Determination of the Cyanide Metabolite, L-2-Amino-2-Thiazoline-4R-Carboxylic Acid (L-ATCA), in Human Urine by Automated Solid Phase Extraction and Isotope Dilution LC-MS-MS
Wed 2:00 PM - Track 3: Emergency Response
William M. Draper
California Dept of Public Health
William M. Draper, Dayananda Rajapaksa, James Chithalen, Donald Wijekoon and Kusum Perera

Sanitation & Radiation Laboratory, California Department of Public Health, Richmond, CA 94804
The determination of urinary ATCA provides certain advantages in cyanide exposure measurement and biomonitoring. In particular, ATCA is a very stable cyanide metabolite in the urine matrix, and urine specimens are easier to obtain and handle than blood. The present paper describes development of a reliable, high throughput method for quantitative analysis of ATCA in urine, based on solid phase extraction (SPE) using an automated 96 well plate apparatus, followed by instrumental analysis using isotope dilution LC-MS-MS. A D3-labeled internal standard was prepared by reaction of D3-cysteine with cyanamide. The chemical and isotopic purity of the product was excellent based on +ESI mass spectrometry and high field NMR spectroscopy. In +ESI ATCA gives an MH+ ion as the base peak (m/z 147) – the major fragment ions of m/z 147 are m/z 104 (C3H5N2S+) and m/z 59 (S=CH2=CH+). Calibration curves for both transitions have high linearity (r2 > 0.99) over the range of 5 to 1,500 ng/mL in human urine – the D3 internal standard avoids interference between isotope peaks that can occur using D1 or D2 labeled internal standards. This phenomenon of lower mass internal standards flattens calibration curves at high analyte concentrations and high analyte:IS ratios, e.g., greater that 10:1. ATCA is extracted from urine using mixed mode cation exchange resins in a 96 well plate format, and ATCA is determined without derivatization using a cyano HPLC column. The method has an estimated throughput of several hundred specimens/8 hr shift.

(This research was supported, in part, by the federal Centers for Disease Control and Prevention of the U. S. Department of Health and Human Services.)