Quantification of glyphosate and glufosinate in human blood using the aqueous layer recovered from QuEChERS extraction.

The Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method is widely used as a pretreatment technique to extract a broad range of drugs and toxicants into the acetonitrile layer. However, the aqueous layer generated during the extraction process has a high-salt content and has not been analytically explored. This study focused on the unused aqueous layer and established a method for detecting the highly polar compounds glyphosate (Gly) and glufosinate (Glu) through derivatization with 9-fluorenylmethoxycarbonyl chloride, followed by liquid chromatography–tandem mass spectrometry. Under optimized conditions, both analytes were effectively separated and detected with favorable peak shapes from 50 µL of whole blood. Calibration curves exhibited good linearity over the range of 0.1–10 µg/mL, with correlation coefficients of 0.993 for Gly and 0.994 for Glu. The lower limit of quantification for both compounds was 0.1 µg/mL. The method also demonstrated acceptable precision, accuracy, process efficiency, and stability. Herein, we demonstrate that the aqueous layer generated during the QuEChERS extraction process can serve as a valuable analytical matrix for determining Gly and Glu in blood. This method can be employed by directly adding the derivatization reagent to the aqueous layer without modifying the conventional QuEChERS extraction process. This method has the ability to reduce sample consumption by employing multiple extraction layers with distinct physicochemical characteristics. This is particularly important when only a limited number of sample volumes is available such as in fatal cases or poisoning incidents.