Single- and Multiple-Laboratory Validation of LC-MS/MS Method for Simultaneous Determination of Fosetyl-Al and Phosphonic Acid in Cereal Grains and Analysis of Rice, Wheat, and Barley

Background In Japan, the residue definition for fosetyl-Al is “sum of fosetyl-Al and phosphonic acid expressed as fosetyl-Al” and its current provisional maximum residue limit (MRL) in cereals is under review. Objective For establishment and enforcement of fosetyl-Al MRL in cereals, a new analytical method for fosetyl-Al and phosphonic acid in cereals should be developed and validated. Method The new method involves water extraction, cleanup using tandem cation- and anion-exchange mini columns, and determination by LC-MS/MS. It was validated in a single laboratory and multiple laboratories. Using the method, 41 samples of rice, wheat, and barley were analyzed. Results In the multiple-laboratory validation: repeatability and reproducibility for three concentrations of fosetyl-Al and phosphonic acid were in ranges of 4.8–20 and 5.9–34%; calculated sum of fosetyl-Al and phosphonic acid, expressed as fosetyl-Al, showed good recoveries; linearity was observed for fosetyl-Al and phosphonic acid in ranges of 0.005–0.4 and 0.025–2.0 mg/kg; and specificity was sufficient. The method was verified for rice matrixes. In 41 samples, phosphonic acid was detected up to 0.2 mg/kg while fosetyl was not. Conclusions The method was successfully validated with high precision, accuracy, linearity, and specificity and capable of analyzing fosetyl-Al and phosphonic acid with a practical LOQ of 0.01 and 0.05 mg/kg. The LOQs and concentrations of phosphonic acid in samples indicate that a potential MRL would be 0.5 mg/kg for fosetyl-Al in cereals. Highlights The validated method was simpler than many methods and did not require derivatization, matrix-matched orisotopically-labeled internal standards.