Inaccurately Reported Statin Use Affects the Assessing of Lipid Profile Measures and Their Association with Coronary Artery Disease Risk.

BACKGROUND: Lipid profiling is central for coronary artery disease (CAD) risk assessment. Nonadherence or unreported use of lipid-lowering drugs, particularly statins, can significantly complicate the association between lipid profile measures and CAD clinical outcomes. By combining medication history evaluation with statin analysis in plasma, we determined the effects of inaccurately reported statin use on lipid profile measures and their association with CAD risk. METHODS: We compared medication history of statin use with statin concentration measurements, by liquid chromatography-tandem mass spectrometry, in 690 participants undergoing coronary angiography (63 ± 11 years of age). Nominal logistic regression was employed to model CAD diagnosis with statin measurements, phenotypic, and lipid profile characteristics. RESULTS: Medication history of statin use was confirmed by statin assay for 81% of the patients. Surprisingly, statins were detected in 46% of patients without statin use records. Nonreported statin use was disproportionately higher among older participants. Stratifying samples by statin history resulted in underestimated LDL-lipid measures. Apolipoprotein B concentrations had a significant inverse CAD association, which became nonsignificant upon re-stratification using the statin assay data. CONCLUSIONS: Our study uncovered prominent discrepancies between medication records and actual statin use measured by mass spectrometry. We showed that inaccurate statin use assessments may lead to overestimation and underestimation of LDL levels in statin user and nonuser categories, exaggerating the reverse epidemiology association between LDL levels and CAD diagnosis. Combining medication history and quantitative statin assay data can significantly improve the design, analysis, and interpretation of clinical and epidemiological studies.

Vitamin E Acetate in Bronchoalveolar-Lavage Fluid Associated with EVALI.

BACKGROUND: The causative agents for the current national outbreak of electronic-cigarette, or vaping, product use-associated lung injury (EVALI) have not been established. Detection of toxicants in bronchoalveolar-lavage (BAL) fluid from patients with EVALI can provide direct information on exposure within the lung. METHODS: BAL fluids were collected from 51 patients with EVALI in 16 states and from 99 healthy participants who were part of an ongoing study of smoking involving nonsmokers, exclusive users of e-cigarettes or vaping products, and exclusive cigarette smokers that was initiated in 2015. Using the BAL fluid, we performed isotope dilution mass spectrometry to measure several priority toxicants: vitamin E acetate, plant oils, medium-chain triglyceride oil, coconut oil, petroleum distillates, and diluent terpenes. RESULTS: State and local health departments assigned EVALI case status as confirmed for 25 patients and as probable for 26 patients. Vitamin E acetate was identified in BAL fluid obtained from 48 of 51 case patients (94%) in 16 states but not in such fluid obtained from the healthy comparator group. No other priority toxicants were found in BAL fluid from the case patients or the comparator group, except for coconut oil and limonene, which were found in 1 patient each. Among the case patients for whom laboratory or epidemiologic data were available, 47 of 50 (94%) had detectable tetrahydrocannabinol (THC) or its metabolites in BAL fluid or had reported vaping THC products in the 90 days before the onset of illness. Nicotine or its metabolites were detected in 30 of 47 of the case patients (64%). CONCLUSIONS: Vitamin E acetate was associated with EVALI in a convenience sample of 51 patients in 16 states across the United States. (Funded by the National Cancer Institute and others.).

The small HDL particle hypothesis of Alzheimer's disease.

We propose the hypothesis that small high-density lipoprotein (HDL) particles reduce the risk of Alzheimer's disease (AD) by virtue of their capacity to exchange lipids, affecting neuronal membrane composition and vascular and synaptic functions. Concentrations of small HDLs in cerebrospinal fluid (CSF) and plasma were measured in 180 individuals ≥60 years of age using ion mobility methodology. Small HDL concentrations in CSF were positively associated with performance in three domains of cognitive function independent of apolipoprotein E (APOE) ε4 status, age, sex, and years of education. Moreover, there was a significant correlation between levels of small HDLs in CSF and plasma. Further studies will be aimed at determining whether specific components of small HDL exchange across the blood, brain, and CSF barriers, and developing approaches to exploit small HDLs for therapeutic purposes.

Confirmation of Statin and Fibrate Use from Small-Volume Archived Plasma Samples by High-Throughput LC-MS/MS Method.

Designing studies for lipid-metabolism-related biomarker discovery is challenging because of the high prevalence of various statin and fibrate usage for lipid-lowering therapies. When the statin and fibrate use is determined based on self-reports, patient adherence to the prescribed statin dose regimen remains unknown. A potentially more accurate way to verify a patient's medication adherence is by direct analytical measurements. Current analytical methods are prohibitive because of the limited panel of drugs per test and large sample volume requirement that is not available from archived samples. A 4-min-long method was developed for the detection of seven statins and three fibrates using 10 µL of plasma analyzed via reverse-phase liquid chromatography and tandem mass spectrometry. The method was applied to the analysis of 941 archived plasma samples collected from patients before cardiac catheterization. When statin use was self-reported, statins were detected in 78.6% of the samples. In the case of self-reported atorvastatin use, the agreement with detection was 90.2%. However, when no statin use was reported, 42.4% of the samples had detectable levels of statins, with a similar range of concentrations as the samples from the self-reported statin users. The method is highly applicable in population studies designed for biomarker discovery or diet and lifestyle intervention studies, where the accuracy of statin or fibrate use may strongly affect the statistical evaluation of the biomarker data.

Apolipoprotein A-I modulates HDL particle size in the absence of apolipoprotein A-II.

Human high-density lipoproteins (HDLs) are a complex mixture of structurally related nanoparticles that perform distinct physiological functions. We previously showed that human HDL containing apolipoprotein A-I (APOA1) but not apolipoprotein A-II (APOA2), designated LpA-I, is composed primarily of two discretely sized populations. Here, we isolated these particles directly from human plasma by antibody affinity chromatography, separated them by high-resolution size-exclusion chromatography and performed a deep molecular characterization of each species. The large and small LpA-I populations were spherical with mean diameters of 109 Å and 91 Å, respectively. Unexpectedly, isotope dilution MS/MS with [15N]-APOA1 in concert with quantitation of particle concentration by calibrated ion mobility analysis demonstrated that the large particles contained fewer APOA1 molecules than the small particles; the stoichiometries were 3.0 and 3.7 molecules of APOA1 per particle, respectively. MS/MS experiments showed that the protein cargo of large LpA-I particles was more diverse. Human HDL and isolated particles containing both APOA1 and APOA2 exhibit a much wider range and variation of particle sizes than LpA-I, indicating that APOA2 is likely the major contributor to HDL size heterogeneity. We propose a ratchet model based on the trefoil structure of APOA1 whereby the helical cage maintaining particle structure has two "settings"-large and small-that accounts for these findings. This understanding of the determinants of HDL particle size and protein cargo distribution serves as a basis for determining the roles of HDL subpopulations in metabolism and disease states.

Composition-function analysis of HDL subpopulations: influence of lipid composition on particle functionality.

The composition-function relationship of HDL particles and its effects on the mechanisms driving coronary heart disease (CHD) is poorly understood. We tested the hypothesis that the functionality of HDL particles is significantly influenced by their lipid composition. Using a novel 3D-separation method, we isolated five different-sized HDL subpopulations from CHD patients who had low preß-1 functionality (low-F) (ABCA1-dependent cholesterol-efflux normalized for preß-1 concentration) and controls who had either low-F or high preß-1 functionality (high-F). Molecular numbers of apoA-I, apoA-II, and eight major lipid classes were determined in each subpopulation by LC-MS. The average number of lipid molecules decreased from 422 in the large spherical α-1 particles to 57 in the small discoid preß-1 particles. With decreasing particle size, the relative concentration of free cholesterol (FC) decreased in α-mobility but not in preß-1 particles. Preß-1 particles contained more lipids than predicted; 30% of which were neutral lipids (cholesteryl ester and triglyceride), indicating that these particles were mainly remodeled from larger particles not newly synthesized. There were significant correlations between HDL-particle functionality and the concentrations of several lipids. Unexpectedly, the phospholipid:FC ratio was significantly correlated with large-HDL-particle functionality but not with preß-1 functionality. There was significant positive correlation between particle functionality and total lipids in high-F controls, indicating that the lipid-binding capacity of apoA-I plays a major role in the cholesterol efflux capacity of HDL particles. Functionality and lipid composition of HDL particles are significantly correlated and probably both are influenced by the lipid-binding capacity of apoA-I.

Evaluation of Bronchoalveolar Lavage Fluid from Patients in an Outbreak of E-cigarette, or Vaping, Product Use-Associated Lung Injury - 10 States, August-October 2019.

CDC, the Food and Drug Administration (FDA), state and local health departments, and multiple public health and clinical partners are investigating a national outbreak of e-cigarette, or vaping, product use-associated lung injury (EVALI). Based on data collected as of October 15, 2019, 86% of 867 EVALI patients reported using tetrahydrocannabinol (THC)-containing products in the 3 months preceding symptom onset (1). Analyses of THC-containing product samples by FDA and state public health laboratories have identified potentially harmful constituents in these products, such as vitamin E acetate, medium chain triglyceride oil (MCT oil), and other lipids (2,3) (personal communication, D.T. Heitkemper, FDA Forensic Chemistry Center, November 2019). Vitamin E acetate, in particular, might be used as an additive in the production of e-cigarette, or vaping, products; it also can be used as a thickening agent in THC products (4). Inhalation of vitamin E acetate might impair lung function (5-7).

Zeptomole per milliliter detection and quantification of edema factor in plasma by LC-MS/MS yields insights into toxemia and the progression of inhalation anthrax.

Inhalation of Bacillus anthracis spores can cause a rapidly progressing fatal infection. B. anthracis secretes three protein toxins: lethal factor (LF), edema factor (EF), and protective antigen (PA). EF and LF may circulate as free or PA-bound forms. Both free EF (EF) and PA-bound-EF (ETx) have adenylyl cyclase activity converting ATP to cAMP. We developed an adenylyl cyclase activity-based method for detecting and quantifying total EF (EF+ETx) in plasma. The three-step method includes magnetic immunocapture with monoclonal antibodies, reaction with ATP generating cAMP, and quantification of cAMP by isotope-dilution HPLC-MS/MS. Total EF was quantified from 5PL regression of cAMP vs ETx concentration. The detection limit was 20 fg/mL (225 zeptomoles/mL for the 89 kDa protein). Relative standard deviations for controls with 0.3, 6.0, and 90 pg/mL were 11.7-16.6% with 91.2-99.5% accuracy. The method demonstrated 100% specificity in 238 human serum/plasma samples collected from unexposed healthy individuals, and 100% sensitivity in samples from 3 human and 5 rhesus macaques with inhalation anthrax. Analysis of EF in the rhesus macaques showed that it was detected earlier post-exposure than B. anthracis by culture and PCR. Similar to LF, the kinetics of EF over the course of infection were triphasic, with an initial rise (phase-1), decline (phase-2), and final rapid rise (phase-3). EF levels were ~ 2-4 orders of magnitude lower than LF during phase-1 and phase-2 and only ~ 6-fold lower at death/euthanasia. Analysis of EF improves early diagnosis and adds to our understanding of anthrax toxemia throughout infection. The LF/EF ratio may also indicate the stage of infection and need for advanced treatments.

Comparability of Lipoprotein Particle Number Concentrations Across ES-DMA, NMR, LC-MS/MS, Immunonephelometry, and VAP: In Search of a Candidate Reference Measurement Procedure for apoB and non-HDL-P Standardization.

BACKGROUND: Despite the usefulness of standard lipid parameters for cardiovascular disease risk assessment, undiagnosed residual risk remains high. Advanced lipoprotein testing (ALT) was developed to provide physicians with more predictive diagnostic tools. ALT methods separate and/or measure lipoproteins according to different parameters such as size, density, charge, or content, and equivalence of results across methods has not been demonstrated. METHODS: Through a split-sample study, 25 clinical specimens (CSs) were assayed in 10 laboratories before and after freezing using the major ALT methods for non-HDL particles (non-HDL-P) or apolipoprotein B-100 (apoB-100) measurements with the intent to assess their comparability in the current state of the art. RESULTS: The overall relative standard deviation (CV) of non-HDL-P and apoB-100 concentrations measured by electrospray differential mobility analysis, nuclear magnetic resonance, immunonephelometry, LC-MS/MS, and vertical autoprofile in the 25 frozen CSs was 14.1%. Within-method comparability was heterogeneous, and CV among 4 different LC-MS/MS methods was 11.4% for apoB-100. No significant effect of freezing and thawing was observed. CONCLUSIONS: This study demonstrates that ALT methods do not yet provide equivalent results for the measurement of non-HDL-P and apoB-100. The better agreement between methods harmonized to the WHO/IFCC reference material suggests that standardizing ALT methods by use of a common commutable calibrator will improve cross-platform comparability. This study provides further evidence that LC-MS/MS is the most suitable candidate reference measurement procedure to standardize apoB-100 measurement, as it would provide results with SI traceability. The absence of freezing and thawing effect suggests that frozen serum pools could be used as secondary reference materials.

Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in Frozen Human Plasma.

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

Optimization of the linear quantification range of an online trypsin digestion coupled liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform.

Tandem mass spectrometry (MS/MS)-based proteomic workflows with a bottom-up approach require enzymatic digestion of proteins to peptide analytes, usually by trypsin. Online coupling of trypsin digestion of proteins, using an immobilized enzyme reactor (IMER), with liquid chromatography (LC) and MS/MS is becoming a frequently used approach. However, finding IMER digestion conditions that allow quantitative analysis of multiple proteins with wide range of endogenous concentration requires optimization of multiple interactive parameters: digestion buffer flow rate, injection volume, sample dilution, and surfactant type/ concentration. In this report, we present a design of experiment approach for the optimization of an integrated IMER-LC-MS/MS platform. With bovine serum albumin as a model protein, the digestion efficacy and digestion rate were monitored based on LC-MS/MS peak area count versus protein concentration regression. The optimal parameters were determined through multivariate surface response modeling and consideration of diffusion controlled immobilized enzyme kinetics. The results may provide guidance to other users for the development of quantitative IMER-LC-MS/MS methods for other proteins.

The effects of apolipoprotein B depletion on HDL subspecies composition and function.

HDL cholesterol (HDL-C) efflux function may be a more robust biomarker of coronary artery disease risk than HDL-C. To study HDL function, apoB-containing lipoproteins are precipitated from serum. Whether apoB precipitation affects HDL subspecies composition and function has not been thoroughly investigated. We studied the effects of four common apoB precipitation methods [polyethylene glycol (PEG), dextran sulfate/magnesium chloride (MgCl2), heparin sodium/manganese chloride (MnCl2), and LipoSep immunoprecipitation (IP)] on HDL subspecies composition, apolipoproteins, and function (cholesterol efflux and reduction of LDL oxidation). PEG dramatically shifted the size distribution of HDL and apolipoproteins (assessed by two independent methods), while leaving substantial amounts of reagent in the sample. PEG also changed the distribution of cholesterol efflux and LDL oxidation across size fractions, but not overall efflux across the HDL range. Dextran sulfate/MgCl2, heparin sodium/MnCl2, and LipoSep IP did not change the size distribution of HDL subspecies, but altered the quantity of a subset of apolipoproteins. Thus, each of the apoB precipitation methods affected HDL composition and/or size distribution. We conclude that careful evaluation is needed when selecting apoB depletion methods for existing and future bioassays of HDL function.

Durable protection from vaginal simian-human immunodeficiency virus infection in macaques by tenofovir gel and its relationship to drug levels in tissue.

A vaginal gel containing 1% tenofovir (TFV) was found to be safe and effective in reducing HIV infection in women when used pericoitally. Because of the long intracellular half-life of TFV and high drug exposure in vaginal tissues, we hypothesized that a vaginal gel containing TFV may provide long-lasting protection. Here, we performed delayed-challenge experiments and showed that vaginal 1% TFV gel protected 4/6 macaques against vaginal simian-human immunodeficiency virus (SHIV) exposures occurring 3 days after gel application, demonstrating long-lasting protection. Despite continued gel dosing postinfection, neither breakthrough infection had evidence of drug resistance by ultrasensitive testing of SHIV in plasma and vaginal lavage. Analysis of the active intracellular tenofovir diphosphate (TFV-DP) in vaginal lymphocytes collected 4 h to 3 days after gel dosing persistently showed high TFV-DP levels (median, 1,810 fmol/10(6) cells) between 4 and 24 h that exceed the 95% inhibitory concentration (IC(95)), reflecting rapid accumulation and long persistence. In contrast to those in peripheral blood mononuclear cells (PBMCs) following oral dosing, TFV-DP levels in vaginal lymphocytes decreased approximately 7-fold by 3 days, exhibiting a much higher rate of decay. We observed a strong correlation between intracellular TFV-DP in vaginal lymphocytes, in vitro antiviral activity, and in vivo protection, suggesting that TFV-DP above the in vitro IC(95) in vaginal lymphocytes is a good predictor of high efficacy. Data from this model reveal an extended window of protection by TFV gel that supports coitus-independent use. The identification of protective TFV-DP concentrations in vaginal lymphocytes may facilitate the evaluation of improved delivery methods of topical TFV and inform clinical studies.

Integrated Quantitative Targeted Lipidomics and Proteomics Reveal Unique Fingerprints of Multiple Metabolic Conditions.

Aberrations in lipid and lipoprotein metabolic pathways can lead to numerous diseases, including cardiovascular disease, diabetes, neurological disorders, and cancer. The integration of quantitative lipid and lipoprotein profiling of human plasma may provide a powerful approach to inform early disease diagnosis and prevention. In this study, we leveraged data-driven quantitative targeted lipidomics and proteomics to identify specific molecular changes associated with different metabolic risk categories, including hyperlipidemic, hypercholesterolemic, hypertriglyceridemic, hyperglycemic, and normolipidemic conditions. Based on the quantitative characterization of serum samples from 146 individuals, we have determined individual lipid species and proteins that were significantly up- or down-regulated relative to the normolipidemic group. Then, we established protein-lipid topological networks for each metabolic category and linked dysregulated proteins and lipids with defined metabolic pathways. To evaluate the differentiating power of integrated lipidomics and proteomics data, we have built an artificial neural network model that simultaneously and accurately categorized the samples from each metabolic risk category based on the determined lipidomics and proteomics profiles. Together, our findings provide new insights into molecular changes associated with metabolic risk conditions, suggest new condition-specific associations between apolipoproteins and lipids, and may inform new biomarker discovery in lipid metabolism-associated disorders.

Effect of the ABCA1 agonist CS-6253 on amyloid-ß and lipoprotein metabolism in cynomolgus monkeys.

BACKGROUND: Inducing brain ATP-binding cassette 1 (ABCA1) activity in Alzheimer's disease (AD) mouse models is associated with improvement in AD pathology. The purpose of this study was to investigate the effects of the ABCA1 agonist peptide CS-6253 on amyloid-ß peptides (Aß) and lipoproteins in plasma and cerebrospinal fluid (CSF) of cynomolgus monkeys, a species with amyloid and lipoprotein metabolism similar to humans. METHODS: CS-6253 peptide was injected intravenously into cynomolgus monkeys at various doses in three different studies. Plasma and CSF samples were collected at several time points before and after treatment. Levels of cholesterol, triglyceride (TG), lipoprotein particles, apolipoproteins, and Aß were measured using ELISA, ion-mobility analysis, and asymmetric-flow field-flow fractionation (AF4). The relationship between the change in levels of these biomarkers was analyzed using multiple linear regression models and linear mixed-effects models. RESULTS: Following CS-6253 intravenous injection, within minutes, small plasma high-density lipoprotein (HDL) particles were increased. In two independent experiments, plasma TG, apolipoprotein E (apoE), and Aß42/40 ratio were transiently increased following CS-6253 intravenous injection. This change was associated with a non-significant decrease in CSF Aß42. Both plasma total cholesterol and HDL-cholesterol levels were reduced following treatment. AF4 fractionation revealed that CS-6253 treatment displaced apoE from HDL to intermediate-density- and low density-lipoprotein (IDL/LDL)-sized particles in plasma. In contrast to plasma, CS-6253 had no effect on the assessed CSF apolipoproteins or lipids. CONCLUSIONS: Treatment with the ABCA1 agonist CS-6253 appears to favor Aß clearance from the brain.

Variant APOL1 protein in plasma associates with larger particles in humans and mouse models of kidney injury.

BACKGROUND: Genetic variants in apolipoprotein L1 (APOL1), a protein that protects humans from infection with African trypanosomes, explain a substantial proportion of the excess risk of chronic kidney disease affecting individuals with sub-Saharan ancestry. The mechanisms by which risk variants damage kidney cells remain incompletely understood. In preclinical models, APOL1 expressed in podocytes can lead to significant kidney injury. In humans, studies in kidney transplant suggest that the effects of APOL1 variants are predominantly driven by donor genotype. Less attention has been paid to a possible role for circulating APOL1 in kidney injury. METHODS: Using liquid chromatography-tandem mass spectrometry, the concentrations of APOL1 were measured in plasma and urine from participants in the Seattle Kidney Study. Asymmetric flow field-flow fractionation was used to evaluate the size of APOL1-containing lipoprotein particles in plasma. Transgenic mice that express wild-type or risk variant APOL1 from an albumin promoter were treated to cause kidney injury and evaluated for renal disease and pathology. RESULTS: In human participants, urine concentrations of APOL1 were correlated with plasma concentrations and reduced kidney function. Risk variant APOL1 was enriched in larger particles. In mice, circulating risk variant APOL1-G1 promoted kidney damage and reduced podocyte density without renal expression of APOL1. CONCLUSIONS: These results suggest that plasma APOL1 is dynamic and contributes to the progression of kidney disease in humans, which may have implications for treatment of APOL1-associated kidney disease and for kidney transplantation.

Comparison of MALDI-TOF-MS and HPLC-ESI-MS/MS for endopeptidase activity-based quantification of Anthrax lethal factor in serum.

Diagnosing and treating anthrax at the earliest stage of disease is critical. We developed a method to diagnose anthrax at early stages of infection by detecting anthrax lethal factor (LF) at the attomol/mL level in plasma or serum. This method uses antibody capture and quantification of LF endoproteinase activity by isotope dilution matrix-assisted laser-desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Many public health laboratories do not use MALDI-TOF-MS; thus, we have adapted the LF method for detection by electrospray ionization (ESI) tandem MS (MS/MS), which allowed comparison of both MS platforms for LF quantification. Calibration curves were linear from 0.05-2.5 ng/mL when measured after 2 h and from 0.005-1.0 ng/mL after 18 h incubation time. The limit of detection was 0.005 ng/mL using a 200 µL sample. The coefficient of variation for quality control samples was 6-12% for both MS platforms. Samples used to perform cross-validation included 158 serum samples from a study in rabbits exposed to anthrax spores by inhalation. Some were treated with anthrax immune globulin before exposure. Concentrations measured by ESI-MS/MS matched those by MALDI-TOF-MS with p = 0.99 (r(2) = 0.997) and -0.25% mean relative difference (±9% standard deviation). This study shows that isotope dilution MALDI-TOF-MS is a robust and precise quantitative MS platform.

Trends in exposure to polyfluoroalkyl chemicals in the U.S. Population: 1999-2008.

Since 2002, practices in manufacturing polyfluoroalkyl chemicals (PFCs) in the United States have changed. Previous results from the National Health and Nutrition Examination Survey (NHANES) documented a significant decrease in serum concentrations of some PFCs during 1999-2004. To further assess concentration trends of perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), perfluorohexane sulfonate (PFHxS), and perfluorononanoate (PFNA), we analyzed 7876 serum samples collected from a representative sample of the general U.S. population ≥12 years of age during NHANES 1999-2008. We detected PFOS, PFOA, PFNA, and PFHxS in more than 95% of participants. Concentrations differed by sex regardless of age and we observed some differences by race/ethnicity. Since 1999-2000, PFOS concentrations showed a significant downward trend, because of discontinuing industrial production of PFOS, but PFNA concentrations showed a significant upward trend. PFOA concentrations during 1999-2000 were significantly higher than during any other time period examined, but PFOA concentrations have remained essentially unchanged during 2003-2008. PFHxS concentrations showed a downward trend from 1999 to 2006, but concentrations increased during 2007-2008. Additional research is needed to identify the environmental sources contributing to human exposure to PFCs. Nonetheless, these NHANES data suggest that sociodemographic factors may influence exposure and also provide unique information on temporal trends of exposure.

Design of online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) hyphenated systems for quantitative analysis of small organic compounds in biological matrices.

Three online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method examples are presented where two different types of chromatographic columns or solvent systems were coupled to meet specific analytical objectives: (i) SPE of target analytes by restricted access media from high ionic strength urine matrix was coupled with reversed phase LC-MS/MS conditions accommodating high ionization potentials of the analytes (urinary bisphenol A and other phenolic derivatives); (ii) strong cation exchange SPE of analytes of diverse polarity and pK(a) was coupled with reversed phase LC-MS/MS analysis (urinary atrazine metabolites); (iii) pre-concentration of low pg per sample analytes by weak anion exchange SPE was hyphenated with ion pair LC-MS analysis (intracellular nucleotide triphosphate analogs). With these examples we suggest a conductive generic work flow for the development of online SPE-LC-MS methods and show how advanced commercial LC devices and software allow for the design of complex yet highly versatile analytical separation systems suited to the unique physicochemical properties of the target analytes.