Urinary Metals Analysis by ICP-MS

We measured 24 urinary metals in urine samples, including lithium, beryllium (Be), aluminum (Al), titanium, vanadium (V), chromium (Cr), manganese, iron (Fe), cobalt, nickel (Ni), Cu, Zn, As, Se, rubidium, strontium, molybdenum (Mo), Cd, indium (In), tin, antimony (Sb), barium, thallium (Tl), and Pb using inductively coupled plasma-mass spectrometry (ICP-MS, NEXION 300X. PerkinElmer Inc. Waltham, Massachusetts, USA). Briefly, urine samples were thawed at room temperature and then centrifuged (4,200 rpm × 10 min) at room temperature. Afterward, 0.5 ml of supernatant from each urine sample was added into a 15 ml polypropylene tube, and then 4.5 ml of 2% nitric acid solution (10 times the diluted urine sample) was added. To assess the accuracy of the measurements, Seronom^TM Trace Elements Urine L-1 (Sero Incorporated Company. Billingstad, Norway), Seronom^TM Trace Elements Urine L-2 (Sero Incorporated Company. Billingstad, Norway), and Trace Elements in Natural Water (SRM1640a) (National Institute of Standards and Technology. Gaithersburg, Maryland, USA) were added as quality control samples for each batch. As shown in Supplementary Table 1, the range values of 66.04–152.86% in urinary metals were considered acceptable for spike recoveries. The limits of detection (LOD) ranged from 0 to 1.66 μg/L for urinary Fe and from 0 to 0.13 μg/L for urinary Zn. The limits of quantification (LOQ) ranged from 0.01 to 5.54 μg/L for urinary Fe and from 0 to 0.44 μg/L for urinary Zn. Urinary concentrations of Be, In, and Sb were excluded from further analysis because the values of Be, In, and Sb in more than 80% of individuals were below the corresponding LOD. Values of urinary metals below the LOQ were replaced by LOQ/2.