The Family Smoking Prevention and Tobacco Control Act of 2009 gave the Food and Drug Administration regulatory authority over tobacco products. In Section 907(a), the Act states that the Secretary of HHS shall establish tobacco product standards. These product standards shall also include provisions for the testing of the tobacco products. In order to determine the effectiveness of these product standards, it is critical that surveillance systems be in place before the standards are enacted to determine whether they impact exposure of people who continue to use tobacco product. NHANES is an ideal source of human samples for assessing smokers in multiple locations around the country. NHANES will provide the most robust dataset possible for identifying changes in exposure of smokers that should result from enactment of tobacco product standards.
We anticipate that the results of this survey of tobacco exposure biomarkers will be used by FDA, other government agencies, and research institutions. The NHANES data will be used to determine the effectiveness of initial product standards and direct whether further changes to product standards need to be made.
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 subsample were eligible. Additionally, to oversample adult smokers, those participants aged 20 years and older, not in the regular one-third subsample, who smoked at least 100 cigarettes in their entire lifetime (SMQ020=1) and now smoke cigarettes every day (SMQ040=1), were also included in this special subsample.
Description of Laboratory Methodology
This method is a quantitative procedure for the measurement of VOC metabolites in human urine using ultra performance liquid chromatography coupled with electro spray tandem mass spectrometry (UPLC-ESIIMSMS) (29). Chromatographic separation is achieved using an Acquity UPLC® HSS T3 (Part no. 186003540, 1.8f 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.
Laboratory Method Files
Volatile Organic Compounds (VOCs) and Metabolites - Urine
(Updated July 2017)
Laboratory Quality Assurance and Monitoring
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 quality assurance and quality control (QA/QC) protocols meet the 1988 Clinical Laboratory Improvement Amendments mandates. Detailed QA/QC instructions are discussed in the NHANES LPM.
Mobile Examination Centers (MECs)
Laboratory team performance is monitored using several techniques. NCHS and contract consultants use a structured competency assessment evaluation during visits to evaluate both the quality of the laboratory work and the QC 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.
NHANES uses several methods to monitor the quality of the analyses performed by CDC and 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 QC protocol for all the contract laboratories which outlined the Westgard rules (Westgard,
et al. 1981) used when testing 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 and Westat 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).
Refer to the 2011-2012 Laboratory Data Overview for general information on NHANES laboratory data.
There are over 800 laboratory tests performed on NHANES participants. However, not all participants provided biospecimens or enough volume for all the tests to be performed. The specimen availability can also vary by age or other population characteristics. For example, in 2011-2012, approximately 79% of children aged 1-17 years who were examined in the MEC provided a blood specimen through phlebotomy, while 95% of examined adults age 18 and older provided a blood specimen. Analysts should evaluate the extent of missing data in the dataset related to the outcome of interest as well as any predictor variables used in the analyses to determine whether additional re-weighting for item non-response is necessary.
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.
Measures of volatile organic compounds in urine were assessed in a subsample of participants aged 20 years and older. Use the special smoking 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 2011-2012 Demographic 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.
Bias Adjustments Applied to VOC Metabolites in Urine Assay in NHANES 2011–2012 cycle
Several systematic biases were discovered in measurements of certain urinary VOC metabolites in the 2005-2006 and 2011-2012 NHANES cycles. Upon further investigation, the following adjustments have been applied to the previously published data
and included in the present dataset.
A. Adjustment to correct calibrator errors of the CDC lab prepared calibration
During the 2005–2006 and 2011–2012 NHANES cycles, calibrators used for tests on
several VOC metabolites were prepared from a multi-analyte stock solution
formulated from neat materials prepared by the testing lab at CDC. Beginning
with the NHANES 2015–2016 cycle, calibrators and the multi-analyte stock
solution were prepared externally. A systematic bias was observed between the
cycles that used in-house calibration materials and the cycle using externally
prepared calibration materials. 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 unadjusted 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
Validations of the externally prepared calibrators suggested that the calibrators previously prepared by the CDC testing lab were inaccurate due to special requirements for handling and preparing accurate solutions from neat materials, some of which are highly hygroscopic. Below is the list of analytes affected and the corresponding adjustment factors used in correction measures.
||m used in correction
||b used in correction
Correction for Mass Fraction of Salt in Neat Compound Standard
Validations conducted by the CDC testing lab confirmed that the large mass fraction of salt in the neat materials was not accounted for during the formulation of 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. See below for the adjustment factors used in correction measures.
||m used in correction
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.
||m used in correction
||b used in correction
B. Adjustment to correct calibrator errors of calibration materials prepared by an external vendor
Correction of N-Acetyl-S-(3-hydroxypropyl-1-methyl)-L-cysteine (URXPMM)
In the 2005-2006 and 2011-2016 NHANES cycles, calibrators used to test N-Acetyl-S-(3-hydroxypropyl-1-methyl)-L-cysteine (URXPMM) were externally prepared, by a vendor, from a multi-analyte stock solution formulated from neat materials. In 2021, accuracy solutions from a new vendor indicated a systematic bias. Validations conducted by the CDC testing lab confirmed that the large mass fraction of salt in the neat materials was not accounted for during formulation of the externally prepared calibration materials from the previous vendor. This discrepancy between the calculated concentration of the calibrators and the actual concentration led to a systematic bias in results for URXPMM. An algebraic correction factor of 0.565 were applied to the original URXPMM values and released in the present file as below:
URXPMM = URXPMMOriginal×0.565
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 ). 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:
||Urinary 2-Aminothiazoline-4-carboxylic acid
||Urinary Mandelic acid
||Urinary 2-Methylhippuric acid
||Urinary 3- and 4-Methylhippuric acid
||Urinary Phenylglyoxylic acid
||Urinary 2-Thioxothiazolidine-4-carboxylic acid
Withdrawn of t,t-Muconic acid (URXMUCA) Data
In November 2016,
the t,t-Muconic acid (URXMUCA) data were withdrawn from the NHANES 2011-2012
cycle report due to an observed issue with the internal standard transition
used for quantitation. During sample measurements, the absolute response of the
internal standard decreased over time resulting in inconsistent intra-day
t,t-Muconic acid (MUCA) concentrations. However, the change in absolute
response in the unlabeled standards was small enough that the correlation
coefficients of the calibration curves passed CDC testing lab’s quality control
requirements. Further investigation by the CDC lab determined that the internal
standard quantitation scheme used was not consistent during the 2011-2012
cycle, and the data could not be recalculated even after the issue was
identified and corrected.