Glyphosate in human urine. Development of a simple analytical method using pre-column derivatization and UHPLC-MS/MS

DEMONTE, LUISINA D.; MICHLIG, MELINA P.; MAGNI, FLORENCIA V.; REPETTI, MARÍA R.

Panamá

Workshop; 9TH Latin American Pesticide Residue Workshop (LAPRW); 2023

Ministerio de Desarrollo Agropecuario de Panamá (MIDA)

The general population is ubiquitously exposed to glyphosate, the most widely used herbicide worldwide. It is currently classified as probably carcinogenic to humans by IARC, and its toxicity remains controversial. In Argentina, an agricultural country where glyphosate is used massively, it is necessary to know the population exposure to this herbicide. Urine is an interesting biological sample for biomonitoring purposes, since it is collected non-invasively and glyphosate is quickly excreted. The main objective of this study was to develop and validate an analytical method based on pre-column derivatization with FMOC and analysis through UHPLC-MS/MS to determine glyphosate in human urine.Determination of glyphosate in urine is a challenge because of its highly polar and amphoteric nature and low molecular weight. Some compounds with similar chemical groups, like creatinine, make determination even more complicated. Urine composition is variable; e.g. creatinine level is within a wide range of concentrations (30-300 mg/dL). The amine functional group of creatinine can also be derivatizated by FMOC; for that reason, an extra amount of FMOC (from 6-50 mg/mL) was necessary to allow the complete derivatization reaction of analytes. Briefly, the analytical method has a first step of protein precipitation with acetonitrile [1], subsequent derivatization with FMOC in basic medium (pH=9) and a clean-up by L-L partition with dichloromethane [2]. Finally, an aqueous phase fraction is injected into the UHPLC-MS/MS system.To improve the instrumental conditions for analyte identification, confirmation and sensitivity, different mobile phases (H2O:MeOH, H2O:MeCN) with ammonium acetate, acetic acid and formic acid as modifier were evaluated.In the validation process, the recoveries obtained were 68-130%. LOD and LOQ were 0.1 and 0.5 μg/L, respectively. The linearity was evaluated by analyzing glyphosate 1,2-13C2 15N within the range of 0.1-25 μg/L (6 levels) in urine samples with different creatinine levels (low, medium and high). For quantitative analysis, each urine sample was spiked with glyphosate 1,2-13C2 15N to obtain 10 µg/L. The method was tested by analyzing 35 human urine samples from volunteers. The developed and validated method has an acceptable performance and can be used in monitoring projects in our country.