bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2023‒11‒19
23 papers selected by
Sofia Costa, Matterworks

  1. BMC Bioinformatics. 2023 Nov 14. 24(1): 431
      BACKGROUND: Liquid chromatography-mass spectrometry is widely used in untargeted metabolomics for composition profiling. In multi-run analysis scenarios, features of each run are aligned into consensus features by feature alignment algorithms to observe the intensity variations across runs. However, most of the existing feature alignment methods focus more on accurate retention time correction, while underestimating the importance of feature matching. None of the existing methods can comprehensively consider feature correspondences among all runs and achieve optimal matching.RESULTS: To comprehensively analyze feature correspondences among runs, we propose G-Aligner, a graph-based feature alignment method for untargeted LC-MS data. In the feature matching stage, G-Aligner treats features and potential correspondences as nodes and edges in a multipartite graph, considers the multi-run feature matching problem an unbalanced multidimensional assignment problem, and provides three combinatorial optimization algorithms to find optimal matching solutions. In comparison with the feature alignment methods in OpenMS, MZmine2 and XCMS on three public metabolomics benchmark datasets, G-Aligner achieved the best feature alignment performance on all the three datasets with up to 9.8% and 26.6% increase in accurately aligned features and analytes, and helped all comparison software obtain more accurate results on their self-extracted features by integrating G-Aligner to their analysis workflow. G-Aligner is open-source and freely available at under a permissive license. Benchmark datasets, manual annotation results, evaluation methods and results are available at CONCLUSIONS: In this study, we proposed G-Aligner to improve feature matching accuracy for untargeted metabolomics LC-MS data. G-Aligner comprehensively considered potential feature correspondences between all runs, converting the feature matching problem as a multidimensional assignment problem (MAP). In evaluations on three public metabolomics benchmark datasets, G-Aligner achieved the highest alignment accuracy on manual annotated and popular software extracted features, proving the effectiveness and robustness of the algorithm.
    Keywords:  Combinatorial optimization; Feature alignment; LC–MS; Multidimensional assignment problem
  2. Chin J Cancer Res. 2023 Oct 30. 35(5): 550-562
      Objective: As an important part of metabolomics analysis, untargeted metabolomics has become a powerful tool in the study of tumor mechanisms and the discovery of metabolic markers with high-throughput spectrometric data which also poses great challenges to data analysis, from the extraction of raw data to the identification of differential metabolites. To date, a large number of analytical tools and processes have been developed and constructed to serve untargeted metabolomics research. The different selection of analytical tools and parameter settings lead to varied results of untargeted metabolomics data. Our goal is to establish an easily operated platform and obtain a repeatable analysis result.Methods: We used the R language basic environment to construct the preprocessing system of the original data and the LAMP (Linux+Apache+MySQL+PHP) architecture to build a cloud mass spectrum data analysis system.
    Results: An open-source analysis software for untargeted metabolomics data (openNAU) was constructed. It includes the extraction of raw mass data and quality control for the identification of differential metabolic ion peaks. A reference metabolomics database based on public databases was also constructed.
    Conclusions: A complete analysis system platform for untargeted metabolomics was established. This platform provides a complete template interface for the addition and updating of the analysis process, so we can finish complex analyses of untargeted metabolomics with simple human-computer interactions. The source code can be downloaded from
    Keywords:  LAMP architecture; Untargeted metabolomics; normalization; reference metabolomics; shiny
  3. J Chromatogr A. 2023 Oct 31. pii: S0021-9673(23)00704-5. [Epub ahead of print]1712 464479
      The analysis of the brain extracellular metabolome is of interest for numerous subdomains within neuroscience. Not only does it provide information about normal physiological functions, it is even more of interest for biomarker discovery and target discovery in disease. The extracellular analysis of the brain is particularly interesting as it provides information about the release of mediators in the brain extracellular fluid to look at cellular signaling and metabolic pathways through the release, diffusion and re-uptake of neurochemicals. In vivo samples are obtained through microdialysis, cerebral open-flow microperfusion or solid-phase microextraction. The analytes of potential interest are typically low in concentration and can have a wide range of physicochemical properties. Liquid chromatography coupled to mass spectrometry has proven its usefulness in brain metabolomics. It allows sensitive and specific analysis of low sample volumes, obtained through different approaches. Several strategies for the analysis of the extracellular fluid have been proposed. The most widely used approaches apply sample derivatization, specific stationary phases and/or hydrophilic interaction liquid chromatography. Miniaturization of these methods allows an even higher sensitivity. The development of chiral metabolomics is indispensable, as it allows to compare the enantiomeric ratio of compounds and provides even more challenges. Some limitations continue to exist for the previously developed methods and the development of new, more sensitive methods remains needed. This review provides an overview of the methods developed for sampling and liquid chromatography-mass spectrometry analysis of the extracellular metabolome.
    Keywords:  Brain; Extracellular fluid; Liquid chromatography-mass spectrometry; Metabolomics; Sample collection
  4. Anal Chem. 2023 Nov 16.
      Identifying and mapping steroids in tissues can provide opportunities for biomarker discovery, the interrogation of disease progression, and new therapeutics. Although separation coupled to mass spectrometry (MS) has emerged as a powerful tool for studying steroids, imaging and annotating steroid isomers remains challenging. Herein, we present a new method based on the fragmentation of silver-cationized steroids in tandem MS, which produces distinctive and consistent fragmentation patterns conferring confidence in steroid annotation at the regioisomeric level without using prior derivatization, separation, or instrumental modification. In addition to predicting the structure of the steroid with isomeric specificity, the method is simple, flexible, and inexpensive, suggesting that the wider community will easily adapt to it. We demonstrate the utility of our approach by visualizing steroids and steroid isomer distributions in mouse brain tissue using silver-doped pneumatically assisted nanospray desorption electrospray ionization mass spectrometry imaging.
  5. Anal Chem. 2023 Nov 14.
      Commonly, in MS-based untargeted metabolomics, some metabolites cannot be confidently identified due to ambiguities in resolving isobars and structurally similar species. To address this, analytical techniques beyond traditional MS2 analysis, such as MSn fragmentation, can be applied to probe metabolites for additional structural information. In MSn fragmentation, recursive cycles of activation are applied to fragment ions originating from the same precursor ion detected on an MS1 spectrum. This resonant-type collision-activated dissociation (CAD) can yield information that cannot be ascertained from MS2 spectra alone, which helps improve the performance of metabolite identification workflows. However, most approaches for metabolite identification require mass-to-charge (m/z) values measured with high resolution, as this enables the determination of accurate mass values. Unfortunately, high-resolution-MSn spectra are relatively rare in spectral libraries. Here, we describe a computational approach to generate a database of high-resolution-MSn spectra by converting existing low-resolution-MSn spectra using complementary high-resolution-MS2 spectra generated by beam-type CAD. Using this method, we have generated a database, derived from the NIST20 MS/MS database, of MSn spectral trees representing 9637 compounds and 19386 precursor ions where at least 90% of signal intensity was converted from low-to-high resolution.
  6. Heliyon. 2023 Nov;9(11): e21610
      An innovative method based on dispersive solid phase extraction (d-SPE) in conjunction with LC-MS/MS had been developed for the simultaneous quantitative determination of three brevetoxins (BTXs), which can result in neurotoxic shellfish poisoning (NSP), in shellfish. The toxins were extracted with a 50 % acetonitrile (v/v) and cleaned by alumina-neutral sorbent. After chromatographic separation on a C18 column, the analytes were qualitatively and quantitatively detected using multiple reaction monitoring (MRM) in positive ionization mode. The created approach was validated by SANTE 11312/2021. The LOQs were 5 μg/kg for each toxin, below the advised regulatory limit of 800 μg BTX-2/kg. The mean recoveries of brevetoxins were in the range of 75.9 %-114.1 %, and the ranges of their intra- and inter-day precisions were 0.9-9.7 % and 0.6-7.2 %, respectively. The matrix effects for three BTXs in four shellfish matrices were in the range of 85.6 %-114.8 %. The method demonstrated great consistency and high sensitivity, and it can meet the requirements of daily monitoring.
    Keywords:  Brevetoxins; Dispersive solid phase extraction; Liquid chromatography-tandem mass spectrometry; Neurotoxic shellfish poisoning; Shellfish
  7. Rapid Commun Mass Spectrom. 2023 Dec 30. 37(24): e9637
      BACKGROUND: The renin-angiotensin system produces a series of biologically active angiotensin (Ang) peptides. These Ang peptides are the major regulators of blood pressure and Na homeostasis, and play a critical role in maintaining cardiovascular and fluid homeostasis. The concentration of Ang peptides in the body is at trace levels, making their detection and quantification a challenge. In this study, a rapid and sensitive analytical method using mass spectrometry coupled with ultrahigh-performance liquid chromatography (UHPLC/MS) was developed to simultaneously quantify 14 Ang peptides.METHODS: UHPLC/MS was employed to quantify 14 Ang peptides in mouse and human plasma. An HSS T3 column (2.1 × 100 mm, 1.8 μm) with an HSS T3 precolumn and triple-quadrupole mass spectrometer combined with an electrospray ionization source were utilized. Sample pretreatment involved a one-step protein precipitation using methanol. The total analysis time was within 7.5 min and the target peptides were detected in positive ion mode and quantified by selected reaction monitoring mode.
    RESULTS: The method was validated for linearity, detection and quantification limits, precision, stability, recovery and matrix effect. The limits of detection of Ang II, Ang III, Ang-(1-7), Ang-(2-7), Ang-(3-7), Ang-(1-9), bradykinin, Asn1 and Val5 -Ang II are all less than 1 pg mL-1 , indicating high sensitivity. The intra-day and inter-day precision was within 15%, and the accuracy was between 85% and 115%. Meanwhile, the sample and reference solution were stable within 48 h, and the recovery and matrix effect met the quantitative requirements.
    CONCLUSIONS: The method is currently reported to allow the largest number of Ang peptide species to be detected at one time. In addition, the proposed method offers a fast and reliable approach for comprehensive analysis of Ang metabolism in biological samples, facilitating research on the physiological and pathological states of cardiovascular, kidney and respiratory diseases.
  8. Se Pu. 2023 Nov;41(11): 976-985
      The discovery and identification of mushroom toxins has long been an important area in the fields of toxicology and food safety. Mushrooms are widely favored for their culinary and medicinal value; however, the presence of potentially lethal toxins in some species poses a substantial challenge in ensuring their safe consumption. Therefore, the development of a robust and sensitive analytical method is necessary for accurately identifying the risks associated with mushroom consumption. The study of mushroom toxins, which are characterized by their diversity and substantial variations in chemical structures, present a considerable challenge for achieving precise and high-throughput analysis. To address this issue, the present study employed a robust approach combining a solid-phase extraction (SPE) purification technique with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to establish an analytical method for the detection and quantification of five amatoxins and two tryptamines (psilocybin and bufotenine) present in some mushrooms. Several optimization procedures were undertaken, including optimizing the chromatographic conditions, mass spectrometric parameters, and sample extraction and purification. The procedure involved the extraction of dry mushroom powder with methanol containing 0.3% formic acid, followed by purification using a strong cation exchange cartridge (SCX). The analytes were separated on a T3 chromatographic column (100 mm×2.1 mm, 1.8 μm) using mobile phases of acetonitrile and 5 mmol/L ammonium acetate solution containing 0.1% formic acid. The multiple reaction monitoring (MRM) mode was employed for data acquisition. Amatoxins were quantified using matrix-matched standard calibration curves, whereas isotopic internal standards were used to quantify tryptamine. The results showed that all seven toxins exhibited good linearities (r2>0.99) within the optimized concentration range. The limits of detection (LODs) for bufotenine, psilocybin, and amatoxins were determined as 2.0, 5.0, and 10 μg/kg, respectively, while the limits of quantification (LOQs) were determined as 5.0, 10, and 20 μg/kg, respectively. The LOD and LOQ values further underscore the ability of the method to detect minute quantities of toxins, making it particularly well suited for screening food samples for potential contamination. Using dried shiitake mushroom powder as the matrix, the recoveries of the two tryptamines ranged from 80.6% to 117%, with relative standard deviations (RSDs) ranging from 1.73% to 5.98%, while the recoveries of amatoxins ranged from 71.8% to 115%, with RSDs varying from 2.14% to 9.92% at the three concentration levels. The consistent and satisfactory recoveries of amatoxins and tryptamines demonstrated the ability of this method to accurately quantify the target analytes even in a complex matrix. Comparison with the results of supplementary test method recognized by State Administration for Market Regulation for food (BJS 202008) demonstrated comparable results, indicating no significant differences (p>0.05) in amatoxin contents. The newly developed method is rapid, accurate, precise, meets the required standards, and is suitable for the detection of seven toxins in wild mushrooms. As part of the application of this method, a comprehensive investigation of the distribution of toxins in wild mushrooms from Fujian Province was undertaken. In this study, 59 wild mushroom samples from nine cities were collected in the Fujian province. Species identification was conducted using rDNA-internal transcribed space (rDNA-ITS) molecular barcode technology, which revealed the presence of toxins in the two samples. Notably, one specimen named Amanita fuligineoides contained α-amanitin, β-amanitin, and phalloidin in quantities of 607, 377, and 69.0 mg/kg, respectively. Additionally, another sample, identified as Tricholomataceae, had a psilocybin concentration of 12.6 mg/kg.
    Keywords:  amanitins; bufotenine; high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS); mushroom; phallotoxins; psilocin
  9. J Ind Microbiol Biotechnol. 2023 Nov 13. pii: kuad039. [Epub ahead of print]
      Gas chromatography-tandem mass spectrometry with electron ionization (GC-EI-MS/MS) provides rich information on stable-isotope labeling for 13C-metabolic flux analysis (13C-MFA). To pave the way for the routine application of tandem MS data for metabolic flux quantification, we aimed to compile a comprehensive library of GC-EI-MS/MS fragments of tert-butyldimethylsilyl (TBDMS) derivatized proteinogenic amino acids. First, we established an analytical workflow which combines high resolution gas chromatography-quadrupole time-of-flight mass spectrometry (GC-EI-QTOFMS) and fully 13C-labeled biomass to identify and structurally elucidate tandem MS amino acid fragments. Application of the high-mass accuracy MS procedure resulted into the identification of 129 validated precursor-product ion pairs of 13 amino acids with 30 fragments being accepted for 13C-MFA. The practical benefit of the novel tandem MS data was demonstrated by a proof-of-concept study which confirmed the importance of the compiled library for high resolution 13C-MFA.
    Keywords:   13C-metabolic flux analysis; GC-MS/MS; isotopomer; metabolism; stable isotope tracer
  10. Se Pu. 2023 Nov;41(11): 960-975
      Various types of milk powder purportedly providing diverse health functions have emerged with the growth of the country's elderly population. Some manufacturers illegally add chemical drugs to their products to achieve their reported benefits, which poses a threat to consumer health. The existing standard methods are inapplicable to such complex sample matrices and require testing based on functional claims and classification. These limitations not only consume manpower and resources but also seriously impede daily regulatory efforts to detect unknown risk substances. In this study, a high-throughput method for the screening and quantitative analysis of 300 illegally added chemical drugs in functional milk powder and an identification strategy for unknown structural analogues were established using Zeno SWATH® data-independent acquisition (DIA) ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) technology combined with a QuEChERS sample purification method. The QuEChERS purification process was developed according to the characteristics of milk powder matrix. The supernatant was separated on a Kinetex F5 column (100 mm×3.0 mm, 2.6 μm) by gradient elution using 5 mmol/L ammonium formate aqueous solution (0.1% (v/v) formic acid, ) and methanol-acetonitrile (1∶1, v/v) as mobile phases. The method was validated in terms of selectivity, linearity, limits of detection and quantification (LODs and LOQs, respectively), matrix effect, accuracy, and precision. Based on a screening database for the 300 target substances, electron-activated dissociation (EAD) fragmentation was applied to obtain rich secondary MS fragmentation information, and unknown structural analogues were identified and confirmed through fragment attribution analysis. The results indicated that all compounds had good linear relationships in certain ranges with correlation coefficients >0.99. The LODs and LOQs were 0.04-2.7 and 0.2-8.0 μg/kg, respectively. The average recoveries at three spiked levels were in the range of 73.1%-125.2%, and the relative standard deviations were ≤14.8% (n=6). When the developed method was applied to detect illegally added chemicals in 60 functional milk powder samples, it detected benzoguanidine and sildenafil and successfully identified ethylphenidate, which is the structural analogue of an amphetamine. The proposed method is simple, sensitive, and accurate; thus, it may have practical application value for the daily supervision and law enforcement of milk powders with reported health functions.
    Keywords:  QuEChERS; functional milk powder; high resolution mass spectrometry (HRMS); illegally added; quadrupole-time of flight-mass spectrometry (QTOF-MS); structural analogues; ultra-high performance liquid chromatography (UHPLC)
  11. Se Pu. 2023 Nov;41(11): 1030-1037
      A method based on ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry was developed for the rapid determination of 13 β-blockers in health foods. The MS fragmentation pathways of the analytes were subsequently investigated. The optimal MS conditions, extraction solvents, mobile phases, and matrix effects were evaluated in detail. The samples were extracted with methanol, filtered by high-speed centrifugation and ultrasonic treatment, and then separated on an Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm) with gradient elution using acetonitrile and 0.1% (v/v) formic acid aqueous solution as mobile phases. MS analysis was conducted in positive-ion mode, and the data were collected using full mass and data-dependent MS2 scans (Full MS/dd-MS2). The efficient separation and high-precision primary and secondary scanning of the 13 β-blockers in health foods were realized within 10 min, and accurate mass numbers and fragment-ion information were obtained. The methodological validation showed good linear relationships in the range of 0.5-100 μg/L, with correlation coefficients (r)≥0.9912. The limits of detection ranged from 1 to 10 μg/kg. When the standard substances were added to the blank sample in the amount of 10-200 μg/kg, the recoveries were in the range of 75.3%-108.4%, and the relative standard deviations ranged from 0.9% to 10.0% (n=6). The method was used to screen 30 batches of commercially available health foods, and none of the 13 β-blockers was detected. The proposed method is fast, accurate, and sensitive, and can be used for the rapid determination of β-blockers in health foods.
    Keywords:  health foods; ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry; β-blockers
  12. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Oct 29. pii: S1570-0232(23)00327-6. [Epub ahead of print]1230 123917
      Janus kinase inhibitors (JAKi) are oral small molecules used in the treatment of a broad spectrum of autoimmune and myeloproliferative diseases. JAKi exhibit significant intra- and inter-individual pharmacokinetic variabilities, due to fluctuations in compliance with oral treatments and their metabolism essentially driven by cytochrome P450 enzymes. Intrinsically, JAKi have dose-response relationship and narrow therapeutic index: therapeutic drug monitoring (TDM) is expected to optimize and adapt their dosage regimen in order to resolve problems of efficacy and tolerance linked to dose and safety. A sensitive analytical method using multiplex high-performance liquid-chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was developed and validated for the simultaneous quantification in plasma of the 6 major currently used JAKi, namely abrocitinib, baricitinib, fedratinib, ruxolitinib, tofacitinib, and upadacitinib. Plasma samples are subjected to protein precipitation with MeOH, using stable isotopically labelled internal standards. The separation of JAKi in supernatants diluted 1:1 with ultrapure H2O was performed using a C18 column Xselect HSS T3 2.5 µm, 2.1x150 mm using a mobile phase composed of formic acid (FA) 0.2% and acetonitrile (+FA 0.1%) in gradient mode. The analytical run time for the multiplex assay was 7 min. JAKi drugs were monitored by electrospray ionization in the positive mode followed by triple-stage quadrupole MS/MS analysis. The method was validated according to SFSTP and ICH guidelines over the clinically relevant concentration ranges (0.5-200 ng/mL for abrocitinib, baricitinib and upadacitinib; 1-400 ng/mL for tofacitinib; 0.5-400 ng/mL for ruxolitinib, and 10-800 ng/mL for fedratinib). This multiplex HPLC-MS/MS assay achieved good performances in term of trueness (91.1-113.5%), repeatability (3.0-9.9%), and intermediate precision (4.5-11.3%). We developed and validated a highly sensitive method for the multiplex quantification of the JAKi abrocitinib, baricitinib, fedratinib, ruxolitinib, tofacitinib, and upadacitinib in human plasma. The method will be applied for prospective clinical pharmacokinetic studies to determine whether TDM programs for JAKi based on residual drug concentrations can be recommended using disease-specific therapeutic ranges.
    Keywords:  Immunosuppressants; Inflammatory diseases; JAK inhibitors; LC-MS/MS; Targeted therapy; Therapeutic Drug Monitoring
  13. Anal Chem. 2023 Nov 15.
      Subtle variations in stable isotope ratios at natural abundance are challenging to measure but can yield critical insights into biological, physical, and geochemical processes. Well-established methods, particularly multicollector, gas-source, or plasma isotope ratio mass spectrometry, are the gold standard for stable isotope measurement, but inherent limitations in these approaches make them ill-suited to determining site-specific and multiply substituted isotopic abundances of all but a few compounds or to characterizing larger intact molecules. Fourier transform mass spectrometry, namely, Orbitrap mass spectrometry, has recently demonstrated the ability to measure natural abundance isotope ratios with chemically informative accuracy and precision. Here, we report the first use of Fourier transform ion cyclotron resonance mass spectrometry for the accurate (<1‰) and precise (<1‰ standard error) simultaneous determination of δ13C and δ15N in caffeine isotopologues and provide a discussion of the critical instrumental parameters necessary to make such measurements. We further report the ability to make these measurements with online liquid chromatography, expanding the ability of this technique to explore mixtures in the future.
  14. Environ Sci Technol. 2023 Nov 15.
      In an increasingly chemically polluted environment, rapidly characterizing the human chemical exposome (i.e., chemical mixtures accumulating in humans) at the population scale is critical to understand its impact on health. High-resolution mass spectrometry (HRMS) profiling of complex biological matrices can theoretically provide a comprehensive picture of chemical exposures. However, annotating the detected chemical features, particularly low-abundant ones, remains a significant obstacle to implementing such approaches at a large scale. We present Scannotation (, an automated and user-friendly suspect screening tool for the rapid pre-annotation of HRMS preprocessed data sets. This software tool combines several MS1 chemical predictors, i.e., m/z, experimental and predicted retention times, isotopic patterns, and neutral loss patterns, to score the proximity between features and suspects, thus efficiently prioritizing tentative annotations to verify. Scannotation and MS-DIAL4 were used to annotate blood serum samples of 75 Breton adolescents. Scannotation's combination of MS1-based chemical predictors allowed us to annotate 89 chemically diverse environmental compounds with high confidence (confirmed by MS2 when available). These compounds included 62% of emerging molecules, for which no toxicological or human biomonitoring data are reported in the literature. The complementarity observed with MS-DIAL4 results demonstrates the relevance of Scannotation for the efficient pre-annotation of large-scale exposomics data sets.
    Keywords:  chemical exposomics; compound identification; high-resolution mass spectrometry; nontargeted analysis; scoring; suspect screening software
  15. Methods Appl Fluoresc. 2023 Nov 13.
      In this study, QuEChERS extraction was combined with dispersive liquid-liquid microextraction (DLLME) to extract pesticides from tropical fruits for determination by a highly accurate and sensitive liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS/MS) system. The QuEChERS method served as a matrix clean-up tool and the DLLME method preconcentrated the analytes for their determination at trace levels. All parameter variables of the DLLME method were optimized to improve the extraction output and lower the limits of detection and quantification (LOD and LOQ) for all the analytes. Under the optimum experimental conditions, the LOD and LOQ values were found in the range of 0.004 - 0.013 and 0.27 - 0.61 µg/L, respectively. The detection limits achieved by direct LC-QTOF-MS/MS analysis were increased by about 10 - 260 folds using the optimized DLLME method. To assess the accuracy and applicability of the developed method, spike recovery experiments on tropical fruits were carried out. The matrix matching calibration method was used to enhance the quantification accuracy of the analytes in kiwi, pineapple, and mango matrices, with percent recoveries ranging between 89 and 117%.
    Keywords:  Dispersive liquid-liquid microextraction; LC-QTOF-MS/MS; Pesticide Residue; QuEChERS
  16. Front Vet Sci. 2023 ;10 1285932
      Introduction: Milbemycin oxime (MBO) and praziquantel (PZQ) have a broad spectrum of biological activity and are commonly used to treat the parasitic infection in the veterinary clinic. In this study, a fast and efficient LC-MS/MS method was established and validated for the simultaneous determination of MBO, PZQ, cis-4-hydroxylated-PZQ (C-4-OH-PZQ) and trans-4-hydroxylated-PZQ (T-4-OH-PZQ) and in cat plasma.Methods: Extraction of analytes and internal standards from cat plasma by acetonitrile protein precipitation, allows rapid processing of large batches of samples. MBO, PZQ, C-4-OH-PZQ, T-4-OH-PZQ, and internal standard (IS) were eluted for 13.5 min on a C18 column with a 0.1% formic acid water/acetonitrile mixture as the mobile phase.
    Results: Results showed that the method had good precision, accuracy, recovery, and linearity. The linearity range was 2.5-250 ng/mL for MBO, and 10-1000 ng/mL for PZQ, C-4-OH-PZQ, and T-4-OH-PZQ. The intra-day and inter-day precision CV values of the tested components were within 15%. The extraction recoveries of the four components ranged from 98.09% to 107.46%. The analytes in plasma remained stable for 6 h at room temperature, 26 h in the autosampler (4 °C), after freeze-thaw (-20°C) cycles, and 60 days in a -20°C freezer. Method sensitivity sufficed for assessing pharmacokinetic parameters of MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ in plasma samples with LLOQ of 2.5 ng/mL for MBO and 10 ng/mL for PZQ, C-4-OH-PZQ, and T-4-OH-PZQ.
    Conclusion: In this study, a selective and sensitive LC-MS/MS method for the simultaneous quantification of MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ in cat plasma was developed and validated.This method had been successfully applied to evaluate the pharmacokinetics of MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ after a single oral administration of 8 mg MBO and 20 mg PZQ in cats.
    Keywords:  LC–MS/MS; cat plasma; milbemycin oxime; pharmacokinetics; praziquantel
  17. J Pharm Biomed Anal. 2023 Nov 04. pii: S0731-7085(23)00601-5. [Epub ahead of print]238 115832
      A reversed phase ultra-high performance liquid chromatography method was developed for the simultaneous quantification of cabotegravir (CAB) and the E-isomer of rilpivirine (RPV) in human EDTA plasma, also considering RPV E-isomer instability. Because of the instability of RPV (and CAB) in all light conditions, the (RPV Z-isomer/total RPV)-isomer ratio of RPV was determined for each stock, calibration curve standard, quality control sample and patient sample. [2H3]-CAB and [13C6]-RPV were used as internal standard. Sample preparation involved protein precipitation of plasma using methanol. An HSS T3 column with a guard column (set at 40 °C) was used for analyte separation. The mobile phase components were 65 % 0.1 % formic acid in water (A) and 35 % 0.1 % formic acid in acetonitrile (B) and the flow rate was 0.5 mL/min. Detection was performed with tandem mass spectrometry (MS/MS) in a total runtime of 3.0 min. The assay was validated over the concentration range of 0.0500 - 10.0 mg/L for CAB and 0.00300 - 3.00 mg/L for RPV. The average within-day and between-day accuracies for the assay in plasma were 101 % and 101 % for CAB and 97.6 % and 98.5 % voor RPV, respectively. Within-day and between-day precision in coefficients of variations (CV) were 5.0 %. Extraction recovery was 99 % and 102 % for CAB and its internal standard and 95 % and 97 % for RPV and its internal standard. As our aim was that the (Z-isomer RPV/total RPV) response ratio in patient samples had to be less than 10 % to give reliable results, the (Z-isomer RPV/total RPV) response ratio in stocks, calibration curve standards and internal quality control samples were also taken into account being maximal 0.9 % and 2.3 % respectively. This assay has been successfully used in our Therapeutic Drug Monitoring (TDM) service for people living with HIV on long-acting injectable therapy with CAB/RPV and will also be used in future pharmacokinetic studies.
    Keywords:  Cabotegravir; Light instability; MS/MS detection; Plasma; Rilpivirine; Ultra-high performance liquid chromatography
  18. J Pharm Biomed Anal. 2023 Nov 07. pii: S0731-7085(23)00616-7. [Epub ahead of print]238 115847
      A LC-ESI/MS/MS method was developed for quantification of up to eighteen cannabinoids, the maximum number published so far. A thorough study of published LC-ESI/MS/MS methods using triple quadrupole mass spectrometers revealed a possible misconception that multiple reaction monitoring (MRM) was able to definitively differentiate structural isomers of cannabinoids, especially Δ8-/Δ9-tetrahydrocannabinol (THC), which explained why many of those methods were developed for a limited number of cannabinoids, as small as two, and did not include Δ8-THC. In this study, the use of a quadrupole time-of-flight (QTOF) mass spectrometer for targeted analysis indicated that MRM could not definitively distinguish structural isomers of Δ9-THC, with a possible exception of cannabicyclol (CBL) for less accurate quantification, so their baseline separation was essential for their accurate quantification. After the developed method was successfully validated according to the ISO 17025 guidelines, it was further applied for the analysis of eighteen hemp-derived products, including drinks, water-soluble oils, topical serum, body lotion, face cream, lip balm, gummies, hard candy, coffee, snacks, and pet treats. The LOQ was 0.00008% (w/w) for drinks with the analysis of 12.5 mg/mL extracts, while the LOQ was 0.008% (w/w) for other samples because 125 μg/mL extracts were analyzed due to higher content of cannabinoids in non-drink samples. For the first-time, extraction recovery and matrix effect were tracked in real-time for each sample being analyzed, obtaining 92.9-106.3% and 91.3-120.2% in triplicate measurements, respectively, by spiking abnormal cannabidiol (ACBD), a cannabinoid not naturally present in hemp, into each sample before extraction and ACBD-d3 into each sample after extraction.
    Keywords:  Cannabinoids; Drinks; Edibles; Hemp; Liquid chromatography electrospray ionization tandem mass spectrometry; Topicals
  19. Anal Chim Acta. 2023 Dec 01. pii: S0003-2670(23)01176-5. [Epub ahead of print]1283 341955
      BACKGROUND: Reliable methods enabling detection of metal ions, and especially heavy metals, in different matrices are necessary in various fields such as ecology, pharmaceuticals and toxicology. As some of the currently used methods suffer from spectral and chemical interferences, this study investigates the applicability of SFC-MS/MS for the determination of metal ions.RESULTS: Effective novel approaches for metal ion analysis using CO2-based mobile phase were developed using three ligands forming metal complexes. As metal-EDTA complexes are prepared by simple addition of EDTA to the solution containing metal ions, this approach to metal ion analysis does not require laborious synthesis and isolation of solid metal-complexes. Besides, two other approaches using diethyldithiocarbamate and acetylacetonate as ligands were compared. Metal complexes of Cu, Co, Cr, Fe, Al, Mn, and Zn with all 3 ligands were synthesized and their identity was confirmed by high-resolution mass spectrometry (HRMS). The suitability of the three developed UHPSFC-MS/MS methods was examined using the determination of calibration range and repeatability of injections. Moreover, the universality of the developed UHPSFC-MS/MS method for the determination of metal-EDTA complexes was proved by analyzing Ni, Bi and Pb as additional metal ions.
    SIGNIFICANCE AND NOVELTY: This study demonstrates the extended range of applicability for SFC based separations. For the first time, the possibility to analyze metal complexes with EDTA using a fast and reliable ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS) method is reported. The three developed UHPSFC-MS/MS methods are able to separate DDC, acac, and EDTA complexes of various metals very efficiently (total cycle times of 5, 2, and 3 min, respectively). They offer a fast and green alternative to chromatographic methods commonly used for metal ion analysis.
    Keywords:  Acetylacetonate; Diethyldithiocarbamate; EDTA; Mass spectrometry; Metal ion complexes; Supercritical fluid chromatography
  20. Anal Chim Acta. 2023 Dec 01. pii: S0003-2670(23)01185-6. [Epub ahead of print]1283 341964
      BACKGROUND: The detection of 25-hydroxyvitamin D (25OHD) from dried blood spots (DBS) has been widely studied. However, the existing pretreatment methods suffer from limitations in terms of throughput (usually exceeding 2 h), complexity (involving liquid-liquid extraction or solid-phase extraction), and contamination (including multiple steps of organic solvent evaporation).RESULTS: We first released 25OHD from DBS samples by 50% acetonitrile solution through ultrasonication. Subsequently, the cold-induced phase separation technique was introduced for in-situ concentration and purification. Afterward, the PTAD derivatization of 25OHD was performed directly, profiting from the high acetonitrile content in the concentrated solution. In the end, the resulting solution was submitted to LC-MS/MS for quantification. The established LC-MS/MS methodology possessed favorable analytical performance, possessing lower limit of quantification of 1 ng/mL pointing to plasma, accuracy of 86.8-110.1% and imprecision of 5.4-16.8%. Method comparison with plasma samples demonstrated that over 93% of the detections met the acceptance limit for cross-validation of ±20%.
    SIGNIFICANCE AND NOVELTY: The novel sample preparation can be finished within 15 min and eliminated the traditional steps of extraction and organic solvent evaporation. Based on this high-throughput, reliable and applicable LC-MS/MS method, the detection of 25OHD in DBS samples can be better achieved for clinical patients and researchers with relevant demands.
    Keywords:  25-Hydroxyvitamin D2; 25-Hydroxyvitamin D3; Cold-induced phase separation (CIPS); Dried blood spot (DBS); LC-MS/MS
  21. J Mass Spectrom. 2023 Dec;58(12): e4984
      Ribociclib is a cyclin-dependent kinase (CDK4/6) inhibitor and is a standard of care for treating metastatic breast cancer. The drug has moderate oral bioavailability and exhibits permeability-controlled absorption. Novel formulations to enhance ribociclib pharmacokinetics are being developed and tested in rats. This requires developing analytical assays for quantifying ribociclib monitoring in rat plasma. We present a fully validated, sensitive, and simple LC-MS/MS method for measuring ribociclib in rat plasma. Ribociclib-D6 was utilized as an internal standard, and a simple protein precipitation procedure with acetonitrile was used in sample preparation. Excellent assay linearity was observed over a standard curve concentration of 1.008-1027.624 ng/mL. Acceptable intra- and inter-day accuracy and reproductivity were demonstrated for ribociclib quality controls (bias and CV% within ±15%). Complete extraction recovery of ribociclib was achieved, and a negligible matrix effect of analyte to internal standard ratio was observed. Ribociclib was stable at various conditions, including bench-top, freeze-thaw, and short-term stability. Overall, the presented method is simple, sensitive, accurate, and precise and was successfully applied to quantify ribociclib in plasma samples from a pharmacokinetic study of ribociclib suspension and nanoparticle formulation in rats.
    Keywords:  formulation; liquid chromatography-tandem mass spectrometry; pharmacokinetics; plasma; rat; ribociclib
  22. Se Pu. 2023 Nov;41(11): 986-994
      Mycotoxins have carcinogenic, mutagenic, hepatotoxic, nephrotoxic, immunotoxic, neurotoxic, and teratogenic properties. Thus, these substances have attracted significant attention because they pose a threat to human health. As research on mycotoxins deepens, new structural analogues of mycotoxins are constantly being discovered. In this study, a method based on high performance liquid chromatography-quadrupole/orbitrap mass spectrometry was established for the simultaneous determination of 22 mycotoxins in milk. A simple, effective, and rapid pretreatment method was optimized by focusing on the solvent type, extractant volume, and extracting salt based on the characteristics of the mycotoxins and sample matrix. The analytes were extracted using 0.5% formic acid acetonitrile solution and added with sodium chloride to separate fats from water. The samples were centrifuged at 8000 r/min (4 ℃) for 5 min using a centrifuge and then concentrated using nitrogen. The dry residue was dissolved with 50% methanol aqueous solution. Twenty-two mycotoxins were separated on a ZORBAX Eclipse Plus C18 chromatographic column (100 mm×2.1 mm, 1.7 μm), and quantitative analysis was performed using the isotope internal standard method. The analytes were determined by liquid chromatography-quadrupole/orbitrap mass spectrometry in positive electrospray ionization mode. Qualitative analyses of the compounds were performed in full mass spectrometry/data-dependent tandem mass spectrometry (MS/dd-MS2) mode. Good linearities in the range of 0.5-100.0 μg/L were observed for the 22 mycotoxins, and the correlation coefficients (R2) were greater than 0.999. The limits of detection (S/N=3) and quantification (S/N=10) ranged from 0.3 to 0.5 μg/kg and from 1.0 to 1.5 μg/kg, respectively. The average recoveries of the 22 mycotoxins at three spiked levels of 1.5, 5.0, and 15 μg/kg were between 84.7% and 100.8%, with relative standard deviations (RSDs) of 1.2%-9.9%. These findings indicate that the method has high sensitivity and accuracy as well as good precision. Finally, the method was applied to the detection and analysis of mycotoxins in 25 actual commercial milk samples. The results revealed that the selected samples were not contaminated with any of the mycotoxins analyzed. Thus, the proposed method is useful as a quick preprocessing and confirmatory method for the simultaneous determination of mycotoxins in milk.
    Keywords:  liquid chromatography-quadrupole/orbitrap mass spectrometry; milk; mycotoxins; rapid pretreatment
  23. Foods. 2023 Nov 06. pii: 4037. [Epub ahead of print]12(21):
      The presence of acrylamide, a known human carcinogen, in various heated foods raises significant concerns among consumers. Therefore, the development of a good analytical method is of paramount interest to the scientific community. Keeping this in view, a rapid, simple, reliable, and low-cost analytical method was developed and validated for acrylamide quantification in black ripe olives. The method consisted of the water extraction of the compounds from crushed olives with the addition of (13C3)acrylamide as an internal standard. The quantification was performed using high-pressure liquid chromatography and mass detection with positive electrospray ionization. The limits of detection and quantification were determined to be 4 and 11 µg/kg, respectively. The developed method exhibited excellent results in terms of accuracy (98.4-104.8%) and intra- and inter-day precision limits, both less than 20%. This new method was carried out by analyzing 15 samples of Spanish commercial black ripe olives, revealing a wide range of values, from 79 to 1068 µg/kg of fruit. The new protocol reduces the analysis time to just one hour per sample versus the minimum 24 h required by gas chromatography and mass detection, meaning that it could be a good option for the routine analysis of acrylamide in black ripe olives, and may be extendable to the analysis of this compound in other foods.
    Keywords:  HPLC-MS; acrylamide; black olive; liquid extraction; validation