HPLC−Accelerator MS Measurement of Atrazine Metabolites in Human Urine after Dermal Exposure

Abstract

Metabolites of atrazine were measured in human urine after dermal exposure using HPLC to separate and identify metabolites and accelerator mass spectrometry (AMS) to quantify them. Ring-labeled [14C]atrazine was applied for 24 h with a dermal patch to human volunteers at low (0.167 mg, 6.45 muCi) and high (1.98 mg, 24.7 muCi) doses. Urine was collected for 7 days. The urine was centrifuged to remove solids, and the supernatant was measured by liquid scintillation counting prior to injection on the HPLC to ensure that < 0.17 Bq (4.5 pCi) was injected on the column. A reversed-phase gradient of 0.1% acetic acid in water and 0.1% acetic acid in acetonitrile became less polar with increasing time and separated the parent compound and major atrazine metabolites over 31 min on an octadecylsilane column. Peaks were identified by coelution with known standards. Elution fractions were collected in 1-min increments; half of each fraction was analyzed by AMS to obtain limits of quantitation of 14 amol. Mercapturate metabolites of atrazine and dealkylated atrazine dominated the early metabolic time points, accounting for approximately 90% of the 14C in the urine. No parent compound was detected. The excreted atrazine metabolites became more polar with increasing time, and an unidentified polar metabolite that was present in all samples became as prevalent as any of the known ring metabolites several days after the dose was delivered. Knowledge of metabolite dynamics is crucial to developing useful assays for monitoring atrazine exposure in agricultural workers.

Ion-optics calculations of the LLNL AMS system for biochemical 14C measurements

“…Experiments have proven that accelerator mass spectrometry can be successfully applied to areas of biochemical interest, including studies of nutrition, toxicology, pharmacokinetics and human carcinogenesis [e.g., [1][2][3][4]. Such experiments were conducted at large multi-purpose accelerator facilities, mainly dedicated to the more the "traditional" fields of AMS research: geochemistry, geochronology, archeology and the environmental sciences.…”

Accelerator mass spectrometry of small biological samples

“…Higher currents facilitate faster data acquisition times, better statistics and consequently higher precision. We have measured 13 C ion current, after a 5 min warm up, from DNA samples from herring sperm (Invitrogen, Carlsbad, CA, USA) in the postacceleration region, after the analyzing magnet, as a function of the amount of carbon in the sample. Figure 2 shows two different measurements using standard sample preparation methods 1 and 2.…”

“…This method has been traditionally applied exclusively to high-performance liquid chromatography (HPLC) fractions, 13 e.g. body fluids, containing 14 C-labeled drugs.…”

“…The vacuum throughout the system is in the order of 3 Á 10 À8 mbar. Isotopic ratios are then measured by single particle counting of 14 C in a solid-state detector and current measurements of 13,12 C in a Faraday cup. The 14 C/ 12 C ratio, R, is presented in units of Modern (M) or as a percentage of Modern (pMC).…”

Attomole level protein sequencing by Edman degradation coupled with accelerator mass spectrometry

“…Unlike liquid scintillation counting (LSC), which counts decay events of a radioisotope, AMS is a mass spectrometric technique that detects individual 14 C nuclei. Over the past decade, AMS has been applied to life science studies and has increased precision and sensitivity of detection for 3 H and 14 C by several magnitudes (12)(13)(14)(15)(16)(17)(18). In this study, we describe an increase in Edman sequencing sensitivity by introducing AMS as a PTH-amino acid detection system.…”