• Component Description
• Eligible Sample
• Description of Laboratory Methodology
• Laboratory Method Files
• Laboratory Quality Assurance and Monitoring
• Data Processing and Editing
• Analytic Notes
• References
• Codebook
  • SEQN - Respondent sequence number
  • WTSA2YR - Subsample A Weight
  • URXCOTT - Total Cotinine, urine (ng/mL)
  • URDCOTLC - Total Cotinine, urine Comment Code
  • URXHCTT - Total Hydroxycotinine, urine (ng/mL)
  • URDHCTLC - Total Hydroxycotinine, urine Comt Code
  • URXANBT - Anabasine, urine (ng/mL)
  • URDANBLC - Anabasine, urine Comment Code
  • URXANTT - Anatabine, urine (ng/mL)
  • URDANTLC - Anatabine, urine Comment Code
  • URXCOXT - Cotinine-n-oxide, urine (ng/mL)
  • URDCOXLC - Cotinine-n-oxide, urine Comment Code
  • URXHPBT - 1-(3P)-1-but-4-carbox acid (ng/mL)
  • URDHPBLC - 1-(3P)-1-but-4-carbox acid, Comment Code
  • URXNICT - Nicotine, urine (ng/mL)
  • URDNICLC - Nicotine, urine Comment Code
  • URXNNCT - Nornicotine, urine (ng/mL)
  • URDNCTLC - Nornicotine, urine Comment Code
  • URXNOXT - Nicotine-1 N-oxide, urine (ng/mL)
  • URDNOXLC - Nicotine-1 N-oxide, urine Comment Code
  • URDTNE2 - TNE - 2 (nmol/mL)
  • URDTNE3 - TNE - 3 (nmol/mL)
  • URDTNE6 - TNE - 6 (nmol/mL)
  • URDTNE7 - TNE - 7 (nmol/mL)

National Health and Nutrition Examination Survey

2015-2016 Data Documentation, Codebook, and Frequencies

Cotinine, Hydroxycotinine, & Other Nicotine Metabolites and Analogs - Urine (UCOT_I)

Data File: UCOT_I.xpt

First Published: September 2019

Last Revised: NA

Component Description

The specific aims of the component are: 1) to measure the prevalence and extent of tobacco use; 2) to estimate the extent of exposure to environmental tobacco smoke (ETS), and determine trends in exposure to ETS; and 3) to describe the relationship between tobacco use (as well as exposure to ETS) and chronic health conditions, including respiratory and cardiovascular diseases.

Nicotine is the primary tobacco-specific alkaloid in tobacco plants and tobacco smoke. Although, nicotine is not a direct cause of most diseases associated with tobacco use, it is highly addictive, which can lead to tobacco product dependence and chronic exposure to the carcinogens and bioactive compounds in tobacco. The presence of nicotine in biological specimens indicates exposure to tobacco, either through the active use of tobacco, or from passive exposure to secondhand smoke (SHS). Trans-3’-hydroxycotinine and cotinine are the two predominant nicotine metabolites in urine. Because their concentrations are greater and their elimination half-lives are longer, these metabolites are generally preferred over nicotine itself as exposure biomarkers. The concentration ratio of hydroxycotinine to cotinine has been used as an index of cytochrome P-450 2A6 activity. The relative concentration of nicotine to its six major metabolites (trans-3’-hydroxycotinine, cotinine, cotinine-N-oxide, nicotine-N-oxide, nornicotine, and 1-(3-Pyridyl)-1-butanol-4-carboxylic acid) is of interest when elucidating differences in metabolic profiles of various ethnic, age, and gender groups (James E. McGuffey, 2013). Furthermore, the sum of these metabolites may more accurately describe the exposure of individuals and groups to tobacco products compared to the most commonly used biomarker, urinary cotinine. Lastly, anatabine and anabasine are nicotine analogs in tobacco product, and are precursors for nitrosamines. Therefore, levels of these two compounds have been used in monitoring compliance of smoking cessation programs as well as in biomonitoring for nitrosamine-related studies.

Eligible Sample

All examined participants aged 3 to 5 years were eligible, and participants aged 6 years and older from a one-third subsample were eligible.

Description of Laboratory Methodology

“Total” urinary nicotine metabolites, including the free and the glucuronide conjugated forms, are measured by two separate isotope dilution high-performance liquid chromatography/tandem mass spectrometric (HPLC-MS/MS) methods based on the cotinine cutoff value of 20 ng/mL so that the markedly different levels of nicotine exposure biomarkers can be optimally quantified in both users and non-users. For samples with cotinine levels above or equal to 20 ng/mL, the “Nicotine Metabolites and Analogs in Urine” method (Urinary Nicotine Metabolites and Analogs Laboratory Procedure Manual) was applied, and nicotine, its six metabolites (cotinine, trans-3'-hydroxycotinine, nicotine-N-oxide, cotinine-N-oxide, nornicotine, and 1-(3-Pyridyl)-1-butanol-4-carboxylic acid), anatabine, and anabasine were measured. Briefly, a 100 µL urine sample is spiked with a labeled internal standard mixture and incubated with beta-glucuronidase to hydrolyze the conjugated analytes. Following the incubation period, proteins and salts in the sample are precipitated with cold acetone. The sample is centrifuged and part of the supernatant is transferred and evaporated to remove acetone. The sample is further diluted approximately 5-fold prior to analysis on a HPLC-MS/MS system using electrospray ionization. Chromatography is performed using a C18 analytical column; mass spectrometric analysis is carried out under positive mode using scheduled multiple reaction monitoring. One quantitation transition, one confirmation transition, and one corresponding internal standard transition are monitored for each of the nine analytes.

For samples with cotinine levels less than 20 ng/mL, the “Cotinine and Hydroxycotinine in Urine” method (Urinary Cotinine and Hydroxycotinine Laboratory Procedure Manual) was applied to measure only the cotinine and trans-3’-hydroxycotinine levels. Briefly, a 200 uL urine sample is spiked with labeled internal standard mixture and incubated with beta-glucuronidase to hydrolyze the conjugated analytes. Following the incubation period, the sample is basified and applied to a supported liquid extraction plate. The two analytes are extracted with 5% (v/v) isopropanol in methylene chloride. The organic extract is concentrated and the residue is injected onto a C18 UHPLC (ultra-high-performance liquid chromatography) column. The eluent from these injections is monitored by atmospheric pressure chemical ionization (APCI)-MS/MS. One quantitation transition, one confirmation transition, and one corresponding internal standard transition are monitored for each of the two analytes.

Sample concentration is derived from the ratios of native-to-labeled compounds in the sample by comparing to a standard curve. Adequate method performance has been confirmed with criteria established internally.

Refer to the Laboratory Method Files section for a detailed description of the laboratory methods used.

There were no changes to the lab method, lab equipment, or lab site for this component in the NHANES 2015-2016 cycle.

Laboratory Method Files

Urinary Cotinine and Hydroxycotinine Laboratory Procedure Manual (September 2019)

Urinary Nicotine Metabolites and Analogs Laboratory Procedure Manual (September 2019)

Laboratory Quality Assurance and Monitoring

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

Detailed instructions on specimen collection and processing are discussed in the NHANES Laboratory Procedures Manual (LPM). Vials are stored under appropriate frozen (–30°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 Act 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 quality-control procedures. Each laboratory staff member is observed for equipment operation, specimen collection and preparation; testing procedures and constructive feedback are given to each staff member. 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 blind split samples collected on “dry run” sessions. In addition, contract laboratories randomly perform repeat testing on 2% of all specimens.

NCHS developed and distributed a quality control protocol for all CDC and contract laboratories, which outlined the use of Westgard rules (Westgard et al., 1981) 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 Division of Laboratory Sciences quality control and quality assurance performance criteria for accuracy and precision, similar to the Westgard rules (Caudill, et al., 2008).

Data Processing and Editing

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

Analytic Notes

Urinary nicotine metabolites and analogs (nicotine, nicotine-N-oxide, cotinine-N-oxide, nornicotine, and 1-(3-Pyridyl)-1-butanol-4-carboxylic acid) and urinary minor tobacco alkaloids (anatabine and anabasine) were not measured if urinary cotinine (URXCOTT) results were less than 20 ng/mL.

Refer to the 2015-2016 Laboratory Data Overview for general information on NHANES laboratory data.

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.

Subsample Weights

Urinary cotinine, trans-3’-hydroxycotinine, and other nicotine metabolites and analogs were assessed in a one-third subsample of persons 6 years and older. 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 using the appropriate survey design and demographic variables. The NHANES 2015-2016 Demographics File contains demographic data, health indicators, and other related information collected during household interviews as well as the sample weight variables. The recommended procedure for variance estimation requires use of stratum and PSU variables (SDMVSTRA and SDMVPSU, respectively) in the demographic data file.  

The laboratory data file can be linked to the other NHANES data files using the unique survey participant identifier (i.e., SEQN).

Total Nicotine Equivalents (TNE) Calculations

Total Nicotine Equivalents (TNE) were calculated for each analysis for which total results were available for all analytes by dividing the concentration in ng/mL by the MW for each analyte.  NIST’s molecular weight, which include 4 digits after the decimal point were used in TNE calculations. If any of the individual analyte values used in the respective formulas are missing, the TNE variable was set to missing.

TNE-2 = (Total Cotinine /176.2151) + (Total Trans-3’-Hydroxycotinine/192.2145) nmol/mL

TNE-3 = (Total Nicotine/162.2316) + (Total Cotinine /176.2151) + (Total Trans-3’-Hydroxycotinine/192.2145) nmol/mL

TNE-6 = (Total Nicotine/162.2316) + (Total Cotinine /176.2151) + (Total Trans-3’-Hydroxycotinine/192.2145) + (Total (S)-Cotinine N-oxide/ 192.2145) + ((1’S,2’S)-Nicotine 1’-oxide/ 178.231) + ((R,S)-Nornicotine/ 148.2050) nmol/mL

TNE-7 = (Total Nicotine/162.2316) + (Total Cotinine /176.2151) + (Total Trans-3’-Hydroxycotinine/192.2145) + (Total (S)-Cotinine N-oxide/ 192.2145) + ((1’S,2’S)-Nicotine 1’-oxide/ 178.231) + ((R,S)-Nornicotine/ 148.2050) + (1-(3-Pyridyl)-1-butanol-4-carboxylic acid)/ 181.1885) nmol/mL

Detection Limits

The detection limits were constant for all of the analytes in the data set. Two variables are provided for each of these analytes. The variable name ending in “LC” (ex., URDCOTLC) 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. URDCOTLC =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., URXCOTT) provides the analytic result for the analyte.

The lower limit of detection (LLOD in ng/mL) for Cotinine, Hydroxycotinine, & Nicotine Metabolites and Analogs in urine is:

References

• Caudill, S.P., Schleicher, R.L., Pirkle, J.L. Multi-rule quality control for the age-related eye disease study. Statist. Med. (2008) 27(20):4094-40106.

• Hukkanen J, Jacob P, Benowitz NL. Metabolism and disposition kinetics of nicotine. Pharmacol Rev. 2005;57(1):79-115.

• McGuffey JE, Wei B, Bernert JT, Morrow JC, Xia B, et al. (2014) Validation of a LC-MS/MS Method for Quantifying Urinary Nicotine, Six Nicotine Metabolites and the Minor Tobacco Alkaloids—Anatabine and Anabasine—in Smokers’ Urine. PLoS ONE 9(7): e101816. doi:10.1371/journal.pone.0101816.

• Wei B, Feng J, Rehmani IJ, et al. A high-throughput robotic sample preparation system and HPLC-MS/MS for measuring urinary anatabine, anabasine, nicotine and major nicotine metabolites. Clin Chim Acta. 2014;436:290-297.

• Westgard J.O., Barry P.L., Hunt M.R., Groth T. A multi-rule Shewhart chart for quality control in clinical chemistry. Clin Chem (1981) 27:493-501.

Codebook and Frequencies