper understanding of carbohydrates' structure.
    Keywords:  Biological tissues; Isomer; Mass spectrometry imaging; Monosaccharides and disaccharide; On-tissue derivatization; Quantification