in 2001, a random one-third sample of chil- dren 6–11 years of age in 2002, and a random one-third sample of people ≥ 12 years of age in 2001 and 2002. Because the subset was a ran- dom selection from the entire set, the ability of the samples tested to accurately represent the U.S. population was maintained. Laboratory methods. The urine for pyre- throid measurements was aliquoted at the collection site, stored cold (2–4°C) or frozen until shipment to CDC’s National Center for Environmental Health laboratory on dry ice. Urinary creatinine concentrations were deter- mined using an automated colorimetric method based on a modified Jaffe reaction (Jaffe 1886) on a Beckman Synchron AS/ASTRA clinical analyzer (Beckman Instruments, Inc., Brea, CA) at the Fairview University Medical Center (Minneapolis, MN). We analyzed urine sam- ples for pyrethroid metabolites using established methodology (Baker et al. 2004; Olsson et al. 2004). Briefly, 2 mL urine was spiked with an internal standard mixture consisting of iso- topically labeled 3PBA and trans-DCCA and incubated with β-glucuronidase/sulfatase to liberate conjugated metabolites. The hydro- lysates were extracted using OASIS HLB (Waters Corp., Milford, MA) mixed-mode solid-phase extraction cartridges. The cartridges were washed with 5% methanol in a 0.1% acetic acid solution, and the metabolites were eluted using methanol. The extracts were con- centrated and analyzed using high-performance liquid chromatography/electrospray chemical ionization/tandem mass spectrometry. 3PBA and trans-DCCA were quantified using iso- tope dilution calibration, whereas 4F3PBA, cis-DCCA, and cis-DBCA were quantified using the labeled 3PBA, labeled trans-DCCA, and labeled trans-DCCA, respectively, as internal standards. Positive and negative control samples represented 10% of the samples analyzed to ensure proper method operation. Metabolite concentrations were adjusted using creatinine concentrations to correct for variable urine dilu- tions in the “spot” urine samples. Both labora- tories and methods were certified according to guidelines set forth in the Clinical Laboratory Improvement Amendment of 1988. Demographic and other covariates. Age was reported at the time of the household interview as the age in years at the last birth- day. Age categories used in our statistical anal- yses were 6–11, 12–19, and 20–59 years for 1999–2002, and ≥ 60 years for 2001–2002. A composite race/ethnicity variable based on self-reported race and ethnicity was created to define three major racial/ethnic groups: non-Hispanic black, non-Hispanic white, and Mexican American. Individuals from other racial/ethnic groups were included in the total estimates reported here; however, no separate demographic breakdown is provided. Traditionally, creatinine concentrations have been used to adjust spot urine samples for variable dilution caused by the different hydra- tion states of the sample donor. Because crea- tinine concentrations vary with age, sex, and race/ethnicity, creatinine adjustment in diverse populations would not be valid for compari- sons of pyrethroid insecticide metabolite con- centrations among the demographic groups (Barr et al. 2005). To overcome this limita- tion and allow for an appropriate compari- son of metabolite concentrations among the demographic groups, we also used creatinine as a covariate in statistical models (Barr et al. 2005). By using this model, we appropriately corrected for covariate effects on the creatinine concentrations while reducing the variability caused by urine dilution of spot samples. Statistical analysis. Survey-specific sam- ple weights calculated for the specified ran- dom subset were used in statistical analyses. SUDAAN (RTI International, Research Triangle Park, NC) incorporates the NHANES sampling weights and adjusts for the complex sample design of the survey. Sample weights take into account the unequal probabilities of selection, resulting from the cluster design and the planned oversampling of certain subgroups. Oversampling of adolescents, the elderly, non- Hispanic blacks, and Mexican Americans necessitated the use of sampling weights in all analyses to produce national estimates of preva- lence and associated variances. Parametric statistics were performed only on metabolites with sufficient frequency of Pyrethroid metabolites in the U.S. population detection (> 60%) to avoid undue influence on the estimates caused by imputed values in the analyses; thus, 3PBA was the only metabo- lite parametrically analyzed. Data distribution, univariate, bivariate, and multivariate statistics were evaluated. Arithmetic means, geometric means (GMs), least-squares geometric means (LSGMs), weighted Pearson correlation coef- ficients, and percentiles of 3PBA, cis-DCCA, and trans-DCCA concentrations were calcu- lated using SAS (release 9.1; SAS Institute Inc., Cary, NC) and SUDAAN (release 9.0). For comparing populations, LSGMs corrected the GMs for the common statistical variables tested (i.e., age group, race/ethnicity, sex, and time of sample collection, and fasting) that showed correlations in bivariate statistics or could potentially influence comparisons. For concen- trations below the analytic limits of detection (LODs), a value equal to the LOD divided by the square root of 2 was used (Hornung and Reed 1990). The LODs were 0.1 ng/mL for 3PBA, 0.2 ng/mL for 4F3PBA, 0.1 ng/mL for cis-DCCA, 0.4 ng/mL for trans-DCCA, and 0.1 ng/mL for cis-DBCA. Differences in LSGMs among demographic groups were con- sidered to be significant when p < 0.05. Results Table 2 shows distribution percentiles of concentrations of 3PBA, both volume based and creatinine adjusted. Of the metabolites measured, 3PBA was the most frequently detected. Overall, 3PBA was detected more frequently in samples collected in 2001 and 2002 (75.4%) than in samples collected in 1999–2000 (66.5%; p = 0.07). Univariate analysis results showed that the GM of the 3PBA concentrations among race (p = 0.003 for 1999–2000; p < 0.001 for 2001–2002) and age group (p = 0.003 for 1999–2000 only) were significantly different. For the 1999–2000 survey period, in the final multivariate model, age group and time of sample collection were the only signifi- cant variables in addition to log-transformed creatinine. The LSGM 3PBA concentration was higher for children than for adolescents (p = 0.009) (Figure 3A). Samples collected ) 0.40 L /gm 0.35 n s ( 0.30 n o ta i 0.25 r t cen 0.20 n co 0.15 A PB 0.10 3 GM 0.05 S L 0 * * * ** * * * Morning Afternoon Evening 0.45 ) L /m 0.40 g (n 0.35 s n ito 0.30 a r tn 0.25 e nc 0.20 o cA 0.15 3PB 0.10 GM 0.05 S 0 L 1999–2000 2001–2002 1999–2002 2001–2002 1999–2002 Figure 3. LSGM concentrations of age (A), sample collection time (B), and race (C: MA, Mexican American; NHB, non-Hispanic black; NHW, non-Hispanic white). *p < 0.05. **0.05 < p < 0.1. Environmental Health Perspectives • volume 118 | number 6 | June 2010 * * NHB NHW MA 745 ) 0.6 L m /ng 0.5 ( s ion 0.4 t a r t enc 0.3 n o cA 0.2 B P 3M 0.1 G S L 0 * ** 1999–2000 * * 6–11 years old 12–19 years old 20–59 years old 1999–2002