Development of a Sensitive High-Resolution Mass Spectrometry Approach for Urea Nitrogen Quantitation in Small Volumes of Bronchoalveolar Lavage Fluid (BALF).

A sensitive, selective, and quantitative method incorporating high-resolution mass spectrometry was developed for the determination of blood urea nitrogen (BUN) in bronchoalveolar lavage fluid. The method requires no sample cleanup or derivatization prior to analysis. High-performance liquid chromatography (HPLC) on a Hypersil Gold PFP column (100 × 3 mm, 3 µm particle size) connected to a C18 guard column was employed for a 10 min chromatographic separation. The detection of urea was achieved using a Thermo Scientific Q-Exactive Plus instrument incorporating selected ion monitoring (SIM) modes for the protonated adduct of urea. The urea analytical measuring range for the method is 0.047-17.134 mg/dL, resulting in a BUN analytical measurement range of 0.022-8.007 mg/dL, which allows for quantitation over 3 orders of magnitude (R2 = 0.999). In addition, the method is suitable for small sample volumes (15 µL) with a high level of accuracy, precision, and specificity.

Quantitation of Trans-Fatty Acids in Children from the National Health and Nutrition Examination Survey.

OBJECTIVES: Trans-fatty acids (TFA) are geometric isomers of naturally occurring cis-fatty acids, formed industrially through partial hydrogenation of vegetable oils or naturally in ruminant animals. TFA intake has been associated with risk factors for cardiovascular disease; therefore, reduced TFA consumption is a major public health objective. Food intake studies have provided estimates for TFA levels in the U.S. and we previously assessed TFA blood concentrations in fasting U.S. adults in the National Health and Nutrition Examination Survey (NHANES), finding an average 54% decrease in TFA between 1999-2000 and 2009-2010. The aim of this study was to determine nationally representative estimates for plasma TFA concentrations in children in the U.S. population. METHODS: We used the CDC isotope dilution-gas chromatography-negative chemical ionization-mass spectrometry (ID-GC-NCI-MS) method to quantitate four major TFA, palmitelaidic acid, elaidic acid, trans-vaccenic acid, and linoelaidic acid, in a nationally representative group of children in the U.S. population using NHANES samples. TFA were derivatized with pentafluorobenzyl-bromide and analyzed by GC-MS in selected ion monitoring mode using NCI with methane reagent gas. Separation was carried out on a Select FAME 200 m column with hydrogen carrier gas. The TFA limits of detection ranged from 0.02 µM to 0.43 µM. The intraday and inter-day % coefficients of variation ranged from 1-11% CV and 6-15% CV respectively. The mean accuracy for all four TFA was 102% (95% CI: 98%-107%). RESULTS: The TFA were measured in 916 participants (age: 6-19 years) from NHANES 1999-2000 and 1718 participants (age: 3-19 years) from NHANES 2009-2010, and were detected in all samples. Geometric means for the sum of the 4 TFAs were 80.7 µmol/l (95% CI: 74.6-87.3 µmol/L) and 30.6 µmol/l (95% CI: 29.0-32.3 µmol/L) in the NHANES 1999-2000 and 2009-2010, respectively. Overall TFA concentrations were 62% lower in NHANES 2009-2010 compared to 1999-2000. CONCLUSIONS: TFA levels in children in the U.S. population are notably lower in NHANES 2009-2010 compared to NHANES 1999-2000 suggesting an overall reduction of TFA levels in children. FUNDING SOURCES: N/A.

High-throughput, simultaneous quantitation of hemoglobin adducts of acrylamide, glycidamide, and ethylene oxide using UHPLC-MS/MS.

Ethylene oxide (EO), acrylamide (AA) and glycidamide (GA) exposures are associated with mammary tumors in animals. Currently available information about human exposure to these chemicals is limited creating the need for analytical methods to assess their exposure. We developed a sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to simultaneously quantitate hemoglobin (Hb) N-terminal valine adducts of AA (HbAA), GA (HbGA), and EO (HbEO) using modified Edman reaction. The limits of detection of this method were 3.9, 4.9 and 12.9 in pmol/g Hb for HbAA, HbGA and HbEO, respectively. The among-day and within-day precision for all analytes determined with three levels of quality control pools ranged from 2.2-13.0% in percent coefficient of variation (%CV). The accuracy determined by standard addition was between 94 and 111% among all analytes. The median HbAA, HbGA and HbEO values in 34 self-reported non-smokers were 64.9, 45.3 and 113.6 pmol/g Hb and in 70 self-reported smokers were 127.8, 69.6 and 237.1 pmol/g Hb, respectively. HbAA, HbGA, and HbEO were detectable in all samples suggesting that the described method is suitable for measuring hemoglobin adducts of AA, GA and EO in the general population. This high throughput method can process 148 samples in 8 h. The HbEO/HbGA ratio appears independent of the HbAA levels in non-smokers and decreases with increasing HbAA concentration in smokers. This new method is suitable for measuring human exposure to AA, GA and EO and can provide further insight into the metabolism of these chemicals in humans.

Among 10 sociodemographic and lifestyle variables, smoking is strongly associated with biomarkers of acrylamide exposure in a representative sample of the U.S. Population.

Hemoglobin adducts of acrylamide (HbAA) and glycidamide (HbGA) have been measured as biomarkers of acrylamide exposure and metabolism in a nationally representative sample of the U.S. population in the NHANES 2003-2004. We assessed the association of sociodemographic (age, sex, race-ethnicity, education, and income) and lifestyle (smoking, alcohol consumption, BMI, physical activity, and dietary supplement use) variables with these biomarkers in U.S. adults (aged ≥ 20 y). We used bivariate and multiple regression models and assessed the magnitude of an estimated change in biomarker concentration with change in a covariable for 2 biomarkers of acrylamide exposure. Smoking was strongly and significantly correlated with HbAA and HbGA concentrations (rs = 0.51 and 0.42, respectively), with biomarker concentrations being 126 and 101% higher in smokers compared with nonsmokers after adjusting for sociodemographic and lifestyle covariates. Age was moderately and significantly correlated with both biomarkers (rs = -0.21 and -0.22, respectively). BMI (rs = -0.11) and alcohol consumption (rs = 0.13) were weakly yet significantly correlated with HbAA concentrations only. The estimated percentage change in biomarker concentration was ≤ 20% for all variables other than smoking after adjusting for sociodemographic and lifestyle covariates. Using multiple regression models, the sociodemographic variables explained 9 and 7% whereas the sociodemographic and lifestyle variables together explained 46 and 25% of the variability in HbAA and HbGA, respectively, showing the importance of considering and adequately controlling for these variables in future studies. Our findings will be useful in the design and analysis of future studies that assess and evaluate exposure to acrylamide and its metabolism to glycidamide.