bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2023‒01‒08
27 papers selected by
Sofia Costa

  1. Anal Bioanal Chem. 2023 Jan 04.
      Direct infusion of lipid extracts into the ion source of a mass spectrometer is a well-established method for lipid analysis. In most cases, nanofluidic devices are used for sample introduction. However, flow injection analysis (FIA) based on sample infusion from a chromatographic pump can offer a simple alternative to shotgun-based approaches. Here, we describe important modification of a method based on FIA and tandem mass spectrometry (MS/MS). We focus on minimizing contamination of the FIA/MS both to render the lipidomic platform more robust and to increase its capacity and applicability for long-sequence measurements required in clinical applications. Robust validation of the developed method confirms its suitability for lipid quantitation in human plasma analysis. Measurements of standard human plasma reference material (NIST SRM 1950) and a set of plasma samples collected from kidney cancer patients and from healthy volunteers yielded highly similar results between FIA-MS/MS and ultra-high-performance supercritical fluid chromatography (UHPSFC)/MS, thereby demonstrating that all modifications have practically no effect on the statistical output. Newly modified FIA-MS/MS allows for the quantitation of 141 lipid species in plasma (11 major lipid classes) within 5.7 min. Finally, we tested the method in a clinical laboratory of the General University Hospital in Prague. In the clinical setting, the method capacity reached 257 samples/day. We also show similar performance of the classification models trained based on the results obtained in clinical settings and the analytical laboratory at the University of Pardubice. Together, these findings demonstrate the high potential of the modified FIA-MS/MS for application in clinical laboratories to measure plasma and serum lipid profiles.
    Keywords:  Direct infusion lipidomics; Flow injection analysis; High-throughput lipidomics; Lipid quantitation; Mass spectrometry; Validation
  2. J Mass Spectrom Adv Clin Lab. 2023 Jan;27 24-32
      Background: Steroids play a key role in numerous physiological processes. Steroid determination is a useful tool to explore various endocrine diseases. Because of its specificity, mass spectrometry is considered to be a reference method for the determination of steroids in serum compared to radioimmunoassay. This technology could progress towards more automation for the optimal organization of clinical laboratories and ultimately for the benefit of patients.Methods: A fully automated ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed and fully validated to determine five steroids in serum. Sample preparation was based on protein precipitation with filtration followed by online solid phase extraction. Chromatographic separation was performed using a biphenyl stationary phase.
    Results: The method was successfully validated according to European Medicine Agency guidelines. Coefficients of variation did not exceed, respectively, 8.4% and 8.1% for intra- and inter-assay precision. Method comparison with radioimmunoassay showed a proportional bias for all compounds, except for testosterone in men. Comparison with another LC-MS/MS method demonstrated acceptable concordance for all steroids, although a small bias was observed for androstenedione.
    Conclusion: The novelty of this method is that it has been fully automated. Automation provides benefits in traceability and allows significant savings in cost and time.
    Keywords:  11DF, 11-deoxycortisol; 17OHP, 17-hydroxyprogesterone; 2D-UHPLC-MS/MS, Two dimensional ultra-high performance liquid chromatography-tandem mass spectrometry; Automation; D4, delta4-androstenedione; DHEA, dehydroepiandrosterone; EMA, European Medicine Agency; GC–MS/MS, Gas chromatography tandem mass spectrometry; LC-MS/MS, Liquid chromatography tandem mass spectrometry; LLE, Liquid-liquid extraction; LLOQ, Lower limit of quantification; Liquid chromatography tandem mass spectrometry; MRM, Multiple reaction monitoring; PTFE, Polytetrafluoroethylene; QC, Quality control; RIA, Radioimmunoassay; Radioimmunoassay; SLE, Supported liquid extraction; SPE, Solid phase extraction; SRM, Standard reference material; Steroids; T, Testosterone; Testosterone; UHPLC, Ultra-high performance liquid chromatography
  3. Anal Chem. 2023 Jan 03.
      Ion mobility (IM) spectrometry provides semiorthogonal data to mass spectrometry (MS), showing promise for identifying unknown metabolites in complex non-targeted metabolomics data sets. While current literature has showcased IM-MS for identifying unknowns under near ideal circumstances, less work has been conducted to evaluate the performance of this approach in metabolomics studies involving highly complex samples with difficult matrices. Here, we present a workflow incorporating de novo molecular formula annotation and MS/MS structure elucidation using SIRIUS 4 with experimental IM collision cross-section (CCS) measurements and machine learning CCS predictions to identify differential unknown metabolites in mutant strains of Caenorhabditis elegans. For many of those ion features, this workflow enabled the successful filtering of candidate structures generated by in silico MS/MS predictions, though in some cases, annotations were challenged by significant hurdles in instrumentation performance and data analysis. While for 37% of differential features we were able to successfully collect both MS/MS and CCS data, fewer than half of these features benefited from a reduction in the number of possible candidate structures using CCS filtering due to poor matching of the machine learning training sets, limited accuracy of experimental and predicted CCS values, and lack of candidate structures resulting from the MS/MS data. When using a CCS error cutoff of ±3%, on average, 28% of candidate structures could be successfully filtered. Herein, we identify and describe the bottlenecks and limitations associated with the identification of unknowns in non-targeted metabolomics using IM-MS to focus and provide insights into areas requiring further improvement.
  4. Anal Methods. 2023 Jan 06.
      Plasma renin activity (PRA) is recommended as the first screening indicator for primary aldosteronism. Immunoassays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed for quantifying PRA, but the interchangeability across assays and laboratories was suboptimal, which predominantly related to the differences in the plasma incubation strategy. This study aims to establish and validate a designed comparison method based on LC-MS/MS. The sensitivity, matrix effect, precision, accuracy, and storage stability were validated according to the Clinical Laboratory Standard Institution (CLSI) C-62A guidelines. The plasma incubation procedure was optimized to achieve maximum PRA results. The short-term stability of PRA plasma was assessed at 4 °C and room temperature (RT) for specific time points. Differences from the baseline were calculated using a one-way analysis of variance. The designed comparison method for PRA measurement exhibits excellent performance characteristics. The results from the 2022 national external quality assessment scheme for PRA showed good consistency of the developed method with other LC-MS/MS methods (relative biases: -6.8% to 4.6%), which demonstrated the reliability of the established method. Two sets of generation buffers were optimized to maximize the renin activity. The acetate buffer was recommended to be used in laboratory practice due to better metrological sensitivity. PRA plasma is stable for one day at 4 °C and RT. In summary, a reliable, traceable, and reproducible LC-MS/MS method for determining PRA was well-established and validated. The recommended incubation protocol is hoped to reduce the discrepancy in Ang1 generation. The evaluated short-term stability for PRA plasma could provide flexibility in clinical practice.
  5. Anal Chem. 2023 Jan 04.
      Data-dependent acquisition (DDA) mode in ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) can provide massive amounts of MS1 and MS/MS information of compounds in untargeted metabolomics and can thus facilitate compound identification greatly. In this work, we developed a new platform called AntDAS-DDA for the automatic processing of UHPLC-HRMS data sets acquired under the DDA mode. Several algorithms, including extracted ion chromatogram extraction, feature extraction, MS/MS spectrum construction, fragment ion identification, and MS1 spectrum construction, were developed within the platform. The performance of AntDAS-DDA was investigated comprehensively with a mixture of standard and complex plant data sets. Results suggested that features in complex sample matrices can be extracted effectively, and the constructed MS1 and MS/MS spectra can benefit in compound identification greatly. The efficiency of compound identification can be improved by about 20%. AntDAS-DDA can take full advantage of MS/MS information in multiple sample analyses and provide more MS/MS spectra than single sample analysis. A comparison with advanced data analysis tools indicated that AntDAS-DDA may be used as an alternative for routine UHPLC-HRMS-based untargeted metabolomics. AntDAS-DDA is freely available at
  6. Chem Pharm Bull (Tokyo). 2023 ;71(1): 10-14
      In this study, an HPLC analysis method using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was developed for the determination of o-phosphoethanolamine (PEA), which is a potential biomarker for the diagnosis of major depressive disorder, in human plasma sample. After PEA was derivatized with AQC under mild conditions, the obtained derivative was subjected to purification with a titanium dioxide-modified monolithic silica spin column (MonoSpin® TiO). The eluate from the MonoSpin® TiO was directly injected into an amide-type hydrophilic interaction liquid chromatography (HILIC) column-equipped HPLC system, and the resulting derivative could be separated on the column under alkaline mobile phase conditions and subsequently detected fluorometrically at excitation and emission wavelengths of 250 and 395 nm, respectively. The limit of detection and limit of quantification for a 10 µL injection volume of PEA were 0.052 and 0.17 µM, respectively. The method was validated at 0.2, 1.0, and 5.0 nmol/mL levels in plasma sample, and the precision values were 2.0-6.6% as relative standard deviation and the correlation coefficient (r) of the calibration curve was 0.9995. Furthermore, applicability of this method was demonstrated by analyzing PEA levels in plasma samples from mental illness patients.
    Keywords:  fluorescence derivatization; human plasma; hydrophilic interaction liquid chromatography (HILIC); o-phosphoethanolamine
  7. Food Res Int. 2023 Jan;pii: S0963-9969(22)01373-4. [Epub ahead of print]163 112315
      LC-HR-MS/MS is the predominant analytical technique in phenolic compound (PC) research. However, the manual interpretation of mass spectra is a heavy nontrivial time-consuming task and depends on mass spectrometry and phenolic compounds fragmentation deep knowledge. We think this manual approach should be partially translated into a practical software that allows users to perform such complicated analyses. In silico fragmentation software have been tested for small molecule identification, MS-FINDER and SIRIUS stood out at identification contests and challenges. We evaluated both software to identify PC from two data categories: 1st MS/MS spectra from 18 phenolic compound standards (PCS) and 2nd phenolic compounds from 8 food samples (FPC) (coffee, green tea, cranberry juice, grape juice, orange juice, apple juice, soy extract and parsley extract). MS-FINDER and SIRIUS were able to correctly identifymore than 90% of the PCS by LC-HR-MS/MS. The main FPC were also correctly identified by MS-FINDER (70%) and SIRIUS (38%). We highlight that these software were unable to differentiate PC isomers. This task is only possible by using additional information, such as chromatographic behavior and manual analysis of the relative intensity of fragments in the MS/MS spectra. Therefore, the combination of initial screening by using MS-FINDER and SIRIUS with manual analyses of additional information is a powerful and efficient approach for identifying phenolic compounds.
    Keywords:  bioinformatics; high-resolution mass spectrometry; identification; phenolic compounds
  8. Talanta. 2022 Dec 28. pii: S0039-9140(22)01014-1. [Epub ahead of print]255 124218
      Anti-doping rule violations related to the abuse of endogenous anabolic androgenic steroids can be currently discovered by the urinary steroidal module of Athlete Biological Passport. Since this powerful tool is still subjected to some limitations due to various confounding factors altering the steroid profile, alternative strategies have been constantly proposed. Among these, the measurement of blood concentrations of endogenous steroid hormones by LC-MS is currently of increasing interest in anti-doping, bringing significant advantages for the detection of testosterone abuse in females and in individuals with deletion of UGT2B17 enzyme. Although various research groups have made significant efforts in method development, there is currently no accepted or harmonized anti-doping method for quantitative analysis of the various testosterone doping markers in blood. In this study we present a UHPLC-MS/MS method for the quantification of major circulating steroid hormones together with an extended panel of glucuro- and sulpho-conjugated phase II metabolites of androgens. Chromatographic setup was optimized by comparing the performance of three different C18 stationary phases and by the careful selection of mobile phases with the aim of separating all the target steroids, including numerous isomeric/isobaric compounds. MS parameters were fine-tuned to obtain the sensitivity needed for measuring the target analytes, that show specific serum concentrations ranging from low pg/mL for less abundant compounds to μg/mL for sulpho-conjugated steroids. Finally, sample preparation protocol was developed for the extraction of steroid hormones from 200 μL of serum and the performance was evaluated in terms of extraction recovery and matrix effect. The final method was then applied to authentic serum samples collected from healthy volunteers (40 males and 40 females) at the Blood Bank of the City of Health and Science University Hospital of Turin. The analysis of these samples allowed to obtain results on serum concentrations of the targeted steroids, with particular emphasis on previously undiscovered phase II metabolites, such as the isomers of 5-androstane-3,17-diol glucuronide. This preliminary application also enabled measuring dihydrotestosterone sulphate in male samples, efficiently separating this analyte from its isomer, epiandrosterone sulphate, which circulates in blood at high concentrations. The promising results of this study are encouraging for the measurement of blood steroid profile markers in serum and plasma samples for Athlete Biological Passport purposes.
    Keywords:  Androgens; Athlete biological passport; Doping; Serum; Steroids; UHPLC-MS/MS
  9. Chem Pharm Bull (Tokyo). 2023 ;71(1): 19-23
      An assay using HPLC with fluorescence (FL) detection method for monitoring native FL of tocilizumab (TCZ) in human serum combined with extremely simple and rapid pretreatment without any antigen-antibody reaction was developed. Good separation of TCZ was achieved within 13 min on a Presto FF-C18 column (100 × 4.6 mm i.d., 2 µm). Simple pretreatment with acetonitrile containing primary and secondary alkylamines having longer than C3 in the alkyl chain removed immunoglobulin G subclass 1 and TCZ could be recovered selectively. The spiked calibration curve of TCZ in human serum showed good linearity in the range of 40-1000 µg/mL (r > 0.997). The lower limit of quantitation (S/N = 10) of the TCZ was 19.7 µg/mL. The accuracy was within 103.5-114.9%, and the intra- and inter-day precisions as relative standard deviations were less than 5.3 and 7.8% (n = 5), respectively. The recovery of TCZ was 42.2 ± 3.4% (n = 3). The TCZ in pretreated sample was confirmed to be stable for 6 h (>95%) at room temperature and 24 h (>95%) at 4 °C. The proposed method is considered extremely superior to the previous methods in terms of time requirement for analysis. Therefore, the developed method may be more useful than conventional methods in urgent situations, such as confirming therapeutic efficacy of cytokine-release syndrome by 2019 coronavirus disease.
    Keywords:  HPLC-fluorescence detection; cytokine-release syndrome; primary alkylamine; selective precipitation; tocilizumab
  10. J Pharm Biomed Anal. 2022 Dec 20. pii: S0731-7085(22)00632-X. [Epub ahead of print]225 115211
      The cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors palbociclib, ribociclib, and abemaciclib were approved by the U.S. Food and Drug Administration (FDA) and European Medicine Agency for the treatment of breast cancer between 2015 and 2018. Oral tumor therapeutics extend the options for cancer therapy, but also challenge physicians and patients. The aim of the present work was to establish a semi-automated liquid-chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of abemaciclib, its active metabolites abemaciclib M20 and M2, palbociclib, and ribociclib in human serum. Detuning of ribociclib enabled the development of a simultaneous quantification method for abemaciclib, M20, M2, palbociclib, and ribociclib in the respective relevant concentration ranges based on semi-automated sample preparation with isotope dilution LC-MS/MS. The method was validated according to the guidance of the FDA. The LC-MS/MS method was successfully validated according to FDA and showed inaccuracies ≤ 10.7% and imprecisions ≤ 8.51%. Linearity was given from 20 to 800 ng/mL for abemaciclib, 15-600 ng/mL for M20, 10-400 ng/mL for M2 and palbociclib, and 100-4000 ng/mL for ribociclib. Normalized matrix factors and process efficiency showed no significant matrix effects regardless of the analytes. To demonstrate the applicability of the method, authentic samples were also analyzed. This novel semi-automated LC-MS/MS method covering all previously approved CDK4/6 inhibitors as well as the similarly pharmacologically active metabolites in human serum simultaneously was developed for potential future use in routine analysis in order to improve personalized therapy, patient safety, and treatment success.
    Keywords:  Abemaciclib; CDK4/6 inhibitor; LCMS/; MS; Palbociclib; Ribociclib; Therapeutic drug monitoring
  11. Food Res Int. 2023 Jan;pii: S0963-9969(22)01228-5. [Epub ahead of print]163 112170
      α-dicarbonyl compounds (α-DCs) serve as potential biomarkers for oxidative stress-related diseases but are difficult to detect.To study the metabolism of carbonyl compounds, we developed a new mass spectrometry probe, 3-benzyl-2-oxo-4λ3-thiazolidine-4-carbohydrazide (BOTC), containing hydrazyl groups for the targeted detection of carbonyl functional groups.In a novel approach, we used BOTC pre-column derivatization with ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to simultaneously detect four kinds of α-DCs in red wine as well as in urine after drinking. The α-DCs were completely separated (R2 ≥ 0.9995), detection was sensitive (detection limit was 12.5-50 fmol), consistent (intraday and interday precision was 0.1-5.7 %), and efficient (average recoveries were 103.3-110.2 %). The method was applied to the analysis of α-DCs in different wines and the dynamic monitoring of transit and excretion in vivo after drinking. Our novel method provides a new strategy for the detection of α-dicarbonyl compounds in red wine and dicarbonyl compounds produced in oxidative stress-related diseases.
    Keywords:  Human urine; Oxidative; Red wine; UHPLC-MS/MS; α-dicarbonyl compound
  12. Anal Chem. 2023 Jan 05.
      Volatolomics offers an opportunity for noninvasive detection and monitoring of human disease. While gas chromatography-mass spectrometry (GC-MS) remains the technique of choice for analyzing volatile organic compounds (VOCs), barriers to wider adoption in clinical practice still exist, including: sample preparation and introduction techniques, VOC extraction, throughput, volatolome coverage, biological interpretation, and quality control (QC). Therefore, we developed a complete pipeline for untargeted urinary volatolomic profiling. We optimized a novel extraction technique using HiSorb sorptive extraction, which exhibited high analytical performance and throughput. We achieved a broader VOC coverage by using HiSorb coupled with a set of complementary chromatographic methods and time-of-flight mass spectrometry. Furthermore, we developed a data preprocessing strategy by evaluating internal standard normalization, batch correction, and we adopted strict QC measures including removal of nonlinearly responding, irreproducible, or contaminated metabolic features, ensuring the acquisition of high-quality data. The applicability of this pipeline was evaluated in a clinical cohort consisting of pancreatic ductal adenocarcinoma (PDAC) patients (n = 28) and controls (n = 33), identifying four urinary candidate biomarkers (2-pentanone, hexanal, 3-hexanone, and p-cymene), which can successfully discriminate the cancer and noncancer subjects. This study presents an optimized, high-throughput, and quality-controlled pipeline for untargeted urinary volatolomic profiling. Use of the pipeline to discriminate PDAC from control subjects provides proof of principal of its clinical utility and potential for application in future biomarker discovery studies.
  13. Anal Methods. 2023 Jan 05.
      There are at least 500 naturally occurring amino acids, of which only 20 standard proteinogenic amino acids are used universally across all organisms in the synthesis of peptides and proteins. Non-standard amino acids can be incorporated into proteins or are intermediates and products of metabolic pathways. While the analysis of standard amino acids is well-defined, the analysis of non-standard amino acids can be challenging due to the wide range of physicochemical properties, and the lack of both reference standards and information in curated databases to aid compound identification. It has been shown that the use of an AccQ·Tag™ derivatization kit along with LC-MS/MS is an attractive option for the analysis of free standard amino acids in complex samples because it is fast, sensitive, reproducible, and selective. It has been demonstrated that the most abundant quantitative transition for MS/MS analysis of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatized amino acids corresponds to the fragmentation of the molecule at the 6-aminoquinoline carbonyl group producing a common m/z 171 fragment ion and occurs at similar mass spectrometry collision energy and cone voltages. In this study, the unique properties of AQC derivatized amino acids producing high intensity common fragment ions, along with chromatographic separation of amino acids under generic chromatography conditions, were used to develop a novel screening method for the detection of trace levels of non-standard amino acids in complex matrices. Structural elucidation was carried out by comparing the MS/MS fragment ion mass spectra generated with in silico predicted fragmentation spectra to enable a putative identification, which was confirmed using an appropriate analytical standard. This workflow was applied to screen human plasma samples for bioactive thiol-group modified cysteine amino acids and S-allylmercaptocysteine (SAMC), S-allylcysteine sulfoxide (SACS or alliin) and S-propenylcysteine (S1PC) are reported for the first time to be present in human plasma samples after the administration of garlic supplements.
  14. Anal Methods. 2023 Jan 03.
      The global market for new psychoactive substances (NPSs) continues to expand, and the range of drugs available on the market has probably never been wider. Synthetic cannabinoids (SCRAs) constitute the largest family of NPSs, and they go unnoticed during illicit drug market control and during routine toxicological-forensic analysis. Membrane-assisted solvent extraction (MASE) has been a novelty proposed for the simultaneous extraction of SCRAs, and urine has been selected as a model forensic-clinical sample. Isolated SCRAs were further determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). An optimised sample pre-treatment procedure consists of using 400 μL of n-hexane as an extraction phase placed inside a polypropylene (PP) membrane, adjusting the donor phase (urine) at a pH value of 5.9. Extraction was assisted by mechanical (orbital-horizontal) stirring in a temperature-controlled chamber at room temperature for 20 min. n-Hexane extracts were evaporated to dryness and re-suspended in 100 μL of mobile phase, which leads to a pre-concentration factor of 50. Method validation showed analytical recoveries higher than 80% for most SCRAs and repeatability (inter-day and intra-day assays) with RSD values lower than 20%. The proposed method was found to be selective and sensitive and limits of quantification (LOQs) between 0.10 and 1.0 μg L-1 were achieved.
  15. Anal Methods. 2023 Jan 04.
      Quantification of metabolites present within exhaled breath is a major challenge for on-line breath analysis. It is also important for gauging the analytical performance, accuracy, reproducibility, reliability, and stability of the measuring technology. Short-chain fatty acids (SCFAs) are of high interest for nutrition and health. Their quantification enables a deep mechanistic understanding of a wide range of biological processes and metabolic pathways, while their high volatility makes them an attractive target for breath analysis. This article reports, for the first time, the development and testing of a modular, dynamic vapor generator for the qualitative and quantitative analysis of volatile SCFAs in the gaseous phase using a secondary electrospray ionization (SESI) source coupled to a high-resolution mass spectrometer. Representative compounds tested included acetic acid, propionic acid, butyric acid, pentanoic acid and hexanoic acid. Gas-phase experiments were performed both in dry and humid (95% relative humidity) conditions from ppt to low ppb concentrations. The results obtained exhibited excellent linearity within the examined concentration range, low limits of detection and quantification down to the lower ppt area. Mixture effects were also investigated and are presented.
  16. J Toxicol Sci. 2023 ;48(1): 15-24
      We developed a derivatization technique that involves microwave heating to reduce the overall forensic analysis time of phosphorus-containing amino acid herbicides (PAAHs). Combined with an extraction method that uses titanium (IV) oxide (TiO2), we were able to obtain a practical analytical method for PAAHs and their metabolites in samples intended for poisoning cases. The optimized derivatization conditions were 700 W power and 5-min irradiation time, which is a significant time-saving. The plasma samples extracted using TiO2-packed Tip columns and derivatized under the optimized conditions had an intra-day accuracy and precision within 9.3% and 9.0%, respectively. The intermediate accuracy and precision were within 8.8% and 8.5%, respectively, and the recoveries were more than 91.2%. Similarly, for urine samples, the intra-day accuracy and precision were within 13.3% and 9.1%, respectively. The intermediate accuracy and precision were within 13.6% and 10.3%, respectively, and finally, the recoveries were more than 88.2%. In addition to reducing the pretreatment time, this method was suitable for reducing the overall labor burden on laboratories responsible for routine analysis because of its stable validation data.
    Keywords:  Extraction; Microwave heating; N-acetyl-O-methyl derivatization; Phosphorus-containing amino acid herbicides; Titanium (IV) oxide
  17. Anal Chem. 2023 Jan 04.
      Region of interest (ROI) extraction is a fundamental step in analyzing metabolomic datasets acquired by liquid chromatography-mass spectrometry (LC-MS). However, noises and backgrounds in LC-MS data often affect the quality of extracted ROIs. Therefore, developing effective ROI evaluation algorithms is necessary to eliminate false positives meanwhile keep the false-negative rate as low as possible. In this study, a deep fused filter of ROIs (dffROI) was proposed to improve the accuracy of ROI extraction by combining the handcrafted evaluation metrics with convolutional neural network (CNN)-learned representations. To evaluate the performance of dffROI, dffROI was compared with peakonly (CNN-learned representation) and five handcrafted metrics on three LC-MS datasets and a gas chromatography-mass spectrometry (GC-MS) dataset. Results show that dffROI can achieve higher accuracy, better true-positive rate, and lower false-positive rate. Its accuracy, true-positive rate, and false-positive rate are 0.9841, 0.9869, and 0.0186 on the test set, respectively. The classification error rate of dffROI (1.59%) is significantly reduced compared with peakonly (2.73%). The model-agnostic feature importance demonstrates the necessity of fusing handcrafted evaluation metrics with the convolutional neural network representations. dffROI is an automatic, robust, and universal method for ROI filtering by virtue of information fusion and end-to-end learning. It is implemented in Python programming language and open-sourced at under BSD License. Furthermore, it has been integrated into the KPIC2 framework previously proposed by our group to facilitate real metabolomic LC-MS dataset analysis.
  18. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Dec 28. pii: S1570-0232(22)00493-7. [Epub ahead of print]1215 123588
      Conventional analysis of microbial bioproducers requires the extraction of metabolites from liquid cultures, where the culturing steps are time consuming and greatly limit throughput. To break through this barrier, the current study aims to directly evaluate microbial bioproduction colonies by way of supercritical fluid extraction-supercritical fluid chromatography-triple quadrupole mass spectrometry (SFE-SFC-MS/MS). The online SFE-SFC-MS/MS system offers great potential for high-throughput analysis due to automated metabolite extraction without any need for pretreatment. This is the first report of SFE-SFC-MS/MS as a method for direct colony screening, as demonstrated in the high-throughput screening of (-)-limonene bioproducers. Compared with conventional analysis, the SFE-SFC-MS/MS system enables faster and more convenient screening of highly productive strains.
    Keywords:  High throughput screening; Limonene; Supercritical fluid chromatography; Supercritical fluid extraction; Triple quadrupole mass spectrometry
  19. Front Cell Dev Biol. 2022 ;10 1075810
      We present the use of conductive spray polymer ionization mass spectrometry (CPSI-MS) combined with machine learning (ML) to rapidly gain the metabolic fingerprint from 1 μl liquid extraction from the biopsied tissue of triple-negative breast cancer (TNBC) in China. The 76 discriminative metabolite markers are verified at the primary carcinoma site and can also be successfully tracked in the serum. The Lasso classifier featured with 15- and 22-metabolites detected by CPSI-MS achieve a sensitivity of 88.8% for rapid serum screening and a specificity of 91.1% for tissue diagnosis, respectively. Finally, the expression levels of their corresponding upstream enzymes and transporters have been initially confirmed. In general, CPSI-MS/ML serves as a cost-effective tool for the rapid screening, diagnosis, and precise characterization for the TNBC metabolism reprogramming in the clinical practice.
    Keywords:  ambient ionization mass spectrometry; biomarkers; clinical screening and diagnosis; metabolomics; triple-negative breast cancer
  20. Methods Mol Biol. 2023 ;2613 289-299
      Glycosphingolipids (GSLs) are glycolipids with ceramide and carbohydrate head groups that play an important role in numerous biological processes. Previously, we performed GSL-glycan analysis of various cell lines and virus-infected cells using a glycoblotting approach. Recently, we developed several methods for sialic acid linkage-specific chemical modification to distinguish sialylated glycan isomers by mass spectrometry. In this chapter, we describe a method for analyzing GSL-glycans in human serum/plasma using glycoblotting combined with aminolysis-SALSA (sialic acid linkage-specific alkylamidation) and lactone-driven ester-to-amide derivatization (LEAD)-SALSA for comprehensive and detailed structural glycomics.
    Keywords:  Glycoblotting; Glycosphingolipid-glycan; Human plasma; Human serum; Lactone-driven ester-to-amide derivatization; Mass spectrometry; Sialic acid linkage-specific alkylamidation
  21. J Pharm Biomed Anal. 2022 Dec 30. pii: S0731-7085(22)00642-2. [Epub ahead of print]225 115221
      Furanocoumarins and flavonoids have various important biological activities and wide application. In the present study, a rapid and reliable supercritical fluid chromatography method was proposed for the separation of 10 target components including 8 furanocoumarins and 2 flavonoids. After detailed condition optimization, the 10 target compounds can be baseline separated on a Trefoil CEL1 (3.0 mm × 150 mm, 2.5 µm) column using gradient elution. A 0.07% (v/v) trifluoroacetic acid in ethanol was determined to be the most proper mobile phase for the separation of target compounds. The column temperature, back pressure, flow rate were set at 36 ℃, 2000 psi, 1.0 mL min-1 to 1.4 mL min-1, respectively. The ten target compounds were analyzed within 24 min using the optimized conditions. Under the optimized conditions, all the target compounds showed good linearity with linear correlation coefficients higher than 0.995, and satisfactory recovery in the range of 83.52-112.92%. All these results showed that the developed ultra-high performance supercritical fluid chromatography method was reliable and effective. Finally, the application of the developed method to cosmetic, Psoraleae fructus and Angelicae dahuricae radix samples were presented. The results highlight the applicability of the ultra-high performance supercritical fluid chromatography method to the analysis of interested compounds in pharmaceutical and cosmetic samples.
    Keywords:  Cosmetic; Flavonoids; Furanocoumarins; Pharmaceutical samples; Supercritical fluid chromatography
  22. Anal Methods. 2023 Jan 05.
      In this work, desorption electrospray ionization and paper spray ionization both with high-resolution mass spectrometry (DESI-HRMS and PSI-HRMS) were explored for the fast and direct analysis of stimulants and diuretics in urine samples. The analysis was performed at a resolution of 70 000 FWHM (m/z 200) using a quadrupole-Orbitrap mass spectrometer in full scan acquisition mode, detecting stimulants and diuretics in positive and negative ion modes, respectively. The most critical parameters affecting the desorption and ionization efficiencies of compounds were optimized, paying particular attention to the optimization of the spray solvent for PSI-HRMS analysis and to the selection of the DESI sample substrate. For stimulants, the PSI-HRMS method performed better than DESI-HRMS, allowing the direct analysis of raw urine samples with better signal-to-noise ratios than DESI. However, results obtained for diuretics were not as satisfactory as we expected. The PSI-HRMS method was applied to the screening of 52 stimulants for doping control purposes, providing satisfactory detectability for most of them at the Minimum Reporting Level (MRL) in less than 2 minutes for each single analysis. Despite the advantages offered by the PSI-HRMS method, in this study is also included a discussion on the limitations observed because of the presence of interference for some compounds.
  23. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Dec 24. pii: S1570-0232(22)00470-6. [Epub ahead of print]1215 123565
      Paralytic shellfish toxins (PSTs) and tetrodotoxins (TTXs) are powerful neurotoxins. Previous research reported that PSTs and TTXs are found together in seafoods and may pose a serious hazard to public health. In this study, a new analytical method combining modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) with high-performance liquid chromatography coupled to Q-Exactive Orbitrap high-resolution mass spectrometry was developed and validated for the quantification of 10 PSTs and 2 TTXs in human serum. Chromatographic separation was achieved using the HILIC TSK-Gel Amide-80 column. The mass spectrometer was operated in full scan/dd-MS2(data-dependent MS2) mode, and for quantification analysis. The dd-MS2 resolution was set to 17,500 fullwidthat halfmaximum (FWHM). Results showed that methanol with 1 % (v/v) acetic acid extraction combined with 50 mg graphitized carbon black (GCB) and 50 mg octadecyl bonded silica gel (C18) was most suitable for purification. The mean recovery for all toxins ranged from 85.3 % to 118.2 % (RSD < 12 %). The limits of detection and quantification for human serum were in the ranges of 0.67-2.61 and 2.23-8.69 ng mL-1, respectively. The method was applied to analyze toxins in serum samples obtained from three poisoned patients in a case of poisoning caused by consumption of toxin-contaminated gastropoda (Bullacta exerata). The study has important application for rapid and accurate diagnosis of PSTs and TTXs toxin poisoning patients in clinic.
    Keywords:  Human serum; Orbitrap HR-MS; Paralytic shellfish toxins(PSTs); QuEChERS; Tetrodotoxins(TTXs)
  24. Anal Chem. 2023 Jan 03.
      In-source fragmentation (ISF) is a naturally occurring phenomenon in various ion sources including soft ionization techniques such as matrix-assisted laser desorption/ionization (MALDI). It has traditionally been minimized as it makes the dataset more complex and often leads to mis-annotation of metabolites. Here, we introduce an approach termed PICA (for pixel intensity correlation analysis) that takes advantage of ISF in MALDI imaging to increase confidence in metabolite identification. In PICA, the extraction and association of in-source fragments to their precursor ion results in "pseudo-MS/MS spectra" that can be used for identification. We examined PICA using three different datasets, two of which were published previously and included validated metabolites annotation. We show that highly colocalized ions possessing Pearson correlation coefficient (PCC) ≥ 0.9 for a given precursor ion are mainly its in-source fragments, natural isotopes, adduct ions, or multimers. These ions provide rich information for their precursor ion identification. In addition, our results show that moderately colocalized ions (PCC < 0.9) may be structurally related to the precursor ion, which allows for the identification of unknown metabolites through known ones. Finally, we propose three strategies to reduce the total computation time for PICA in MALDI imaging. To conclude, PICA provides an efficient approach to extract and group ions stemming from the same metabolites in MALDI imaging and thus allows for high-confidence metabolite identification.
  25. Anal Chem. 2023 Jan 02.
      Multi-omics analysis is a powerful and increasingly utilized approach to gain insight into complex biological systems. One major hindrance with multi-omics, however, is the lengthy and wasteful sample preparation process. Preparing samples for mass spectrometry (MS)-based multi-omics involves extraction of metabolites and lipids with organic solvents, precipitation of proteins, and overnight digestion of proteins. These existing workflows are disparate and laborious. Here, we present a simple, efficient, and unified approach to prepare lipids, metabolites, and proteins for MS analysis. Our approach, termed the Bead-enabled Accelerated Monophasic Multi-omics (BAMM) method, combines an n-butanol-based monophasic extraction with unmodified magnetic beads and accelerated protein digestion. We demonstrate that the BAMM method affords comparable depth, quantitative reproducibility, and recovery of biomolecules as state-of-the-art multi-omics methods (e.g., Matyash extraction and overnight protein digestion). However, the BAMM method only requires about 3 h to perform, which saves 11 steps and 19 h on average compared to published multi-omics methods. Furthermore, we validate the BAMM method for multiple sample types and formats (biofluid, culture plate, and pellet) and show that in all cases, it produces high biomolecular coverage and data quality.
  26. J Am Soc Mass Spectrom. 2023 Jan 03.
      In the present contribution, a novel approach based on multivariate curve resolution and deep learning (DL) is proposed for quantitative mass spectrometry imaging (MSI) as a potent technique for identifying different compounds and creating their distribution maps in biological tissues without need for sample preparation. As a case study, chlordecone as a carcinogenic pesticide was quantitatively determined in mouse liver using matrix-assisted laser desorption ionization-MSI (MALDI-MSI). For this purpose, data from seven standard spots containing 0 to 20 picomoles of chlordecone and four unknown tissues from the mouse livers infected with chlordecone for 1, 5, and 10 days were analyzed using a convolutional neural network (CNN). To solve the lack of sufficient data for CNN model training, each pixel was considered as a sample, the designed CNN models were trained by pixels in training sets, and their corresponding amounts of chlordecone were obtained by multivariate curve resolution-alternating least-squares (MCR-ALS). The trained models were then externally evaluated using calibration pixels in test sets for 1, 5, and 10 days of exposure, respectively. Prediction R2 for all three data sets ranged from 0.93 to 0.96, which was superior to support vector machine (SVM) and partial least-squares (PLS). The trained CNN models were finally used to predict the amount of chlordecone in mouse liver tissues, and their results were compared with MALDI-MSI and GC-MS methods, which were comparable. Inspection of the results confirmed the validity of the proposed method.
    Keywords:  convolutional neural network; deep learning; mass spectrometry imaging; multivariate curve resolution
  27. J Pharm Anal. 2022 Dec;12(6): 815-823
      In recent years, scientific researchers have increasingly become interested in noninvasive sampling methods for therapeutic drug monitoring and disease diagnosis. As a result, dried saliva spot (DSS), which is a sampling technique for collecting dried saliva samples, has been widely used as an alternative matrix to serum for the detection of target molecules. Coupling the DSS method with a highly sensitive detection instrument improves the efficiency of the preparation and analysis of biological samples. Furthermore, dried blood spots, dried plasma spots, and dried matrix spots, which are similar to those of the DSS method, are discussed. Compared with alternative biological fluids used in dried spot methods, including serum, tears, urine, and plasma, saliva has the advantage of convenience in terms of sample collection from children or persons with disabilities. This review aims to provide integral strategies and guidelines for dried spot methods to analyze biological samples by illustrating several dried spot methods. Herein, we summarize recent advancements in DSS methods from June 2014 to March 2021 and discuss the advantages and disadvantages of the key aspects of this method, including sample preparation and method validation. Finally, we outline the challenges and prospects of such methods in practical applications.
    Keywords:  Disease diagnosis; Dried saliva spot; Human saliva; Therapeutic drug monitoring