National Health and Nutrition Examination Survey

Exposure to volatile organic compounds (VOCs) is ubiquitous. Chronic exposure to extremely high levels of some VOCs can lead to cancer and neurocognitive dysfunction. Urinary metabolites of VOCs can be detectable in urine for a longer period of time than the parent VOCs can be detected in blood.

Nearly 200 air toxics have been associated with adverse health effects in occupational studies or laboratory studies, but have not been monitored in general population groups. Information on levels of exposure to these compounds, as measured by their metabolite levels in urine, is essential to determine the need for regulatory mechanisms to reduce the levels of hazardous air pollutants to which the general population is exposed.

Examined participants aged 6 years and older from a one-third sample were eligible.

This method is a quantitative procedure for the measurement of VOC metabolites in human urine using ultra performance liquid chromatography coupled with electrospray tandem mass spectrometry (UPLC-ESI/MSMS) as described by Alwis et al (2012). Chromatographic separation is achieved using an Acquity UPLC® HSS T3 (Part no. 186003540, 1.8 µm x 2.1 mm x 150 mm, Waters Inc.) column with 15 mM ammonium acetate and acetonitrile as the mobile phases. The eluent from the column is ionized using an electrospray interface to generate and transmit negative ions into the mass spectrometer. Comparison of relative response factors (ratio of native analyte to stable isotope labeled internal standard) with known standard concentrations yields individual analyte concentrations.

Refer to NHANES 2011-2012 Lab Methods for VOC Metabolites for detailed description of the laboratory method used.

Volatile Organic Compounds (VOCs) Metabolites (Updated July 2017)

Urine specimens are processed, stored, and shipped to the Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta GA for analysis.

Detailed instructions on specimen collection and processing are discussed in the NHANES 2011-2012 Laboratory Procedures Manual (LPM). Vials are stored under appropriate frozen (–20°C) conditions until they are shipped to National Center for Environmental Health for testing.

The NHANES QA/QC protocols meet the 1988 Clinical Laboratory Improvement Act mandates. Detailed QA/QC instructions are discussed in the NHANES Laboratory Procedures Manual (LPM).

Mobile Examination Centers (MECs)
Laboratory team performance is monitored using several techniques. NCHS and contract consultants use a structured quality assurance evaluation during unscheduled visits to evaluate both the quality of the laboratory work and the quality-control procedures. Each laboratory staff person is observed for equipment operation, specimen collection and preparation; testing procedures and constructive feedback are given to each staff. Formal retraining sessions are conducted annually to ensure that required skill levels were maintained.

Analytical Laboratories
NHANES uses several methods to monitor the quality of the analyses performed by the contract laboratories. In the MEC, these methods include performing second examinations on previously examined participants and blind split samples collected on “dry run” sessions.

NCHS developed and distributed a quality control protocol for all the contract laboratories which outlined the Westgard rules used when running NHANES specimens. Progress reports containing any problems encountered during shipping or receipt of specimens, summary statistics for each control pool, QC graphs, instrument calibration, reagents, and any special considerations are submitted to NCHS quarterly. The reports are reviewed for trends or shifts in the data. The laboratories are required to explain any identified areas of concern.

All QC procedures recommended by the manufacturers were followed. Reported results for all assays meet the CDC/NCEH Tobacco and Volatile Branch quality control and quality assurance performance criteria for accuracy and precision, similar to the Westgard rules (Caudill et al., 2008).

The data were reviewed. Incomplete data or improbable values were sent to the performing laboratory for confirmation.

Refer to the 2011-2012 Laboratory Data Overview for general information on NHANES laboratory data.

Subsample Weights

VOC metabolites in urine were assessed in a subsample of participants aged 6 years and older. Use the special one-third weights included in this data file when analyzing data. Special sample weights are required to analyze these data properly. Specific sample weights for this subsample are included in this data file and should be used when analyzing these data.

Demographic and Other Related Variables

The analysis of NHANES laboratory data must be conducted with the key survey design and basic demographic variables. The NHANES Demographic Data File contains demographic and sample design variables. The recommended procedure for variance estimation requires use of stratum and PSU variables (SDMVSTRA and SDMVPSU, respectively) in the demographic data file.

This laboratory data file can be linked to the other NHANES data files using the unique survey participant identifier SEQN.

Special Analytic Note: Bias Adjustments Applied to VOC Metabolites in Urine Assay in NHANES 2011–2012 cycle

Adjustments have been applied to the previously published data.

During routine review, several systematic biases were discovered in measurements of certain urinary VOC metabolites in the 2005 – 2006 and 2011 – 2012 NHANES cycles. The systematic nature of these biases permitted retrospective adjustment through a statistical approach based on linear regression modeling where adjusted measurements are predicted from unadjusted measurements using suitable data. The “statistical recalibrations” implemented here have the general form of a univariate linear regression equation (accounting for dilution factors routinely used during measurement):

where exp[·] and ln(·) are the natural exponential and natural logarithm functions, respectively. Natural log-transformation of both the un-adjusted and adjusted measurements moderated adverse influence on model fit from extreme measurements. The estimated regression slope and intercept are m and b, respectively, which are tabulated below for each analyte. The dilution factor was accounted for only once for each analyte that underwent more than one statistical recalibration (e.g., URXMB3). Statistical recalibration for the mass fraction of salt (URXHP2, URXGAM) only required a regression slope (i.e., b=0).

The explanatory power of the linear regressions was excellent (R²≥0.95), and residuals exhibited distributions with good approximation to normality, as well as stable variance over the observed range of measurements. These favorable diagnostics demonstrate the efficacy of statistical recalibration for the retrospective amelioration of systematic bias.

Correction for Variation in Preparation of Neat Native Metabolites Standards

During the 2005–2006 and 2011–2012 NHANES cycles, calibrators were prepared from a multi-analyte stock solution formulated from neat materials in-house. Beginning with the NHANES 2015–2016 cycle, calibrators and the multi-analyte stock solution were prepared externally. A systematic bias between the cycles that used in-house calibration materials and the cycle using externally prepared calibration materials was observed for analytes listed in the table below. Further validation of the externally prepared calibrators suggested that the in-house calibrators were inaccurate due to special requirements for handling and preparing accurate solutions from neat materials, some of which are highly hygroscopic.

Correction for Mass Fraction of Salt in Neat Compound Standard

During the 2005–2006 and 2011–2012 NHANES cycles, calibrators were prepared from a multi-analyte stock solution formulated from neat materials in-house. Beginning with the NHANES 2015–2016 cycle, calibrators and the multi-analyte stock solution were prepared externally. A systematic bias between the cycles that used in-house calibration materials and the cycle using externally prepared calibration materials was observed for analytes listed in the table below. Further validation confirmed that the large mass fraction of salt in the neat materials was not accounted for during formulation of in-house calibration materials. This discrepancy between the calculated concentration of the calibrators and the actual concentration led to a systematic bias in results for URXHP2 and URXGAM.

 Correction for Stereoisomerism in Urine Samples

UXMB3 occurs in human urine predominantly in the cis isomer, but quantitation in the NHANES 2005–2006 and NHANES 2011–2012 cycles used standards containing the trans isomer. This discrepancy led to a systematic bias in the URXMB3 results. Neat material that is purely cis isomer cannot be readily obtained, but a predictive regression equation can account for these quantitative differences.

Detection limits

The detection limits were constant for the analytes in the data set. Two variables are provided for each of these analytes. The variable named ending in “LC” (ex., URDAAMLC) indicates whether the result was below the limit of detection: the value “0” means that the result was at or above the limit of detection, “1” indicates that the result was below the limit of detection. For analytes with analytic results below the lower limit of detection (ex., URDAAMLC=1), an imputed fill value was placed in the analyte results field. This value is the lower limit of detection divided by the square root of 2 (LLOD/sqrt [2]). The other variable prefixed URX (ex., URXAAM) provides the analytic result for the analyte.

The lower limit of detection (LLOD, in µg/L) for urinary VOC metabolites:

Please refer to the NHANES Analytic Guidelines and the on-line NHANES Tutorial for further details on the use of sample weights and other analytic issues.