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

Abstract

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.

Fig. 1

Definition of abbreviations used for atrazine derivatives with different 1-X, 3-amino-R₁ and 5-amino-R₂ substitutions on the triazine ring.

Fig. 2

Tubing connection diagram with a cascade of three two-position (1–2 and 1–10) ten-port valves. Each valve holds a different HPLC column; Valve I: SCX-t trapping column; Valve II: SCX-a analytical column; Valve III: RP-a analytical column. Each valve in 1–2 position allows the flow from Pump I (1.0 mL/min), while in 1–10 position allows the flow from Pump II (1.0 mL/min) through the corresponding HPLC column.

Fig. 3

Separation periods and concurrent loading/elution processes on the SCX-t SPE column, on the RP-a reversed phase analytical column, and on the SCX-a analytical column. Dual color indicates mixing of flows from two pumps.

Fig. 4

MS/MS chromatograms collected from a 50 μg/L spiked urine sample by connecting the mass spectrometer after the SCX-t SPE column without analytical columns (A), and by connecting the MS instrument after the analytical columns with the final 2D-HPLC–MS/MS method (B).

Fig. 5

Analyte-specific effect of the solid phase extraction parameters on MS/MS signal intensity using fractional factorial experimental design. Parameter effect contrasts expressed in percent of maximum signal intensity found for each analyte.

Fig. 6

Four-day stability study of DAA, DIA, DEA, ATZ, ATZ-Mer, and ATZ-OH in urine acidified with 1% formic acid. DAA, DEA, DIA, and ATZ hydrolyzed to their hydroxyl analogs in 4 days. ATZ, ATZ-Mer, and ATZ-OH levels decreased without producing desethyl and desisopropyl derivatives.