bims-metlip 2024-06-23 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–06–23
29 papers selected by
Sofia Costa, Matterworks

Identification of Stress-Responsive Metabolites in Plants Using an Untargeted Metabolomics Approach.
An Optimized Method for LC-MS-Based Quantification of Endogenous Organic Acids: Metabolic Perturbations in Pancreatic Cancer.
Optimal transport for automatic alignment of untargeted metabolomic data.
Investigation of variability in the matrix effect on stable isotope-labeled internal standards in liquid chromatography-tandem mass spectrometry analysis of 25 pesticides in vegetables.
Automated online solid-phase extraction-tandem mass spectrometry detection for simultaneous analysis of acidic and alkaline catecholamines and their metabolites in human urine.
Development and evaluation of a liquid chromatography-tandem mass spectrometry method for simultaneous measurement of toxic aldehydes from brain tissue.
Analysis of Globotriaosylceramide (Gb3) in Liquid Urine: A Straightforward Assay Using Tandem Mass Spectrometry.
Determination of nine bisphenol analogues in human urine by high-throughput solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry analysis.
Method development with high-throughput enhanced matrix removal followed by UHPLC-QqQ-MS/MS for analysis of grape polyphenol metabolites in human urine.
omicsMIC: a comprehensive benchmarking platform for robust comparison of imputation methods in mass spectrometry-based omics data.
Non-Targeted Detection of Synthetic Oligonucleotides in Equine Serum Using Liquid Chromatography-High-Resolution Mass Spectrometry.
Analysis of highly polar anionic pesticides in food of plant and animal origin by ion chromatography and tandem mass spectrometry with emphasis on addressing adverse effects caused by matrix co-extractives.
Establishment and clinical application of a candidate reference measurement procedure for quantification of urinary vanillylmandelic acid and homovanillic acid using ID-LC-MS/MS method.
Rapid differentiation of xylazine metabolites using SLIM IM-MS.
Organophosphate esters in human serum: a relatively simple and efficient liquid chromatography-mass spectrometry method.
Impact of three different peak picking software tools on the quality of untargeted metabolomics data.
Resolving Challenges in Detection and Quantification of D-2-hydroxyglutarate and L-2-hydroxyglutarate via LC/MS.
Development of a high-throughput dual-stream liquid chromatography-tandem mass spectrometry method to screen for inhibitors of glutamate carboxypeptidase II.
Improving the intestinal lipidome coverage in a gnotobiotic mouse model using UHPLC-MS-based approach through optimization of mobile phase modifiers and column selection.
Mass Spectrometry Imaging Techniques: Non-Ambient and Ambient Ionization Approaches.
Recent advances and challenges in the analysis of natural toxins.
Method optimization for a simultaneous determination of neonicotinoid, carbamate/thiocarbamate, triazole, organophosphate and pyrethroid pesticides and their metabolites in urine using UPLC-MS/MS.
Single-Cell Proteomics Analysis with Tecan Uno and SCREEN Workflow.
Cyanoacetohydrazide as a Novel Derivatization Agent for the Determination of UHPLC-HRMS Steroids in Urine.
Pharmacokinetics and Tissue Distribution of Itampolin A following Intragastric and Intravenous Administration in Rats Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.
Ambient ionization mass spectrometry provides screening of selective androgen receptor modulators.
Development and validation of an ultra-performance liquid chromatography tandem mass spectrometry for target determination of bioactive compounds in Dendrobium spp.
Quick and high-throughput quantification of β-agonist residues in bovine liver, meat, milk, kidney, poultry, and egg using dispersive solid phase extraction.
Toxicologic analysis of 35 drugs in post mortem human blood samples with focus on antihypertensive and antiarrhythmic drugs.

Methods Mol Biol. 2024 ;2832 171-182

Identification of Stress-Responsive Metabolites in Plants Using an Untargeted Metabolomics Approach.

Sajedeh Rezaei Cherati, Mariya V Khodakovskaya.

  Stress can affect different groups of plant metabolites and multiple signaling pathways. Untargeted metabolomics enables the collection of whole-spectrum data for the entire metabolite content present in plant tissues at that point in time. We present a thorough approach for large-scale, untargeted metabolomics of plant tissues using reverse-phase liquid chromatography connected to high-resolution mass spectrometry (LC-MS) of dilute methanolic extract. MZmine is a specialized computer software that automates the alignment and baseline modification of all derived mass peaks across all samples, resulting in precise information on the relative abundance of hundreds of metabolites reflected by thousands of mass signals. Further processing with statistic and bioinformatic techniques will provide a comprehensive perspective of the variations and connections among groups of samples.

Keywords:  Internal Standard; Liquid Chromatography-Mass Spectrometry; MZmine; MetaboAnalyst; Metabolite extraction; Plant Metabolomics

DOI:  https://doi.org/10.1007/978-1-0716-3973-3_12
Int J Mol Sci. 2024 May 28. pii: 5901. [Epub ahead of print]25(11):

An Optimized Method for LC-MS-Based Quantification of Endogenous Organic Acids: Metabolic Perturbations in Pancreatic Cancer.

Shreyans K Jain, Shivani Bansal, Sunil Bansal, Baldev Singh, William Klotzbier, Khyati Y Mehta, Amrita K Cheema.

  Accurate and reliable quantification of organic acids with carboxylic acid functional groups in complex biological samples remains a major analytical challenge in clinical chemistry. Issues such as spontaneous decarboxylation during ionization, poor chromatographic resolution, and retention on a reverse-phase column hinder sensitivity, specificity, and reproducibility in multiple-reaction monitoring (MRM)-based LC-MS assays. We report a targeted metabolomics method using phenylenediamine derivatization for quantifying carboxylic acid-containing metabolites (CCMs). This method achieves accurate and sensitive quantification in various biological matrices, with recovery rates from 90% to 105% and CVs ≤ 10%. It shows linearity from 0.1 ng/mL to 10 µg/mL with linear regression coefficients of 0.99 and LODs as low as 0.01 ng/mL. The library included a wide variety of structurally variant CCMs such as amino acids/conjugates, short- to medium-chain organic acids, di/tri-carboxylic acids/conjugates, fatty acids, and some ring-containing CCMs. Comparing CCM profiles of pancreatic cancer cells to normal pancreatic cells identified potential biomarkers and their correlation with key metabolic pathways. This method enables sensitive, specific, and high-throughput quantification of CCMs from small samples, supporting a wide range of applications in basic, clinical, and translational research.

Keywords:  4-Chloro-o-phenylenediamine; CCMs; LC–MRM; pancreatic cancer; quantification

DOI:  https://doi.org/10.3390/ijms25115901
Elife. 2024 Jun 18. pii: RP91597. [Epub ahead of print]12

Optimal transport for automatic alignment of untargeted metabolomic data.

Marie Breeur, George Stepaniants, Pekka Keski-Rahkonen, Philippe Rigollet, Vivian Viallon.

  Untargeted metabolomic profiling through liquid chromatography-mass spectrometry (LC-MS) measures a vast array of metabolites within biospecimens, advancing drug development, disease diagnosis, and risk prediction. However, the low throughput of LC-MS poses a major challenge for biomarker discovery, annotation, and experimental comparison, necessitating the merging of multiple datasets. Current data pooling methods encounter practical limitations due to their vulnerability to data variations and hyperparameter dependence. Here, we introduce GromovMatcher, a flexible and user-friendly algorithm that automatically combines LC-MS datasets using optimal transport. By capitalizing on feature intensity correlation structures, GromovMatcher delivers superior alignment accuracy and robustness compared to existing approaches. This algorithm scales to thousands of features requiring minimal hyperparameter tuning. Manually curated datasets for validating alignment algorithms are limited in the field of untargeted metabolomics, and hence we develop a dataset split procedure to generate pairs of validation datasets to test the alignments produced by GromovMatcher and other methods. Applying our method to experimental patient studies of liver and pancreatic cancer, we discover shared metabolic features related to patient alcohol intake, demonstrating how GromovMatcher facilitates the search for biomarkers associated with lifestyle risk factors linked to several cancer types.

Keywords:  Gromov-Wasserstein; LC-MS; cancer biology; cancer metabolism; computational biology; data integration; human; optimal transport; systems biology; untargeted metabolomics

DOI:  https://doi.org/10.7554/eLife.91597
J Pestic Sci. 2024 May 20. 49(2): 65-76

Investigation of variability in the matrix effect on stable isotope-labeled internal standards in liquid chromatography-tandem mass spectrometry analysis of 25 pesticides in vegetables.

Arisa Banno, Yoshinori Yabuki, Motohiro Sonoda, Shinji Tanimori.

  The matrix effects (ME) in simultaneous analysis of pesticide residue using liquid chromatography-tandem mass spectrometry (LC-MS/MS) were evaluated by comparing the slopes of matrix-matched and reagent-only calibrations of four types of vegetable samples. Both the sampling and measurement variances of the ME were also determined using one-way analysis of variance. Substantial ion suppression (ME<-20%) was observed in komatsuna, spinach, and tomato when a modified Japanese official method was implemented. The ME magnitude varied significantly due to sample variability for some pesticides, but it varied by no more than 4% as a result of analytical procedure variance. This study also showed that the addition of stable isotope-labeled internal standards at low concentrations improved the recovery of pesticides from samples at various residue levels. The findings of this study highlight the importance and practical application of internal standards and the matrix-matched calibration method in residue analysis using LC-MS/MS.

Keywords:  internal standard; liquid chromatography-tandem mass spectrometry; matrix effect; pesticide residue; simultaneous analysis

DOI:  https://doi.org/10.1584/jpestics.D23-060
J Pharm Biomed Anal. 2024 Jun 06. pii: S0731-7085(24)00332-7. [Epub ahead of print]248 116292

Automated online solid-phase extraction-tandem mass spectrometry detection for simultaneous analysis of acidic and alkaline catecholamines and their metabolites in human urine.

Shunyan Deng, Yu Wang, Xia Huang, Yan Zhou, Tianxun Wang, Xiquan Chen, Lan Xiong, Wenlin Wu, Bing Xia.

  Metabolic dysregulation of catecholamines (CAs) is implicated in various human diseases. Simultaneously analyzing these acidic and alkaline CAs and their metabolites poses a significant challenge for clinical detection. This study introduces an efficient method employing automated online solid-phase extraction coupled with tandem mass spectrometry (aoSPE-MS/MS). The method employs weak cation exchange (WCX) and mixed-mode anion exchange (MAX) adsorbents to fabricate an on-line solid-phase extraction (SPE) column, along with an automated injection and multi-valve switching capabilities. The setup allows for automated extraction and analysis of urine samples in 15 minutes while retaining a wide range of acidic and basic CAs and their metabolites. The applicability of this method was demonstrated by optimising the adsorbent dosage volume, extraction solvent, and extraction rate. The limits of detection (LODs) and limits of quantitation (LOQs) for the 8 CAs and their metabolites were determined using the aoSPE-MS/MS approach, with ranges of 0.0625 ∼ 62.5 ng/mL and 0.125 ∼ 125 ng/mL, respectively. Additionally, assessments were made on the linearity, accuracy, and precision within and between batches, as well as matrix and ionic effects, and spiked recoveries. The study discovered that the aoSPE-MS/MS technique simplifies operation, increases efficiency, saves time, and has low detection and quantification limits when detecting a wide range of acid and alkaline CAs and their metabolites in urine. The study successfully demonstrated the high-throughput and automated detection of the 8 CAs and their metabolites with varying acidity and alkalinity in human urine samples. This method is expected to be a potential powerful tool for clinical detection.

Keywords:  Automation; Catecholamine; On-line solid phase extraction; Urine

DOI:  https://doi.org/10.1016/j.jpba.2024.116292
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jun 13. pii: S1570-0232(24)00217-4. [Epub ahead of print]1242 124208

Development and evaluation of a liquid chromatography-tandem mass spectrometry method for simultaneous measurement of toxic aldehydes from brain tissue.

Yuanyuan Ji, Yulemni Morel, Anh Q Tran, Marta M Lipinski, Chinmoy Sarkar, Jace W Jones.

  Reactive aldehydes are a class of electrophilic low molecular weight compounds that play an essential role in physiological function and lipid peroxidation. These molecules are implicated in many diseases, especially cardiovascular and neurodegenerative diseases, and are potential endogenous markers of lipid peroxidation. However, there are limited options to accurately quantify multiple reactive aldehydes in brain tissue. This study developed and validated a 3-nitrophenylhydrazine derivatization-based LC-MS/MS method to quantify four reactive aldehydes: malondialdehyde, acrolein, 4-hydroxy-2-hexenal and 4-hydroxy-2-nonenal. Method development involved comparing the sensitivity of detection between widely used derivatization reagents: 2,4-dinitrophenylhydrazine and 3-nitrophenylhydrazine. Our data showed that 3-nitrophenylhydrazine resulted in greater sensitivity. Additional method development included evaluation of hydrolysis sample pretreatment, selection of protein precipitation reagent, and optimization of derivatization conditions. The optimized conditions included no hydrolysis and use of 20 % trichloroacetic acid as the protein precipitation reagent. The optimized derivatization condition was 25 mM 3-nitrophenylhydrazine reacted at 20 °C for 30 min. The chromatographic conditions were optimized to reduce matrix effects, ion suppression, and efficient analysis time (<7-minute analytical run). The four aldehyde species were accurately quantified in brain tissue using stable-labeled internal standards. Application of this assay to a traumatic brain injury mouse model revealed significant accumulation of acrolein, 4-hydroxy-2-hexenal, and 4-hydroxy-2-nonenal at 28 days post injury. Overall, a validated method was developed to rapidly quantify the most prominent reactive aldehydes associated with lipid peroxidation during injury progression following acute brain trauma.

Keywords:  Accurate quantitation; Derivatization; LC-MS/MS; Reactive aldehydes; Traumatic Brain Injury

DOI:  https://doi.org/10.1016/j.jchromb.2024.124208
Curr Protoc. 2024 Jun;4(6): e1087

Analysis of Globotriaosylceramide (Gb3) in Liquid Urine: A Straightforward Assay Using Tandem Mass Spectrometry.

Michel Boutin, Bruno Maranda, Paula J Waters.

  Fabry disease (FD) is a lysosomal storage disorder caused by variants in the GLA gene encoding α-galactosidase A, an enzyme required for catabolism of globotriaosylceramide (Gb3). Accumulation of Gb3 in patients' cells, tissues, and biological fluids causes clinical manifestations including ventricular hypertrophy, renal insufficiency, and strokes. This protocol describes a methodology to analyze urinary Gb3 and creatinine. Samples are diluted with an internal standard solution containing Gb3(C17:0) and creatinine-D3, centrifuged, and directly analyzed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) using an 8.7-min method. Eight Gb3 isoforms [C16:0, C18:0, C20:0, C22:1, C22:0, C24:1, C24:0, and (C24:0)OH] are analyzed and the total is normalized to creatinine. Confirmation ions are monitored to detect potential interferences. The Gb3 limit of quantification is 0.023 µg/ml. Its interday coefficients of variation (3 concentrations measured) are ≤15.4%. This method minimizes matrix effects (≤6.5%) and prevents adsorption or precipitation of Gb3. Urine samples are stable (bias <15%) for 2 days at 21°C, 7 days at 4°C, and 4 freeze/thaw cycles, whereas prepared samples are stable for 5 days at 21°C, and 14 days at 4°C. The Gb3/creatinine age-related upper reference limits (mean + 2 standard deviations) are 29 mg/mol creatinine (<7 years) and 14 mg/mol creatinine (≥7 years). This simple, robust protocol has been fully validated (ISO 15189) and provides a valuable tool for diagnosis and monitoring of FD patients. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Analysis of urinary globotriaosylceramide (Gb3) and creatinine by UHPLC-MS/MS Support Protocol 1: Preparation of the urinary quality controls Support Protocol 2: Preparation of the urine matrix used for the Gb3 calibration curve Support Protocol 3: Preparation of the Gb3 calibrators Support Protocol 4: Preparation of the working solution containing the internal standards Support Protocol 5: Preparation of the creatinine calibrators Support Protocol 6: Preparation of the UHPLC solutions and mobile phases.

Keywords:  Fabry disease; Gb3; UHPLC‐MS/MS; creatinine; globotriaosylceramide; mass spectrometry

DOI:  https://doi.org/10.1002/cpz1.1087
J Chromatogr A. 2024 Jun 16. pii: S0021-9673(24)00470-9. [Epub ahead of print]1730 465096

Determination of nine bisphenol analogues in human urine by high-throughput solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry analysis.

Weiyi Jin, Siyu Yang, Longlong Yin, Yeqing Jia, Xinghua Li, Kang An, Jianping Li, Yi Zhang.

  Bisphenol analogues (BPs) are a class of typical environmental endocrine-disrupting chemicals (EDCs). This study aimed to establish a highly sensitive and high-throughput method utilizing 96-well solid-phase extraction (96-well SPE) in conjunction with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) employing multiple reaction monitoring (MRM), information-dependent acquisition (IDA), and enhanced product ion (EPI) scan modes for the identification and quantitative analysis of nine BPs in human urine. Urine samples were initially thawed to room temperature, followed by digestion using β-glucuronidase in an ammonium acetate buffer solution at 37 °C overnight. Subsequently, they were purified using 96-well SPE and finally analyzed by UHPLC-MS/MS. The limits of detection (LOD) for the nine BPs ranged from 0.05 μg∙kg-1 to 0.3 μg kg-1. Average recoveries fell within the range of 92.8 % to 111.7 %. Moreover, both the intra-day and inter-day precisions were satisfactory, with relative standard deviations (RSDs) ranging from 2.2 % to 6.7 % and 3.5 % to 6.3 %, respectively. The targets in the samples exhibited a perfect match, with a purity fit value exceeding 70 % from the self-built library. The analytical method developed in this study demonstrates high accuracy and sensitivity. In addition, the MRM-IDA-EPI mode can effectively identifies the target BPs and prevents false positive detection of analytes in the urine.

Keywords:  96-well SPE; Bisphenol analogues; MRM-IDA-EPI; UHPLC-MS/MS; Urine

DOI:  https://doi.org/10.1016/j.chroma.2024.465096
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jun 03. pii: S1570-0232(24)00198-3. [Epub ahead of print]1242 124189

Method development with high-throughput enhanced matrix removal followed by UHPLC-QqQ-MS/MS for analysis of grape polyphenol metabolites in human urine.

Weiting Lyu, Zhiya Yin, Lingjun Xie, Giulio M Pasinetti, James W Murrough, Maxine Marchidan, Elizabeth Karpman, Matthew Dobbs, Mario G Ferruzzi, James E Simon, Qingli Wu.

  Grape and grape derived products contain many bioactive phenolics which have a variety of impacts on health. Following oral ingestion, the phenolic compounds and their metabolites may be detectable in human urine. However, developing a reliable method for the analysis of phenolic compounds in urine is challenging. In this work, we developed and validated a new high-throughput, sensitive and reproducible analytical method for the simultaneous analysis of 31 grape phenolic compounds and metabolites using Oasis PRiME HLB cleanup for sample preparation combined with ultra-performance liquid chromatography with triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). Using this new method, the accuracy achieved was 69.3 % ∼ 134.9 % (except for six compounds), and the recovery achieved was 52.4 % ∼ 134.7 % (except for two very polar compounds). For each of the 31 target analytes, the value of intra-day precision was less than 14.3 %. The value of inter-day precision was slightly higher than intra-day precision, with a range of 0.7 % ∼ 19.1 %. We report for the first time on the effect of gender and BMI on the accuracy and recovery of human urine samples, and results from analysis of variance (ANOVA), and principal component analysis (PCA) indicated there was no difference in the value of accuracy and recovery between different gender or BMI (>30) using our purposed cleanup and UHPLC-QqQ-MS/MS method. Overall, this newly developed method could serve as a powerful tool for analyzing grape phenolic compounds and metabolites in human urine samples.

Keywords:  Metabolomics; Polyphenols; Solid phase extraction; Urine metabolites

DOI:  https://doi.org/10.1016/j.jchromb.2024.124189
NAR Genom Bioinform. 2024 Jun;6(2): lqae071

omicsMIC: a comprehensive benchmarking platform for robust comparison of imputation methods in mass spectrometry-based omics data.

Weiqiang Lin, Jiadong Ji, Kuan-Jui Su, Chuan Qiu, Qing Tian, Lan-Juan Zhao, Zhe Luo, Chong Wu, Hui Shen, Hongwen Deng.

Mass spectrometry is a powerful and widely used tool for generating proteomics, lipidomics and metabolomics profiles, which is pivotal for elucidating biological processes and identifying biomarkers. However, missing values in mass spectrometry-based omics data may pose a critical challenge for the comprehensive identification of biomarkers and elucidation of the biological processes underlying human complex disorders. To alleviate this issue, various imputation methods for mass spectrometry-based omics data have been developed. However, a comprehensive comparison of these imputation methods is still lacking, and researchers are frequently confronted with a multitude of options without a clear rationale for method selection. To address this pressing need, we developed omicsMIC (mass spectrometry-based omics with Missing values Imputation methods Comparison platform), an interactive platform that provides researchers with a versatile framework to evaluate the performance of 28 diverse imputation methods. omicsMIC offers a nuanced perspective, acknowledging the inherent heterogeneity in biological data and the unique attributes of each dataset. Our platform empowers researchers to make data-driven decisions in imputation method selection based on real-time visualizations of the outcomes associated with different imputation strategies. The comprehensive benchmarking and versatility of omicsMIC make it a valuable tool for the scientific community engaged in mass spectrometry-based omics research. omicsMIC is freely available at https://github.com/WQLin8/omicsMIC.

DOI: https://doi.org/10.1093/nargab/lqae071
Int J Mol Sci. 2024 May 25. pii: 5752. [Epub ahead of print]25(11):

Non-Targeted Detection of Synthetic Oligonucleotides in Equine Serum Using Liquid Chromatography-High-Resolution Mass Spectrometry.

Emily Helmes, Jacob Montgomery, Gwendolyne Alarcio, Herra G Mendoza, Jeffrey A Blea, Peter A Beal, Benjamin C Moeller.

  There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which makes targeted analysis for these agents problematic. Accordingly, we have developed a non-targeted approach to detect the presence of specific product ions that are not naturally present in ribonucleic acids. Briefly, serum samples were extracted using solid-phase extraction with a mixed-mode cartridge following the disruption of protein interactions to isolate the oligonucleotides. Following the elution and concentration steps, chromatographic separation was achieved utilizing reversed-phase liquid chromatography. Following an introduction to a Thermo Q Exactive HF mass spectrometer using electrospray ionization, analytes were detected utilizing a combination of full-scan, parallel reaction monitoring and all ion fragmentation scan modes. The limits of detection were determined along with the accuracy, precision, stability, recovery, and matrix effects using a representative 13mer oligonucleotide. Following method optimization using the 13mer oligonucleotide, the method was applied to successfully detect the presence of specific product ions in three unique oligonucleotide sequences targeting equine-specific transcripts.

Keywords:  gene doping; liquid chromatography–mass spectrometry; oligonucleotide; phosphorothioate; serum

DOI:  https://doi.org/10.3390/ijms25115752
Anal Bioanal Chem. 2024 Jun 19.

Analysis of highly polar anionic pesticides in food of plant and animal origin by ion chromatography and tandem mass spectrometry with emphasis on addressing adverse effects caused by matrix co-extractives.

Ann-Kathrin Schäfer, Walter Vetter, Michelangelo Anastassiades.

  Residues of various highly polar pesticides and their metabolites are commonly found in numerous food products. Some of these compounds, such as glyphosate, are not only used in large amounts in agriculture, but are also controversially discussed in public. Here, we present a method, employing ion chromatography (IC) coupled to tandem mass spectrometry (IC-MS/MS), for the analyses of glyphosate, aminomethyl phosphonic acid (AMPA), N-acetyl-glyphosate (NAGly), fosetyl, and 10 further highly polar pesticides and metabolites in various plant and animal matrices following a minimal sample preparation by means of the QuPPe method. Thorough investigations showed that an AS19 column enabled the analysis of all 14 compounds within 30 min. The best sensitivity could be obtained with the make-up solvent acetonitrile being admixed to the mobile phase at a 1:2 flow rate ratio. Matrix effects were thoroughly studied in terms of ion suppression and retention time shifts. Conductivity detection was used to monitor elution profiles of matrix co-extractives in comparison with matrix effect profiles obtained by continuous post-column infusion of a mix with 13 highly polar pesticides and metabolites. These tests indicated that a fivefold dilution of QuPPe extracts was suitable for the routine analysis of samples for MRL-conformity, as it considerably reduced matrix effects maintaining sufficient sensitivity and high recovery rates in eight different commodities. The suitability of the final method for its application in routine analysis was verified by the analysis of >130 samples containing incurred residues where the results were compared with two existing LC-MS/MS methods.

Keywords:  Glyphosate; IC-MS/MS; Ion chromatography; Matrix effect; Polar pesticide; Tandem mass spectrometry

DOI:  https://doi.org/10.1007/s00216-024-05389-4
J Pharm Biomed Anal. 2024 Jun 12. pii: S0731-7085(24)00351-0. [Epub ahead of print]248 116311

Establishment and clinical application of a candidate reference measurement procedure for quantification of urinary vanillylmandelic acid and homovanillic acid using ID-LC-MS/MS method.

Lintao Yu, Shiyue Kang, Long Cheng, Qiaoxuan Zhang, Fen Ouyang, Liqiao Han, Min Zhan, Dezheng Liao, Pengwei Zhang, Jun Yan, Xianzhang Huang.

  Neuroblastoma (NB), an embryonic tumor of the autonomous nervous system, poses a significant threat to the health and lives of children. Accurate measurement of vanillylmandelic acid (VMA) and homovanillic acid (HVA) in human urine is crucial for screening and diagnosis of NB. Although various laboratories have developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect VMA and HVA, the comparability between the results obtained from different laboratories and methods was poor. The absence of reference method for VMA and HVA hinders the standardization of their measurements. Therefore, a candidate reference measurement procedure (cRMP) based on isotope dilution LC-MS/MS (ID-LC-MS/MS) for the detection of VMA and HVA in human urine was established. Urine samples were spiked with VMA-d3 and HVA-d5 as internal standards and extracted using a protein precipitation method. The cRMP exhibited desirable precision with the total imprecision below 5 %. The accuracy of this cRMP was demonstrated by the high analytical recovery (98.64 % - 102.22 % and 98.41 % - 100.97 % for VMA and HVA, respectively), and comparability between different reference systems. The limit of detection for HVA and VMA were 15.625 ng/mL and 3.906 ng/mL, respectively; the quantification limits were 62.5 ng/mL and 7.813 ng/mL, respectively, which can meet the clinical detection requirements. The linear range was from 78.125 ng/mL to 20 μg/mL. Specificity evaluations showed no corresponding interference from structurally similar analogs. In conclusion, we have established a cRMP based on ID-LC-MS/MS for the measurement of VMA and HVA in urine samples, demonstrating well-defined method performance including accuracy, precision, and specificity. This newly established cRMP is suitable for routine assay standardization and evaluation of clinical samples. Furthermore, this method has the potential to significantly enhance the diagnostic accuracy for neuroblastoma.

Keywords:  Candidate reference measurement procedure; Homovanillic acid; ID-LC-MS/MS; Vanillylmandelic acid

DOI:  https://doi.org/10.1016/j.jpba.2024.116311
Anal Methods. 2024 Jun 13.

Rapid differentiation of xylazine metabolites using SLIM IM-MS.

Ralph Aderorho, Shadrack Wilson Lucas, Christopher D Chouinard.

Xylazine represents an increased threat to the recreational drug market. In this study, we present a rapid strategy for identifying xylazine and differentiating its common isomeric metabolites using Structures for Lossless Ion Manipulations (SLIM) ion mobility coupled to high-resolution/tandem mass spectrometry (IM-HRMS/MS). Chemical derivatization using dansyl chloride also assisted with separations and led to identification of resolvable reaction product atropisomers.

DOI: https://doi.org/10.1039/d4ay00801d
Anal Methods. 2024 Jun 18.

Organophosphate esters in human serum: a relatively simple and efficient liquid chromatography-mass spectrometry method.

Zhiyuan Du, Xiaoyi Wei, Xiaohua Hu, Yijing Zhao, Guanghua Chen, Xiushuai Du, Jialing Li, Ming Zhan, Weiwei Zheng.

Organophosphate esters (OPEs) are widely used as flame retardants and plasticizers, which are of growing concern due to their endocrine-disrupting effects, developmental toxicity, and potential carcinogenicity. However, data on human exposure to OPEs is still scarce. In this study, a relatively simple and efficient method with less serum consumption for the detection of OPEs in human serum was developed and validated. Nine OPEs in 200 μL of human serum were extracted by an acetonitrile-formic acid system and analyzed using ultra-high-performance liquid chromatography-quadrupole tandem time-of-flight high-resolution mass spectrometry. Several experiments were conducted to optimize the chromatographic and mass spectrometric conditions as well as sample preparation to obtain a more sensitive and efficient analytical protocol. The proposed method was examined in terms of its linearity, accuracy, precision, detection limit, and matrix effect. The matrix-spiked recoveries of the target OPEs ranged from 83.3% to 111.1%, with relative standard deviations between 2.7% and 16.6%. The detection limits were within (0.002 to 0.029) ng mL-1, while the quantification limits were within (0.007 to 0.098) ng mL-1. The internal standard-corrected matrix effects varied from 82.7% to 113.9%. Finally, the method was applied to detect OPEs in actual human serum samples. All nine OPEs were detected in 269 serum samples to varying degrees, with the average concentrations ranging from (0.08 to 1.77) ng mL-1. After validation, the method was found to be simple in pretreatment, high in sensitivity, good in practicality, and suitable for exposure evaluation of OPEs in populations.

DOI: https://doi.org/10.1039/d4ay00787e
J Pharm Biomed Anal. 2024 Jun 10. pii: S0731-7085(24)00342-X. [Epub ahead of print]248 116302

Impact of three different peak picking software tools on the quality of untargeted metabolomics data.

Yannick Wartmann, Martina I Boxler, Thomas Kraemer, Andrea E Steuer.

  Data quality and control parameters are becoming more important in metabolomics. For peak picking, open-source or commercial solutions are used. Other publications consider different software solutions or data acquisition types for peak picking, a combination, including proposed and new quality parameters for the process of peak picking, does not exist. This study tries to examine the performance of three different software in terms of reproducibility and quality of their output while also considering new quality parameters to gain a better understanding of resulting feature lists in metabolomics data. We saw best recovery of spiked analytes in MS-DIAL. Reproducibility over multiple projects was good among all software. The total number of features found was consistent for DDA and full scan acquisition in MS-DIAL but full scan data leading to considerably more features in MZmine and Progenesis Qi. Feature linearity proved to be a good quality parameter. Features in MS-DIAL and MZmine, showed good linearity while Progenesis Qi produced large variation, especially in full scan data. Peak width proved to be a very powerful filtering criteria revealing many features in MZmine and Progenesis Qi to be of questionable peak width. Additionally, full scan data appears to produce a disproportionally higher number of short features. This parameter is not yet available in MS-DIAL. Finally, the manual classification of true positive features proved MS-DIAL to perform significantly better in DDA data (62 % true positive) than the two other software in either mode. We showed that currently popular solutions MS-DIAL and MZmine perform well in targeted analysis of spiked analytes as well as in classic untargeted analysis. The commercially available solution Progenesis Qi does not hold any advantage over the two in terms of quality parameters, of which we proposed peak width as a new parameter and showed that already proposed parameters such as feature linearity in samples of increasing concentration are advisable to use.

Keywords:  (Un)targeted metabolomics; Comparison; DDA; DIA; LC-MS/MS; MS-DIAL; MZmine

DOI:  https://doi.org/10.1016/j.jpba.2024.116302
bioRxiv. 2024 Apr 26. pii: 2024.04.26.591335. [Epub ahead of print]

Resolving Challenges in Detection and Quantification of D-2-hydroxyglutarate and L-2-hydroxyglutarate via LC/MS.

Tyrone Dowdy, Mioara Larion.

D-2-Hydroxyglutarate and L-2-Hydroxyglutarate (D-2HG/L-2HG) are typically metabolites of non-specific enzymatic reactions that are kept in check by the housekeeping enzymes, D-2HG /L-2HG dehydrogenase (D-2HGDH/L-2HGDH). In certain disease states, such as D-2HG or L-2HG aciduria and cancers, accumulation of these biomarkers interferes with oxoglutarate-dependent enzymes that regulate bioenergetic metabolism, histone methylation, post-translational modification, protein expression and others. D-2HG has a complex role in tumorigenesis that drives metabolomics investigations. Meanwhile, L-2HG is produced by non-specific action of malate dehydrogenase and lactate dehydrogenase under acidic or hypoxic environments. Characterization of divergent effects of D-2HG/L-2HG on the activity of specific enzymes in diseased metabolism depends on their accurate quantification via mass spectrometry. Despite advancements in high-resolution quadrupole time-of-flight mass spectrometry (HR-QTOF-MS), challenges are typically encountered when attempting to resolve of isobaric and isomeric metabolites such as D-2HG/L-2HG for quantitative analysis. Herein, available D-2HG/L-2HG derivatization and liquid chromatography (LC) MS quantification methods were examined. The outcome led to the development of a robust, high-throughput HR-QTOF-LC/MS approach that permits concomitant quantification of the D-2HG and L-2HG enantiomers with the benefit to quantify the dysregulation of other intermediates within interconnecting pathways. Calibration curve was obtained over the linear range of 0.8-104 nmol/mL with r 2 ≥ 0.995 for each enantiomer. The LC/MS-based assay had an overall precision with intra-day CV % ≤ 8.0 and inter-day CV % ≤ 6.3 across the quality control level for commercial standard and pooled biological samples; relative error % ≤ 2.7 for accuracy; and resolution, R s = 1.6 between 2HG enantiomers (m/z 147.030), D-2HG and L-2HG (at retention time of 5.82 min and 4.75 min, respectively) following chiral derivatization with diacetyl-L-tartaric anhydride (DATAN). Our methodology was applied to disease relevant samples to illustrate the implications of proper enantioselective quantification of both D-2HG and L-2HG. The stability of the method allows scaling to large cohorts of clinical samples in the future.

DOI: https://doi.org/10.1101/2024.04.26.591335
Rapid Commun Mass Spectrom. 2024 Jun 12. e9772

Development of a high-throughput dual-stream liquid chromatography-tandem mass spectrometry method to screen for inhibitors of glutamate carboxypeptidase II.

Nate Hoxie, Yixuan Qiu, Stephen C Kales, Rick Schneider, Xin Hu, Anu Dalal, Stephanie L Ford-Scheimer, Robyn Wiseman, Takashi Tsukamoto, Huijun Wei, Barbara S Slusher, John S Janiszewski, Matthew D Hall.

RATIONALE: Glutamate carboxypeptidase II (GCPII) catalyzes the hydrolysis of N-acetylaspartylglutamate (NAAG) to yield glutamate (Glu) and N-acetylaspartate (NAA). Inhibition of GCPII has been shown to remediate the neurotoxicity of excess Glu in a variety of cell and animal disease models. A robust high-throughput liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was needed to quantify GCPII enzymatic activity in a biochemical high-throughput screening assay.
METHODS: A dual-stream LC/MS/MS method was developed. Two parallel eluent streams ran identical HILIC gradient methods on BEH-Amide (2 × 30 mm) columns. Each LC channel was run independently, and the cycle time was 2 min per channel. Overall throughput was 1 min per sample for the dual-channel integrated system. Multiply injected acquisition files were split during data review, and batch metadata were automatically paired with raw data during the review process.
RESULTS: Two LC sorbents, BEH-Amide and Penta-HILIC, were tested to separate the NAAG cleavage product Glu from isobaric interference and ion suppressants in the bioassay matrix. Early elution of NAAG and NAA on BEH-Amide allowed interfering species to be diverted to waste. The limit of quantification was 0.1 pmol for Glu. The Z-factor of this assay averaged 0.85. Over 36 000 compounds were screened using this method.
CONCLUSIONS: A fast gradient dual-stream LC/MS/MS method for Glu quantification in GCPII biochemical screening assay samples was developed and validated. HILIC separation chemistry offers robust performance and unique selectivity for targeted positive mode quantification of Glu, NAA, and NAAG.

DOI: https://doi.org/10.1002/rcm.9772
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jun 14. pii: S1570-0232(24)00197-1. [Epub ahead of print]1242 124188

Improving the intestinal lipidome coverage in a gnotobiotic mouse model using UHPLC-MS-based approach through optimization of mobile phase modifiers and column selection.

Habiba Selmi, Alesia Walker, Laurent Debarbieux, Philippe Schmitt-Kopplin.

  Lipidomics is focusing on the screening of lipid species in complex mixtures using mass spectrometry-based approaches. In this work, we aim to enhance the intestinal lipidome coverage within the Oligo-Mouse-Microbiota (OMM12) colonized mouse model by testing eight mobile phase conditions on five reversed-phase columns. Our selected mobile phase modifiers included two ammonium salts, two concentrations, and the addition of respective acids at 0.1 %. We compared two columns with hybrid surface technology, two with ethylene bridged hybrid technology and one with core-shell particles. Best performance was attained for standards and intestinal lipidome, using either ammonium formate or acetate in ESI(+) or ammonium acetate in ESI(-) for all column technologies. Notably, a concentration of 5 mM ammonium salt showed optimal results for both modes, while the addition of acids had a negligible effect on lipid ionization efficiency. The HST BEH C18 column improved peak width and tailing factor parameters compared to other technologies. We achieved the highest lipid count in colon and ileum content, including ceramides, phosphatidylethanolamines and phosphatidylcholines, when using 5 mM ammonium acetate in ESI(-). Conversely, in ESI(+) 5 mM ammonium formate demonstrated superior coverage for diacylglycerols and triacylglycerols.

Keywords:  Hybrid surface technology; Mobile phase modifiers; Non-targeted lipidomics; Oligo-Mouse-Microbiota

DOI:  https://doi.org/10.1016/j.jchromb.2024.124188
Crit Rev Anal Chem. 2024 Jun 18. 1-54

Mass Spectrometry Imaging Techniques: Non-Ambient and Ambient Ionization Approaches.

Shundi Hu, Ahsan Habib, Wei Xiong, La Chen, Lei Bi, Luhong Wen.

  Molecular information can be acquired from sample surfaces in real time using a revolutionary molecular imaging technique called mass spectrometry imaging (MSI). The technique can concurrently provide high spatial resolution information on the spatial distribution and relative proportion of many different compounds. Thus, many scientists have been drawn to the innovative capabilities of the MSI approach, leading to significant focus in various fields during the past few decades. This review describes the sampling protocol, working principle and applications of a few non-ambient and ambient ionization mass spectrometry imaging techniques. The non-ambient techniques include secondary ionization mass spectrometry and matrix-assisted laser desorption ionization, while the ambient techniques include desorption electrospray ionization, laser ablation electrospray ionization, probe electro-spray ionization, desorption atmospheric pressure photo-ionization and femtosecond laser desorption ionization. The review additionally addresses the advantages and disadvantages of ambient and non-ambient MSI techniques in relation to their suitability, particularly for biological samples used in tissue diagnostics. Last but not least, suggestions and conclusions are made regarding the challenges and future prospects of MSI.

Keywords:  Non-ambient and ambient ionization; mass spectrometry imaging

DOI:  https://doi.org/10.1080/10408347.2024.2362703
Adv Food Nutr Res. 2024 ;pii: S1043-4526(24)00033-0. [Epub ahead of print]110 67-144

Recent advances and challenges in the analysis of natural toxins.

Ids B Lemmink, Leonie V Straub, Toine F H Bovee, Patrick P J Mulder, Han Zuilhof, Gert Ij Salentijn, Laura Righetti.

  Natural toxins (NTs) are poisonous secondary metabolites produced by living organisms developed to ward off predators. Especially low molecular weight NTs (MW<∼1 kDa), such as mycotoxins, phycotoxins, and plant toxins, are considered an important and growing food safety concern. Therefore, accurate risk assessment of food and feed for the presence of NTs is crucial. Currently, the analysis of NTs is predominantly performed with targeted high pressure liquid chromatography tandem mass spectrometry (HPLC-MS/MS) methods. Although these methods are highly sensitive and accurate, they are relatively expensive and time-consuming, while unknown or unexpected NTs will be missed. To overcome this, novel on-site screening methods and non-targeted HPLC high resolution mass spectrometry (HRMS) methods have been developed. On-site screening methods can give non-specialists the possibility for broad "scanning" of potential geographical regions of interest, while also providing sensitive and specific analysis at the point-of-need. Non-targeted chromatography-HRMS methods can detect unexpected as well as unknown NTs and their metabolites in a lab-based approach. The aim of this chapter is to provide an insight in the recent advances, challenges, and perspectives in the field of NTs analysis both from the on-site and the laboratory perspective.

Keywords:  Mass spectrometry; Mycotoxins; Natural toxins and producers; Non-targeted analysis; On-site screening; Phycotoxins; Plant toxins; Sample preparation; Separations; Targeted analysis

DOI:  https://doi.org/10.1016/bs.afnr.2024.05.001
J Chromatogr A. 2024 Jun 19. pii: S0021-9673(24)00428-X. [Epub ahead of print]1730 465054

Method optimization for a simultaneous determination of neonicotinoid, carbamate/thiocarbamate, triazole, organophosphate and pyrethroid pesticides and their metabolites in urine using UPLC-MS/MS.

Carolina M Bustamante, Natalia Bravo, Paula Ruiz, Joan O Grimalt, Mercè Garí.

  An accurate and sensitive method for the determination of a total of 23 pesticides and their metabolites in human urine has been optimised. The methodology is based on a previously published method based on solid-phase extraction with methanol and acetone followed by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) in the selected reaction mode (SRM) with both positive and negative electrospray ionization (ESI+/-). The detection settings of the previous method, which allowed to determine the metabolites from 6 organophosphate and 2 pyrethroid pesticides, were optimised in order to include further pesticide groups, such as 11 neonicotinoids, 3 carbamates/thiocarbamates and 2 triazoles. The 5-windows method enduring 22 min was optimized with acceptable results in relation to accuracy (recoveries >75 %), precision (coefficients of variation <26 %) and linearity (R2> 0.9915). The limits of detection ranged between 0.012 ng/mL and 0.058 ng/mL. Samples from the German External Quality Assessment Scheme (G-EQUAS) encompassing 2 pyrethroids, 2 organophosphate and one neonicotinoid (6-chloronicotinic acid, a common metabolite of imidacloprid and acetamiprid) were analysed, and the latter, included in this newest optimization, provided good reference results. The method is optimal as a human biomonitoring tool for health risk assessment in large population surveys.

Keywords:  Human biomonitoring; Method optimization; Neonicotinoid pesticides; UPLC-MS/MS; Urinary metabolites

DOI:  https://doi.org/10.1016/j.chroma.2024.465054
Methods Mol Biol. 2024 ;2817 157-175

Single-Cell Proteomics Analysis with Tecan Uno and SCREEN Workflow.

Michael Lewandowski, Shad Morton, Matthew Blake, Erica Squires, Rushdy Ahmad, David R Walt, Bogdan Budnik.

  With advances in sample preparation, small-volume liquid dispensing technologies, high-resolution MS/MS instrumentation, and data acquisition methodologies, it has become increasingly possible to confidently investigate the heterogeneous proteome found within individual cells. In this chapter, we present an automated high-throughput sample preparation workflow based on the Tecan Uno instrument for quantitative single-cell mass spectrometry-based proteomics. Cells are analyzed by the Single-Cell Proteome Analysis platform (SCREEN), which was introduced earlier and provides deeper proteome coverage across single cells.

Keywords:  Automation; High-throughput sample preparation; SCREEN method; Tecan Uno

DOI:  https://doi.org/10.1007/978-1-0716-3934-4_13
Molecules. 2024 May 22. pii: 2433. [Epub ahead of print]29(11):

Cyanoacetohydrazide as a Novel Derivatization Agent for the Determination of UHPLC-HRMS Steroids in Urine.

Azamat Temerdashev, Maria Zorina, Yu-Qi Feng, Elina Gashimova, Victor V Dotsenko, Vitalij Ioutsi, Sanka N Atapattu.

  The possibility of cyanoacetohydrazide usage as a novel derivatizing agent is demonstrated in the presented article, and a comparison with hydroxylamine as the most commonly used reagent is provided. Optimal conditions for steroid derivatization with cyanoacetohydrazide are provided. According to the collected data, the maximum yield of derivatives was observed at pH 2.8 within 70 min at 40 °C with 5 ng/mL limit of detection for all investigated analytes. It was shown that cyanoacetohydrazide derivatives produces both syn- and anti-forms as well as hydroxylamine, and their ratios were evaluated and shown in presented work. An efficiency enchantment from two to up to five times was achieved with a novel derivatization reagent. Its applicability for qualitative analysis of steroids in urine was presented at real samples. Additionally, the reproducible fragmentation of the derivatizing agent in collision-induced dissociation offers opportunities for simplified non-targeted steroidomic screening. Furthermore, cyanoacetohydrazide increases ionization efficiency in positive mode, which can eliminate the need for redundant high-resolution instrument runs required for both positive and negative mode analyses.

Keywords:  LC-HRMS; cyanoacetohydrazide; derivatization; non-targeted screening; steroidomics; steroids; testosterone

DOI:  https://doi.org/10.3390/molecules29112433
Molecules. 2024 Jun 04. pii: 2652. [Epub ahead of print]29(11):

Pharmacokinetics and Tissue Distribution of Itampolin A following Intragastric and Intravenous Administration in Rats Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Qi Sun, Jingwei Liang, Qingyu Zhang, Xuezhen Wang, Nan Zhao, Fanhao Meng.

  Itampolin A, a natural brominated tyrosine alkaloid isolated from the sponge Iotrochota purpurea, has been shown to have good inhibitory effects in lung cancer cells as a p38α inhibitor. A simple, sensitive, and reliable ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been established, validated, and applied to the study of the pharmacokinetics and tissue distribution of itampolin A following intragastric and intravenous administration. Itampolin A and theophylline (internal standard, IS) were extracted by the simple protein precipitation technique using methanol as the precipitating solvent. Chromatographic separation was achieved by using the optimized mobile phase of a 0.1% formic acid aqueous solution and acetonitrile in the gradient elution mode. Itampolin A and IS were detected and quantified using positive electrospray ionization in the multiple reaction monitoring mode with transitions of m/z 863.9 → 569.1 for itampolin A and m/z 181.1 → 124.1 for IS, respectively. The assay exhibited a linear dynamic range of 1-1600 ng/mL for itampolin A in biological samples and the low limit of quantification was 1 ng/mL. Non-compartmental pharmacokinetic parameters indicated that itampolin A was well-absorbed into the systemic circulation and rapidly eliminated after administration. The apparent distribution volume of itampolin A was much higher after intragastric administration than that after intravenous administration. A tissue distribution study showed that itampolin A could be detected in different tissues and maintained a high concentration in the lung, which provided a material basis for its effective application in lung cancer. The pharmacokinetic process and tissue distribution characteristics of imtapolin A were expounded in this study, which can provide beneficial information for the further research and clinical application of itampolin A.

Keywords:  UHPLC-MS/MS; itampolin A; pharmacokinetics; tissue distribution

DOI:  https://doi.org/10.3390/molecules29112652
Talanta. 2024 Jun 06. pii: S0039-9140(24)00737-9. [Epub ahead of print]277 126358

Ambient ionization mass spectrometry provides screening of selective androgen receptor modulators.

Alžběta Nemeškalová, Jitka Konvalinková, Magdaléna Vágnerová, Martin Kuchař, Jan Buček, Vladimír Vrkoslav, David Sýkora, Josef Cvačka, Michael Volny.

  Ambient ionization mass spectrometry allows for analysis of samples in their natural state, i.e., with no sample pre-treatment. It can be viewed as a fast, simple, and economical analysis, but its main disadvantages include a lower analytical performance due to the presence of complex sample matrix and the lack of chromatographic separation prior to the introduction of the sample into the mass spectrometer. Here we present an application of two ambient ionization mass spectrometry techniques, i.e., Desorption Atmospheric Pressure Photoionization and Dielectric Barrier Discharge Ionization, for the analysis of known Selective Androgen Receptor Modulators, which represent common compounds of abuse in professional and semiprofessional sport. Eight real samples of illegal food supplements, seized by the local law enforcement, were used to test the performance of the ambient mass spectrometry and the results were validated against a newly developed targeted LC-UV-MS/MS method performed in multiple reaction monitoring mode with an external calibration for each analyte. In order to decide whether or not the compound can be declared as present, we proposed a system of rules for the interpretation of the obtained spectra. The criteria are based on mass spectrum matching (5-10 ppm accuracy from the theoretical exact mass and a correct isotopic pattern), duration of the mass signal (three or five consecutive scans, depending on the instrumentation used), and intensity above the background noise (threefold increase in intensity and absolute intensity above 5E4 or 1E5, depending on the instrumentation). When applying these criteria, good agreement was found between the tested methods. Ambient ionization techniques were effective at detecting SARMs at pharmacologically relevant doses, i.e., approximately above 1 mg per capsule, although they may fail to detect lower levels or isomeric species. It is demonstrated that when adhering to a set of clear and consistent rules, ambient mass spectrometry can be employed as a qualitative technique for the screening of illegal SARMs with sufficient confidence and without the necessity to perform a regular LC-MS analysis.

Keywords:  Ambient ionization; LC-MS; Mass spectrometry; SARMs

DOI:  https://doi.org/10.1016/j.talanta.2024.126358
Heliyon. 2024 Jun 15. 10(11): e32124

Development and validation of an ultra-performance liquid chromatography tandem mass spectrometry for target determination of bioactive compounds in Dendrobium spp.

Jingjing Liang, Ruyan Chen, Wenping Zhang, Lei Luo, Yun Wang, Qing Shen.

  As a medicine-food homology herb, Dendrobium spp. has versatile applications in modern medicine and food industry. Herein, an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based method was established for simultaneous quantification of six active components, including gigantol, erianin, naringenin, quercetin, rutin, and p-coumaric acid in Dendrobium spp., on the basis of optimized sample preparation, mass spectrometry conditions, and chromatography conditions. Sample extraction was carried out using methanol at a temperature of 60 °C, followed by separation on a T3 C18 column utilizing a gradient eluting program. The results demonstrated excellent linearity (r > 0.999) for the six active components within a specified concentration range. The average recovery rates ranged from 84.7 % to 106.9 %, and the precision (RSD) was within 7.4 %. The detection and quantification limits of this method ranged from 0.34 to 4.17 ng mL-1 and 1.12-13.91 ng mL-1, respectively. The established method demonstrates high accuracy and reliability and is applicable in practical sample detection. Different Dendrobium spp. exhibit specific variations in compound composition, with D. fimbriatum Hook. having a higher content of benzyl compounds and D. crystallinum. Rchb. f. having a higher content of flavonoids. This study provides experimental evidence for the quality and safety regulation of Dendrobium spp.

Keywords:  Bioactive compounds; Dendrobium spp.; Quality and safety regulation; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.heliyon.2024.e32124
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jun 13. pii: S1570-0232(24)00216-2. [Epub ahead of print]1242 124207

Quick and high-throughput quantification of β-agonist residues in bovine liver, meat, milk, kidney, poultry, and egg using dispersive solid phase extraction.

Omar Khaled, Lamia Ryad, Nermine Gad.

  A reliable liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (LC-Q-Orbitrap HRMS) method was developed for the simultaneous identification and quantification of 13 β-agonist residues in bovine liver, meat, milk, kidney, poultry, and egg. Dispersive-solid phase extraction (d-SPE) using acetonitrile (ACN) was used to prepare the samples. The analyte in the extracts was separated on a reversed-phase Accucore aQ (50 mm × 2.1 mm, 2.6 μm) using a mobile phase of an aqueous solution containing 2 mM ammonium acetate and acetonitrile (ACN) 0.1 % formic acid. The method was validated in accordance with Commission Implementing Regulation (CIR) EU 2021/808 at six different concentrations ranging from 0.1 to 5 μg/kg. The mean recoveries ranged from 65 to 94 %, while repeatability and reproducibility values were all below 13 %. The linearity, as correlation coefficients (R2) ranged from 0.9955 to 0.9999. The decision limit (CCα) and detection capability (CCβ) ranges were 0.11-0.13 µg/kg and 0.12-0.15 µg/kg, respectively. The limits of detection (LOD) and limits of quantification (LOQ) were in the range of 0.004-0.048 μg/kg and 0.010-0.075 μg/kg, respectively. Of the 180 samples that were collected from local markets in Egypt, 21.11 % had β-agonist residues. The mean concentration (µg/kg) and detection frequency (%) of the most frequently found β-agonist in the samples were as follows: terbutaline (2.63 µg/kg and 90 %), ractopamine (5.14 µg/kg and 23.3 %). The method's applicability was verified by successfully completing two rounds of proficiency testing (PT).

Keywords:  Egg; Food of animal origin; LC-Q-Orbitrap HRMS; Method validation; Poultry meat; β-agonist residues

DOI:  https://doi.org/10.1016/j.jchromb.2024.124207
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jun 10. pii: S1570-0232(24)00205-8. [Epub ahead of print]1242 124196

Toxicologic analysis of 35 drugs in post mortem human blood samples with focus on antihypertensive and antiarrhythmic drugs.

Julian Bickel, Hilke Jungen, Alexander Müller, Anne Szewczyk, Benjamin Ondruschka, Stefanie Iwersen-Bergmann.

  Antiarrhythmic and antihypertensive drugs are frequently encountered in post mortem analysis, and the question may arise as to whether they were administered in therapeutic doses, and if they were taken in accidental, intentional, or suicidal overdose scenarios. Therefore, a novel analytical method was developed and validated for the quantification of 35 drugs with toxicological relevance, including antihypertensive and antiarrhythmic drugs (ajmaline, amlodipine, amiodarone, atenolol, bisoprolol, carvedilol, clonidine, desethylamiodarone, diltiazem, donepezil, doxazosin, dronedarone, esmolol, flecainide, lercanidipine, lidocaine, metoprolol, nebivolol, nimodipine, pindolol, prajmaline, propafenone, propranolol, sotalol, urapidil, and verapamil), as well as other medications commonly found in combination (sildenafil, tadalafil, atorvastatin, clopidogrel, dapoxetine, memantine, pentoxifylline, rivastigmine, and ivabradine). The method enables simultaneous identification and quantification in blood samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Validation exhibited excellent linearity across the concentration range for all analytes. Precision and accuracy were within acceptable limits, with bias and relative standard deviation (RSD) values consistently below 9 % and 10 %, respectively. Selectivity and specificity assessments confirmed the absence of any interference from contaminants or co-extracted drugs. The method demonstrated very high sensitivity, with limits of detection (LOD) as low as 0.01 ng/ml and limits of quantification (LOQ) as low as 0.04 ng/ml. Extraction recovery exceeded 57.5 % for all analytes except atenolol, and matrix effects were <17 % for all analytes except pindolol. Processed sample stability evaluations revealed consistent results with acceptable deviations for all analytes. In addition, the method was specifically tested for the use in post mortem analysis. The applicability of our method was demonstrated by the analysis of two authentic human autopsy blood samples.

Keywords:  Antiarrhythmic drugs; Antihypertensive drugs; Forensic toxicology; LC/MS/MS; Post mortem analysis

DOI:  https://doi.org/10.1016/j.jchromb.2024.124196