The primary objective of this study was to characterize serum concentrations of selected perfluorinated chemicals ( PFCs), including perfluoroctanoate (PFOA) and perfluorooctanesulfonate (PFOS), in a representative random 1/3 subset of the non-occupationally exposed US population from NHANES 1999-2000, so that we could obtain national population levels for these compounds over this two-year period.
Participants aged 12 years and older who do not meet any of the exclusion criteria were eligible.
Description of Laboratory Methodology
The measurements of PFCs were performed at the Division of Laboratory Sciences (DLS), National Center for Environmental Health (NCEH), Centers for Disease Control and Prevention (CDC). Through a multiple reaction monitoring experiment, the following analytes were measured: perfluorooctane sulfonamide (PFOSA), 2-(N-ethyl-perfluorooctane sulfonamido) acetic acid (Et-PFOSA-AcOH), 2-(N-methyl-perfluorooctane sulfonamido) acetic acid (Me-PFOSA-AcOH), perfluorohexane sulfonic acid (PFHxS), PFOS, PFOA, perfluorohexanoic acid (PFHxA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDeA), perfluoroundecanoic acid (PFUA), and perfluorododecanoic acid (PFDoA). The analytical method used has been described in detail (Kuklenyik et al. 2005). Briefly, without protein precipitation, only dilution with 0.1 M formic acid, one aliquot of 100 μL of serum was injected into a commercial column switching system allowing for concentration of the analytes on a C18 solid-phase extraction column. This column was placed automatically in front of a C8 analytical high-performance liquid chromatography column for chromatographic separation of the analytes. Detection and quantification were done using negative-ion TurboIonSpray ionization, a variant of electrospray ionization, tandem mass spectrometry. Three isotope-labeled internal standards were used for quantification: 18
O2-PFOS, and 13
C2-PFOA. To compensate for the lack of isotope-labeled internal standards for the other analytes and account for matrix effects, the calibration standards were spiked into calf serum. Spiked serum was analyzed repeatedly to determine the limit of detection (LOD), accuracy, and precision of the method. LOD was calculated as 3S0, where S0 is the standard deviation as the concentration approaches zero (Taylor 1987). LOD was 0.2 nanograms per milliliter (ng/mL), except for PFHxS (0.1 ng/mL) and PFOSA (0.05 ng/mL). The standard accuracies (77%-109%) and their relative standard deviations (5%-24%) were obtained at three spike levels (LOD, 1.25 ng/mL and 12.5 ng/mL) (Kuklenyik et al. 2005). To correct for the endogenous PFOS present in the calf serum, we increased the calculated PFOS concentrations by 0.6 ng/mL. No corrections were applied to the other PFC.concentrations (Kuklenyik et al. 2005).
Data Processing and Editing
Specimens were processed, stored, and shipped to DLS, NCEH, CDC (Atlanta, Georgia). Detailed specimen collection and processing instructions are discussed in the NHANES LPM. Read the LABDOC file for detailed data processing and editing protocols. The analytical method is described in detail in a peer-reviewed publication (Kuklenyik et al. 2005).
Laboratory Quality Assurance and Monitoring
CDC’s laboratory is CLIA ‘88 certified and practices all quality control (QC) and assurance procedures dictated by this certification. Serum concentrations of PFCs currently have no known clinical significance other than indicating exposure to PFCs prior to specimen collection in 1999-2000. Therefore, no reports will be forwarded to NHANES survey participants.
QC procedures included the daily analysis of characterized serum pools and the periodic analysis of proficiency testing materials. Low-concentration (QCL; ~3 ng/mL to ~9 ng/mL, depending upon the analyte) and high-concentration (QCH; ~10 ng/mL to ~30 ng/mL, depending upon the analyte) QC materials were prepared from a base calf serum pool, dispensed in 3-mL aliquots and stored at −20 °C. QC materials were characterized through repeated measurements spanning at least 3 weeks, to define the mean concentrations and the 95% and 99% control limits of PFCs. The coefficients of variation of 30 repeated measurements for each serum pool ranged between 6% and 16% for all analytes (Kuklenyik et al. 2005). Each analytical batch of NHANES samples also included 9 calibration standards, 2 QCH, 2 QCL, 2 reagent blanks, and 1 serum blank. The concentrations of the two QCH and the two QCL were averaged to obtain one measurement of QCH and of QCL per batch; these concentrations were evaluated using standard statistical probability rules.
The analysis of NHANES 1999–2000 PFCs data must be conducted with the key survey design and basic demographic variables. The NHANES 1999–2000 Household Questionnaire Data files contain demographic data, health indicators, and other related information collected during household interviews. They also contain all survey design variables and sample weights. The phlebotomy file includes auxiliary information such as the conditions precluding venipuncture. The household questionnaire and phlebotomy files may be linked to the laboratory data file using the unique survey participant identifier SEQN.