Honors Program


Document Type


Date of this Version

Spring 2023


Billings, Carolyn. Extraction and Analytical Method for N-Nitrosoatrazine Using Liquid Chromatography Paired with Tandem Mass Spectrometry. Undergraduate Honors Thesis. University of Nebraska-Lincoln. 2023.


Copyright Carolyn Billings 2023


Atrazine is an herbicide used in the United States to control weeds. When combined with nitrite, atrazine can be nitrosated to form N-nitrosoatrazine (NNAT) in vivo and in vitro under acidic conditions. Atrazine can also be metabolized to atrazine mercapturate, deethylatrazine (DEA), didealkylatrazine (DDA), and deisopropylatrazine (DIA). Atrazine is monitored in drinking water due to its possible carcinogenicity. The objective of this study was to develop extraction and analytical methods for NNAT, atrazine, and atrazine metabolites. An instrument detection limit, extraction methods comparison, and extraction method detection limit were determined. To compare methods, Fischer 344 rat tissue samples were spiked with analytes and internal standard (13C3 atrazine). Method 1 consisted of acetonitrile solvent extraction, mixing by centrifuge, and filtering. Method 2 consisted of acetonitrile solvent extraction, mixing by shaker followed by centrifuge, and filtering. Method 1 had a higher NNAT recovery (26.8%) compared to method 2, so was used for all extraction method detection limit tests. Several extraction method adaptations were performed to improve NNAT recovery and determine the method detection limit. Sand samples were spiked with analytes, surrogate (terbuthylazine), and internal standard (13C3 atrazine). The samples were subjected to sequential solvent extraction using acetonitrile and were filtered and analyzed using liquid-chromatography paired with tandem mass spectrometry (LC-MS-MS). The average recoveries in sand spikes were 149, 140, 153, 124, 135, and 75.4 percent for atrazine, atrazine mercapturate, DDA, DEA, DIA, and NNAT, respectively. After further evaluations with spiked tissues, this method will be used to quantify placental transfer of these compounds in animal models.