Phthalate acid esters (phthalates) have been used extensively as plasticizers in a wide range of applications such as children’s toys, food packaging, personal care products, and medical supplies. Because some of these compounds have been shown to bind to estrogen receptors and have been associated with estrogenic effects in laboratory animals, increasing concerns have been expressed about phthalate usage in food packaging materials, personal care products, and children’s toys. Measurement of urinary phthalate metabolites in humans is useful to evaluate recent exposure and potential human health effects from these chemicals.
Participants aged 6 years and older who met the subsample requirements.
Description of Laboratory Methodology
The test principle utilizes high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) for the quantitative detection in urine of the following phthalate metabolites: monomethyl phthalate (mMP), monoethyl phthalate (mEP), monobutyl phthalate (mBP), mono-isobutyl phthalate (miBP), mono (3-carboxypropyl) phthalate (mCPP), monocyclohexyl phthalate (mCHP), mono (2-ethylhexyl) phthalate (mEHP), monooctyl phthalate (mOP), monobenzyl phthalate (mBzP), monoisononyl phthalate (mNP), mono(2-ethyl-5-oxohexyl) phthalate (mEOHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (mEHHP), and mono(2-ethyl-5-carboxypentyl) phthalate (mECPP) . Urine samples are processed using enzymatic deconjugation of the glucuronidated phthalate monoesters followed by on-line solid phase extraction (SPE) coupled with reversed phase HPLC-ESI-MS/MS. Assay precision is improved by incorporating isotopically-labeled internal standards for each of the phthalate metabolites. In addition, 4-methyl umbelliferone glucuronide is used to monitor deconjugation efficiency. This selective method allows for rapid detection of up to fifteen monoester metabolites of commonly used phthalate diesters in human urine with limits of detection in the low parts per billion (ppb) ranges.
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 for analysis.
Detailed specimen collection and processing instructions are discussed in the NHANES Laboratory/Medical Technologists Procedures Manual (LPM). Vials are stored under appropriate frozen (–20°C) conditions until they are shipped to National Center for Environmental Health for testing.
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.
The NHANES QA/QC protocols meet the 1988 Clinical Laboratory Improvement Act mandates. Detailed QA/QC instructions are discussed in the NHANES LPM.
NHANES uses several methods to monitor the quality of the analyses performed by the 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 2.0% of all specimens.
NCEH developed and distributed a quality control protocol for all the laboratories which outlined the Westgard rules used when running NHANES specimens. Any problems encountered during shipping or receipt of specimens, instrument calibration, reagents, and any special considerations are submitted to NCHS and Westat. Summary statistics for each control pool, QC graphs, are reviewed by NCHS 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 specifications outlined by Westgard (1981).
Mono-ethyl phthalate (URXMEP), Mono-benzyl phthalate (URXMZP), Mono-n-octyl phthalate (URXMOP), Mono-isononyl phthalate (URXMNP), and Mono-cyclohexyl phthalate (URXMCP):
NHANES recently learned that analytical standards used since 1999 and used for several metabolites were of insufficient purity. As a result of further investigation, a correction factor was applied to each value for the 5 metabolites.
Measures of urinary environmentals were measured in a one third subsample of persons 6 years and over. 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.
The analysis of NHANES 2003-2004 laboratory data must be conducted with the key survey design and basic demographic variables. The NHANES 2003-2004 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.
Links to NHANES Data Files
This laboratory data file can be linked to the other NHANES 2003-2004 data files using the unique survey participant identifier SEQN.
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 named URD___LC indicates whether the result was below the limit of detection. There are two values: “0” and “1””. “0” means that the result was at or above the limit of detection. “1” indicates that the result was below the limit of detection.
The other variable named URX___ provides the analytic result for that analyte.
The lower limit of detections for the environmental phthalates is:
||Limit of Detection (LLOD)
|Mono-n-butyl phthalate (ng/mL)
|Mono-(3-carboxypropyl) phthalate (ng/mL)
|Mono-cyclohexyl phthalate (ng/mL)
|Mono-ethyl phthalate (ng/mL)
|Mono-(2-ethyl)-hexyl phthalate (ng/mL)
|Mono-isobutyl phthalate (ng/mL)
|Mono-n-methyl phthalate (ng/mL)
|Mono-isononyl phthalate (ng/mL)
|Mono-benzyl phthalate (ng/mL)
Please refer to the Analytic Guidelines for further details on the use of sample weights and other analytic issues.