Isotope Dilution Ultrahigh Performance Liquid Research Articles

Assessing the performance of a targeted absolute quantification isotope dilution liquid chromatograhy tandem mass spectrometry assay versus a commercial nontargeted relative quantification assay for detection of three major perfluoroalkylsin human blood.

Isotope dilution ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) is commonly used for trace analysis of polyfluoroalkyl and perfluoroalkyl substances (PFAS) in difficult matrices. Commercial nontargeted analysis of major PFAS where relative concentrations are obtained cost effectively is rapidly emerging and is claimed to provide comparable results to that of absolute quantification using matrix matched calibration and isotope dilution UHPLC-MS/MS. However, this remains to be demonstrated on a large scale. We aimed to assess the performance of a targeted absolute quantification isotope dilution LC-MS/MS assay versus a commercial nontargeted relative quantification assay for detection of three major PFAS in human blood. We evaluated a population-based cohort of 503 individuals. Correlations were assessed using Spearman's rank correlation coefficients (rho). Precision and bias were assessed using Bland-Altman plots. For perfluorooctane sulfonic acid, the median concentrations were 5.10ng/mL (interquartile range [IQR] 3.50-7.24 ng/mL), the two assays correlated with rho 0.83. For perfluorooctanoic acid, the median concentrations were 2.14 ng/mL (IQR 1.60-3.0ng/mL), the two assays correlated with rho 0.92. For perfluorohexanesulfonate, the median concentrations were 5.5ng/mL (IQR 2.50-11.61 ng/mL), the two assays correlated with rho 0.96. The Bland-Altman statistical test showed agreement of the mean difference for the majority of samples (97-98%) between the two assays. Absolute plasma concentrations of PFAS obtained using matrix matched calibration and isotope dilution UHPLC-MS/MS show agreement with relative plasma concentrations from a nontargeted commercial platform by Metabolon. We observed striking consistency between the two assays when examining the associations of the three PFAS with cholesterol, offering additional confidence in the validity of utilizing the nontargeted approach for correlations with various health phenotypes.

Certification and long-term stability monitoring of low-content folic acid and 5-methyltetrahydrofolate in human plasma certified reference material by isotope dilution ultra-high performance liquid chromatography/tandem mass spectrometry.

A reliable measurement of blood folate levels is necessary in the clinical field to prevent serious diseases such as cognitive impairment and neural tube defects. Herein, the certification of the low-content folic acid (FA) and 5-methyltetrahydrofolate (5-Me-THF) in human plasma certified reference material (KRISS CRM 111-01-018) was performed. A human plasma pool obtained from the Korean Red Cross was used as a CRM candidate. The certification of the human plasma CRM was performed by isotope dilution ultra-performance liquid chromatography/tandem mass spectrometry. Two-dimensional liquid chromatography was employed to confirm the validity of the analytical method for FA due to the susceptibility of FA to matrix effects because of its limited quantity. The CRM stability was evaluated at -20 °C for 2 months and at -70 °C for up to 12 months to determine the certified value of the CRM. The certified value of the CRM was (84.6 ± 4.3) ng kg-1 and (5.80 ± 0.47) μg kg-1 for FA and 5-Me-THF, respectively. The homogeneity of the CRM was 1.64% and 3.10% for FA and 5-Me-THF, respectively. Further long-term stability assessments were conducted, indicating that the CRM remains valid for at least 58 months at -70 °C for FA and 48 months for 5-Me-THF. Compared to other blood-based CRMs, this CRM has lower folate levels, making it helpful in establishing analytical methods for a broader range of folate levels.

Quantification of Resveratrol in Peanut Oils Using Stable Isotope Dilution Ultra-high Performance Liquid Chromatography-Mass Spectrometry

Quantification of Resveratrol in Peanut Oils Using Stable Isotope Dilution Ultra-high Performance Liquid Chromatography-Mass Spectrometry

Liquid Chromatography-Mass Spectrometry Analysis of Frataxin Proteoforms in Whole Blood as Biomarkers of the Genetic Disease Friedreich's Ataxia.

Friedreich's ataxia (FRDA) is caused primarily by expanded GAA repeats in intron 1 of both alleles of the FXN gene, which causes transcriptional silencing and reduced expression of frataxin mRNA and protein. FRDA is characterized by slowly progressive ataxia and cardiomyopathy. Symptoms generally appear during adolescence, and patients slowly progress to wheelchair dependency usually in the late teens or early twenties with death on average in the 4th decade. There are two known mature proteoforms of frataxin. Mitochondrial frataxin (frataxin-M) is a 130-amino acid protein with a molecular weight of 14,268 Da, and there is an alternatively spliced N-terminally acetylated 135-amino acid form (frataxin-E) with a molecular weight of 14,953 Da found in erythrocytes. There is reduced expression of frataxin in the heart and brain, but frataxin is not secreted into the systemic circulation, so it cannot be analyzed in serum or plasma. Blood is a readily accessible biofluid that contains numerous different cell types that express frataxin. We have found that pig blood can serve as an excellent surrogate matrix to validate an assay for frataxin proteoforms because pig frataxin is lost during the immunoprecipitation step used to isolate human frataxin. Frataxin-M is expressed in blood cells that contain mitochondria, whereas extra-mitochondrial frataxin-E is found in erythrocytes. This means that the analysis of frataxin in whole blood provides information on the concentration of both proteoforms without having to isolate the individual cell types. In the current study, we observed that the distributions of frataxin levels for a sample of 25 healthy controls and 50 FRDA patients were completely separated from each other, suggesting 100% specificity and 100% sensitivity for distinguishing healthy controls from FRDA cases, a very unusual finding for a biomarker assay. Additionally, frataxin levels were significantly correlated with the GAA repeat length and age of onset with higher correlations for extra-mitochondrial frataxin-E than those for mitochondrial frataxin-M. These findings auger well for using frataxin levels measured by the validated stable isotope dilution ultrahigh-performance liquid chromatography-multiple reaction monitoring/mass spectrometry assay to monitor therapeutic interventions and the natural history of FRDA. Our study also illustrates the utility of using whole blood for protein disease biomarker discovery and validation.

Assessment of the impact of hydrolysis on bound sulfonamide residue determination in honey using stable isotope dilution ultrahigh performance liquid chromatography tandem mass spectrometry

In this study, an analytical method based on isotope dilution-liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) was developed as a candidate reference method for the determination of sulfonamides (SAs) in honey. To guarantee the accuracy and authenticity, the impact of hydrolysis on bound SA residues was first investigated by enabling (i) identification of sugar-bound SAs, (ii) clarifying the binding reaction rule between the SAs and sugar, (iii) detection of free SAs and sugar-bound SAs, and (iv) preparation of SA-contaminated honey. Thus, the efficiency of different hydrolysis conditions was assessed by comparing the bound SA content before and after hydrolysis. In addition, optimization of the sample pretreatment procedures and LC conditions to minimize matrix effects by separation from significant matrix interferences was also performed. Satisfactory results in terms of hydrolysis efficiency (approximately 88.3%–99.2%), extraction efficiency (84.2%–105.3%), recovery (95.9%–103.1%), and limit of quantification (0.6–1.5 μg·kg−1) were obtained.

Highly sensitive analytical method for the accurate determination of 5-methyltetrahydrofolic acid monoglutamate in various volumes of human plasma using isotope dilution ultra-high performance liquid chromatography-mass spectrometry

One predominant and bioactive folate vitamer circulating in the blood is 5-methyltetrahydrofolate (5-Me-THF). In this study, a method for the accurate determination of 5-Me-THF in human plasma samples of various volumes was established using isotope dilution ultra-high performance liquid chromatography-mass spectrometry (ID-UPLC-MS). For this purpose, 500 μL of homogeneous human plasma was initially employed, and the 5-Me-THF and the 13C5-5-Me-THF standard solutions prepared using 1% ascorbic acid in water gave the calibration solution and spiking sample. The desired amount of 13C5-5-Me-THF standard solution was spiked into the sample followed by sample pretreatment. The method was validated for its repeatability, reproducibility, recovery, and limits of detection and quantification. Subsequently, it was applied to smaller volumes of human plasma samples (i.e., 50 and 10 μL), the results of which corresponded well with those obtained using 500 μL. The feasibility of the method was further confirmed using 10 μL of a standard reference material, SRM 3949, which is a human serum sample containing three different levels of 5-Me-THF. The established ID-UPLC-MS method was successfully applied to various volumes of human plasma or serum ranging from 500 to 10 μL, which exhibited particularly good sensitivity in addition to reliable results for the quantification of 5-Me-THF. Our method therefore expands on the ability to obtain accurate quantitative results for 5-Me-THF using small volumes of blood.

Development of an Isotope Dilution UHPLC-QqQ-MS/MS-Based Method for Simultaneous Determination of Typical Advanced Glycation End Products and Acrylamide in Baked and Fried Foods.

In this work, a stable isotope dilution ultrahigh-performance liquid chromatography triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) method was developed and validated for simultaneous determination of Nε-(carboxymethyl)lysine (CML), Nε-(carboxyethyl)lysine (CEL), and acrylamide (AA) in baked and fried foods. Ground food samples were extracted with acetone followed by two parallel assays. In assay A, a cleanup procedure based on dispersive solid-phase extraction was conducted for AA, free CML, and CEL analysis using the supernatant. In assay B, a multistep process including reduction, protein precipitation, acid hydrolysis, and solid-phase extraction was conducted for bound CML and CEL analysis using precipitation. The developed method was validated in terms of linearity, sensitivity (limit of detection, LOD; limit of quantitation, LOQ), accuracy, and precision. The results showed that the method had a wide linear range (0.25-500 ng/mL for CML and CEL, 0.5-500 ng/mL for AA), low LOD and LOQ (0.47-0.94 and 1.52-1.91 μg/kg, respectively), and good linearity (R2 > 0.999). The recovery test on baby biscuit and French fries samples showed the recovery rates of 90.2-108.3% for CML, 89.0-106.1% for CEL, and 94.5-112.3% for AA with satisfactory precision (relative standard deviation (RSD) < 10%). Finally, the developed method was successfully applied to 11 baked and fried food samples, and total CML, CEL, and AA contents varied in the ranges of 4.07-35.88 mg/kg, 1.99-14.49 mg/kg, and 5.56-506.64 μg/kg, respectively. Therefore, the isotope dilution UHPLC-QqQ-MS/MS method developed herein is promising for routine analysis of CML, CEL, and AA in baked and fried foods.

High-Throughput and Sensitive Analysis of Free and Total 8-Isoprostane in Urine with Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry.

Oxidative stress (OS) plays a major role in the pathogenesis of various diseases in humans. OS is a result of an imbalance between reactive oxygen species (ROS) and the biologically available antioxidants that prevent or repair damage that ROS inflict on the host cells. ROS are naturally generated during normal mitochondrial respiration and by oxidative burst during the immune response. Many factors may influence OS, including genetics, diet, exercise, and exposure to environmental toxicants (e.g., tobacco smoke). A nonenzymatic peroxidation product of arachidonic acid (AA), 8-iso-PGF2α (8-isoprostane), is a validated biomarker of OS that is present in urine as both glucuronide conjugate and free acid. Previous studies report that the conjugated forms of 8-isoprostane can vary between 30 and 80% of the total 8-isoprostane levels. By hydrolyzing the conjugated forms, it is possible to obtain a total (free + conjugated) measurement of 8-isoprostane in urine samples. Here, we describe a robust, automated, and high-throughput method for measuring total urinary 8-isoprostane using a polymeric weak anion-exchange solid-phase extraction (SPE) and isotope-dilution ultrahigh performance liquid chromatography electrospray ionization–tandem mass spectrometry (UHPLC–MS/MS). This method, using a 96-well plate platform, showed good sensitivity (8.8 pg/mL LOD) and used only 400 μL of the sample volume with a cycle time of 11 min. The inter- and intraday precision, calculated from 20 repeated measurements of two quality control pools, varied from 4 to 10%. Accuracy, calculated from the recovery percentage at three spiking levels, ranged from 92.7 to 106.7%. We modified this method to allow for the exclusive measurement of free 8-isoprostane by removing the hydrolysis step. We measured both free and total 8-isoprostane in urine collected from 30 cigarette smokers (free: 460 ± 78.8 pg/mL; total: 704 ± 108 pg/mL) and 30 nonusers of tobacco products (free: 110 ± 24.2 pg/mL; total: 161 ± 38.7 pg/mL). This method is robust, accurate, and easily adaptable for large population studies.

Dual-vortex-assisted matrix solid-phase dispersion coupled with isotope-dilution ultrahigh-performance liquid chromatography-high resolution mass spectrometry for the rapid determination of parabens in indoor dust samples

A reliable and straightforward method was developed for the rapid determination of nine parabens (methyl-, ethyl-, propyl-, butyl-, isopropyl-, isobutyl-, pentyl-, hexyl-, and benzyl-parabens) in indoor dust by a mortar/pestle-free and column-free dual-vortex-assisted matrix solid-phase dispersion (DVA-MSPD) technique. After that, they were determined by isotope-dilution ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-qTOF-MS, or called UHPLC-HRMS) and operating in negative electrospray ionization mode. Optimization of the DVA-MSPD was done using Box-Behnken Design along with response surface methodology. Validation was done by measuring and calculating selectivity, limits of detection (LOD), limits of quantitation (LOQs), precision and trueness (accuracy) of intra- and inter-day analysis. The LOQs of the method ranged from 0.9 to 2.8 ng/g. High precisions for both intra- and inter-day analysis were obtained ranging from 1 to 8%. Excellent trueness (or mean extraction recovery) varied from 93 to 104%. The DVA-MSPD combined with isotope-dilution UHPLC-qTOF-MS was successfully applied to determine parabens in indoor dust samples from office rooms and private houses, and the total concentrations ranged from 55 to 686 ng/g.

The quantitation of 7beta-hydroxy-epiandrosterone in the plasma and seminal plasma of men with different degrees of fertility.

7beta-hydroxy-epiandrosterone (7beta-OH-EpiA) is an endogenous androgen metabolite that has been shown to exert neuroprotective, anti-inflammatory and anti-estrogenic effects. However, to the best of our knowledge no information is available about this androgen steroid in relation to sperm quality. We analyzed 7beta-OH-EpiA in plasma and seminal plasma using a newly developed isotope dilution ultra-high performance liquid chromatography - mass spectrometry method. Validation met the requirements of FDA guidelines. Levels of 7beta-OH-EpiA were measured in 191 men with different degrees of infertility. One-way analysis of variance followed by multiple comparison and correlation analysis adjusted for age, BMI and abstinence time were performed to evaluate the relationships between this steroid and sperm quality. Concentrations of 7beta-OH-EpiA in seminal plasma were significantly higher in severely infertile men in comparison with healthy men and slightly infertile men. The same trend was found when blood plasma was evaluated. Furthermore, plasma 7beta-OH-EpiA negatively correlated with sperm concentration (-0.215; p<0.01) and total count (-0.15; p<0.05). Seminal 7beta-OH-EpiA was negatively associated with motility (-0.26; p<0.01), progressively motile spermatozoa (-0.233; p<0.01) and nonprogressively motile spermatozoa (-0.188; p<0.05). 7beta-OH-EpiA is associated with lower sperm quality and deserves more research in that respect.

Simultaneous determination of nine neonicotinoids in human urine using isotope-dilution ultra-performance liquid chromatography–tandem mass spectrometry

Neonicotinoids (neonics), a class of systemic insecticides, have been frequently detected in pollen, vegetables, and fruits. Recently, an increasing concern has been aroused for human exposure to neonics. However, biological monitoring for quantifying body burden of neonics has rarely been reported. In this study, we developed an isotope-dilution ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method to simultaneously quantify nine neonics, including acetamiprid (ACE), thiamethoxam (THIAM), imidacloprid (IMIP), clothianidin (CLO), flonicamid (FLO), thiacloprid (THIAC), dinotefuran (DIN), nitenpyram (NIT), and imidaclothiz (IMIT) in urine. The limits of quantification were 0.1 μg/L for ACE, FLO, DIN, NIT and IMIT, and 0.2 μg/L for THIAM, IMIP, CLO, and THIAC. The overall recoveries were 80.8–103%, 81.5–91.7% and 83.0–92.3% for QA/QC samples fortifying at 1, 25, and 100 μg/L levels, respectively. UPLC/MS/MS method was used to analyze urine samples obtained from 10 children in Hangzhou, China. The detection frequencies were 80% for ACE and IMIP, 70% for THIAM and CLO, 20% for DIN and IMIT and 10% for THIAC. FLO and NIT were not detected in those urine samples. The data provided here will be helpful for conducting biological monitoring of neonics exposure in the future.

Simultaneous determination of 21 trace perfluoroalkyl substances in fish by isotope dilution ultrahigh performance liquid chromatography tandem mass spectrometry

Perfluoroalkyl substances (PFASs) have been identified as emerging environmental contaminants. In this study, an efficient and accurate ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous determination of 21 PFASs in fish. Acetonitrile was used for sample extraction. Solid phase extraction (SPE) by WAX cartridges and then freezing at −30 °C were adopted as cleanup strategies. Strict measurements were performed to control background contamination during the whole procedure. Matrix effects were evaluated by external standard and isotope dilution mass spectrometry (IDMS) methods. IDMS can compensate the matrix effects to a large extent. The method detection limits (MDLs) ranged from 2 pg/g to 10 pg/g except for PFBA (120 pg/g). The method quantitation limits (MQLs) ranged from 5 pg/g to 30 pg/g except for PFBA (300 pg/g). The matrix spiked recoveries of three spiked levels were in the range of 79.6%–109%. The intra-day relative standard deviation (RSD) and inter-day RSD were from 0.94% to 13.9% and 0.36% to 11.2% respectively. Two fish tissue reference materials were analyzed by the developed method. The results of reference materials were within the uncertainty of the given value. The quantitative results of IDMS and standard addition (SA) - IDMS were comparable. The developed UHPLC-MS/MS method was applied for PFASs detection in 20 marine fish samples. 9 PFASs were detected in the samples with the ∑9PFASs concentration range of 0.04 to 2.14 ng/g wet weight (ww).

Determination of patulin in apple juice by single-drop liquid-liquid-liquid microextraction coupled with liquid chromatography-mass spectrometry

Quick and simple analytical methodology has been developed for the measurement of patulin in apple juice by combining single-drop liquid-liquid-liquid microextraction with isotope dilution ultra-high performance liquid chromatography-mass spectrometry. After systematic parameters optimization, the whole sample pretreatment only consisted of single extraction that consumed 1.5 mL ethyl acetate. While, other methods usually needed complicated pretreatment, including extraction, purification, evaporation and redissolution. This sample pretreatment method could greatly lower the interferences from sugar-rich matrix, and the limit of detection was 0.5 μg/L and limit of quantification was 2 μg/L in apple juice. Moreover, linear range covered three orders of magnitude from 2 to 2000 μg/L. The proposed method is promising for patulin extraction and detection in apple juice, which will opens a new perspective in the enrichment of trace contaminations in high sugar complex matrix.

Quantification of Neurotransmitters from Intact and Regenerating Planarians Using UHPLC-MS/SRM Method.

Freshwater planarian species S. mediterranea is an emerging stem cell model because of its capability of regenerating large portions of missing body parts. It is one of the best model systems available to address the basic biological mechanisms in the regeneration processes. Absolute quantification of metabolites from planarians is imperative to understand their role in the regeneration processes. Here we describe a stable isotope dilution ultrahigh performance liquid chromatography/mass spectrometry/selected reaction monitoring (UHPLC-MS/SRM) assay for a sensitive and quantitative assessment of neurotransmitters (NTs) in planaria. We used this method for the simultaneous quantification of 16 NTs from both intact and regenerating planarians.

Accurate analysis of parabens in human urine using isotope-dilution ultrahigh-performance liquid chromatography-high resolution mass spectrometry

An analytical method that utilizes isotope-dilution ultrahigh-performance liquid chromatography coupled with hybrid quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS or called UHPLC-HRMS) was developed, and validated to be highly precise and accurate for the detection of nine parabens (methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, hexyl-, and benzyl-parabens) in human urine samples. After sample preparation by ultrasound-assisted emulsification microextraction (USAEME), the extract was directly injected into UHPLC-HRMS. By using negative electrospray ionization in the multiple reaction monitoring (MRM) mode and measuring the peak area ratios of both the natural and the labeled-analogues in the samples and calibration standards, the target analytes could be accurately identified and quantified. Another use for the labeled-analogues was to correct for systematic errors associated with the analysis, such as the matrix effect and other variations. The limits of quantitation (LOQs) were ranging from 0.3 to 0.6 ng/mL. High precisions for both repeatability and reproducibility were obtained ranging from 1 to 8%. High trueness (mean extraction recovery, or called accuracy) ranged from 93 to 107% on two concentration levels. According to preliminary results, the total concentrations of four most detected parabens (methyl-, ethyl-, propyl- and butyl-) ranged from 0.5 to 79.1 ng/mL in male urine samples, and from 17 to 237 ng/mL in female urine samples. Interestingly, two infrequently detected pentyl- and hexyl-parabens were found in one of the male samples in this study.

Deoxynivalenol and its acetyl derivatives in bread and biscuits in Shandong province of China

ABSTRACTIn the present study, 100 commercial breads and biscuits were investigated for the occurrence of deoxynivalenol (DON) and its acetylated derivatives 3-acetyldeoxynivalenol (3-Ac-DON) and 15-acetyldeoxynivalenol (15-Ac-DON). The target mycotoxins were determined by isotope dilution ultrahigh performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). DON was determined in 95% of the test samples with a mean value of 153.3 µg/kg. Compared with DON, 3-Ac-DON and 15-Ac-DON were far less frequently detected, with mean values of 1.14 and 0.37 µg/kg, respectively. The estimated daily intakes of the sum of DON and its derivatives in breads and biscuits were 0.0059 and 0.0313 µg/kg bw/day, respectively, which was within the group provisional tolerable daily intake of 1.0 µg/kg bw/day set by the Joint FAO/WHO Expert Committee on Food Additives. In the future, systematic monitoring programmes of DON and its derivatives in relevant foodstuffs are still necessary for food safety and human health.

Biomarker analysis of hemoglobin adducts of acrylamide and glycidamide enantiomers for mid-term internal exposure assessment by isotope dilution ultra-high performance liquid chromatography tandem mass spectrometry

Hemoglobin (Hb) adducts of acrylamide (AA) and its oxidative metabolite glycidamide (GA) are important biomarkers for evaluating the mid-term exposure of acrylamide toxicity in vivo. Taking pentafluoro-2-methylphenyl isothiocyanates of N-(2-carbamoylethyl)valine (AAVal-PFPTH) and N-(2-carbamoyl-2-hydroxyethy)valine (GAVal-PFPTH) as target analytes, we developed an isotope dilution ultra-high performance liquid chromatograph tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of AA and GA hemoglobin (Hb) adducts under the electroscopy ionization negative (ESI‾) mode in the present work. Among them, the enantiomer pair of GA-Hb adducts was firstly identified and successfully separated at baseline level. The method achieved high sensitivity with the LOD and LOQ ranging 1.43–5.05pmol/g Hb and 4.78–16.82pmol/g Hb, respectively. The recovery rates with low, intermediate and high spiking levels were calculated as 97.0–105.2%, 97.4–106.4% and 100.3–111.2%, respectively. Acceptable within-laboratory reproducibility (RSD < 13.7%) substantially supported the robustness of current UHPLC-MS/MS method, which was successfully applied to measure the hemoglobin adducts of acrylamide and glycidamide enantiomers in blood of both rats and humans. A linear exposure assessment model was developed for estimating the daily exposure to acrylamide in humans via considering acrylamide hemoglobin adducts as variables, indicating a novel connect between biomarker-based internal exposure and dietary-based external exposure. Overall, the present instrumental analysis and related internal exposure assessment model provide a substantially methodological support for profiling the internal biological exposure and estimating the external dietary exposure to acrylamide.

Dispersive micro solid phase extraction (DMSPE) using polymer anion exchange (PAX) as the sorbent followed by UPLC–MS/MS for the rapid determination of four bisphenols in commercial edible oils

The present work presents a novel and rapid analytical method for the simultaneous analysis of bisphenol A (BPA), bisphenol B (BPB), bisphenol F (BPF) and bisphenol S (BPS) in edible oil based on dispersive micro solid phase extraction (DMSPE) for the first time followed by isotope dilution-ultra high performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). The edible oil sample was dispersed by n-hexane and extracted with ammoniated methanol-water solution. Then the target analytes were dispersedly absorbed using the polymer anion exchange (PAX) as the sorbent and eluted by acidic methanol. After that, four bisphenols were separated on a C18 column by gradient elution with methanol and 0.05% ammonium hydroxide in water as mobile phase, detected by MS/MS under multiple reactions monitoring (MRM) mode and quantified by internal standard method. The PAX amounts, adsorption time, concentrations of formic acid in the elution solvent and volume of elution solvent for the DMSPE technique were optimized. The limit of detection and quantitation (LOD and LOQ), matrix effect, recovery and precision of the developed method were investigated. Results indicated that BPS and the rest three bisphenols displayed excellent linearity in the concentration ranges of 0.1–50μg/L and 0.5–250μg/L, respectively, with correlation coefficients (R2) all larger than 0.998. Achieved MLODs (S/N=3) varied between 0.1–0.4μg/kg for all bisphenols. The mean recoveries at three spiked levels in edible oil were in the range of 87.3–108%. Intra-day precision (n=6) and inter-day precision (n=5) were <9% and <11%, respectively. This method is of rapid-and-simple pretreatment, accurate and sensitive, and suitable for the simultaneous determination of bisphenols in edible oil.

Quantitative Analysis of Ingenol in Euphorbia species via Validated Isotope Dilution Ultra-high Performance Liquid Chromatography Tandem Mass Spectrometry.

Various species of the Euphorbia genus contain diterpene ingenol and ingenol mebutate (ingenol-3-angelate), a substance found in the sap of the plant Euphorbia peplus and an inducer of cell death. A gel formulation of the drug has been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the topical treatment of actinic keratosis. To develop a rapid and reliable method for quantification of ingenol in various plant extracts. Methanolic extracts of 38 species of the Euphorbia genus were analysed via ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) after methanolysis and solid-phase extraction (SPE) purification. The 18 O-labelled ingenol analogue was prepared and used as an internal standard for ingenol content determination and method validation. The highest ingenol concentration (547mg/kg of dry weight) was found in the lower leafless stems of E. myrsinites. The screening confirms a substantial amount of ingenol in species studied previously and furthermore, reveals some new promising candidates. The newly established UHPLC-MS/MS method shows to be an appropriate tool for screening of the Euphorbia genus for ingenol content and allows selection of species suitable for raw material production and/or in vitro culture initiation. Copyright © 2017 John Wiley & Sons, Ltd.

Abstract 4247: Quantitative measurement of aflatoxin-serum albumin adducts reveals substantial exposure in Guatemala, a country with high rates of liver cancer mortality in men and women

Abstract Low-income countries across the world are increasingly burdened with growing rates of cancer incidence and mortality. Opportunities for prevention in these populations may be realized most readily for cancer sites with known etiologies that are amenable to intervention. Liver cancer is the sixth-most commonly occurring cancer worldwide and is the second-most common contributor to global cancer mortality. Aflatoxin B1 (AFB1) is a known and potent human hepatocarcinogen, with exposure occurring via ingestion of grains - especially maize - contaminated with Aspergillus flavus. Previous work in regions with endemic levels of liver cancer has shown that reducing AFB1 exposure attenuated liver cancer mortality - even in the presence of other known etiological exposures, such as hepatitis B virus. Guatemala experiences high rates of liver cancer - which is uniquely distributed equally between men and women - and consumes maize as a dietary staple, making the assessment of AFB1 exposure a high priority for cancer prevention efforts in this country. Therefore, we measured circulating levels of the AFB1-albumin adduct, an established biomarker of AFB1 exposure, in adults residing in five geographically and ecologically dispersed departments of Guatemala (Guatemala, Suchitepéquez, Sololá, Escuintla, and Quiché). Healthy men and women aged 40 and older were recruited for the study. Pregnant women or individuals with implanted cardiac devices were excluded from participation. Subjects (n=436) gave informed consent and provided demographic information and a venipuncture blood sample during a clinic visit. Serum was separated from whole blood, digested with protease, subjected to solid-phase extraction, and analyzed with isotope dilution ultrahigh-performance liquid chromatography-triple quadrupole mass spectrometry. Serum albumin was measured by ELISA. AFB1-albumin adducts (pg AFB1-lysine adduct/mg albumin) were detectable in 100% of samples, with a mean ± SEM of 21.6 ± 2.5 and range of 0.2 - 814.8. These levels are similar to those observed in regions of China in the 1980s, prior to reductions in AFB1 exposure and liver cancer mortality. Stratification by demographic factors revealed significant differences in aflatoxin levels in urban (9.6 ± 1.3) versus rural (28.8 ± 3.9) subjects (p ≤ 0.001). Accordingly, AFB1-albumin adduct levels were lowest in the urban municipality of Escuintla (5.8 ± 1.1) and highest in rural Suchitepéquez (36.4 ± 4.8). Exposure was significantly different by sex, with men having higher levels compared to women (30.0 ± 5.4 vs. 15.3 ± 1.3, p = 0.01). These data show that AFB1 exposure in Guatemalan adults is similar to levels previously shown to increase liver cancer risk. Larger cohort and prospective studies are warranted to assess the relationship between liver cancer incidence and AFB1 exposure in this population. Citation Format: Joshua W. Smith, Patricia A. Egner, Mariana Lazo, Alvaro Rivera, Fernanda Kroker, Neal D. Freedman, Katherine A. McGlynn, Manuel Ramirez-Zea, Eliseo Guallar, John D. Groopman. Quantitative measurement of aflatoxin-serum albumin adducts reveals substantial exposure in Guatemala, a country with high rates of liver cancer mortality in men and women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4247. doi:10.1158/1538-7445.AM2017-4247