bims-metlip 2024-03-17 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–03–17
eightteen papers selected by
Sofia Costa, Matterworks

Hydrophilic Interaction Liquid Chromatography-Hydrogen/Deuterium Exchange-Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics.
MALDI-MSI-LC-MS/MS Workflow for Single-Section Single Step Combined Proteomics and Quantitative Lipidomics.
Comparisons of different extraction methods and solvents for saliva samples.
Perspective: use and reuse of NMR-based metabolomics data: what works and what remains challenging.
Determination of 27 bovine plasma amino acids and metabolites using zwitterionic-hydrophilic interaction liquid chromatography coupled with isotope dilution electrospray ionization triple quadrupole liquid chromatography-mass spectrometry and the effect of deproteinization timing.
Quantitative mass spectrometry imaging (qMSI): A tutorial.
Multiplexed quantification of venlafaxine and metabolites in human plasma by liquid chromatography-tandem mass spectrometry.
Liquid chromatography-tandem mass spectrometry method for mycophenolic acid and its glucuronide determination in saliva samples from children with nephrotic syndrome.
Using differential mobility spectrometry to improve the specificity of targeted measurements of 2,3-dinor 11β-Prostaglandin F2α.
A microfluidic chip-based capillary zone electrophoresis-mass spectrometry method for measuring adenosine 5'-Triphosphate and its similar nucleotide analogues.
Simultaneous Analysis of Nicarbazin, Diclazuril, Toltrazuril, and Its Two Metabolites in Chicken Muscle and Eggs by In-Syringe Dispersive Solid-Phase Filter Clean-Up Followed by Liquid Chromatography-Tandem Mass Spectrometry.
Development and Validation of an HPLC-UV Method for the Quantification of Acyclovir and Ganciclovir in the Plasma of Pediatric Immunocompromised Patients.
A Simple and Rapid LC-MS/MS Method for the Quantification of Nirmatrelvir/Ritonavir in Plasma of Patients with COVID-19.
The underappreciated diversity of bile acid modifications.
Comprehensive analysis of vitamin D3 impurities in oily drug products using supercritical fluid chromatography-mass spectrometry.
Rat Pharmacokinetics and In Vitro Metabolite Identification of KM-819, a Parkinson's Disease Candidate, Using LC-MS/MS and LC-HRMS.
Targeted Metabolomics on Rare Primary Cells.
Detection of 26 Drugs of Abuse and Metabolites in Quantitative Dried Blood Spots by Liquid Chromatography-Mass Spectrometry.

Int J Mol Sci. 2024 Mar 01. pii: 2899. [Epub ahead of print]25(5):

Hydrophilic Interaction Liquid Chromatography-Hydrogen/Deuterium Exchange-Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics.

Tomas Cajka, Jiri Hricko, Stanislava Rakusanova, Kristyna Brejchova, Michaela Novakova, Lucie Rudl Kulhava, Veronika Hola, Michaela Paucova, Oliver Fiehn, Ondrej Kuda.

  Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time-m/z pairs) in biological samples per analysis, yet the metabolite annotation rate remains low, with 90% of signals classified as unknowns. To enhance the metabolite annotation rates, researchers employ tandem mass spectral libraries and challenging in silico fragmentation software. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) may offer an additional layer of structural information in untargeted metabolomics, especially for identifying specific unidentified metabolites that are revealed to be statistically significant. Here, we investigate the potential of hydrophilic interaction liquid chromatography (HILIC)-HDX-MS in untargeted metabolomics. Specifically, we evaluate the effectiveness of two approaches using hypothetical targets: the post-column addition of deuterium oxide (D2O) and the on-column HILIC-HDX-MS method. To illustrate the practical application of HILIC-HDX-MS, we apply this methodology using the in silico fragmentation software MS-FINDER to an unknown compound detected in various biological samples, including plasma, serum, tissues, and feces during HILIC-MS profiling, subsequently identified as N1-acetylspermidine.

Keywords:  hydrogen/deuterium exchange; liquid chromatography; mass spectrometry; metabolomics; unknown identification

DOI:  https://doi.org/10.3390/ijms25052899
Anal Chem. 2024 Mar 12. 96(10): 4266-4274

MALDI-MSI-LC-MS/MS Workflow for Single-Section Single Step Combined Proteomics and Quantitative Lipidomics.

Tim F E Hendriks, Kasper K Krestensen, Ronny Mohren, Michiel Vandenbosch, Steven De Vleeschouwer, Ron M A Heeren, Eva Cuypers.

We introduce a novel approach for comprehensive molecular profiling in biological samples. Our single-section methodology combines quantitative mass spectrometry imaging (Q-MSI) and a single step extraction protocol enabling lipidomic and proteomic liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis on the same tissue area. The integration of spatially correlated lipidomic and proteomic data on a single tissue section allows for a comprehensive interpretation of the molecular landscape. Comparing Q-MSI and Q-LC-MS/MS quantification results sheds new light on the effect of MSI and related sample preparation. Performing MSI before Q-LC-MS on the same tissue section led to fewer protein identifications and a lower correlation between lipid quantification results. Also, the critical role and influence of internal standards in Q-MSI for accurate quantification is highlighted. Testing various slide types and the evaluation of different workflows for single-section spatial multiomics analysis emphasized the need for critical evaluation of Q-MSI data. These findings highlight the necessity for robust quantification methods comparable to current gold-standard LC-MS/MS techniques. The spatial information from MSI allowed region-specific insights within heterogeneous tissues, as demonstrated for glioblastoma multiforme. Additionally, our workflow demonstrated the efficiency of a single step extraction for lipidomic and proteomic analyses on the same tissue area, enabling the examination of significantly altered proteins and lipids within distinct regions of a single section. The integration of these insights into a lipid-protein interaction network expands the biological information attainable from a tissue section, highlighting the potential of this comprehensive approach for advancing spatial multiomics research.

DOI: https://doi.org/10.1021/acs.analchem.3c05850
Metabolomics. 2024 Mar 09. 20(2): 38

Comparisons of different extraction methods and solvents for saliva samples.

Lingli Fang, Qiming Zhai, Hua Zhang, Ping Ji, Chang Chen, Hongmei Zhang.

   INTRODUCTION: Changes in the categories and concentrations of salivary metabolites may be closely related to oral, intestinal or systemic diseases. To study salivary metabolites, the first analytical step is to extract them from saliva samples as much as possible, while reducing interferences to a minimum. Frequently used extraction methods are protein precipitation (PPT), liquid-liquid extraction (LLE) and solid-phase extraction (SPE), with various organic solvents. The types and quantities of metabolites extracted with different methods may vary greatly, but few studies have systematically evaluated them.
OBJECTIVES: This study aimed to select the most suitable methods and solvents for the extraction of saliva according to different analytical targets.
METHODS: An untargeted metabolomics approach based on liquid chromatography-mass spectrometry was applied to obtain the raw data. The numbers of metabolites, repeatability of the data and intensities of mass spectrometry signals were used as evaluation criteria.
RESULTS: PPT resulted in the highest coverage. Among the PPT solvents, acetonitrile displayed the best repeatability and the highest coverage, while acetone resulted in the best signal intensities for the extracted compounds. LLE with the mixture of chloroform and methanol was the most suitable for the extraction of small hydrophobic compounds.
CONCLUSION: PPT with acetonitrile or acetone was recommended for untargeted analysis, while LLE with the mixture of chloroform and methanol was recommended for small hydrophobic compounds.

Keywords:  LC-MS; Metabolomics; Salivary metabolites; Sample preparation

DOI:  https://doi.org/10.1007/s11306-024-02105-2
Metabolomics. 2024 Mar 13. 20(2): 41

Perspective: use and reuse of NMR-based metabolomics data: what works and what remains challenging.

Goncalo Jorge Gouveia, Thomas Head, Leo L Cheng, Chaevien S Clendinen, John R Cort, Xiuxia Du, Arthur S Edison, Candace C Fleischer, Jeffrey Hoch, Nathaniel Mercaldo, Wimal Pathmasiri, Daniel Raftery, Tracey B Schock, Lloyd W Sumner, Panteleimon G Takis, Valérie Copié, Hamid R Eghbalnia, Robert Powers.

   BACKGROUND: The National Cancer Institute issued a Request for Information (RFI; NOT-CA-23-007) in October 2022, soliciting input on using and reusing metabolomics data. This RFI aimed to gather input on best practices for metabolomics data storage, management, and use/reuse.
AIM OF REVIEW: The nuclear magnetic resonance (NMR) Interest Group within the Metabolomics Association of North America (MANA) prepared a set of recommendations regarding the deposition, archiving, use, and reuse of NMR-based and, to a lesser extent, mass spectrometry (MS)-based metabolomics datasets. These recommendations were built on the collective experiences of metabolomics researchers within MANA who are generating, handling, and analyzing diverse metabolomics datasets spanning experimental (sample handling and preparation, NMR/MS metabolomics data acquisition, processing, and spectral analyses) to computational (automation of spectral processing, univariate and multivariate statistical analysis, metabolite prediction and identification, multi-omics data integration, etc.) studies.
KEY SCIENTIFIC CONCEPTS OF REVIEW: We provide a synopsis of our collective view regarding the use and reuse of metabolomics data and articulate several recommendations regarding best practices, which are aimed at encouraging researchers to strengthen efforts toward maximizing the utility of metabolomics data, multi-omics data integration, and enhancing the overall scientific impact of metabolomics studies.

Keywords:  Best practices; Mass spectrometry; Metabolomics; NMR; Use and reuse of metabolomics datasets

DOI:  https://doi.org/10.1007/s11306-024-02090-6
JDS Commun. 2024 Mar;5(2): 118-123

Determination of 27 bovine plasma amino acids and metabolites using zwitterionic-hydrophilic interaction liquid chromatography coupled with isotope dilution electrospray ionization triple quadrupole liquid chromatography-mass spectrometry and the effect of deproteinization timing.

A F Ortega, M E Van Amburgh.

The use of zwitterionic-hydrophilic interaction liquid chromatography (Z-HILIC) columns for analysis of underivatized analytes has allowed simpler sample preparation of bovine plasma for sensitive and selective analysis, when coupled with mass spectrometry (MS). The objective of this study was to evaluate and validate this analytical technique to measure AA and metabolites in bovine plasma at 2 deproteinization times. A robust method using Z-HILIC coupled to a triple quadrupole MS (TQMS) was evaluated and validated to quantitatively analyze 19 AA using isotope dilution and 8 AA metabolites qualitatively in bovine deproteinized plasma. The timing of deproteinization was investigated to determine if plasma should be deproteinized upon collection (on-site) or immediately before analysis (in-lab). Analytes were separated using a Z-HILIC column in a 21 min run and analyzed with a TQMS in positive electrospray ionization for identification and quantification. The method was validated for standard curve linearity, limits of detection (LOD) and quantification (LOQ), intra- and interday precision (% coefficient of variation; CV), recovery (%), and freeze-thaw stability (% CV) after 1 mo. Coefficients of determination (R2) were over 0.993, and LOD and LOQ were below measured values for all AA. The CV for the intraday and interday precision were below 18%, except for cystine (Cys2) and Orn in-lab. Recoveries on-site and in-lab ranged from 75% to 120% for all analytes except Cys2 in-lab. Most analytes were stable after 1 mo of freezing regardless of deproteinization timing, CV <25%, except for hydroxyproline (Hyp). The concentration of Cys2 was affected by deproteinization in-lab compared with on-site, and even though Glu and Hyp were different between the 2 deproteinization timings, the concentrations between the 2 timings were within the standard deviation.

DOI: https://doi.org/10.3168/jdsc.2023-0449
J Mass Spectrom. 2024 Apr;59(4): e5009

Quantitative mass spectrometry imaging (qMSI): A tutorial.

Russell R Kibbe, David C Muddiman.

  Mass spectrometry imaging (MSI) is an analytical technique that enables the simultaneous detection of hundreds to thousands of chemical species while retaining their spatial information; usually, MSI is applied to biological tissues. Combining these elements can create ion images, which allows for the identification and localization of multiple chemical species within the sample. Being able to produce molecular images of biological tissues has already impacted the study of health and disease; however, the next logical step is being able to combine MSI with quantitative mass spectrometry methods to both quantify and determine the localization of disease progression or drug action. In this tutorial, we will detail the main factors to consider when designing a qMSI experiment and highlight the methods that have been developed to overcome these added complexities, specifically for those newer to the field of MSI.

Keywords:  absolute quantification; mass spectrometry imaging; qMSI; relative quantification

DOI:  https://doi.org/10.1002/jms.5009
J Pharm Biomed Anal. 2024 Mar 07. pii: S0731-7085(24)00122-5. [Epub ahead of print]243 116082

Multiplexed quantification of venlafaxine and metabolites in human plasma by liquid chromatography-tandem mass spectrometry.

Aashish Pandey, Amelia Price, Nadia Ayala-Lopez, Kyana Y Garza, Mark A Marzinke, Claire E Knezevic.

   BACKGROUND: Venlafaxine (VEN) and its O-demethylated metabolite, O-desmethylvenlafaxine (ODV), are commonly prescribed serotonin-norepinephrine reuptake inhibitors, approved for the treatment of depression and anxiety. Both are metabolized to inactive metabolites via cytochrome P450 enzymes. While previous studies have focused on quantifying VEN and ODV, bioanalytical methods for the simultaneous measurement of all metabolites are needed to fully characterize the pharmacology of VEN and ODV.
METHODS: K2EDTA plasma was spiked with VEN, ODV, N-desmethylvenlafaxine (NDV), N,O-didesmethylvenlafaxine (NODDV), and N,N-didesmethylvenlafaxine (NNDDV). Drugs and metabolites were extracted via protein precipitation and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The multiplexed assay was validated in accordance with regulatory recommendations, and evaluated in remnant plasma samples from persons prescribed venlafaxine.
RESULTS: The analytical measuring range for venlafaxine and all four metabolites was 5-800 ng/mL. Standard curves were generated via weighted quadratic (NNDDV) or linear (VEN, ODV, NDV, NODDV) regression of calibrators. Inter-assay imprecision was between 1.9-9.3% for all levels of all analytes. Minor matrix effects were observed, and both recovery efficiency and process efficiency were >96% for all analytes. All other assay validation assessments met acceptance criteria. Drug concentrations measured from remnant plasma specimens obtained from patients with current venlafaxine prescriptions (37.5-450 mg/day) yielded NDDV, NDV, and NODDV metabolite concentrations in 6/21, 14/21, and 20/21 samples, respectively. The ratio of active to inactive analytes ranged from 0.74 to 14.5, with a median of 6.39.
CONCLUSIONS: An efficient and accurate LC-MS/MS method was developed and validated for the quantification of VEN, ODV, and all three inactive metabolites in plasma. The assay met all acceptance criteria, and may be used in future studies of the pharmacokinetics of these drugs.

Keywords:  Antidepressant; Mass spectrometry; Metabolites; Venlafaxine

DOI:  https://doi.org/10.1016/j.jpba.2024.116082
Pharmacol Rep. 2024 Mar 15.

Liquid chromatography-tandem mass spectrometry method for mycophenolic acid and its glucuronide determination in saliva samples from children with nephrotic syndrome.

Joanna Sobiak, Matylda Resztak, Weronika Sikora, Jacek Zachwieja, Danuta Ostalska-Nowicka.

   BACKGROUND: Saliva sampling is one of the methods of therapeutic drug monitoring for mycophenolic acid (MPA) and its metabolite, mycophenolic acid glucuronide (MPAG). The study describes the liquid chromatography tandem mass spectrometry (LC-MS/MS) method developed for saliva MPA and MPAG determination in children with nephrotic syndrome.
METHODS: The mobile phase consisted of methanol and water at gradient flow, both with 0.1% formic acid. Firstly, 100 µL of saliva was evaporated at 45 °C for 2 h to dryness, secondly, it was reconstituted in the mobile phase, and finally 10 µL was injected into the LC-MS/MS system. Saliva from ten children with nephrotic syndrome treated with mycophenolate mofetil was collected with Salivette®.
RESULTS: For MPA and MPAG, within the 2-500 ng/mL range, the method was selective, specific, accurate and precise within-run and between-run. No carry-over and matrix effects were observed. Stability tests showed that MPA and MPAG were stable in saliva samples if stored for 2 h at room temperature, 18 h at 4 °C, and at least 5 months at - 80 °C as well as after three freeze-thaw cycles, in a dry extract for 16 h at 4 °C, and for 8 h at 15 °C in the autosampler. The analytes were not adsorbed onto Salivette® cotton swabs. For concentrations above 500 ng/mL, the samples may be diluted twofold. In children, saliva MPA and MPAG were within the ranges of 4.6-531.8 ng/mL and 10.7-183.7 ng/mL, respectively.
CONCLUSIONS: The evaluated LC-MS/MS method has met the validation requirements for saliva MPA and MPAG determination in children with nephrotic syndrome. Further studies are needed to explore plasma-saliva correlations and assess their potential contribution to MPA monitoring.

Keywords:  Mycophenolate mofetil; Nephrotic syndrome; Pediatric patients; Saliva; Therapeutic drug monitoring

DOI:  https://doi.org/10.1007/s43440-024-00574-9
Clin Biochem. 2024 Mar 08. pii: S0009-9120(24)00039-0. [Epub ahead of print]126 110745

Using differential mobility spectrometry to improve the specificity of targeted measurements of 2,3-dinor 11β-Prostaglandin F2α.

Kayla Moehnke, Jennifer Kemp, Michelle R Campbell, Ravinder J Singh, Anne E Tebo, Anthony Maus.

   INTRODUCTION: 2,3-dinor 11β-Prostaglandin F2α (BPG) is an arachidonic acid derivative and the most abundant metabolic byproduct of prostaglandin D2, which is released during mast cell activation. Therefore, measurements of BPG in urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a noninvasive method for evaluation and management of mast cell disorders. Measurements obtained by LC-MS/MS exhibit a high prevalence of chromatographic interferences resulting in challenges with optimal determination of BGP. In this investigation, differential mobility spectrometry (DMS) is utilized to overcome the limitations of current testing.
METHODS: Urine samples were extracted using an automated solid-phase extraction method. Samples were then analyzed with and without DMS devices installed on two commercially available mass spectrometry platforms to assess the benefits of DMS. Following promising results from a preliminary analytical evaluation, LC-DMS-MS/MS measurements of BPG in urine were fully validated to assess the analytical implications of using this technology.
RESULTS AND DISCUSSION: The addition of DMS devices to the LC-MS/MS systems evaluated in this investigation significantly reduced interferences observed in the chromatograms. Concomitantly, DMS reduced the number of discordant quantifier/qualifier fragment ion results that significantly exceeded the ± 20 % limits, suggesting greater analytical specificity. The validation studies yielded low interday imprecision, with %CVs less than 6.5 % across 20 replicate measurements. Validation studies assessing other aspects of analytical performance also met acceptance criteria.
CONCLUSIONS: Incorporating DMS devices greatly improved the specificity of BPG measurements by LC-MS/MS, as evidenced by the comparison of chromatograms and fragment ion results. Validation studies showed exceptional performance for established analytical metrics, indicating that this technology can be used to minimize the impact of interferences without adversely impacting other aspects of analytical or clinical performance.

Keywords:  Differential mobility spectrometry; Ion mobility; LC-MS/MS; Tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.clinbiochem.2024.110745
Anal Chim Acta. 2024 Apr 15. pii: S0003-2670(24)00201-0. [Epub ahead of print]1298 342400

A microfluidic chip-based capillary zone electrophoresis-mass spectrometry method for measuring adenosine 5'-Triphosphate and its similar nucleotide analogues.

Cristina Di Poto, Xiang Tian, Scott Mellors, Sanna Rosengren, Sabina Issop, Sara J Bonvini, Sonja Hess, Erik L Allman.

   BACKGROUND: Extracellular ATP is involved in disorders that cause inflammation of the airways and cough, thus limiting its release has therapeutic benefits. Standard luminescence-based ATP assays measure levels indirectly through enzyme degradation and do not provide a simultaneous readout for other nucleotide analogues. Conversely, mass spectrometry can provide direct ATP measurements, however, common RPLC and HILIC methods face issues because these molecules are unstable, metal-sensitive analytes which are often poorly retained. These difficulties have traditionally been overcome using passivation or ion-pairing chromatography, but these approaches can be problematic for LC systems. As a result, more effective analytical methods are needed.
RESULTS: Here, we introduce a new application that uses microfluidic chip-based capillary zone electrophoresis-mass spectrometry (μCZE-MS) to measure ATP and its analogues simultaneously in biofluids. The commercially available ZipChip Interface and a High-Resolution Bare-glass microchip (ZipChip, HRB, 908 Devices Inc.) coupled to a Thermo Scientific Tribrid Orbitrap, were successfully used to separate and detect various nucleotide standards, as well as ATP, ADP, AMP, and adenosine in plasma and BALF obtained from naïve Brown Norway rats. The findings demonstrate that this approach can rapidly and directly detect ATP and its related nucleotide analogues, while also highlighting the need to preserve these molecules in biofluids with chelators like EDTA. In addition, we demonstrate that this μCZE-MS method is also suitable for detecting a variety of metabolites, revealing additional potential future applications.
SIGNIFICANCE: This innovative μCZE-MS approach provides a robust new tool to directly measure ATP and other nucleotide analogues in biofluids. This can enable the study of eATP in human disease and potentially contribute to the creation of ATP-targeting therapies for airway illnesses.

Keywords:  Adenosine 5′-triphosphate; Bronchoalveolar lavage fluid; Capillary zone electrophoresis mass spectrometry; Chronic cough; Ethylenediaminetetraacetic acid; Metabolomics

DOI:  https://doi.org/10.1016/j.aca.2024.342400
Foods. 2024 Feb 29. pii: 754. [Epub ahead of print]13(5):

Simultaneous Analysis of Nicarbazin, Diclazuril, Toltrazuril, and Its Two Metabolites in Chicken Muscle and Eggs by In-Syringe Dispersive Solid-Phase Filter Clean-Up Followed by Liquid Chromatography-Tandem Mass Spectrometry.

Yuxin An, Xiaodong Pan, Zengxuan Cai, Meijia Xu, Dingguo Jiang, Xiaomin Xu.

  Nicarbazin (NICA) and triazine anticoccidial drugs (diclazuril (DIZ) and toltrazuril (TOZ)) are the primary strategy for preventing and treating coccidiosis. To prevent the development of drug resistance and mitigate the potential chronic toxicity to humans resulting from prolonged exposure, a liquid chromatography-tandem mass spectrometry method with high reliability and sensitivity was developed to determine NICA, DIZ, TOZ, and its two metabolites in chicken muscle and eggs. Upon establishing the extraction conditions involving 10 mL of acetonitrile and 10 min of sonication, in-syringe dispersive solid-phase extraction with silica was performed in combination with n-hexane clean-up. The selection of isotope peaks of precursor ions and low-mass range scanning allowed the two transitions for the quantification of all compounds. The limits of detection for DIZ and NICA were both 0.1 μg/kg, and for TOZ and metabolites, they were 0.3 μg/kg; the limits of quantitation were 0.3 and 1 μg/kg, respectively. The linear range was 0.25-50 ng/mL with a correlation coefficient r > 0.999. The average recoveries at three spiking levels in muscle and eggs were 90.1-105.2% and 94.0-103.7% with the relative standard deviations of 3.0-8.1% and 3.1-14.4%, respectively. The precision, accuracy, and stability were evaluated by three quality control samples.

Keywords:  anticoccidial drugs; chicken muscle; eggs; in-syringe dispersive solid-phase clean-up; liquid chromatography; tandem mass spectrometry

DOI:  https://doi.org/10.3390/foods13050754
Int J Mol Sci. 2024 Feb 26. pii: 2685. [Epub ahead of print]25(5):

Development and Validation of an HPLC-UV Method for the Quantification of Acyclovir and Ganciclovir in the Plasma of Pediatric Immunocompromised Patients.

Martina Franzin, Rachele Ruoso, Rossella Del Savio, Riccardo Addobbati.

  Acyclovir and ganciclovir comprise the prophylaxis and treatment of herpesvirus and cytomegalovirus infections occurring in immunocompromised patients. Their therapeutic drug monitoring is fundamental because of interindividual variability leading to side effects and drug resistance and is performed through several techniques, such as liquid chromatography coupled with UV spectrophotometry (HPLC-UV) or mass spectrometry (LC-MS/MS). Therefore, we developed and validated a low-cost, non-time-consuming, and low-sample-consuming HPLC-UV method. Briefly, 100 µL of sample was used for sample preparation, mainly consisting of precipitation through organic solvent. In total, 20 µL was injected into the instrument. Chromatographic separation was obtained eluting mobile phases A (10 mM ammonium formiate 0.01% formic acid) and B (acetonitrile) on a Poroshell 120 SB-C8 2.1 × 150 mm, 2.7 µm for 12 min isocratically (97:3; A:B) at a flow rate of 0.2 mL/min. The linearity range (0.5-40 mg/L) of the method allowed us to quantify both the Cmin and Cmax of acyclovir and ganciclovir. Plasma concentrations measured on a small cohort of patients undergoing acyclovir (31) and ganciclovir (9) treatment by the proposed method and the LC-MS/MS methods, already in use, were significantly correlated. The proposed HPLC-UV method may be implemented in diagnostics as an alternative method in case of the unavailability of the LC-MS/MS system.

Keywords:  acyclovir; ganciclovir; pediatric; therapeutic drug monitoring

DOI:  https://doi.org/10.3390/ijms25052685
Int J Anal Chem. 2024 ;2024 6139928

A Simple and Rapid LC-MS/MS Method for the Quantification of Nirmatrelvir/Ritonavir in Plasma of Patients with COVID-19.

Xiujing Zhu, Lin Li, Bing Dai, Zhijun Liu, Zhipeng Wang, Lili Cui, Shouhong Gao, Wansheng Chen, Xia Tao, Deduo Xu.

The combined prescriptions of nirmatrelvir/ritonavir and other drugs are limited due to potential drug-drug interactions, so therapeutic drug monitoring (TDM) becomes particularly important. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for determination of the nirmatrelvir/ritonavir in plasma of patients with COVID-19, providing technical and theoretical support for the TDM. Plasma samples were processed by protein precipitation using acetonitrile, and analytes were separated on an Agilent Poroshell 120 SB-C18 (2.1 × 75 mm, 2.7 μm) column at 35°C. Acetonitrile and 0.1% formic acid in water (52 : 48) were utilized as the mobile phases at a flow rate of 0.3 mL/min. In the multiple reaction monitoring (MRM) mode, nirmatrelvir and ritonavir were monitored using precursor/product ions: m/z 500.2/110.1 and 721.3/296.1, respectively, with selinexor as the internal standard. The linear range of both analytes was 2.0 ng/mL to 5000 ng/mL with good inter- and intraday precision and accuracy, and the recovery was 92.0%-107% for nirmatrelvir and 85.7%-106% for ritonavir. Finally, this method was successfully applied to monitor the exposure levels of nirmatrelvir/ritonavir in plasma samples from hemodialysis patients.

DOI: https://doi.org/10.1155/2024/6139928
Cell. 2024 Mar 06. pii: S0092-8674(24)00185-5. [Epub ahead of print]

The underappreciated diversity of bile acid modifications.

Ipsita Mohanty, Helena Mannochio-Russo, Joshua V Schweer, Yasin El Abiead, Wout Bittremieux, Shipei Xing, Robin Schmid, Simone Zuffa, Felipe Vasquez, Valentina B Muti, Jasmine Zemlin, Omar E Tovar-Herrera, Sarah Moraïs, Dhimant Desai, Shantu Amin, Imhoi Koo, Christoph W Turck, Itzhak Mizrahi, Penny M Kris-Etherton, Kristina S Petersen, Jennifer A Fleming, Tao Huan, Andrew D Patterson, Dionicio Siegel, Lee R Hagey, Mingxun Wang, Allegra T Aron, Pieter C Dorrestein.

  The repertoire of modifications to bile acids and related steroidal lipids by host and microbial metabolism remains incompletely characterized. To address this knowledge gap, we created a reusable resource of tandem mass spectrometry (MS/MS) spectra by filtering 1.2 billion publicly available MS/MS spectra for bile-acid-selective ion patterns. Thousands of modifications are distributed throughout animal and human bodies as well as microbial cultures. We employed this MS/MS library to identify polyamine bile amidates, prevalent in carnivores. They are present in humans, and their levels alter with a diet change from a Mediterranean to a typical American diet. This work highlights the existence of many more bile acid modifications than previously recognized and the value of leveraging public large-scale untargeted metabolomics data to discover metabolites. The availability of a modification-centric bile acid MS/MS library will inform future studies investigating bile acid roles in health and disease.

Keywords:  GABA; MassQL; agmatine; bile acids; diet; fastMASST; microbial; polyamines; putrescine; spectral resource

DOI:  https://doi.org/10.1016/j.cell.2024.02.019
Drug Test Anal. 2024 Mar 14.

Comprehensive analysis of vitamin D3 impurities in oily drug products using supercritical fluid chromatography-mass spectrometry.

Hugues Jambo, Amandine Dispas, Leticia Pérez-Mayán, Isaac Rodríguez, Éric Ziemons, Philippe Hubert.

  Vitamin D3 , an essential micronutrient, often requires supplementation via medicines or food supplements, which necessitate quality control (QC). This study presents the development of a method for detecting and quantifying seven impurities of vitamin D3 in oily drug products using supercritical fluid chromatography-mass spectrometry (SFC-MS). Targeted impurities include two esters of vitamin D3 and five non-esters including four that are isobaric to vitamin D3 . Firstly, a screening study highlighted the Torus 1-AA column and acetonitrile modifier as adequate for the separation, followed by optimization of the SFC conditions. Secondly, make-up solvent composition and MS settings were optimized to reach high sensitivity. For both the separation and MS response, the screening design of experiments proved useful. Lastly, a fast saponification and liquid-liquid extraction method was developed, enabling efficient sample cleanup and impurities recovery from the complex oily matrix. The SFC-MS method suitability was assessed in two validation studies. The first study employed the ICH Q2 guideline for impurity limit test to demonstrate method specificity and establish a limit of detection (LOD) and a limit of quantification (LOQ) at 0.2% and 0.5%, respectively, for ester impurities. The second study conducted a comprehensive quantitative assessment for three non-ester impurities using a total error approach, determining method validity through accuracy profiles. The validated method exhibited reliable performance across impurity concentrations from 0.1% to 2.0%, with estimated LODs ranging from 2 to 7 ng/mL. This study further promotes SFC-MS as a valuable, versatile, and green tool for routine pharmaceutical QC.

Keywords:  SFC-MS; impurities; quality control; validation; vitamin D3

DOI:  https://doi.org/10.1002/dta.3670
Molecules. 2024 Feb 25. pii: 1004. [Epub ahead of print]29(5):

Rat Pharmacokinetics and In Vitro Metabolite Identification of KM-819, a Parkinson's Disease Candidate, Using LC-MS/MS and LC-HRMS.

Hae-In Choi, Taeheon Kim, Jin Woo Kim, Gi Ju Lee, Jinyoung Choi, Yoon-Jee Chae, Eunhee Kim, Tae-Sung Koo.

  FAF1 (FAS-associated factor 1) is involved in the activation of Fas cell surface death receptors and plays a role in apoptosis and necrosis. In patients with Parkinson's disease, FAF1 is overexpressed in dopaminergic neurons in the substantia nigra. KM-819, an FAF1 inhibitor, has shown potential for preventing dopaminergic neuronal cell death, promoting the degradation of α-synuclein and preventing its accumulation. This study aimed to develop and validate a quantitative analytical method for determining KM-819 levels in rat plasma using liquid chromatography-tandem mass spectrometry. This method was then applied to pharmacokinetic (PK) studies in rats. The metabolic stability of KM-819 was assessed in rat, dog, and human hepatocytes. In vitro metabolite identification and metabolic pathways were investigated in rat, dog, and human hepatocytes. The structural analog of KM-819, namely N-[1-(4-bromobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl]-2-(phenylsulfanyl) acetamide, served as the internal standard (IS). Proteins were precipitated from plasma samples using acetonitrile. Analysis was carried out using a reverse-phase C18 column with a mobile phase consisting of 0.1% formic acid in distilled water and 0.1% formic acid in acetonitrile. The analytical method developed for KM-819 exhibited linearity within the concentration range of 0.002-10 μg/mL in rat plasma. The precision and accuracy of the intra- and inter-day measurements were <15% for the lower limit of quantification (LLOQ) and all quality control samples. KM-819 demonstrated stability under various sample storage conditions (6 h at room temperature (25 °C), four weeks at -20 °C, three freeze-thaw cycles, and pretreated samples in the autosampler). The matrix effect and dilution integrity met the criteria set by the Food and Drug Administration and the European Medicines Agency. This sensitive, rapid, and reliable analytical method was successfully applied in pharmacokinetic studies in rats. Pharmacokinetic analysis revealed the dose-independent kinetics of KM-819 at 0.5-5 mg/kg, with a moderate oral bioavailability of ~20% in rats. The metabolic stability of KM-819 was also found to be moderate in rat, dog, and human hepatocytes. Metabolite identification in rat, dog, and human hepatocytes resulted in the discovery of six, six, and eight metabolites, respectively. Glucuronidation and mono-oxidation have been proposed as the major metabolic pathways. Overall, these findings contribute to a better understanding of the pharmacokinetic characteristics of KM-819, thereby aiding future clinical studies.

Keywords:  FAF1 inhibitor; KM-819; LC-HRMS; LC-MS/MS; metabolite identification; pharmacokinetics

DOI:  https://doi.org/10.3390/molecules29051004
J Vis Exp. 2024 Feb 23.

Targeted Metabolomics on Rare Primary Cells.

Katharina M Glaser, Mirijam Egg, Sebastian Hobitz, Michael Mitterer, Dominik Schain-Zota, Katharina Schönberger, Konrad Schuldes, Nina Cabezas-Wallscheid, Tim Lämmermann, Angelika Rambold, Joerg M Buescher.

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule intermediates. However, obtaining accurate and reliable measurements of cellular metabolism, particularly in rare cell types like hematopoietic stem cells, has traditionally required pooling cells from multiple animals. A protocol now enables researchers to measure metabolites in rare cell types using only one mouse per sample while generating multiple replicates for more abundant cell types. This reduces the number of animals that are required for a given project. The protocol presented here involves several key differences over traditional metabolomics protocols, such as using 5 g/L NaCl as a sheath fluid, sorting directly into acetonitrile, and utilizing targeted quantification with rigorous use of internal standards, allowing for more accurate and comprehensive measurements of cellular metabolism. Despite the time required for the isolation of single cells, fluorescent staining, and sorting, the protocol can preserve differences among cell types and drug treatments to a large extent.

DOI: https://doi.org/10.3791/65690
Molecules. 2024 Feb 23. pii: 975. [Epub ahead of print]29(5):

Detection of 26 Drugs of Abuse and Metabolites in Quantitative Dried Blood Spots by Liquid Chromatography-Mass Spectrometry.

Thomas Meikopoulos, Helen Gika, Georgios Theodoridis, Olga Begou.

  A method was developed for the determination of 26 drugs of abuse from different classes, including illicit drugs in quantitative dried blood spots (qDBSs), with the aim to provide a convenient method for drug testing by using only 10 μL of capillary blood. A satisfactory limit of quantification (LOQ) of 2.5 ng/mL for 9 of the compounds and 5 ng/mL for 17 of the compounds and a limit of detection (LOD) of 0.75 ng/mL for 9 of the compounds and 1.5 ng/mL for 17 of the compounds were achieved for all analytes. Reversed-phase liquid chromatography was applied on a C18 column coupled to MS, providing selective detections with both +ESI and -ESI modes. Extraction from the qDBS was performed using AcN-MeOH, 1:1 (v/v), with recovery ranging from 84.6% to 106%, while no significant effect of the hematocrit was observed. The studied drugs of abuse were found to be stable over five days under three different storage conditions (at ambient temperature 21 °C, at -20 °C, and at 35 °C), thus offering a highly attractive approach for drug screening by minimally invasive sampling for individuals that could find application in forensic toxicology analysis.

Keywords:  LC–MS/MS analysis; blood micro sampling; drug screening; drugs of abuse; quantitative dried blood spot

DOI:  https://doi.org/10.3390/molecules29050975