Two-dimensional high performance liquid chromatography separation and tandem mass spectrometry detection of atrazine and its metabolic and hydrolysis products in urine

Atrazine [6-chloro-N-ethyl-N′-(1-methylethyl)-1,3,5-triazine-2,4-diamine] is the most widely used herbicide in the United States. In recent years, there has been controversy about atrazine's potential endocrine/reproductive and neurological adverse effects in wildlife and humans. The controversy triggered several environmental and epidemiologic studies, and it generated needs for sensitive and selective analytical methods for the quantification of atrazine, atrazine metabolites, and degradation or hydrolysis products. We developed a two-dimensional high performance liquid chromatography (2D-HPLC) method with isotope dilution tandem mass spectrometry detection to measure atrazine in urine, along with 11 atrazine metabolites and hydrolysis products, including 6-chloro (Cl), 6-mercapto (Mer) and 6-hydroxy (OH) derivatives, and their desethyl, desisopropyl and diamino atrazine analogs (DEA, DIA and DAA, respectively). The 2D-HPLC system incorporated strong cation exchange and reversed phase separation modes. This versatile approach can be used for the quantitative determination of all 12 compounds in experimental animals for toxicological studies. The method requires only 10 μL of urine, and the limits of detection (LODs) range from 10 to 50 μg/L. The method can also be applied to assess atrazine exposure in occupational settings by measurement of 6-Cl and 6-Mer analogs, which requires only 100 μL of urine with LODs of 1–5 μg/L. Finally, in combination with automated off-line solid phase extraction before 2D-HPLC, the method can also be applied in non-occupational environmental exposure studies for the determination of −6-Cl and 6-Mer metabolites, using 500 μL of urine and LODs of 0.1–0.5 μg/L.

► Simultaneous measurement of metabolites of atrazine in human urine by HPLC–MS/MS.
► 2D HPLC method design based on both pKa and polarity of atrazine derivatives.
► Development of a 2D HPLC method incorporating both SCX–RP and SCX–SCX separation.
► Method validation for application in animal toxicology studies and biomonitoring.