bims-metlip 2024-09-01 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024‒09‒01
twenty papers selected by
Sofia Costa, Matterworks

Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Applications for Metabolomics.
[Determination of ten bisphenols and five parabens in urine by solid supported liquid-liquid extraction and liquid chromatography-tandem mass spectrometry].
Identification of Plant Compounds with Mass Spectrometry Imaging (MSI).
[Rapid determination of chlorate and perchlorate in tea by ion exchange chromatography-tandem mass spectrometry].
[Determination of eight neonicotinoid pesticides in wastewater by solid phase extraction combined with liquid chromatography-tandem mass spectrometry].
[Simultaneous determination of 61 hormones in water by solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry].
High-coverage identification of hydroxyl compounds based on pyridine derivatization-assisted liquid chromatography mass spectrometry.
Machine learning methods for compound annotation in non-targeted mass spectrometry-A brief overview of fingerprinting, in silico fragmentation and de novo methods.
Simultaneous detection of 10 cancer-associated amino acids and polyamines by high-performance liquid chromatography-high resolution mass spectrometry in pleural effusion cells obtained from lung adenocarcinoma patients.
Dried plasma spot as an innovative approach to therapeutic drug monitoring of apixaban: Development and validation of a novel liquid chromatography-tandem mass spectrometry method.
A novel ultra-high-performance supercritical fluid chromatography hyphenated to tandem mass spectrometry method for the analysis of urinary endogenous steroids in the anti-doping context.
Development and clinical implementation of an LC-HRMS method for ivacaftor, lumacaftor, tezacaftor and elexacaftor in human plasma and breast milk.
An Ensemble Spectral Prediction (ESP) model for metabolite annotation.
Identification of 3-hydroxyaspartate with two chiral centers by capillary electrophoresis-mass spectrometry and vibrational circular dichroism independent of single enantiomer standard.
Gas Chromatography-Mass Spectrometry-Based Analyses of Fecal Short-Chain Fatty Acids (SCFAs): A Summary Review and Own Experience.
Studying drug excretion into exhaled breath aerosol - A workflow based on an impaction sampling device and LC-HRMS/MS analysis.
Enhancement of weak signals by applying a suppression method to high-intense methyl and methylene signals of lipids in NMR spectroscopy.
Determination of Taurine in Infant Formula and Adult/Pediatric Nutritionals by Ultra High Performance Liquid Chromatography with Multiple Reaction Monitoring Mass Spectrometry: Single Laboratory Validation, First Action 2022.08.
Determination of three ephedrine psychoactive substances in sewage using solid-phase extraction-ultra-performance liquid chromatography-tandem mass spectrometry.
Collision cross-section measurements of small molecules via transient decay profiles observed in Orbitrap mass analyzers.

Biomedicines. 2024 Aug 06. pii: 1786. [Epub ahead of print]12(8):

Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Applications for Metabolomics.

Darcy Cochran, Robert Powers.

  Metabolomics is an interdisciplinary field that aims to study all metabolites < 1500 Da that are ubiquitously found within all organisms. Metabolomics is experiencing exponential growth and commonly relies on high-resolution mass spectrometry (HRMS). Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is a form of HRMS that is particularly well suited for metabolomics research due to its exceptionally high resolution (105-106) and sensitivity with a mass accuracy in parts per billion (ppb). In this regard, FT-ICR-MS can provide valuable insights into the metabolomics analysis of complex biological systems due to unique capabilities such as the easy separation of isobaric and isomeric species, isotopic fine structure analysis, spatial resolution of metabolites in cells and tissues, and a high confidence (<1 ppm mass error) in metabolite identification. Alternatively, the large and complex data sets, long acquisition times, high cost, and limited access mainly through national mass spectrometry facilities may impede the routine adoption of FT-ICR-MS by metabolomics researchers. This review examines recent applications of FT-ICR-MS metabolomics in the search for clinical and non-human biomarkers; for the analysis of food, beverage, and environmental samples; and for the high-resolution imaging of tissues and other biological samples. We provide recent examples of metabolomics studies that highlight the advantages of FT-ICR-MS for the detailed and reliable characterization of the metabolome. Additionally, we offer some practical considerations for implementing FT-ICR-MS into a research program by providing a list of FT-ICR-MS facilities and by identifying different high-throughput interfaces, varieties of sample types, analysis methods (e.g., van Krevelen diagrams, Kendrick mass defect plot, etc.), and sample preparation and handling protocols used in FT-ICR-MS experiments. Overall, FT-ICR-MS holds great promise as a vital research tool for advancing metabolomics investigations.

Keywords:  FT-ICR-MS; imaging; mass spectrometry; metabolomics

DOI:  https://doi.org/10.3390/biomedicines12081786
Se Pu. 2024 Sep;42(9): 827-836

[Determination of ten bisphenols and five parabens in urine by solid supported liquid-liquid extraction and liquid chromatography-tandem mass spectrometry].

Yu-Fan Xue, Ting Shang, Jun-Tao Cui, Ling-Juan Zhao, Pei Li, Xiang-Ying Zeng, Zhi-Qiang Yu.

  Bisphenols (BPs) and parabens (PBs) are of great concern for environmental pollution and human health because of their endocrine-disrupting effects and potential health hazards. Urinary biomonitoring of BPs and PBs can provide basic data for human internal exposure evaluation, which is a prerequisite for accurately assessing their health risks. In this study, we developed a new pretreatment procedure based on solid supported liquid-liquid extraction (SLE) for the simultaneous separation of ten BPs and five PBs in human urine, followed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. In the instrumental analysis, the HPLC conditions and MS/MS parameters were comprehensively optimized. Accurate qualitative and quantitative determination of ten BPs and five PBs was achieved by introducing a ternary gradient elution system of water, methanol, and acetonitrile for LC separation. During sample pretreatment, the extraction solvent and elution volume were optimized. Specifically, urine samples were held at room temperature and centrifuged at 3000 r/min for 10 min. The supernatant (2 mL) was then transferred to a glass tube, and the pH was adjusted to 5.0 using HCl (0.5 mL; 0.1 mol/L) and NaAc-HAc buffer (1.5 mL). Thereafter, β-glucuronidase-arylsulfatase (20 μL) and surrogate standard solutions (10 ng;13C12-BPS,13C12-BPAF,13C6-MeP, and 13C6-BuP) were added, and the mixture was incubated in a shaker bath in the dark at 37 ℃ for 16 h. After incubation, the hydrolyzed sample (4 mL) was loaded onto an SLE cartridge and equilibrated for a minimum of 5 min to ensure the solution was completely absorbed by the packing material. Subsequently, the target chemicals were eluted with a mixed ethyl acetate/n-hexane solution (3∶7, v/v; 15 mL). Separation of the targets was performed on a ZORBAX SB-C18 reversed-phase column (250 mm×4.6 mm, 5 μm) using an acetonitrile-methanol-water system as the mobile phase. The method was verified by spiking mixed urine samples at three levels (1, 5, and 50 μg/L), with the recoveries ranging from 84.3% to 119.8%. Except for bisphenols (BPS), whose matrix effect was calculated as -21.8%, the matrix effects of other analytes were lower than 20%, indicating low matrix interference. The linear ranges of the analytes varied from 0.1-500 μg/L to 1-500 μg/L, with correlation coefficients higher than 0.995. The method limits of quantification for target chemicals ranged from 0.03 to 0.30 μg/L, and the relative standard deviations of intra- and inter-day experiments were 1.4%-8.4% and 5.7%-14.6%, respectively, suggesting high stability and reproducibility. The method was successfully applied to the determination of ten BPs and five PBs in 10 urine samples from a general population. The concentrations of target chemicals in the human urine samples varied. Methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), and bisphenol A (BPA) were detected in all samples, with median mass concentrations of 1.10, 0.60, 0.21, and 0.55 μg/L, respectively. The detection rates of the other chemicals were less than 50%, which may be related to the production and use of specific chemicals, their bioavailability, and biological metabolism in humans.

Keywords:  bisphenols (BPs); liquid chromatography-tandem mass spectrometry (LC-MS/MS); parabens (PBs); solid supported liquid-liquid extraction (SLE); urine

DOI:  https://doi.org/10.3724/SP.J.1123.2024.01001
Metabolites. 2024 Jul 30. pii: 419. [Epub ahead of print]14(8):

Identification of Plant Compounds with Mass Spectrometry Imaging (MSI).

Nancy Shyrley García-Rojas, Carlos Daniel Sierra-Álvarez, Hilda E Ramos-Aboites, Abigail Moreno-Pedraza, Robert Winkler.

  The presence and localization of plant metabolites are indicative of physiological processes, e.g., under biotic and abiotic stress conditions. Further, the chemical composition of plant parts is related to their quality as food or for medicinal applications. Mass spectrometry imaging (MSI) has become a popular analytical technique for exploring and visualizing the spatial distribution of plant molecules within a tissue. This review provides a summary of mass spectrometry methods used for mapping and identifying metabolites in plant tissues. We present the benefits and the disadvantages of both vacuum and ambient ionization methods, considering direct and indirect approaches. Finally, we discuss the current limitations in annotating and identifying molecules and perspectives for future investigations.

Keywords:  compound identification; mass spectrometry imaging; plant metabolomics

DOI:  https://doi.org/10.3390/metabo14080419
Se Pu. 2024 Sep;42(9): 875-880

[Rapid determination of chlorate and perchlorate in tea by ion exchange chromatography-tandem mass spectrometry].

Ming-Li Ye, Guo-Hua Zhao, Yong Wang, Ji-Run Lin, Wen-Xin Liu, Jie Lu, Yong-Gang Zhao, Ban Cao.

  Ion exchange chromatography-tandem mass spectrometry (IEC-MS/MS) has recently become the preferred method for detecting ionic substances in tea. In this study, an IEC-MS/MS method was developed for the rapid determination of chlorate and perchlorate residues in tea samples. The optimal sample extraction process, pretreatment column, and chromatographic and mass spectrometric conditions were systematically investigated. In the optimal process, the tea samples were ultrasonically extracted with methanol-water (13∶7, v/v), and a PRiME HLB SPE column was used to purify the sample extract. An AceChrom Hybri-A IEC column (150 mm×2.1 mm, 5.0 μm) was used for separation, and 100 mmol/L ammonium acetate-acetonitrile (40∶60, v/v) was used as the mobile phase for isocratic elution. The flow rate was 0.3 mL/min, the column temperature was 40 ℃, and the injection volume was 5.0 μL. The mass spectrometric data were collected in negative electrospray ionization mode combined with multiple reaction monitoring (MRM) mode to achieve the rapid and accurate separation and qualitative analysis of the desired chemical components. Quantification was performed using the internal standard (IS) method. The measurement results showed a good linear relationship when the mass concentrations of chlorate and perchlorate were between 2.00-200 and 1.00-100 μg/L, respectively, with correlation coefficients (r2) greater than 0.9990. The average recoveries of chlorate and perchlorate at three spiked levels of low, medium, and high ranged from 88.54% to 97.25% with relative standard deviations (RSDs, n=7) of 3.2%-5.2%. The limits of detection for chlorate and perchlorate were 12.0 and 8.0 μg/kg, respectively, while the limits of quantification were 40.0 and 26.6 μg/kg, respectively. The results of tests conducted to assess the linearity, specificity, accuracy, precision, and applicability of the method to the analysis of chlorate and perchlorate in 15 tea samples collected from a local market demonstrated its validity for the routine analysis of tea samples. The proposed method is simple, rapid, sensitive, and accurate, and can meet requirements for the rapid screening and quantitative analysis of residual trace chlorate and perchlorate in large quantities of tea samples.

Keywords:  chlorate; ion exchange chromatography-tandem mass spectrometry (IEC-MS/MS); perchlorate; tea

DOI:  https://doi.org/10.3724/SP.J.1123.2023.10026
Se Pu. 2024 Sep;42(9): 856-865

[Determination of eight neonicotinoid pesticides in wastewater by solid phase extraction combined with liquid chromatography-tandem mass spectrometry].

Hai-Tang Wang, Han-Yin Li, Qi-Wei Lu, Shi-Long He.

  Neonicotinoid pesticides are a relatively new class of pesticides that have garnered significant attention owing to their potential ecological risks to nontarget organisms. A method combining solid phase extraction with liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) was developed for the rapid and accurate detection of eight neonicotinoid pesticides (dinotefuran, E-nitenpyram, thiamethoxam, clothianidin, imidacloprid, imidaclothiz, acetamiprid, and thiacloprid) in wastewater. The chromatographic mobile phase and MS parameters were selected, and a single-factor method was used to determine the optimal column type, extraction volume, sample loading speed, and pH for SPE. The optimal parameters were as follows: column type, HLB column (500 mg/6 mL); sample extraction volume, 500 mL; sample loading speed, 10 mL/min; and sample pH, 6-8. The matrix effects of the wastewater samples were reduced by optimizing the chromatographic gradient-elution program, examining the dilution factor of the samples, and using the isotope internal standard calibration method. Prior to analysis, the wastewater samples were diluted 5-fold with ultrapure water for pretreatment. Subsequently, 2 mmol/L ammonium acetate aqueous solution containing 0.1% (v/v) formic acid and methanol was used as mobile phases for gradient elution on a ZORBAX Eclipse Plus C18 column (100 mm×2.1 mm, 1.8 μm). The samples were quantified using positive-ion multiple reaction monitoring (MRM) mode for 10 min. Imidacloprid-d4 was used as the isotope internal standard. The SPE process was further optimized by applying response surface methodology to select the type and mass of rinsing and elution solvents. The optimal pretreatment of the SPE column included rinsing with 10% methanol aqueous solution and elution with methanol-acetonitrile (1∶1, v/v) mixture (7 mL). The eight neonicotinoid pesticides showed satisfactory linearity within the relevant range, with linear correlation coefficients (r) all greater than 0.9990. The method detection limits (MDLs) ranged from 0.2 to 1.2 ng/L, and the method quantification limits (MQLs) ranged from 0.8 to 4.8 ng/L. The average recoveries of the eight neonicotinoid pesticides were in the range of 82.6%-94.2% at three spiked levels, with relative standard deviations (RSDs) ranging from 3.9% to 9.4%. Finally, the optimized method was successfully applied to analyze wastewater samples collected from four sewage treatment plants. The results indicated that the eight neonicotinoid pesticides could be generally detected at concentrations ranging from not detected (ND) to 256 ng/L. The developed method has a low MDL and high accuracy, rendering it a suitable choice for the trace detection of the eight neonicotinoid pesticides in wastewater when compared with other similar methods. The proposed method can be utilized to monitor the environmental impact and assess the potential risks of neonicotinoid pesticides in wastewater, thus promoting the protection of nontarget organisms and the sustainable use of these pesticides in agriculture.

Keywords:  liquid chromatography-tandem mass spectrometry (LC-MS/MS); matrix effect; neonicotinoid pesticides; response surface methodology (RSM); solid phase extraction (SPE); wastewater

DOI:  https://doi.org/10.3724/SP.J.1123.2023.11010
Se Pu. 2024 Sep;42(9): 866-874

[Simultaneous determination of 61 hormones in water by solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry].

Yue-Qin Chen, Ming Ma, Hong-Dan Xu, Chun-Yan Pan.

  Concerns over the emergence of steroid hormones as pollutants in water have grown. Steroid hormone compounds present challenges in the simultaneous detection of total residual hormones owing to their analogous structures and diverse types. In this study, we established a rapid and high-throughput continuous online method based on solid phase extraction (SPE) coupled with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the simultaneous determination of 61 hormone components, including 48 glucocorticoids, 1 mineralocorticoid, 4 androgens, and 8 progesterones, in water. Various SPE columns were investigated to assess their extraction efficiency for enriching and purifying target compounds in a large sample volume (1 L). An HC-C18 SPE column was selected because of its superior performance. Acetonitrile was used as a washing solution during SPE to ensure that the majority of the tested substances achieved recoveries exceeding 70% and effectively avoid interferences from water-soluble components. Various C8 and C18 columns were tested, and the optimal HPLC conditions for hormone retention were established. We systematically evaluated different UPLC columns and mobile phases, including methanol-water and acetonitrile-water systems with 0.1% formic acid added to the aqueous phase. The optimized UPLC conditions were as follows: BEH C18 column (100 mm×2.1 mm, 1.7 μm); column temperature, 40 ℃; flow rate, 0.3 mL/min; injection volume, 5 μL; mobile phase A: 0.1% formic acid aqueous phase; mobile phase B: acetonitrile. Gradient elution was performed as follows: 0-0.5 min, 30%B; 0.5-15.0 min, 30%B-75%B; 15.0-18.0 min, 75%B-98%B; 18.0-19.0 min, 98%B; 19.0-19.1 min, 98%B-30%B; 19.1-20.0 min, 30%B. Both positive- and negative-ion modes were explored in the UPLC-MS/MS experiment to obtain the full scan of the parent ions, and positive mode was finally selected for electrospray ionization (ESI). Two product ions exhibiting strong signals and minimal interference were selected for quantitative and qualitative ion analyses, using an external standard method for quantification. MS/MS was performed in positive-ion (ESI+) mode with multiple reaction monitoring (MRM) scanning. The MS/MS parameters were as follows: atomizing gas pressure, 379 kPa; curtain air pressure, 241 kPa; spray voltage, 5500 V; desolvation temperature, 550 ℃; collision exit voltage (CXP), 13 V; intake voltage (EP), 10 V; and residence time of each ion pair, 0.5 ms. Other instrument settings, such as the collision energy and declustering voltage, were also optimized. The 61 hormones exhibited excellent linear relationships within their corresponding concentration ranges, with correlation coefficients greater than 0.99. The method detection limits (MDLs) were in the range of 0.05-1.50 ng/L. The average recoveries of the 61 hormones across three spiked levels ranged from 62.3% to 125.2%, with relative standard deviations (RSDs, n=6) of 1.1%-10.5%. Using this method, we successfully detected 10 hormone components (cortisone, fluticasone propionate, ciclesonide, betamethasone dipropionate, clobetasone butyrate, diflucortolone valerate, halobetasol propionate, isoflupredone, difluprednate, and hydroxyprogesterone caproate) in various surface water and groundwater samples collected from the Taihu Basin region. The SPE-UPLC-MS/MS method presented in this paper is simple, highly sensitivity, and exceptionally accurate. Thus, it exhibits promising potential for tracing targeted hormone residues in water and will be of great value in monitoring and ensuring water safety. Finally, a regional analysis was conducted on the hormone levels in water, and suggestions were made for the targeted treatment of hormone residues in future sewage treatment processes.

Keywords:  hormone; solid phase extraction (SPE); ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS); water

DOI:  https://doi.org/10.3724/SP.J.1123.2023.11014
Anal Chim Acta. 2024 Sep 15. pii: S0003-2670(24)00866-3. [Epub ahead of print]1322 343065

High-coverage identification of hydroxyl compounds based on pyridine derivatization-assisted liquid chromatography mass spectrometry.

Xiaoyu Xie, Xiuqiong Zhang, Tiantian Chen, Di Yu, Ming Ma, Xin Lu, Guowang Xu.

  Hydroxyl compounds are widely present in plants and play essential roles in plant growth and development. High-coverage detection of hydroxyl compounds is crucial for understanding the physiological processes of plants. Despite the prevalence of chemical derivatization-assisted liquid chromatography-high resolution mass spectrometry (CD-LC-HRMS) in high-coverage detection of compounds with diverse functional groups, the confident identification of these compounds after derivatization remains a significant challenge. Herein, a novel method was developed for the identification of pyridine (PY)-derivatized hydroxyl compounds by comparing the MS/MS similarity of derivatized and corresponding underivatized compounds. Fragmentation rules of standards were summarized, and theoretical calculations have demonstrated the MS/MS similarity of PY-derivatized hydroxyl compounds with their underivatized counterparts. The effectiveness of the developed method was demonstrated by identifying PY-derivatized authentic standards. A total of 90 hydroxyl compounds were putatively identified in maize using the proposed method. This method can significantly enhance ionization efficiency with minimal impact on the quality of the MS/MS spectra, enabling the effective utilization of mass spectra databases for the identification of hydroxyl compounds.

Keywords:  Chemical derivatization; Hydroxyl compounds; Identification; Liquid chromatography; Mass spectrometry

DOI:  https://doi.org/10.1016/j.aca.2024.343065
Rapid Commun Mass Spectrom. 2024 Oct 30. 38(20): e9876

Machine learning methods for compound annotation in non-targeted mass spectrometry-A brief overview of fingerprinting, in silico fragmentation and de novo methods.

Francesco F Russo, Yannek Nowatzky, Carsten Jaeger, Maria K Parr, Phillipp Benner, Thilo Muth, Jan Lisec.

Non-targeted screenings (NTS) are essential tools in different fields, such as forensics, health and environmental sciences. NTSs often employ mass spectrometry (MS) methods due to their high throughput and sensitivity in comparison to, for example, nuclear magnetic resonance-based methods. As the identification of mass spectral signals, called annotation, is labour intensive, it has been used for developing supporting tools based on machine learning (ML). However, both the diversity of mass spectral signals and the sheer quantity of different ML tools developed for compound annotation present a challenge for researchers in maintaining a comprehensive overview of the field. In this work, we illustrate which ML-based methods are available for compound annotation in non-targeted MS experiments and provide a nuanced comparison of the ML models used in MS data analysis, unravelling their unique features and performance metrics. Through this overview we support researchers to judiciously apply these tools in their daily research. This review also offers a detailed exploration of methods and datasets to show gaps in current methods, and promising target areas, offering a starting point for developers intending to improve existing methodologies.

DOI: https://doi.org/10.1002/rcm.9876
J Pharm Biomed Anal. 2024 Aug 14. pii: S0731-7085(24)00458-8. [Epub ahead of print]251 116418

Simultaneous detection of 10 cancer-associated amino acids and polyamines by high-performance liquid chromatography-high resolution mass spectrometry in pleural effusion cells obtained from lung adenocarcinoma patients.

Tingting Wang, Yina Pan, Xiuhong Hu, Wenbo Ren, Li Li, Jing Huang.

  The deregulation of amino acid and polyamine metabolism is a hallmark of malignancy that regulates cancer cell proliferation, angiogenesis, and invasion. A sensitive mass spectrometry method was developed to simultaneously quantify 10 cancer-associated metabolites in pleural effusion cells for the diagnosis of malignancy and to complement conventional pleural cytology. Analytes were detected by high-performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) using C8-reversed-phase HPLC for separation and sequential window acquisition of all theoretical fragment ion spectra (SWATH) acquisition for obtaining high-resolution quantitative MS/MS chromatograms. This method was validated and applied to pleural effusion cells from patients with lung adenocarcinoma (LUAD, n = 48) and those from benign controls (n = 23). The range of the above metabolites was 2-200 ng/mL for proline, aspartate, ornithine, creatine, glutamine, glutamate, arginine, citrulline, and spermine and 10-1000 ng/mL for putrescine. The intra-assay and inter-assay coefficient of variation was below 13.70 % for all analytes. The joint detection of these metabolites in pleural effusion cells achieved a clinical sensitivity of 75.0 % and specificity of 95.7 % differentiating LUAD patients from benign controls. This assay enabled the detection of 10 cancer-associated metabolites in pleural effusion cells, and the increased concentration of these metabolites was correlated with the presence of LUAD.

Keywords:  Cancer-associated metabolites; High-performance liquid chromatography-high resolution mass spectrometry; Lung adenocarcinoma; Pleural effusion

DOI:  https://doi.org/10.1016/j.jpba.2024.116418
J Mass Spectrom. 2024 Sep;59(9): e5081

Dried plasma spot as an innovative approach to therapeutic drug monitoring of apixaban: Development and validation of a novel liquid chromatography-tandem mass spectrometry method.

Alessia Cafaro, Manuela Stella, Giammarco Baiardi, Sebastiano Barco, Federica Pigliasco, Roberto Bandettini, Luca Nanni, Francesca Mattioli, Giuliana Cangemi.

  Apixaban, a direct oral anticoagulant drug (DOAC), typically does not require routine therapeutic drug monitoring (TDM), yet recent guidelines propose its use in specific clinical scenarios. While various antifactor Xa (anti-FXa) chromogenic assays serve as useful proxies for measuring plasma exposure to apixaban in emergencies, they lack specificity compared with chromatographic methods. This research project is intended to the development and validation of a standardized protocol of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conformity with the ICH guidelines M10 for the measurement of apixaban in both plasma and dried plasma spots (DPSs). Samples preparation included protein precipitation after the addition of a deuterated internal standard (IS), and the chromatographic separation was carried out on a Thermo Scientific™ Accucore™ Polar Premium column (50 mm × 2.1 mm, i.d. 2.6 m). The newly developed LC-MS/MS method for apixaban mesurement from both plasma and DPS resulted linear over a wide concentration range (31.25-500 ng/mL), accurate, and reproducible without matrix effects, allowing for specific and rapid quantification. Stability was assessed on quality controls and a real sample, allowing the setting up of a robust TDM protocol that was applied to five anonymized plasma samples obtained from adult patients undergoing apixaban treatment at steady-state. In conclusion our novel LC-MS/MS method is adequate for accurate apixaban quantitation from both plasma and DPS matrixes, and may thus facilitate the guidelines suggested implementation of apixaban TDM, even in peripheral hospitals through shipment of DPS at reference laboratories.

Keywords:  DOACs; apixaban; dried plasma spot; drug monitoring; liquid chromatography‐mass spectrometry; microsampling

DOI:  https://doi.org/10.1002/jms.5081
J Chromatogr A. 2024 Aug 08. pii: S0021-9673(24)00598-3. [Epub ahead of print]1734 465224

A novel ultra-high-performance supercritical fluid chromatography hyphenated to tandem mass spectrometry method for the analysis of urinary endogenous steroids in the anti-doping context.

Tobias Langer, Raul Nicoli, Davy Guillarme, Carine Schweizer-Grundisch, Serge Rudaz, Silke Grabherr, Tiia Kuuranne, Alessandro Musenga.

  The first step in the detection of testosterone (T) doping is to measure the urinary steroid profile for the athlete biological passport (ABP). To harmonise the analysis between anti-doping laboratories, urinary steroid profiling is parametrised in deep detail and shall be performed by gas chromatography hyphenated to mass spectrometry (GC-MS). However, due to its requirement for extensive sample preparation, alternatives to GC-MS are being actively pursued. The aim of this study was the evaluation of Ultra-High-Performance Supercritical Fluid Chromatography hyphenated to tandem Mass Spectrometry (UHPSFC-MS/MS) as an alternative for the quantification of endogenous urinary steroids. In this context, we developed a high throughput sample extraction method, followed by a novel UHPSFC-MS/MS method for the analysis of 10 endogenous urinary steroids which are relevant for doping control analysis. Depending on the steroid, the herein presented method is capable of quantification from 0.5 ng/mL up to 10 µg/mL. After validation, the applicability of the method was evaluated by analysing 132 authentic urine samples, which demonstrated results similar to classical GC-MS analysis. Steroid concentrations determined by UHPSFC-MS/MS were slightly overestimated in comparison with GC-MS, but the ratios had <10 % difference between the two methods. As the ABP considers the steroid ratios for passport evaluation, the herein presented method could be used for steroid profiling without reducing the sensitivity of the ABP. Thus, we would propose to consider UHPSFC-MS/MS as an alternative to GC-MS after more tests would have been performed to support our findings. Furthermore, we have also investigated the potential of this technology for sample purification prior to Isotope Ratio Mass Spectrometry (IRMS) for the differentiation between exogenous and endogenous origin of T and its metabolites. While the achieved separation was sufficient to purify urine samples for IRMS analysis in our proof-of-concept study, the instrumental parameters should be further refined for future use.

Keywords:  Anti-doping analysis; Athlete biological passport; IRMS; UHPSFC-MS/MS; Urinary steroid profiling

DOI:  https://doi.org/10.1016/j.chroma.2024.465224
Anal Bioanal Chem. 2024 Aug 28.

Development and clinical implementation of an LC-HRMS method for ivacaftor, lumacaftor, tezacaftor and elexacaftor in human plasma and breast milk.

Anna B Hansson, Hjalmar Wadström, Erik Eliasson, Mahasin Al Shakirchi, Isabelle de Monestrol, Victoria Barclay.

  The four cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators, ivacaftor, lumacaftor, tezacaftor, and elexacaftor, have revolutionised the treatment of CF by direct action on the protein target behind the disease's development. The aim was to develop and validate a quantification method for these CFTR modulators in plasma and breast milk to better understand inter-patient variability in pharmacokinetics and treatment outcome, including the risk of adverse drug reactions. The ability to monitor CFTR modulators in breast milk enables the estimation of the exposure of breastfed infant, with a potential concern for CFTR modulator-induced liver injury. The analysis was performed on a Thermo Vanquish Flex Binary UHPLC system coupled to a high-resolution mass spectrometer (HRMS), Thermo Q Exactive. The analytes were detected using positive electrospray ionisation in full scan mode. After sample preparation by protein precipitation, the supernatant was injected onto the LC system and the analytes were separated using a Zorbax SB-C18 Rapid Res HPLC column (3.5 µm, 4.6 × 75 mm). This is the first published method for CFTR modulators in breast milk. The validated quantification range for ivacaftor is 0.0050-10 µg/mL with a coefficient of variation < 6% and a mean accuracy of 97-106%; for lumacaftor, tezacaftor, and elexacaftor, the validated quantification range is 0.050-100 µg/mL with a coefficient of variation < 8% and a mean accuracy 93-106%. A simple and sensitive quantification method for CFTR modulators has been developed and used for routine analysis of human plasma and breast milk samples since 2022.

Keywords:  Breast milk; CFTR modulators; High-resolution mass spectrometry; Human plasma; Therapeutic drug monitoring

DOI:  https://doi.org/10.1007/s00216-024-05496-2
Bioinformatics. 2024 Aug 02. pii: btae490. [Epub ahead of print]40(8):

An Ensemble Spectral Prediction (ESP) model for metabolite annotation.

Xinmeng Li, Yan Zhou Chen, Apurva Kalia, Hao Zhu, Li-Ping Liu, Soha Hassoun.

MOTIVATION: A key challenge in metabolomics is annotating measured spectra from a biological sample with chemical identities. Currently, only a small fraction of measurements can be assigned identities. Two complementary computational approaches have emerged to address the annotation problem: mapping candidate molecules to spectra, and mapping query spectra to molecular candidates. In essence, the candidate molecule with the spectrum that best explains the query spectrum is recommended as the target molecule. Despite candidate ranking being fundamental in both approaches, limited prior works incorporated rank learning tasks in determining the target molecule.RESULTS: We propose a novel machine learning model, Ensemble Spectral Prediction (ESP), for metabolite annotation. ESP takes advantage of prior neural network-based annotation models that utilize multilayer perceptron (MLP) networks and Graph Neural Networks (GNNs). Based on the ranking results of the MLP- and GNN-based models, ESP learns a weighting for the outputs of MLP and GNN spectral predictors to generate a spectral prediction for a query molecule. Importantly, training data is stratified by molecular formula to provide candidate sets during model training. Further, baseline MLP and GNN models are enhanced by considering peak dependencies through label mixing and multi-tasking on spectral topic distributions. When trained on the NIST 2020 dataset and evaluated on the relevant candidate sets from PubChem, ESP improves average rank by 23.7% and 37.2% over the MLP and GNN baselines, respectively, demonstrating performance gain over state-of-the-art neural network approaches. However, MLP approaches remain strong contenders when considering top five ranks. Importantly, we show that annotation performance is dependent on the training dataset, the number of molecules in the candidate set and candidate similarity to the target molecule.
AVAILABILITY AND IMPLEMENTATION: The ESP code, a trained model, and a Jupyter notebook that guide users on using the ESP tool is available at https://github.com/HassounLab/ESP.

DOI: https://doi.org/10.1093/bioinformatics/btae490
Anal Chim Acta. 2024 Sep 22. pii: S0003-2670(24)00826-2. [Epub ahead of print]1323 343025

Identification of 3-hydroxyaspartate with two chiral centers by capillary electrophoresis-mass spectrometry and vibrational circular dichroism independent of single enantiomer standard.

Sen Yang, Li Wu, Yujian He, Xiangjun Li.

  BACKGROUND: 3-Hydroxyaspartate(3-HA) is a non-proteinaceous amino acid whose four stereoisomers have different biological functions, making it meaningful to separate and analyze them. A combination of capillary zone electrophoresis-mass spectrometry (CZE-MS) and electron circular dichroism enables the separation and analysis of the four stereoisomers of 3-HA, but relies on enantiomeric standards. In contrast, the new method based on vibrational circular dichroism (VCD) can successfully confirm the peak order of the four enantiomers simultaneously without a single enantiomeric standard. This method is suitable for molecules without UV absorption, as well as for molecules with polychiral centers, and does not require enantiomeric standards.RESULTS: We present a new analytical method based on CZE-MS coupled with VCD for the simultaneous determination of four stereoisomers of 3-HA with two chiral centers (D-threo-3-hydroxyaspartate, D-THA; L-threo-3-hydroxyaspartate, L-THA; D-erythro-3-hydroxyaspartate, D-EHA; L-erythro-3-hydroxyaspartate, L-EHA) in the absence of an optically pure single enantiomeric standard. The semipreparations of the non-racemic mixtures of DL-THA and DL-EHA were performed using high performance liquid chromatography (HPLC), with enantiomeric excess values reaching 90 %. By comparing the experimental VCD spectra of the DL-THA/DL-EHA mixture with the theoretical VCD spectra of the single L-THA/L-EHA enantiomer, the configuration of the dominant enantiomer in the nonracemic mixture was determined, where the two characteristic peaks in the 1250-1750 cm-1 spectral range fitted well. Finally, combined with the comparison of CE peak areas, the four stereoisomers were identified successfully.
SIGNIFICANCE: This is the first combined CZE-MS and VCD method for the simultaneous separation and analysis of four stereoisomers of 3-HA without relying on enantiomeric standards. This method is simple and reliable, and provides a new idea for the separation and analysis of chiral compounds with polychiral centers, which are difficult to obtain from enantiomeric standards.

Keywords:  3-Hydroxyaspartate; Capillary zone electrophoresis-mass spectrometry; DFT calculations; Vibrational circular dichroism

DOI:  https://doi.org/10.1016/j.aca.2024.343025
Biomedicines. 2024 Aug 20. pii: 1904. [Epub ahead of print]12(8):

Gas Chromatography-Mass Spectrometry-Based Analyses of Fecal Short-Chain Fatty Acids (SCFAs): A Summary Review and Own Experience.

Paweł Czarnowski, Michał Mikula, Jerzy Ostrowski, Natalia Żeber-Lubecka.

  The gut microbiome, crucial to human health, changes with age and disease, and influences metabolic profiles. Gut bacteria produce short-chain fatty acids (SCFAs), essential for maintaining homeostasis and modulating inflammation. Dysbiosis, commonly due to poor diet or lifestyle, disrupts the integrity of the intestinal barrier and may contribute to conditions such as obesity, diabetes, and non-alcoholic fatty liver disease (NAFLD). Analytical methods such as gas chromatography-mass spectrometry (GC/MS) are vital for SCFA analysis, with various preparation and storage techniques improving the accuracy. Advances in these methods have improved the reliability and sensitivity of SCFA quantification, which is crucial for the identification of disease biomarkers. Evidence from GC/MS-based studies has revealed that accurate SCFA quantification requires meticulous sample preparation and handling. The process begins with the extraction of SCFAs from biological samples using methods such as direct solvent extraction or solid-phase microextraction (SPME), both of which require optimization for maximum recovery. Derivatization, which chemically modifies SCFAs to enhance volatility and detectability, is a crucial step, typically involving esterification or silylation. Following this, the cleanup process removes impurities that might interfere with the analysis. Although recent advances in GC/MS technology have significantly improved SCFA-detection sensitivity and specificity, proper sample storage, with acid preservatives and the avoidance of repeated thawing, is essential for maintaining SCFA integrity.

Keywords:  GC/MS; SCFA; stool metabolites

DOI:  https://doi.org/10.3390/biomedicines12081904
Anal Chim Acta. 2024 Sep 22. pii: S0003-2670(24)00792-X. [Epub ahead of print]1323 342991

Studying drug excretion into exhaled breath aerosol - A workflow based on an impaction sampling device and LC-HRMS/MS analysis.

Juel Maalouli Schaar, Michael Kunz, Lea Wagmann, Olof Beck, Felix Mahfoud, Markus R Meyer.

  BACKGROUND: Exhaled breath (EB) aerosol was in principle shown to be a suitable matrix for bioanalysis of volatile but also non-volatile compounds. This attracted particular interest in the field of drug analysis. However, a big gap still exists in the understanding how and which drugs and/or their metabolites are excreted into exhaled breath and could thus actually be detected. The current study aimed to develop an analytical workflow for the qualitative detection of non-volatile drugs in EB aerosol microparticles.RESULTS: The analyte selection covered different drug classes such as antihypertensives, anticonvulsants or opioid analgesics to investigate and understand the excretion of drugs and their metabolites into EB aerosol. A device for collecting aerosol particles from the lung through impaction was used for the non-invasive sampling procedure. Three expiration cycles per participant and device were collected. The sample preparation consisted of a collector extraction with methanol. Qualitative method development and validation were performed using reversed-phase liquid chromatography (LC) coupled to orbitrap-based high-resolution mass spectrometry (HRMS). Qualitative method validation was done according to published recommendations and international guidelines. Parameters such as selectivity, carry-over, limits of detection and identification, recovery, matrix effects, and long-term stability were evaluated. The limits of detection ranged from 100 pg/collector to 10,000 pg/collector. The procedure was finally used to analyze a total of 31 patient EB samples and demonstrated that e.g., tilidine and its metabolite nortilidine as well as tramadol and its active metabolite O-desmethyltramadol can be detected in EB aerosol.
SIGNIFICANCE AND NOVELTY: The work shows a comprehensive workflow for elucidating drug excretion into exhaled breath aerosol. This bioanalytical strategy and the corresponding novel data from this study are the foundation for further method development and to better understand, which drugs and their metabolites can be addressed by exhaled breath aerosol bioanalysis.

Keywords:  Application testing; Drug excretion; Exhaled breath; LC-HRMS; Method development; Qualitative method validation

DOI:  https://doi.org/10.1016/j.aca.2024.342991
RSC Adv. 2024 Aug 22. 14(37): 26873-26883

Enhancement of weak signals by applying a suppression method to high-intense methyl and methylene signals of lipids in NMR spectroscopy.

Upendra Singh, Abdul-Hamid Emwas, Mariusz Jaremko.

Lipids play crucial roles in human biology, serving as energy stores, cell membranes, hormone production, and signaling molecules. Accordingly, their study under lipidomics has advanced the study of living organisms. 1-Dimensional (D) and 2D NMR methods, particularly 1D 1H and 2D 1H-1H Total Correlation Spectroscopy (TOCSY), are commonly used in lipidomics for quantification and structural identification. However, these NMR methods suffer from low sensitivity, especially in cases of low concentrated molecules such as protons attached to hydroxy, esters, aliphatic, or aromatic unsaturated carbons. Such molecules are common in complex mixtures such as dairy products and plant oils. On the other hand, lipids have highly populated fractions of methyl and methylene groups that result in intense peaks that overwhelm lower peaks and cause inhomogeneities in 2D TOCSY spectra. In this study, we applied a method of suppression to suppress these intense peaks of methyl and methylene groups to detect weaker peaks. The suppression method was investigated on samples of cheese, butter, a mixture of lipids, coconut oil, and olive oil. A significant improvement in peak sensitivity and visibility of cross-peaks was observed, leading to enhanced comparative quantification and structural identification of a greater number of lipids. Additionally, the enhanced sensitivity reduced the time required for the qualitative and comparative quantification of other lipid compounds and components. This, in turn, enables faster and more reliable structural identification and comparative quantification of a greater number of lipids. Additionally, it reduces the time required for the qualitative, and comparative quantification due to the enhancement of sensitivity.

DOI: https://doi.org/10.1039/d4ra03019b
J AOAC Int. 2024 Aug 24. pii: qsae068. [Epub ahead of print]

Determination of Taurine in Infant Formula and Adult/Pediatric Nutritionals by Ultra High Performance Liquid Chromatography with Multiple Reaction Monitoring Mass Spectrometry: Single Laboratory Validation, First Action 2022.08.

Geert A Ten Kate, Bertus Popma, Gerrit van der Pruik, Martin Roeleveld, Martine P van Gool.

BACKGROUND: Taurine (2-aminoethanesulfonic acid) is an essential β-amino acid which is one of the most abundant intracellular amino acid-like components in humans. The level of free taurine in human milk is higher than in bovine milk. Since taurine is considered important for the development of a newborn, fortification of taurine to infant nutrition is common. This publication describes a rapid method for the determination of taurine in infant formula and adult/pediatric nutritionals.OBJECTIVE: This publication demonstrates the suitability and applicability of the method to determine taurine content in infant formula and adult/pediatric nutritionals.
METHODS: Test portions are deproteinized by acid in aqueous environment prior to analysis. The level of taurine is analyzed by Hydrophilic Interaction Chromatography (HILIC) coupled with Multiple Reaction Monitoring Mass Spectrometry (MRM-MS). Quantitation is accomplished using a stable isotope labeled internal standard (SILS) for taurine.
RESULTS: The accuracy of the method was validated using standard reference materials and spike recovery studies. The average recoveries ranged between 92-105%. The repeatability and intermediate precision were in the range of 0.7-4.6% (RSDr) and 1.3-6.7% (RSDiR). The limit of quantification was below the requirement of 0.5 mg/100 g.
CONCLUSIONS: The single laboratory validation (SLV) of the method for determination of taurine content in infant formula and adult/pediatric nutritionals demonstrates that it meets the requirements as per AOAC SMPR 2014.013.
HIGHLIGHTS: A fast method to selectively determine the content of taurine in infant formula and adult/pediatric nutritionals was evaluated and found to be fit for purpose for routine product compliance testing. Based on the results of this SLV, the method was approved by the AOAC Stakeholder Program on Infant Formula and Adult Nutritionals (SPIFAN) Expert Review Panel for SPIFAN Nutrient Methods for Official Methods First Action status.

DOI: https://doi.org/10.1093/jaoacint/qsae068
Rapid Commun Mass Spectrom. 2024 Oct 30. 38(20): e9877

Determination of three ephedrine psychoactive substances in sewage using solid-phase extraction-ultra-performance liquid chromatography-tandem mass spectrometry.

Yiqin Xu, Fang Yang, Hong Ye, Qingqiang Tang, Yanwen Chen, Zhigang Gao, Shuhua Wang, Fang Zhang, Xiaojing Li.

RATIONALE: In recent years, ephedrine psychoactive substances have attracted much attention due to their prevalence in water bodies and potential threat to aquatic ecosystems. Psychoactive substances have been considered as a new type of environmental pollutant due to their unpredictable potential risks to the behavior and nervous system of non-target organisms. A rapid, sensitive, selective, and robust method for the quantification of three ephedrine psychoactive substances in sewage is needed.METHODS: An ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of three ephedrine psychoactive substances in water. The optimal processing conditions were determined by optimizing the chromatography-mass spectrometry and solid-phase extraction (SPE) conditions (e.g., the SPE column, sample pH, washing, and elution), and the treatment conditions were determined; this was achieved via positive ion scanning in multiple reaction monitoring mode. Poly-Sery MCX was selected as the extraction column, with samples loaded at pH 3. And 4-mL solution of 2% formic acid (FA) aqueous solution was used as the eluent; the target compounds were eluted with 5 mL of 5% NH4OH in acetonitrile (ACN) solution. The best results were obtained when the residue was resolubulization in ACN after nitrogen evaporation.
RESULTS: The developed UPLC-MS/MS showed a good linear relationship in the range of 0-50.00 μg/L, with determination coefficients (R2) greater than 0.9990. The detection limit and quantitation limit were 0.05-0.10 and 0.20-0.50 μg/L, respectively. Recovery rates of the target compounds in blank sewage at three different concentrations ranged from 92.37% to 106.31%, with relative standard deviations (RSDs) of 0.77%-4.83% (n = 7).
CONCLUSIONS: This method has been successfully applied to the analysis of surface water and domestic sewage, and the samples were processed stably, indicating that the method is practical for the determination of ephedrine psychoactive drugs in water bodies.

DOI: https://doi.org/10.1002/rcm.9877
Rapid Commun Mass Spectrom. 2024 Oct 30. 38(20): e9887

Collision cross-section measurements of small molecules via transient decay profiles observed in Orbitrap mass analyzers.

Ziqin Ni, Ricardo Arevalo.

Collision cross section (CCS) of organic compounds can be measured via Fourier transform-based mass spectrometry (MS) by modeling the decay rate of transient signals in the analyzer. Deriving CCS values of low-mass molecules (mass < 2000 Da and CCS < 500 Å2) with Orbitrap MS is challenging due to their high axial frequencies and small absolute variances in cross-sectional profiles. Here, we acquired mass spectra of progressively more complex low-mass analytes using commercial Orbitrap mass spectrometers. The transient signals were processed using Fast Fourier transform (FFT) and short-time Fourier transform (StFFT) to derive decay constants of multiple select ionic species from a single MS full-scan experiment. Decay constants were translated into CCS values using at least two internal standards in the same mass spectrum. Our results suggest target ionic species should have high S/N in order to derive CCS values with ≤0.5% uncertainty. Limitations in the precision of CCS measurements reflect local space charge effects that disturb ion motion in the analyzer. The derived CCS values of polymer like fragments of Ultramark 1621 and small molecules such as individual protonated amino acids can achieve average ±1% error with selection of internal standards across a wide mass range. Future studies need to optimize the strategy to select internal standards in order to improve the precision and accuracy of CCS measurements for small molecules via Orbitrap MS.

DOI: https://doi.org/10.1002/rcm.9887