LC-MS Method Research Articles

Quantification of Urine and Plasma Porphyrin Precursors Using LC-MS in Acute Hepatic Porphyrias: Improvement in Routine Diagnosis and in the Monitoring of Kidney Failure Patients.

The quantification of delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) in urine are the first-line tests for diagnosis and monitoring of acute hepatic porphyrias (AHP). Ion-exchange chromatography (IEC), which is time- and staff-consuming and limited to urine, is still the preferred method in many specialized laboratories, despite the development of mass spectrometry-based methods. We describe a new LC-MS method that allows for rapid and simple quantification of ALA and PBG in urine and plasma with an affordable instrument that was used to analyze 2260 urine samples and 309 blood samples collected in 2 years of routine activity. The results were compared to those obtained with IEC, and urine reference ranges and concentrations in asymptomatic carriers were determined. Plasma concentrations were measured in healthy subjects and subgroups of symptomatic and asymptomatic AHP carriers. In urine, the clinical decision limits were not impacted by the change of method despite discrepancies in low absolute concentrations, leading to lower normal values. Two-thirds of asymptomatic AHP carriers (with the exception of coproporphyria carriers) showed an increased urine PBG concentration. Urine and plasma levels showed a good correlation except in patients with kidney disease in whom the urine/plasma ratio was relatively low. We described an LC-MS based method for the routine diagnosis and monitoring of AHP that allows for the detection of more asymptomatic carriers than the historical method. Blood analysis appears to be particularly relevant for patients with kidney disease, where urine measurement underestimates the increase in ALA and PBG levels.

LC-MS methods combination for identification and quantification of trans-sinapoylquinic acid regioisomers in green coffee.

The present study aims to both identify and quantify trans-sinapoylquinic acid (SiQA) regioisomers in green coffee by combined UHPLC-ESI-QqTOF-MS/MS and UHPLC-ESI-QqQ-MS/MS methods. Among the various mono-acyl chlorogenic acids found in green coffee, SiQA regioisomers are the least studied despite having been indicated as unique phytochemical markers of Coffea canephora (known as Robusta). The lack of commercially available authentic standards has been bypassed by resorting to the advantages offered by high-resolution LC-MS as far as the identification is concerned. SiQA regioisomers have been identified in several samples of Robusta and Coffea arabica (known as Arabica) commercial lots from different geographical origin and, for the first time, in different samples of coffee wild species (Coffea liberica and Coffea pseudozanguebariae). Quantification (total SiQA ranging from 3 to 5mg/100 g) let to reconsider these chlorogenic acids as unique phytochemical markers of Robusta being present in the same quantity and distribution in C.liberica as well. Gardeniae Fructus samples (fruits of Gardenia jasminoides) have additionally been characterized as this matrix is recognized as one of the few naturally occurring SiQA sources. The SiQA regioisomer content (total SiQA about 80 mg/100 mg) fully supports the proposal to use this matrix as a surrogate standard for further studies.

A innovative application of lactose cross-linked adhesive in plywood using hyperbranched network structure with high performance

The use of biomass as raw material to prepare green biomass-based resins for wood adhesives is an effective way to alleviate the oil resource crisis and environmental problems. However, the reported biomass-based adhesives suffer from poor mechanical properties and water resistance, limiting their application to some extent. It is therefore of great interest to investigate new synthetic methods for the preparation of biomass-based adhesives with good mechanical properties and water resistance. In this paper, in order to improve the mechanical properties of biomass-based adhesives, this can be achieved through chemical modification of biomass and hyperbranched network cross-linking strategies. A new biomass-based hyperbranched adhesive, L-PEI, was prepared by co-crosslinking lactose and PEI through air oxidation strategy. The molecular structure of L-PEI was determined using FTIR, 13C NMR, X-ray photoelectron spectroscopy, LC-MS, GPC and other characterisation methods. During the modification process, the addition of PEI leads to the formation of a super branched network structure between lactose and PEI, increasing the crosslinking density and serving as a water barrier. The results of the study show that L-PEI has good adhesive properties and water resistance. Wet shear strength is 1.13 MPa after 3 h in hot water at 63 °C and after soaking in boiling water for 3 h, it is 1.1 MPa. In summary, L-PEI is a green, easy to synthesize and high performance wood adhesive.

Green magnetic snail shell hydroxyapatite sorbent for reliable solid-phase extraction of pesticides from water samples.

To address sustainability issues, the green synthesis of nanomaterials has recently received considerable attention. This article addresses a novel and cost-effective adsorbent for the extraction of eight phenyl-N-methylcarbamate insecticides from water samples. We first synthesized a magnetite/hydroxyapatite nanocomposite using snail shell powder via an environmental friendly approach. The morphology and physicochemical properties of magnetic hydroxyapatite were characterized by Fourier transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, and scanning electron microscopy. Magnetic extraction parameters were optimized using a Doehlert matrix. Under optimum conditions, the magnetic extraction coupled with a LC-MS method shows good linearity with R2 ≥ 0.9982, suitable intra- and interday precision, and limits of detection and quantification in the range of 0.052-0.093μg/L and 0.11-0.31μg/L, respectively. Satisfactory relative recoveries of all carbamates were achieved from fortified water samples in the range of 93.89-101.01%.

Preparation of 18O-labelled azaspiracids for accurate quantitation using liquid chromatography–mass spectrometry

Azaspiracids (AZAs) are a group of polyether marine algal toxins known to accumulate in shellfish, posing a risk to human health and the seafood industry. Analysis of AZAs is typically performed using LC–MS, which can suffer from matrix effects that significantly impact the accuracy of measurement results. While the use of isotopic internal standards is an effective approach to correct for these effects, isotopically labelled standards for AZAs are not currently available. In this study, 18O-labelled AZA1, AZA2, and AZA3 were prepared by reaction with H218O under acidic conditions, and the reaction kinetics and sites of incorporation were studied using LC–HRMS/MS aided by mathematical analysis of their isotope patterns. Analysis of the isotopic incorporation in AZA1 and AZA3 indicated the presence of four exchangeable oxygen atoms. Excessive isomerization occurred during preparation of 18O-labelled AZA2, suggesting a role for the 8-methyl group in the thermodynamic stability of AZAs. Neutralized mixtures of 18O-labelled AZA1 and AZA3 were found to maintain their isotopic and isomeric integrities when stored at −20 °C and were used to develop an isotope-dilution LC–MS method which was applied to reference materials of shellfish matrices containing AZAs, demonstrating high accuracy and excellent reproducibility. Preparation of isotopically labelled compounds using the isotopic exchange method, combined with the kinetic analysis, offers a feasible way to obtain isotopically labelled internal standards for a wide variety of biomolecules to support reliable quantitation.Graphical

Safe food through better labelling; a robust method for the rapid determination of caprine and bovine milk allergens.

Accidental milk cross-contamination is one of the most common causes for costly food recalls. Yet, quantifying trace-levels of allergen is time-consuming and current methods are not adapted for routine analyses making quality control for trace-level allergen content impractical. This perpetuates voluntary "may-contain" statements that are unhelpful for people suffering from food allergies. Here, we developed a rapid LC-MS method enabling milk allergen quantification by comparing all tryptic-peptides of major milk allergens. The bovine-specific αS-2 casein peptide and allergen-epitope NAVPITPTLNR provided excellent performance in sensitivity (LOD 1mg.kg-1; LOQ 2mg.kg-1) across various dairy products, good recovery rates in baked croissants (77% with a 10% inter-day RSD) and a linear range of 2-2,000mg.kg-1. The method can be used for routine determination of trace-contamination with bovine milk allergen and the adulteration of high-value caprine dairy products with lower-value bovine milk products, protecting consumer trust and the growing population suffering from food allergies.

A Sensitive Liquid Chromatography-Mass Spectrometric Method for Determination of Bisoprolol in Rat Serum after Pre-Column Derivatization.

Two derivatization regents were reacted with bisoprolol (BIS), followed by liquid-chromatography-mass spectrometric analysis. 3-Bromomethyl-propyphenazone (BMP) and dansyl-chloride (Dns-Cl) were reacted via the secondary amino group using a catalyst to accelerate the reaction progress and completeness with minimal reaction byproducts. The sensitivity and the ionization efficiency of both BIS-methyl-propyphenazone (BIS-MP) and BIS-Dns via electrospray ionization were studied. The sensitivity of BIS-MP was superior to BIS-Dns. The derivatization procedure, extraction procedure, and LC-MS method were optimized and validated to achieve the monitoring of BIS in rat serum at a picogram scale. The calibration curve showed a regression coefficient value of 0.999 within a concentration spanning a range of 10-500pg/mL. The detection limit and quantitation were 4 and 10pg/mL, respectively. The intraday and inter-day precision values (% relative standard deviation) ranged from 0.53 to 6.91%, whereas the accuracy values (expressed as % error) ranged from -4.20 to -0.77%. The pharmacokinetic parameters were: 15,280pg/mL for a maximum concentration of BIS (Cmax) at the maximum time (Tmax) of 1 h. BIS's elimination half-life (t1/2) was determined to be 3 h. The value of the area under the concentration-time curve (AUC0 - t) was 34,370pg/mLh.

Hidden Hunger and Hidden Danger: Regional Gradients in Rice Grain Nutrient Elements, Vitamins B and E and Toxicants Arsenic and Cadmium Along a North–South Transect of Western Bangladesh

Here is presented a study to investigate the geographic variation in rice grain nutrients (trace and macro-elements and vitamins) and toxicants (arsenic species and cadmium) across a contiguous strip of 36 districts that constitute western Bangladesh. The survey collected ~ 500 market rice samples, averaging 15 samples and 10.7 cultivars per district. New LC-MS methods were developed for rice relevant, B and E complex compounds. Cadmium and zinc decreased southward, while copper, DMA, inorganic arsenic and oryzanols decreased northwards. There was a longitudinal gradient for iron, potassium, and vitamin B6. The greatest changes ~ twofold for cadmium and vitamin B6, and 1.5 for zinc across these gradients. The gradients may be driven by climate, geographical setting, soils, or cultivar, or a combination of all. The most obvious gradient was the transition from high to low altitude and from Pleistocene to Holocene soils as land transitioned from the upland plains of the north to sea-level in the south. Rice is a very important source of copper, phosphorus, vitamin B1, and zinc, and to a lesser extent iron, B3, B6, potassium. It is a poor source of vitamin E and calcium.

A novel method for quantification of cefazolin local tissue concentration in blood, fat, synovium, and bone marrow using liquid chromatography − mass spectrometry

To be effective, the concentration of antibiotic used must exceed the minimum inhibitory concentration (MIC) against infecting organisms at and in the surgical site. Few studies follow antibiotic levels for tissues that are manipulated during surgery. The aim of this work was to develop and validate a novel LC-MS method as well as an efficient extraction technique for the quantification of cefazolin in local tissues and whole blood. This method uses the same efficient extraction method across multiple tissue types affected by orthopedic surgery: blood, fat, synovium, and bone marrow. The ability to quantify cefazolin in these tissues will help identify surgical techniques and antibiotic dosing protocols that better protect patients from infection. The internal standard, 13C2,15N-cefazolin, co-elutes with cefazolin, and was used in calibration curves and tissue extracts as well as for cefazolin recovery and matrix effects. The protocol was rigorously tested, including measurements of reproducibility and calibration curve quality. The recovery of the extraction method ranges from 94% to 113% across all sample types. There is little to no matrix effect on cefazolin signal (98–120%). The developed method was used to determine cefazolin concentrations in tissues of 10 patients undergoing a total knee replacement. Cefazolin blood concentrations were approximately 500 times higher than in adipose, synovium, and bone marrow tissues. This clinical data shows that although the minimum inhibitory concentration is largely surpassed in blood, the concentration of cefazolin in fat, synovium, and bone marrow could be insufficient during a knee replacement. This method of cefazolin quantification will help surgeons optimize antibiotic concentrations in the local tissues during knee replacement surgery and potentially reduce serious post-surgical infections.

Establishment and clinical application evaluations of a deep mining strategy of plasma proteomics based on nanomaterial protein coronas

Research on plasma proteomics has received extensive attention, because human plasma is an important sample for disease biomarker research due to its easy clinical accessibility and richness in biological information. Plasma samples contain a large number of leaked proteins from different tissues in the body, immune proteins and communication signal proteins. However, MS signal suppression from high-abundance proteins results in a large number of proteins that are present in low abundance in plasma not being detected by the LC-MS method. This situation makes it more difficult to study neurological diseases, where tissue sampling is difficult and body fluid samples such as plasma or cerebrospinal fluid are both affected by signal suppression. A large number of methods have been developed to deeply mine plasma proteomics information; however, their application limitations remain to some extent. Traditional immuno- or affinity-based depletion, fractionation and subproteome enrichment methods cannot meet the challenges of large clinical cohort applications due to limited time efficiency.In this study, a deep mining strategy of plasma proteomics was established by combing the protein corona formed by deep mining beads (DMB beads, hereafter referred to as magnetic covalent organic frameworks Fe3O4@TpPa-1), DIA-MS detection and the DIA-NN library searching method. By optimizing the enrichment step, mass spectrometry acquisition and data processing, the evaluation results of the deep mining strategy showed the following: depth, the strategy identified and quantified results of 2000+ proteins per plasma sample; stability, more than 87% of the enriched low-abundance proteins had CV < 20%; accuracy, good agreement between measured and theoretical values (1.81/2, 8.68/10, 38.36/50) for the gradient addition of E. coli proteins to a plasma sample; time efficiency, the processing time was reduced from >12h in the traditional method to <5h (incubation 30 min, washing 15 min, reductive/alkylation/digestion/desalting 4 h), and more importantly, 96 samples can be processed simultaneously in combination with the magnetic module of the automated device. The optimal strategy enables greater enrichment of neurological disease-related proteins, including SNCA and BDNF. Finally, the deep mining strategy was applied in a pilot study of multiple system atrophy (MSA) for biomarker discovery. The results showed that a total of 215 proteins were upregulated and 184 proteins were downregulated (p < 0.05) in the MSA group compared with the healthy control group. Eighteen proteins of these differentially expressed proteins were reported to be associated with neurological diseases or expressed specifically in brain tissue, 8 and 4 of which have reference concentrations of μg/L and ng/L, respectively. The alterations of ENPP2 and SLC2A1/Glut1 were reanalyzed by ELISA, further supporting the results of mass spectrometry. In conclusion, the results of the evaluation and application of the deep mining strategy showed promise for clinical research applications.

Determination of Constituents of Extract of Celtis tournefortii Lam. by LC-MS/MS, Investigation of Enzyme Inhibition, Antimicrobial and Anticancer Effects

Phytochemicals found in extracts obtained from plants are very important bioactive constituents. In this study, phytochemicals in extract content obtained from Celtis tournefortii .Lam. tree (CT) leaves were determined by a LC-MS method. The constituents with the major concentrations was found rutin (2479.89 µg ml-1), coumarin (1241.68 µg ml-1), biochanin A (1026.42 µg ml-1), shikimic acid (477.32 µg ml-1), chlorogenic acid (300.76 µg ml-1). The suppressive effects of CT extract on the growth of pathogenic strains were studied by microdilution method. It was observed that it caused suppression on the strains in the concentration range of 2.00-8.00 µg ml-1. The inhibition effects of the extract on acetyl cholinesterase and glutathione-S transferase enzyme activities were investigated, and 50% inhibitory values of enzyme activity were found to be 13.58 and 13.86, respectively. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay method, the cell viability suppressive effects of CT leaf extract were studied in healthy skin fibroblast cells as well as ovarian, colon and brain cancer cells. It was observed that they created a 42%, 4.27%, and 14.29% suppression in cells, respectively.

The colonic metabolism differences of main alkaloids in normal and colitis mice treated with Coptis chinensis Franch. and Sophora flavescens Ait. herbal pair using liquid chromatography-high resolution mass spectrometry method combined with chemometrics.

Coptis chinensis Franch. and Sophora flavescens Ait. is a herbal pair frequently used in treating ulcerative colitis. However, the bio-disposition profile of the major components in the inflamed gut remains unclear, which is essential to understand the pharmacological material basis of this herb pair. Here we established an integral quantitative and chemometric method to deduce the colonic metabolism differences of this herbal pair in normal and colitis mice. With this LC-MS method, a total of 41 components have been found in the Coptis chinensis Franch. and Sophora flavescens Ait. extract, and 28 metabolites were found in the colon after oral administration. Alkaloid and its phase I metabolites were the main components in the colon of normal and colitis mice. The results of principal component analysis at 6 h after oral administration showed significant colonic metabolism differences between normal and colitis mice. Heamap results showed that colitis induced significant changes in the colonic bio-disposition of this herbal pair extract. In particular, in the context of colitis, the phase I metabolism of berberine, coptisine, jatrorrhizine, palmatine,and epiberberine has been inhibited. These results may provide a basis for understanding the pharmacological material basis of Coptis chinensis Franch. and Sophora flavescens Ait. in treating ulcerative colitis.

Migration of Dihydroxy Alkylamines and Their Possible Impurities from Packaging into Foods and Food Simulants: Analysis and Safety Evaluation.

Alkyl diethanolamines are a group of compounds commonly used as antistatic agents in plastic food packaging materials. These additives and their possible impurities have the ability to transfer into the food; hence, the consumer may be exposed to these chemicals. Recently, scientific evidence of unknown adverse effects associated with these compounds was reported. N,N-bis(2-hydroxyethyl)alkyl (C8-C18) amines as well as other related compounds and their possible impurities were analyzed in different plastic packaging materials and coffee capsules using target and non-target LC-MS methods. N,N-bis(2-hydroxyethyl)alkyl amines, precisely, C12, C13, C14, C15, C16, C17 and C18, 2-(octadecylamino)ethanol and octadecylamine, among others, were identified in most of the analyzed samples. It should be emphasized that the latter compounds are not listed in the European Regulation 10/2011 and 2-(octadecylamino)ethanol was classified as high toxicity according to Cramer rules. Migration tests were carried out in foods and in the food simulants Tenax and 20% ethanol (v/v). The results showed that stearyldiethanolamine migrated into the tomato, salty biscuits, salad and Tenax. Lastly, as a crucial step in the risk assessment process, the dietary exposure to stearyldiethanolamine transferred from the food packaging into the food was determined. The estimated values ranged from 0.0005 to 0.0026 µg/kg bw/day.

N/S-Co-doped carbon dot-based FRET ratiometric fluorescence aptasensing platform modulated with entropy-driven DNA amplifier for ochratoxin A detection.

This study proposes a nitrogen and sulfur co-doped carbon dot (N/S-CD)-based FRET ratiometric fluorescence aptasensing strategy modulated with entropy-driven DNA amplifier for sensitive and accurate detection of ochratoxin A (OTA). In the strategy, a duplex DNA probe containing OTA aptamer and complementary DNA (cDNA) is designed as arecognition and transformation element. Upon sensing of target OTA, the cDNA was liberated, and triggered a three-chain DNA composite-based entropy-driven DNA circuit amplification, making CuO probes anchor on amagnetic bead (MB). The CuO-encoded MB complex probe is finally turned into abundant Cu2+, which oxidizes o-phenylenediamine (oPD) to generate 2,3-diaminophenazine (DAP) with yellow fluorescence and further triggers FRET between the blue fluorescent N/S-CDs and DAP. The changes in ratiometric fluorescence are related to the OTA concentration. Originating from the synergistic amplifications from the entropy-driven DNA circuits and Cu2+ amplification, the strategy dramatically enhanced detection performance. A limit of detection as low as 0.006 pg/mL of OTA was achieved. Significantly, the aptasensor can visually evaluate the OTA via on-site visual screening. Moreover, the high-confidence quantification of the OTA in real samples with results consistent with that of the LC-MS method indicated that the proposed strategy has practical application prospects for sensitive and accurate quantification in food safety.

Application of novel plasma separation filter cards for quantification of nucleoside/nucleotide reverse transcriptase inhibitor di/triphosphates in dried blood spots using LC-MS.

Background: A rapid and sensitive LC-MS method has been developed and validated for the quantification of nucleoside di/triphosphates using a novel plasma separation card (HemaSep). Materials & methods: Cards were spotted with whole blood and stored at -80°C. Metabolites were extracted using 70:30 MeOH:20% formic acid, followed by weak anion exchange SPE and eluted using a Biobasic-AX column. Quantification was performed using a triple quadrupole mass spectrometer with a calibration range of 1.25-250pmol/sample. Results: The recovery of metabolites was high (>93%). Precision and accuracy were acceptable and metabolites remained stable on the card after 29days (stored at ambient temperature). Conclusion: HemaSep dried blood spots are a useful microsampling tool and offer an alternative to liquid plasma as they maintain stability over time.

Comprehensive Understanding of the Kinetic Behaviors of Main Protease from SARS-CoV-2 and SARS-CoV: New Data and Comparison to Published Parameters.

The main protease (Mpro) is a promising drug target for inhibiting the coronavirus due to its conserved properties and lack of homologous genes in humans. However, previous studies on Mpro's kinetic parameters have been confusing, hindering the selection of accurate inhibitors. Therefore, obtaining a clear view of Mpro's kinetic parameters is necessary. In our study, we investigated the kinetic behaviors of Mpro from SARS-CoV-2 and SARS-CoV using both FRET-based cleavage assay and the LC-MS method, respectively. Our findings indicate that the FRET-based cleavage assay could be used for preliminary screening of Mpro inhibitors, while the LC-MS method should be applied to select the effective inhibitors with higher reliability. Furthermore, we constructed the active site mutants (H41A and C145A) and measured the kinetic parameters to gain a deeper understanding of the atomic-level enzyme efficiency reduction compared to the wild type. Overall, our study provides valuable insights for inhibitor screening and design by offering a comprehensive understanding of Mpro's kinetic behaviors.

LC-UV and LC-MS Characterization of Stress Degradation Behavior of Levocetirizine Using a Green Mobile Phase

Introduction: The current investigation's objective was to develop an HPLC method along with a validated stability-indicating method and analyzing the behavior of levocetirizine degradation under various ICH-suggested stress conditions using LC-UV and LC-MS with a triple quadrupole mass analyzer. Methods: Levocetirizine was exposed to various stressors while performing degradation studies. Using LC-UV and LC-MS methods, the degradation products formed in different stress environments were studied. The drug and degradation products were successfully separated using a Thermo Hypersil BDSC18 column having a dimension of 250 mm length, 4.6 mm internal diameter, and a particle size of 5.0 μm. The isocratic green mobile phase contained methanol:water in the proportion of 65:35 at a flow rate of 1 mL/min, and the selected wavelength for the UV detector was 230 nm. Results: Significant degradation was found in acidic, alkaline, and oxidative conditions, whereas in photo and neutral conditions, no degradation products were formed. The drug was fairly stable in the solid state. Conclusion: The values of m/z obtained from LC-MS after exposing the drug to different stress conditions were used to study fragmentation patterns, characterize the structure of the degradation product, and design the degradation pathway.

LC-MS Method Development and Optimization for Small Drug Analysis in Urine

Both amphetamine and methamphetamine are considered to be illegal chemicals, and hence, the purchase, possession, and use of these drugs is forbidden in many nations. Within the fields of forensic and clinical toxicology, there has been a recent uptick in the detection and quantification of illicit substances within urine samples. Objective: To detect and quantify both drugs in urine samples utilizing caffeine as an internal standard with an optimized liquid-liquid extraction procedure. Methods: An alternative rapid and efficient method of liquid chromatography – electron spray ionization – Tandem mass spectrometry (LC – ESI – TMS) was developed and optimized. The chromatographic separation was carried out using an isocratic high-performance liquid chromatography (HPLC) system, and the eluent that was applied was a mixture of 20% acetonitrile and 80% buffer with a pH of 2.6 that included 10mM ammonium acetate and 0.1% trifluoroacetic acid. The run duration was 9 minutes, and the detection was accomplished at 210 nm with a flow rate of 1 mL/min utilizing triple quadruple MSMS to validate ionic transitions following direct infusion and fragmentation of analytes. Results: An excellent linearity was seen in the calibration curves of amphetamine and methamphetamine in urine samples across the concentration range of 0-10 mg/L, with a regression coefficient of 0.91 and 0.97, respectively, for each of these substances. Conclusions: More compounds are able to be identified in urine as chromatographic techniques, such as high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS), continue to improve in terms of their sensitivity

Profiling of branched chain and straight chain saturated fatty acids by ultra-high performance liquid chromatography-mass spectrometry

Branched chain fatty acids (BCFAs) are one of the important sub categories of fatty acids (FAs) which have unique functions in nature. They are commonly analyzed by GC–MS after derivatization to methyl esters (FAMEs). On the other hand, there is a lack of isomer-selective LC-MS methods which allow the distinction of different isomers with wide coverage of carbon chain length. In this work, a systematic retention and isomer selectivity study on seven commercially available UHPLC columns (six polysaccharide columns Chiralpak IA-U, IB-U, IC-U, ID-U, IG-U and IH-U; one Acquity UPLC CSH C18 column) was performed. Various experimental factors were evaluated including column temperatures, gradient profiles and flow rates to elucidate their effects on the separation ability of homologous series of BCFAs with distinct chain lengths, different branching types and branching positions. In general, IG-U outperformed the other columns in terms of isomer selectivity especially for the short and medium-chain BCFA isomers while RP C18 showed good potential in terms of selectivity for long-chain BCFA isomers. Furthermore, after the evaluation of the chromatographic retention pattern on the various columns and method optimization, we report a methodology for untargeted isomer-selective BCFA profiling without precolumn derivatization with UHPLC-ESI-MS/MS by quadrupole-time-of-flight instrument with SWATH acquisition. The best method provides selectivity for constitutional isomers of BCFAs covering distinct chain length (C5-C20) with different branching types (methyl or ethyl) and branching positions (2Me, 3Me, 4Me, 6Me, anteiso and iso-BCFAs) with an optimized LC condition on Acquity UPLC CSH C18 column. Finally, the optimized method was applied for the BCFAs profiling in lipid extracts of Staphylococcus aureus samples. Besides, pooled human platelets and pooled human plasma were evaluated as mammalian samples for presence of BCFAs as well. The new method showed strong potential for BCFA profiling in bacterial samples including different isomers anteiso and iso-BCFAs, which could be a useful tool for related subdisciplines in metabolomics and lipidomics in particular in combination with electron-activated dissociation MS. Compared to GC, the presented isomer selective LC methods can be also of great utility for preparative purposes. Equivalent (carbon) chain length numbers were calculated for RP18 and Chiralpak IG-U and compared to those of FAMEs obtained by GC.

Metabolism characterization and chemical and plasma stability of casearin B and caseargrewiin F.

Oral preparations of Casearia sylvestris (guacatonga) are used as antacid, analgesic, anti-inflammatory and antiulcerogenic activities. The clerodane diterpenes casearin B (cas B) and caseargrewiin F (casg F) are major active compounds in vitro and in vivo. The oral bioavailability and metabolism of cas B and casg F were not previously investigated. We aimed to assess the stability of cas B and casg F in physiological conditions and their metabolism in human liver microsomes (HLM). The compounds were identified by UHPLC-QTOF-MS/MS and quantified by validated LC-MS methods. The stability of cas B and casg F in physiological conditions was assessed in vitro. Both diterpenes showed a fast degradation (p < 0.05) in simulated gastric fluid. Their metabolism is not mediated by cytochrome P-450 enzymes, but the depletion was inhibited by the esterase inhibitor NaF. Both diterpenes and their dialdehydes showed log P in the range of 3.6 to 4.0, suggesting high permeability. Metabolism kinetic data were fitted to the Michaelis-Menten profile with KM values of 61.4 μM and 66.4 μM and Vmax values of 327 and 648 nmol/min/mg of protein for cas B and casg F, respectively. Metabolism parameters in HLM were extrapolated to predict human hepatic clearance and suggest that casg F and cas B have a high hepatic extraction ratio. In conclusion, our data suggest that casg F and cas B present low oral bioavailability due to extensive gastric degradation and high hepatic extraction.