2.2. Measurement of Caffeine Metabolites in Urine

Spot urine samples were collected by experienced operators at the MECs. Recorded documents included the date and time of sampling and the volume of urine collection. Samples were stored at ≤−70 °C based on the Laboratory Procedures Manual before transportation to the National Center for Environmental Health (Centers for Disease Control and Prevention, Atlanta, GA, USA) for testing. Urinary metabolite quantification was determined by ultra-high performance liquid chromatography–electrospray ionization–tandem quadrupole mass spectrometry (UHPLC–ESI–MS/MS) (Agilent Technologies, Palo Alta, CA, USA) with stable isotope-labeled internal standards. More detailed methods are reported on the NHANES website.

Caffeine and 14 of its urinary metabolites, 15 in total, were examined, including 1-methyluric acid, 3-methyluric acid, 7-methyluric acid, 1,3-dimethyluric acid, 1,7-dimethyluric acid, 3,7-dimethyluric acid, 1,3,7-trimethyluric acid, 1-methylxanthine, 3-methylxanthine, 7-methylxanthine, 1,3-dimethylxanthine (theophylline), 1,7-dimethylxanthine (paraxanthine), 3,7-dimethylxanthine (theobromine), 1,3,7-trimethylxanthine (caffeine), and 5-acetylamino-6-amino-3-methyluracil (AAMU). AAMU is the decomposition product of the relatively unstable caffeine metabolite 5-acetylamino-6-formylamino-3-methyluracil (AFMU). Samples were allowed to incubate for at least 30 min at room temperature so that conversion of all AFMU to the more stable AAMU was ensured.

The lower limit of detection (LLOD in umol/L) for caffeine and caffeine metabolites can be obtained from the NHANES website. For analytes with results below the lower limit of detection, the value is the lower limit of detection divided by the square root of 2 (LLOD/sqrt [2]). All presented data satisfied quality control (QC) procedures, which were performed by a multirule quality control system. Samples examined were collected from 3 QC pools (low-, medium-, and high-quality control pools). Urine analyte concentrations were adjusted to urinary creatinine (uCr) by dividing urine concentration of metabolites by uCr values.