LC-tandem MS Research Articles

Rapid quantitative estimation of metformin and ertugliflozin in rat plasma by liquid chromatography-tandam mass spectroscopy and its application to pharmacokinetic studies

Background The development of sound bioanalytical liquid chromatography-mass spectroscopy (LC-MS) method(s) is of paramount importance during the process of drug discovery and development, eventually culminating in marketing approval. The use of oral antidiabetic agents has been increased significantly from past decades, and till now, no bioanalytical method is available for quantitation of metformin (MET) and ertugliflozin (ERT) in the biological matrix that can be applied in bioequivalence studies using LC-MS/MS. Objective To study the use of highly responsive simple liquid–liquid extraction method development using deuterated MET and deuterated ERT, LC-MS/MS method for gradation of MET and ERT in the rat plasma. Materials and methods The chromatographic condition involves isocratic mode using Waters XBridge C18 3.5 μ (150×4.6 mm) column. Mobile phase was 0.1% orthophosphoric acid and acetonitrile in the ratio of 80 : 20 v/v. Detection was carried out on a triple quadrapole MS employing electrospray ionization technique, operating multiple reactions, monitoring with the transitions of m/z 258.2→174.1, m/z 250.1→210.2, m/z 258.2→174.1, and m/z 260.3→210.2 for MET, ERT, deuterated MET, and deuterated ERT, respectively, in the positive ion mode. Results and conclusion The method has been validated, and the linearity was observed in the range of 10–150 ng/ml and 0.1–1.5 ng/ml for MET and ERT, respectively. For intraday and interday %RSD, the values were found to be within the acceptable limits. Recovery studies for MET and ERT obtained, mean recovery of 99.5 and 98.6%, respectively. A battery of stability studies like bench-top stability, autosampler stability, freeze-thaw stability, and long-term stability were performed. Highly responsive simple LC-tandem MS assay method was developed and witnessed for the gradation of MET and ERT in the rat plasma; the developed method was applied to pharmacokinetic studies.

Identification of Microorganisms by Liquid Chromatography-Mass Spectrometry (LC-MS1) and in Silico Peptide Mass Libraries

Over the past decade, modern methods of MS (MS) have emerged that allow reliable, fast and cost-effective identification of pathogenic microorganisms. Although MALDI-TOF MS has already revolutionized the way microorganisms are identified, recent years have witnessed also substantial progress in the development of liquid chromatography (LC)-MS based proteomics for microbiological applications. For example, LC-tandem MS (LC-MS2) has been proposed for microbial characterization by means of multiple discriminative peptides that enable identification at the species, or sometimes at the strain level. However, such investigations can be laborious and time-consuming, especially if the experimental LC-MS2 data are tested against sequence databases covering a broad panel of different microbiological taxa. In this proof of concept study, we present an alternative bottom-up proteomics method for microbial identification. The proposed approach involves efficient extraction of proteins from cultivated microbial cells, digestion by trypsin and LC-MS measurements. Peptide masses are then extracted from MS1 data and systematically tested against an in silico library of all possible peptide mass data compiled in-house. The library has been computed from the UniProt Knowledgebase covering Swiss-Prot and TrEMBL databases and comprises more than 12,000 strain-specific in silico profiles, each containing tens of thousands of peptide mass entries. Identification analysis involves computation of score values derived from correlation coefficients between experimental and strain-specific in silico peptide mass profiles and compilation of score ranking lists. The taxonomic positions of the microbial samples are then determined by using the best-matching database entries. The suggested method is computationally efficient - less than 2 mins per sample - and has been successfully tested by a test set of 39 LC-MS1 peak lists obtained from 19 different microbial pathogens. The proposed method is rapid, simple and automatable and we foresee wide application potential for future microbiological applications.

Antifungal evaluation of fengycin isoforms isolated from Bacillus amyloliquefaciens PPL against Fusarium oxysporum f. sp. lycopersici

Bacillus amyloliquefaciens PPL is known to have a broad spectrum antifungal activity against plant pathogenic fungi. We focused on the cyclic lipopeptides (CLPs) extracted from the culture broth that are known to promote the ability and the efficiency of B. amyloliquefaciens PPL to control fungal diseases in pepper and tomato. In this study, the PPL strain exhibited enhanced culture yield and increased production of fengycin lipopeptides upon lecithin supplementation. The purified iturin A fraction from strain PPL exhibited higher antifungal activity (73 – 80%) against pepper anthracnose than fengycin fraction in vitro and in vivo. However, the control of tomato Fusarium wilt by the PPL strain was mainly attributed to fengycin lipopeptides. A comparison of liquid chromatography-mass spectrometry (LC-MS) and LC-tandem MS analysis of the filtrate, we found that the antifungal compounds against Fusarium wilt present in the strain PPL culture filtrate were a series of isoforms of fengycin (type F1, F2, and F3). The purified fengycin F1 type showed better antifungal activity against Fusarium wilt compared the other isoforms. To the best of our knowledge, this is the first study to report the antifungal activity of fengycin isoform types in the context of Fusarium wilt. The CLPs produced by the PPL strain are potential candidates for controlling fungal disease in tomato and pepper plants.

A Novel Urinary Biomarker Approach Reveals Widespread Exposure to Multiple Low-CalorieSweeteners in Adults

Observational investigations into the health impacts of low-calorie sweeteners (LCSs) in humans fail to adequately identify or fully characterize LCS consumption. We aimed to utilize a novel biomarker approach to investigate exposure to 5 LCSs and to test whether reported low-calorie sweetened beverage (LCSB) consumption effectively identifies exposure to LCSs in adults. In this cross-sectional analysis, 2 population studies were conducted in adults. Urinary excretions of 5 LCSs, namely acesulfame-K, saccharin, cyclamate, sucralose, and steviol glycosides, were simultaneously determined using LC tandem-MS. In Study 1, previously collected 24-h urine samples (n=357) were analyzed. In Study 2, previously collected 24-h urine samples (n=79) were analyzed to compare urinary excretions of LCSs with self-reported LCSB consumption for identifying LCS exposure. Exposure to LCSs was characterized using descriptive statistics and chi-square tests were performed to assess associations between age-groups and LCS excretion, and to assess the proportion of individuals identified as LCS consumers using biomarker data or reported LCSB consumption. A total of 341 adults (45% men) and 79 adults (39% men) were included in the final analysis of Studies 1 and 2, respectively. In Study 1, >96% of samples contained ≥1 LCS and almost 60% contained ≥3 LCSs. A greater proportion of younger adults (<40 y old) excreted ≥3 LCSs than older adults (>40 y old) (P <0.001). In Study 2, a much higher prevalence of LCS consumption was observed using biomarker data (92%) than reported LCSB consumption (6%) (P <0.001). This work indicates widespread exposure to LCSs, suggesting that population-based research to date into LCS exposure and health may be flawed. Therefore, a urinary biomarker approach offers considerable potential for more robust investigations in this area.

Comprehensive structural glycomic characterization of the glycocalyxes of cells and tissues.

The glycocalyx comprises glycosylated proteins and lipids and fcorms the outermost layer of cells. It is involved in fundamental inter- and intracellular processes, including non-self-cell and self-cell recognition, cell signaling, cellular structure maintenance, and immune protection. Characterization of the glycocalyx is thus essential to understanding cell physiology and elucidating its role in promoting health and disease. This protocol describes how to comprehensively characterize the glycocalyx N-glycans and O-glycans of glycoproteins, as well as intact glycolipids in parallel, using the same enriched membrane fraction. Profiling of the glycans and the glycolipids is performed using nanoflow liquid chromatography-mass spectrometry (nanoLC-MS). Sample preparation, quantitative LC-tandem MS (LC-MS/MS) analysis, and data processing methods are provided. In addition, we discuss glycoproteomic analysis that yields the site-specific glycosylation of membrane proteins. To reduce the amount of sample needed, N-glycan, O-glycan, and glycolipid analyses are performed on the same enriched fraction, whereas glycoproteomic analysis is performed on a separate enriched fraction. The sample preparation process takes 2-3 d, whereas the time spent on instrumental and data analyses could vary from 1 to 5 d for different sample sizes. This workflow is applicable to both cell and tissue samples. Systematic changes in the glycocalyx associated with specific glycoforms and glycoconjugates can be monitored with quantitation using this protocol. The ability to quantitate individual glycoforms and glycoconjugates will find utility in a broad range of fundamental and applied clinical studies, including glycan-based biomarker discovery and therapeutics.

Contribution of Vitamin D2 and D3 and Their Respective 25-Hydroxy Metabolites to the Total Vitamin D Content of Beef and Lamb.

ABSTRACTBackgroundRed meat and meat products can contribute meaningfully to the mean daily intake of vitamin D. Beef and lamb can contain vitamin D3 and 25-hydroxyvitamin D3 [25(OH)D3] but also potentially vitamin D2 and 25-hydroxyvitamin D2 [25(OH)D2], all of which contribute to meat's vitamin D activity.ObjectivesWe aimed to measure the vitamin D3, vitamin D2, 25(OH)D3, and 25(OH)D2 content of Irish beef and lamb.MethodsFull striploin steaks (longissimus dorsi) (n = 39) from beef cattle slaughtered in winter, spring, summer, and autumn as well as lamb steaks (hind leg) from sheep slaughtered in autumn (n = 8) were sourced and homogenized. The contents of all 4 vitamin D–related compounds were analyzed using an LC-tandem MS method in conjunction with the National Institute of Standards and Technology's standard reference material no. 1546a-Meat Homogenate. The total vitamin D activity of meat was defined as: {vitamin D3 + [25(OH)D3 × 5] + vitamin D2 + [25(OH)D2 × 5]}.ResultsThe median (IQR) total vitamin D activity of striploin beef steak (n = 39, irrespective of season) was 0.56 (0.37–0.91) μg/100 g. The content of all 4 vitamin D compounds in beef steak varied significantly (P < 0.0001) with season (n = 8–11/season group). Median total vitamin D activity of beef steak increased in a stepwise manner (P < 0.0001) from winter to the following autumn (increasing from 0.31 to 1.07 μg/100 g). The mean total vitamin D activity of lamb samples (n = 8) from autumn was 0.47 μg/100 g.ConclusionsAbout one-third of the total vitamin D activity of Irish beef was attributable to its combined vitamin D2 and 25(OH)D2 content, estimates of which are largely or completely missed in food composition tables. There was significant seasonal variation in all 4 vitamin D compounds as well as in total vitamin D activity, which has implications for vitamin D nutrient claims for beef.

Effect of a lifestyle intervention program with energy-restricted Mediterranean diet and exercise on the serum polyamine metabolome in individuals at high cardiovascular disease risk: a randomized clinical trial

Many food items included in the Mediterranean diet (MedDiet) are rich in polyamines, small aliphatic amines with potential cardioprotective effects. The consumption of a MedDiet could increase polyamine concentrations. Based on experimental models, polyamine concentrations may be also influenced by physical activity (PA). We aimed to evaluate whether an intervention based on an energy-restricted MedDiet (er-MedDiet) and PA promotion, in comparison with an energy-unrestricted MedDiet and traditional health care, influences the serum pattern of polyamines and related metabolites in subjects at high risk of cardiovascular disease (CVD). This was a substudy from the PREDIMED-Plus trial, an ongoing randomized clinical trial including 6874 participants allocated either to an intensive weight-loss lifestyle intervention based on er-MedDiet, PA promotion, and behavioral support (er-MedDiet+PA group), or to an energy-unrestricted MedDiet and traditional health care group (MedDiet group). A total of 75 patients (n=38, er-MedDiet+PA group; n=37, MedDiet group) were included in this study. Serum concentrations of arginine, ornithine, polyamines, and acetyl polyamines at baseline and 26 wk of intervention were measured by an ultra-high-performance LC-tandem MS platform. At week 26, study groups had similar adherence to the MedDiet but patients randomly assigned to the er-MedDiet+PA group showed significantly lower mean energy intake (-340.3 kcal/d; 95% CI: -567.3, -113.4 kcal/d; P=0.004), higher mean PA (1290.6; 95% CI: 39.9, 2541.3 metabolic equivalent tasks · min/d; P=0.043), and higher mean decrease in BMI (in kg/m2) (-1.3; 95% CI: -1.8, -0.6; P<0.001) than the MedDiet group. However, no significant differences in serum polyamines or related metabolites were found between study groups after 26 wk of intervention and no significant between-group differences were found in glycated hemoglobin, HDL-cholesterol, or triglyceride concentrations. In individuals at high CVD risk, an er-MedDiet with increased PA did not result in significant changes of serum concentrations of polyamines or related metabolites in comparison with an energy-unrestricted MedDiet and no increase in PA. This trial was registered at isrctn.com as ISRCTN89898870.

Summer Season and Recommended Vitamin D Intake Support Adequate Vitamin D Status throughout Pregnancy in Healthy Canadian Women and Their Newborns

Vitamin D deficiency in pregnancy is reported as a prevalent public health problem. We aimed to evaluate, in pregnant Canadian women, 1) vitamin D intake, 2) maternal and cord serum 25-hydroxycholecalciferol [25(OH)D] and maternal 1,25-dihydroxycholecalciferol [1,25(OH)2D], and 3) factors associated with maternal serum 25(OH)D. Women (n=187; mean prepregnancy BMI 24.4kg/m2, mean age 31y) recruited to the Be Healthy in Pregnancy study provided fasting blood samples and nutrient intake at 12-17 (early) and 36-38 (late) weeks of gestation, and cord blood. Vitamin D intakes (Nutritionist Pro™) and serum 25(OH)D and 1,25(OH)2D concentrations (LC-tandem MS) were measured. Vitamin D intake was comparable in early and late pregnancy [median (IQR) = 586 (459, 859) compared with 689 (544, 974) IU/d; P=0.83], with 71% consumed as supplements. Serum 25(OH)D was significantly higher in late pregnancy (mean±SD: 103.1±29.3nmol/L) than in early pregnancy (82.5±22.5nmol/L; P <0.001) and no vitamin D deficiency (<30nmol/L) occurred. Serum 1,25(OH)2D concentrations were significantly higher in late pregnancy (101.1±26.9pmol/L) than in early pregnancy (82.2±19.2 pmol/L, P <0.001, n=84). Cord serum 25(OH)D concentrations averaged 55% of maternal concentrations. In adjusted multivariate analyses, maternal vitamin D status in early pregnancy was positively associated with summer season (est.β: 13.07; 95% CI: 5.46, 20.69; P <0.001) and supplement intake (est.β: 0.01; 95% CI: 0.00, 0.01; P <0.001); and in late pregnancy with summer season (est.β: 24.4; 95% CI: 15.6, 33.2; P <0.001), nonmilk dairy intake (est.β: 0.17; 95% CI: 0.02, 0.32; P=0.029), and supplement intake (est.β: 0.01; 95% CI: 0.00, 0.01; P=0.04). Summer season and recommended vitamin D intakes supported adequate vitamin D status throughout pregnancy and in cord blood at >50nmol/L in healthy Canadian pregnant women. This trial was registered at clinicaltrials.gov as NCT01693510.

Detection and Quantitation of Selected Food Allergens by Liquid Chromatography with Tandem Mass Spectrometry: First Action 2017.17.

In response to a need for accurate and reliable methods for food allergen regulatory compliance, a method for the detection and quantitation of whole egg, whole milk, peanut, and hazelnut in eight food matrices was developed and evaluated in a single-laboratory validation. The matrices include cookies, cookie dough, bread, breakfast cereal, salad dressing, ice cream, and red wine. The method was compared with Standard Method Performance Requirements (SMPR) 2016.002 established by the AOAC Stakeholder Panel on Strategic Food Analytical Methods. The method involves tryptic digestion of allergen proteins in food matrices incurred or spiked with allergen standards [reference materials (RMs), Standard RMs (SRMs), or in-house prepared standard] and uses labeled peptide internal standards. LC-tandem MS analysis of the signature tryptic peptides of the four allergens is performed using multiple reaction monitoring. For 10 allergen/matrix combinations, the method demonstrated adequate sensitivity with a minimum quantitation limit of 3 mg/kg for whole egg and 10 mg/kg for milk, peanut, and hazelnut allergens. Repeatability precision across 3 days of analyses was <17% with analytical range of 10-1000 mg/kg. Recovery from incurred and spiked matrix-matched standards varied from 60 to 118%. The method met the minimum performance requirements of SMPR 2016.002 for whole egg in cookies, bread, cookie dough, and salad dressing; whole milk in cookies and red wine; peanut in breakfast cereal; and hazelnut in cookies. The ERP determined that the data presented met the SMPR and accordingly recommended the method to be granted First Action status. In September 2017, the Official Methods Board approved the method as First Action.

Liquid Chromatography-Mass Spectrometry Technique-A Review

Liquid Chromatography/Mass Spectrometry (LC/MS) is quickly becoming the preferred tool of liquid chromatographers. It is a powerful analytical technique that agglutinate the resolving powerful of liquid chromatography with the detection specificities of mass spectrometry. Liquid chromatography-mass spectrometry (LC-MS) is now became routine technique with the advancement of electro spray ionisation (ESI) provide a simple and robust interface. Since the newly developed API-based methods produce mild ionization, for structural elucidation studies it can be complemented by invoking fragmentation-induced collisions in the interface itself or by recourse to LC-tandem MS as realized with the help of a triple quadrupole system. It is applicable for of biological molecules and the use of tandem MS and stable isotope internal standards allows highly sensitive and accurate methods to be enlarge through some method optimisation to minimise ion repression effects. Method validation is of important between the process of drug discovery and development. The LC/MS data may be used to provide information about the molecular weight, structure, identity and quantity of particular sample components.

Development and Characterization of a Multimycotoxin Reference Material

Abstract Background: Matrix-matched reference materials (RMs) are critical for adequate quality assurance of extraction, digestion, separation, and/or detection processes for analytes of interest in foods and dietary supplements. The accurate determination of mycotoxins in foods is an international concern. While RMs for mycotoxins are available from a variety of RM producers, these mainly address a single mycotoxin or group of mycotoxins and therefore require the use of multiple RMs for multitarget methods. Objective: To address the increasing needs of laboratories moving toward LC-MS-based multimycotoxin analysis, the U.S. National Institute of Standards and Technology (NIST) collaborated with the U.S. Food and Drug Administration (FDA) to produce a naturally incurred RM for multiple mycotoxins in corn. Methods: Homogeneity of the RM has been assessed using a stratified random sampling of the final product based on mycotoxin mass fractions measured by the FDA and NIST. Multiple sample sizes were evaluated to maximize homogeneity in the obtained results. The mycotoxin levels in the final materials have been evaluated via interlaboratory comparison and isotope dilution LC–tandem MS measurements made at the FDA and NIST. The final value assignment combined results from these data sets. Conclusions: The study successfully developed a certified RM, SRM 1565 Mycotoxins in Corn, and a workflow for the future development of multimycotoxin RMs in different matrices.

Quantitation of Mycotoxins in Four Food Matrices Comparing Stable Isotope Dilution Assay (SIDA) with Matrix-Matched Calibration Methods by LC–MS/MS

Abstract Background: Mycotoxins are big concerns in food safety. Analytical methods are important for the evaluation of mycotoxins in different food commodities. Objective: In this study, stable isotope dilution assay (SIDA) was compared with a matrix-matched calibration method for the quantification of mycotoxins in four different commercially available commodities and two reference materials. Methods: All samples were extracted with water–acetonitrile (50+50, v/v), followed by filtration and LC–tandem MS analysis. Results: SIDA calibration accuracies ranged from 78.6 to 112% with relative SDs (RSDs) ≤16% across all four matrices. The majority of the recoveries across all matrices ranged from 70 to 120% with RSDs &amp;lt;11%. Of the four mycotoxins in the reference materials analyzed, only three had 13C-internal standard (IS), whereas the fourth was quantified using a closely eluting 13C-IS for a different mycotoxin. Mycotoxins paired with their corresponding 13C-IS had accuracies &amp;gt;90%, whereas accuracies for the mismatched mycotoxin/13C-IS were &amp;lt;14%. Conclusions: When 13C-IS are not available, matrix-matched calibration was also evaluated as an alternative to quantitating target mycotoxins. The use of 13C-IS is the best way to dynamically account for prevalent matrix effects, but matrix matching provides a viable alternative. Highlights: The study compared SIDA and matrix-matched calibration methods in terms of recovery, efficiency, advantages, and limitations for LC-MS based mycotoxin analysis.

Application of Stable Isotope Dilution and Liquid Chromatography Tandem Mass Spectrometry for Multi-Mycotoxin Analysis in Edible Oils

Abstract Background: Mycotoxin contamination in oils remains an important food safety issue. To monitor the occurrence of mycotoxins in edible oils, it is important to develop analytical methods that can determine multiple mycotoxins in oil products. A stable isotope dilution LC-tandem MS (LC-MS/MS) method for the simultaneous determination of 12 mycotoxins in five edible oil matrixes (canola, corn, olive, peanut, and soybean oil) was developed and validated. Methods: Prior to extraction, the oil samples were fortified with ¹³C uniformly labeled internal standards (¹³C-IS) for 12 target mycotoxins, followed by extraction and LC-MS/MS analysis. Quantitation was achieved using solvent-only calibration standards, relative response factors of ¹³C-IS, and target mycotoxins. Results: The majority of recoveries in oil for ochratoxin A and aflatoxins B1, B2, G1, and G2 fortified at 1, 10, and 100 ng/g as well as deoxynivalenol; fumonisins B1, B2, and B3; T-2 toxin; HT-2 toxin; and zearalenone fortified at 10, 100, and 1000 ng/g ranged from 80 to 120% with RSDs of &amp;lt;20%. The method LOQs ranged from 0.1 ng/g (aflatoxin B1) to 6.4 ng/g (zearalenone). Among 16 U.S. market samples, zearalenone was detected in three corn oil samples at 37, 185, and 317 ng/g, respectively. T-2 toxin was found in two corn oil samples at 7 and 10 ng/g, respectively. Conclusions: The method provides sufficient selectivity, sensitivity, accuracy, and repeatability to screen edible oils for regulated mycotoxins such as aflatoxins at low nanogram per gram concentrations without using conventional standard addition or matrix-matched calibration standards to correct for matrix effects.

Advances in LC–MS/MS Methods for Allergen Testing, Meat Speciation, and Gelatin Speciation

Abstract Background: Food authenticity is demanded by the consumer at all times. The consumer places trust in the manufacturer that the food product is genuine in terms of what is recorded on the packaging label. Objective: Recent advancements in LC–tandem MS methodology in the detection of allergens, meat, and gelatin speciation in raw food products and processed foods are detailed in this paper. Method: For each of the three methods, initial proteome analysis and the screening leading to the determination of unique tryptic peptides were conducted using a high-resolution, accurate tandem mass spectrometer. Having identified the unique markers, the method was transferred to a tandem quadrupole mass spectrometer for a higher-sensitivity quantitative study, multiple reaction monitoring transition analysis. Results: For the allergens method a detection limit of at least 10 ppm was attained across the 12 allergen peptides in this workflow. In the gluten workflow the resulting chromatograms show good detection down to 5 ppm, with no interference from the food matrices. The meat speciation method details that signature peptides could be readily identified at 1% w/w with no matrix interference. Conclusions: These single-injection workflows with cycle-time optimization enable wide coverage of analytes to identify multiple species within challenging matrix samples.

Metabolomic study on bleomycin and polyhexamethylene guanidine phosphate-induced pulmonary fibrosis mice models.

Polyhexamethylene guanidine phosphate (PHMG) has been used as a disinfectant and biocide, and was known to be harmless and non-toxic. However, in 2011, PHMG used as a humidifier disinfectant was reported to be associated with lung diseases, such as, fibrosis in the toxicant studies on pulmonary fibrosis by PHMG. However, no metabolomics study has been performed in PHMG-induced mouse models of pulmonary fibrosis. We performed a metabolomic study to understand the biochemical events that occur in bleomycin (BLM)- and PHMG-induced mouse models of pulmonary fibrosis using gas chromatography-mass spectrometry (GC-MS), LC-tandem MS, and GC-tandem MS. The levels of 61 metabolites of 30 amino acids, 13 organic acids, 12 fatty acids, 5 polyamines, and oxidized glutathione were determined in the pulmonary tissues of mice with BLM- and PHMG-induced pulmonary fibrosis and in normal controls. Principal component analysis and partial least squares discriminant analysis used to compare level of these 61 metabolites in pulmonary tissues. Levels of metabolites were significantly different in the BLM and PHMG groups as compared with the control group. In particular, the BLM- and PHMG-induced pulmonary fibrosis models showed elevated collagen synthesis and oxidative stress and metabolic disturbance of TCA related organic acids including fumaric acid by NADPH oxidase. In addition, polyamine metabolism showed severe alteration in the PHMG group than that of the BLM group. This result suggests PHMG will be able to induce pulmonary fibrosis by arginine metabolism and NADPH oxidase signaling.

A Dilute-and-Shoot Based UPLC-MS/MS Method for Multimycotoxins Analysis in Astragali radix.

Background: Astragali radix is prone to be contaminated by various mycotoxins, leading to unpredictable threats on the quality and safety of Astragali radix and the health of the consumers. Therefore, the determination of multimycotoxins is imperative. Objective: To develop an efficient, sensitive, fast, and multianalyte method for detecting multimycotoxins in Astragali radix. Methods: A selective dilute-and-shoot pretreatment procedure based ultra-performance LC-tandem MS (UPLC-MS/MS) method was developed for sensitive determination of multimycotoxins in Astragali radix, including aflatoxin (AF) B1, B₂, G1, G₂, and ochratoxin A. The five mycotoxins were extracted by the dilute-and-shoot pretreatment procedure followed by UPLC-MS/MS detection. Types of sample extraction solvent, mobile-phase compositions and MS/MS parameters, and dilute-and-shoot conditions were optimized. Results: The optimized chromatographic and mass spectrometric conditions allowed the separation and detection of the five mycotoxins within 5 min. The validated UPLC-MS/MS method exhibited good sensitivity with LOD and LOQ lower than 0.2 and 0.5 μg/kg, respectively. RSD values for method precision were lower than 9%. Recoveries obtained were between 90.87 and 108.44% for all the analytes with RSDs of 3.96-8.03%. The developed method was applied for the detection of the 5 mycotoxins in 18 batches of Astragali radix with good determination performance and no matrix interferences. Two samples collected from Shanxi province and Neimenggu Autonomous Region in China were positive for AFB1 at 3.24 and 2.69 μg/kg, respectively. Conclusions: The dilute-and-shoot procedure allowed for the extraction of the mycotoxins with advantages of simple pretreatment, small extraction time, high selectivity and accuracy, as well as being cost effective and easy to operate without any clean-up steps. Highlights: This is the first report on dilute-and-shoot approach for mycotoxins extraction and detection in Chinese medicinal material matrixes.

Development and Characterization of a Multimycotoxin Reference Material.

Background: Matrix-matched reference materials (RMs) are critical for adequate quality assurance of extraction, digestion, separation, and/or detection processes for analytes of interest in foods and dietary supplements. The accurate determination of mycotoxins in foods is an international concern. While RMs for mycotoxins are available from a variety of RM producers, these mainly address a single mycotoxin or group of mycotoxins and therefore require the use of multiple RMs for multitarget methods. Objective: To address the increasing needs of laboratories moving toward LC-MS-based multimycotoxin analysis, the U.S. National Institute of Standards and Technology (NIST) collaborated with the U.S. Food and Drug Administration (FDA) to produce a naturally incurred RM for multiple mycotoxins in corn. Methods: Homogeneity of the RM has been assessed using a stratified random sampling of the final product based on mycotoxin mass fractions measured by the FDA and NIST. Multiple sample sizes were evaluated to maximize homogeneity in the obtained results. The mycotoxin levels in the final materials have been evaluated via interlaboratory comparison and isotope dilution LC-tandem MS measurements made at the FDA and NIST. The final value assignment combined results from these data sets. Conclusions: The study successfully developed a certified RM, SRM 1565 Mycotoxins in Corn, and a workflow for the future development of multimycotoxin RMs in different matrices.

4-Methylumbelliferyl glucuronide contributes to hyaluronan synthesis inhibition

4-Methylumbelliferone (4-MU) inhibits hyaluronan (HA) synthesis and is an approved drug used for managing biliary spasm. However, rapid and efficient glucuronidation is thought to limit its utility for systemically inhibiting HA synthesis. In particular, 4-MU in mice has a short half-life, causing most of the drug to be present as the metabolite 4-methylumbelliferyl glucuronide (4-MUG), which makes it remarkable that 4-MU is effective at all. We report here that 4-MUG contributes to HA synthesis inhibition. We observed that oral administration of 4-MUG to mice inhibits HA synthesis, promotes FoxP3+ regulatory T-cell expansion, and prevents autoimmune diabetes. Mice fed either 4-MUG or 4-MU had equivalent 4-MU:4-MUG ratios in serum, liver, and pancreas, indicating that 4-MU and 4-MUG reach an equilibrium in these tissues. LC-tandem MS experiments revealed that 4-MUG is hydrolyzed to 4-MU in serum, thereby greatly increasing the effective bioavailability of 4-MU. Moreover, using intravital 2-photon microscopy, we found that 4-MUG (a nonfluorescent molecule) undergoes conversion into 4-MU (a fluorescent molecule) and that 4-MU is extensively tissue bound in the liver, fat, muscle, and pancreas of treated mice. 4-MUG also suppressed HA synthesis independently of its conversion into 4-MU and without depletion of the HA precursor UDP-glucuronic acid (GlcUA). Together, these results indicate that 4-MUG both directly and indirectly inhibits HA synthesis and that the effective bioavailability of 4-MU is higher than previously thought. These findings greatly alter the experimental and therapeutic possibilities for HA synthesis inhibition.

Direct Determination of Glyphosate and its Metabolite AMPA in Soil Using Mixed-Mode Solid-Phase Purification and LC-MS/MS Determination on a Hypercarb Column.

Background: Although glyphosate is widely used in agriculture, information on its residue level in soils remains scarce partly because of the difficulty in its analysis. Objective: Develop and validate a method to directly analyze glyphosate and its metabolite aminomethylphosphonic acid (AMPA) in soil. Method: Soils were extracted with 0.6 M KOH solution, and coextracted interferences were removed using a mixed-mode Bond Elut Plexa PAX®. The extracts were analyzed by LC-tandem MS fitted with a Hypercarb column and isotope-labeled (13C,15N) glyphosate and AMPA were used as internal standards. Results: LOQs were 0.05 mg/kg for both glyphosate and AMPA in soils. Correlation coefficients were ≥0.99, residuals were below 20%, and calibrations were linear in the range 0.02-1.0 μg/mL. The method was validated on five contrasting soils (Vertosol, Calcarosol, Chromosol, Sodosol, and Tenosol) commonly used for grain production in Australia. The recoveries for glyphosate and AMPA in the soils were 96-121 and 91-118%, respectively, with RSD in the range of 3-16%. Conclusions: This paper presents using the validated method in analysis glyphosate and AMPA in soils collected from crop production paddocks in Australia. The survey data showed that glyphosate and AMPA were detected in all collected soils, with concentrations ranging between 0.05 and 1.2 mg/kg. Highlights: The study demonstrates that the mixed-mode solid-phase extraction is effective in removing interferences and validates the use of Hypercarb as an alternative stationary phase for glyphosate and AMPA analysis from soils.

Analytical Method of Multi-Mycotoxins in Table-Ready Foods for a Total Diet Study Using Stable Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry.

Background: Determining the multi-mycotoxins present in table-ready foods is necessary for a total diet study. However, so far, most methods of analyzing multi-mycotoxins apply to raw foods. Therefore, a reliable method for analyzing multi-mycotoxins in table-ready foods is needed. Objective: We developed and validated methods of multi-mycotoxin analysis that employed stable isotope dilution with LC-tandem MS (LC-MS/MS) using two representative matrices. Methods: Samples were fortified with [13C]-labeled mycotoxins as internal standards and extracted with 50% acetonitrile in water for high-carbohydrate foods and 3% formic acid in acetonitrile for high-protein and/or high-fat foods, cleaned up with n-hexane and the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method, and followed up by LC-MS/MS. Results: Validation of the methods was performed, and the results were as follows: the correlation coefficient, R², was 0.99; method detection limit, 0.01-2.4 μg/kg; recoveries, 83.6-114%; precision, 0.8-10 (intraday) and 3.1-22% (interday); interlaboratory reproducibility, ≤15%; and uncertainty, 3.5-19%error. The applicability of the methods was evaluated by analyzing table-ready foods spiked with standards. Conclusions: These methods were successfully evaluated and deemed appropriate for determining the multi-mycotoxins in table-ready foods. Highlights: This work demonstrates that stable isotope dilution with LC-MS/MS can be effectively used to analyze multi-mycotoxins simultaneously in a total diet study.