• Component Description
• Eligible Sample
• Description of Laboratory Methodology
• Data Processing and Editing
• Laboratory Quality Assurance and Monitoring
• Analytic Notes
• References
• Codebook
  • SEQN - Respondent sequence number
  • WTSBMEL - Melamine Subsample Weight
  • SSMEL - Melamine
  • SSCYA - Cyanuric acid

National Health and Nutrition Examination Survey

2003-2004 Data Documentation, Codebook, and Frequencies

Melamine - Urine (Surplus) (SSMEL_C)

Data File: SSMEL_C.xpt

First Published: June 2009

Last Revised: April 2022

Component Description

Measurement of cyanuric acid in a 500 sample subset of NH2003-2004 urine samples

Eligible Sample

Participants ≥ 6 years of age from NHANES 2003-2004

Description of Laboratory Methodology

Measurement of Urinary Melamine 

   A 1mL urine sample was spiked with 20 µL ISTD, diluted with ammonium hydroxide and mixed. The sample was purified on an Oasis MAX Strong Anion solid phase extraction (SPE) cartridge. The cartridge was conditioned with 2 mL MeOH and then equilibrated with 2 mL of deionized water. The urine sample was loaded onto the cartridge. The cartridge was rinsed with 2 mL of 2ammonium hydroxide in methanol to wash away any contaminants from the urine matrix that possibly adhered to the sorbent. The analytes were eluted from the column with 2 mL of 1%forminc acid in methanol. The eluent was evaporated to dryness (approximately 20 minutes) in a TurboVap LV Evaporator (Caliper Life Sciences, Framingham, MA) under nitrogen (15 psi) at 40^oC. The residue was reconstituted with 100 µL of 1% formic acid. 

Instrumentation

   Cyanuric acid and its isotopically labeled analogue were separated on a Zir-Chrome column. The column was connected to an Agilent 1200 Series high performance liquid chromatography system (HPLC), consisting of an autosampler, binary pump, degasser, and thermostattable column compartment (Agilent Technologies, Waldbronn, Germany). The HPLC system was interfaced to an API 5000 triple quadrupole tandem mass spectrometer (MS/MS; Applied Biosystems, Foster City, CA); with an atmospheric pressure chemical ionization source (APCI). 

HPLC-MS-MS Conditions

    Melamine and its isotopically labeled analogue were separated using an isocratic elution at a constant flow rate of 1 mL/min with 10 nM ammonium acetate in methanol. The total run time was 20 minutes. The injection volume was 15 µL. The column compartment was set at 20^oC. 

The collision gas was 5 psi. The curtain gas was 30 psi. Ion source gasses 1 and 2 were set at 65 and 80 psi, respectively. The ionspray voltage was 5500, and the heater was set at 700^oC.

The acquisition method operated in positive ion mode. One scan period with multiple reaction monitoring (MRM) was used to detect the quantification and confirmation ions listed below.

MS/MS Method

    Positive APCI was used as an interface. All parameters were optimized to yield the optimum intensity of the compound. The two MRM transitions, including the isotopic internal standard (L), were as followed: Q [m/z] 128?142, C [m/z] 128?84 and L [m/z] 134?144, respectively. The limit of detection was 0.09 ng/mL.

Data Processing and Editing

Data was received after all the laboratory testing was complete. The data were not edited.
Data Access: All data are publicly available.

Laboratory Quality Assurance and Monitoring

The laboratory and method were certified according to the Clinical Laboratory Improvement Amendments (1988) guidelines (Clinical Laboratory Improvement Amendment, 1988).

Quality Control

Urine was collected from multiple (> 15) donors, combined, diluted with water (1:1 v/v) to reduce endogenous levels of the analytes of interest, and mixed overnight at 4°C. Our protocol for anonymous collection of urine was reviewed and approved by CDC’s Institutional Review Board (IRB). The urine pool was pressure-filtered with a 0.2-µm filter capsule and divided into four pools. The first pool (QCL), the second pool (QCM), and the third pool (QCH) were spiked with the native standard stock solution to yield concentrations of 5 ng/ml, 15 ng/ml and 30 ng/ml, respectively. The fourth pool was not spiked. After being screened for possible endogenous analytes, the fourth pool was used as matrix material for calibration standards and blanks.

Just before each analytical run, calibration standards were prepared by diluting the working standard stock solutions in blank urine. The concentrations of the nine calibration standards ranged from 0.125 to 50 ng/ml for each of the analytes. To each run was added the nine calibration samples, two sets of three quality control samples (QCL, QCM, and QCH), and one blank urine sample; these were extracted and analyzed in parallel with the unknown samples.

All QC pools were characterized before use to determine the mean and 99^th and 95^th control limits by consecutively analyzing at least 20 samples from each QC pool. QC samples were analyzed in runs with 2 replicates in 10 runs over 10 days. After establishment of the control limits of the pools, individual QC samples contained within each analytical run were evaluated for validity by use of a multi-rule quality control (Caudill et al., 2008).

Quality Assurance

A quantification and a confirmation product (MS/MS) ion are monitored for each analyte. In order to be quantified, the peaks must coelute with its isotopically labeled internal standard and possess both the quantification and confirmation ion in a specified ratio of abundance.

Blanks or negative controls were analyzed concurrently with the samples and were subtracted from the analytical data

Analytic Notes

There are 2 variables:

Column 1: sequence number

Column 2: Cyanuric acid ng/mL

References

• Caudill SP, Schleicher RL, Pirkle JL. 2008. Multi-rule quality control for the age-related eye disease study. Stat Med 27:4094-4106.
• Clinical Laboratory Improvement Amendment. Public Law 100-578. 1988. 10-31-1988.

Codebook and Frequencies