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Among the extracellular vesicles, apoptotic bodies (ABs) are only formed during the apoptosis and perform a relevant role in the pathogenesis of different diseases. Recently, it has been demonstrated that ABs from human renal proximal tubular HK-2 cells, either induced by cisplatin or by UV light, can lead to further apoptotic death in naïve HK-2 cells. Thus, the aim of this work was to carry out a non-targeted metabolomic approach to study if the apoptotic stimulus (cisplatin or UV light) affects in a different way the metabolites involved in the propagation of apoptosis. Both ABs and their extracellular fluid were analyzed using a reverse-phase liquid chromatography-mass spectrometry setup. Principal components analysis showed a tight clustering of each experimental group and partial least square discriminant analysis was used to assess the metabolic differences existing between these groups. Considering the variable importance in the projection values, molecular features were selected and some of them could be identified either unequivocally or tentatively. The resulting pathways indicated that there are significant, stimulus-specific differences in metabolites abundancies that may propagate apoptosis to healthy proximal tubular cells; thus, we hypothesize that the share in apoptosis of these metabolites might vary depending on the apoptotic stimulus, This research was funded by the Spanish Ministry of Science and Innovation (project PID2019-104913GB-I00, Agencia Estatal de Investigación, Referencia del Proyecto/AEI/10.13039/ 501100011033) and the Community of Madrid, I+D Biomedicine Activities Program 2022 (General Direction of Research and Technological Innovation (project P2022/BMD7223 CIFRA_CORCM)

Oxidized low-density lipoproteins (oxLDLs) induce oxidative stress in the liver tissue, leading to hepatic steatosis, inflammation, and fibrosis. Precise information on the role of oxLDL in this process is needed to establish strategies for the prevention and management of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Here, we report the effects of native LDL (nLDL) and oxLDL on lipid metabolism, lipid droplet formation, and gene expression in a human liver-derived C3A cell line. The results showed that nLDL induced lipid droplets enriched with cholesteryl ester (CE) and promoted triglyceride hydrolysis and inhibited oxidative degeneration of CE in association with the altered expression of LIPE, FASN, SCD1, ATGL, and CAT genes. In contrast, oxLDL showed a striking increase in lipid droplets enriched with CE hydroperoxides (CE-OOH) in association with the altered expression of SREBP1, FASN, and DGAT1. Phosphatidylcholine (PC)-OOH/PC was increased in oxLDL-supplemented cells as compared with other groups, suggesting that oxidative stress increased hepatocellular damage. Thus, intracellular lipid droplets enriched with CE-OOH appear to play a crucial role in NAFLD and NASH, triggered by oxLDL. We propose oxLDL as a novel therapeutic target and candidate biomarker for NAFLD and NASH.

Drying fruits and vegetables is a long-established preservation method, and for tomatoes, in most cases sun-drying is preferred. Semi-drying is relatively a new application aimed to preserve better the original tomato properties. We have assessed the effects of different drying methods on the phytochemical variation in tomato products using untargeted metabolomics and targeted analyses of key compounds. An LC-MS approach enabled the relative quantification of 890 mostly semi-polar secondary metabolites and GC–MS analysis in the relative quantification of 270 polar, mostly primary metabolites. Metabolite profiles of sun-dried and oven-dried samples were clearly distinct and temperature-dependent. Both treatments caused drastic changes in lycopene and vitamins with losses up to > 99% compared to freeze-dried controls. Semi-drying had less impact on these compounds. In vitro bioaccessibility analyses of total phenolic compounds and antioxidants in a gastrointestinal digestion protocol revealed the highest recovery rates in semi-dried fruits. Semi-drying is a better way of preserving tomato phytochemicals, based on both composition and bioaccessibility results. © 2022 Elsevier Ltd

Background The measurement of plasma concentrations of retinol binding protein is a component of nutritional assessment in neonatal intensive care. However, serial testing in newborns is hampered by the limited amount of blood that can be sampled. Limitations are most severe with preterm infants, for whom close monitoring may be most important. Methods We developed an assay to quantify retinol binding protein using trypsin digestion and liquid chromatography-tandem mass spectrometry, which requires a serum or plasma volume of 5 µl. Additionally, we validated the method according to current recommendations and performed comparison with a standard nephelometry platform in clinical use. Results The assay demonstrated linearity from below 1 mg/dL (0.48 µM) to more than 20 mg/dL (9.7 µM), and an imprecision of 11.8% at 0.43 mg/dL (0.21 µM). The distribution of results observed with the new method was different when compared with nephelometry. Conclusion Liquid chromatography-tandem mass spectrometry facilitated testing a smaller sample volume, thereby increasing the ability to monitor key nutritional markers in premature infants. The differences in results compared with a commercially-available nephelometric assay revealed questionable results for lower concentrations by immunoassay.

Background Lack of clinically specific biomarkers has impeded the precise diagnosis of schizophrenia, meanwhile, limited comprehending of pathogenesis for schizophrenia has restricted the effective treatment. Method An integrated multi-omic approach, combining metabolomic platform (LC-MS and 1H-NMR) and transcriptomic platform, was established to differentiate healthy subjects from schizophrenia patients. Based on filtered metabolites and genes, characteristic spectrums were further built. Then, representative metabolites and genes were screened out through Boruta algorithm. Moreover, characteristic diagnostic formulas were established via LASSO regression analysis. Result As a result, 86 differential metabolites (in line with amino acid metabolism, etc.) and 189 differential expression genes (involving in amino acid metabolic process, etc.) were obtained as potential biomarkers for schizophrenia. The latent interaction between metabolites with genes, such as HMGCLL1 with energy metabolism, etc., was further studied through the analysis of pathway-based integration. Moreover, fine predictive ability was attributed to characteristic metabolomic/transcriptomic diagnostic spectrums/formulas. Conclusion The functional relationships of filtered metabolites and genes were studied, which could elaborate the pathological process of schizophrenia more systemically, supplying more precise information on mechanism description and diagnostic evidence of schizophrenia.

A simple, selective and rapid ultra performance liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous estimation of dolutegravir, lamivudine and tenofovir in bulk and tablet dosage form. Chromatographic separation was attained on Acquity Ethylene Bridged Hybrid (BEH) C18 column (50 × 2.1 mm, 3.5 µm), using a mixture of acetonitrile and 0.1% formic acid in water (60:40, v/v) as a mobile phase at a flow rate of 0.12 mL/min. The total run time of analysis was 3.5 min. The analytes were detected using tandem mass spectrometry, operating in positive ionization and multiple reaction monitoring modes. The method's linearity was determined to be in the range of 10–150 ng/mL with r2 > 0.99. The proposed method was validated as per the International Council for Harmonization (ICH) guidelines, and the results were found well within the acceptance limits. The method was successfully applied for the simultaneous quantification of all the three analytes in the combined tablet dosage form.

Metabolite profiling is an indispensable part of drug discovery and development, enabling a comprehensive understanding of the drug's metabolic behavior. Liquid chromatography-mass spectrometry facilitates metabolite profiling by reducing sample complexity and providing high sensitivity. This review discusses the in vivo metabolite profiling involving LC-MS/MS and the utilization of QTOF, QQQ mass analyzers with a particular emphasis on a mass filter. Further, a summary of sample extraction procedures in biological matrices such as plasma, urine, feces, serum and hair as in vivo samples are outlined. toward the end, we present 15 case studies in biological matrices and their LC-MS/MS conditions to understand the metabolic disposition.

Breast milk is an emerging matrix for vitamin D assessment of breastfed infants and their mothers. It is considered a more reliable indicator of infant intake than the assessment of maternal circulating vitamin D. With the improved sensitivity of mass spectrometry-based technologies, this method principle has been the recent mainstay for the quantitation of various vitamin D metabolites in breast milk for population-based clinical trials. There are still several areas across the total testing process (pre-analytical, analytical and post-analytical) to be defined and harmonised to translate breast milk vitamin D measurement by liquid chromatography-tandem mass spectrometry (LC-MS/MS) from population-based research to routine clinical use and public health applications. Pre-analytically, the determination of the best form of vitamin D to measure in breast milk requires more evidence. Analytically, standardisation of the methods to allow for comparability of results is required. Post analytically, breast milk vitamin D decision limits are needed to turn the individual numerical outputs into clinically meaningful results. This review aims to synthesise the current evidence and utility of measurement of breast milk vitamin D by LC-MS/MS and to lead a future discussion on best practices to allow for its clinical utility beyond its current research-based use.

The toxic effects of mercury in earthworms and the potential alleviation effect of hydrogen-rich water (HRW) using ATR-FTIR and LC–MS analysis methods were investigated. Different concentrations of mercury chloride (H1: 5 µg/mL, H2: 10 µg/mL, H3: 20 µg/mL, H4: 40 µg/mL, and C1: control) and mercury chloride prepared in hydrogen-rich water (H5: 5 µg/mL, H6: 10 µg/mL, H7: 20 µg/mL, H8: 40 µg/mL, and C2: control) were injected into earthworms. The changes and reductions in some bands representing proteins, lipids, and polysaccharides (3280 cm−1, 2922 cm−1, 2855 cm−1, 1170 cm−1, and 1047 cm−1) showed that protective effects could occur in groups prepared with hydrogen-rich water. In the FTIR results, it was found that these bands in the H3 group were more affected and decreased by the influence of mercury on earthworms than the H7 group prepared with hydrogen. LC–MS analysis showed that the changes in some ions of the highest dose groups (H4 and H8) were different, and mercury caused oxidative DNA damage in earthworms. When the high-level application groups of mercury, i.e., H4 and H8 were compared with the controls, the ion exchange ([M + H] + ; m/z 283.1) representing the 8-Oxo-dG level in earthworms was higher in the H4 group than the H8 group. This reveals that HRW exhibited the potential ability to alleviate the toxic effects of mercury; however, a longer period of HRW treatment may be necessary to distinguish an obvious effect. The ATR-FTIR spectroscopy provided a rapid and precise method for monitoring the changes in biological tissues caused by a toxic compound at the molecular level.

For routine analytical purpose a method based on a combination of automated solid-phase extraction (SPE) clean-up and detection by Ultra-Performance Liquid Chromatography/Tandem Mass Spectrometry (UPLC-MS/MS) was developed for the determination of pyrrolizidine alkaloids (PA) in various food commodities. In this survey, honey, tea and herbal infusion samples from local retailers collected in 2012-2015 were obtained and analysed for their PA content. PA concentrations were found in 30% of the honey samples and in 42% of the tea and herbal infusion samples with levels up to 595 µg/kg. The survey included 17 individual PA, and their sum is also reported for each sample.

Objectives Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators have revolutionized the therapeutic landscape in CF treatment. These vital drugs are extensively metabolized via CYP3A, so caution must be exercised in multimodal CF therapy because of the risk of adverse drug interactions. Our goal was to develop a highly sensitive assay for the purpose of therapeutic drug monitoring (TDM) in diagnostic laboratories. Methods After protein precipitation, the CFTR modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their metabolites ivacaftor-M1, ivacaftor-M6, and tezacaftor-M1 were separated with a two-dimensional chromatography setup within 5 min, and quantified with stable isotope-labeled internal standards. The method was validated according to the European Medicines Agency (EMA) guideline on bioanalytical method validation and applied to CF patient samples. Results Inaccuracy was ≤7.0% and the imprecision coefficient of variation (CV) was ≤8.3% for all quality controls (QCs). The method consistently compensated for matrix effects, recovery, and process efficiency were 105–115 and 96.5–103%, respectively. Analysis of CF serum samples provided concentrations comparable to the pharmacokinetic profile data reported in the EMA assessment report for the triple combination therapy Kaftrio. Conclusions We hereby present a robust and highly selective isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) assay for the simultaneous quantification of the so far approved CFTR modulators and their metabolites in human serum. The assay is suitable for state-of-the-art pharmacovigilance of CFTR modulator therapy in CF patients, in order to maximize safety and efficacy, and also to establish dose-response relationships in clinical trials.

Achieving simultaneous determination of antibiotic multiresidues in environmental waters by solid phase extraction (SPE) coupled with ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) with detection limits ≤ ng L-1 is still a huge challenge. Moreover, the offline SPE procedure was performed manually, costly, and time-consuming, while the online SPE required precision pretreatment instruments that require highly-skilled personnel. In this paper, a fully automated SPE coupled with UHPLC-MS/MS method was developed for analysis of antibiotics (sulfonamides, quinolones, and macrolides) in water matrices. Sample preparation optimization included SPE materials and configuration (HLB disks), sample volume (500-1000 mL), and pH (pH = 3) with a flow rate at 2~5 mL min-1, and an elution procedure with 2 × 6 mL methanol, and 2 × 6 mL acetone. Meanwhile, the parameters for UHPLC-MS/S detection of analytes were optimized, including LC retention time, and MS parameters. The instrumental limits of detection (LOD) and quantification (LOQ) of analytes ranged from 0.01-0.72 μg L-1 and 0.05-2.39 μg L-1, respectively, with satisfactory linear calibration (R2 > 0.995) and precision (< 9.9%). Recoveries in spike samples ranged between 77.5-104.9% in pure water, 59.4-97.8% in surface water (SW), and 58.2-108.6% in wastewater effluent (WWE) with relative standard deviations ≤ 12.8%. The matrix effects observed for most analytes were suppression (0-28.1%) except for five analytes having presented enhancement (0-14.6 %) in SW or WWE. This method can basically meet the needs of trace antibiotic residues detection in waters, with examples of concentrations of detected antibiotics being lower than LOQ (LLQ) -94.47 ng L-1 in WWEs and LLQ-15.47 ng L-1 in SW in the lower reaches of the Yangtze River Basin.

Background The circulating concentration of 1α,25-dihydroxyvitamin D [1α,25(OH)2D] is very low, and the presence of multiple isomers may lead to inaccurate quantitation if not separated prior to analysis. Antibody-based immunoextraction procedures are sometimes used to remove structurally related isomers of 1α,25(OH)2D prior to an LC-MS/MS analysis. However, immunoextraction increases sample preparation time and cost. In addition, some dihydroxyvitamin D metabolites are not completely removed by immunoextraction. Method We developed an HPLC method using a phenyl-hexyl column to investigate interfering isomers of 1α,25(OH)2D. Result Using this method, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) derivatization product of 1α,25(OH)2D was found to be present as 2 epimers, which were separated chromatographically with an area ratio of 2:1. PTAD derivatized metabolite of 25-hydroxyvitamin D3 [i.e., 4β,25-dihydroxyvitamin D3 (4β,25(OH)2D3)] eluted out between 6R and 6S epimers of derivatized 1α,25(OH)2D3. If not chromatographically resolved, 4β,25(OH)2D can affect 1α,25(OH)2D quantitation. In a method comparison study, it was found that the presence of 4β,25(OH)2D produced positive bias up to 127% on 1α,25(OH)2D3 quantitation. Conclusion The LC-MS/MS method we developed without an immunoextraction procedure was able to resolve the major interference peak from 1α,25(OH)2D and achieved reliable quantitation of 1α,25(OH)2D.

Background Total gentamicin is a sum of five congeners C1, C1a, C2, C2a and minor C2b, which differ from each other in their methylation on the purpurosamine ring. Liquid chromatography with mass detection (LC-MS/MS) and specified calibration material enables the concentration of total gentamicin and its individual congeners to be analysed. Methods 50 µL serum was precipitated with acetonitrile in the presence of 0.5 mol/L formic acid. A RP BEH C18 1.7 µm 2.1x50 mm column maintained at 30 °C and tobramicin as the internal standard were used. Mass detection was performed in positive electrospray. The gentamicin results were compared with fluorescence polarization immunoassay (FPIA) and chemiluminiscent microparticle immunoassay (CMIA). Passing-Bablock regression analysis and Bland-Altman analysis were used. Results Calibration curves for individual gentamicin congeners were linear with correlation coefficients between 0.997 and 0.998. Recovery was 91.6–102.0% and the coefficients of variation 1.4–8.4%. The total gentamicin concentration was compared with immunoassay FPIA (LC-MSgen = 0.9798xPFIAgen) and CMIA (LC MSgen = 0.9835xCMIAgen) both with significant correlation (p Conclusion The LC-MS/MS method is fast and precise and can be applied to routine TDM in patients. Comparing it to immunoassays makes it possible to measure concentration of gentamicin congeners, which may be important in the case of their different pharmacokinetics.

The aim of this study was to demonstrate the metabolic profile of post-culture medium as an expression of cell suspension metabolic activity of the tree fern Cyathea delgadii Sternb. The molecular profile of the tree fern’s cell culture has been never described, according to our knowledge. The cell suspension was established using ½ MS medium supplemented with various concentrations of 2,4-D and BAP. The optimal concentrations were 2.0 mg·L−1 and 0.2 mg·L−1, respectively. The cell suspension initially showed an organized system of cell division and later unorganized cell proliferation. LC-MS and GC-MS were used to identify the chemical composition of the post-culture medium. The LC-MS analysis results suggested that the color of liquid medium could be due to the presence of flavonoid derivatives, as this group of compounds was represented by eight compounds. After GC-MS analysis based on retention indexes and thanks to mass spectra comparison, 130 natural products were recognized, belonging to various classes of primary and secondary metabolites.

Metabolomics has become an important tool for clinical research, especially for analyzing inherited metabolic disorders (IMDs). The purpose of this study was to explore the performance of metabolomics in diagnosing IMDs using an untargeted metabolomic approach. A total of 40 urine samples were collected: 20 samples from healthy children and 20 from pediatric patients, of whom 13 had confirmed IMDs and seven had suspected IMDs. Samples were analyzed by Orbitrap mass spectrometry in positive and negative mode alternately, coupled with ultra-high liquid chromatography. Raw data were processed using Compound Discovery 2.0 ™ and then exported for partial least squares discriminant analysis (PLS-DA) by SIMCA-P 14.1. After comparing with m/zCloud and chemSpider libraries, compounds with similarity above 80% were selected and normalized for subsequent relative quantification analysis. The uncommon compounds discovered were analyzed based on the Kyoto Encyclopedia of Genes and Genomes to explore their possible metabolic pathways. All IMDs patients were successfully distinguished from controls in the PLS-DA. Untargeted metabolomics revealed a broader metabolic spectrum in patients than what is observed using routine chromatographic methods for detecting IMDs. Higher levels of certain compounds were found in all 13 confirmed IMD patients and 5 of 7 suspected IMD patients. Several potential novel markers emerged after relative quantification. Untargeted metabolomics may be able to diagnose IMDs from urine and may deepen insights into the disease by revealing changes in various compounds such as amino acids, acylcarnitines, organic acids, and nucleosides. Such analyses may identify biomarkers to improve the study and treatment of IMDs.

INTRODUCTION Direct oral anticoagulant (DOAC)-inhibiting factor Xa (FXa-DOAC) are being increasingly used as prophylaxis of venous thromboembolism and for prevention of stroke in patients with atrial fibrillation. In contrast to vitamin K antagonists, DOACs do not require monitoring in general. However, it is sometimes of value in the acute setting, for instance when considering a reversal agent in uncontrolled bleeding in patients on DOAC. METHODS We evaluated if a low-molecular weight heparin (LMWH)-calibrated anti-factor Xa assay could be used to estimate FXa-DOAC concentration in the concentration range

INTRODUCTION Portulaca oleracea is a commonly used nutritional vegetable and traditional herbal medicine with plenty of nutrients and manifold pharmacological activities. However, the potential active ingredients for its remarkable antioxidant, hypoglycemic and hypolipidemic activities remain unexplored. OBJECTIVES The present study aims to systematically evaluate the antioxidant activities of different extracts of P. oleracea and screen bioactive ligands that can interact with α-glucosidase, pancreatic lipase, and superoxide dismutase (SOD). METHODS In this research, the antioxidant activities of different parts of P. oleracea and their corresponding total phenolic content (TPC) and total flavonoid content (TFC) were systematically determined. Subsequently, a multi-target affinity ultrafiltration method was developed using affinity ultrafiltration with SOD, α-glucosidase, and pancreatic lipase coupled to liquid chromatography-mass spectrometry (UF-LC-MS). Later, molecular docking was used to further investigate the possible interaction mechanism between these ligands and target enzymes. RESULTS Among them, the ethyl acetate (EA) fraction showed the highest antioxidant activity along with the highest TPC and TFC, and four compounds in the EA fraction were quickly retrieved as potential SOD, α-glucosidase, and pancreatic lipase ligands, respectively. Molecular docking revealed that these potential ligands exhibited strong binding ability and inhibitory activities on SOD, α-glucosidase, and pancreatic lipase. CONCLUSION The present study revealed that P. oleracea can be used as a functional food with excellent antioxidant, hypoglycemic and hypolipidemic effects. Meanwhile, the integrated strategy based on multi-target UF-LC-MS and molecular docking also provided a powerful tool and a multidimensional perspective for further exploration of active ingredients in P. oleracea responsible for the antioxidant, hypoglycemic and hypolipidemic activities.

Objectives Fat-soluble vitamins are measured to identify deficiencies that may lead to rickets, osteomalacia, night blindness, and reversible motor and sensory neuropathies. We present a rapid liquid chromatography–mass spectrometry (LC-MS/MS) method that simultaneously measures 25-hydroxyvitamin D3 (25[OH]D3), epi-25(OH)D3, 25(OH)D2, vitamin A, α-tocopherol, and γ-tocopherol. Methods We mixed 100 µL serum with internal standard and extracted it by using supported liquid extraction plates. Reconstituted specimens were analyzed by LC-MS/MS with a 10-minute gradient. Results The method was linear, covering physiological levels with r2 > 0.99, and the total precision was less than 15% at all quality control levels. The lower limit of the measuring intervals for 25(OH)D3, epi-25(OH)D3, 25(OH)D2, vitamin A, α-tocopherol, and γ-tocopherol were 4 ng/mL, 4 ng/mL, 4 ng/mL, 1 µg/dL, 0.2 µg/mL, and 0.2 µg/mL, respectively, with coefficient of variation of 20% or less. Recoveries were between 92% and 111% for National Institute of Standards and Technology reference materials and 81% and 122% for spike-recovery studies. Comparison studies for vitamin D total, vitamin A, and α-tocopherol demonstrated slopes between 1.04 and 1.11 and r2 between 0.94 and 0.96. Minimal matrix effect was observed for all analytes. Conclusions We developed and validated a rapid LC-MS/MS method for the simultaneous measurement of 25(OH)D3, epi-25(OH)D3, 25(OH)D2, vitamin A, α-tocopherol, and γ-tocopherol.

A simultaneous determination method for caffeine, theobromine, and theophylline in chocolate was developed. Three compounds were ultrasonically extracted twice (15 min at 50℃) in an acetonitrile-water (1 : 1, v/v). The extract was purified using Oasis HLB SPE cartridge, and the purified processed by LC-MS. The method exhibited recoveries of 97.4-100.2%, RSDs of repeatability of 1.0-2.8%, and RSDs of within-laboratory reproducibility of 2.0-7.9%. This method was simpler and more selective than existing methods, and was practical for caffeine, theobromine, and theophylline analysis in chocolate.

Background Urinary 5-hydroxyindoleacetic acid (5-HIAA) is a first-line investigation for gastrointestinal neuroendocrine tumours that secrete serotonin. It also has clinical utility for monitoring disease progression and therapeutic response. Aim To develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for urinary 5-hydroxyindoleacetic acid that incorporates a supported liquid extraction and 13C-labelled internal standard. Methods Samples were diluted in ammonium acetate containing a 13C-labelled internal standard (5-hydroxyindole-3a,4,5,6,7,7a-13C6-3-acetic acid). Supported liquid extraction was performed followed by chromatographic separation using the 2.1 × 30 mm CORTECS® UPLC® T3 column. Mass spectrometry detection (Waters Xevo TQ-XS) was performed in electrospray positive mode using the transitions 192.3 > 146.4 m/z (quantifier) and 192.3 > 118.4 m/z (qualifier) for 5-hydroxyindoleacetic acid and 198.2 > 152.4 m/z for 13C-5-HIAA. Results A well-defined 5-hydroxyindoleacetic acid peak was observed at 0.8 min with a run time of 2.4 min. The assay was linear (r2 > 0.99) to 382 µmol/L, with a lower limit of quantification of 5.3 µmol/L (CV Conclusion We have developed a robust LC-MS/MS method with semi-automated extraction that offers an improved run time and performance over the existing, labour-intensive, HPLC method. The method was quick, precise, showed good agreement with UKNEQAS external quality assurance material and is in routine service for clinical samples.

Purpose. To establish a liquid chromatography/mass spectrometry method to compare the tissue distribution and metabolism of AN1284 after subcutaneous and oral administration at doses causing maximal reductions in IL-6 in plasma and tissues of mice. Methods. Lipopolysaccharide activated RAW 264.7 macrophages were used to detect the anti-inflammatory activity of AN1284 and its metabolites. Mice were given AN1284 by injection or gavage, 15 min before lipopolysaccharide. The reduction of IL-6 measured after 4h. Results. AN1284 is metabolized to the indole (AN1422), a 7-OH derivative and its glucuronide. AN1422 had weaker anti-inflammatory activity than AN1284 in LPS-activated macrophages and in mice. Maximal reductions in IL-6 in plasma, brain and liver were seen after subcutaneous injection of (0.5 mg/kg). This dose was also most effective in reducing IL-6 in the liver after oral drug administration but 2.5 mg/kg was needed for the same reductions in plasma and brain. Peak concentrations after oral administration of AN1284 (2.5 mg/kg) were 5.5-fold higher in the liver but 7-, 11 and 19-fold lower in plasma, brain and kidneys than after injection of 0.5 mg/kg. Similar concentrations in the liver were achieved by AN1284 (1 mg/kg/day) administered in the drinking fluid, and 2.5 mg/kg/day, via subcutaneously-implanted mini-pumps. Although drug levels were only 12% of the peak seen after acute injection of 0.5 mg/kg, they significantly decreased hepatocellular damage, liver triglycerides and cholesterol in diabetic mice. Conclusion. AN1284 can be given orally to treat chronic liver disease and its preferential concentration in the liver should limit potential adverse effects.

To support a phase III randomized trial of the multi-targeted tyrosine kinase inhibitor cabozantinib in neuroendocrine tumors, we developed a high-performance liquid chromatography mass spectrometry method to quantitate cabozantinib in 50 μL of human plasma. After acetonitrile protein precipitation, chromatographic separation was achieved with a Phenomenex synergy polar reverse phase (4 μm, 2 × 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and 0.1% formic acid in water over a 5-min run time. Detection was performed on a Quattromicro quadrupole mass spectrometer with electrospray, positive-mode ionization. The assay was linear over the concentration range 50–5000 ng/mL and proved to be accurate (103.4–105.4%) and precise (

Humans are constantly exposed to formaldehyde (FA) of both exogenous and endogenous sources, and FA exposure is associated with the development of many human diseases, including cancers. Marker molecules that can provide information on exposure history and amounts will assist disease risk assessment and early interventions. To develop marker signatures of FA exposure, we explored in this study the conjugation reaction of FA with γ-glutamylcysteine (GGC), one of the precursors to glutathione biosynthesis, under physiologically relevant conditions. The results showed that the reaction produced a stable metabolite of FA, (S)-1-((((R)-2-amino-2-carboxyethyl)thio)methyl)-5-oxopyrrolidine-2-carboxylic acid (COCA). Using liquid chromatography-tandem mass spectrometry coupled to a stable isotope-dilution method, we then quantitated for the first time the formation of this novel metabolite in FA- and Fe2+-EDTA-exposed human cells. The results revealed the exposure time- and concentration-dependent formation of COCA in FA- or Fe2+-EDTA-exposed cells, suggesting that COCA may serve as a biomarker of FA and oxidative stress exposure. Furthermore, the study sheds light on a previously unknown protective role of GGC against FA and oxidative stress.

Bushra T ALquadeib,1,&ast; Nouf M Aloudah,2,&ast; Alanood S Almurshedi,1 Iman M ALfagih,1 Basmah N ALdosari,1 Adim S ALmeleky,3 Nour M Almubyedh3 1Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, 11671, Saudi Arabia; 2Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh, 11671, Saudi Arabia; 3College of Pharmacy, King Saud University, Riyadh, 11671, Saudi Arabia&ast;These authors contributed equally to this workCorrespondence: Bushra T ALquadeib Email bquadeib@ksu.edu.saObjective: Metformin (MET), an oral biguanide agent, can improve insulin resistance and decrease hepatic glucose production, leading to a reduction in blood-sugar levels. The objective of the present study was to develop and validate simple and rapid LC-MS/MS method for analysis of MET in dried blood spot (DBS) sample for patient monitoring studies purposes (drug adherence).Methods: The chromatographic separation was achieved with Waters HSS-T3 column using gradient elution of mobile phases of two solvents: 1) solvent A, consisted of 10mM ammonium formate, 0.2% formic acid 1%; and 2) acetonitrile solvent B, contained 0.2% formic acid in acetonitrile at a flow rate of 0.2 mL/min. The total run time was 3.0 min. The effectiveness of chromatographic conditions was optimized, and afatinib was used as the internal standard. The assay method was validated using USP 26 and the ICH guidelines.Results: The method showed good linearity in the range 8– 48 ng/mL for MET with correlation coefficient (r) > 0.9907. The intra- and inter‑day precision values for MET met the acceptance criteria as per regulatory guidelines. MET was stable during the stability studies at ambient temperature 25 °C, at refrigerator 4 °C, at 10 °C autosampler, freeze/thaw cycles and 30 days storage in a freezer at − 30 ± 0.5 °C.Conclusion: This method has successfully fulfilled all validation requirements referring to EMA and FDA guidelines, and successfully can be applied for MET adherence study. All the six studied patients were approved to metformin adherence.Keywords: metformin, dried blood spot, liquid chromatography–tandem mass spectrometry, method validation, medication adherence

Cortisol is considered a particularly relevant biomarker in the context of stress evaluation. This study aims to review of the available literature on the determination of cortisol in hair using LC-MS/MS. Currently, there is no standardized procedure for the measurement of cortisol concentrations in hair, and different sample preparation, chromatographic separation and mass spectrometric detection conditions were described. Simple methanolic extraction, reversed-phase separation and MRM detection in negative ion mode are the most common employed analytical approaches. Reported assays presented acceptable sensitivity for clinical purposes. The increasing use of mass spectrometry in clinical laboratories may contribute to the establishment of LC-MS/MS as the method of choice for the determination of cortisol concentrations in hair.

Nephrotic syndrome (NS) is a kidney illness characterized by excessive proteinuria, hypoalbuminemia, edema, and hyperlipidemia, which may lead to kidney failure and necessitate renal transplantation. End-stage renal disease, cardiovascular issues, and mortality are much more common in those with NS. Therefore, the present study aimed to identify potential new biomarkers associated with the pathogenesis and diagnosis of NS. The liquid chromatography–mass spectrometry (LC–MS) metabolomics approach was applied to profile the metabolome of human serum of patients with NS. A total of 176 metabolites were significantly altered in NS compared to the control. Arginine, proline, and tryptophan metabolism; arginine, phenylalanine, tyrosine, and tryptophan biosynthesis were the most common metabolic pathways dysregulated in NS. Furthermore, alanyl-lysine and isoleucyl-threonine had the highest discrimination between NS and healthy groups. The candidate biomarkers may lead to understanding the possible metabolic alterations associated with NS and serve as potential diagnostic biomarkers.

Macrophages synthesize active vitamin D (1,25-dihydroxy-vitamin D) and express the vitamin D receptor in the nucleus; however, vitamin D metabolism in relation to macrophage polarization and function is not well understood. We studied monocyte-derived macrophages (MDMs) from human buffy coats polarized into M0, M1 (LPS + IFNγ), M2a (IL4 + IL13) and M2c (IL10) macrophage subtypes stimulated with 25-hydroxy-vitamin D (1000 and 10,000 nanomolar). We measured vitamin D metabolites (25-hydroxy-vitamin D, 1,25-dihydroxy-vitamin D, 24,25-dihydroxy-vitamin D and 3-epi-25-hydroxy-vitamin D) in cell media with liquid chromatography-mass spectrometry-mass spectrometry. The mRNA expression (CYP27B1, CYP24A1 and CYP24A1-SV) was measured with qPCR. We found that reparative MDMs (M2a) had significantly more 1,25-dihydroxy-vitamin D compared to the other MDMs (M0, M1 and M2c). All MDMs were able to produce 3-epi-25-hydroxy-vitamin D, but this pathway was almost completely attenuated in inflammatory M1 MDMs. All MDM subtypes degraded vitamin D through the 24-hydroxylase pathway, although M1 MDMs mainly expressed an inactive splice variant of CYP24A1, coding the degrading enzyme. In conclusion, this study shows that vitamin D metabolism is highly dependent on macrophage polarization and that the C3-epimerase pathway for vitamin D is active in macrophages.

The Yongyou series of indica-japonica hybrid rice has excellent production potential and storage performance. However, little is known about the underlying mechanism of its storage resistance. In this study, Yongyou 1540 rice (Oryza sativa cv. yongyou 1540) was stored at different temperatures, and the storability was validated though measuring nutritional components and apparent change. In addition, a broad-targeted metabolomic approach coupled with liquid chromatography-mass spectrometry was applied to analyze the metabolite changes. The study found that under high temperature storage conditions (35 °C), Yongyou 1540 was not significantly worse in terms of fatty acid value, whiteness value, and changes in electron microscope profile. A total of 19 key differential metabolites were screened, and lipid metabolites related to palmitoleic acid were found to affect the aging of rice. At the same time, two substances, guanosine 3′,5′-cyclophosphate and pipecolic acid, were beneficial to enhance the resistance of rice under harsh storage conditions, thereby delaying the deterioration of its quality and maintaining its quality. Significant regulation of galactose metabolism, alanine, aspartate and glutamate metabolism, butyrate metabolism, and arginine and proline metabolism pathways were probably responsible for the good storage capacity of Yongyou 1540.

Carboxylic acids participate in many metabolic pathways including tricarboxylic acid (TCA) cycle. Therefore,there have been ongoing attempts to develop sensitive liquid chromatography-mass spectrometry methods overthe last decades. Derivatization of the carboxylic acids with 3-nitrophenylhydrazine presents a well-establishedmethodology, and yet the derivatized species of polycarboxylic acids and their fragmentation in collisioninduceddissociation have not been fully studied before. In our study, we elucidated how annotation of mostabundant 3-nitrophenylhydrazine derivatives and optimization of their fragmentation in multiple reactionmonitoring can boost the sensitivity, especially for polycarboxylic acids. Finally, the optimized liquidchromatography-tandem mass spectrometry method allowed for low detection limits ranging from 10 pM for 2-oxoglutaric acid to 800 pM for pyruvic acid. All TCA carboxylates were quantified in 20 μL of human plasma andthe targeted method was validated in the same matrix. The same methodology with a modified gradient elutionwas also applied to untargeted screening of fatty acids by using high-resolution mass spectrometry enablingidentification of 29 medium- to long-chain fatty acids in human plasma. The TCA carboxylates were alsoquantified in 105 of C2C12 mouse myuotube cells grown under different treatments to proof applicability of themethodology to biological studies in a wider sense. However, unfortunately all the TCA carboxylates were alsofound in the derivatized blanks in substantial amounts, which prevents from using the methodology for quantificationof the carboxylates in less than 105 cells. Carboxylic acids participate in many metabolic pathways including tricarboxylic acid (TCA) cycle. Therefore, there have been ongoing attempts to develop sensitive liquid chromatography-mass spectrometry methods over the last decades. Derivatization of the carboxylic acids with 3-nitrophenylhydrazine presents a well-established methodology, and yet the derivatized species of polycarboxylic acids and their fragmentation in collision-induced dissociation have not been fully studied before. In our study, we elucidated how annotation of most abundant 3-nitrophenylhydrazine derivatives and optimization of their fragmentation in multiple reaction monitoring can boost the sensitivity, especially for polycarboxylic acids. Finally, the optimized liquid chromatography-tandem mass spectrometry method allowed for low detection limits ranging from 10 pM for 2-oxoglutaric acid to 800 pM for pyruvic acid. All TCA carboxylates were quantified in 20 µL of human plasma and the targeted method was validated in the same matrix. The same methodology with a modified gradient elution was also applied to untargeted screening of fatty acids by using high-resolution mass spectrometry enabling identification of 29 medium- to long-chain fatty acids in human plasma. The TCA carboxylates were also quantified in 105 of C2C12 mouse myuotube cells grown under different treatments to proof applicability of the methodology to biological studies in a wider sense. However, unfortunately all the TCA carboxylates were also found in the derivatized blanks in substantial amounts, which prevents from using the methodology for quantification of the carboxylates in less than 105 cells.

The present study describes a simple and rapid method for the determination of aflatoxins in almonds using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Aflatoxins were extracted using a modified QuEChERS method with little sample preparation, excluding the use of laborious purification procedures. Extracts were frozen overnight to separate the majority of lipids. The method was successfully validated for almonds. Linearity was demonstrated in the range 0.125-20 µg/kg. Limits of quantification (LOQ) ranged from 0.34 to 0.5 μg/kg. Matrix effect was not significant for the aflatoxins. Satisfactory recoveries were obtained at spike levels below 1 μg/kg and between 1 and 10 μg/kg. Relative standard deviations (RSDs) of repeatability and reproducibility were below 15%. The method was successfully tested with two proficiency tests in almond powder and peanut paste, with acceptable

The anticancer effects of rosmarinic acid (RA) are a hotspot of current research. In order to enhance its pharmacological activity, N-substituted RA was prepared, and it has been shown to exhibit notable antitumor effects. In order to elucidate the underlying mechanisms of action, pharmacokinetic analysis is necessary. In the present study, liquid chromatography–tandem mass spectrometric method, was used to determine the concentrations of RA and its analog, (E)-3-(3,4-dihydroxyphenyl)-2-(3-(3,4-dihydroxyphenyl)acrylamido)propanoic acid (A2) in plasma from rats. The analyses were divided into a C18 column (1.9 μm, 2.1 mm × 100 mm) with a security guard C18 column (5 μm, 2.1 mm × 10 mm) and a triple-quadrupole mass spectrometry with an electrospray ionization ion-source generates ions. The sample pretreatment is relevant to the one-step protein precipitation with isopropanol:ethyl acetate (v/v, 1:1) This method presented a linear association within ranges at the concentration of 5–2000 ng/mL for A2 and RA. Relative standard deviations in daily courses were

A liquid chromatography tandem mass spectrometry method (LC-MS/MS) preceded by a rapid and simple QuEChERS-based sample preparation was developed and validated for the determination of acrylamide in non-centrifugal sugar (NCS), a solid product obtained from sugarcane juice without centrifugation, up to a concentration greater than 90 ºBrix. Adequate linearity in calibration curves, analytical selectivity and sensitivity were achieved. A limit of detection (LOD) and a limit of quantitation (LOQ) of 10 and 20 µg kg

Gibberellic acid (GA3) is widely used in agriculture and maybe transfer with groundwater flow, which is an endocrine disruptor, but few studies have focused on the transformation pathway and toxicity assessment of GA3 and its products. Here, GA3 and its transformation products in aqueous solution were identified and quantified by liquid chromatography mass spectrometry hybrid ion trap time-of-flight (LCMS-IT-TOF) and high-performance liquid chromatography (HPLC), respectively. The results showed that the half-life of GA3 transformation in ultrapure water was 16.1-24.6 days at pH=2.0-8.0, with the lowest half-life occurring at pH=8.0 and highest half-life occurring at pH=3.3. Isomerized gibberellic acid (Iso-GA3) and gibberellenic acid (GEA) were the main transformation products with a little hydroxy gibberellic acid (OH-GA3). In North China groundwater, the mass balance of GA3 and its products was 76.2%, including Iso-GA3 (58%), GEA (7.9%), GA3 (7.3%) and OH-GA3 (3%) after reaching transformation equilibrium. Using Gaussian 09 for chemical computation, it was found that the transformation mechanism of GA3 was dependent upon the bond energy and the stereochemical feature of its molecular structure. GA3 always isomerized from the γ-lactone ring due to the lowest bond energy between the oxygen terminus of the γ-lactone ring and A ring. While GA3 and its transformation products all had developmental toxicity, the predicated LC50 (96 hr) and LD50 of the main products of GA3 were much lower than those of GA3, indicating GA3 would be transformed into higher toxicity derivatives in water environments, posing a significant health risk to humans and the environment.

International audience; Cross-contamination of animal feed with antibiotics may occur during manufacturing in feed mills, because shared production lines can be used for medicated and non-medicated feed, but may also occur during transport, storage and at the farm level. This is a major issue in the current context where antimicrobial usage must be controlled in order to maintain their effectiveness. A LC-MS/MS method was developed for the determination of colistin, bacitracin A and virginiamycin M1 in feed for pigs, poultry and rabbits at concentrations similar to those encountered in cross-contamination. After investigating various issues related to colistin behaviour and matrix effects, we successfully validated this method according to the requirements of European regulations in terms of linearity, trueness, precision, limit of quantification and limit of decision. Trueness ranged 88.6–107.8% and precision ranged 12.6–21.2%. We then applied this method to the analysis of medicated pig feed to check the performance of the method on “real” samples of medicated feed. We subsequently analysed non-medicated pig, and rabbit feed samples, collected directly on farms, to check the rate of cross-contamination. No samples were contaminated by colistin, bacitracin, or virginiamycin.

Cells are continually exposed to endogenous reactive oxygen, nitrogen, and halogen species, causing damage to biomolecules. Among them, peroxynitrite and hypochlorous acid are not only oxidants but also biological nitrating and chlorinating agents, leading to the formation of 3-nitrotyrosine and 3-chlorotyrosine, respectively, in proteins. 3-Nitrotyrosine has been detected in vivo under several pathophysiological conditions, including breast cancer. Studies show that the concentrations of 3-nitrotyrosine in plasma proteins and platelets were significantly elevated in breast cancer patients. Compared to blood serum albumin, hemoglobin adducts represent biomonitoring of exposure with a longer lifetime. In this study, human hemoglobin was freshly isolated from blood and digested into peptides with trypsin, and the levels of protein adducts, including nitration, nitrosylation, and chlorination of tyrosine as well as oxidation of methionine residues, were simultaneously quantified by nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) with selected reaction monitoring. The results demonstrated that the relative extents of nitration at α-Tyr-42 and β-Tyr-130, nitrosylation at α-Tyr-24, and chlorination at α-Tyr-24 and β-Tyr-130 are significantly higher in globin of 25 breast cancer patients compared to those in 25 healthy subjects (p 0.8. While the age of the subjects is correlated with the extents of some of these adducts, the body mass index does not have an effect on any of them. Starting with 1 drop of blood, our results indicated that this highly sensitive and specific nanoLC-NSI/MS/MS is useful in investigating the role of reactive nitrogen oxide species and reactive chlorine species in the etiology of breast cancer.

Background The current first-line screening test for primary hyperaldosteronism is the plasma aldosterone:renin ratio; however, renin assays have several disadvantages and the ARR is affected by medications and physiological factors. Angiotensin II is a key biologically active hormone in the renin-angiotensin-aldosterone system. It has been suggested that measurement of equilibrium levels of this peptide, involving an in vitro incubation of serum prior to analysis, may provide a better marker of renin-angiotensin-aldosterone system activity than renin. Methods An eqAng II LC-MS/MS assay was developed, optimized and validated. Serum samples were incubated at 37°C for 45 min prior to stabilization with cold EDTA solution, solid phase extraction and LC-MS/MS analysis. Stability in whole blood and the effect of cryoactivation were assessed. For comparison to the current screening test, 150 anonymized patients’ samples were analysed for eqAng II, renin activity and aldosterone (all by LC-MS/MS). Results The assay had good precision, minimal bias and acceptable recovery. EqAng II did not change significantly when whole blood samples were stored for up to 72 h, and cryoactivation was only observed for pregnant patients. EqAng II was significantly correlated with renin, and the aldosterone:eqAng II ratio had a strong positive correlation with the aldosterone:renin ratio. Conclusions An LC-MS/MS assay for eqAng II has been developed which shows promise as an alternative screening test for primary hyperaldosteronism. Compared to renin assays, it is quicker, simpler and less likely to be affected by anti-hypertensive medications. Further clinical validation in hypertensive patients would be required prior to implementation.

Background We developed and compared two liquid chromatography methods, one with UV/Visible spectrophotometric detection (HPLC) and the other with mass spectrometric detection (LC-MS), for quantifying very-long chain fatty acids (VLCFA) in human plasma. Association of VLCFA with various cardiovascular risk factors were evaluated. Method Fasting blood samples were collected from 541 human volunteers (242 men and 299 women; mean age ±SD, 58.9 ± 12.4 years), including 429 and 112 individuals with and without hypertriglyceridemia, respectively. Esterified VLCFA were saponified and derivatized with 2-nitrophenylhydrazine. Separation of VLCFA species was achieved with C4 Mightysil column (HPLC) and Ascentis Express Phenyl-Hexyl column (LC-MS) followed by spectrophotometric and selected-reaction monitoring mode of mass spectrometric detection, respectively. Results The HPLC assay of VLCFA was precise with intra-assay imprecision of 2.5% to 6.9% and inter-assay imprecision of 3.2% to 9.5%. Moreover, there was an excellent correlation (r > 0.96) between HPLC and LC-MS methods. The 95 percentile reference intervals (RI; upper limit) of VLCFA were determined to be 41.3 µmol/L in healthy volunteers. Plasma VLCFA were significantly correlated with triglycerides (Spearman’s ρ = 0.306, P < 0.001) and total cholesterol (Spearman’s ρ = 0.251, P < 0.001). All species of VLCFA were significantly elevated in hypertriglyceridaemic individuals compared with control. Conclusion We established LC-based assays of VLCFA with either spectrophotometry or mass spectrometry as a detection system. Hypertriglyceridaemia is significantly associated with elevated concentration of each species of VLCFA.

A rapid, simple and sensitive liquid chromatography tandem mass spectrometry method for the determination of methohexital in human whole blood was developed and validated. Ethyl acetate/n-hexane (9:1) was used as extraction solvent while aprobarbital was used as internal standard. Methohexital was recovered by liquid–liquid extraction from 100 μL of human whole blood. The mobile phase was water-acetonitrile, and an ACQUITY BEH C18 (2.1 × 100 mm, 1.7 μm) column was adopted. Negative electrospray ionization source and multiply reaction monitoring mode were applied. The transitions of m/z were 261.2/42.2 and 261.2/119.0 for methohexital. The limit of detection was 0.5 ng/mL, which was lower than the previous methods. Wide linear range (2–2,000 ng/mL) with a good correlation coefficient (r > 0.99) was also obtained. The intra- and inter- day precisions represented by relative standard deviation were 79.67%. This analytical method involved small sample volume and had been proven to be rapid, easy, sensitive and specific. Therefore, it could be used for the clinical analysis of methohexital.

INTRODUCTION Giant garlic is a functional food that contains different kinds of bioactive molecules with beneficial effects on chronic noncommunicable diseases like diabetes and cardiovascular conditions. Considering biosynthesis pathways, abundance, and biological activity, alliin and S-allyl-cysteine were used as chemical markers of organosulphur compounds present in giant garlic. OBJECTIVE To establish a chemometric optimisation of pressurised liquid extraction for the determination of alliin and S-allyl-cysteine in giant garlic by liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODOLOGY Samples were blanched (ca. 90°C for 10 min) to inactivate alliinase and γ-glutamyl transpeptidase enzymes and then freeze-dried. Chemometric optimisation was performed via response surface methodology based on central composite design (CCD). Organosulphur compound yields were determined applying a validated LC-MS/MS method in multiple reaction monitoring (MRM) mode using the following transitions: for alliin m/z 178 → 74 and for S-allyl-cysteine m/z 162 → 41. RESULTS According to CCD results, under constant conditions of pressure (1500 psi) and time (20 min), the optimal conditions for pressurised liquid extraction of alliin and S-allyl-cysteine were 70.75 and 68.97% v/v of ethanol in extraction solvent and 76.45 and 98.88°C as extraction temperature, respectively. Multiple response optimisation for the simultaneous extraction of both organosulphur compounds was established via desirability function. Under these conditions, 2.70 ± 0.27 mg g-1 dry weight (DW) of alliin and 2.79 ± 0.22 mg g-1 DW of S-allyl-cysteine were extracted. CONCLUSIONS These results clearly demonstrated that pressurised liquid extraction is an efficient green technique to extract bioactive organosulphur compounds from giant garlic. Extraction yields were significantly (p

This study was carried out to evaluate the effect of extraction methods (direct maceration (DM) and successive maceration (SM)) and solvents (dichloromethane (DCM), ethyl acetate (EAc), butanol (Bu...

Peptide drug leads possess unusual structural features that allow them to exert their unique biological activities and ideal physicochemical properties. In particular, these peptides often have D-amino acids, and therefore the absolute configurations of the component amino acids have to be elucidated during the structural determination of newly isolated peptide drug leads. Recently, we developed the highly sensitive labeling reagents D/L-FDVDA and D/L-FDLDA for the structural determination of the component amino acids in peptides. In an LC-MS-based structural study of peptides, these reagents enabled us to detect infinitesimal amounts of amino acids derived from mild degradative analysis of the samples. Herein, we firstly report the improved LC-MS protocols for the highly sensitive analyses of amino acids. Second, two new labeling reagents were synthesized and their detection sensitivities evaluated. These studies increase our understanding of the structural basis of these highly sensitive labeling reagents, and should provide opportunities for future on-demand structural modifications of the reagents to enhance their hydrophobicity, stability, and affinity for applications to specialized HPLC columns.

Background: Profiling the whole metabolome with a single injection is not an easy process because the chemical and physical properties of metabolites are totally different with each other and the analytical methodologies and data mining procedures need lots of effort to make such an approach in real. This reality leads researchers to select an already applied methodology for metabolite profiling and analyze the samples through identical techniques. Objective: In this study, it was focused on answer the question the sample preparation techniques on human blood samples prior to Q-TOF LC/MS analysis affect the number of detectable peaks and analyze the matched metabolites for these peaks. The results were compared with each other. Methods: Precipitation of proteins with methanol, ultrafiltration (Amicon® Ultra 3 kDa 0.5 mL Centrifugal Filters), liquid-phase extraction (EXtrelut® NT 3 cartridges) and solid-phase extraction (Supelco HybridSPE®-Phospholipid Cartridge) were used for sample preparation on commercial pooled plasma samples. C18 column (Agilent Zorbax 1.8 μM, 50 x 2.1 mm) was used as the chromatography column. Q-TOF LC/MS analysis was performed on positive ionization mode. XCMS and MetaboAnalyst 4.0 - MS Peaks to Pathways utility were used to evaluate the raw data. Results: Although the number of detected peaks through precipitation of proteins with methanol was the highest one (624 peaks), the detected peaks observed through ultrafiltration- based sample preparation technique matched with the highest number of metabolite peaks (151 metabolites). The number of the matched peaks with metabolites on liquid phase extraction (81 metabolites) was higher than the ones for solid phase extraction (29 metabolites). Conclusion: The results in this study may provide a novel perspective for analytical chemists working with clinicians to select their sample preparation technique prior to Q-TOF LC/MS based untargeted metabolomic approaches.

Aim: We aimed to develop an easy, low-cost and versatile mass spectrometric method for the bioanalysis of a therapeutic monoclonal antibody (mAb) in human serum that employs peptide adsorption-controlled (PAC)-LC/MS using selected reaction monitoring mode (LC–MS/MS-SRM). Materials & methods: Rituximab was used as a model mAb. To apply the method to human serum samples, a peptide of the complementarity-determining region was selected as the surrogate peptide. The usefulness of PAC-LC–MS/MS-SRM was evaluated by a collaborative study. Results: The calibration curve ranged from 0.5 (or 1.0) to 1000.0 μg/ml. The selectivity, linearity, accuracy and precision met the predefined acceptance criteria. Conclusion: Our method could be a useful bioanalytical method for the quantification of mAbs in clinical samples.

Background: Surrogate monoclonal antibodies (mAbs) used in preclinical in vivo studies can be challenging to quantify due to lack of suitable immunoaffinity reagents or unavailability of the mAb protein sequence. Generic immunoaffinity reagents were evaluated to develop sensitive LC–MS/MS assays. Peptides of unknown sequence can be used for selective LC–MS quantification. Results: anti-mouse IgG1 was found to be an effective immunoaffinity reagent, enabling quantification of mouse IgG1 mAbs in mouse serum. Selective peptides of unknown sequence were applied for multiplex LC–MS quantification of two rat mAbs co-dosed in mouse. Conclusion: Generic anti-mouse IgG subtype-specific antibodies can be used to improve assay sensitivity and peptides of unknown sequence can be used to quantify surrogate mAbs when the mAb protein sequence in unavailable.

An analytical protocol was developed to quantify six-category pharmaceuticals in surface water under paralleling pHs using a combination of solid phase extraction (SPE) and high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Of 121 compound standards commercially, 94 compounds were reliably quantified using a paralleling pH combination at 3 and 9 with a sound recovery-rate of 50-150%. The quantified compounds covered 6 categories as 37 antibiotics, 16 central nervous system drugs (CNs), 14 cardiovascular drugs (CVs), 12 endocrine and family planning drugs (EFs), 8 antiparasitic drugs (APs), and 7 non-steroidal anti-inflammatory drugs (NSAIDs). The protocol achieved limits of detection and quantitation in ranges of 0.001–11.9 ng L–1 and 0.003–39.7 ng L–1, respectively. Serial field surface water samples from Jiulong River in Fujian, China were testified using the protocol with 41 quantified compounds of the above-mentioned 6 categories from < LODs to 1,389 ng L−1 (caffeine). The key procedure prior to the SPE is to split filtrate of each surface water sample into two portions, adjusted to pH 3 and 9 separately and both spiked with 0.2 g Na2EDTA.

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful technique for visualizing lipids in biological tissues. Phosphatidylinositol (PI), a phospholipid in pork, is a major source of inositol in animal-derived foods believed to be protective against diseases related to pregnancy and cancer. However, the distribution of PI molecular species in pork is not well understood. Here, we performed MALDI-MSI analysis to investigate the distribution and composition of PI molecular species in pork chop comprising Longissimus thoracis et lumborum muscle (loin), intermuscular fat tissue, transparent tissue, and spinalis muscle. Twelve diacyl-PI molecular species were identified using liquid chromatography-electrospray ionization-tandem mass spectrometry (MS/MS) and MALDI-MS/MS analysis and visualized using MALDI-MSI. Spinalis muscle had the highest amount of identified PI molecular species, followed by loin, transparent tissue, and intermuscular fat tissue. The diacyl-PI molecular species containing hexadecadienoic, oleic, linoleic and eicosadienoic acids at the sn-2 position were mainly abundant in the loin and spinalis muscle, whereas those containing mead, arachidonic, docosatetraenoic, and docosapentaenoic acids at the sn-2 position were mainly abundant in both muscles as well as transparent tissues. Notably, the balance of PI molecular species differed among the tissues depending on fatty acid compositions at the sn-2 position. These results suggested that MALDI-MSI is a promising tool for assessing the association between individual pork tissues and the protective effects of PI molecular species against diseases related to pregnancy and cancer. To the best of our knowledge, this is the first report showing tissue-specific distributions of PI molecular species in pork chop using MALDI-MSI.