bims-metlip 2022-09-11 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2022–09–11
34 papers selected by
Sofia Costa, Matterworks

Instrument-type effects on chemical isotope labeling LC-MS metabolome analysis: Quadrupole time-of-flight MS vs. Orbitrap MS.
Advances in mass spectrometry imaging for spatial cancer metabolomics.
Liquid Chromatography-Mass Spectrometry (LC-MS) Derivatization-Based Methods for the Determination of Fatty Acids in Biological Samples.
A re-calibration procedure for interoperable lipid collision cross section values measured by traveling wave ion mobility spectrometry.
Short-chain fatty acids profiling in biological samples from a mouse model of Sjögren's syndrome based on derivatized LC-MS/MS assay.
A Novel and Sensitive Method for the Analysis of Fatty Acid Biosignatures by Capillary Electrophoresis-Mass Spectrometry.
A Direct Infusion Probe for Rapid Metabolomics of Low-Volume Samples.
Spatially resolved characterization of tissue metabolic compartments in fasted and high-fat diet livers.
Development and Validation of a Highly Sensitive and Rapid LC-MS3 Strategy to Determine Oxcarbazepine and Its Active Metabolite in the Serum of Patients with Epilepsy and Its Application in Therapeutic Drug Monitoring.
Development and Validation of a Modified QuEChERS Method for Simultaneous Analysis of 250 Flavor Constituents in Tobacco by Gas Chromatography Tandem Mass Spectrometry.
Development and application of a High-Resolution mass spectrometry method for the detection of fentanyl analogs in urine and serum.
A rapid and standardized workflow for functional assessment of bacterial biosensors in fecal samples.
Free fatty acid profiling of Greek yogurt by liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis.
Improved Spatial Resolution of Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) Mass Spectrometry Imaging (MSI) Using a Reflective Objective.
Simultaneous quantification of 106 drugs or their metabolites in nail samples by UPLC-MS/MS with high-throughput sample preparation: Application to 294 real cases.
Development of the Method for Nusinersen and Its Metabolites Identification in the Serum Samples of Children Treated with Spinraza for Spinal Muscular Atrophy.
Isomer and Conformer-Specific Mass Distribution-Based Isotopic Shifts in High-Resolution Cyclic Ion Mobility Separations.
Volumetric Absorptive Microsampling (VAMS) for Targeted LC-MS/MS Determination of Tryptophan-Related Biomarkers.
Optimization of capillary electrophoresis coupled to negative mode electrospray ionization-mass spectrometry using polyvinyl alcohol coated capillaries. Application to a study on non-small cell lung cancer.
Rapid determination of perfluorinated compounds in pork samples using a molecularly imprinted phenolic resin adsorbent in dispersive solid phase extraction-liquid chromatography tandem mass spectrometry.
Quantification of monoamine biomarkers in cerebrospinal fluid: comparison of a UHPLC-MS/MS method to a UHPLC coupled to fluorescence detection method.
Multi-DNA Adduct and Abasic Site Quantitation In Vivo by Nano-Liquid Chromatography/High-Resolution Orbitrap Tandem Mass Spectrometry: Methodology for Biomonitoring Colorectal DNA Damage.
Bioanalysis of Trifluoperazine in human plasma by LC-MS/MS: Application to disposition kinetics.
Quantification of Gut Microbiota Dysbiosis-Related Organic Acids in Human Urine Using LC-MS/MS.
Establishment and validation of a LC-MS/MS method for the determination of anlotinib in human plasma: application to therapeutic drug monitoring.
Novel molecularly imprinted phenolic resin-dispersive filter extraction for rapid determination of perfluorooctanoic acid and perfluorooctane sulfonate in milk.
Oligonucleotides Isolation and Separation-A Review on Adsorbent Selection.
New Insights into Vitamin K-From Its Natural Sources through Biological Properties and Chemical Methods of Quantitative Determination.
Development and Validation of a Sensitive and Specific LC-MS/MS Method for IWR-1-Endo, a Wnt Signaling Inhibitor: Application to a Cerebral Microdialysis Study.
Liquid chromatography-mass spectrometry measurements of blood diphosphoinositol pentakisphosphate levels.
Untargeted ultra-high-resolution mass spectrometry metabolomic profiling of blood serum in bladder cancer.
Development of a mass spectrometry-based metabolomics workflow for traceability of wild and cultivated Cordyceps sinensis.
DNA photoproducts released by repair in biological fluids as biomarkers of the genotoxicity of UV radiation.
Comparison of LC-MS3 and LC-MRM Method for Quantifying Voriconazole and Its Application in Therapeutic Drug Monitoring of Human Plasma.

Anal Chim Acta. 2022 Sep 15. pii: S0003-2670(22)00826-1. [Epub ahead of print]1226 340255

Instrument-type effects on chemical isotope labeling LC-MS metabolome analysis: Quadrupole time-of-flight MS vs. Orbitrap MS.

Chu-Fan Wang, Liang Li.

  Chemical isotope labeling (CIL) LC-MS is a powerful tool for metabolome analysis with markedly improved metabolomic coverage and quantification accuracy over the conventional LC-MS technique. In addition, with differential isotope labeling, each labeled metabolite is detected as a peak pair in the mass spectra, offering the possibility of differentiating true metabolite peaks from the singlet noise or background peaks. In this study, we examined the effects of instrument type on the detectability of true metabolites with a focus on the comparison of quadrupole time-of-flight (QTOF) and Orbitrap mass spectrometers. Using the same ultra-high-performance liquid chromatography setup and optimized running conditions for QTOF and Orbitrap, we compared the total number of peak pairs detected and identified from the two instruments using human urine and serum as the test samples. Many common peak pairs were detected from the two instruments; however, there were a significant number of unique peak pairs detected in each type of instrument. By combining the datasets obtained using QTOF and Orbitrap, the total number of peak pairs detected could be significantly increased. We also examined the effect of mass resolving power on peak pair detection in Orbitrap (60,000 vs. 120,000 resolution). The observed differences in peak pair detectability were much less than those of QTOF vs. Orbitrap. However, the type of peak pairs detected using different resolutions could be somewhat different, offering the possibility of increasing the overall number of peak pairs by combining the two datasets obtained at two different resolutions. The results from this study clearly indicate that instrument type can have a profound effect on metabolite detection in CIL LC-MS. Therefore, comparison of metabolome data generated using different instruments needs to be carefully done. Moreover, future research (e.g., hardware modifications) is warranted to minimize the differences in order to generate more reproducible metabolome data from different types of instruments.

Keywords:  Chemical isotope labeling; Liquid chromatography; Mass spectrometry; Metabolomics; Orbitrap; QTOF

DOI:  https://doi.org/10.1016/j.aca.2022.340255
Mass Spectrom Rev. 2022 Sep 06. e21804

Advances in mass spectrometry imaging for spatial cancer metabolomics.

Xin Ma, Facundo M Fernández.

  Mass spectrometry (MS) has become a central technique in cancer research. The ability to analyze various types of biomolecules in complex biological matrices makes it well suited for understanding biochemical alterations associated with disease progression. Different biological samples, including serum, urine, saliva, and tissues have been successfully analyzed using mass spectrometry. In particular, spatial metabolomics using MS imaging (MSI) allows the direct visualization of metabolite distributions in tissues, thus enabling in-depth understanding of cancer-associated biochemical changes within specific structures. In recent years, MSI studies have been increasingly used to uncover metabolic reprogramming associated with cancer development, enabling the discovery of key biomarkers with potential for cancer diagnostics. In this review, we aim to cover the basic principles of MSI experiments for the nonspecialists, including fundamentals, the sample preparation process, the evolution of the mass spectrometry techniques used, and data analysis strategies. We also review MSI advances associated with cancer research in the last 5 years, including spatial lipidomics and glycomics, the adoption of three-dimensional and multimodal imaging MSI approaches, and the implementation of artificial intelligence/machine learning in MSI-based cancer studies. The adoption of MSI in clinical research and for single-cell metabolomics is also discussed. Spatially resolved studies on other small molecule metabolites such as amino acids, polyamines, and nucleotides/nucleosides will not be discussed in the context.

Keywords:  DESI; MALDI; SIMS; cancer; glycans; lipids; mass spectrometry imaging; spatial metabolomics

DOI:  https://doi.org/10.1002/mas.21804
Molecules. 2022 Sep 05. pii: 5717. [Epub ahead of print]27(17):

Liquid Chromatography-Mass Spectrometry (LC-MS) Derivatization-Based Methods for the Determination of Fatty Acids in Biological Samples.

Christiana Mantzourani, Maroula G Kokotou.

  Fatty acids (FAs) play pleiotropic roles in living organisms, acting as signaling molecules and gene regulators. They are present in plants and foods and may affect human health by food ingestion. As a consequence, analytical methods for their determination in biological fluids, plants and foods have attracted high interest. Undoubtedly, mass spectrometry (MS) has become an indispensable technique for the analysis of FAs. Due to the inherent poor ionization efficiency of FAs, their chemical derivatization prior to analysis is often employed. Usually, the derivatization of the FA carboxyl group aims to charge reversal, allowing detection and quantification in positive ion mode, thus, resulting in an increase in sensitivity in determination. Another approach is the derivatization of the double bond of unsaturated FAs, which aims to identify the double bond location. The present review summarizes the various classes of reagents developed for FA derivatization and discusses their applications in the liquid chromatography-MS (LC-MS) analysis of FAs in various matrices, including plasma and feces. In addition, applications for the determination of eicosanoids and fatty acid esters of hydroxy fatty acids (FAHFAs) are discussed.

Keywords:  charge reversal; derivatization reagents; fatty acids; liquid chromatography; mass spectrometry

DOI:  https://doi.org/10.3390/molecules27175717
Anal Chim Acta. 2022 Sep 15. pii: S0003-2670(22)00807-8. [Epub ahead of print]1226 340236

A re-calibration procedure for interoperable lipid collision cross section values measured by traveling wave ion mobility spectrometry.

Anaïs C George, Isabelle Schmitz-Afonso, Vincent Marie, Benoit Colsch, François Fenaille, Carlos Afonso, Corinne Loutelier-Bourhis.

  Collision cross sections (CCS) have been described as relevant molecular descriptors in metabolomics and lipidomics analyses for ascertaining compound identity. Ion mobility spectrometry (IMS) allows to determine CCS with different techniques, such as drift tube ion mobility spectrometry (DTIMS), traveling wave ion mobility spectrometry (TWIMS) or trapped ion mobility spectrometry (TIMS). In contrast with DTIMS where CCS can be obtained directly with measured drift times and mathematical relationship, TWIMS and TIMS techniques require an additional step of calibration to obtain CCS values. However, literature reports significantly disparate CCS values depending on the calibrant used (often more than 10%), as no consensus has been reached to define a universal CCS reference standard or harmonized calibration procedure. Therefore, publicly available CCS databases cannot be regarded as readily interoperable and exchangeable. Here, we performed a comprehensive evaluation of 11 distinct CCS calibrants in a traveling wave ion mobility spectrometry-mass spectrometry (TWIMS-MS) instrument. We showed that, using lipids from plasma as model compounds, CCS determination drastically fluctuates from one calibrant to the other with up to 25% differences, which precludes direct CCS comparison. Using the large panel of calibration curves generated, we showed that any CCS value can be efficiently re-calibrated relatively to the calibration curve made with the widely used Tune Mix solution whatever the calibration procedure originally used. The re-calibrated CCS values for each calibrant constitute a database which allows to correct any deviation on lipid CCS values whatever the calibrant originally used. Resulting corrected CCS values from plasma lipids were thus efficiently matched to those previously reported in the literature (with deviations<2%). Therefore, this work shows that unique and comparable CCS values can be obtained upon re-calibration relatively to Tune Mix CCS values, while also paving the way for the establishment of a universal CCS database of various metabolite or lipid classes.

Keywords:  Collision cross section; Ion mobility calibration; Ion mobility-mass spectrometry; Lipidomics; Metabolomics; TWIMS

DOI:  https://doi.org/10.1016/j.aca.2022.340236
J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Aug 27. pii: S1570-0232(22)00336-1. [Epub ahead of print]1210 123432

Short-chain fatty acids profiling in biological samples from a mouse model of Sjögren's syndrome based on derivatized LC-MS/MS assay.

Ryosuke Nagatomo, Haruki Kaneko, Shihori Kamatsuki, Mayuko Ichimura-Shimizu, Naozumi Ishimaru, Koichi Tsuneyama, Koichi Inoue.

  An analytical platform is required to characterize the short-chain fatty acids (SCFAs) in a mouse model of pathological immune conditions. Therefore, liquid chromatography tandem mass spectrometry combined with 2-picolylamine derivatization and a comprehensive study of SCFAs distribution based on serum, saliva, feces, liver, and brain from a mouse model of Sjögren's syndrome (SS) is performed. The design of experiments is used to achieve efficient 2-picolylamine derivatization, and optimize the reaction conditions. Twelve SCFAs are derivatized, and separated on a reversed-phase C18 column. All SCFAs show high linearity (r2 > 0.995) and intra/inter-day accuracy values from 71.6% to 115.6% (precision < 13.7%). This method was used to determine SCFAs concentrations in the serum, saliva, feces, liver, and brain of an SS model mice, and isobutyric acid, valeric acid, isovaleric acid, and 2-methylbutyric acid in liver from SS were significantly different compared with control group. Moreover, the preliminary evaluation of propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid in saliva is conducted based on the respective SS stages and are correlated with these histological scores. This analytical platform for the widely SCFAs profiling in several tissues can be a clue for studying unclear immune pathophysiology.

Keywords:  Biological sample; Derivatization; Design of experiments; Liquid chromatography-tandem mass spectrometry; Short-chain fatty acids; Sjögren’s syndrome; Stable isotope

DOI:  https://doi.org/10.1016/j.jchromb.2022.123432
Anal Chem. 2022 Sep 06.

A Novel and Sensitive Method for the Analysis of Fatty Acid Biosignatures by Capillary Electrophoresis-Mass Spectrometry.

Miranda G M Kok, Maria F Mora, Aaron C Noell, Ceth W Parker, Peter A Willis.

Fatty acids are a well-established class of compounds targeted as biosignatures for future missions to look for evidence of life on ocean worlds such as Europa and Enceladus. In order to establish their abiotic or biotic origin, we need to separate and quantify fatty acids to determine their relative abundances within a sample. In this study, we demonstrate the high potential of capillary electrophoresis coupled to mass spectrometry (CE-MS) for the efficient separation and sensitive detection of a wide variety of fatty acids. Three derivatization strategies were evaluated to allow the detection of fatty acids by positive ionization mode MS. Furthermore, CE-MS conditions were optimized to provide maximum separation efficiencies and detection sensitivities for the analysis of saturated and unsaturated fatty acids with even- and odd-numbered carbon chain lengths. Optimum separation and detection were obtained using a background electrolyte of 2 M acetic acid in 45% acetonitrile, after derivatization of the fatty acids with 2-picolylamine or N,N-diethylethylenediamine. The limits of detection for the derivatized fatty acids using the optimized method ranged from 25 to 250 nM. The optimized method was also used for the analysis of fatty acids in cell cultures and natural samples. Two distinctive biosignatures were obtained for the microorganisms Halobacillus halophilus and Pseudoalteromonas haloplanktis. In addition, multiple fatty acids were detected in a natural sample from Mono Lake, California.

DOI: https://doi.org/10.1021/acs.analchem.2c02716
Anal Chem. 2022 Sep 07.

A Direct Infusion Probe for Rapid Metabolomics of Low-Volume Samples.

Cátia Marques, Liangwen Liu, Kyle D Duncan, Ingela Lanekoff.

Targeted and nontargeted metabolomics has the potential to evaluate and detect global metabolite changes in biological systems. Direct infusion mass spectrometric analysis enables detection of all ionizable small molecules, thus simultaneously providing information on both metabolites and lipids in chemically complex samples. However, to unravel the heterogeneity of the metabolic status of cells in culture and tissue a low number of cells per sample should be analyzed with high sensitivity, which requires low sample volumes. Here, we present the design and characterization of the direct infusion probe, DIP. The DIP is simple to build and position directly in front of a mass spectrometer for rapid metabolomics of chemically complex biological samples using pneumatically assisted electrospray ionization at 1 μL/min flow rate. The resulting data is acquired in a square wave profile with minimal carryover between samples that enhances throughput and enables several minutes of uniform MS signal from 5 μL sample volumes. The DIP was applied to study the intracellular metabolism of insulin secreting INS-1 cells and the results show that exposure to 20 mM glucose for 15 min significantly alters the abundance of several small metabolites, amino acids, and lipids.

DOI: https://doi.org/10.1021/acs.analchem.2c02918
PLoS One. 2022 ;17(9): e0261803

Spatially resolved characterization of tissue metabolic compartments in fasted and high-fat diet livers.

Sylwia A Stopka, Jiska van der Reest, Walid M Abdelmoula, Daniela F Ruiz, Shakchhi Joshi, Alison E Ringel, Marcia C Haigis, Nathalie Y R Agar.

Cells adapt their metabolism to physiological stimuli, and metabolic heterogeneity exists between cell types, within tissues, and subcellular compartments. The liver plays an essential role in maintaining whole-body metabolic homeostasis and is structurally defined by metabolic zones. These zones are well-understood on the transcriptomic level, but have not been comprehensively characterized on the metabolomic level. Mass spectrometry imaging (MSI) can be used to map hundreds of metabolites directly from a tissue section, offering an important advance to investigate metabolic heterogeneity in tissues compared to extraction-based metabolomics methods that analyze tissue metabolite profiles in bulk. We established a workflow for the preparation of tissue specimens for matrix-assisted laser desorption/ionization (MALDI) MSI that can be implemented to achieve broad coverage of central carbon, nucleotide, and lipid metabolism pathways. Herein, we used this approach to visualize the effect of nutrient stress and excess on liver metabolism. Our data revealed a highly organized metabolic tissue compartmentalization in livers, which becomes disrupted under high fat diet. Fasting caused changes in the abundance of several metabolites, including increased levels of fatty acids and TCA intermediates while fatty livers had higher levels of purine and pentose phosphate-related metabolites, which generate reducing equivalents to counteract oxidative stress. This spatially conserved approach allowed the visualization of liver metabolic compartmentalization at 30 μm pixel resolution and can be applied more broadly to yield new insights into metabolic heterogeneity in vivo.

DOI: https://doi.org/10.1371/journal.pone.0261803
Molecules. 2022 Sep 02. pii: 5670. [Epub ahead of print]27(17):

Development and Validation of a Highly Sensitive and Rapid LC-MS3 Strategy to Determine Oxcarbazepine and Its Active Metabolite in the Serum of Patients with Epilepsy and Its Application in Therapeutic Drug Monitoring.

Zhengchao Ji, Tingting Li, Xin Zhao, Wei Ma, Yanyan Li, Jing Huang.

  A sensitive and rapid bioanalytical method based on the LC-triple-stage fragmentation (LC-MS3) strategy on a hybrid triple quadrupole-linear ion trap mass spectrometer in combination with protein precipitation extraction for sample pretreatment has been developed and validated for the simultaneous determination of the antiepileptic drug oxcarbazepine (OXC) and its main active metabolite (MHD) in human serum. The separation was performed on a Waters XBridge BEH C18 column (2.5 µm, 2.1 × 50 mm) in isocratic elution with 0.1% formic acid in water and methanol (50:50, v:v) as the mobile phase. The run time for each sample was 2.0 min. The calibration curves ranging from 25 to 1600 ng/mL for OXC and from 0.5 to 32 μg/mL for MHD showed correlation coefficients (r) better than 0.99. All of the validation data, such as precision, accuracy and other parameters, fit the requirements of the current bioanalytical method validation guidelines. The LC-MS3 method for quantitation of OXC and MHD was compared with the LC-MRM based method. Passing-Bablok regression coefficients and Bland-Altman plots showed that the developed LC-MS3 method is a reliable method for quantitative analysis of OXC and MHD. The proposed LC-MS3 method was successfully applied to determine the serum concentrations of OXC and MHD to support a clinical study.

Keywords:  10-hydroxycarbazepine; LC-MS3; oxcarbazepine; therapeutic drug monitoring; validation

DOI:  https://doi.org/10.3390/molecules27175670
J AOAC Int. 2022 Sep 07. pii: qsac105. [Epub ahead of print]

Development and Validation of a Modified QuEChERS Method for Simultaneous Analysis of 250 Flavor Constituents in Tobacco by Gas Chromatography Tandem Mass Spectrometry.

Li Chen, Tiancai Shi, Jiaxing Zhao, Zongcan Ren, Huapeng Cui, Fuwei Xie, Meijuan Fan, Shaofeng Liu, Cong Nie, Huimin Liu.

BACKGROUND: Flavor constituents play an important role in the flavor characteristics of tobacco leaves and cigarettes. Sensitive, selective and high-throughput multi-analyte analytical methods are needed to satisfy the demand for analyzing trace level flavor constituents in tobacco. However, trace analysis of multi-targets in complex tobacco matrix is significantly challenging.
OBJECTIVE: This study was undertaken to develop and validate a fast, selective, sensitive and accurate GC-MS/MS method for the simultaneous analysis of 250 flavor constituents in tobacco using a modified QuEChERS extraction procedure and backflushing technique.
METHODS: The samples were extracted with a mixture of acetonitrile and phosphate buffer. GC-MS/MS served as a reliable tool to quantify the flavor constituents due to its high sensitivity, selectivity, and good repeatability.
RESULTS: Our evaluation showed that 243 flavor constituents presented good linearity. Average recoveries of 216 target compounds in tobacco ranged from 70 to 120% with relative standard deviations less than 20% at three fortification levels. The limits of quantitation of 225 and 25 compounds were in the range of 2-50 and 51-112 μg/kg, respectively. Then, this method was successfully applied to the analysis of 15 commercial cigarette samples with different style characteristics.
CONCLUSION: The modified QuEChERS method worked very well for a wide range of flavor constituents that were never reported by QuEChERS pretreatment previously, and the use of concurrent backflushing offered significant increase in system robustness and sample throughput. The method greatly improved the detection performance and the range of the flavor constituents, and proved to be more accurate, sensitive, selective, convenient and practical than the reported method, and thus, can be applied in routine analysis.
HIGHLIGHTS: A validated QuEChERS based GC-MS/MS method for multiple flavor constituents analysis in tobacco was reported for the first time. The use of concurrent backflushing markedly improved the instrument robustness and sample throughput.

DOI: https://doi.org/10.1093/jaoacint/qsac105
J Mass Spectrom Adv Clin Lab. 2022 Nov;26 1-6

Development and application of a High-Resolution mass spectrometry method for the detection of fentanyl analogs in urine and serum.

Yu Zhang, John C Halifax, Christina Tangsombatvisit, Cassandra Yun, Shaokun Pang, Shirin Hooshfar, Alan H B Wu, Kara L Lynch.

   Introduction: The use of illicitly manufactured synthetic opioids, specifically fentanyl and its analogs, has escalated exponentially in the United States over the last decade. Due to the targeted nature of drug detection methods in clinical laboratories and the ever-evolving list of synthetic opioids of concern, alternative analytical approaches are needed.
Methods: Using the fentanyl analog screening (FAS) kit produced by the Centers for Disease Control and Prevention (CDC), we developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) synthetic opioid spectral library and data acquisition method using information dependent acquisition of product ion spectra. Chromatographic retention times, limits of detection and matrix effects, in urine and serum, for the synthetic opioids in the FAS kit (n = 150) were established. All urine and serum specimens sent to a clinical toxicology laboratory for comprehensive drug testing in 2019 (n = 856) and 2021 (n = 878) were analyzed with the FAS LC-HRMS library to determine the prevalence of fentanyl analogs and other synthetic opioids, retrospectively (2019) and prospectively (2021).
Results: The limit of detection (LOD) of each opioid ranged from 1 to 10 ng/mL (median, 2.5 ng/mL) in urine and 0.25-2.5 ng/mL (median, 0.5 ng/mL) in serum. Matrix effects ranged from -79 % to 86 % (median, -37 %) for urine, following dilution and direct analysis, and -80 % to 400 % (median, 0 %) for serum, following protein precipitation. The prevalence of fentanyl/fentanyl analogs in serum samples increased slightly from 2019 to 2021 while it remained the same in urine. There were only 2 samples identified that contained a fentanyl analog without the co-occurrence of fentanyl or fentanyl metabolites. Analysis of the established MS/MS spectral library revealed characteristic fragmentation patterns in most fentanyl analogs, which can be used for structure elucidation and drug identification of future analogs.
Conclusions: The LC-HRMS method was capable of detecting fentanyl analogs in routine samples sent for comprehensive drug testing. The method can be adapted to accommodate testing needs for the evolving opioid epidemic.

Keywords:  CDC, Centers for Disease Control and Prevention; FAS, fentanyl analog screening;; Fentanyl analogs; GC–MS, gas chromatography-mass spectrometry; High resolution mass spectrometry; LC-HRMS, liquid chromatography-high resolution mass spectrometry; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LOD, limit of detection; Liquid chromatography; RT, retention time; Synthetic opioids; TOF-MS, time-of-flight mass spectrometry

DOI:  https://doi.org/10.1016/j.jmsacl.2022.07.005
Front Bioeng Biotechnol. 2022 ;10 859600

A rapid and standardized workflow for functional assessment of bacterial biosensors in fecal samples.

Ana Zúñiga, Geisler Muñoz-Guamuro, Lucile Boivineau, Pauline Mayonove, Ismael Conejero, Georges-Philippe Pageaux, Romain Altwegg, Jerome Bonnet.

  Gut metabolites are pivotal mediators of host-microbiome interactions and provide an important window on human physiology and disease. However, current methods to monitor gut metabolites rely on heavy and expensive technologies such as liquid chromatography-mass spectrometry (LC-MS). In that context, robust, fast, field-deployable, and cost-effective strategies for monitoring fecal metabolites would support large-scale functional studies and routine monitoring of metabolites biomarkers associated with pathological conditions. Living cells are an attractive option to engineer biosensors due to their ability to detect and process many environmental signals and their self-replicating nature. Here we optimized a workflow for feces processing that supports metabolite detection using bacterial biosensors. We show that simple centrifugation and filtration steps remove host microbes and support reproducible preparation of a physiological-derived media retaining important characteristics of human feces, such as matrix effects and endogenous metabolites. We measure the performance of bacterial biosensors for benzoate, lactate, anhydrotetracycline, and bile acids, and find that they are highly sensitive to fecal matrices. However, encapsulating the bacteria in hydrogel helps reduce this inhibitory effect. Sensitivity to matrix effects is biosensor-dependent but also varies between individuals, highlighting the need for case-by-case optimization for biosensors' operation in feces. Finally, by detecting endogenous bile acids, we demonstrate that bacterial biosensors could be used for future metabolite monitoring in feces. This work lays the foundation for the optimization and use of bacterial biosensors for fecal metabolites monitoring. In the future, our method could also allow rapid pre-prototyping of engineered bacteria designed to operate in the gut, with applications to in situ diagnostics and therapeutics.

Keywords:  diagnostics; engineered bacteria; gut microbiome; metabolite detection; synthetic biology; whole-cell biosensor

DOI:  https://doi.org/10.3389/fbioe.2022.859600
Food Res Int. 2022 Oct;pii: S0963-9969(22)00809-2. [Epub ahead of print]160 111751

Free fatty acid profiling of Greek yogurt by liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis.

Christiana Mantzourani, Charikleia S Batsika, Maroula G Kokotou, George Kokotos.

  Yogurt is a fermented dairy product of high nutritional value, very popular in many parts of the world. Free fatty acids (FFAs), which are formed during fermentation, may cause changes in organoleptic properties of yogurt, and thus, the determination of FFAs is of importance. We present a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method, which allows the simultaneous determination of a large set of common and uncommon FFAs in yogurt samples, avoiding any derivatization step. Twenty-five common saturated and unsaturated FAs, together with 21 saturated hydroxy fatty acids (SHFAs) and 17 saturated oxo fatty acids (SOFAs), were analyzed in 26 cow and 7 sheep Greek yogurt samples. A detailed analysis of bioactive SHFAs and SOFAs was carried out in yogurt samples for the first time. Differences at the concentrations of six common FAs and five oxidized FAs between the cow and sheep samples were observed. Based on these FAs, Principal Component Analysis (PCA) allows the discrimination of cow from sheep yogurt samples.

Keywords:  Free fatty acids; High resolution mass spectrometry; Hydroxy fatty acids; Oxo fatty acids; Yogurt

DOI:  https://doi.org/10.1016/j.foodres.2022.111751
Rapid Commun Mass Spectrom. 2022 Sep 04. e9392

Improved Spatial Resolution of Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) Mass Spectrometry Imaging (MSI) Using a Reflective Objective.

Alena N Joignant, Hongxia Bai, Jeffrey G Manni, David C Muddiman.

RATIONALE: The level of visual detail of a mass spectrometry image is dependent on the spatial resolution with which it is acquired, which is largely determined by the focal diameter in infrared laser ablation-based techniques. While the use of mid-IR light for mass spectrometry imaging (MSI) has advantages, it results in a relatively large focal diameter and spatial resolution. The continual advancement of infrared matrix assisted electrospray ionization (IR-MALDESI) for MSI warranted novel methods to decrease laser ablation areas and thus improve spatial resolution.
METHODS: In this work, a Schwarzschild-like reflective objective was incorporated into the novel NextGen IR-MALDESI source and characterized on both burn paper and mammalian tissue using an ice matrix. Ablation areas, mass spectra, and annotations obtained using the objective were compared against the current optical train on the NextGen system without modification.
RESULTS: The effective resolution was determined to be 55 μm by decreasing the step size until oversampling was observed. Use of the objective improved the spatial resolution by a factor of three as compared against the focus lens.
CONCLUSIONS: A Schwarzschild-like reflective objective was successfully incorporated into the NextGen source and characterized on mammalian tissue using an ice matrix. The corresponding improvement in spatial resolution facilitates the future expansion of IR-MALDESI applications to include those that require fine structural detail.

DOI: https://doi.org/10.1002/rcm.9392
Anal Chim Acta. 2022 Sep 15. pii: S0003-2670(22)00741-3. [Epub ahead of print]1226 340170

Simultaneous quantification of 106 drugs or their metabolites in nail samples by UPLC-MS/MS with high-throughput sample preparation: Application to 294 real cases.

Peipei Liu, Wenjuan Liu, Hongwei Qiao, Shan Jiang, Youmei Wang, Jie Chen, Mengxiang Su, Bin Di.

  The nail is an alternative matrix to complement hair analysis in proving drug intake over several months in forensic toxicology investigations. However, because of the high hardness and toughness of nails, the existing pretreatment procedures for nails have the disadvantages of either a high degree of time consumption (from hours to days), or low extraction recoveries. This study aims to propose a high-throughput nail sample preparation method and provide a quantitative analytical method for 106 drugs and their metabolites present in nail. We developed cryogenic grinding, coupled with high-speed grinding in the extraction solvent method, which could improve the extraction recovery by thoroughly destroying the nail keratin for approximately 18 min. Subsequently, an ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed for the identification and quantification of 34 synthetic cannabinoids, 26 fentanyls, 18 synthetic cathinones, 10 phenylethylamines, eight opioids, three phencyclidine, two tryptamines, two piperazine, cocaine, benzoylecgonine, and tetrahydrocannabinol (THC). Nail samples were collected from people with a history of drug abuse from five different regions of China. The analysis of 294 authentic samples resulted in 213 detected samples, and showed a broad concentration range including 5.04-67.26 pg/mg for nine synthetic cannabinoids, 109.29-250.29 pg/mg for a synthetic cathinone, 5.06-434291 pg/mg for four phenylethylamines, 5.06-464278 pg/mg for three phencyclidine, 5.50-192195 pg/mg for six opioids, 19.44-36.11 pg/mg for cocaine, and 50.53 pg/mg for THC in nail. Furthermore, up to 10 different compounds were detected in a single nail sample. This nail analysis method serves as a useful tool for the large-scale surveillance of illicit drugs abuse.

Keywords:  Forensic toxicology; Illicit drugs; Nail analysis; New psychoactive substances; Ultra-high performance liquid chromatography-tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.aca.2022.340170
Int J Mol Sci. 2022 Sep 05. pii: 10166. [Epub ahead of print]23(17):

Development of the Method for Nusinersen and Its Metabolites Identification in the Serum Samples of Children Treated with Spinraza for Spinal Muscular Atrophy.

Sylwia Studzińska, Maria Mazurkiewicz-Bełdzińska, Bogusław Buszewski.

  The application of oligonucleotides as drugs for different genetic diseases is increasing rapidly. Since 2016 they are used during spinal muscular atrophy treatment with the use of nusinersen oligonucleotide. The purpose of this study was to improve methods for the analysis of serum samples of patients treated with nusinersen. The results showed that liquid-liquid extraction (with phenol/chloroform) is insufficient and an additional purification step using solid-phase extraction is necessary. The best results were obtained for microextraction by packed sorbents. Important parameters in the optimization of the method were mainly the type of amine in the mobile phase and the stationary phase. Both influenced the selectivity of metabolite separation and thus their correct identification; while amine type impacted also the intensity of signals. Finally, the highest resolution of separation and the highest peak areas were obtained for N,N-dimethylbutylamine or N,N-diisopropylthylamine with an octadecyl column with a terminal aryl group. Over a dozen of metabolites were successfully identified with the use of methods developed during the study. The 3' exonucleases and 5' exonucleases were mainly responsible for nusinersen metabolism, consequently, 3'end shortmers, and 5'end shortmers were observed, as well as metabolites with simultaneous loss of bases at both ends of the sequence. However, some depurination and depyrimidination products were also identified. To the best of our knowledge, this is the first report on nusinersen and its metabolite identification in serum samples by liquid chromatography and mass spectrometry.

Keywords:  extraction; ion pair chromatography; mass spectrometry; metabolites; nusinersen; oligonucleotides

DOI:  https://doi.org/10.3390/ijms231710166
Anal Chem. 2022 Sep 06.

Isomer and Conformer-Specific Mass Distribution-Based Isotopic Shifts in High-Resolution Cyclic Ion Mobility Separations.

David L Williamson, Gabe Nagy.

Herein, we present the use of mass distribution-based isotopic shifts in high-resolution cyclic ion mobility spectrometry-mass spectrometry (cIMS-MS)-based separations to characterize various isomeric species as well as conformers. Specifically, by using the observed relative arrival time values for the isotopologues found in the isotopic envelope after long pathlength cIMS-MS separations, we were able to distinguish dibromoaniline, dichloroaniline, and quaternary ammonium salt isomers, as well as a pair of 25-hydroxyvitamin D3 conformers based on their respective mass distribution-based shifts. Our observed shifts were highly reproducible and broadly applied to the isotopologues of various atoms (i.e., Cl, Br, and C). Additionally, through a control experiment, we determined that such shifts are indeed pathlength-independent, thus demonstrating that our presented methodology could be readily extended to other high-resolution IMS-MS platforms. These results are the first characterization of conformers using mass distribution-based IMS-MS shifts, as well as the first use of a commercial cIMS-MS platform to characterize isomers via their mass distribution-based shifts. We anticipate that our methodology will have broad applicability for biological analytes and that mass distribution-based shifts could potentially act as an added dimension of analysis in existing IMS-MS workflows in omics-based research. Specifically, we envision that the development of a database of these mass distribution-based shifts could, for example, enable the identification of unknown metabolites in complex matrices.

DOI: https://doi.org/10.1021/acs.analchem.2c02991
Molecules. 2022 Sep 01. pii: 5652. [Epub ahead of print]27(17):

Volumetric Absorptive Microsampling (VAMS) for Targeted LC-MS/MS Determination of Tryptophan-Related Biomarkers.

Michele Protti, Marco Cirrincione, Roberto Mandrioli, James Rudge, Luca Regazzoni, Valeria Valsecchi, Claudia Volpi, Laura Mercolini.

  L-Tryptophan (TRP) metabolites and related biomarkers play crucial roles in physiological functions, and their imbalances are implicated in central nervous system pathologies and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, schizophrenia and depression. The measurement of TRP metabolites and related biomarkers possesses great potential to elucidate the disease mechanisms, aid preclinical drug development, highlight potential therapeutic targets and evaluate the outcomes of therapeutic interventions. An effective, straightforward, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of 24 TRP-related compounds in miniaturised murine whole blood samples. Sampling and sample pretreatment miniaturisation were achieved thanks to the development of a volumetric dried blood microsampling approach. Volumetric absorptive microsampling (VAMS) allows the accurate sampling of microvolumes of blood with advantages including, but not limited to, minimal sampling invasiveness, logistical improvements, method sustainability in terms of solvents and energy consumption, and improvement of animal studies in the framework of the 3Rs (Replacement, Reduction and Refinement) principles on animal welfare. The VAMS-LC-MS/MS method exhibited good selectivity, and correlation coefficient values for the calibration curves of each analyte were >0.9987. The limits of quantitation ranged from 0.1 to 25 ng/mL. The intra- and inter-day precisions in terms of RSD were <9.6%. All analytes were stable in whole blood VAMS samples stored at room temperature for at least 30 days with analyte losses < 14%. The developed method was successfully applied to the analysis of biological samples from mice, leading to the unambiguous determination of all the considered target analytes. This method can therefore be applied to analyse TRP metabolites and related biomarkers levels to monitor disease states, perform mechanistic studies and investigate the outcomes of therapeutic interventions.

Keywords:  LC-MS/MS; biomarkers; neurodegenerative diseases; tryptophan catabolism; volumetric absorptive microsampling (VAMS)

DOI:  https://doi.org/10.3390/molecules27175652
Anal Chim Acta. 2022 Sep 15. pii: S0003-2670(22)00830-3. [Epub ahead of print]1226 340259

Optimization of capillary electrophoresis coupled to negative mode electrospray ionization-mass spectrometry using polyvinyl alcohol coated capillaries. Application to a study on non-small cell lung cancer.

Ángeles López-López, Michal Ciborowski, Jacek Niklinski, Coral Barbas, Ángeles López-Gonzálvez.

  Despite recent developments in separation techniques, the analysis of relatively small highly polar negatively charged analytes (e.g. small organic acids, phosphorylated sugars, and underivatized amino acids) remains challenging. Capillary electrophoresis coupled to mass spectrometry (CE-MS) has been included in the untargeted metabolomics toolbox, although mostly in positive polarity. The aim of this study was to assess the use of CE-MS to analyze highly polar and negatively charged metabolites at physiological levels without the need for derivatization. After a preliminary selection, conditions regarding CE (buffers, applied potential, injection time and applied pressure), electrospray parameters (sheath liquid flow, temperature and drying gas flow, nebulizer, and capillary voltage), and fragmentor voltage were optimized using a capillary coated with polyvinyl alcohol (PVA) for the metabolic profiling of anionic compounds compared to fused silica as the reference capillary. In addition, a database of 240 metabolites with two relative migration times (RMT) obtained against methionine sulfone and 2-morpholinoethanesulfonic acid (MES) as internal standards (IS) has been compiled. Finally, the optimized method has been used to characterize the metabolic profile of blood plasma in patients with non-small cell lung cancer (NSCLC). The identified compounds are mostly amino acids and their derivatives, carboxylic acids and organic compounds from the TCA cycle, and sugars and their phosphoderivates. In addition, we performed a comparative study to find significant differences between non-small cell lung cancer (NSCLC) vs non-cancer individuals, and squamous cell carcinoma (SCC) and adenocarcinoma (ADC) vs non-cancer individuals, respectively, searching for differences between the various types of NSCLC.

Keywords:  Anionic metabolites; Biological sample; CE-MS negative Polarity; untargeted metabolomics

DOI:  https://doi.org/10.1016/j.aca.2022.340259
Anal Chim Acta. 2022 Sep 15. pii: S0003-2670(22)00842-X. [Epub ahead of print]1226 340271

Rapid determination of perfluorinated compounds in pork samples using a molecularly imprinted phenolic resin adsorbent in dispersive solid phase extraction-liquid chromatography tandem mass spectrometry.

Deshuai Zou, Pengfei Li, Chunliu Yang, Dandan Han, Hongyuan Yan.

  Perfluorinated compounds (PFCs) that bioaccumulate enter the food chain through animals-derived foods and ultimately harm human health. However, these compounds are extensively used, and consequently, it is important to focus on the residual PFCs content in various animal-derived foods. In this work, a molecularly imprinted polymer with surface grooves and rapid mass transfer ability was prepared hydrothermally, applied as an adsorbent to a needle filter device, and combined with LC-MS/MS for the rapid extraction and determination of trace PFCs in pork. This method requires simple equipment (syringe filter), small amount of adsorbent (2 mg), and short operation time (6-8 min) and has a low detection limit (0.011-0.08 ng g-1), good recovery (89.3-116.3%), and satisfactory intra-day (≤4.0%) and inter-day (≤8.6%) precision. This study provides a new method for assessing the exposure risk of PFCs in complex samples.

Keywords:  Animal-derived foods; Dispersive solid phase extraction; Perfluorinated compounds; Rapid analysis

DOI:  https://doi.org/10.1016/j.aca.2022.340271
Biomed Chromatogr. 2022 Sep 09. e5502

Quantification of monoamine biomarkers in cerebrospinal fluid: comparison of a UHPLC-MS/MS method to a UHPLC coupled to fluorescence detection method.

Ayoub Boulghobra, Taous Abar, Fathi Moussa, Bruno Baudin, Diana Rodriguez, Antoine Pallandre, Myriam Bonose.

  Inborn errors of monoamine neurotransmitter metabolism are rare genetic diseases classified as catecholamine and serotonin metabolism disorders or neurotransmitter transportopathies. To diagnose these orphan diseases, monoamine metabolites have been identified and validated as cerebrospinal fluid (CSF) biomarkers: 5-hydroxy-tryptophane, 5-hydroxy-indol-acetic acid, 3-ortho-methyl-DOPA, homovanillic acid, and 3-methoxy-4-hydroxyphenylglycol. The present work presents a UHPLC-MS/MS method developed for the quantification of these metabolites in CSF and compares it to a previously described UHPLC-FD method. MS/MS detection was performed in positive electrospray ionization and multiple reaction monitoring (MRM) mode. The UHPLC-MS/MS and UHPLC-FD methods were validated in terms of accuracy, linearity, precision, and matrix effect. The lower limits of quantification (LLOQ) were ranging between 0.5 nM and 10 nM and between 1 and 5 nM for the UHPLC-MS/MS method and the UHPLC-FD one, respectively. We verified the applicability of both methods by analyzing 30 CSF samples. The measured concentrations were comparable to the reference values described in the literature. The two methods allowed to distinguish pathological samples from healthy ones for clinical diagnosis. UHPLC-MS/MS and UHPLC-FD methods exhibit very close LLOQs. As UHPLC-MS/MS method is more selective, it allows faster analysis with 6 minutes per run versus 10 minutes for the UHPLC-FD method.

Keywords:  Cerebrospinal fluid; Fluorescence detection; MS/MS; Monoamine biomarkers; UHPLC

DOI:  https://doi.org/10.1002/bmc.5502
Chem Res Toxicol. 2022 Sep 06.

Multi-DNA Adduct and Abasic Site Quantitation In Vivo by Nano-Liquid Chromatography/High-Resolution Orbitrap Tandem Mass Spectrometry: Methodology for Biomonitoring Colorectal DNA Damage.

Dmitri Konorev, Lihua Yao, Robert J Turesky.

Epidemiological and mechanistic studies suggest that processed and red meat consumption and tobacco smoking are associated with colorectal cancer (CRC) risk. Several classes of carcinogens, including N-nitroso compounds (NOCs) in processed meats and heterocyclic aromatic amines (HAAs) and polycyclic aromatic hydrocarbons (PAHs) in grilled meats and tobacco smoke, undergo metabolism to reactive intermediates that may form mutation-inducing DNA adducts in the colorectum. Heme iron in red meat may contribute to oxidative DNA damage and endogenous NOC formation. However, the chemicals involved in colorectal DNA damage and the paradigms of CRC etiology remain unproven. There is a critical need to establish physicochemical methods for identifying and quantitating DNA damage induced by genotoxicants in the human colorectum. We established robust nano-liquid chromatography/high-resolution accurate mass Orbitrap tandem mass spectrometry (LC/HRAMS2) methods to measure DNA adducts of nine meat and tobacco-associated carcinogens and lipid peroxidation products in the liver, colon, and rectum of carcinogen-treated rats employing fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues. Some NOCs form O6-carboxymethyl-2'-deoxyguanosine, O6-methyl-2'-deoxyguanosine, and unstable quaternary N-linked purine/pyrimidine adducts, which generate apurinic/apyrimidinic (AP) sites. AP sites were quantitated following derivatization with O-(pyridin-3-yl-methyl)hydroxylamine. DNA adduct quantitation was conducted with stable isotope-labeled internal standards, and method performance was validated for accuracy and reproducibility. Limits of quantitation ranged from 0.1 to 1.1 adducts per 108 bases using 3 μg of DNA. Adduct formation in animals ranged from ∼1 in 108 to ∼1 in 105 bases, occurring at comparable levels in fresh-frozen and FFPE specimens for most adducts. AP sites increased by 25- to 75-fold in the colorectum and liver, respectively. Endogenous lipid peroxide-derived 3-(2-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) and 6-oxo-M1dG adduct levels were not increased by carcinogen dosing but increased in FFPE tissues. Human biomonitoring studies can implement LC/HRAMS2 assays for DNA adducts and AP sites outlined in this work to advance our understanding of CRC etiology.

DOI: https://doi.org/10.1021/acs.chemrestox.2c00177
Biomed Chromatogr. 2022 Sep 05. e5499

Bioanalysis of Trifluoperazine in human plasma by LC-MS/MS: Application to disposition kinetics.

Viritha Bezawada, Padma Mogili, Sireesha Dodda, Ramakrishna Gajula, Suhasini Ponnada.

  A sensitive, selective and rapid bioanalytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the quantification of Trifluoperazine in human plasma. Trifluoperazine-D8 was used as the internal standard and the extraction from human plasma was performed by liquid-liquid extraction technique using tertiary butyl methyl ether as the solvent. Chromatographic separation was carried out on Zodiac C18 column (50 x 4.6 mm, 3 μm) employing a mixture of acetonitrile, methanol and 5mM ammonium bicarbonate buffer in water (85:10:5, v/v/v) at a flow rate of 0.55 mL/min. The linearity was established within the concentration range of 5-1250 pg/mL with r2 >0.99. Results of all the validation parameters as per USFDA guidelines were within the acceptance limits. Pharmacokinetics of Trifluoperazine after oral administration of syrup of 1 mg dose under fasting conditions was performed by successful application of the present method.

Keywords:  LC-MS/MS; Trifluoperazine; bioanalysis; liquid-liquid extraction; pharmacokinetic study

DOI:  https://doi.org/10.1002/bmc.5499
Molecules. 2022 Aug 23. pii: 5363. [Epub ahead of print]27(17):

Quantification of Gut Microbiota Dysbiosis-Related Organic Acids in Human Urine Using LC-MS/MS.

Yu-Tsung Lee, Sui-Qing Huang, Ching-Hao Lin, Li-Heng Pao, Chun-Hui Chiu.

  Urine organic acid contains water-soluble metabolites and/or metabolites-derived from sugars, amino acids, lipids, vitamins, and drugs-which can reveal a human's physiological condition. These urine organic acids-hippuric acid, benzoic acid, phenylacetic acid, phenylpropionic acid, 4-hydroxybenzoic acid, 4-hydroxyphenyl acetic acid, 3-hydroxyphenylpropionic acid, 3,4-dihydroxyphenyl propionic acid, and 3-indoleacetic acid-were the eligible candidates for the dysbiosis of gut microbiota. The aim of this proposal was to develop and to validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis method for the nine organic acids in human urine. Stable-labeled isotope standard (creatinine-d3) and acetonitrile were added to the urine sample. The supernatant was diluted with deionized water and injected into LC-MS/MS. This method was validated with high selectivity for the urine sample, a low limit of quantification (10-40 ng/mL), good linearity (r > 0.995), high accuracy (85.8-109.7%), and high precision (1.4-13.3%). This method simultaneously analyzed creatinine in urine, which calibrates metabolic rate between different individuals. Validation has been completed for this method; as such, it could possibly be applied to the study of gut microbiota clinically.

Keywords:  LC-MS/MS; gut microbiota; human urine; organic acid

DOI:  https://doi.org/10.3390/molecules27175363
Biomed Chromatogr. 2022 Sep 09. e5501

Establishment and validation of a LC-MS/MS method for the determination of anlotinib in human plasma: application to therapeutic drug monitoring.

Mingjie Yu, Zaiming Liang, Lirong Xiong, Fang Liu, Lin Cheng, Changsheng Jia, Xiaowei Qi, Fengjun Sun.

  Anlotinib is a novel small molecule multitarget tyrosine kinase inhibitor (TKI) for the treatment of several cancers. We developed and validated a highly sensitive, rapid and stable high-performance liquid chromatography-mass spectrometry (LC-MS/MS) method for the determination of anlotinib in human plasma with anlotinib-d5 as a stable isotopically labeled internal standard (SIL-IS). To explore the feasibility of therapeutic drug monitoring (TDM) in the treatment of tumors with anlotinib, human plasma samples were prepared by protein precipitation (PPT). The mobile phase consisted of A (5.0 mM NH4 AC aqueous solution containing 0.1% formic acid) and B (100% methanol containing 0.1% formic acid). A gradient mobile phase system was adopted for chromatographic separation using a BEH C18 (2.1 mm× 50 mm, 1.7 μm). A positive ion pattern was chosen for quantification under multiple reaction monitoring. The ion pairs were detected at m/z 408.2→339.1 and m/z 413.4→344.3 for anlotinib and anlotinib-d5 (SIL-IS), respectively. The total run time was 5.0 min. The calibration curve was found to be linear within a plasma concentration range of 2-400 ng·mL-1 .The precision and accuracy, matrix effect, extraction recovery and stability were all validated and met the requirements of international guidelines. The proposed methods were successfully applied to support TDM breast and thyroid Cancer patients receiving anlotinib for therapy. Clinical data showed that in the 12 mg dose group, the mean plasma concentrations of anlotinib in breast cancer (BC)patients and thyroid cancer (TC) patients were 87.1 and 118.8ng·mL-1 , respectively. The data demonstrate that the peak concentration of anlotinib it may be related to the different tumor types of patients.

Keywords:  LC-MS/MS; SIL-IS,anlotinib,tumor; TDM

DOI:  https://doi.org/10.1002/bmc.5501
Food Chem. 2022 Sep 01. pii: S0308-8146(22)02024-6. [Epub ahead of print]400 134062

Novel molecularly imprinted phenolic resin-dispersive filter extraction for rapid determination of perfluorooctanoic acid and perfluorooctane sulfonate in milk.

Jing Ren, Yanke Lu, Yehong Han, Fengxia Qiao, Hongyuan Yan.

  Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are emerging pollutants that threaten food safety. Herein, a rapid, accurate, and selective method for determination of PFOA and PFOS in milk was established by using new molecularly imprinted phenolic resin (MIP-PR) as the adsorbent of dispersive filter extraction (DFE) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MIP-PR was synthesized at room-temperature using m-aminophenol, glutaraldehyde, and perfluorononanoic acid as the monomers, cross-linkers, and virtual templates, respectively, and exhibited rapid mass transfer (30 s), high selectivity (imprinted factors > 3.7), and large adsorption capacity (>54.6 mg/g). Compared with reported methods, the developed MIP-PR-DFE method is fast, selective, inexpensive, and shows good purification and enrichment efficiency. The proposed MIP-PR-DFE-LC-MS/MS exhibited low limits of detection (0.006-0.022 ng/mL), high recoveries (94.7-109 %), and good precision (RSDs ≤ 9.5 %). This study provides a new idea for the development of imprinted resin adsorbents for perfluorinated compound, and a new method for sample pretreatment for monitoring of PFOA and PFOS in food.

Keywords:  Food analysis; Liquid chromatography-tandem mass spectrometry; Molecularly imprinted polymer; Perfluorinated compounds; Sample preparation

DOI:  https://doi.org/10.1016/j.foodchem.2022.134062
Int J Mol Sci. 2022 Aug 23. pii: 9546. [Epub ahead of print]23(17):

Oligonucleotides Isolation and Separation-A Review on Adsorbent Selection.

Sylwia Studzińska, Łukasz Nuckowski, Bogusław Buszewski.

  Oligonucleotides have many important applications, including as primers in polymerase chain reactions and probes for DNA sequencing. They are proposed as a diagnostic and prognostic tool for various diseases and therapeutics in antisense therapy. Accordingly, it is necessary to develop liquid chromatography and solid phase extraction methods to separate oligonucleotides and isolate them from biological samples. Many reviews have been written about the determination of these compounds using the separation technique or sample preparation for their isolation. However, presumably, there are no articles that critically review the adsorbents used in liquid chromatography or solid phase extraction. The present publication reviews the literature from the last twenty years related to supports (silica, polymers, magnetic nanoparticles) and their modifications. The discussed issues concern reversed phase (alkyl, aromatic, cholesterol, mixed ligands), ion-exchange (strong and weak ones), polar (silica, polyhydroxy, amide, zwitterionic), and oligonucleotide-based adsorbents.

Keywords:  antisense oligonucleotides; biotransformation products; hydrophobic; impurities; ion-exchange groups; polar; solid support

DOI:  https://doi.org/10.3390/ijms23179546
Crit Rev Anal Chem. 2022 Sep 09. 1-21

New Insights into Vitamin K-From Its Natural Sources through Biological Properties and Chemical Methods of Quantitative Determination.

Dorota Wianowska, Iryna Bryshten.

  Vitamin K is one of the many health-promoting substances whose impact on the human body has been underestimated until recently. However, recently published research results have changed this situation, prompting some researchers to consider it a new panacea for diseases of old age. The result is a significant increase in interest in the accurate analysis of vitamin K in various types of samples, ranging from food, through dietary supplements, to biological matrices and clinical trials, both observational and interventional. This review summarizes the current state of knowledge about the proven and speculated biological activity of vitamin K and its importance for the world's aging societies, including the methods used for its isolation and analysis in various matrices types. Of all the analytical methods, the currently preferred methods of choice for the direct analysis of vitamin K are chromatographic methods, in particular liquid chromatography-tandem mass spectrometry. This technique, despite its sensitivity and selectivity, requires an appropriate stage of sample preparation. As there is still room for improvement in the efficiency of these methods, especially at the sample preparation stage, this review shows the directions that need to be taken to make these methods faster, more efficient and more environmentally friendly.

Keywords:  Chromatographic analysis; isolation methods; miniaturized extraction methods; sample preparation; vitamin K vitamers

DOI:  https://doi.org/10.1080/10408347.2022.2121599
Molecules. 2022 Aug 25. pii: 5448. [Epub ahead of print]27(17):

Development and Validation of a Sensitive and Specific LC-MS/MS Method for IWR-1-Endo, a Wnt Signaling Inhibitor: Application to a Cerebral Microdialysis Study.

Sreenath Nair, Abigail Davis, Olivia Campagne, John D Schuetz, Clinton F Stewart.

  IWR-1-endo, a small molecule that potently inhibits the Wnt/β-catenin signaling pathway by stabilizing the AXIN2 destruction complex, can inhibit drug efflux at the blood-brain barrier. To conduct murine cerebral microdialysis research, validated, sensitive, and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were used to determine IWR-1-endo concentration in the murine plasma and brain microdialysate. IWR-1-endo and the internal standard (ISTD) dabrafenib were extracted from murine plasma and microdialysate samples by a simple solid-phase extraction protocol performed on an Oasis HLB µElution plate. Chromatographic separation was executed on a Kinetex C18 (100A, 50 × 2.1 mm, 4 µm particle size) column with a binary gradient of water and acetonitrile, each having 0.1% formic acid, pumped at a flow rate of 0.6 mL/min. Detection by mass spectrometry was conducted in the positive selected reaction monitoring ion mode by monitoring mass transitions 410.40 > 344.10 (IWR-1-endo) and 520.40 > 307.20 (ISTD). The validated curve range of IWR-1-endo was 5-1000 ng/mL for the murine plasma method (r2 ≥ 0.99) and 0.5-500 ng/mL for the microdialysate method (r2 ≥ 0.99). The lower limit of quantification (LLOQ) was 5 ng/mL and 0.5 ng/mL for the murine plasma and microdialysate sample analysis method, respectively. Negligible matrix effects were observed in murine plasma and microdialysate samples. IWR-1-endo was extremely unstable in murine plasma. To improve the stability of IWR-1-endo, pH adjustments of 1.5 were introduced to murine plasma and microdialysate samples before sample storage and processing. With pH adjustment of 1.5 to the murine plasma and microdialysate samples, IWR-1-endo was stable across several tested conditions such as benchtop, autosampler, freeze-thaw, and long term at -80 °C. The LC-MS/MS methods were successfully applied to a murine pharmacokinetic and cerebral microdialysis study to characterize the unbound IWR-1-endo exposure in brain extracellular fluid and plasma.

Keywords:  IWR-1-endo; LC-MS/MS; Wnt signaling inhibitor; bioanalysis; cerebral microdialysis; pharmacokinetics; solid-phase extraction

DOI:  https://doi.org/10.3390/molecules27175448
J Chromatogr A. 2022 Aug 27. pii: S0021-9673(22)00642-2. [Epub ahead of print]1681 463450

Liquid chromatography-mass spectrometry measurements of blood diphosphoinositol pentakisphosphate levels.

Akihiro Kobayashi, Shin-Ichi Abe, Masanori Watanabe, Yusuke Moritoh.

  Diphosphoinositol pentakisphosphate (InsP7) is an inositol pyrophosphate generated by inositol hexakisphosphate kinases (IP6Ks) that regulate diverse biological functions in cells. To date, we have a limited understanding of the InsP7 biology owing to limited data on InsP7 levels in blood or other tissues. Given the significant role of InsP7 in maintaining biological homeostasis, further advancement in InsP7 measurement is essential. In this study, we report a highly sensitive liquid chromatography with tandem mass spectrometry method for determining InsP7 levels in whole blood, which is an easily accessible tissue and for which knowledge on InsP7 levels is limited. We applied a perchloric acid-based method to increase the extraction efficiency of InsP7 from the cells. Subsequently, we combined a YMC-Triart C18 metal-free column, ion-pair reagents, EDTA, methylenediphosphonic acid, and N,N,N',N'-Ethylenediaminetetrakis(methylenephosphonic acid) to minimize InsP7 adsorption to the detection system. We prepared blood quality control samples that were highly exposed to IP6K inhibitor, showing minimum InsP7 levels, to decrease the lower quantification limit of InsP7. Furthermore, we applied rat plasma, which was found to show no InsP7 levels, as a surrogate matrix. This setting resulted in the highly sensitive detection of InsP7 levels in blood obtained from rats, with a quantification sensitivity of 1 ng of InsP7 per mL of blood. The current method demonstrated acceptable accuracy (100 ± 15%) and precision (coefficient of variation ≤ 15%) in rat blood. Using this method, we revealed endogenous basal levels of InsP7 (37.4 ng/mL) in blood obtained from normal rats, and decreased levels of InsP7 (4.1-21.7 ng/mL) in blood obtained from IP6K inhibitor-administered rats. In summary, we established a highly sensitive method for measuring InsP7 and revealed its levels in rat blood. The current findings may help in understanding InsP7 biology.

Keywords:  Blood; Inositol hexakisphosphate kinase; Inositol polyphosphate; InsP(7); Liquid chromatography-tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.chroma.2022.463450
Sci Rep. 2022 Sep 07. 12(1): 15156

Untargeted ultra-high-resolution mass spectrometry metabolomic profiling of blood serum in bladder cancer.

Joanna Nizioł, Krzysztof Ossoliński, Aneta Płaza-Altamer, Artur Kołodziej, Anna Ossolińska, Tadeusz Ossoliński, Tomasz Ruman.

Bladder cancer (BC) is a common urological cancer of high mortality and recurrence rates. Currently, cystoscopy is performed as standard examination for the diagnosis and subsequent monitoring for recurrence of the patients. Frequent expensive and invasive procedures may deterrent patients from regular follow-up screening, therefore it is important to look for new non-invasive methods to aid in the detection of recurrent and/or primary BC. In this study, ultra-high-performance liquid chromatography coupled with ultra-high-resolution mass spectrometry was employed for non-targeted metabolomic profiling of 200 human serum samples to identify biochemical signatures that differentiate BC from non-cancer controls (NCs). Univariate and multivariate statistical analyses with external validation revealed twenty-seven metabolites that differentiate between BC patients from NCs. Abundances of these metabolites displayed statistically significant differences in two independent training and validation sets. Twenty-three serum metabolites were also found to be distinguishing between low- and high-grade of BC patients and controls. Thirty-seven serum metabolites were found to differentiate between different stages of BC. The results suggest that measurement of serum metabolites may provide more facile and less invasive diagnostic methodology for detection of bladder cancer and recurrent disease management.

DOI: https://doi.org/10.1038/s41598-022-19576-9
Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2022 Sep 07. 1-12

Development of a mass spectrometry-based metabolomics workflow for traceability of wild and cultivated Cordyceps sinensis.

Bo Ding, Hanxiang Li, Hongbo Huang, Jianjun Xie, Zhiyuan Wang, Wenrui Chen, Yiwen Tao.

  Cordyceps sinensis, as an expensive traditional Chinese medicine and edible fungus mycelium, lacks an effective quality evaluation method, especially and cultivated Cordyceps sinensis. In this study, a feasible workflow method was developed for traceability evaluation of wild and cultivated Cordyceps sinensis, based on mass spectrometry-based metabolomics. Mass spectrometry data were firstly acquired from Cordyceps sinensis, samples by liquid chromatography-quadrupole and time of flight mass spectrometry. Characteristic mass spectrometry peaks were extracted by applying the MZmine. Then significant markers were obtained from Cordyceps sinensis samples by orthogonal partial least square discriminant analysis. Then, identification of significant markers were identified by MS-FINDER data analytics. The results showed that Changdu, the other four wild origins (Naqu, Xinghai, Yushu and Guoluo) and cultivated samples could be significantly distinguished. This identified significant markers of Cordyceps sinensis, including 174 special significant markers for the wild samples, 204 special significant markers for the cultivated samples and 87 share significant markers. Number of 87 shared significant markers were identified in the wild and cultivated Cordyceps sinensis, especially 28 confident significant compounds, such as adenosine, riboflavin, tyrosine, arginine and glutamine. These shared significant markers might support the quality control of multi-targets of Cordyceps sinensis, compared with a single target in the Chinese Pharmacopoeia. The special significant markers indicated that cultivated Cordyceps sinensis was different from the wild based on mass spectrometry-based metabolomics. In the comparison of chromatographic fingerprint technology, it was found that the established feasible workflow method was easy to acquire significant markers and traceability of Cordyceps sinensis. This feasible workflow method has great potential to be successful for comprehensive and traceability evaluation of the wild and cultivated Cordyceps sinensis.

Keywords:  Cordyceps sinensis; OPLS-DA; feature MS peaks; mass spectrometry-based metabolomics; significant markers; traceability

DOI:  https://doi.org/10.1080/19440049.2022.2118864
Anal Bioanal Chem. 2022 Aug 31.

DNA photoproducts released by repair in biological fluids as biomarkers of the genotoxicity of UV radiation.

Noémie Reynaud, Laura Belz, David Béal, Daniel Bacqueville, Hélène Duplan, Camille Géniès, Emmanuel Questel, Gwendal Josse, Thierry Douki.

  UV-induced formation of photoproducts in DNA is a major initiating event of skin cancer. Consequently, many analytical tools have been developed for their quantification in DNA. In the present work, we extended our previous liquid chromatography-mass spectrometry method to the quantification of the short DNA fragments containing photoproducts that are released from cells by the repair machinery. We designed a robust protocol including a solid-phase extraction step (SPE), an enzymatic treatment aimed at releasing individual photoproducts, and a liquid chromatography method combining on-line SPE and ultra-high-performance liquid chromatography for optimal specificity and sensitivity. We also added relevant internal standards for a better accuracy. The method was validated for linearity, repeatability, and reproducibility. The limits of detection and quantification were found to be in the fmol range. The proof of concept of the use of excreted DNA repair products as biomarkers of the genotoxicity of UV was obtained first in in vitro studies using cultured HaCat cells and ex vivo on human skin explants. Further evidence was obtained from the detection of pyrimidine dimers in the urine of human volunteers collected after recreational exposure in summer. An assay was designed to quantify the DNA photoproducts released from cells within short fragments by the DNA repair machinery. These oligonucleotides were isolated by solid-phase extraction and enzymatically hydrolyzed. The photoproducts were then quantified by on-line SPE combined with UHPLC-MS/MS with isotopic dilution.

Keywords:  Biological fluids; Biomarkers; DNA damage; Solid-phase extraction; UHPLC-MS/MS

DOI:  https://doi.org/10.1007/s00216-022-04302-1
Molecules. 2022 Aug 31. pii: 5609. [Epub ahead of print]27(17):

Comparison of LC-MS3 and LC-MRM Method for Quantifying Voriconazole and Its Application in Therapeutic Drug Monitoring of Human Plasma.

Wenbo Ren, Lei Yin, Gaixia Zhang, Taiyu Zhai, Jing Huang.

  The TDM of voriconazole which exhibits wide inter-individual variability is indispensable for treatment in clinic. In this study, a method that high-performance liquid chromatography tandem mass spectrometry cubed (HPLC-MS3) is first built and validated to quantify voriconazole in human plasma. The system is composed of Shimadzu Exion LCTM UPLC coupled with a Qtrap 5500 mass spectrometer. The separation of voriconazole is performed on a Poroshell 120 SB-C18 column at a flow rate of 0.8 mL/min remaining 7 min for each sample. The calibration curves are linear in the concentration range of 0.25-20 μg/mL. Intra-day and inter-day accuracies and precisions are within 8.0% at three concentrations, and the recoveries and matrix effect are all within accepted limits. In terms of stability, there is no significant degradation of voriconazole under various conditions. The HPLC-MS3 and HPLC-MRM (multiple reaction monitoring) methods are compared in 42 patients with Passing-Bablok regression and Bland-Altman plots, and the results show no significant difference between the two methods. However, HPLC-MS3 has a higher S/N (signal-to-noise ratio) and response than the MRM. Finally, the HPLC-MS3 assay is successfully applied to monitor the TDM (therapeutic drug monitoring) of voriconazole in human plasma, and this verifies that the dosing guidelines for voriconazole have been well implemented in the clinic and patients have received excellent treatment.

Keywords:  HPLC-MS3; therapeutic drug monitoring; voriconazole

DOI:  https://doi.org/10.3390/molecules27175609