bims-metlip 2024-11-17 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–11–17
34 papers selected by
Sofia Costa, Matterworks

Strategies for Using Postcolumn Infusion of Standards to Correct for Matrix Effect in LC-MS-Based Quantitative Metabolomics.
Triple acquisition mass spectrometry (TRAM) combining targeted and non-targeted metabolomics in a single run.
A Micro-Flow Liquid Chromatography-Mass Spectrometry Method for the Quantification of Oxylipins in Volume-Limited Human Plasma.
Rapid Liquid Chromatography-Tandem Mass Spectrometry Method for Determination of Total and Free Testosterone in Human Serum and Its Application to Monitoring Biomarker Response of Elite Athletes.
Development and validation of a rapid HPLC-MS/MS method for simultaneous determination of cyclosporine A and tacrolimus in whole blood for routine therapeutic drug monitoring in organ transplantation.
Advancing Targeted Metabolomics Using Cyanopropyl-Based Liquid Chromatography Tandem Mass Spectrometry.
A Simple HPLC-DAD Method for the Therapeutic Monitoring of Clozapine and Related Metabolites in Human Plasma and Urine Samples.
Hair and Saliva Endocannabinoid and Steroid Hormone Analysis by Liquid Chromatography Paired with Tandem Mass Spectrometry.
The validation of an LC-MS/MS method for the quantification of vitamin D metabolites in human milk and their biological variability in Gambian women.
From historical drugs to present perils: UHPLC-QqQ-MS/MS determination of methaqualone and its designer analogs (NPS) with comprehensive fragmentation pathways study (QTOF).
An orthogonal approach for analysis of underivatized steroid hormones using ultrahigh performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS).
Multi-class cyanobacterial toxin analysis using hydrophilic interaction liquid chromatography-mass spectrometry.
UHPLC-QTOFMS Urine Drug Screening With Dilute-and-Shoot Sample Preparation and Vacuum-Insulated Probe-Heated Electrospray Ionization.
Polarity Gradient Solvent Confinement Membrane Cartridge to Broaden Metabolite Coverage of Plasma Untargeted Analysis.
Limitations in LC-MS/MS application for adagrasib and pembrolizumab quantification in rat plasma.
Towards the non-invasive determination of estradiol levels: Development and validation of an LC-MS/MS assay for quantification of salivary estradiol at sub-pg/mL level.
Simultaneous Determination of Methotrexate and 7-Hydroxy-Methotrexate by UHPLC-MS3 Assay Coupled With Multiple Stage Fragmentation to Enhance Sensitivity.
A complementary approach for detecting biological signals through a semi-automated feature selection tool.
Obtaining 20 μm Spatial Resolution with a 2940 nm Laser by IR-MALDESI Mass Spectrometry Imaging.
Quantitative sampling by IR-MALDESI is not susceptible to tissue heterogeneity in a multi-organ model.
A novel, sensitive, and fast ultra-high-performance liquid chromatography tandem mass spectrometry method for TNG908 determination in dog plasma and pharmacokinetic study.
Boosting miniaturization in clinical analysis: determination of bisphenols in human serum and urine by miniaturized stir bar sorptive dispersive microextraction.
High-throughput glycosaminoglycan extraction and UHPLC-MS/MS quantification in human biofluids.
A Streamlined Workflow for Microscopy-Driven MALDI Imaging Mass Spectrometry Data Collection.
Development and validation of an LC-MS/MS method for the detection of sodium pentachlorophenolate residues on cutting boards.
Design-assisted HPLC-UV method for therapeutic drug monitoring of pholcodine, ephedrine, and guaifenesin in biological fluids.
Comparative metabolic profiling and quantitative analysis of metabolites in different tissues of Ajuga turkestanica by ESI-UHPLC-QqTOF-MS and NMR.
Towards high throughput analysis using 96-well plate solid phase extraction to determine sedatives and β-blocker residues in food control monitoring.
Enhanced differentiation between 3-hydroxyglutaric and 2-hydroxyglutaric acids facilitates diagnostic testing for glutaric aciduria type 1.
A Highly Sensitive UPLC-MS/MS Method for the Quantification of the Organic Cation Transporters' Mediated Metformin Uptake and Its Inhibition in Cells.
Investigation on the Retention and Separation of Glucosinolates With a Mixed-Mode Reversed-Phase/Weak Anion-Exchange Column.
Age and sex effects of a validated LC-MS/MS method for the simultaneous quantification of testosterone, allopregnanolone, and its isomers in human serum.
Toxicokinetics and Tissue Distribution of the Hepatotoxic Triterpenoid Saponin Pterocephin A in Rats Using the Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) Method.
Development and validation of HPLC-UV and LC-MS/MS methods for the quantitative determination of a novel aminothiazole in preclinical samples.

J Am Soc Mass Spectrom. 2024 Nov 15.

Strategies for Using Postcolumn Infusion of Standards to Correct for Matrix Effect in LC-MS-Based Quantitative Metabolomics.

Anne-Charlotte Dubbelman, Bo van Wieringen, Lesley Roman Arias, Michael van Vliet, Roel Vermeulen, Amy C Harms, Thomas Hankemeier.

The matrix effect limits the accuracy of quantitation of the otherwise popular metabolomics technique liquid chromatography coupled to mass spectrometry (LC-MS). The gold standard to correct for this phenomenon, whereby compounds coeluting with the analyte of interest cause ionization enhancement or suppression, is to quantify an analyte based on the peak area ratio with an isotopologue added to the sample as an internal standard. However, these stable isotopes are expensive and sometimes unavailable. Here, we describe an alternative approach: matrix effect correction and quantifying analytes using a signal ratio with a postcolumn infused standard (PCIS). Using an LC-MS/MS method for eight endocannabinoids and related metabolites in plasma, we provide strategies to select, optimize, and evaluate PCIS candidates. Based on seven characteristics, the structural endocannabinoid analogue arachidonoyl-2'-fluoroethylamide was selected as a PCIS. Three methods to evaluate the PCIS correction vs no correction showed that PCIS correction improved values for the matrix effect, precision, and dilutional linearity of at least six of the analytes to within acceptable ranges. PCIS correction also resulted in parallelization of calibration curves in plasma and neat solution, for six of eight analytes even with higher accuracy than peak area ratio correction with their stable isotope labeled internal standard, i.e., the gold standard. This enables quantification based on neat solutions, which is a significant step toward absolute quantification. We conclude that PCIS has great, but so far underappreciated, potential in accurate LC-MS quantification.

DOI: https://doi.org/10.1021/jasms.4c00408
Anal Chim Acta. 2024 Dec 01. pii: S0003-2670(24)01115-2. [Epub ahead of print]1331 343314

Triple acquisition mass spectrometry (TRAM) combining targeted and non-targeted metabolomics in a single run.

Lisa Panzenboeck, Harald Schoeny, Bruno Stelzer, Elisabeth Foels, Marvin Glas, Marlene Pühringer, Dorian Hirschmann, Daniela Loetsch, Christian Dorfer, Evelyn Rampler, Gunda Koellensperger.

   BACKGROUND: We introduce TRAM, a triple acquisition strategy on a high-speed quadrupole time-of-flight mass spectrometer for merging non-targeted and targeted metabolomics into one run. TRAM stands for "quasi-simultaneous" acquisition of (1) a full scan MS1, (2) top 30 data-dependent MS2 (DDA), and (3) targeted scheduled MS2 for multiple reaction monitoring (MRM) within measurement cycles of ∼1 s. TRAM combines the selectivity and sensitivity of state-of-the-art targeted MRM-based methods with the full scope of non-targeted analysis enabled by high-resolution mass spectrometry.
RESULTS: In this work, we deploy a workflow based on hydrophilic interaction liquid chromatography (HILIC). For a broad panel of metabolites, we provide chromatographic retention times, and optimized conditions as a basis for targeted MRM experiments, listing accurate masses and sum formulas for fragment ions (including fully 13C labeled analogs). Validation experiments showed that TRAM offered (1) linear working ranges and limits of quantification comparable to MRM-only methods, (2) enabled accurate quantification in SRM 1950 human plasma reference material, and (3) was equivalent to DDA-only approaches in non-targeted metabolomics. Metabolomics in human cerebrospinal fluid showcased the power of the strategy, emphasizing the need for high coverage/high throughput metabolomics in clinical studies.
SIGNIFICANCE: Acquiring up to 30 data-dependent spectra per MS cycle while still offering gold standard absolute quantification down to low nanomolar concentrations, TRAM allows in-depth profiling and reduces required sample volume, time, cost, and environmental impact.

Keywords:  Absolute quantification; HILIC; Liquid chromatography-mass spectrometry; Meningioma; Non-targeted metabolomics; Targeted metabolomics; ZenoTOF 7600

DOI:  https://doi.org/10.1016/j.aca.2024.343314
Electrophoresis. 2024 Nov 11.

A Micro-Flow Liquid Chromatography-Mass Spectrometry Method for the Quantification of Oxylipins in Volume-Limited Human Plasma.

Bingshu He, Rawi Ramautar, Marian Beekman, P Eline Slagboom, Amy Harms, Thomas Hankemeier.

  Oxylipins are well-known lipid mediators in various inflammatory conditions. Their endogenous concentrations range from low picomolar to nanomolar, and there are growing demands to determine their concentrations in low-volume matrices for pathological studies, including blood, cerebrospinal fluids from animal disease models, infants, and microsampling devices. Most of the published quantification methods for comprehensive profiling of oxylipins still require more than 50 µL plasma as a starting volume to detect these low levels. The aim of our study is to develop a sensitive and reliable method for the quantification of oxylipins in volume-limited human plasma samples. We established and validated a micro-liquid chromatography (LC)-mass spectrometry (MS)/MS method that requires only 5 µL of human plasma for the determination of 66 oxylipins. The optimized micro-LC-MS/MS method utilized a flow rate of 4 µL/min with a 0.3-mm inner diameter column. With an injection volume of 3 µL, our method provides limits of detection in the range from 0.1 to 91.9 pM, and limits of quantification range from 0.3 to 306.2 pM. The sensitivity enhancement compared to conventional flow ranged from 1.4 to 180.7 times for 51 compounds depending on their physical-chemical properties. After validation, the method was applied to analyze 40 plasma samples from a healthy aging study to demonstrate robustness and sensitivity.

Keywords:  micro–liquid chromatography (LC)–mass spectrometry (MS); oxylipins; sensitivity enhancement; volume‐limited plasma

DOI:  https://doi.org/10.1002/elps.202400151
Molecules. 2024 Oct 23. pii: 5007. [Epub ahead of print]29(21):

Rapid Liquid Chromatography-Tandem Mass Spectrometry Method for Determination of Total and Free Testosterone in Human Serum and Its Application to Monitoring Biomarker Response of Elite Athletes.

Jianli Zhang, Hang Yu, Yulin Shen, Xingya Yang, Yan Wang.

  Total testosterone (TT) and free testosterone (FT) are important biochemical markers for anabolism of the human body, and can also serve as early screening indicators for overtraining syndrome (OTS). Presently, there is no fast and reliable serum TT and FT determination method in the field of sport science that can meet the requirements of sports research. Thus, a rapid and accurate determination method for serum TT and FT to fill the gap is needed urgently in sports training. Herein, a simple and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of TT and FT in serum was developed and fully validated, followed by the application of professional athletes in training monitoring. Efficient pretreatments based on only one-step liquid-liquid extraction (LLE) for TT and one-step LLE after a 20 min ultrafiltration for FT were adopted in this study, and the isotope internal standard of testosterone-13C3 was used to ensure the reliability of the whole procedure. A linear range of four orders of magnitude with 0.02-100 ng/mL can meet the concentration range requirement between a higher limit for male TT and a lower limit for female FT. The accuracy, precision, stability, and matrix effect were all within the limits of the guidelines. The serum TT and FT levels of 200 professional athletes (98 male athletes and 102 female athletes) were investigated by this method. Serum TT, FT, and FT/TT levels of professional athletes were significantly higher than the general population, and serum TT levels were significantly higher by LC-MS/MS than by a chemiluminescence immunoassay. In conclusion, the LC-MS/MS method for TT and FT measurement developed in this study is time-saving and easy to operate, which can be used as a reliable method for the determination of serum TT and FT in sports training, offering valuable information for monitoring anabolism of athletes and screening OTS in the early stage.

Keywords:  LC-MS/MS; athlete; biomarker; free testosterone; total testosterone

DOI:  https://doi.org/10.3390/molecules29215007
Rapid Commun Mass Spectrom. 2025 Jan 30. 39(2): e9932

Development and validation of a rapid HPLC-MS/MS method for simultaneous determination of cyclosporine A and tacrolimus in whole blood for routine therapeutic drug monitoring in organ transplantation.

Fei-Fei Han, Hong-Chuan Liu, Ting Hu, Peng-Fei Li, Rui Zhao, Zhuo-Ling An.

BACKGROUND: Therapeutic drug monitoring is an integral part of organ transplantation. A rapid, simple, economical, and robust high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneously determining the immunosuppressants cyclosporine A and tacrolimus might increase detection efficiency.
METHODS: In this study, we developed and validated a rapid HPLC-MS/MS method. Whole blood samples of 100 μL were prepared by protein precipitation with acetonitrile and 0.5 mol. L-1 ZnSO4. Chromatography was performed on a pre-column using a gradient elution with 20 mmol. L-1 ammonium formate and 0.1% (v/v) formic acid in water (mobile phase A) and 0.1% (v/v) formic acid in methanol (mobile phase B) at a flow rate of 1.5 mL.min-1. The analysis time was 2.2 min. Electrospray ionization and multiple reaction monitoring were performed. The lower limit of quantification was set at 1 ng. L-1 for tacrolimus and 50 ng. L-1 for cyclosporine A.
RESULTS: The method showed adequate accuracy and precision with a sufficient linear range. The calibration curve range of tacrolimus and cyclosporine A was 1-30 and 50-1500 ng·mL-1, respectively. All correlation coefficients were >0.99.
CONCLUSIONS: The developed HPLC-MS/MS is rapid and can be used for simultaneous monitoring of tacrolimus and cyclosporine A.

DOI: https://doi.org/10.1002/rcm.9932
Anal Chem. 2024 Nov 15.

Advancing Targeted Metabolomics Using Cyanopropyl-Based Liquid Chromatography Tandem Mass Spectrometry.

Wan-Rou Liao, Jiun-Tsai Lin, Pei-Chen Lin, Chin-Chen Chen, Chun-Fang Huang, Han-Min Chen, Sung-Fang Chen.

The change of metabolic pathways is recognized as the key to disease discovery prompting the development of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-based quantitative platforms to explore the dynamic metabolite profiles of organisms. In this study, a liquid chromatography method based on cyanopropyl (CN) was developed. By adjusting the pH environment of the column, we achieved the elution of 51 metabolites spanning the most comprehensive set of biological pathways currently known. Offering rapid chromatography, efficient separation, and green chemistry benefits, the method encompasses nucleosides and nucleotides, the oxidative-redox metabolome, the glycolysis pathway, the pentose phosphate pathway, the purine de novo pathway, amino acids, and neurological disorder-related metabolites. The mass spectrometry was equipped with electrospray ionization in both positive and negative modes with scheduled multiple reactions monitoring. The validation of the method involved a comprehensive assessment of linearity, accuracy, precision, and matrix effect. The linear range was from 1.0 to 2000 ng mL-1 with a high correlation coefficient (r > 0.99). The LOD ranged from 0.1 to 10 ng mL-1, and the LOQ ranged from 0.1 to 25 ng mL-1. The overall recovery ranged from 81.3% to 117.8%, with RSD < 15.1%. Subsequently, an analysis of metabolites was conducted in dSH-SY5Y neuroblastoma cells with 6-hydroxydopamine, a commonly used neurotoxin in neurodegenerative diseases. The results demonstrate that neurotoxin-induced mitochondrial damage significantly altered related analytes, corroborating previous estimates and validating the feasibility and reliability of the bioanalytical platform.

DOI: https://doi.org/10.1021/acs.analchem.4c01939
Molecules. 2024 Oct 25. pii: 5039. [Epub ahead of print]29(21):

A Simple HPLC-DAD Method for the Therapeutic Monitoring of Clozapine and Related Metabolites in Human Plasma and Urine Samples.

Mircea-Alexandru Comănescu, Dana-Maria Preda, Dalia-Simona Miron, Flavian-Ștefan Rădulescu, Victor Voicu, Andrei-Valentin Medvedovici.

  Clozapine and its metabolites require close therapeutic monitoring (TDM) in patients due to poor correlation between the administrated doses and resulting plasma concentrations, the narrow therapeutic interval, high inter-individual variability, and the risk of serious side effects once toxic levels are exceeded. The aim of the study was to develop a simple (relatively cheap) LC-UV method for the quantification of clozapine and its metabolites in plasma and urine samples. For sample preparation, liquid-liquid extraction (LLE) in n-octanol was more efficient and less limiting in injection volumes compared to the in-situ formation of SUPRAS. When analyzing urine, an alkalinization step before extraction was required. The proposed method produced linear concentration responses with/without internal standard (IS) for the target analytes, with LLOQs within the targeted range of 50 ppb and %RSD within the acceptable 15% range. Furthermore, sample stability studies proved that pre-extracted samples were stable for the short term at room temperature and long-term when frozen.

Keywords:  HPLC-DAD; LLE; SUPRAS; clozapine; metabolites; n-octanol

DOI:  https://doi.org/10.3390/molecules29215039
Methods Mol Biol. 2025 ;2868 135-147

Hair and Saliva Endocannabinoid and Steroid Hormone Analysis by Liquid Chromatography Paired with Tandem Mass Spectrometry.

Madeline Jarvis, Khalisa Amir Hamzah, David Nichols, Luke J Ney.

  Endocannabinoids are lipid neurotransmitters that play an important part in human health. Recent methods have found that quantification of endocannabinoids in hair and saliva samples is possible using liquid chromatography paired with tandem mass spectrometry (LC-MS/MS). This chapter describes two simple sample preparation methods that can be used to prepare hair and saliva samples for analysis using LC-MS/MS. Our LC-MS/MS method can be applied to both hair and saliva samples and is sufficiently sensitive for endocannabinoid, as well as steroid hormone, quantification in both of these sample matrices. This chapter provides a comprehensive description of how this can be achieved and provides tips and tricks for troubleshooting problems users may experience.

Keywords:  Endocannabinoids; Hair; Hormones; Mass spectrometry; Saliva

DOI:  https://doi.org/10.1007/978-1-0716-4200-9_8
J Steroid Biochem Mol Biol. 2024 Nov 13. pii: S0960-0760(24)00181-X. [Epub ahead of print] 106633

The validation of an LC-MS/MS method for the quantification of vitamin D metabolites in human milk and their biological variability in Gambian women.

Kerry S Jones, Sarah R Meadows, Georgia Billing, Albert Koulman, Ann Prentice.

  Vitamin D is required for healthy growth and development, but data on human milk vitamin D content is limited. We describe a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the analysis of vitamin D metabolites in human milk, and its application in samples collected on two consecutive days from women in rural Gambia. Vitamin D compounds were extracted from 1mL of milk by liquid-liquid extraction and derivatised with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) prior to analysis by LC-MS/MS. The limit of quantification was 0.05 nmol/L for vitamin D2, 0.025 nmol/L for vitamin D3 and 0.1 nmol/L for 25(OH)D2 and 25(OH)D3. Within- and between-day imprecision was <12% for all analytes except vitamin D2 (14%). From all data combined, geometric mean (-/+ 1SD) vitamin D3 concentration was 0.94 (0.43, 1.80) nmol/L and for 25(OH)D3 0.32 (0.23, 0.42) nmol/L. The intra-individual coefficient of variation (%CV) was 32% and 12% for vitamin D3 and 25(OH)D3, respectively. Inter-individual %CVs were 89% and 34% for vitamin D3 and 25(OH)D3, respectively. There was no significant association between vitamin D metabolite concentrations and milk fat (creamatocrit). Mean vitamin D content of human milk as ARA averaged 42 IU/L with 25(OH)D3 responsible for around two-thirds of the biological activity. In conclusion, this work describes a reliable LC-MS/MS method for quantification of vitamin D and 25(OH)D in low volumes of human milk providing a platform for future work. This study contributes to current understanding of variability of milk vitamin D content.

Keywords:  Lactation; biological variation; vitamin D

DOI:  https://doi.org/10.1016/j.jsbmb.2024.106633
Toxicol Mech Methods. 2024 Nov 15. 1-11

From historical drugs to present perils: UHPLC-QqQ-MS/MS determination of methaqualone and its designer analogs (NPS) with comprehensive fragmentation pathways study (QTOF).

Kaja Tusiewicz, Olga Wachełko, Paweł Szpot, Marcin Zawadzki.

  Methaqualone, introduced in the 1960s as a sedative-hypnotic alternative to barbiturates, was withdrawn from the market due to its side effects and growing recreational use. Despite this, interest in methaqualone and its analogs remains high, raising concerns about potential abuse in the future. An ultra-high-performance liquid chromatography method coupled with triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) was developed to determine nine methaqualone-related compounds simultaneously. Biological samples were prepared using liquid-liquid extraction with ethyl acetate at pH9; quantification was performed in blood using multiple reaction monitoring (MRM) mode. Methaqualone-d7 served as an internal standard. The limit of quantification (LOQ) ranged from 0.1 to 0.2 ng/mL, with precision and accuracy within 20%. Recovery ranged from 84.2% to 113.7%. The developed method allowed chromatographic separation of all compounds tested, including two structural isomers: methylmethaqualone and etaqualone. The mass spectra acquired from quadrupole time-of-flight mass spectrometer allowed for the elucidation of comprehensive fragmentation study of methaqualone derivatives. The described situation poses a significant problem from the analytical point of view, as well as interpretation and forensic toxicological expertise. The developed method will contribute to increased analytical capabilities and enhanced detection of compounds from the methaqualone group that may appear on the illicit market.

Keywords:  New psychoactive substances; UHPLC–MS/MS; isomers separation; methaqualones; sedative-hypnotic compounds

DOI:  https://doi.org/10.1080/15376516.2024.2426582
J Neural Transm (Vienna). 2024 Nov 15.

An orthogonal approach for analysis of underivatized steroid hormones using ultrahigh performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS).

Perry Devo, Victoria Cretu, Harsha Radhakrishnan, Darren Hamilton-Pink, Stergios Boussios, Saak V Ovsepian.

  The crucial role of steroid hormones in health and diseases merits their high-throughput, accurate and affordable measurements in biological specimens. Despite advances in analytical methods, sensing and quantifying steroid hormones remains challenging. Immunoassays offer excellent sensitivity but are inherently labour-intensive, costly, and prone to false positives. Mass spectrometry (MS) has been increasingly utilised, with the main hurdle being the isobaric tendencies of similar analytes, which complicates their separation and accurate quantification. This study compares ultrahigh-performance supercritical fluid chromatography separation (UHPSFC) and ultra-high-performance liquid chromatography (UHPLC) for MS detection. It optimises the column chemistry, temperature, and pressure to provide an operational protocol for the resolution and quantification of analytes. It presents the systematic characterisation of UHPSFC-MS performance by investigating spiked blood samples using Solid-Phase Extraction (SPE) and describes the matrix effects associated with MS measurements. Although both separation methods showed adequate resolution, specificity, and retention time, UHPSFC-MS was superior for five out of seven columns tested. With added high-throughput capacities, UHPSFC-MS, thus, offers an optimal solution for the analysis of steroid hormones for research, medical chemistry, and clinical diagnostics.

Keywords:  Biomarkers; Solid phase extraction; Steroid hormones; Supercritical fluid; UHPLC-MS; UHPSFC-MS

DOI:  https://doi.org/10.1007/s00702-024-02862-3
J Chromatogr A. 2024 Nov 14. pii: S0021-9673(24)00857-4. [Epub ahead of print]1738 465483

Multi-class cyanobacterial toxin analysis using hydrophilic interaction liquid chromatography-mass spectrometry.

Krista M Thomas, Elliott J Wright, Daniel G Beach, Pearse McCarron.

  Cyanobacteria produce diverse classes of toxins including microcystins, nodularins, anatoxins, cylindrospermopsins and saxitoxins, encompassing a range of chemical properties and mechanisms of toxicity. Comprehensive analysis of these toxins in cyanobacterial, environmental and biological samples generally requires multiple methods of extraction and analysis. In this work, a method was developed for the major classes of cyanotoxins, which comprised of a three-step liquid-solid extraction method using 75 % CH3CN with 0.1 % HCOOH and a hydrophilic interaction liquid chromatography (HILIC) elution gradient that provided retention of the less polar microcystins through to the highly polar saxitoxins. Detection was performed by tandem mass spectrometry in selected reaction monitoring mode with positive and negative polarity switching. Identification criteria included matching retention times and product ion ratios with available standards. In-house validation demonstrated good performance of the method including precision ranging from 1.5 (microcystin-LA) to 5.8 (gonyautoxin-2) % RSDs, and detection limits ranging from 0.01 (cylindrospermopsin) to 0.99 (gonyautoxin-3) µg/g in freeze dried material cyanobacteria. Recovery was assessed using spiked non-toxic cyanobacterial samples (Aphanizomenon sp.) and ranged from 83 (neosaxitoxin) to 107 % ([Dha7]microcystin-LR). As a demonstration of application, toxin profiles in cyanobacterial cultures, benthic and planktonic cyanobacteria field samples, and shellfish reference materials were successfully evaluated. The procedure is also amenable for extension to other polar toxin classes including domoic acid and guanitoxin. With increasing reports of cyanobacterial blooms globally, the method represents a powerful quantitative screening tool for measuring cyanotoxins across a broad range of samples.

Keywords:  Cyanotoxins; Harmful algae; LC–MS; Polar toxins; Screening

DOI:  https://doi.org/10.1016/j.chroma.2024.465483
Drug Test Anal. 2024 Nov 12.

UHPLC-QTOFMS Urine Drug Screening With Dilute-and-Shoot Sample Preparation and Vacuum-Insulated Probe-Heated Electrospray Ionization.

Mira Sundström, Pirkko Kriikku, Ilkka Ojanperä, Carsten Baessmann, Anna Pelander.

  We developed a method for comprehensive urine drug screening by applying dilute-and-shoot extraction and vacuum-insulated probe-heated electrospray ionization with ultra-high performance liquid chromatography high-resolution quadrupole time-of-flight mass spectrometry (DS-UHPLC-VIP-HESI-QTOFMS). The method involved five-fold post-hydrolysis dilution of urine samples and chromatography on a C18 UHPLC column prior to QTOFMS analysis. The recently introduced VIP-HESI ion source was chosen due to its enhanced ionization efficiency and compatibility with UHPLC-QTOFMS. Extensive data was acquired in positive ion mode with a low collision energy (7 eV) and an elevated collision energy (30 eV), using the broadband collision-induced dissociation data acquisition scan mode that continuously generated high-resolution and accurate mass for parent and fragment qualifier ions, and parent ion isotopic patterns. Compound identification was performed against an in-house database with 1263 compound entries, using an automated post-run reverse target database search with preset identification criteria. Method validation with 56 different drugs showed acceptable results for the limit of identification (median 5 ng/mL), matrix effects (70-130%), repeatability of retention times (< 1%), mass accuracy (< 1 mDa), as well as for specificity and stability. As compared with an established UHPLC-QTOFMS method relying on solid-phase extraction and conventional electrospray ionization, DS-UHPLC-VIP-HESI-QTOFMS produced comparable results from authentic clinical urine samples for most drugs, but showed clearly improved detectability for pregabalin, gabapentin, and ritalinic acid. We anticipate that the new method will be a step forward for laboratories performing routine urine drug screening due to its fast turnaround time, reduced manual workload, cost efficiency, and broad substance coverage.

Keywords:  dilute‐and‐shoot; high‐resolution mass spectrometry; time‐of‐flight mass spectrometry; urine drug screening; vacuum‐insulated probe‐heated electrospray ionization (VIP‐HESI)

DOI:  https://doi.org/10.1002/dta.3830
Anal Chem. 2024 Nov 12.

Polarity Gradient Solvent Confinement Membrane Cartridge to Broaden Metabolite Coverage of Plasma Untargeted Analysis.

Chunyu Yu, Jiaxin Zhang, Xiaohan Zong, Xiangzi Jin, Lu Liu, Yilin Zou, Yifan Jiao, Meihui Tong, Meiyu Cui, Huwei Liu, Donghao Li.

Various polarity chemicals exist in complex samples, such as plasma; nontargeted comprehensive analysis naturally requires multiple polar-extracted solvents; consequently, the polarity of the solvent plays a crucial role in the extraction efficiency of analytes from complex samples. In the present study, based on the diffusion behavior and nanoconfinement effect of solvents in the nanoconfined space, the polarity gradient solvent confinement liquid-phase nanoextraction (PGSC-NLPNE) protocol aimed to perform a one-step nontargeted analysis of a wide range of metabolites in plasma was established. The continuously wide range of extracted solvent polarities on carbon nanofibers/carbon fiber (CNFs/CF) membranes was achieved using a mixture of hexane, dichloromethane, methanol, and water as nanoconfined solvents. The polarities (Log P) of gradient solvents ranged from -1.38 to 3.94. Correlational analyses indicated that metabolites with Log P values ranging from -1.90 to 3.84 were closely related according to similarity-intermiscibility theory. Coupled with a homemade modified guard column device, CNFs/CF membrane cartridge (CCMC), a PGSC-NLPNE-UHPLC-MS online protocol was established and applied in plasma untargeted analysis. By comparing metabolome coverage, reproducibility, and extraction recovery with protein precipitation and two-step liquid-liquid extraction commonly used in untargeted analysis, the PGSC-NLPNE-CCMC protocol demonstrated higher reproducibility and recovery. This protocol has shown great potential for ultrafast analysis of plasma untargeted metabolomics with broader metabolome coverage. It could be a potential tool to rapidly screen out valuable biomarkers related to diseases in the clinic.

DOI: https://doi.org/10.1021/acs.analchem.4c04400
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Oct 31. pii: S1570-0232(24)00363-5. [Epub ahead of print]1248 124354

Limitations in LC-MS/MS application for adagrasib and pembrolizumab quantification in rat plasma.

Zvonimir Mlinarić.

  An article by Harsha Shi et al. raises multiple concerns regarding sample preparation, the presence of the analyte in the analyzed solution, authenticity of the MS spectra, administration of drugs and blood collection, choice of internal standard, obtained pharmacokinetic parameters and usage of chromatographic column and solvents. While the research topic is interesting and the development of bioanalytical methods for novel drugs is crucial, this article does not seem to meet the analytical and methodological standards for the reliable determination of adagrasib and pembrolizumab in plasma samples. A detailed explanation is given in the letter.

Keywords:  Liquid chromatography; Mass spectrometry; Method development

DOI:  https://doi.org/10.1016/j.jchromb.2024.124354
Anal Chim Acta. 2024 Dec 01. pii: S0003-2670(24)01114-0. [Epub ahead of print]1331 343313

Towards the non-invasive determination of estradiol levels: Development and validation of an LC-MS/MS assay for quantification of salivary estradiol at sub-pg/mL level.

David Fabregat-Safont, Élida Alechaga, Noemí Haro, Àlex Gomez-Gomez, Eric R Velasco, Jaime F Nabás, Raül Andero, Oscar J Pozo.

   BACKGROUND: Estradiol (E2) is a female sex hormone involved in several biological processes. Although E2 levels are commonly measured in blood samples, the use of non-invasive techniques (e.g. determination of salivary E2) would allow for the collection of repeated samples and the inclusion of a greater number of participants. Immunoassay-based techniques to measure salivary E2 failed to accurately mirror the variations observed in the plasmatic concentrations of E2 during the menstrual cycle probably due to the high sensitivity required (in the sub-pg/mL range). Therefore, sensitive and rugged analytical methods for the determination of salivary E2 are required. For this, we developed and validated an analytical methodology for the accurate determination of salivary E2.
RESULTS: The method is based on chemical derivatization with 1,2-dimethyl-1H-imidazole-5-sulphonyl chloride and liquid chromatography-tandem mass spectrometry analysis by summing highly-specific SRM transitions. This strategy allowed for increasing the sensitivity of the method. The validation of the method showed an accurate and precise quantification of E2 in 1 mL of saliva even at 250 fg/mL (97 % accuracy and 15 % RSD intra-day, and 104 % accuracy and 18 % RSD inter-day). In order to evaluate its efficacy, we analysed saliva samples from 5 healthy female volunteers collected during a whole menstrual cycle. Our analyses showed that the variations in the concentration of E2 in the measured samples mirrored those expected during a complete menstrual cycle. Additionally, we validated the suitability of our method for determining salivary E2 levels during pregnancy.
SIGNIFICANCE: To the best of our knowledge, this is the first method that allows to precisely and accurately measuring E2 in saliva samples along the whole menstrual cycle of healthy females. It is also suitable for the determination of estradiol during pregnancy. Its high sensitivity makes this strategy ideal for the evaluation of the role of hormone production in women's health.

Keywords:  Bioanalysis; Estradiol; LC-MS/MS; Non-invasive; Saliva; Ultrasensitive

DOI:  https://doi.org/10.1016/j.aca.2024.343313
J Sep Sci. 2024 Nov;47(21): e70020

Simultaneous Determination of Methotrexate and 7-Hydroxy-Methotrexate by UHPLC-MS3 Assay Coupled With Multiple Stage Fragmentation to Enhance Sensitivity.

Huaqiang Li, Xujian Duan, Feifei Wu, Lei Yin.

  A selective ultra-high performance liquid chromatography tandem mass spectrometry cubed (UHPLC/MS3) assay for simultaneous determination of methotrexate (MTX) and 7-hydroxy-methotrexate (7-OH-MTX) in human plasma was developed and validated. After protein precipitation with methanol, chromatographic separation of MTX, MTX-d3, and 7-OH-MTX was performed on a Waters AcQuity UPLC-BEH column (2.1 × 50 mm I.D., 1.7 µm) with gradient elution. MRM3 transition was used for the detection of MTX (m/z 455.2→308.0→175.1) and 7-OH-MTX (m/z 471.3→191.0→148.1). The linear range of UHPLC/MS3 assay for the determination of MTX and 7-OH-MTX was 0.5-300 ng/mL (R2 ≥ 0.99) and 5-1500 ng/mL (R2 ≥ 0.99), respectively. The enhanced selectivity and sensitivity are the novelties of the developed UHPLC/MS3 assay. The analytical method was successfully applied to simultaneous determination of MTX and its major metabolite 7-OH-MTX in real human plasma samples.

Keywords:  7‐hydroxy‐methotrexate; UHPLC‐MS3; methotrexate; quantification; therapeutic drug monitoring

DOI:  https://doi.org/10.1002/jssc.70020
Front Chem. 2024 ;12 1477492

A complementary approach for detecting biological signals through a semi-automated feature selection tool.

Gabriel Santos Arini, Luiz Gabriel Souza Mencucini, Rafael de Felício, Luís Guilherme Pereira Feitosa, Paula Rezende-Teixeira, Henrique Marcel Yudi de Oliveira Tsuji, Alan Cesar Pilon, Danielle Rocha Pinho, Letícia Veras Costa Lotufo, Norberto Peporine Lopes, Daniela Barretto Barbosa Trivella, Ricardo Roberto da Silva.

   Introduction: Untargeted metabolomics is often used in studies that aim to trace the metabolic profile in a broad context, with the data-dependent acquisition (DDA) mode being the most commonly used method. However, this approach has the limitation that not all detected ions are fragmented in the data acquisition process, in addition to the lack of specificity regarding the process of fragmentation of biological signals. The present work aims to extend the detection of biological signals and contribute to overcoming the fragmentation limits of the DDA mode with a dynamic procedure that combines experimental and in silico approaches.
Methods: Metabolomic analysis was performed on three different species of actinomycetes using liquid chromatography coupled with mass spectrometry. The data obtained were preprocessed by the MZmine software and processed by the custom package RegFilter.
Results and Discussion: RegFilter allowed the coverage of the entire chromatographic run and the selection of precursor ions for fragmentation that were previously missed in DDA mode. Most of the ions selected by the tool could be annotated through three levels of annotation, presenting biologically relevant candidates. In addition, the tool offers the possibility of creating local spectral libraries curated according to the user's interests. Thus, the adoption of a dynamic analysis flow using RegFilter allowed for detection optimization and curation of potential biological signals, previously absent in the DDA mode, being a good complementary approach to the current mode of data acquisition. In addition, this workflow enables the creation and search of in-house tailored custom libraries.

Keywords:  chemometrics; data dependent acquisition; mass spectrometry; natural products; untargeted metabolomics

DOI:  https://doi.org/10.3389/fchem.2024.1477492
J Am Soc Mass Spectrom. 2024 Nov 08.

Obtaining 20 μm Spatial Resolution with a 2940 nm Laser by IR-MALDESI Mass Spectrometry Imaging.

Seth M Eisenberg, Alena N Joignant, Kevan T Knizner, Jeffrey G Manni, David C Muddiman.

  High spatial resolution is a key parameter in mass spectrometry imaging (MSI), enabling a greater understanding of system biology and cellular processes. Using a novel IR laser with good Gaussian beam quality (M2 = 4) coupled with spatial filtering and a reflective objective, 20 μm spatial resolution was obtained by IR-MALDESI. The optical train was optimized on burn paper before demonstrating feasibility for imaging of liver tissue. Finally, a mouse brain was analyzed using nested regions of interest at 20 and 140 μm spatial resolution, detecting neurotransmitters and lipids with high spatial resolution on the corpus callosum and surrounding brain tissue.

Keywords:  IR-MALDESI; mass spectrometry imaging; mid-IR laser; spatial resolution

DOI:  https://doi.org/10.1021/jasms.4c00276
Anal Bioanal Chem. 2024 Nov 13.

Quantitative sampling by IR-MALDESI is not susceptible to tissue heterogeneity in a multi-organ model.

Alena N Joignant, Emily C Hector, Morgan M Barnes, Seth W Kullman, David C Muddiman.

  Quantitative mass spectrometry imaging (qMSI) provides the relative or absolute analyte quantities in a biological specimen in a spatially resolved manner. However, the chemical complexity and physical structure of biological specimens often require one to precisely account for matrix effects in qMSI platforms. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) completely ablates a volume of cryosectioned tissue. This enables the use of a normalization standard that is sprayed underneath the tissue for qMSI applications. Complete sampling has shown to be a significant advantage for qMSI by IR-MALDESI; however, the impact of high tissue heterogeneity has not been systematically studied or quantified. The bias introduced by tissue heterogeneity was investigated by uniformly spraying standards beneath and on top of a whole-body zebrafish section. The quantitative relationship between the signals of the two standards was investigated across this multi-organ model to serve future qMSI experiments by IR-MALDESI and other laser ablation-based sampling methods. The overall ratio between the standards sprayed on top of and beneath the tissue sections remained constant across the entire whole-body section despite significant tissue heterogeneity (e.g., gills, heart, and liver). Additionally, we noted that thinner and/or sucrose-embedded tissues improved these ratios, which will inform future qMSI investigations.

Keywords:  IR-MALDESI; Mass spectrometry imaging; Quantitative; Zebrafish

DOI:  https://doi.org/10.1007/s00216-024-05653-7
Biomed Chromatogr. 2024 Nov 07. e6039

A novel, sensitive, and fast ultra-high-performance liquid chromatography tandem mass spectrometry method for TNG908 determination in dog plasma and pharmacokinetic study.

Weiwei Zhu, Huiying Zhang, Fan Li.

  TNG908 is a potent and selective protein arginase methyltransferase 5 (PRMT5) inhibitor that is currently going through phase I/II clinical development for the treatment of non-small cell lung cancer. To facilitate pharmacokinetic and toxicokinetic studies of TNG908, here, we reported an ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the detection of TNG908 in dogs. The dog plasma samples were precipitated by acetonitrile and analyzed using a Waters ACQUITY BEH C18 column combined with a Thermo triple quadrupole mass spectrometer. The mobile phase consisted of 0.1% formic acid solution and acetonitrile, at a flow rate of 0.3 mL/min. TNG908 and internal standard were monitored by selective reaction monitoring (SRM) with m/z 410.2 > 150.1 and m/z 394.2 > 278.1, respectively. The method demonstrated excellent linearity over the concentration range of 1-1000 ng/mL, with a correlation coefficient greater than 0.995. Acetonitrile-mediated protein precipitation showed high extraction efficiency and a recovery above 80%. The validated assay was further applied to measure TNG908 in dog plasma after oral and intravenous administration and achieved success. The obtained pharmacokinetic parameters indicated low clearance of TNG908 (3.7 ± 0.8 mL/min/kg) and moderate oral bioavailability (>36.4%).

Keywords:  PRMT5 inhibitor; TNG908; UPLC‐MS/MS; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.6039
Anal Bioanal Chem. 2024 Nov 13.

Boosting miniaturization in clinical analysis: determination of bisphenols in human serum and urine by miniaturized stir bar sorptive dispersive microextraction.

Guillem Peris-Pastor, Evelin E Lara-Molina, Juan L Benedé, Alberto Chisvert.

  In this work, a miniaturized and sustainable method for the determination of endocrine-disrupting bisphenols in human serum and urine employing the miniaturized stir bar sorptive dispersive microextraction (mSBSDME) approach has been developed. As bisphenols are conjugated in the human body to their glucorinated and sulfated forms, an enzymolysis employing a commercial mixture of β-glucuronidase and arylsulfatase was carried out prior to the microextraction procedure to determine their total content. A magnetic covalent organic framework (COF) was employed as the sorbent to carry out the extraction of the analytes from the biological matrixes, showing good extraction performance due to its hydrophobic, π-π, and dipole-dipole interactions with the analytes. As instrumental detection, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to achieve good sensitivity and selectivity. The method was validated for both matrixes, showing good linearity at least up to 100 ng mL-1, limits of detection in the low ng mL-1 range, good precision values (relative standard deviations below 15%), and good accuracy (relative recoveries between 80 and 127%). In order to show the applicability of the developed method, five samples from female volunteers were analyzed with the final aim of offering a practical tool for monitoring the female population's exposure to these highly endocrine-disrupting compounds. This new procedure enhances the implementation of miniaturized sample preparation approaches in biological samples for clinical analysis, giving special relevance to the sustainability of the method.

Keywords:  Bioanalysis; Bisphenols; Low-volume samples; Magnetic covalent organic framework; Miniaturized stir bar sorptive dispersive microextraction

DOI:  https://doi.org/10.1007/s00216-024-05634-w
Nat Protoc. 2024 Nov 14.

High-throughput glycosaminoglycan extraction and UHPLC-MS/MS quantification in human biofluids.

Nicola Volpi, Fabio Galeotti, Francesco Gatto.

Glycosaminoglycans (GAGs) are linear, unbranched heteropolysaccharides whose structural complexity determines their function. Accurate quantification of GAGs in biofluids at high throughput is relevant for numerous biomedical applications. However, because of the structural variability of GAGs in biofluids, existing protocols require complex pre-analytical procedures, have limited throughput and lack accuracy. Here, we describe the extraction and quantification of GAGs by using ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-MS/MS). Designed for 96-well plates, this method enables the processing of up to 82 study samples per plate, with the remaining 14 wells used for calibrators and controls. Key steps include the enzymatic depolymerization of GAGs, their derivatization with 2-aminoacridone and their quantification via UHPLC-MS/MS. Each plate can be analyzed in a single UHPLC-MS/MS run, offering the quantitative and scalable analysis of 17 disaccharides from chondroitin sulfate, heparan sulfate and hyaluronic acid, with a level of precision and reproducibility sufficient for their use as biomarkers. The procedure from sample thawing to initiating the UHPLC-MS/MS run can be completed in ~1.5 d plus 15 min of MS runtime per sample, and it is structured to fit within ordinary working shifts, thus making it a valuable tool for clinical laboratories seeking high-throughput analysis of GAGs. The protocol requires expertise in UHPLC-MS/MS.

DOI: https://doi.org/10.1038/s41596-024-01078-9
J Am Soc Mass Spectrom. 2024 Nov 07.

A Streamlined Workflow for Microscopy-Driven MALDI Imaging Mass Spectrometry Data Collection.

Allison B Esselman, Megan S Ward, Cody R Marshall, Ellie L Pingry, Martin Dufresne, Melissa A Farrow, Matthew Schrag, Jeffrey M Spraggins.

  Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a rapidly advancing technology for biomedical research. As spatial resolution increases, however, so do acquisition time, file size, and experimental cost, which increases the need to perform precise sampling of targeted tissue regions to optimize the biological information gleaned from an experiment and minimize wasted resources. The ability to define instrument measurement regions based on key tissue features and automatically sample these specific regions of interest (ROIs) addresses this challenge. Herein, we demonstrate a workflow using standard software that allows for direct sampling of microscopy-defined regions by MALDI IMS. Three case studies are included, highlighting different methods for defining features from common sample types─manual annotation of vasculature in human brain tissue, automated segmentation of renal functional tissue units across whole slide images using custom segmentation algorithms, and automated segmentation of dispersed HeLa cells using open-source software. Each case minimizes data acquisition from unnecessary sample regions and dramatically increases throughput while uncovering molecular heterogeneity within targeted ROIs. This workflow provides an approachable method for spatially targeted MALDI IMS driven by microscopy as part of multimodal molecular imaging studies.

Keywords:  HeLa cells; high spatial resolution imaging; high-throughput; human brain; human kidney; molecular imaging; multimodal; single-cell analysis; targeted; targeted sampling

DOI:  https://doi.org/10.1021/jasms.4c00365
Sci Prog. 2024 Oct-Dec;107(4):107(4): 368504241300638

Development and validation of an LC-MS/MS method for the detection of sodium pentachlorophenolate residues on cutting boards.

Shicheng Liao, Wanna Xu, Jie Jiang, Honghe Liu, Zhuoying Zeng.

   OBJECTIVES: The objective of this study was to develop and validate an automated solid-phase extraction (SPE) coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the detection of sodium pentachlorophenolate (PCP-Na) residues on cutting boards. Given the potential hazards and environmental persistence of PCP-Na, a sensitive and reliable method is crucial for monitoring its residues in food contact materials to ensure consumer safety.
METHODS: Wood shavings from cutting boards were extracted using 10% methanol in water, followed by purification using an automated SPE system. The eluent was concentrated, reconstituted, and analyzed by UPLC-MS/MS. An isotope-labeled internal standard was used to mitigate matrix effects, enhancing detection sensitivity. The method was validated by assessing linearity, limit of detection (LOD), limit of quantification (LOQ), recovery rates, and relative standard deviations (RSDs) across various concentration levels.
RESULTS: The method demonstrated excellent linearity over a concentration range of 0 to 100 μg/L with a regression equation of Y = 1.035X-0.7771 and an R² of 0.9996. The LOD and LOQ were determined to be 0.4 and 1.0 μg/kg, respectively. Recovery rates ranged from 71.75% to 96.50% with RSDs between 5.19% and 16.66%. When applied to 30 market cutting board samples, PCP-Na residues were detected in 50% of the samples, with concentrations ranging from 0 to 83,990 µg/kg.
CONCLUSION: This study presents a robust UPLC-MS/MS method for the detection of PCP-Na on cutting boards, offering improved sensitivity and simplified sample preparation. The high detection rate in commercial samples underscores the need for stringent monitoring and regulatory measures to mitigate the exposure risk to consumers.

Keywords:  Sodium pentachlorophenolate; UPLC-MS/MS; cutting boards; food safety; method validation.; solid-phase extraction

DOI:  https://doi.org/10.1177/00368504241300638
Sci Rep. 2024 11 14. 14(1): 27933

Design-assisted HPLC-UV method for therapeutic drug monitoring of pholcodine, ephedrine, and guaifenesin in biological fluids.

Aya Roshdy, Randa Abdel Salam, Ghada Hadad, Fathallah Belal, Heba Elmansi.

  Drug-drug interactions may amplify or diminish their intended effects, or even produce entirely new effects. Multicomponent mixture HPLC analysis offers a thorough and effective method for comprehending the makeup and behavior of complicated materials, advancing research and development across a range of scientific and industrial domains. A novel experimental design-assisted HPLC methodology for the concurrent investigation of the drug-drug interaction of pholcodine, ephedrine, and guaifenesin in biological fluids has been established. Rather than the routine methodology, the application of the factorial design-HPLC method offers a powerful and efficient tool for the analysis of these compounds. Both mixed and full factorial designs were employed to assess the impact of variable factors on chromatographic results. Utilizing an isocratic elution mode on a C18 column, the chromatographic separation was carried out. 15% Methanol, 5% acetonitrile, and 80% phosphate buffer with 0.1%(v/v) triethylamine set to pH 3 make up the mobile phase flowing at rate 1.0 mL/min. The calibration curves of the drugs show excellent linearity over a concentration ranges: 0.20-13.0 µg/mL for PHO, 0.50-20.0 µg/mL for EPH and 0.70-20.0 µg/mL for GUA with LOQ values of 0.18, 0.38 and 0.50. The fast separation and quantitation in less than 6 min is an advantage. Also, the method includes a robust sample preparation protocol for the analysis of complex biological samples, ensuring high selectivity and precision. The ease, speed and cost-effectiveness of the method are ideal for supporting in vitro studies, including drug-drug interaction investigations, especially in bioanalytical labs.

Keywords:  Drug interactions; Ephedrine; Experimental design; Guaifenesin; Pholcodine

DOI:  https://doi.org/10.1038/s41598-024-78793-6
Sci Rep. 2024 Nov 15. 14(1): 28179

Comparative metabolic profiling and quantitative analysis of metabolites in different tissues of Ajuga turkestanica by ESI-UHPLC-QqTOF-MS and NMR.

Nilufar Z Mamadalieva, Michal Šoral, Elana Kysil, Pauline Stark, Andrej Frolov, Ludger A Wessjohann.

  Ajuga turkestanica preparations are used as anti-aging cosmeceuticals and for medicinal purposes. Herein we describe the characterization and quantification of its metabolites in different organs using UHPLC-MS and NMR spectroscopy. A total of 51 compounds belonging to various phytochemical classes (11 flavonoids, 10 ecdysteroids, 9 diterpenes, 6 fatty acids, 5 iridoids, 3 phenylpropanoids, 3 sugars, 2 phenolics, 1 coumarin, 1 triterpene) were annotated and tentatively identified by UHPLC-ESI-QqTOF-MS/MS of methanolic extracts obtained separately from the organs. 1D and 2D NMR spectroscopy independently confirmed the identity of six major compounds. The abundances of these main constituents in flowers, fruits, leaves, roots, seeds, and stems were compared and quantified using 1H NMR. The results showed that 8-O-acetylharpagide, 20-hydroxyecdysone (ecdysterone) and ajugachin B were the most abundant constituents in the species. The two major compounds, 8-O-acetylharpagide and 20-hydroxyecdysone, were chosen as the markers for the quality assessment of A. turkestanica material. The methanolic extract of the aerial parts of A. turkestanica showed no noteworthy anthelmintic (antihelmintic), antifungal, or cytotoxic effect in in vitro assays.

Keywords:   Ajuga turkestanica ; Biological activity; Metabolite annotation; Metabolite profiling; NMR; UHPLC-MS

DOI:  https://doi.org/10.1038/s41598-024-71546-5
Anal Chim Acta. 2024 Dec 01. pii: S0003-2670(24)01136-X. [Epub ahead of print]1331 343335

Towards high throughput analysis using 96-well plate solid phase extraction to determine sedatives and β-blocker residues in food control monitoring.

Ane Arrizabalaga-Larrañaga, Dieke van Doorn, Saskia S Sterk.

   BACKGROUND: Veterinary drugs are widely used in animal production to prevent infections and treat diseases but, this may cause a risk to consumers. Due to the high number of food samples required to monitor yearly, simple, fast, sensitive and selective analytical methods are needed in control laboratories to ensure consumers safety. Nevertheless, many analytical methodologies available in these laboratories include multiple steps and therefore are time-consuming and hinder the analysis throughput requiring significant amounts of solvents and reagents.
RESULTS: This work developed a 96-well plate solid phase extraction (SPE) method for the extraction of seven sedatives and a β-blocker in animal kidney by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The developed method was validated based on Implementing Regulation (EU) 2021/808 in kidney, meat and fish. The performance characteristics of the validation (1-5 μg kg-1) showed good linearity (R2 > 0.998) while decision limits (CCα) were between 1 and 1.2 μg kg-1. Trueness and precision were determined at three levels (n = 7) and the results showed values ranging from 85 to 103 % and from 1 to 9 %, respectively. The feasibility of the method was demonstrated for the residue control requirement established by EU. Method was applied to 201 samples of kidney, meat and fish.
SIGNIFICANCE: This study is the first to present an optimized and validated holistic method for sedatives and β-blocker using 96-well plate SPE and UHPLC-MS/MS. The method showed good performance in kidney, meat and fish samples being an universal method for any species along the same type of sample. The fast, easy, efficient, reliable and universal method showed high throughput and so reduced the analysis time by nine-fold and the required solvent amount by four times fulfilling the green chemistry principals.

Keywords:  Animal tissues; Food safety; LC-MS/MS; Sample preparation; Sedatives

DOI:  https://doi.org/10.1016/j.aca.2024.343335
JIMD Rep. 2024 Nov;65(6): 433-441

Enhanced differentiation between 3-hydroxyglutaric and 2-hydroxyglutaric acids facilitates diagnostic testing for glutaric aciduria type 1.

Denis Cyr, Michel Boutin, Bruno Maranda, Paula J Waters.

  Glutaric aciduria type 1 (GA1) is an inherited neurometabolic disorder, in which deficiency of glutaryl-CoA dehydrogenase leads to accumulation of glutaric acid (GA) and 3-hydroxyglutaric acid (3-HG). Some low excretors may exhibit only slight elevation of urinary 3-HG, with normal urinary GA, yet are at significant risk of severe clinical disease. Accurate quantitation of urinary 3-HG is crucial in diagnostic workup for GA1, but in this context, current gas chromatography-mass spectrometry (GC-MS) methods have inherent analytical challenges. Co-elution and spectral similarities of the 3-HG and 2-HG structural isomers can cause difficulties in quantitation of slightly elevated 3-HG. Our laboratory recently acquired a gas chromatography system coupled to a triple quadrupole mass spectrometer (GC-MS/MS), and we took advantage of its increased sensitivity and specificity to improve our existing GC-MS method. A stable isotope dilution process is used, with sample treatment consisting of a double liquid-liquid extraction followed by a trimethylsilyl derivatization. The transitions m/z 349 → 333 for 3-HG and m/z 349 → 321 for 2-HG were selected to differentiate these two isobaric molecules based on their characteristic fragments, thus minimizing interferences despite co-elution. Method validation demonstrated satisfactory precision and accuracy. Using GC-MS/MS instead of GC-MS allowed us to decrease the required specimen volume, number of sample processing steps, chromatographic run time, and instrument maintenance. This enhanced assay facilitates clinical laboratory testing for GA1, both in confirmatory protocols following positive newborn screening and in diagnostic investigation of patients with suggestive signs or symptoms.

Keywords:  2‐hydroxyglutaric acid; 3‐hydroxyglutaric acid; gas chromatography–tandem mass spectrometry; glutaric acid; glutaric acidemia type 1; glutaric aciduria type 1

DOI:  https://doi.org/10.1002/jmd2.12447
Molecules. 2024 Oct 31. pii: 5162. [Epub ahead of print]29(21):

A Highly Sensitive UPLC-MS/MS Method for the Quantification of the Organic Cation Transporters' Mediated Metformin Uptake and Its Inhibition in Cells.

Gzona Bajraktari-Sylejmani, Cindy Bay, Lukas Gebauer, Jürgen Burhenne, Johanna Weiss, Max Sauter.

  Metformin is the gold standard substrate for evaluating potential inhibitors of the organic cation transporters (OCTs). Here, we established a UPLC-MS/MS assay to quantify metformin in cell pellets with a range of 0.05-50 ng/mL using 6-deuterated metformin as an internal standard. We used an ion-pairing chromatographic approach with heptafluorobutyric acid, making use of a reverse-phase column, and overcame the associated ion-suppression via previously established post-column injection of aqueous ammonia. The assay was validated according to the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) recommendations for bioanalytical methods. The established extraction procedure was simple, very fast and ensured almost 100% recovery of the analyte. The exceptionally sharp peak form and retention of the ion-pairing chromatography are superior to other methods and allow us to measure as sensitively as 0.05 ng/mL. We used the herein established and validated method to develop a cellular OCT inhibition assay by using metformin as a substrate and human embryonic kidney cells (HEK) overexpressing the OCTs 1-3. The method presented may be useful for identifying new OCT inhibitors, but also for drug-drug interactions and other pharmacokinetic studies, where accurate quantification of low metformin amounts in relevant tissues is mandatory.

Keywords:  HEK293; OCT; UPLC-MS/MS; metformin; organic cation transporters; verapamil

DOI:  https://doi.org/10.3390/molecules29215162
J Sep Sci. 2024 Nov;47(21): e70007

Investigation on the Retention and Separation of Glucosinolates With a Mixed-Mode Reversed-Phase/Weak Anion-Exchange Column.

Yanfang Li, Pei Chen, Mengliang Zhang, Jianghao Sun.

  Glucosinolates, a crucial group of secondary metabolites in Brassica vegetables, present significant chromatographic separation challenges due to their anionic form, structure diversities, and co-existence of other phenolic compounds. This study comparatively investigated the retention and separation of seven glucosinolates using a mixed-mode reversed-phase/weak anion-exchange column and a conventional reversed-phase C18 column. Separation factors for each glucosinolate with its adjacent peaks were over 1.0 on the mixed-mode column, while co-eluting was observed on the C18 column. The effects of mobile phase additives and pH on the separation and retention of glucosinolates were also investigated. Results showed that glucosinolate retention was inversely related to both buffer concentration and pH. The optimized method for the mix-mode column was applied to the complex Brassica vegetable samples. In addition to the 17 well-resolved glucosinolate peaks, 34 peaks for phenolic compounds were identified in broccoli microgreen, suggesting the successful application scenarios for qualitative analysis in comparison with the single mode reverse phase C18 column. This study demonstrates that the mixed-mode reversed-phase/weak anion-exchange column can be used as a promising separation tool for organic anions in a complex sample matrix.

Keywords:  chromatographic separation; glucosinolate; mixed‐mode column; retention; reversed‐phase/weak anion‐exchange column

DOI:  https://doi.org/10.1002/jssc.70007
Sci Rep. 2024 11 13. 14(1): 27777

Age and sex effects of a validated LC-MS/MS method for the simultaneous quantification of testosterone, allopregnanolone, and its isomers in human serum.

Khalisa Amir Hamzah, Leisa-Maree Toms, Nathaniel Kucharski, Julia Orr, Peter Hobson, David S Nichols, Luke J Ney.

  Despite the great relevance of the neurosteroid allopregnanolone and related isomers to various health conditions, quantification typically involves immunoassay, which suffers from serious issues with cross-reactivity of closely related molecules. This article describes the development and partial validation of a liquid chromatography coupled with tandem mass spectrometry assay for the simultaneous quantification of allopregnanolone, pregnanolone, isopregnanolone, epi-allopregnanolone, and testosterone in the human serum of healthy males and females aged 5-85 years. 1-amino-4-methylpiperazine (AMP) was used as a derivatisation reagent to enhance the ionisation signal. Linearity was calculated at 0.99 with a lower limit of quantification of 10.08 pg/mL for allopregnanolone, along with a linearity of 0.98 and a lower limit of quantification of 42.32 pg/mL for testosterone. Application of the method showed sex and age effects across the lifespan for both allopregnanolone and testosterone, whereas a comparative immunoassay for allopregnanolone was not able to detect differences in the same samples. Our partial validation of this method should provide a new tool for researchers to discover the role of allopregnanolone and its isomers in human health, and how it compares to testosterone and sex hormones.

Keywords:  Age and sex effects; Allopregnanolone; Liquid chromatography tandem mass spectrometry; Neurosteroids; Testosterone

DOI:  https://doi.org/10.1038/s41598-024-78807-3
Molecules. 2024 Oct 25. pii: 5044. [Epub ahead of print]29(21):

Toxicokinetics and Tissue Distribution of the Hepatotoxic Triterpenoid Saponin Pterocephin A in Rats Using the Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) Method.

Yiran Xiong, Zhaoyue Dong, Hongxu Zhou, Jingxin Mao, Lingjiang Zeng, Yunbin Jiang, Fancheng Meng, Zhihua Liao, Min Chen.

  Pterocephin A is a natural triterpenoid saponin isolated from Pterocephalus hookeri, a traditional Tibetan medicine with slight toxicity, which can induce liver injury in rats. This study aimed to establish a sensitive and reliable UPLC-MS/MS method for exploring the toxicokinetics and tissue distribution of pterocephin A following single intravenous and intragastric administration. Pterocephin A and prosapogenin 1C (internal standard, IS) were extracted using a simple protein precipitation technique with methanol as the precipitant for plasma samples and methanol/acetonitrile = 1:1 (v/v) for tissue samples. UPLC separation was achieved by gradient elution with 0.3 mL/min and a mobile phase consisting of 5 mM ammonium formate (A) and acetonitrile (B) (0-2 min 30% B; 2-4 min: 30-80% B; 4-5 min: 80-98% B; 5-6.5 min: 98% B; 6.5-7 min: 98-30% B; and 7-8 min: 30% B, v/v) with a column temperature of 35 °C. MS spectrometry adopted negative ion scanning mode, primary MS spectrometry adopted full scan monitoring mode, and secondary MS spectrometry adopted targeted MS2 scan monitoring mode. The assay exhibited a linear dynamic range of 0.02-15 μg/mL for pterocephin A in biological samples, with the low limit of quantification set at 0.02 μg/mL. Non-compartmental toxicokinetic parameters indicated that pterocephin A was well absorbed into the systemic circulation and had a long residual time after intravenous (10 mg/kg) and intragastric (60 mg/kg) administration, as it could still be detected after 72 h. Tissue distribution analysis revealed detectable levels of pterocephin A in various tissues, and a high concentration was maintained in the liver after intravenous (10 mg/kg) administration, with the highest concentration being 610.95 ± 25.73 ng/mL and a specific distribution pattern of liver > lung > kidney > intestine > spleen > testes > heart > stomach. The toxicokinetic process and tissue distribution characteristics of pterocephin A were expounded in this study, which can provide relevant data support for further research and clinical application of pterocephin A with its slight toxicity.

Keywords:  Pterocephalus hookeri; UPLC-MS/MS; pterocephin A; tissue distribution; toxicokinetics

DOI:  https://doi.org/10.3390/molecules29215044
BMC Chem. 2024 Nov 07. 18(1): 220

Development and validation of HPLC-UV and LC-MS/MS methods for the quantitative determination of a novel aminothiazole in preclinical samples.

Vinay N Basavanakatti, Mohammad Ali, D R Bharathi, Sheikh Murtuja, Barij Nayan Sinha, Venkatesan Jayaprakash, Faiyaz Shakeel.

  Aminothiazoles are the important class of chemical groups which have proven their broad range of biological activities. A novel aminothiazole (21MAT) was quantified in analytical solutions using a high-performance liquid chromatography (HPLC) approach that was developed and partially validated for the analysis of in vitro experimental samples. An isocratic elution on reverse phase Phenomenex® Luna C18 (50 mm × 4.6 mm, 5 μm) column with 55% 0.1% v/v orthophosphoric acid in water and 45% of orthophosphoric acid in acetonitrile at a flow rate of 1 mL/min was used. The analyte was detected at 272 nm. Similar to this, a robust bioanalytical technique, LC-mass spectrometry (LC-MS/MS) was created and verified to measure 21MAT in rat plasma for use in in vitro screening study samples and early-stage pharmacokinetic research. The protein precipitation method was used to extract 21MAT from plasma. The mixture of 95: 5% v/v methanol: acetonitrile and 0.1% v/v formic acid, along with 15% of 5 mM ammonium formate solution, was used to separate the mixture on a reverse phase Waters Xterra RP® C18 (150 mm × 4.6 mm, 5 μm) column at a flow rate of 1 mL/min. Using electro spray ionisation mode in multiple reaction monitoring mode, the analyte and internal standard (a structural analogue) were both identified. According to current criteria, all validation parameters (specificity, selectivity, accuracy, precision, recovery, matrix factor, hemolysis effect, and stability) were evaluated in rat plasma. The area response of 21MAT was found to be linear over the concentration range of 1.25-1250 ng/mL in rat plasma. Both techniques are suitable for use in any format of preclinical research and were sufficiently reliable to measure 21MAT precisely in various matrices. In silico prediction helped in understanding absorption, distribution, metabolism, excretion, and toxicity (ADMET) behaviour of the molecule. Both developed LC-MS/MS and HPLC-UV methods were successfully used to quantify the analyte in in vitro screening study samples.

Keywords:   In silico ; 2-Aminothiazole; ADMET; ADMETLab2.0; HPLC-UV; Isocratic; LC-MS/MS

DOI:  https://doi.org/10.1186/s13065-024-01321-0