Development and validation of a liquid chromatography-fluorescence-mass spectrometry method to measure glyphosate and aminomethylphosphonic acid in rat plasma.

A simple and fast method has been developed and validated to measure glyphosate (GLYP) and aminomethylphosphonic acid (AMPA) in rat plasma based on reversed-phase high performance liquid chromatography (RP-HPLC) coupled to fluorescence (FLD) and electrospray ionization mass spectrometry (ESI-MS) detection. After protein precipitation with acetonitrile, GLYP and AMPA were derivatized with 9-fluorenylmethylchloroformate (FMOC-Cl) and then separated on a C(12) column (250mm×4.60mm i.d.) using a gradient of an ammonium formate (20mM, pH 8.5) and acetonitrile mobile phase. Selected ion monitoring (SIM) mode of the MS was used to obtain maximum sensitivity when quantifying GLYP and AMPA. The validation shows the method to be consistent and reliable, with an intra- and inter-day precision for GLYP and AMPA>9% for both detectors. For both compounds the accuracy ranged from 2.1% to 7.8% for the intra-day readings, and from 4.1% to 8.6% for the inter-day values. The efficacy of GLYP extraction ranged from 87% to 93% and it was between 76% and 88% for AMPA. Moreover, the limits of quantification (LOQ) for GLYP and AMPA were 5 and 10ng/mL, respectively with FLD, and 0.4 and 2ng/mL with ESI-MS. The method was successfully applied to simultaneously measure both compounds in rat plasma samples several days after oral administration of glyphosate.