Isotope-dilution Chromatography-tandem Mass Research Articles

Hair product use and urinary biomarker concentrations of non-persistent endocrine disrupting chemicals among reproductive-aged Black women

BackgroundStudies have shown an association between hair product use and adverse health outcomes. Scientists have hypothesized that exposure to endocrine-disrupting chemicals (EDCs) drives these associations, but few studies have directly evaluated associations between hair product use and biomarkers of EDCs. Even more limited are studies of Black women, who frequently use EDC-containing products (e.g., hair relaxers). ObjectiveWe estimated associations between hair product use and EDC biomarker concentrations. MethodsWe leveraged cross-sectional data from the Study of Environment, Lifestyle, and Fibroids, a cohort of females aged 23–34 years who self-identified as Black/African American from the Detroit-metropolitan area (USA; n = 425). On structured questionnaires, participants reported their past 24-h and past 12-month use of hair products, including relaxers/straighteners/perms, styling products, moisturizers, oils, and hair food. We quantified urinary concentrations of 19 phthalate/phthalate alternative metabolites, 7 phenols, and 4 parabens using high performance liquid chromatography isotope dilution tandem mass spectrometry. EDC biomarker concentrations were creatinine-adjusted and natural log-transformed. We used multivariable linear regression to estimate mean percent differences in EDC biomarker concentrations and 95% confidence intervals (CIs) associated with hair product use, adjusting for sociodemographic confounders. ResultsHair product use was associated with greater concentrations of multiple EDC biomarkers. Notably, use of hair products in the previous 24 h (compared with non-use) was associated with 16.2% (95% CI = 0.7%, 35.9%), 35.0% (95% CI = 2.6%, 77.6%), and 32.3% (95% CI = 8.8%, 92.0%) higher concentrations of mono-isobutyl phthalate, methyl paraben, and ethyl paraben, respectively. Use of hair relaxers/straighteners/perms, styling products, moisturizers, oils, and hair food in the past 12 months was also associated with higher concentrations of multiple phthalate, phenol, and paraben biomarkers. ConclusionHair product use was associated with higher biomarker concentrations of multiple phthalates, phenols, and parabens. These findings suggest that hair products are potentially important exposure sources for hormonally-active chemicals among Black women.

Unexpected sensitivity enhancement in analysing alfatoxin M1 using LC-IDMS

Aflatoxin M1 (AFM1) and patulin (PAT) are regulated mycotoxins, which are generally analyzed in food samples alone (“one analyte at a time”). This paper presents the development of a liquid chromatography isotope dilution tandem mass spectrometric (LC-IDMS) method for the determination of AFM1 in milk-based products, which method was then successfully applied to PAT in apple-based food samples. The method involved a selective solid-phase extraction clean-up at acidic pH employing mixed-mode cation exchange cartridges. The purification allowed the adequate adsorption of neutral target compounds and basic matrix constituents of the sample to the reversed-phase (RP) and to the cation exchange phase of the cartridge, respectively. The sample elution with pure methanol yielded an extract free from basic matrices. The remaining acidic and neutral matrix constituents of the samples were separated from the analytes using mixed-mode anion exchange HPLC column. Mobile phases without any modifiers were used for the highest sensitivity and better selectivity. The intensity enhancement obtained by neutral eluent composition was inter-laboratory tested and confirmed on same type of instruments. The sample preparation requires low amount of organic solvent, thus adhered to green analytical chemistry. The developed method for analyzing AFM1 resulted in low matrix effect of LC-IDMS separation −4.9-(−1.5%) and high precision (2.7–5.9%), calculated under method validations at spiking levels between 0.005 and 0.050 µg/kg. Method performance characteristics for analyzing PAT were also evaluated at 10 and 25 µg/kg levels. Finally, the advantageous features of the newly developed method were briefly compared and discussed with the existing approaches.

Determination of 3-monoiodotyrosine and 3,5-diiodotyrosine in newborn urine and dried urine spots by isotope dilution tandem mass spectrometry.

High levels of 3-mono- and 3,5-diiodotyrosine (MIT and DIT, respectively) in urine have been related to iodotyrosine dehalogenase 1 deficiency, a type of congenital hypothyroidism. However, the determination of MIT and DIT in urine is not included in newborn screening programs performed in clinical laboratories to detect inborn errors of metabolism. We report here on the development of an analytical method for the determination of MIT and DIT in newborn urine and dried urine spots (DUS) by Liquid Chromatography Isotope Dilution tandem Mass Spectrometry (LC-IDMSMS). The development included the synthesis of 15N-monoiodotyrosine and 13C2-diiodotyrosine through the iodination of 15N-tyrosine and 13C2-tyrosine, respectively, using bis(pyridine)iodonium(I) tetrafluoroborate (IPy2BF4). Both labelled analogues were added at the beginning of the sample preparation procedure and used to develop both single- and double-spike LC-IDMS methods for the determination of MIT and DIT. The developed double spike methodology was able to quantify and correct possible MIT ↔ DIT interconversions throughout the sample preparation, which was observed for concentrated urine samples but not for DUS. Suppression matrix effects on the absolute signals of MIT and DIT were observed in urine samples but did not affect the IDMS results as recoveries on urine samples at different dilution factors could be considered quantitative. Method detection limits were 0.018 and 0.046 ng g-1 (limits of quantification 0.06 and 0.15 ng g-1) by single-spike IDMS, for MIT and DIT, respectively, in the analysis of urine samples and 0.07 and 0.05 ng g-1 (limits of quantification 0.23 and 0.17 ng g-1) for MIT and DIT, respectively, in the analysis of DUS. No significant differences were obtained for MIT concentrations in the analysis of the same newborn samples stored as liquid urine or DUS when the results were corrected for the creatinine content. Finally, 36 DUS samples from healthy newborns were analyzed and MIT was detected in all samples at low ng mg-1creatinine levels.

Development and certification of a reference material for zearalenone in maize germ oil

Zearalenone (ZEN), an estrogenic mycotoxin produced by several species of Fusarium fungi, is a common contaminant of cereal-based food worldwide. Due to frequent occurrences associated with high levels of ZEN, maize oil is a particular source of exposure. Although a European maximum level for ZEN in maize oil exists according to Commission Regulation (EC) No. 1126/2007 along with a newly developed international standard method for analysis, certified reference materials (CRM) are still not available. To overcome this lack, the first CRM for the determination of ZEN in contaminated maize germ oil (ERM®-BC715) was developed in the frame of a European Reference Materials (ERM®) project according to the requirements of ISO Guide 35. The whole process of CRM development including preparation, homogeneity and stability studies, and value assignment is presented. The assignment of the certified mass fraction was based upon an in-house study using high-performance liquid chromatography isotope dilution tandem mass spectrometry. Simultaneously, to support the in-house certification study, an interlaboratory comparison study was conducted with 13 expert laboratories using different analytical methods. The certified mass fraction and expanded uncertainty (k = 2) of ERM®-BC715 (362 ± 22) μg kg−1 ZEN are traceable to the SI. This reference material is intended for analytical quality control and contributes to the improvement of consumer protection and food safety.Graphical abstract

Quantification of glyphosate and other organophosphorus compounds in human urine via ion chromatography isotope dilution tandem mass spectrometry

Organophosphorus pesticides are the most used pesticides in the United States. Most organophosphorus pesticides are composed of a phosphate (or phosphorothioate or phosphorodithioate) moiety and a variable organic group. Organophosphorus pesticides are scrutinized by regulatory bodies and agencies because of their toxicity or suspected carcinogenicity. Upon exposure, organophosphorus pesticides and their metabolites eliminate in urine; these urinary biomarkers are useful to evaluate human exposure. We developed a method using stable isotope dilution, ion chromatography tandem mass spectrometry for quantification in urine of 6 O,O-dialkylphosphates, metabolites of organophosphorus insecticides, and glyphosate, the most used herbicide in the United States. With simple and minimal sample preparation, the analytical method is selective and sensitive (limits of detection are 0.2-0.8 μg/L), accurate (>85%) and precise (relative standard deviation <20%), depending on the analyte. To assess the suitability of the method in real exposure scenarios, we analyzed samples collected anonymously from subjects with suspected exposure to pesticides (n = 40) or who had been on an organic diet (n = 50). We detected glyphosate in 80% of subjects reporting an organic diet and in 78% of those with suspected glyphosate exposure; concentrations ranged from <0.2 to 28.6 μg/L. Median concentrations were 0.39 μg/L for the organic diet group and 0.40 μg/L for individuals with suspected exposure. Interestingly, interquartile ranges were considerably higher among those reporting pesticide exposure (0.63 μg/L) than those consuming organic diets (0.42 μg/L). These data suggest that the method meets typical validation benchmark values and is sensitive to investigate background exposures in the general population.

Association Between Bisphenol A Exposure and Risk of All-Cause and Cause-Specific Mortality in US Adults

Bisphenol A (BPA) is a major public health concern because of its high-volume industrial production, ubiquitous exposure to humans, and potential toxic effects on multiple organs and systems in humans. However, prospective studies regarding the association of BPA exposure with long-term health outcomes are sparse. To examine the association of BPA exposure with all-cause mortality and cause-specific mortality among adults in the United States. This nationally representative cohort study included 3883 adults aged 20 years or older who participated in the US National Health and Nutrition Examination Survey 2003-2008 and provided urine samples for BPA level measurements. Participants were linked to mortality data from survey date through December 31, 2015. Data analyses were conducted in July 2019. Urinary BPA levels were quantified using online solid-phase extraction coupled to high-performance liquid chromatography-isotope dilution tandem mass spectrometry. Mortality from all causes, cardiovascular disease, and cancer. This cohort study included 3883 adults aged 20 years or older (weighted mean [SE] age, 43.6 [0.3] years; 2032 women [weighted, 51.4%]). During 36 514 person-years of follow-up (median, 9.6 years; maximum, 13.1 years), 344 deaths occurred, including 71 deaths from cardiovascular disease and 75 deaths from cancer. Participants with higher urinary BPA levels were at higher risk for death. After adjustment for age, sex, race/ethnicity, socioeconomic status, dietary and lifestyle factors, body mass index, and urinary creatinine levels, the hazard ratio comparing the highest vs lowest tertile of urinary BPA levels was 1.49 (95% CI, 1.01-2.19) for all-cause mortality, 1.46 (95% CI, 0.67-3.15) for cardiovascular disease mortality, and 0.98 (95% CI, 0.40-2.39) for cancer mortality. In this nationally representative cohort of US adults, higher BPA exposure was significantly associated with an increased risk of all-cause mortality. Further studies are needed to replicate these findings in other populations and determine the underlying mechanisms.

Associations of Perfluoroalkyl Substances with Incident Natural Menopause: The Study of Women's Health Across the Nation.

Previous epidemiologic studies of per- and polyfluoroalkyl substances (PFASs) and menopausal timing conducted in cross-sectional settings were limited by reverse causation because PFAS serum concentrations increase after menopause. To investigate associations between perfluoroalkyl substances and incident natural menopause. A prospective cohort of midlife women, the Study of Women's Health Across the Nation, 1999-2017. 1120 multiracial/ethnic premenopausal women aged 45-56 years. Serum concentrations of perfluoroalkyls were quantified by high-performance liquid chromatography isotope dilution tandem mass spectrometry. Natural menopause was defined as the bleeding episode prior to at least 12 months of amenorrhea not due to surgery or hormone use. Cox proportional hazards models were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs). Participants contributed 5466 person-years of follow-up, and 578 had incident natural menopause. Compared with the lowest tertile, women at the highest tertile of baseline serum concentrations had adjusted HR for natural menopause of 1.26 (95% CI: 1.02-1.57) for n-perfluorooctane sulfonic acid (n-PFOS) (Ptrend = .03), 1.27 (95% CI: 1.01-1.59) for branched-PFOS (Ptrend = .03), and 1.31 (95% CI: 1.04-1.65) for n-perfluorooctanoic acid (Ptrend = .01). Women were classified into four clusters based on their overall PFAS concentrations as mixtures: low, low-medium, medium-high, and high. Compared with the low cluster, the high cluster had a HR of 1.63 (95% CI: 1.08-2.45), which is equivalent to 2.0 years earlier median time to natural menopause. This study suggests that select PFAS serum concentrations are associated with earlier natural menopause, a risk factor for adverse health outcomes in later life.

Determination of Alternaria Toxins in Sunflower Oil by Liquid Chromatography Isotope Dilution Tandem Mass Spectrometry.

Alternaria toxins have gained attention as a potential health risk and can be classified as emerging mycotoxins. As a result, they are candidates to be regulated by the European Commission. This paper describes a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for analyzing five Alternaria toxins in sunflower oil, which is a rather different type of sample to those matrices investigated in earlier published papers. An optimal sample preparation condition was achieved when samples were dissolved in n-hexane and extracted with methanol/water mixture, followed by sample pre-concentration with solvent evaporation. This study is the first focusing only on this lipophilic matrix and in using all corresponding isotopically labeled internal standards (ISTD) to compensate the matrix effect that strongly influences the LC-MS/MS analysis of toxins. Target compounds were separated on Zorbax Extend C-18 column enabling the analysis at alkaline pH of 8.8 that was necessary to obtain appropriate peak shape of tenuazonic acid and to separate the analytes at baseline. The method was validated according to the EU 2002/657/EC Decision and all the analytical performance characteristics met the requirements. The recovery was between 74% and 122% in fortified sunflower oil samples and the precision varied from 9% to 22%. The method was successfully demonstrated for sunflower seed quality check (QC) samples. Finally, 16 different sunflower oil samples were measured; and tenuazonic acid and tentoxin toxins were detected at levels close to LOQ concentrations.

Establishment and application of an isotope dilution chromatography tandem mass spectrometry reference method for Hcy quantification

Objective To establish a reference measurement procedure for the determination of human serum homocysteine by isotope dilution high performance liquid chromatography-tandem mass spectrometry (ID-LC/MS/MS), and to apply it to establish sample target values for external quality assessment (EQA) in clinical laboratories. Methods The reference method of Hcyquantification in our laboratory was establishedaccording to the method recommended by the Joint Committee for Traceability in Laboratory Medicine (JCTLM). The precision, trueness, specificity, residue and matrix effect of the method were evaluated. The reference method was applied to establish Hcy target values for samples of the second EQA in Shanghai of 2018. Results The method detects 12.5μmol/L and 37.4μmol/L samples in three batches in three days, and the CV between batches is 1.03% and 2.10%, respectively. The measured values of Standard reference material (SRM) 1955 of National Institute of Standards and Technology (NIST) were within the specified uncertainty range. No matrix effect and carryover were observed. The second EQA data in 2018 showed that the average value of domestic reagent group was lower than that of reference method, and that of imported reagent group was higher than that of reference method. Conclusion Thereference measurement procedure of ID-LC/MS/MS was successfully established to determine the human serum homocysteine. It is expected to play a role in tracing the quantities of Hcy in clinical laboratories. Key words: Homocysteine; Chromatography, liquid; Tandem mass spectrometry; Reference values; Reference standards

Complete validation according to current international criteria of a confirmatory quantitative method for the determination of nitrofuran metabolites in seafood by liquid chromatography isotope dilution tandem mass spectrometry

Despite the ban of nitrofurans (NFs) for use in food production in many countries in the 1990s, NF metabolites in food are still regularly detected during import control testing. We have developed a confirmatory routine method for the detection and quantification of NF metabolites in seafood using LC-MS/MS and validated the method according to the strict criteria in European legislation and Codex Alimentarius. Method characteristics were found to fulfill the criteria. We report for the first time a new false positive for 1-amino-2,4-imidazolidinedione (AHD), the metabolite of Nitrofurantoin (NFT). By using optimized washing procedures, the non tissue bound false positives can be minimized. The results from the validation on both lean and fatty fish and crustaceans, results from proficiency tests and routine use over many years, demonstrates that the method is fit for purpose to determine NF metabolites in the seafood category.

A Mediterranean-style Eating Pattern Lower in Lean Red Meat Reduced Plasma Trimethylamine N-Oxide in Adults Classified as Overweight or Obese (P08-030-19)

ObjectivesTo determine the effect of adopting a Mediterranean-style eating pattern with different quantities of lean unprocessed red meat on the plasma concentration of Trimethylamine N-Oxide (TMAO), an emerging potential cardiovascular disease risk factor, in middle-aged adults classified as overweight or obese. MethodsThirty-nine adults (12 males and 27 females; age 46 ±10 years, BMI 30.6 ± 3.6 kg/m2; mean ± SD) participated in a randomized, crossover and controlled feeding trial. Each participant consumed a Mediterranean-style eating pattern for two 5-week intervention periods separated by ∼4 weeks of consuming their unrestricted, self-selected diet (washout). The Mediterranean-style eating pattern contained either a commonly recommended amount of red meat (∼200 g/wk, Med-200) or the average amount consumed in the U.S. (∼500 g/wk, Med-500). Plasma samples were collected before (baseline) and at the end (post) of the two intervention periods and TMAO was measured by stable isotope dilution chromatography tandem mass spectrometry. Geometric means were compared with mixed model tests in SAS. ResultsBaseline TMAO concentrations were not different between interventions (Med-200 = 4.4 ± 2.7 and Med-500 = 5.2 ± 4.5 μM, P = 0.476). These concentrations are similar to values reported in healthy adults. Post-intervention, TMAO was lower in Med-200 vs. Med-500 (3.1 ± 1.2 and 5.0 ± 2.6 μM, P = 0.001). ConclusionsThe 2015–2020 Dietary Guidelines for Americans encourages adopting a Mediterranean-style eating pattern to promote cardiovascular health, including reduced blood pressure and improved lipid-lipoprotein profile. The current results suggest that adopting a Mediterranean-study eating pattern may also reduce the cardiovascular disease risk factor TMAO, but only when it contains a lower, recommended, amount of lean unprocessed red meat. The influences of animal- and plant-based foods consumed with a Mediterranean-style and other healthy eating patterns on emerging cardiovascular disease risk factors remain to be determined. This trial was registered at clinicaltrials.gov as NCT02573129. Funding SourcesSupported by theBeef Checkoff, the Pork Checkoff, the NIH pre-doctoral training grant 5T32DK076540-08, the NIH-supported Indiana Clinical and Translational Sciences Institute and the USDA-ARS-Western Human Nutrition Research Center.

A Randomized Controlled-feeding Trial Based on the Dietary Guidelines for Americans Does Not Affect Plasma Trimethylamine N-oxide Levels in Women (P08-031-19)

ObjectivesTrimethylamine N-oxide is a gut-mediated metabolite associated with cardiovascular disease. Acute diet challenges demonstrate that circulating TMAO concentrations are diet-responsive; however, long-term trials investigating how different dietary patterns affect circulating TMAO concentrations are sparse. The objective of the current study was to determine whether consumption of a diet patterned after the USDA’s 2010 Dietary Guidelines for Americans (DGA), as compared to the typical American Diet (TAD), would reduce plasma TMAO levels. MethodsA randomized, double-blind, controlled 8-wk intervention was conducted in overweight and obese women selected according to indexes of insulin resistance or dyslipidemia. Women were randomly assigned to the DGA or TAD group (n = 28 DGA and 24 TAD). The TAD diet was based on average adult intake from the NHANES 2009–2010. All foods and beverages were provided during the intervention and matched to each participant’s energy requirement to ensure maintenance of initial body weight. Plasma samples were collected at baseline, 1 wk of their typical diet, and after 2 and 8 weeks of dietary intervention, in an overnight-fasted condition, and utilized to quantitate circulating TMAO concentrations using stable isotope dilution chromatography tandem mass spectrometry. Statistical analysis were performed in SAS using analysis of covariance and partial correlation. ResultsFollowing 2 or 8 weeks of dietary intervention, plasma TMAO concentrations were not different between the DGA and TAD diets (3.45 ± 0.41 vs 2.91 ± 0.38 μM at week 2; 3.48 ± 0.41 vs 3.00 ± 0.40 μM at week 8, Mean ± SE, respectively). Post-intervention TMAO concentration was correlated to initial TMAO concentrations and body mass index but not age. Furthermore, we did not observe significant correlations between TMAO and measures of endothelial function (Endopat) or insulin resistance (HOMA-IR) which may reflect the relative health of the overall population enrolled in the study. ConclusionsConsumption of a diet based on the USDA’s 2010 Dietary Guidelines for Americansfor 8 weeks did not reduce plasma TMAO levels in this study. Funding SourcesSupported by National Dairy Council; Campbell Soup Co.; USDA-ARS Projects 2032-51530-022-00D and 6026-51000-010-05S.

Liquid chromatography–isotope dilution tandem mass spectrometry method for the measurement of urea in human serum and assignment of reference values to external quality assessment samples

Reference measurement procedures are essential for the accurate measurement of urea in human serum. The widely used gas chromatography-isotope dilution mass spectrometry (GC–IDMS) involves tedious derivatization processes. We developed and validated a simple liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) method for the measurement of urea in human serum.In this method, 13C,15N2-urea was spiked into serum samples as the internal standard, and pure urea certified reference material (CRM) was used as the calibration standard to ensure the traceability of the measurements to the International System of Units (SI). The sample cleanup procedure involved a simple protein precipitation step, followed by centrifugation and filtration. The developed LC-IDMS/MS method was found to be accurate and highly reproducible. The coefficient of variation (CV) was generally below 1%. Good accuracy was demonstrated by measuring serum CRMs from two National Metrology Institutes (NMIs) with relative deviations from the certified values being close to or below 1%, and measurement uncertainties of 2.0%–2.8%. The method was used to assign the reference values of serum urea in ten cycles of an external quality assessment (EQA) program for clinical laboratories over a period of four years. The robust mean of the reported results from clinical laboratories using automated analyzers were close to the assigned values from LC-IDMS/MS method.With good accuracy and precision, as well as simple sample cleanup procedure, the developed LC-IDMS/MS method can be used as a reference measurement procedure for the measurement of urea in human serum.

Lewisite Metabolites in Urine by Solid Phase Extraction-Dual Column Reversed-Phase Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry.

Lewisite (2-chlorovinyldichloroarsine) is a chemical warfare agent developed during World War I. A quantitative method using solid phase extraction (SPE) followed by dual column liquid chromatography (LC)-isotope dilution tandem mass spectrometry(MS-MS) was developed for the determination of (2-chlorovinyl)arsonic acid (CVAOA), a metabolite of Lewisite, in human urine. The sample was treated with hydrogen peroxide to oxidize any (2-chlorovinyl)arsonous acid (CVAA) that remained in the trivalent arsenic oxidation state. There was 1.19% (arsenic purity) of bis-(2-chlorovinyl)arsinic acid (BCVAOA), a minor Lewisite metabolite, in the stock CVAA material. The high-throughput method qualitatively assessed BCVAOA simultaneously utilizing normal-phase silica SPE followed by reversed-phase C18 LC for an orthogonal separation. The chromatographic method results in a5.8-min cycle time with adequate retention (k'=2.4) of CVAOA. The mass spectrometer was operated in positive electrospray ionization mode with quantitativem/z 186.9→61.0 and confirmation 186.9→91.0 mass transitions. This selective method demonstrated linearity, accuracy and reproducibility for the clinically relevant calibration range (25-3,200 µg/L as CVAA). The method detection limit was 3.3 µg/L as CVAA from a 10µL injection. This LC-MS-MS emergency response method has a throughput of >240 samples(2.5 extracted 96-well plates) per day.

Monitoring exposure to polycyclic aromatic hydrocarbons in an Australian population using pooled urine samples

Integrated exposure to polycyclic aromatic hydrocarbons (PAHs) can be assessed through monitoring of urinary mono-hydroxylated PAHs (OH-PAHs). The aim of this study was to provide the first assessment of exposure to PAHs in a large sample of the population in Queensland, Australia including exposure to infant (0–4years). De-identified urine specimens, obtained from a pathology laboratory, were stratified by age and sex, and pooled (n=24 pools of 100) and OH-PAHs were measured by gas chromatography–isotope dilution–tandem mass spectrometry. Geometric mean (GM) concentrations ranged from 30ng/L (4-hydroxyphenanthrene) to 9221ng/L (1-naphthol). GM of 1-hydroxypyrene, the most commonly used PAH exposure biomarker, was 142ng/L. The concentrations of OH-PAHs found in this study are consistent with those in developed countries and lower than those in developing countries. We observed no association between sex and OH-PAH concentrations. However, we observed lower urinary concentrations of all OH-PAHs in samples from infants (0–4years), children (5–14years) and the elderly (>60year old) compared with samples from other age groups (15–29, 30–44 and 45–59years) which may be attributed to age-dependent behaviour-specific exposure sources.

An ameliorative protocol for the quantification of purine 5',8-cyclo-2'-deoxynucleosides in oxidized DNA.

5′,8-Cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) are lesions resulting from hydroxyl radical (HO·) attack on the 5′H of the nucleoside sugar moiety and exist in both 5′R and 5′S diastereomeric forms. Increased levels of cdA and cdG are linked to Nucleotide Excision Repair (NER) mechanism deficiency and mutagenesis. Discrepancies in the damage measurements reported over recent years indicated the weakness of the actual protocols, in particular for ensuring the quantitative release of these lesions from the DNA sample and the appropriate method for their analysis. Herein we report the detailed revision leading to a cost-effective and efficient protocol for the DNA damage measurement, consisting of the nuclease benzonase and nuclease P1 enzymatic combination for DNA digestion followed by liquid chromatography isotope dilution tandem mass spectrometry analysis.

Simplified Method for Quantifying Sulfur Mustard Adducts to Blood Proteins by Ultrahigh Pressure Liquid Chromatography−Isotope Dilution Tandem Mass Spectrometry.

Sulfur mustard binds to reactive cysteine residues, forming a stable sulfur-hydroxyethylthioethyl [SHETE]adduct that can be used as a long-term biomarker of sulfur mustard exposure in humans. The digestion of sulfur mustard-exposed blood samples with proteinase K following total protein precipitation with acetone produces the tripeptide biomarker [S-HETE]-Cys-Pro-Phe. The adducted tripeptide is purified by solid phase extraction, separated by ultra high pressure liquid chromatography, and detected by isotope dilution tandem mass spectrometry. This approach was thoroughly validated and characterized in our laboratory. The average interday relative standard deviation was ≤ 9.49%, and the range of accuracy was between 96.1 and 109% over a concentration range of 3.00 to 250. ng/mL with a calculated limit of detection of1.74 ng/mL. A full 96-well plate can be processed and analyzed in 8 h, which is 5 times faster than our previous 96-well plate method and only requires 50 μL of serum, plasma, or whole blood. Extensive ruggedness and stability studies and matrix comparisons were conducted to create a robust, easily transferrable method. As a result, a simple and high-throughput method has been developed and validated for the quantitation of sulfur mustard blood protein adducts in low volume blood specimens which should be readily adaptable for quantifying human exposures to other alkylating agents.

Quantitation of 4,4′-methylene diphenyl diisocyanate human serum albumin adducts

4,4′-Methylene diphenyl diisocyanate (herein 4,4′-MDI) is used in the production of polyurethane foams, elastomers, coatings, adhesives and the like for a wide range of commercial products. Occupational exposure to MDI levels above current airborne exposure limits can elicit immune mediated hypersensitivity reactions such as occupational asthma in sensitive individuals. To accurately determine exposure, there has been increasing interest in developing analytical methods to measure internal biomarkers of exposure to MDI. Previous investigators have reported methodologies for measuring MDI diamine metabolites and MDI-Lysine (4,4′-MDI-Lys) adducts. The purpose of this study was to develop and validate an ultra performance liquid chromatography isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS) quantitation method via a signature peptide approach to enable biomonitoring of 4,4′-MDI adducted to human serum albumin (HSA) in plasma. A murine, anti-4,4′-MDI monoclonal IgM antibody was bound to magnetic beads and utilized for enrichment of the MDI adducted HSA. Following enrichment, trypsin digestion was performed to generate the expected 414 site (primary site of adduction) 4,4′-MDI-adducted HSA signature peptide that was quantified by UPLC-ID/MS/MS. An Agilent 6530 UPLC/quadrupole time of flight MS (QTOF) system was utilized for intact adducted protein analysis and an Agilent 6490 UPLC/MS/MS system operated in multiple reaction monitoring (MRM) mode was utilized for quantification of the adducted signature peptide biomarker both for in chemico and worker serum samples. Worker serum samples were initially screened utilizing the previously developed 4,4′-MDI-Lys amino acid method and results showed that 12 samples were identified as quantifiable for 4,4′-MDI-Lys adducts. The signature peptide adduct approach was applied to the 12 worker samples identified as quantifiable for 4,4′-MDI-Lys adducts. Results indicated no positive results were obtained above the quantification limit by the signature peptide approach. If the 414 site of lysine adduction accounted for 100% of the 4,4′-MDI adductions in the signature peptide adduct approach, the three highest quantifiable samples by the 4,4′-MDI-Lys method should have at least been detectable by the signature peptide method. Results show that although the 4,4′-MDI signature peptide approach is more selective, it is 18 times less sensitive than the 4,4′-MDI-Lys method, thus limiting the ability to detect adduct levels relative to the 4,4′-MDI-Lys amino acid method.

Automated on-line column-switching high performance liquid chromatography isotope dilution tandem mass spectrometry method for the quantification of bisphenol A, bisphenol F, bisphenol S, and 11 other phenols in urine

Human exposure to bisphenol A (BPA) is widespread. However, in recent years, bisphenol analogs such as bisphenol S (BPS) and bisphenol F (BPF) are replacing BPA in the production of some consumer products. Because human exposure to these alternative bisphenols may occur, biomonitoring of these bisphenol analogs is warranted. In the present study, we developed and validated a sensitive and selective method that uses on-line solid phase extraction coupled to high performance liquid chromatography-isotope dilution tandem mass spectrometry with peak focusing to measure BPA, BPF, BPS, and 11 other environmental phenols in urine. The method required a small amount of sample (100μL) and minimal sample pretreatment. The limits of detection were 0.03ng/mL (BPS), 0.06ng/mL (BPF), 0.10ng/mL (BPA), and ranged from 0.1ng/mL to 1.0ng/mL for the other 11 phenols. In 100 urine samples collected in 2009-2012 from a convenience group of anonymous adults in the United States, of the three bisphenols, we detected BPA at the highest frequency and median concentrations (95%, 0.72ng/mL), followed by BPS (78%, 0.13ng/mL) and BPF (55%, 0.08ng/mL). This sensitive, rugged, and labor and cost-effective method could be used for the analysis of large number of samples for epidemiologic studies.

On-line solid phase extraction-high performance liquid chromatography–isotope dilution–tandem mass spectrometry approach to quantify N,N-diethyl-m-toluamide and oxidative metabolites in urine

Human exposure to N,N-diethyl-m-toluamide (DEET) occurs because of the widespread use of DEET as an active ingredient in insect repellents. However, information on the extent of such exposure is rather limited. Therefore, we developed a fast on-line solid phase extraction–high performance liquid chromatography–isotope dilution tandem mass spectrometry (HPLC-MS/MS) method to measure in urine the concentrations of DEET and two of its oxidative metabolites: N,N-diethyl-3-(hydroxymethyl)benzamide and 3-(diethylcarbamoyl)benzoic acid (DCBA). To the best of our knowledge, this is the first HPLC-MS/MS method for the simultaneous quantification of DEET and its select metabolites in human urine. After enzymatic hydrolysis of the conjugated species in 0.1mL of urine, the target analytes were retained and pre-concentrated on a monolithic column, separated from each other and from other urinary biomolecules on a reversed-phase analytical column, and detected by atmospheric pressure chemical ionization in positive ion mode. The limits of detection ranged from 0.1ngmL−1 to 1.0ngmL−1, depending on the analyte. Accuracy ranged between 90.4 and 104.9%, and precision ranged between 5.5 and 13.1% RSD, depending on the analyte and the concentration. We tested the usefulness of this method by analyzing 75 urine samples collected anonymously in the Southeastern United States in June 2012 from adults with no known exposure to DEET. Thirty eight samples (51%) tested positive for at least one of the analytes. We detected DCBA most frequently and at the highest concentrations. Our results suggest that this method can be used for the analysis of a large number of samples for epidemiological studies to assess human exposure to DEET.