Ultrahigh-performance Liquid Chromatography-tandem Mass Spectrometry Research Articles

UHPLC–MS/MS method for the determination of bisphenol A and its chlorinated derivatives, bisphenol S, parabens, and benzophenones in human urine samples

In the present work, a new method based on a sample treatment by dispersive liquid-liquid microextraction (DLLME) for the extraction of six bisphenols (bisphenol A, bisphenol S, and monochloro-, dichloro-, trichloro-, and tetrachlorobisphenol A), four parabens (methyl-, ethyl-, propyl-, and butylparaben), and six benzophenones (benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-6, benzophenone-8, and 4-hydroxybenzophenone) in human urine samples, followed by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis, is validated. An enzymatic treatment allows determining the total content of the target EDCs. The extraction parameters were accurately optimized using multivariate optimization strategies. Ethylparaben ring-(13)C6, benzophenone-d10, and bisphenol A-d16 were used as surrogates. Limits of quantification ranging from 0.1 to 0.6 ng mL(-1) and interday variabilities (evaluated as relative standard deviations) from 2.0 to 13.8% were obtained. The method was validated using matrix-matched standard calibration followed by a recovery assay with spiked samples. Recovery rates ranged from 94 to 106%. A good linearity, for concentrations up to 300 ng mL(-1) for parabens and 40 ng mL(-1) for benzophenones and bisphenols, was also obtained. The method was satisfactorily applied for the determination of target compounds in human urine samples from 20 randomly selected individuals.

Folate Profiling in Potato (Solanum tuberosum) Tubers by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

An ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the profiling of six folate species in potatoes. The calibration curves cover a wide, linear range (the lower and upper limits of quantitation range between 0.22-0.24 and 216.07-242.28 μg/100 g of fresh weight), allowing sensitive determination in small amounts of potato flesh. With a single exception, the acceptance criteria for intra- and interday precision and accuracy were met: for all quality controls, the percent relative standard deviation and the percent bias were lower than 15% (or 20% at the lower limit of quantitation). Application of the method on tubers at different stages of maturation demonstrated the large variability within a single variety: the folate content and polyglutamylation rate varied between 10.35 and 24.01 μg/100 g of fresh weight and between 4.96% and 60.49%, respectively. Additionally, the two-dimensional folate profiling of mature tubers demonstrated an increase in folate from center to peel, combined with a stable species distribution and polyglutamylation rate.

Quantitative determination of β-hydroxymethylbutyrate and leucine in culture media and microdialysates from rat brain by UHPLC-tandem mass spectrometry

The main objective of the present work was to develop a method to determine β-hydroxymethylbutyrate (HMB) and leucine (Leu) in culture media and brain microdialysates. An accurate, selective, and cost-effective method, based on the use of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), was developed for the identification and quantification of both compounds. The method consisted of sample dilution, direct injection onto the chromatographic equipment, and quantification with a triple quadrupole mass spectrometer using an electrospray ionization interface in positive mode. The procedure and the UHPLC-MS/MS parameters were accurately optimized to achieve the highest recoveries and to enhance the analytical characteristics of the method. For chromatographic separation, an Acquity UPLC BEH Hilic column using acetonitrile-water gradient with formic acid as additive was employed. The total run time was 4 min. The limits of detection (LODs) obtained ranged from 0.01 to 0.04 μg mL(-1), and the limits of quantification (LOQs) ranged from 0.04 to 0.12 μg mL(-1). Precision (expressed as relative standard deviation) was lower than 15 %, and the determination coefficient (R (2)) was higher than 99.0 % with a residual deviation for each calibration point lower than ±25 %. Mean recoveries were between 85 and 115 %. The method was successfully applied to the analysis of both compounds, HMB and Leu, in samples obtained from an experiment of blood-brain barrier (BBB) passage in vitro and to an experiment of brain microdialysis in rats in vivo after an oral challenge with HMB to detect its appearance in the brain.

A Practical Limited Sampling Strategy to Predict Free Prednisolone Exposure and Glycemia in Kidney Transplant Recipients

Despite significant interindividual variability in prednisolone pharmacokinetics and potentially serious consequences with inadequate or excessive exposure, monitoring of prednisolone levels is not employed to guide therapy. As ultrahigh-performance liquid chromatography-tandem mass spectrometry methods can now measure the active free fraction of prednisolone, this study aimed to evaluate the performance of 15 published limited sampling strategies (LSSs) for predicting free prednisolone exposure in adult kidney transplant recipients and to examine the relationship between free/total prednisolone exposure and plasma glucose. The study was performed in 11 subjects without diabetes 3-4 weeks postkidney transplantation. Area under the concentration time curve profiles of total and free prednisolone from 0 to 12 hours postdose (AUC₀₋₁₂) were determined and compared with predicted AUC₀₋₁₂ values calculated from published LSSs. Venous glucose was measured concurrently with the 13 sampling time points. The mean (±SD) age of subjects was 52 ± 12 years, 5 were men and the median (interquartile range) daily prednisolone dose was 20.0 mg (20.0-22.5). Interindividual variation in dose-adjusted free and total prednisolone exposure was 1.9- and 3.2-fold, respectively. All 15 free prednisolone LSSs exhibited good correlation (r ≥ 0.83), with bias and imprecision less than 15%. An LSS incorporating 1.25- and 3-hour samples had the highest predictive power (r = 0.97, bias 1.2%, imprecision 5.6%). Free prednisolone AUC₀₋₁₂ correlated with peak glucose levels (r = 0.65, P = 0.037), as did predicted AUC₀₋₁₂ from 14/15 LSSs. Biologically active free prednisolone exposure can be accurately predicted postkidney transplantation by LSSs incorporating 2-point concentration sampling. Peak plasma glucose concentration correlated well with prednisolone exposure.

Rapid analysis of aminoglycoside antibiotics in bovine tissues using disposable pipette extraction and ultrahigh performance liquid chromatography–tandem mass spectrometry

A high-throughput qualitative screening and identification method for 9 aminoglycosides of regulatory interest has been developed, validated, and implemented for bovine kidney, liver, and muscle tissues. The method involves extraction at previously validated conditions, cleanup using disposable pipette extraction, and analysis by a 3min ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method. The drug analytes include neomycin, streptomycin, dihydrosptreptomycin, and spectinomycin, which have residue tolerances in bovine in the US, and kanamicin, gentamicin, apramycin, amikacin, and hygromycin, which do not have US tolerances established in bovine tissues. Tobramycin was used as an internal standard. An additional drug, paromomycin also was validated in the method, but it was dropped during implementation due to conversion of neomycin into paromomycin. Proposed fragmentation patterns for the monitored ions of each analyte were elucidated with the aid of high resolution MS using a quadrupole-time-of-flight instrument. Recoveries from spiking experiments at regulatory levels of concern showed that all analytes averaged 70–120% recoveries in all tissues, except hygromycin averaged 61% recovery. Lowest calibrated levels were as low as 0.005μg/g in matrix extracts, which approximately corresponded to the limit of detection for screening purposes. Drug identifications at levels <0.05μg/g were made in spiked and/or real samples for all analytes and tissues tested. Analyses of 60 samples from 20 slaughtered cattle previously screened positive for aminoglycosides showed that this method worked well in practice. The UHPLC–MS/MS method has several advantages compared to the previous microbial inhibition screening assay, especially for distinguishing individual drugs from a mixture and improving identification of gentamicin in tissue samples.

Comprehensive Analysis of Nonenzymatic Post-Translational β-Lactoglobulin Modifications in Processed Milk by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

Nonenzymatic post-translational protein modifications (nePTMs) result in changes of the protein structure that may severely influence physiological and technological protein functions. In the present study, ultrahigh-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) was applied for the systematic identification and site-specific analysis of nePTMs of β-lactoglobulin in processed milk. For this purpose, β-lactoglobulin, which had been heated with lactose under conditions to force nePTM formation (7 d/60 °C), was screened for predicted modifications by using full scans and enhanced resolution scan experiments combined with enhanced product ion scans. Thus, the main glycation, glycoxidation, oxidation, and deamidation products of lysine, arginine, methionine, cysteine, tryptophan, and asparagine, as well as the N-terminus, were identified. Using these MS data, a very sensitive scheduled multiple reaction monitoring method suitable for the analysis of milk products was developed. Consequently, 14 different PTM structures on 25 binding sites of β-lactoglobulin were detected in different milk products.

Determination of Multiple Mycotoxins in Dietary Supplements Containing Green Coffee Bean Extracts Using Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry (UHPLC-MS/MS)

An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination of 34 mycotoxins in dietary supplements containing green coffee bean (GCB) extracts was developed, evaluated, and used in the analysis of 50 commercial products. A QuEChERS-like procedure was used for isolation of target analytes from the examined matrices. Average recoveries of the analytes were in the range of 75-110%. The precision of the method expressed as relative standard deviation was below 12%. Limits of detection (LODs) and limits of quantitation (LOQs) ranged from 1.0 to 50.0 μg/kg and from 2.5 to 100 μg/kg, respectively. Due to matrix effects, the method of standard additions was used to ensure accurate quantitation. Ochratoxin A, ochratoxin B, fumonisin B1 and mycophenolic acid were found in 36%, 32%, 10%, and 16% of tested products, respectively. Mycotoxins occurred in the following concentration ranges: ochratoxin A, <1.0-136.9 μg/kg; ochratoxin B, <1.0-20.2 μg/kg; fumonisin B1, <50.0-415.0 μg/kg; mycophenolic acid, <5.0-395.0 μg/kg. High-resolution mass spectrometry operated in full MS and MS/MS mode was used to confirm the identities of the reported compounds.

Analysis and Fate of Emerging Pollutants during Water Treatment

Emerging pollutants defined as compounds that are not currently covered by existing water-quality regulations all over the world, have not been studied widely before, and are thought to be potential threats to environmental ecosystems and human health. This special issue compiles 5 exciting papers, which are very meticulously performed researches. Generally, emerging pollutants encompass a diverse group of compounds, including pharmaceuticals, drugs of abuse, personal-care products (PCPs), steroids and hormones, surfactants, perfluorinated compounds (PFCs), flame retardants, industrial additives and agents, gasoline additives, new disinfection byproducts (DBPs), nanomaterials, and the toxic minerals. The analysis methods, occurrence, and fate of hormonal and endocrine disruptors compounds (EDCs) were discussed in two papers of this special issue. R. Guedes-Alonso et al. determine the hormonal residues in treated water by ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS) and evaluate the efficiency of the conventional wastewater treatment for the removal of hormonal compounds. Moreover, Y. Liu et al. study another kinds of PPCPs, named as phthalate esters that is typical kind of EDCs. The occurrence in a surface water and the removal efficiency in a traditional drinking water treatment palnt are studied. According to results of the two papers, the occurrence and fate of hormonal and EDCs in water or wastewater treatment are very clear. J. Xing et al. report a new wastewater treatment technology, bioflocculation, for the removal of sulfamethoxazole that is a typical pharmaceutical in wastewater. The performance and the reaction mechanism of the biodegradation of sulfamethoxazole by the bioflocculation are discussed in depth. Moreover, the optimum reaction condition is obtained. The heavy metal and toxic mineral are also important pollutants in the environment, especially in the mining area. Two papers of this issue are focused on this. K. Naeemullah et al. report a green preconcentration method for the determination of cobalt and lead in water. Y. Liu et al. do a novel research on the electrochemical reaction of pyrite as a simulation of the natural environmental. By compiling this special issue, we hope to enrich our readers and researchers on the analysis and fate of emerging pollutants during water treatment. Fei Qi Guang-Guo Ying Kaimin Shih Jolanta Kumirska Elif Pehlivanoglu-Mantas Xiaojun Luo

Fast screening of 88 pharmaceutical drugs and metabolites in whole blood by ultrahigh-performance liquid chromatography–tandem mass spectrometry

Forensic investigations involving acute or lethal intoxication, drug-facilitated sexual assault, driving or workplace impairment frequently require the analysis of fresh or postmortem blood samples to check out a wide variety of pharmaceutical and illicit drugs, even after single-dose consumption. A sensitive and selective ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) screening method was developed for fast screening of 88 psychoactive drugs and metabolites in blood samples, including the ones most frequently involved in acute intoxications and forensic investigations in Italy. The new method allows short sample processing and analysis time (the whole procedure can be accomplished in less than 30 min) together with the simultaneous monitoring of a large number of pharmaceutical substances. These features represent crucial factors in the approach of acute intoxications, when the patient requires urgent and appropriate therapy. Blood sample treatment was limited to protein precipitation. Two UHPLC-MS/MS runs in positive and negative electrospray ionization modes were performed. The data were acquired at unit mass resolution in the selected reaction monitoring mode. According to international guidelines, linearity range, precision, trueness, detection and quantification limits, recovery, selectivity, specificity, carryover, and matrix effect phenomena were determined. Despite the limited sample purification and the inherent decreased chance of eliminating any potential interference, the present multiresidue screening method proved extremely effective and sensitive, allowing the detection of all tested drugs, even those belonging to structurally different classes of substances. Moreover, the developed method is easily susceptible to further expansion to encompass more drugs, either new or those becoming important for criminal investigation. This protocol was also applied to the analysis of authentic blood samples collected from victims of various crimes in routine casework, whose relevance in forensic investigations is presented in five cases.

Ruggedness testing and validation of a practical analytical method for >100 veterinary drug residues in bovine muscle by ultrahigh performance liquid chromatography–tandem mass spectrometry

In this study, optimization, extension, and validation of a streamlined, qualitative and quantitative multiclass, multiresidue method was conducted to monitor >100 veterinary drug residues in meat using ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). Optimization centered on extensive ruggedness evaluation of the method. Various clean-up sorbents were tested and the amount of co-extractives were weighed, matrix effects were measured using post-column infusion of representative analytes, the effect of extract dilution before injecting was studied, and analyte recoveries and reproducibilities were determined. In order to extend our previous method, more drug analytes were added that possessed a wider range of chemical properties, and a re-appraisal of different types of C18 in dispersive solid-phase extraction clean-up and mobile phases in UHPLC–MS/MS was done. Ultimately, end-capped C18 and post-column infusion of ammonium formate as an ionization enhancer for the late-eluting anthelmintics were found to give improved qualitative results for greater analytical scope. A multi-day, multi-analyst validation demonstrated that the final method is suitable for screening of 113 analytes, identifying 98 and quantifying (recoveries between 70–120% and RSD<25%) 87 out of the 127 tested drugs at or below US regulatory tolerance levels in bovine muscle.

A One-Step Solid Phase Extraction Method for Bioanalysis of a Phosphorothioate Oligonucleotide and Its 3′ n-1 Metabolite from Rat Plasma by uHPLC–MS/MS

Oligonucleotide therapeutics have emerged as a promising class of drugs to treat a wide range of diseases caused by genetic abnormalities. Replacement of the phosphodiester linkage with a phosphorothioate is one of the most successful modifications made to oligonucleotides to enhance their in vivo stability. The longer elimination phase of phosphorothioates and other modified oligonucleotides requires sensitive and selective methods to quantify the parent drug and their metabolites simultaneously. Liquid chromatography tandem mass spectrometry has excellent selectivity between the parent drug and its metabolites and a wide dynamic range. However, the biological sample extraction remains a formidable challenge in developing quantitative LC-MS methods for oligonucleotides. Protein precipitation, protein digestion, liquid-liquid extraction, reversed phase solid phase extraction (SPE), strong anion exchange SPE, and combinations of them have been reported to extract oligonucleotides from biological matrices. Unfortunately, these methods either have low recoveries or present potential problems for applications with chromatography due to the large amount of matrix substances in the resulting solutions. In this study, a weak anion exchange SPE method was optimized. The recovery ranged from 60% to 80% depending on the concentration. This is the first report of a one-step SPE method with recoveries greater than 60% across the method dynamic range. This sample extraction procedure was used in combination with ultrahigh-performance liquid chromatography-tandem mass spectrometry. The lower limit of quantitation was 10ng/mL (1.3nM), and the dynamic range was 10-1,000ng/mL. The intra- and inter-day precision and accuracy were within 8.4% and 10.5%, respectively.

Rapid Determination of Organonitrogen, Organophosphorus and Carbamate Pesticides in Tea by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS)

A rapid and effective multiresidue method has been developed for the determination of 18 pesticide residues (selected from organonitrogen, organophosphorus, and carbamate pesticides) in tea by ultrahigh-performance liquid chromatography–tandem mass spectrometry. Tea samples were extracted with acetonitrile and purified by a novel multilayer solid-phase extraction cartridge—Cleanet TPT. Evaluation of the influence of the mobile phase on the ionization efficiency, resolution, and sensitivity was carried out by comparing the acetonitrile–water and methanol–water with different modifiers. The recoveries of all the pesticides varied from 70 to 110 % with a relative standard deviation of less than 15 %, and the determination coefficient for each pesticide was R 2 > 0.99. Matrix-matched standard calibration curve was used to reduce errors associated with matrix-induced enhancement or suppression effects. The limit of detection for all targeted pesticides ranged from 0.03 to 1.5 μg/kg. Applicability of this analytical approach was confirmed by successful determination of tea samples from different regions of China.

Determination of Tetracyclines and Their Epimers in Agricultural Soil Fertilized with Swine Manure by Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry

A rapid, sensitive and specific ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS) method was developed for the analysis of tetracycline antibiotics, including tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC) and their 4-epimers (4-epiTCs) in agricultural soil fertilized with swine manure. Soil samples were extracted and cleaned-up with 10 mL EDTA-McIlvaine buffer solution (pH 4.0), then cleaned-up and pre-concentrated using the Oasis MAX cartridge and then eluted with 1 mL solution by mixing formic acid, methanol and water at a ratio of 2:15:83 (v/v/v). The purified samples were separated by an ACQUITY UPLC BEH C18 column using acetonitrile and water containing 0.1% formic acid mobile phase and detected by a single quadrupole MS. The limits of detection for the soil extraction method (LODsoil) ranged from 0.6-2.5 μg kg−1 with recoveries from 23.3-159.2%. Finally, the method was applied to an agricultural field in an area with intensive pig-fattening farming. Tetracyclines were detected in soil from 2.8 to 42.4 μg kg−1 soil. These results demonstrate that soil from swine farms can become severely contaminated with tetracycline antibiotics and their metabolites.

Simultaneous Quantitation of 2-Acetyl-4-tetrahydroxybutylimidazole, 2- and 4-Methylimidazoles, and 5-Hydroxymethylfurfural in Beverages by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

An ultrahigh-performance liquid chromatography (UHPLC) tandem mass spectrometric (MS/MS) method was developed for the simultaneous quantification of 2-acetyl-4-tetrahydroxybutylimidazole (THI), 2- and 4-methylimidazoles (2-MI and 4-MI), and 5-hydroxymethylfurfural (HMF) in beverage samples. A C30 reversed-phase column was used in this method, providing sufficient retention and total resolution for all targeted analytes, with an MS/MS instrument operated in selected reaction monitoring (SRM) mode for sensitive and selective detection using isotope-labeled 4-methyl-d(3)-imidazole (4-MI-d(3)) as the internal standard (IS). This method demonstrates lower limit of quantification (LLOQ) at 1 ng/mL and coefficient of determination (r(2)) >0.999 for each analyte with a calibration range established from 1 to 500 ng/mL. This method also demonstrates excellent quantification accuracy (84.6-105% at 5 ng/mL, n = 7), precision (RSD < 7% at 5 ng/mL, n = 7), and recovery (88.8-99.5% at 10, 100, and 200 ng/mL, n = 3). Seventeen carbonated beverage samples were tested (n = 2) in this study including 13 dark-colored beverage samples with different flavors and varieties and 4 light-colored beverage samples. Three target analytes were quantified in these samples with concentrations in the range from 284 to 644 ng/mL for 4-MI and from 706 to 4940 ng/mL for HMF. THI was detected in only one sample at 6.35 ng/mL.

Occurrence of psychoactive compounds and their metabolites in groundwater downgradient of a decommissioned sewage farm in Berlin (Germany)

Psychoactive compounds-meprobamate, pyrithyldione, primidone, and its metabolites, phenobarbital, and phenylethylmalonamide-were detected in groundwater within the catchment area of a drinking water treatment plant located downgradient of a former sewage farm in Berlin, Germany. The aim of this study was to investigate the distribution of the psychoactive compounds in anoxic groundwater and to assess the risk of drinking water contamination. Groundwater age was determined to achieve a better understanding of present hydrogeological conditions. A large number of observation and production wells were sampled. Samples were analyzed using solid-phase extraction and ultrahigh-performance liquid chromatography-tandem mass spectrometry. Groundwater age was estimated using the helium-tritium ((3)He-(3)H) dating method. Concentrations of psychoactive compounds up to 1 μg/L were encountered in the contamination plume. Generally, concentrations of phenobarbital and meprobamate were the highest. Elevated concentrations of the analytes were also detected in raw water from abstraction wells located approximately 2.5 km downgradient of the former sewage farm. Concentrations in the final drinking water were below the limit of quantification owing to dilution. The age of shallow groundwater samples ranged from years to a decade, whereas groundwater was up to four decades old at 40 m below ground. Concentrations of the compounds increased with groundwater age. Elevated concentrations of psychoactive drugs indicate a strong persistence of these compounds in the environment under anoxic aquifer conditions. Results suggest that the heritage of sewage irrigation will affect raw water quality in the area for decades. Therefore, further monitoring of raw and final drinking water is recommended to ensure that contaminant concentrations remain below the health-based precautionary value.

Monitoring Metformin in Cardiac Patients Exposed to Contrast Media Using Ultra–High-Performance Liquid Chromatography Tandem Mass-Spectrometry

There is no evidence that the use of contrast media (CM) in diabetic patients with serum creatinine <130 μmole/L leads to metformin accumulation and subsequent lactic acidosis. Therefore, the objective of this investigation was to monitor cardiac patients for the effects of CM on their metformin plasma concentration and serum creatinine clearance (ClCr). Metformin plasma concentrations were measured by a new, fully validated specific, precise, and accurate ultra-high-performance liquid chromatography tandem mass-spectrometric assay. The detection was performed using positive electrospray ionization in the multiple reaction monitoring mode. Fifty patients with serum creatinine levels <130 μmole/L were monitored for the effect of CM exposure on metformin concentration and ClCr. Pharmacokinetic parameters were calculated in 8 of these patients, and metformin accumulation was monitored in 10 patients before and after their exposure to CM. Linear response (r ≥ 0.998) was observed over the range of 5-2000 ng/mL of metformin, with the lower limit of quantification of 2.3 ng/mL. The intraday and interday precision (relative standard deviation) values were <13%, and the accuracy (relative error) was <-10% for metformin concentrations. The assay was sensitive to follow the pharmacokinetics of metformin in humans during a dosing interval after an oral dose at steady state. Metformin pharmacokinetic parameters were estimated in 8 patients exposed to CM. The mean C(max) of 1.9 ± 0.6 mg/L was attained at 4.1 ± 1.9 hours. There was no evidence of any drug accumulation or altered elimination due to the exposure to CM in the current population. ClCr showed no significant difference (P > 0.05) before (92.8 ± 11.3 mL/min) and after 48 hours (90.5 ± 10.5 mL/min) of exposure to CM. Our data suggest that the recommendation to withhold metformin in diabetic patients during CM exposure could be revised to withholding the drug only in patients with moderate to severe renal dysfunction.

Ultrahigh-Performance Liquid Chromatographic–Tandem Mass Spectrometric Multimycotoxin Method for Quantitating 26 Mycotoxins in Maize Silage

A multianalyte method was developed to identify and quantitate 26 mycotoxins simultaneously in maize silage by means of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The extraction and cleanup procedure consists of two extraction steps followed by purification on a Waters Oasis HLB column. The method developed was validated with the requirements of Commission Decision 2002/657/EC taken into account. The limit of detection and quantitation ranges were 5-348 and 11-695 ng/g, respectively. Apparent recovery varied between 61 and 116%, whereas repeatability and reproducibility were within the ranges of 3-45 and 5-49%, respectively. The method developed was successfully applied for maize silage samples taken at the cutting surface and 1 m behind that surface. Mainly Fusarium toxins (beauvericin, deoxynivalenol, enniatins, fumonisins, fusaric acid, and zearalenone) were detected, but postharvest toxins such as mycophenolic acid and roquefortine C were identified as well.

Influence of High-Pressure Processing on the Profile of Polyglutamyl 5-Methyltetrahydrofolate in Selected Vegetables

In plants, folate occurs predominantly as 5-methyltetrahydrofolate (5MTHF) polyglutamyl forms. Differences in stability and bioavailability of food folate compared to synthetic folic acid have been attributed to the presence of the polyglutamyl chain. High-pressure processing (HPP) was tested for whether it might shorten polyglutamyl chains of 5MTHF species in fresh vegetables by enabling action of native γ-glutamylhydrolase (GGH). A validated ultrahigh-performance reversed-phase liquid chromatography-tandem mass spectrometry method using stable isotope as internal standard was applied for characterizing 5MTHF polyglutamyl profiles. HPP conditions included 300, 450, and 600 MPa at 30 °C for 0 or 5 min, and vegetables were vacuum-packed before treatment. Investigated vegetables included cauliflower (Brassica oleracea), baby carrots (Daucus carota), and carrot greens (D. carota). HPP treatment caused conversion of polyglutamyl 5MTHF species to short-chain and monoglutamyl forms. Maximal conversion of polyglutamyl folate to monoglutamyl folate occurred at the highest pressure/time combination investigated, 600 MPa/30 °C/5 min. Under this condition, cauliflower monoglutamyl folate increased nearly 4-fold, diglutamyl folate 32-fold, and triglutamyl folate 8-fold; carrot monoglutamyl increased 23-fold and diglutamyl 32-fold; and carrot greens monoglutamyl increased 2.5-fold and the diglutamyl form 19-fold. Although some folate degradation was observed at certain intermediate HPP conditions, total 5MTHF folate was largely preserved at 600 MPa/5 min. Thus, HPP of raw vegetables is a feasible strategy for enhancing vegetable monoglutamate 5MTHF.

Development and validation of an ultra-high performance liquid chromatography–tandem mass-spectrometry (UHPLC–MS/MS) method for the simultaneous determination of neurotransmitters in rat brain samples

A simple method for the simultaneous determination of glutamate, γ-aminobutyric acid (GABA), choline, acetylcholine, dopamine, 5-hydroxyindole-3-acetic (5-HIAA), serotonin, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was developed by using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS). These compounds are analysed in a single chromatographic run in less than 8 min, adding heptafluorobutyric acid (HFBA) in the mobile phase to improve the separation of the selected neurotransmitters. The analytes were detected using electrospray ionization (ESI)-MS/MS in positive mode with multiple reaction monitoring (MRM). Good linearity was obtained ( R 2 > 0.98) and the intra and inter-day precision of the method (expressed as relative standard deviation) were lower than 26%. Limits of quantification were lower than 2.440 μg/g of brain in all the cases, allowing the sensitive determination of these compounds in rat brain extracts. Therefore, the method was successfully applied for the quantitative determination of neurotransmitters in several rat brain regions (prefrontal cortex, striatum, nucleus accumbens and amygdala), detecting glutamate, GABA and choline at concentrations higher than 1000 μg/g, 30 μg/g and 100 μg/g respectively, whereas the other compounds were found at lower concentrations.

813 HEPATITIS C VIRUS-SPECIFIC CELLULAR IMMUNITY DOES NOT PROTECT AGAINST FUTURE HCV INFECTION IN ANTI-HCV NEGATIVE INJECTING DRUG USERS

performed using a non-targeted multiple platform methodologycombining ultrahigh performance liquid chromatography/tandemmass spectrometry (UHPLC/MS) and gas chromatography/massspectrometry (GC/MS). Metabolites were identified by automatedcomparison of ion features to a reference of chemical standardentries. Welch’s two-sample t-tests were used to identifybiochemicals that differed significantly between HCV infected andun-infected cells.Results: A total of 250 metabolites were detected and quantified,of which 73 were differentially regulated. At the 24-hour timepoint, there was a significant increase in a number of metabolitesinvolved in nucleotide synthesis and RNA replication. NAD levelswere also significantly increased along with several aminoacids. A number of lipid metabolic pathways were disruptedby HCV infection, resulting in an increase in cholesterol andsphingolipid levels, altered phospholipid metabolism and a possibledisruption in mitochondrial fatty acid transport. Fluctuations inmethylthioadenosine (MTA) levels were also noted, along withalterations in the glutathione synthesis pathway.Conclusions: These results suggest that elevated metabolism andaltered energy status are associated with early HCV infection. Thesefindings also provide new information on the effect HCV infectionhas on the disruption of lipid metabolism.813HEPATITIS C VIRUS-SPECIFIC CELLULAR IMMUNITY DOESNOT PROTECT AGAINST FUTURE HCV INFECTION IN ANTI-HCVNEGATIVE INJECTING DRUG USERSR. Sacks-Davis