bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2024–07–28
thirty-six papers selected by
Sofia Costa, Matterworks
  1. Determination of tetrodotoxin in human plasma and urine using online MCX SPE column cleanup coupled with liquid chromatography-tandem mass spectrometry.
  2. A candidate reference measurement procedure for quantification of glycocholic acid in human serum based on isotope dilution liquid chromatography-tandem mass spectrometry.
  3. Bridging Targeted (Zeno MRM-HR) and Untargeted (SWATH) LC-HRMS in a Single Run for Sensitive Exposomics.
  4. Development of a cyclic ion mobility spectrometry-mass spectrometry-based collision cross-section database of permethylated human milk oligosaccharides.
  5. Development and validation of a simultaneous quantitative analytical method for two Alternaria toxins and their metabolites in plasma and urine using ultra-high-performance liquid chromatography-tandem mass spectrometry.
  6. A radioactivity-mass spectrometry calibration method coupled with biosynthesis to generate a metabolite standard for enzyme kinetics studies.
  7. Automated Liquid Handling Extraction and Rapid Quantification of Underivatized Amino Acids and Tryptophan Metabolites from Human Serum and Plasma Using Dual-Column U(H)PLC-MRM-MS and Its Application to Prostate Cancer Study.
  8. Exploring Ion Mobility Mass Spectrometry Data File Conversions to Leverage Existing Tools and Enable New Workflows.
  9. Combined matrix-assisted laser desorption/ionisation-mass spectrometry imaging with liquid chromatography-tandem mass spectrometry for observing spatial distribution of lipids in whole Caenorhabditis elegans.
  10. Development of LC-MS/MS method for quantification of Lurasidone using volumetric absorptive microsampling (VAMS); a comparative study between dried blood and plasma samples.
  11. Online Charge-Generation Derivatization by Electrochemical Radical Cations of Thianthrene: Mass Spectrometry Imaging of Estrogens in Biological Tissues.
  12. Simultaneous analysis of DL-Amino acids in foods and beverages using a highly sensitive chiral resolution labeling reagent.
  13. A Novel UHPLC-MS/MS Based Method for Isomeric Separation and Quantitative Determination of Cyanogenic Glycosides in American Elderberry.
  14. Determination of 465 psychoactive substances, drugs and their metabolites in urine by LC-MS/MS.
  15. Assessment of remdesivir and its nucleoside metabolite in beagle dogs and healthy humans by liquid chromatography coupled with triple quadrupole mass spectrometry.
  16. Latest Developments in Direct and Non-Direct LC-MS Methods Based on Liquid Electron Ionization (LEI).
  17. Evaluation and comparison of liquid chromatography/high-resolution mass spectrometry platforms for the separation and characterization of ginsenosides from the leaves of Panax ginseng.
  18. Separation of weak acids, neutral compounds, and permanent anions using sequential elution liquid chromatography with tandem columns.
  19. Development of UPLC-MS/MS method for the simultaneous quantification of valproic acid and phenytoin in human plasma and application to study pharmacokinetic interaction in epilepsy patients.
  20. Formation of Micelles by Nonionic Detergent Molecules Leads to the Breakthrough Peak in Reversed-Phase Ultraperformance Liquid Chromatography (UPLC).
  21. Skin Surface Sebum Analysis by ESI-MS.
  22. Microfluidic Sample Preparation for Multiplexed Single-Cell Proteomics Using a Nested Nanowell Chip.
  23. Potency analysis of twelve cannabinoids in industrial hemp via ultrahigh-performance liquid chromatography-tandem mass spectrometry.
  24. A Bottom-Up Liquid Chromatography-Tandem Mass Spectrometry Method for Therapeutic Drug Monitoring of Infliximab: Method Development, Comparison With 2 Enzyme-Linked Immunosorbent Assay Methods, and Evaluation of Anti-Drug Antibody Interference.
  25. Direct Enantiomer Differentiation of Drugs and Drug-Like Compounds via Noncovalent Copper-Amino Acid Complexation and Ion Mobility-Mass Spectrometry.
  26. QuEChERS Extraction and Simultaneous Quantification in GC-MS/MS of Hexahydrocannabinol Epimers and Their Metabolites in Whole Blood, Urine, and Oral Fluid.
  27. BreathXplorer: Processing Online Breathomics Data Generated from Direct Analysis Using High-Resolution Mass Spectrometry.
  28. Monitoring Epoxidized Soybean Oil Degradation Using Liquid Chromatography-Mass Spectrometry and In Silico Spectral Libraries.
  29. Effects of Relative Humidity and Phase on the Molecular Detection of Nascent Sea Spray Aerosol Using Extractive Electrospray Ionization.
  30. A robust ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of 10 components in glutathione cycle.
  31. External Data Systems Enable Enhanced (and Sustainable) Fourier Transform Mass Spectrometry Imaging for Legacy Hybrid Linear Ion Trap-Orbitrap Platforms.
  32. 12-Plex Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tag-Enabled High-Throughput Quantitative Glycomics.
  33. Conversion of 5α-Androstane-3α,17β-diol to bis[(4-dimethylamino)phenyl carbamate] derivative for sensitive determination of its rat brain level by LC/ESI-MS/MS.
  34. Detection of thyroid hormones in urine by liquid chromatography coupled to tandem mass spectrometry.
  35. Development of the headspace gas chromatography-tandem mass spectrometry method for ethylene oxide and ethylene chlorohydrin residue in medical devices.
  36. Uncertainty management for In Silico screening of reversed-phase liquid chromatography methods for small compounds.
  1. J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jun 14. pii: S1570-0232(24)00183-1. [Epub ahead of print]1244 124174
    Determination of tetrodotoxin in human plasma and urine using online MCX SPE column cleanup coupled with liquid chromatography-tandem mass spectrometry.
    Li Fang, Fengmei Qiu, Yuchao Wang.
      An efficient technique for quantitative analysis of tetrodotoxin (TTX) in human plasma and urine has been developed, which combines liquid chromatography-tandem mass spectrometry (LC-MS/MS) with online MCX solid phase extraction (SPE) cleanup. Sample preparation, including extraction with acetonitrile containing 0.5 % acetate acid, centrifugation, and filtration, was followed by online SPE cleanup. The whole run-time was less than 15 min, including online cleanup, chromatographic separation, and re-equilibration of the online SPE - LC-MS/MS system. The parameters of sample extraction, purification, separation, and detection were optimized. The matrix-matched internal standard calibration standard curves with linear regression coefficients larger than 0.9990 were established for quantification. The LOD and LOQ for this approach were determined to be 0.1 ng/mL and 0.3 ng/mL, respectively. The recoveries for varied concentrations of TTX in human plasma and urine were 84.9-104.2 % and 89.2-109.6 %, respectively. The matrix effects of TTX in human plasma and urine matrices were 85.5 % and 74.3 %, respectively, and both the inter- and intra-day precision values were less than 9.5 %. This analytical method was successfully employed for detecting TTX in biological samples from a poisoned patient who accidentally ingested the nassarius glans.
    Keywords:  Online SPE cleanup; Plasma; Poisoning; Tetrodotoxin; Urine
    DOI:  https://doi.org/10.1016/j.jchromb.2024.124174
  2. Anal Bioanal Chem. 2024 Jul 24.
    A candidate reference measurement procedure for quantification of glycocholic acid in human serum based on isotope dilution liquid chromatography-tandem mass spectrometry.
    Pingping Zhang, Huimin Wang, Man Liang, Zhifang Wang, Chunlong Liu, Yanlin Han.
      Accurate measurement of serum glycocholic acid (GCA) is crucial for evaluating the activity of chronic hepatitis. Moreover, GCA is a novel identified biomarker for hepatocellular carcinoma. Although some laboratories have used the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure GCA in recent years, the problem of potential interference of GCA analogues has not been solved well yet. Neither reference measurement procedures nor reference materials for GCA have been listed in the Joint Committee for Traceability in Laboratory Medicine (JCTLM) database. For standardization of GCA, it is urgent to establish a candidate measurement procedure for GCA. In this study, a candidate reference measurement procedure for the quantification of GCA in human serum based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) by a two-step sample pretreatment of protein precipitation and MAX solid-phase extraction was developed and validated. GCA can be completely separated from its structural analogues with gradient elution in 9 min compared with short time gradients published in previous literature by Huang's group. Method validation indicated perfect quantitation precision with intra-day and inter-day values that were ≤1.30% and ≤1.80%, respectively. The method showed excellent linearity with high regression coefficients (R2 > 0.999) over a range of 0.92 ng/g-38.38 μg/g and perfect recoveries at three spiked levels (99.87-100.43%). No interference, matrix effect, and carryover were observed. Moreover, the cRMP was successfully applied to measure GCA in serum samples and compared with two immunoassays in a clinical laboratory. As a candidate reference method, this method can promote a GCA standardization program.
    Keywords:  Candidate reference measurement procedure; Glycocholic acid; Hepatobiliary disease; Isotope dilution liquid chromatography-tandem mass spectrometry
    DOI:  https://doi.org/10.1007/s00216-024-05449-9
  3. Anal Chem. 2024 Jul 26.
    Bridging Targeted (Zeno MRM-HR) and Untargeted (SWATH) LC-HRMS in a Single Run for Sensitive Exposomics.
    Vinicius Verri Hernandes, Benedikt Warth.
      Traditionally, chemical exposure has been assessed by low-resolution mass spectrometry via targeted approaches due to the typically extremely low concentration of such compounds in biological samples. Nevertheless, untargeted approaches are now becoming a promising tool for a broader investigation of the exposome, covering additional compounds, their biotransformation products, and possible metabolic alterations (metabolomics). However, despite broad compound coverage, untargeted metabolomics still underperforms in ultratrace biomonitoring analysis. To overcome these analytical limitations, we present the development of the first combined targeted/untargeted LC-MS method, merging MRM-HR and SWATH experiments in one analytical run, making use of Zeno technology for improved sensitivity. Multiple reaction monitoring transitions were optimized for 135 highly diverse toxicants including mycotoxins, plasticizers, PFAS, personal care products ingredients, and industrial side products as well as potentially beneficial xenobiotics such as phytohormones. As a proof of concept, standard reference materials of human plasma (SRM 1950) and serum (SRM 1958) were analyzed with both Zeno MRM-HR + SWATH and SWATH-only methodologies. Results demonstrated a significant increase in sensitivity represented by the detection of lower concentration levels in spiked SRM materials (mean value: 2.2 and 3 times lower concentrations for SRMs 1950 and 1958, respectively). Overall, the detection frequency was increased by 68% (19 to 32 positive detections) in the MRM-HR + SWATH mode compared to the SWATH-only. This work presents a promising avenue for addressing the outstanding key challenge in the small-molecule omics field: finding a balance between high sensitivity and broad chemical coverage. It was demonstrated for exposomic applications but might be transferred to lipidomics and metabolomics workflows.
    DOI:  https://doi.org/10.1021/acs.analchem.4c01630
  4. J Mass Spectrom. 2024 Aug;59(8): e5076
    Development of a cyclic ion mobility spectrometry-mass spectrometry-based collision cross-section database of permethylated human milk oligosaccharides.
    Sanaz C Habibi, Victoria R Bradford, Sophie C Baird, Shadrack Wilson Lucas, Christopher D Chouinard, Gabe Nagy.
      Human milk oligosaccharides (HMOs) are an important class of biomolecules responsible for the healthy development of the brain-gut axis of infants. Unfortunately, their accurate characterization is largely precluded due to a variety of reasons - there are over 200 possible HMO structures whereas only 10s of these are available as authentic analytical standards. Furthermore, their isomeric heterogeneity stemming from their many possible glycosidic linkage positions and corresponding α/β anomericities further complicates their analyses. While liquid chromatography coupled to tandem mass spectrometry remains the gold standard for HMO analyses, it often times cannot resolve all possible isomeric species and thus warrants the development of other orthogonal approaches. High-resolution ion mobility spectrometry coupled to mass spectrometry has emerged as a rapid alternative to condensed-phase separations but largely has remained limited to qualitative information related to the resolution of isomers. In this work, we have assessed the use of permethylation to improve both the resolution and sensitivity of HMO analyses with cyclic ion mobility separations coupled with mass spectrometry. In addition to this, we have developed the first-ever high-resolution collision cross-section database for permethylated HMOs using our previously established calibration protocol. We envision that this internal reference database generated from high-resolution cyclic ion mobility spectrometry-mass spectrometry will greatly aid in the accurate characterization of HMOs and provide a valuable, orthogonal, approach to existing liquid chromatography-tandem mass spectrometry-based methods.
    Keywords:  collision cross‐section; human milk oligosaccharides; ion mobility spectrometry; mass spectrometry; separation science
    DOI:  https://doi.org/10.1002/jms.5076
  5. J Sep Sci. 2024 Jun;47(12): e2400223
    Development and validation of a simultaneous quantitative analytical method for two Alternaria toxins and their metabolites in plasma and urine using ultra-high-performance liquid chromatography-tandem mass spectrometry.
    Dan Zhang, Bolin Liu, Tingting Xiao, Yan Wang, Ziwei Zhao, Ji'an Xie, Weidong Li, Rui Li, Jie Cui.
      Much more attention has been paid to the contamination of Alternaria toxins because of food contamination and the threat to human health. In this study, an ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous detection of the prototypical alternariol, alternariol monomethylether, and the metabolites 4-oxhydryl alternariol, and alternariol monomethylether 3-sulfate ammonium salt of Alternaria toxins. The positive samples were used as matrix samples to optimize the different experimental conditions. 0.01% formic acid solution and acetonitrile were used as the mobile phase, and analytes were scanned in negative electron spray ionization under multiple reaction monitoring, and quantitative determination by isotope internal standard method. Application of this method to samples of human plasma and urine showed the detection of the above analytes. The results showed that the recoveries were from 80.40% to 116.4%, intra-day accuracy was between 0.6% and 8.0%, and inter-day accuracy was between 1.1% and 12.1%. The limit of detection of the four analytes ranged from 0.02 to 0.6 µg/L in urine, and 0.02 to 0.5 µg/L in plasma, respectively. Thus, the developed method was rapid and accurate for the simultaneous detection of analytes and provided a theoretical basis for the risk assessment of Alternaria toxins for human exposure.
    Keywords:  Alternaria; UHPLC‐MS/MS; mycotoxins; plasma; urine
    DOI:  https://doi.org/10.1002/jssc.202400223
  6. J Pharm Sci. 2024 Jul 23. pii: S0022-3549(24)00264-8. [Epub ahead of print]
    A radioactivity-mass spectrometry calibration method coupled with biosynthesis to generate a metabolite standard for enzyme kinetics studies.
    Ting Wang, Cody Thomas, Bachir Latli, Matt Hrapchak, Mitchell E Taub.
      In early stages of drug development, the absence of authentic metabolite standards often results in semi-quantitative measurements of metabolite formation in reaction phenotyping studies using mass spectrometry (MS), leading to inaccuracies in the determination of enzyme kinetic parameters, such as the Michaelis constant (Km). Moreover, it is impossible to ascertain the maximum rate of enzyme-catalyzed reactions (kcat or Vmax). The use of radiolabeled parent compounds can circumvent this problem. However, radiometric detection exhibits significantly lower sensitivity compared to MS. To address these challenges, we have developed a stepwise approach that leverages biosynthesized radiolabeled and non-radiolabeled metabolites as standards, enabling accurate determination of Km, kcat or Vmax without the need for authentic metabolite standards. This approach, using the carbon-14 [14C] labeled metabolite to calibrate the unlabeled metabolite (14C calibration method), combines radiometric with LC-MS/MS detection to generate both [14C]-labeled and unlabeled metabolite standard curves to ensure that the sample concentrations measured are accurately quantitated. Two case studies were presented to demonstrate the utility of this method. We first compared the accuracy of the 14C calibration method to the use of authentic standards for quantitating imipramine metabolites. Next, we biosynthesized and quantitated the metabolites of BI 894416 using 14C calibration method and evaluated the enzyme kinetics of metabolite formation. The Km values of the metabolite formation demonstrated substantially improved accuracy compared to MS semi-quantitation. Moreover, the 14C calibration method offers a streamlined approach to prepare multiple metabolite standards from a single biosynthesis, reducing the time required for structure elucidation and metabolite synthesis.
    Keywords:  (14)C calibration; HLM; LC-MS/MS; authentic standards; drug metabolism; enzyme kinetics; in vitro; metabolite standards; radiometric detection; small molecule
    DOI:  https://doi.org/10.1016/j.xphs.2024.07.019
  7. Metabolites. 2024 Jun 30. pii: 370. [Epub ahead of print]14(7):
    Automated Liquid Handling Extraction and Rapid Quantification of Underivatized Amino Acids and Tryptophan Metabolites from Human Serum and Plasma Using Dual-Column U(H)PLC-MRM-MS and Its Application to Prostate Cancer Study.
    Tobias Kipura, Madlen Hotze, Alexa Hofer, Anna-Sophia Egger, Lea E Timpen, Christiane A Opitz, Paul A Townsend, Lee A Gethings, Kathrin Thedieck, Marcel Kwiatkowski.
      Amino acids (AAs) and their metabolites are important building blocks, energy sources, and signaling molecules associated with various pathological phenotypes. The quantification of AA and tryptophan (TRP) metabolites in human serum and plasma is therefore of great diagnostic interest. Therefore, robust, reproducible sample extraction and processing workflows as well as rapid, sensitive absolute quantification are required to identify candidate biomarkers and to improve screening methods. We developed a validated semi-automated robotic liquid extraction and processing workflow and a rapid method for absolute quantification of 20 free, underivatized AAs and six TRP metabolites using dual-column U(H)PLC-MRM-MS. The extraction and sample preparation workflow in a 96-well plate was optimized for robust, reproducible high sample throughput allowing for transfer of samples to the U(H)PLC autosampler directly without additional cleanup steps. The U(H)PLC-MRM-MS method, using a mixed-mode reversed-phase anion exchange column with formic acid and a high-strength silica reversed-phase column with difluoro-acetic acid as mobile phase additive, provided absolute quantification with nanomolar lower limits of quantification within 7.9 min. The semi-automated extraction workflow and dual-column U(H)PLC-MRM-MS method was applied to a human prostate cancer study and was shown to discriminate between treatment regimens and to identify metabolites responsible for discriminating between healthy controls and patients on active surveillance.
    Keywords:  LC-MS; amino acids; automation; mixed-mode chromatography; prostate cancer; tryptophan metabolites analysis
    DOI:  https://doi.org/10.3390/metabo14070370
  8. J Am Soc Mass Spectrom. 2024 Jul 26.
    Exploring Ion Mobility Mass Spectrometry Data File Conversions to Leverage Existing Tools and Enable New Workflows.
    Sarah M Stow, Bryson C Gibbons, Leonard C Rorrer Iii, Lauren Royer, Rebecca S Glaskin, Gordon W Slysz, Ruwan T Kurulugama, John C Fjeldsted, Daniel DeBord, Aivett Bilbao.
      Ion mobility (IM) is often combined with LC-MS experiments to provide an additional dimension of separation for complex sample analysis. While highly complex samples are better characterized by the full dimensionality of LC-IM-MS experiments to uncover new information, downstream data analysis workflows are often not equipped to properly mine the additional IM dimension. For many samples the data acquisition benefits of including IM separations are all that is necessary to uncover sample information and the full dimensionality of the data is not required for data analysis. Postacquisition reduction and adaptation of the dimensions of LC-IM-MS and IM-MS experiments into an LC-MS format opens the possibility to use a plethora of existing software tools. In this work, we developed data file conversion tools to reduce the complexity of IM data analysis. Three data file transformations are introduced in the PNNL PreProcessor software: (1) mapping the IM axis to the LC axis for IM-MS data, (2) converting the drift time vs m/z space to CCS/z vs m/z space, and (3) transforming All Ions IM/MS mobility aligned fragmentation data to a standard LC-MS DDA data file format. These new data file conversions are demonstrated with corresponding lipidomics and proteomics workflows that leverage existing LC-MS data analysis software to highlight the benefits of the data transformations.
    Keywords:  all ions IM/MS fragmentation; data-dependent acquisition (DDA); data-independent acquisition (DIA); ion mobility; mobility aligned fragmentation (MAF); preprocessing software
    DOI:  https://doi.org/10.1021/jasms.4c00220
  9. Rapid Commun Mass Spectrom. 2024 Sep;38(17): e9850
    Combined matrix-assisted laser desorption/ionisation-mass spectrometry imaging with liquid chromatography-tandem mass spectrometry for observing spatial distribution of lipids in whole Caenorhabditis elegans.
    Michiel Vandenbosch, Erika R Amstalden van Hove, Ronny Mohren, Isabeau Vermeulen, Henry Dijkman, Ron M A Heeren, Pim E G Leonards, Samantha Hughes.
       RATIONALE: Matrix-assisted laser desorption/ionisation-mass spectrometry imaging (MALDI-MSI) is a powerful label-free technique for biomolecule detection (e.g., lipids), within tissue sections across various biological species. However, despite its utility in many applications, the nematode Caenorhabditis elegans is not routinely used in combination with MALDI-MSI. The lack of studies exploring spatial distribution of biomolecules in nematodes is likely due to challenges with sample preparation.
    METHODS: This study developed a sample preparation method for whole intact nematodes, evaluated using cryosectioning of nematodes embedded in a 10% gelatine solution to obtain longitudinal cross sections. The slices were then subjected to MALDI-MSI, using a RapifleX Tissuetyper in positive and negative polarities. Samples were also prepared for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using an Exploris 480 coupled to a HPLC Vanquish system to confirm the MALDI-MSI results.
    RESULTS: An optimised embedding method was developed for longitudinal cross-sectioning of individual worms. To obtain longitudinal cross sections, nematodes were frozen at -80°C so that all worms were rod shaped. Then, the samples were defrosted and transferred to a 10% gelatine matrix in a cryomold; the worms aligned, and the whole cryomold submerged in liquid nitrogen. Using MALDI-MSI, we were able to observe the distribution of lipids within C. elegans, with clear differences in their spatial distribution at a resolution of 5 μm. To confirm the lipids from MALDI-MSI, age-matched nematodes were subjected to LC-MS/MS. Here, 520 lipids were identified using LC-MS/MS, indicating overlap with MALDI-MSI data.
    CONCLUSIONS: This optimised sample preparation technique enabled (un)targeted analysis of spatially distributed lipids within individual nematodes. The possibility to detect other biomolecules using this method thus laid the basis for prospective preclinical and toxicological studies on C. elegans.
    DOI:  https://doi.org/10.1002/rcm.9850
  10. J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jul 22. pii: S1570-0232(24)00264-2. [Epub ahead of print]1244 124255
    Development of LC-MS/MS method for quantification of Lurasidone using volumetric absorptive microsampling (VAMS); a comparative study between dried blood and plasma samples.
    Madhura Rajadhyaksha, Vaishali Londhe.
      The ecological impact of biological, chemical, and analytical research practices, including toxic reagents and biohazardous waste, has led to the development of alternative sampling and extraction techniques for bioanalysis. Microsampling (sample volume < 50 µL) aligns with the 3Rs principle, allowing multiple sampling points from the same animal at different time points and improving animal welfare. A bioanalytical method was developed to investigate factors related to bioanalytical challenges and the implementation of microsampling techniques. An LC-MS/MS method for Volumetric Absorptive Microsampling (VAMS), 20 µL, was developed for quantifying Lurasidone using a liquid-liquid extraction technique. The method uses a C18, Phenomenex column for chromatographic separation and a mobile phase composition of Methanol, Acetonitrile, and Water with 0.1 % HFBA. The method was validated over a concentration range of 5.0 to 1200.0 ng/mL and achieved acceptable precision and accuracy. The recovery for analyte from VAMS was approximately 40% at four different concentrations and is consistent (%CV < 15), with no significant differences among HCT levels. The matrix factor ranged between 85.00 and 115.00 %, showing no substantial issues with reduced or enhanced signal. The stability data showed no significant degradation of LUR in VAMS samples when stored at room temperature for 15 days. The newly established method for Lurasidone confirmed the use of VAMS sampling method and its analysis on LC-MS/MS. Further, the data obtained from microsampling techniques was compared with conventional (plasma) technique, as proof-of-concept, and it confirms the agreement between the two methods. The study supports the advantages of microsampling in protecting the environment and animals while maintaining scientific judgement.
    Keywords:  Bioanalytical method validation; Dried matrix methods; Microsampling; VAMS
    DOI:  https://doi.org/10.1016/j.jchromb.2024.124255
  11. Anal Chem. 2024 Jul 20.
    Online Charge-Generation Derivatization by Electrochemical Radical Cations of Thianthrene: Mass Spectrometry Imaging of Estrogens in Biological Tissues.
    Yingchao Liu, Jiahui Bai, Xiaoxia Dong, Yuqi Cao, Mingmai Bao, Yingjie Lu, Hui Zeng, Lixing Zhan, Yinlong Guo.
      Estrogens play a significant role in endocrinology and oncology. Although separation methods coupled with mass spectrometry (MS) have emerged as a powerful tool for studying estrogens, imaging the spatial distributions of estrogens is crucial but remains challenging due to its low endogenous concentration and poor ionization efficiency. Charge-generation derivatization, such as N-alkylpyridinium quaternization and S-methyl thioetherification, represents a method wherein neutral molecules involving analytes and derivatization reagents undergo chemical reactions to establish permanent charges directly onto the analytes to improve detection sensitivity. Here, we developed a novel derivatization reagent, thianthrene (TT), which enabled oxidization to radical cations ([TT]•+) using an electrochemical method and completed the online charge-generation derivatization of estrogens on a mass spectrometry imaging platform. In this strategy, [TT]•+ can efficiently and selectively derivatize estrogens via an electrophilic aromatic substitution reaction. Results indicated that derivatization with [TT]•+ can significantly enhance imaging sensitivity (3 orders of magnitude), enabling the visualization of estrogen and its metabolites in ovarian and breast tissues. Furthermore, a higher mass intensity of these estrogens was captured in breast para-cancerous tissues than in cancerous tissues, which might provide estrogens spatial dimension information for further research on the initiation and progression of breast cancer.
    DOI:  https://doi.org/10.1021/acs.analchem.4c02086
  12. J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jul 20. pii: S1570-0232(24)00248-4. [Epub ahead of print]1244 124239
    Simultaneous analysis of DL-Amino acids in foods and beverages using a highly sensitive chiral resolution labeling reagent.
    Makoto Ozaki, Tomomi Nakade, Motoshi Shimotsuma, Akari Ikeda, Takefumi Kuranaga, Hideaki Kakeya, Tsunehisa Hirose.
      Amino acids with various functions are abundant in living organisms and foods. Recent advances in analytical technology show that trace amounts of D-amino acids exist in living organisms and foods. In addition, studies show that these amino acids are involved in various physiological functions that differ from those of L-amino acids. Thus, a technique for analyzing DL-amino acids is required. However, the simultaneous separation and highly sensitive detection of DL-amino acids are complicated; therefore, highly sensitive analytical methods that can rapidly separate and identify compounds are required. We previously developed our original chiral resolution labeling reagents for the separation and highly sensitive detection of DL-amino acids. Here, we developed a simple method for the rapid separation and highly sensitive detection of DL-amino acids in various foods and beverages by liquid chromatography-mass spectrometry (LC-MS) using an octadecyl (C18) column after labeling with 1-fluoro-2,4-dinitrophenyl-5-D-leucine-N,N-dimethylethylenediamineamide (D-FDLDA; enantiomeric excess > 99.9 %). In addition, we synthesized a stable isotope (13C6)-labeled D-FDLDA (13C6-D-FDLDA) and established an analytical method that can accurately identify the peak of each DL-amino acid. MS sensitivity of DL-amino acids labeled with our labeling reagent was higher than that of conventional labeling reagents (Marfey's reagents). The labeling reagent was neither desorbed from each DL-amino acid nor degraded for at least 1 week at 4 °C. Furthermore, we determined the DL-amino acid contents in foods and beverages using the proposed method, and differences in the total amino acid content and D/L ratio in each food and beverage were observed.
    Keywords:  Chiral resolution labeling reagent; D-Amino acid; High sensitivity; Liquid chromatography–mass spectrometry (LC–MS)
    DOI:  https://doi.org/10.1016/j.jchromb.2024.124239
  13. Metabolites. 2024 Jun 26. pii: 360. [Epub ahead of print]14(7):
    A Novel UHPLC-MS/MS Based Method for Isomeric Separation and Quantitative Determination of Cyanogenic Glycosides in American Elderberry.
    Deepak M Kasote, Zhentian Lei, Clayton D Kranawetter, Ashley Conway-Anderson, Barbara W Sumner, Lloyd W Sumner.
      LC-MS/MS analyses have been reported as challenging for the reliable separation and quantification of cyanogenic glycosides (CNGs), especially (R)-prunasin and sambunigrin isomers found in American elderberry (Sambucus nigra L. subsp. canadensis (L.) Bolli). Hence, a novel multiple reaction monitoring (MRM)-based ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated in the present study for simultaneous separation and quantification of five CNGs, including amygdalin, dhurrin, linamarin, (R)-prunasin, and (S)-prunasin (commonly referred to as sambunigrin). Initially, the role of ammonium formate was investigated as an aqueous mobile-phase additive in developing MRM-based UHPLC-MS/MS. Later, chromatographic conditions for the resolved separation of (R)-prunasin and sambunigrin were identified. Validation studies confirmed that the developed method has good linearity and acceptable precision and accuracy. A noticeable matrix effect (mainly signal enhancement) was observed in leaf samples only. This method was used to detect and quantify CNGs, including (R)-prunasin and sambunigrin, in leaf and fruit samples of American elderberry. Among the studied CNGs, only (R)-prunasin was detected in the leaf samples. Interestingly, (S)-prunasin (sambunigrin) was not detected in the samples analyzed, even though it has been previously reported in elderberry species.
    Keywords:  (R)-prunasin; (S)-prunasin; UHPLC-MS/MS; cyanogenic glycosides; elderberry; isomeric separation; mobile phase additives; sambunigrin
    DOI:  https://doi.org/10.3390/metabo14070360
  14. Anal Methods. 2024 Jul 22.
    Determination of 465 psychoactive substances, drugs and their metabolites in urine by LC-MS/MS.
    Jakub Czarny, Jadwiga Musiał, Jolanta Powierska-Czarny, Michał Raczkowski, Natalia Galant, Bogusław Buszewski, Renata Gadzała-Kopciuch.
      Due to the emergence of novel psychoactive substances on the drug market, there is a growing demand for analytical methods allowing identification and determination of as many psychoactive substances as possible in the shortest possible time, which can be easily expanded to include additional analytes appearing in street trade. Immunochemical methods are not sufficient to meet constantly growing requirements. Therefore, the aim of the study was to develop an analytical method enabling quick analysis of urine samples for psychoactive substances, drugs and their metabolites. Liquid-liquid extraction (LLE) and liquid chromatography coupled with mass spectrometry (LC-MS/MS) were used for this purpose. Using available analytical standards, the operating parameters of the mass spectrometer were selected for each of the 477 analytes, and MRM (multiple reaction monitoring) acquisition was selected for each of them. The use of analytical standards allowed for the identification of analytes separated on the chromatography column. The exceptions are methylmethcathinone isomers (3-MMC and 4-MMC), which we were unable to separate using the gradient elution method used. Extraction using acetonitrile with the addition of ammonium formate and formic acid allowed us to obtain high recoveries without the use of β-glucuronidase. Recovery values ranged from 18.43 to 119.94%. The matrix effect was eliminated by obtaining a calibration curve in the matrix. The developed analytical method was validated in accordance with SWGTOX guidelines. Only 12 substances did not meet the validation criteria (CV: ±20% and bias: ±20%). Thus the validated method makes it possible to determine 465 psychoactive substances in just 30 minutes. In the validation process, values such as the limit of detection (LOD) and the limit of quantification (LOQ) were also determined. The LODs are in the range of 3-30 ng mL-1, and the LOQs are in the range of 10-100 ng mL-1. The method was successfully applied to toxicological analyses of urine samples, which was an opportunity to develop it further to meet the needs of toxicology.
    DOI:  https://doi.org/10.1039/d4ay00777h
  15. Biomed Chromatogr. 2024 Jul 22. e5965
    Assessment of remdesivir and its nucleoside metabolite in beagle dogs and healthy humans by liquid chromatography coupled with triple quadrupole mass spectrometry.
    Naveen Kumar Dubey, Peeyush Jain, Ankit Raj, Sandeep Tiwari.
      The aim of this study was to assess the pharmacokinetics of the existing remdesivir intravenous formulation (100 mg dose) against the newly developed oral formulation (20 mg dose) for remdesivir and its active nucleoside metabolite (GS-441524) in beagle dogs followed by healthy human volunteers. A quantification method for remdesivir and its active nucleoside metabolite (GS-441524) in beagle dog and human plasma has been developed and validated using liquid chromatography coupled to triple quadrupole mass spectrometry detection. The analytical methods for beagle dogs and human differ in the calibration curve range, plasma matrix, processing volume, reconstitution volume and injection volume; however all other parameters were same in both methods. A simple protein precipitation extraction was carried out using acetonitrile containing the internal standard remdesivir D5. Remdesivir and GS-441524 were separated on an Endurus C-18P, 100 × 4.6 mm, 3 μm column and detected using a mass spectrometer with electrospray ionization in positive ion mode. The ion transitions used were m/z 603.1 → m/z 200.0 for remdesivir, m/z 292.0 → m/z 202.2 for GS-441524 and m/z 608.2 → m/z 205.1 for remdesivir D5. The calibration curve results were linear in beagle dog plasma (2.0-2,000.8 ng/ml range for remdesivir and 2.0-1,500.4 ng/ml for GS-441524) and human plasma (30.0-4,503.9 ng/ml range for remdesivir and 2.0-200.4 ng/ml for GS-441524). The recovery was >90% in beagle dog and human plasma. These methods were successfully used to determine the pharmacokinetic parameters of the intravenous injection and subcutaneous tablets dosage forms in beagle dogs and healthy humans.
    Keywords:  LC–MS/MS; beagle dog plasma; human plasma; nucleoside metabolite (GS‐441524); remdesivir
    DOI:  https://doi.org/10.1002/bmc.5965
  16. Crit Rev Anal Chem. 2024 Jul 24. 1-18
    Latest Developments in Direct and Non-Direct LC-MS Methods Based on Liquid Electron Ionization (LEI).
    Genny Grasselli, Adriana Arigò, Pierangela Palma, Giorgio Famiglini, Achille Cappiello.
      Mass spectrometry (MS) enables precise identification and quantification of molecules, particularly when combined with chromatography. The advent of atmospheric pressure ionization (API) techniques allowed the efficient coupling of liquid chromatography with MS (LC-MS), extending analyses to nonvolatile and thermolabile compounds. API techniques present limitations such as low informative capacity and reproducibility of mass spectra, increasing instrument complexity and costs. Other challenges include analyzing poorly polar molecules and matrix effects (ME), which negatively impact quantitative analyses, necessitating extensive sample purification or using expensive labeled standards. These limitations prompted the exploration of alternative solutions, leading to the development of the Liquid Electron Ionization (LEI) interface. The system has demonstrated excellent robustness and reproducibility. LEI has been employed to analyze various compounds, including pesticides, drugs of abuse, phenols, polycyclic aromatic hydrocarbons (PAHs), phthalates, and many others. Its versatility has been validated with single quadrupole, triple quadrupole, and QToF detectors, operating in electron ionization (EI) or chemical ionization (CI) modes and with both reverse phase liquid chromatography (RPLC) and normal phase liquid chromatography (NPLC). LEI has also been successfully integrated with the Microfluidic Open Interface (MOI), Membrane Introduction Mass Spectrometry (MIMS), and Microfluidic Water-Assisted Trap Focusing (M-WATF), broadening its application scope and consistently demonstrating promising results in terms of sensitivity and identification power. The most recent advancement is the development of Extractive-Liquid Sampling Electron Ionization-Mass Spectrometry (E-LEI-MS), a surface sampling and real-time analysis technique based on the LEI concept. This review article offers a comprehensive and up-to-date picture of the potential of LEI.
    Keywords:  E-LEI; LEI; Liquid chromatography; electron ionization; mass spectrometry
    DOI:  https://doi.org/10.1080/10408347.2024.2381543
  17. J Sep Sci. 2024 Jul;47(14): e2400354
    Evaluation and comparison of liquid chromatography/high-resolution mass spectrometry platforms for the separation and characterization of ginsenosides from the leaves of Panax ginseng.
    Xiaohang Li, Yadan Zou, Huizhen Cheng, Mengxiang Ding, Yang Yang, Lili Hong, Ying Xiong, Min Zhang, Xue Li, Qinhua Chen, Hongda Wang, Yuanwu Cui, Wenzhi Yang.
      The measurement of data repeatability in small-molecule metabolites acquired within and among different liquid chromatography-mass spectrometry (LC-MS) platforms is crucial for data sharing or data transfer in natural products research. This work was designed to investigate and evaluate the separation and detection performance of three commercial high-resolution LC-MS platforms (e.g., Agilent 6550 QTOF, Waters Vion IM-QTOF, and Thermo Scientific Orbitrap Exploris 120) using 68 ginsenoside references and the extract of Panax ginseng leaf. The retention time (tR), measured on these three platforms (under the same chromatography condition), showed good stability in different concentration tests, and within/among different instruments for both intra-day and inter-day precision examinations. Correlation in tR of ginsenosides was also highly determined on these three platforms. In spite of the different mass analyzers involved, these three platforms gave the accurate mass determination ability, especially enhanced resolution gained because of the ion mobility (IM) separation facilitated by IM-quadrupole time-of-flight. The current study has systematically evaluated the separation and MS detection performance enabled by three high-resolution LC-MS platforms taking ginsenosides as the template, and the reported findings can benefit the researchers for the selection of analytical platforms and the purpose of data sharing or data transfer.
    Keywords:  ginsenoside; liquid chromatography/mass spectrometry; reproducibility; retention time; stability
    DOI:  https://doi.org/10.1002/jssc.202400354
  18. J Chromatogr A. 2024 Jul 20. pii: S0021-9673(24)00552-1. [Epub ahead of print]1731 465178
    Separation of weak acids, neutral compounds, and permanent anions using sequential elution liquid chromatography with tandem columns.
    Lauren Kline Lovejoy, Joe P Foley.
      This paper discusses the development of an analytical method by an alternative separation approach, sequential elution liquid chromatography (SE-LC), to separate permanently charged ions (anions), weak acids, and neutral compounds using anion exchange and reversed-phase columns in tandem. SE-LC separates classes of compounds by group by employing two or more elution modes. Advantages to using SE-LC over conventional HPLC are a greater peak capacity and a reduced separation disorder. Importantly, the same HPLC as used for a conventional HPLC separation may be used to afford a successful SE-LC separation. Mobile phase selection and gradient optimization are integral for a successful SE-LC class separation of permanent anions, weak acids, and neutral compounds and will be discussed in detail in this paper. The most successful (best resolution and repeatability) SE-LC separation was achieved by applying isocratic elution at low pH to elute the weak acids, followed by an acetonitrile gradient to elute the neutral compounds, and last a sodium methanesulfonate gradient to elute the anionic compounds using a superficially porous C18 column coupled with a strong anion exchange (SAX) column. Repeatability (RSD) in the retention times and peak areas of the analytes was less than 0.25 % and 1.5 %, respectively.
    Keywords:  Anion-exchange chromatography; Reversed-phase chromatography; Separation disorder; Sequential elution liquid chromatography; Tandem-column liquid chromatography
    DOI:  https://doi.org/10.1016/j.chroma.2024.465178
  19. Heliyon. 2024 Jul 15. 10(13): e33630
    Development of UPLC-MS/MS method for the simultaneous quantification of valproic acid and phenytoin in human plasma and application to study pharmacokinetic interaction in epilepsy patients.
    Sil Thanh Nguyen, Duy Nguyen Ho, Thi Anh Huynh Huynh, Huyen Thu Thi Nguyen, Ngan Kim Thi Ly, Minh Van Le, Tho Vinh Minh Chau Do.
      Valproic acid and phenytoin are two prevalent antiepileptic medications known for their narrow indices and propensity for cardiovascular and respiratory system toxicity. Therefore, therapeutic drug monitoring (TDM) of valproic acid (VAL) and phenytoin (PHE) concentrations in patient plasma is extremely beneficial for improving clinical choices, avoiding adverse reactions, and optimizing treatment for individual patients. In this study, a rapid and sensitive ultra-performance liquid chromatographic tandem mass spectrometer (UPLC-MS/MS) method was developed and validated for the simultaneous quantitative determination of valproic acid (VAL) and phenytoin (PHE) in human plasma. Negative electron spray ionization (ESI-) mode with selective ion recording (SIR) was employed to determine the transitions of m/z 142.98 and m/z 250.93 for VAL and PHE, respectively. The internal standard (IS) betamethasone (BETA) was ionized using positive electron spray ionization (ESI+) and detected by multi-reaction monitoring (MRM) mode to obtain precursor ions and specific fragment ions for quantification, and the MRM transition was chosen to be m/z 393.17 → 355.16. The separation was performed using a Phenomenex Synergi Hydro-RP (4 μm, 250 × 4.6 mm, I.D.) with an isocratic mobile phase consisting of acetonitrile - water (75:25, v/v) at a flow rate of 0.8 mL/min. The column temperature was maintained at 25 °C. The lower limit of quantification of VAL and PHE was 3.6 μg/mL and 0.72 μg/mL, respectively, which resulted in a recovery of more than 85 % for most analytes. According to US-FDA bioanalytical technique validation, the specificity, intra- and inter-day precision and accuracy, matrix effect, carryover, dilution, and stability of all analytes were within acceptable ranges. This analytical method was successful in evaluating the levels of valproic acid and phenytoin in human plasma from epileptic patients.
    Keywords:  Epilepsy; Human plasma; Phenytoin; UPLC-MS/MS; Valproic acid
    DOI:  https://doi.org/10.1016/j.heliyon.2024.e33630
  20. Anal Chem. 2024 Jul 20.
    Formation of Micelles by Nonionic Detergent Molecules Leads to the Breakthrough Peak in Reversed-Phase Ultraperformance Liquid Chromatography (UPLC).
    Jinxin Zhang, Athula B Attygalle.
      A peculiar phenomenon known as "breakthrough" occurs under reversed-phase ultraperformance liquid chromatography (UPLC) conditions and has been under scrutiny for decades. This effect takes place when a large volume of analyte solution, prepared in a solvent with an eluotropic strength significantly higher than that of the initial mobile phase solvent, is injected. According to the literature, under specific experimental conditions, a substantial portion of solutes is carried by the mobile phase and detected near the dead time of the chromatographic system. This phenomenon is typically observed when the injected volume of a particular analyte is sufficiently large. However, the underlying physicochemical principles governing this phenomenon have remained elusive. We present evidence demonstrating that breakthroughs can occur even when injecting a sample of a neat solvent devoid of any solute. By mass spectrometric analysis, we identified the breakthrough peak to represent the nonionic detergent Triton. When columns are equilibrated with water, Triton molecules, present as impurities in filtered water, accumulate on the nonpolar stationary phase. Upon the introduction of a solvent with a stronger elution strength, Triton molecules retained on the stationary phase are removed. As detergents, these Triton molecules aggregate into micelles featuring a hydrophobic inner core and a hydrophilic outer shell. These hydrophilic micelles are carried by the polar mobile phase and detected as the breakthrough peak at the dead time of the chromatographic system. When analytes are present, a portion of the injected solutes is captured by the micelles and transported with the breakthrough plug. This assertion was verified and confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis of a methanolic solution of perfluorooctanoic acid (PFOA). The mass spectra corresponding to the breakthrough plug featured a peak for the PFOA anion (m/z 413) in addition to those for Triton.
    DOI:  https://doi.org/10.1021/acs.analchem.4c02375
  21. Biomolecules. 2024 Jul 03. pii: 790. [Epub ahead of print]14(7):
    Skin Surface Sebum Analysis by ESI-MS.
    Madeline Isom, Heather Desaire.
      The skin surface is an important sample source that the metabolomics community has only just begun to explore. Alterations in sebum, the lipid-rich mixture coating the skin surface, correlate with age, sex, ethnicity, diet, exercise, and disease state, making the skin surface an ideal sample source for future noninvasive biomarker exploration, disease diagnosis, and forensic investigation. The potential of sebum sampling has been realized primarily via electrospray ionization mass spectrometry (ESI-MS), an ideal approach to assess the skin surface lipidome. However, a better understanding of sebum collection and subsequent ESI-MS analysis is required before skin surface sampling can be implemented in routine analyses. Challenges include ambiguity in definitive lipid identification, inherent biological variability in sebum production, and methodological, technical variability in analyses. To overcome these obstacles, avoid common pitfalls, and achieve reproducible, robust outcomes, every portion of the workflow-from sample collection to data analysis-should be carefully considered with the specific application in mind. This review details current practices in sebum sampling, sample preparation, ESI-MS data acquisition, and data analysis, and it provides important considerations in acquiring meaningful lipidomic datasets from the skin surface. Forensic researchers investigating sebum as a means for suspect elimination in lieu of adequate fingerprint ridge detail or database matches, as well as clinical researchers interested in noninvasive biomarker exploration, disease diagnosis, and treatment monitoring, can use this review as a guide for developing methods of best-practice.
    Keywords:  ESI-MS; biomarkers; fingerprints; lipidomics; lipids; machine learning; mass spectrometry; noninvasive sampling; sebum; skin
    DOI:  https://doi.org/10.3390/biom14070790
  22. Methods Mol Biol. 2024 ;2823 141-154
    Microfluidic Sample Preparation for Multiplexed Single-Cell Proteomics Using a Nested Nanowell Chip.
    Junho Park, Tommy K Cheung, Ying Zhu.
      Mass spectrometry-based single-cell proteomics has undergone rapid progress and has become an active research area. However, because of the ultralow amount of proteins in single cells, it is still highly challenging to achieve efficient sample preparation and sensitive LC-MS detection. Here, we provide a detailed protocol for isobaric labeling-based single-cell proteomics relying on a microfluidic droplet-based sample processing technology. The protocol allows for processing both single cells and carrier samples in separate microchips using a commercially available platform (cellenONE) with high sample recovery and high throughput. We also provide an optimized LC-MS method for sensitive and robust data collection.
    Keywords:  Droplet; Mass spectrometry; Microfluidic; Multiplexed labeling; NanoPOTS; Sample preparation; Single-cell proteomics
    DOI:  https://doi.org/10.1007/978-1-0716-3922-1_10
  23. Rapid Commun Mass Spectrom. 2024 Sep;38(18): e9871
    Potency analysis of twelve cannabinoids in industrial hemp via ultrahigh-performance liquid chromatography-tandem mass spectrometry.
    Youxi Cai, Ruiting Zhang, Hao Zhang, Xiaolei Li.
       RATIONALE: With an increasing appreciation for the unique pharmacological properties associated with distinct, individual cannabinoids of Cannabis sativa, there is demand for accurate and reliable quantification for a growing number of them. In this study, we developed rapid, sensitive, selective, accurate, and validated liquid chromatography-tandem mass spectrometry for the quantification of cannabinoids.
    METHODS: Crushed industrial hemp flower and leaf sample was extracted by 95% methanol aqueous, sonicated for 30 min. UPLC-MS/MS analysis using Waters Acquity BEH-C18 column and electrospray ionization(ESI) mass spectrometry detector.
    RESULTS: The method was validated to demonstrate its reproducibility and precision, linearity, recovery investigation, and investigation of matrix effect. The concentration-response relationship for all analyzed cannabinoids were linear with R2 values >0.99, with intra- and inter-day precision and relative errors below 12%. The recovery and matrix effect were measured as 66.1%-104.1% and 70.42%-110.75%.
    CONCLUSIONS: This study established a UHPLC-MS/MS method for the simultaneous and rapid quantitative determination of twelve cannabinoids in industrial hemp flowers and leaves in 11 min. The method was used to analyze 43 industrial hemp flower and leaf samples, with the data being statistically analyzed. Based on the statistical analysis of the cannabinoids, hemp from different regions and different varieties were well distinguished by the PLS-DA model, with the main contributing substances being cannabidiol, Δ9-tetrahydrocannabinol, and Δ8-tetrahydrocannabinol.
    DOI:  https://doi.org/10.1002/rcm.9871
  24. Arch Pathol Lab Med. 2024 Jul 23.
    A Bottom-Up Liquid Chromatography-Tandem Mass Spectrometry Method for Therapeutic Drug Monitoring of Infliximab: Method Development, Comparison With 2 Enzyme-Linked Immunosorbent Assay Methods, and Evaluation of Anti-Drug Antibody Interference.
    Sang-Mi Kim, Hyeonju Oh, Sung Noh Hong, Mi Jin Kim, Yon Ho Choe, Soo-Youn Lee.
       CONTEXT.—: Therapeutic drug monitoring is recommended to optimize infliximab use and improve outcome in chronic inflammatory disorders.
    OBJECTIVE.—: To describe a simple and affordable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure infliximab in serum.
    DESIGN.—: Infliximab was measured using winged stable isotope-labeled peptides as internal standards. Linearity, lower limit of measuring interval, limit of detection, precision, accuracy, carryover, and ion suppression were evaluated. Method comparison against 2 enzyme-linked immunosorbent assay (ELISA) methods (Remsima Monitor and IDKmonitor Infliximab) and anti-drug antibody (ADA) interference were evaluated using clinical specimens from inflammatory bowel disease patients (N = 237).
    RESULTS.—: Analytical run time and sample preparation time were 5 minutes per sample and 3 hours per batch, respectively. Analytical measurement interval and limit of detection were 0.50 to 50.0 μg/mL (R2 = 0.998) and 0.25 μg/mL, respectively. The intraday and interday imprecision percentage coefficients of variation were less than 6.1%. Accuracy was 94.2% to 98.7%. No significant ion suppression or carryover was observed. Infliximab concentrations measured by LC-MS/MS showed good agreement with those measured by Remsima Monitor (mean percentage difference, 5.7%; 95% CI, -1.2% to 12.6%) but were markedly lower than those measured by IDKmonitor (-32.6%; -35.8% to -29.4%), demonstrating significant bias between ELISAs. Although a good agreement between LC-MS/MS and ELISA was observed for ADA-negative samples (-3.5%; -12.8% to 5.9%), a significant bias was observed for ADA-positive samples (13.6%; 1.7% to 25.6%).
    CONCLUSIONS.—: This simple, fast, and affordable LC-MS/MS method for infliximab quantitation could improve standardization of infliximab quantitation and optimization of infliximab use in patients with high-titer ADA.
    DOI:  https://doi.org/10.5858/arpa.2023-0573-OA
  25. Anal Chem. 2024 Jul 25.
    Direct Enantiomer Differentiation of Drugs and Drug-Like Compounds via Noncovalent Copper-Amino Acid Complexation and Ion Mobility-Mass Spectrometry.
    Benjamin K Blakley, Emanuel Zlibut, Rashi M Gupta, Jody C May, John A McLean.
      Drug enantiomers can possess vastly different pharmacological properties, yet they are identical in their chemical composition and structural connectivity. Thus, resolving enantiomers poses a great challenge in the field of separation science. Enantiomer separations necessitate interaction of the analyte with a chiral environment─in mass spectrometry-based analysis, a common approach is through a three-point interaction with a chiral selector commonly introduced during sample preparation. In select cases, the structural difference imparted through noncovalent complexation results in enantiomer-specific structural differences, facilitating measurement using a structurally selective analytical technique such as ion mobility-mass spectrometry (IM-MS). In this work, we investigate the direct IM-MS differentiation of chiral drug compounds using mononuclear copper complexes incorporating an amino acid chiral selector. A panel of 20 chiral drugs and drug-like compounds were investigated for separation, and four l-amino acids (l-histidine, l-tryptophan, l-proline, and l-tyrosine) were evaluated as chiral selectors (CS) to provide the chiral environment necessary for differentiation. Enantiomer differentiation was achieved for four chiral molecule pairs (R/S-thalidomide, R/S-baclofen, R/S-metoprolol, and d/l-panthenol) with two-peak resolution (Rp-p) values ranging from 0.7 (>10% valley) to 1.5 (baseline separation). Calibration curves relating IM peak areas to enantiomeric concentrations enabled enantiomeric excess quantitation of racemic thalidomide and metoprolol with residuals of 5.7 and 2.5%, respectively. Theoretical models suggest that CuII and l-histidine complexation around the analyte chiral center is important for gas-phase stereoselectivity. This study demonstrates the potential of combining enantioselective noncovalent copper complexation with structurally selective IM-MS for differentiating chiral drugs and drug-like compounds.
    DOI:  https://doi.org/10.1021/acs.analchem.4c02710
  26. Molecules. 2024 Jul 22. pii: 3440. [Epub ahead of print]29(14):
    QuEChERS Extraction and Simultaneous Quantification in GC-MS/MS of Hexahydrocannabinol Epimers and Their Metabolites in Whole Blood, Urine, and Oral Fluid.
    Annagiulia Di Trana, Giorgia Sprega, Giorgi Kobidze, Omayema Taoussi, Alfredo Fabrizio Lo Faro, Giulia Bambagiotti, Eva Montanari, Maria Sofia Fede, Jeremy Carlier, Anastasio Tini, Francesco Paolo Busardò, Alessandro Di Giorgi, Simona Pichini.
      Recently, hexahydrocannabinol (HHC) was posed under strict control in Europe due to the increasing HHC-containing material seizures. The lack of analytical methods in clinical laboratories to detect HHC and its metabolites in biological matrices may result in related intoxication underreporting. We developed and validated a comprehensive GC-MS/MS method to quantify 9(R)-HHC, 9(S)-HHC, 9αOH-HHC, 9βOH-HHC, 8(R)OH-9(R)-HHC, 8(S)OH-9(S)HHC, 11OH-9(R)HHC, 11OH-9(S)HHC, 11nor-carboxy-9(R)-HHC, and 11nor-carboxy-9(S)-HHC in whole blood, urine, and oral fluid. A novel QuEChERS extraction protocol was optimized selecting the best extraction conditions suitable for all the three matrices. Urine and blood were incubated with β-glucuronidase at 60 °C for 2 h. QuEChERS extraction was developed assessing different ratios of Na2SO4:NaCl (4:1, 2:1, 1:1, w/w) to be added to 200 µL of any matrix added with acetonitrile. The chromatographic separation was achieved on a 7890B GC with an HP-5ms column, (30 m, 0.25 mm × 0.25 µm) in 12.50 min. The analytes were detected with a triple-quadrupole mass spectrometer in the MRM mode. The method was fully validated following OSAC guidelines. The method showed good validation parameters in all the matrices. The method was applied to ten real samples of whole blood (n = 4), urine (n = 3), and oral fluid (n = 3). 9(R)-HHC was the prevalent epimer in all the samples (9(R)/9(S) = 2.26). As reported, hydroxylated metabolites are proposed as urinary biomarkers, while carboxylated metabolites are hematic biomarkers. Furthermore, 8(R)OH-9(R)HHC was confirmed as the most abundant metabolite in all urine samples.
    Keywords:  GC-MS/MS; QuEChERS; cannabinoids; hexahydrocannabinol epimers; hexahydrocannabinol metabolites; new psychoactive substances
    DOI:  https://doi.org/10.3390/molecules29143440
  27. J Am Soc Mass Spectrom. 2024 Jul 25.
    BreathXplorer: Processing Online Breathomics Data Generated from Direct Analysis Using High-Resolution Mass Spectrometry.
    Yukai Wang, Zhifeng Tang, Tingting Zhao, Jianming Yang, Wei Zhang, Xue Li, Tao Huan.
      Nontargeted breath analysis in real time using high-resolution mass spectrometry (HRMS) is a promising approach for high coverage profiling of metabolites in human exhaled breath. However, the information-rich and unique non-Gaussian metabolic signal shapes of real-time HRMS-based data pose a significant challenge for efficient data processing. This work takes a typical real-time HRMS technique as an example, i.e. secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS), and presents BreathXplorer, an open-source Python package designed for the processing of real-time exhaled breath data comprising multiple exhalations. BreathXplorer is composed of four main modules. The first module applies either a topological algorithm or a Gaussian mixture model (GMM) to determine the start and end points of each exhalation. Next, density-based spatial clustering of applications with noise (DBSCAN) is employed to cluster m/z values belonging to the same metabolic feature, followed by applying an intensity relative standard deviation (RSD) filter to extract real breath metabolic features. BreathXplorer also offers functions of (1) feature alignment across the samples and (2) associating MS/MS spectra with their corresponding metabolic features for downstream compound annotation. Manual inspection of the metabolic features extracted from SESI-HRMS breath data suggests that BreathXplorer can achieve 100% accuracy in identifying the start and end points of each exhalation and acquire accurate quantitative measurements of each breath feature. In a proof-of-concept study on exercise breathomics using SESI-HRMS, BreathXplorer successfully reveals the significantly changed metabolites that are pertinent to exercise. BreathXplorer is publicly available on GitHub (https://github.com/HuanLab/breathXplorer). It provides a powerful and convenient-to-use tool for the researchers to process breathomics data obtained by directly analysis using HRMS.
    DOI:  https://doi.org/10.1021/jasms.4c00152
  28. J Am Soc Mass Spectrom. 2024 Jul 23.
    Monitoring Epoxidized Soybean Oil Degradation Using Liquid Chromatography-Mass Spectrometry and In Silico Spectral Libraries.
    Paul D Hutchins, Camila A Saez Cabezas, Joshua S Enokida, Yushan Hu, Yuming Lai, Victoria Mazure, Marie Martin, Kelly Setula, John R Stutzman, James H Wade.
      Epoxidized soybean oil (ESO) is routinely used as a bioderived plasticizer and stabilizer in polyvinyl chloride (PVC), as it prolongs material integrity during dehydrochlorination. During this process, the epoxide moieties of ESO are progressively converted to chlorohydrins, which amplify ESO's inherent structural complexity. Past characterization efforts utilized separation-mass spectrometry (MS) analysis of the hydrolyzed acyl chains to simplify the complexity. However, this approach significantly increases the complexity of sample preparation and cannot directly monitor the chlorination of individual ESO species during aging. Here, we present a comprehensive LC-MS/MS data acquisition and in silico spectral library identification workflow optimized for intact ESO byproduct analysis. Detailed MS/MS fragmentation rules derived from synthesized standards were coupled with improved fragment ion intensity modeling capabilities to generate a high-fidelity spectral library for rapid ESO byproduct identification. Identification confidence was further bolstered by using retention time modeling to filter spurious MS/MS matches. Finally, we paired this informatic approach with an optimized extraction procedure and reversed-phase separation to generate a detailed timeline of more than 400 ESO species and byproducts during PVC thermal aging. These developments significantly improve our ability to detect, characterize, and understand ESO degradation in complex PVC formulations with new levels of molecular resolution.
    DOI:  https://doi.org/10.1021/jasms.4c00212
  29. Anal Chem. 2024 Jul 24.
    Effects of Relative Humidity and Phase on the Molecular Detection of Nascent Sea Spray Aerosol Using Extractive Electrospray Ionization.
    Samantha M Kruse, Paul R Tumminello, Alexia N Moore, Christopher Lee, Kimberly A Prather, Jonathan H Slade.
      Online mass spectrometry techniques, such as extractive electrospray ionization mass spectrometry (EESI-MS), present an attractive alternative for analyzing aerosol molecular composition due to reduced aerosol sample collection and handling times and improved time resolution. Recent studies show a dependence of EESI-MS sensitivity on particle size and mixing state. This study measured authentic sea spray aerosol (SSA) components generated during a phytoplankton bloom, specifically glycerol, palmitic acid, and potassium ions. We demonstrate temporal variability and trends dependent on specific biological processes occurring in seawater. We found that the EESI-MS sensitivity, after adjusting for pressure variations at the inlet and normalizing to the reagent ion, critically depends on the sample's relative humidity. Relevant SSA species exhibited heightened sensitivity at an elevated relative humidity near the deliquescence relative humidity of sea salt and poorer sensitivity with sparse detection below the efflorescence relative humidity. Modeling the reagent ion's diffusive depth demonstrates that the sample aerosol particle viscosity governs the relative humidity dependence because it modulates the particle's coagulation efficiency and distance the reagent ion diffuses and reacts with components in the particle bulk. The effects of particle size and mixing state are discussed, revealing improved sensitivity of phase-separated components present along the particle surface. This work highlights the importance of the particle phase state in detecting and quantifying molecular components within authentic and complex aerosol particles and the utility of EESI-MS for measuring SSA composition.
    DOI:  https://doi.org/10.1021/acs.analchem.4c02871
  30. J Sep Sci. 2024 Jun;47(12): e2400247
    A robust ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of 10 components in glutathione cycle.
    Xiang-Ling Qin, Si-Yu Wang, Qi-Long Li, Jun-Jie Wang, Zhong-Wei Yao, Jin-Hao Zhu, Lin-Xia Chen, Jie-Ge Huo, Song-Lin Li, Jing Zhou, He Zhu.
      Glutathione (GSH) is an important antioxidant that is generated and degraded via the GSH cycle. Quantification of the main components in the GSH cycle is necessary to evaluate the process of GSH. In this study, a robust ultra-performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of 10 components (GSH; γ-glutamylcysteine; cysteinyl-glycine; n-acetylcysteine; homocysteine; cysteine; cystine; methionine; glutamate; pyroglutamic acid) in GSH cycle was developed. The approach was optimized in terms of derivative, chromatographic, and spectrometric conditions as well as sample preparation. The unstable thiol groups of GSH, γ-glutamylcysteine, cysteinyl-glycine, n-acetylcysteine, cysteine, and homocysteine were derivatized by n-ethylmaleimide. The derivatized and underivatized analytes were separated on an amino column with gradient elution. The method was further validated in terms of selectivity (no interference), linearity (R2 > 0.99), precision (% relative standard deviation [RSD%] range from 0.57 to 10.33), accuracy (% relative error [RE%] range from -3.42 to 10.92), stability (RSD% < 5.68, RE% range from -2.54 to 4.40), recovery (RSD% range from 1.87 to 7.87) and matrix effect (RSD% < 5.42). The validated method was applied to compare the components in the GSH cycle between normal and oxidative stress cells, which would be helpful in clarifying the effect of oxidative stress on the GSH cycle.
    Keywords:  GSH; UPLC‐MS/MS; glutathione; glutathione cycle; oxidative stress
    DOI:  https://doi.org/10.1002/jssc.202400247
  31. J Am Soc Mass Spectrom. 2024 Jul 20.
    External Data Systems Enable Enhanced (and Sustainable) Fourier Transform Mass Spectrometry Imaging for Legacy Hybrid Linear Ion Trap-Orbitrap Platforms.
    Franklin E Leach, Konstantin O Nagornov, Anton N Kozhinov, Yury O Tsybin.
      Legacy Fourier transform (FT) mass spectrometers provide robust platforms for bioanalytical mass spectrometry (MS) yet lack the most modern performance capabilities. For many laboratories, the routine investment in next generation instrumentation is cost prohibitive. Field-based upgrades provide a direct path to extend the usable lifespan of MS platforms which may be considered antiquated based on performance specifications at the time of manufacture. Here we demonstrate and evaluate the performance of a hybrid linear ion trap (LTQ)-Orbitrap mass spectrometer that has been enhanced via an external high-performance data acquisition and processing system to provide true absorption mode FT processing during an experimental acquisition. For the application to mass spectrometry imaging, several performance metrics have been improved including mass resolving power, mass accuracy, and dynamic range to provide an FTMS system comparable to current platforms. We also demonstrate, perhaps, the unexpected ability of these legacy platforms to detect usable time-domain signals up to 5 s in duration to achieve a mass resolving power 8× higher than the original platform specification.
    Keywords:  FTMS; external data acquisition and processing; imaging; sustainability; ultrahigh resolution
    DOI:  https://doi.org/10.1021/jasms.4c00145
  32. Methods Mol Biol. 2024 ;2823 155-172
    12-Plex Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tag-Enabled High-Throughput Quantitative Glycomics.
    Miyang Li, Yu Feng, Min Ma, Lingjun Li.
      Glycans, which are ubiquitously distributed on most proteins and cell surfaces, are a class of important biomolecules playing crucial roles in various biological processes such as molecular recognition and cellular communication. Modern mass spectrometry (MS) coupled with novel chemical probe labeling strategies has greatly advanced analysis of glycans. However, the requirement of high-throughput and robust quantitative analysis still calls for the development of more advanced tools. Recently, we devised isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags for 4-plex N-glycan analysis. To further improve the throughput, we utilized the mass-defect strategy and expanded the multiplexing capacity to 12 channels without changing the chemical structure of the SUGAR tag, achieving a threefold enhancement in throughput compared with the original design and managing to perform high-throughput N-glycan analysis in a single LC - MS/MS injection. Herein, we present detailed methods for the synthesis of 12-plex SUGAR isobaric tags, the procedure to release and label the N-glycans from proteins, and the analysis by high-resolution LC-MS/MS, as well as data processing to achieve multiplexed quantitative glycomics.
    Keywords:  Isobaric labeling; Mass defect; Mass spectrometry; Multiplexed quantification; Quantitative glycomics; SUGAR; Tag synthesis
    DOI:  https://doi.org/10.1007/978-1-0716-3922-1_11
  33. Rapid Commun Mass Spectrom. 2024 Oct 15. 38(19): e9875
    Conversion of 5α-Androstane-3α,17β-diol to bis[(4-dimethylamino)phenyl carbamate] derivative for sensitive determination of its rat brain level by LC/ESI-MS/MS.
    Tatsuya Higashi, Asuka Tanaka, Shiho Tsubura, Shoichi Nishimoto-Kusunose.
       RATIONALE: 5α-Androstane-3α,17β-diol (3α,5α-Adiol) is a testosterone-derived neurosteroid and has anxiolytic and analgesic effects via γ-aminobutyric acid type A receptors as with the progesterone-derived neurosteroid, allopregnanolone (AP). Although the psychotropic drug-evoked changes in the brain AP concentration have been intensively studied, those in the brain 3α,5α-Adiol concentration remain poorly understood. One of the causes for this is the limited availability of a validated method for quantifying the brain 3α,5α-Adiol with a sufficient sensitivity and specificity, which is described in this study.
    METHODS: To enhance the detectability of 3α,5α-Adiol by electrospray ionization-tandem mass spectrometry (ESI-MS/MS), derivatization with 4-dimethylaminobenzoyl azide was employed. The brain sample was purified by solid-phase extraction and the recovered 3α,5α-Adiol and the deuterated internal standard were derivatized, then measured by liquid chromatography (LC)/ESI-MS/MS with selected reaction monitoring.
    RESULTS: The derivatized 3α,5α-Adiol, i.e., the bis[(4-dimethylamino)phenyl carbamate] derivative, provided the intense doubly-protonated molecule as the precursor ion, then the specific product ion containing the 3α,5α-Adiol-skeleton by collision-induced dissociation. The detectability of 3α,5α-Adiol was eventually increased 1000-fold by derivatization. Separation of the derivatized 3α,5α-Adiol from its stereoisomers and interfering brain components was achieved using a SunShell Biphenyl column with an isopropyl alcohol-containing mobile phase. A good linearity in the sufficient concentration range, acceptable precision and accuracy, and negligible matrix effect were demonstrated by the validation tests. The animal (rat) study using this method revealed that the brain 3α,5α-Adiol levels were unaffected by the administration of fluoxetine (FLX) and clozapine (CLZ), in contrast to the significant increase of the AP levels.
    CONCLUSION: An LC/ESI-MS/MS method capable of quantifying 3α,5α-Adiol in the rat brain using a 20-mg tissue was developed and validated. The brain levels of 3α,5α-Adiol had an entirely different behavior from those of AP due to FLX and CLZ administration.
    DOI:  https://doi.org/10.1002/rcm.9875
  34. Drug Test Anal. 2024 Jul 22.
    Detection of thyroid hormones in urine by liquid chromatography coupled to tandem mass spectrometry.
    Dayamin Martinez Brito, Patrizia Leogrande, Xavier de la Torre, Francesco Botrè.
      Recently, the trend of thyroid hormones (TH) consumption in the sports community has been published. It is known the capacity of the exogenously administered TH to enhance metabolism, being an attractive feature for athletes, who search for weight control and increased caloric expenditure. This paper aimed the validation of a method to measure TH and related compounds in urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was applied to urine samples collected before and after the administration of a diiodothyronine (3,5-T2) supplement. A method to detect nine TH included an enzymatic hydrolysis, liquid-liquid extraction, and solid-phase extraction. The extracts were analyzed by LC-MS/MS. Validated parameters showed good results for accuracy (85%-104%), precision (3%-16%), LOD (10-40 pg/mL, except for thyronacetic acids that was 200 pg/mL), and the combined uncertainty (2.2%-22%). Maximum concentration of 3,5-T2 in pre-administration samples was 0.71 ng/mL, and after 30 h of the last administration, concentrations returned to pre-administration values. Maximum values of ratios between the analyte and thyronine, T3, and T4 were 0.09, 0.19, and 0.12, respectively, and after 30 h of the last administration, the ratios reached back the basal values. Acidic or basic metabolites were not found in urine at least at the method LOD. A proposed method to assess TH in urine was validated, and as a proof of concept, its efficacy was demonstrated with an excretion study of 3,5-diiodothyronine. The consumption of 3,5-T2 was detected in urine measuring the analyte concentration and ratios between the analyte and thyronine, T3, and T4.
    Keywords:  LC‐MS/MS; metabolism; sport; thyroid hormones; urine
    DOI:  https://doi.org/10.1002/dta.3764
  35. Rapid Commun Mass Spectrom. 2024 Oct 15. 38(19): e9869
    Development of the headspace gas chromatography-tandem mass spectrometry method for ethylene oxide and ethylene chlorohydrin residue in medical devices.
    Yi Chun Chiang, Ming Chih Fang, Chuan Kai Yang, Sheng Wei Wang, Su Hsiang Tseng.
       RATIONALE: Ethylene oxide (EO) sterilization is commonly employed for the sterilization of medical devices and has a very high market share. However, EO and its metabolite ethylene chlorohydrin (ECH) are toxic to humans. In compliance with the classification and residue limits of medical devices defined by ISO 10993-7, our study established two extraction methods for the testing of EO and ECH.
    METHODS: The first method involves simulated-use extraction using water as the extraction solvent. While the second, exhaustive extraction, directly extracts sample through headspace sampling analysis. Gas chromatography-tandem mass spectrometry in multiple reaction monitoring mode was utilized, requiring only 16 min. Then, the developed method was applied to assess 10 commercially available medical devices sterilized by EO.
    RESULTS: In simulated-use extraction, calibration curves were evaluated in the range of 1-100 and 5-500 μg for EO and ECH, respectively (r > 0.999). Inter-day recoveries ranged from 85.0% to 95.2% and from 94.8% to 102.4%. In exhaustive extraction, calibration curves spanned 0.5-50 and 2-200 μg for EO and ECH, respectively (r > 0.999). Inter-day recoveries ranged from 101.6% to 102.1% for EO and from 98.1% to 102.2% for ECH. After analysis of the 10 commercially available medical devices, two cotton swabs were found to have ECH of 35.1 and 28.4 μg per device, and four medical devices were found to have EO with concentration below the limit of quantification. Meanwhile, we found that the EO internal standard (propylene oxide) recommended by ISO 10993-7 had interference problems with other similar substances and was not suitable as an internal standard for EO.
    CONCLUSIONS: This study offers a sensitive and straightforward analytical approach to EO and ECH residues in a variety of medical devices. In addition, the results show that the EO or ECH content of these types of medical devices in our study falls below the regulatory limits, therefore instilling confidence among consumers regarding their safe use.
    DOI:  https://doi.org/10.1002/rcm.9869
  36. J Pharm Biomed Anal. 2024 Jul 20. pii: S0731-7085(24)00413-8. [Epub ahead of print]249 116373
    Uncertainty management for In Silico screening of reversed-phase liquid chromatography methods for small compounds.
    Thomas Van Laethem, Priyanka Kumari, Bruno Boulanger, Philippe Hubert, Marianne Fillet, Pierre-Yves Sacré, Cédric Hubert.
      The process of developing new reversed-phase liquid chromatography methods can be both time-consuming and challenging. To meet this challenge, statistics-based strategies have emerged as cost-effective, efficient and flexible solutions. In the present study, we use a Bayesian response surface methodology, which takes advantage of the knowledge of the pKa values of the compounds present in the analyzed sample to model their retention behavior. A multi-criteria decision analysis (MCDA) was then developed to exploit the uncertainty information inherent in the model distributions. This strategic approach is designed to integrate seamlessly with quantitative structure retention relationship (QSRR) models, forming an initial in-silico screening phase. Of the two methods presented for MCDA, one showed promising results. The method development process was carried out with the optimization phase, generating a design space that corroborates the results of the selection phase.
    Keywords:  Method development; Multi-criteria decision analysis; Response surface methodology; Reversed-phase liquid chromatography; Small pharmaceutical compounds
    DOI:  https://doi.org/10.1016/j.jpba.2024.116373
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