Biomonitoring of glyphosate and its metabolite, aminomethylphosphonic acid (AMPA), in human urine

Using the QTRAP 6500+ system

P. Ruiz¹ , P. Dualde1 , J. Lliberia² and A. Morla^2
1Public Health Laboratory of Valencia, Spain; ²SCIEX, France

Introduction

Glyphosate (N-(phosphonomethyl)-glycine, Gly) is an herbicide that was introduced to the market in 1974 and has become the most widely used agricultural herbicide in the world in the last decade. It has been related to several types of cancer¹ and is classified as “probably carcinogenic to humans” (Group 2A) by the International Agency for Research on Cancer (IARC, 2017). ² Conversely, the European Food Safety Authority (EFSA) claimed that Gly is “unlikely to pose a carcinogenic hazard to humans,” establishing an acceptable daily intake (ADI) value of 0.5 mg/kg of bodyweight (BW) per day (mg/kg BW/day) (EFSA, 2015). Another potential source of toxicity is aminomethylphosphonic acid (AMPA). AMPA is a metabolite of Gly and has a similar toxicological profile to Gly. The structural similarities of the compounds involved are shown in Figure 1. 

AMPA can be detected in urine primarily due to its consumption in food and water and, to a lesser extent, due to human metabolism of Gly.³ AMPA accumulates in soil and therefore can be found in the environment. ⁴ For biomonitoring studies, the parent compound in urine is an appropriate biomarker for Gly exposure. The presence of the Gly metabolite, AMPA, in urine is a biomarker of direct exposure to AMPA and the small fraction of Gly that is bio-transformed to AMPA. In this study, the levels of Gly and AMPA present in the urine of Spanish breastfeeding mothers⁵ was assessed.

Key features of the QTRAP 6500+ system 
 

• Simple solid phase extraction to isolate target analytes from human urine
  
  
• Eight-minute runtime to analyze the target analytes
  
  
• QTRAP 6500+ system for high sensitivity assays in bioanalysis
  
  
• IonDrive Turbo V ion source for improved ionization efficiency at high flows and more robustness

Methods

Sample preparation: Urine samples (5 mL) were extracted by solid phase extraction (SPE) with SAX cartridges, a process which briefly consisted of these tasks:

• Condition cartridges with methanol and water 
• Load samples onto wet cartridges 
• Wash with methanol and dry under vacuum 
• Elute with strong acid solution
• Evaporate eluates to dryness
• Reconstitute in water with 0.005% formic acid and transfer to vials for injection

Liquid chromatography: Chromatographic separation was accomplished with a Metrosep A Supp 5 (100 mm × 4.0 mm, 5 µm) column equipped with a Metrosep A Supp 5 Guard/4.0 column, at a flow rate of 0.7 mL/min. Mobile phase A was 0.02% formic acid in water and mobile phase B was 100 mM ammonium bicarbonate. A 200 μL sample of extract was injected into the UHPLC system. 

Mass spectrometry: MS/MS detection was performed using the QTRAP 6500+ system equipped with the IonDrive Turbo V ion source and operated with the electrospray ionization (ESI) interface in negative ionization mode. Multiple reaction monitoring (MRM) mode and QTRAP MS3 mode were employed. Dwell and cycle times were optimized according to the scan modes used. Mass spectrometry parameters are shown in Table 1.

Data processing: Data were acquired using Analyst software and processed using SCIEX OS software.

Analytical performance

Chromatographic conditions were optimized and good retention of the target analytes was demonstrated (Figure 2). Several identification criteria were implemented for Gly and AMPA. First, the maximum retention time deviation between each analyte and its internal isotope-labeled standards (ILIS) in each sample had to be ±0.1 min. Second, for Gly, at least 2 ion transitions were required for quantification and confirmation purposes, respectively. For the ILIS, only the quantitative ion was required and only 1 transition was achieved for AMPA. Third, the signal-to-noise ratio of the peaks had to be ≥3. Fourth, the analyte peaks of both products' ions had to overlap in the chromatograms. Last, the ion ratio had to be within ±30% of the ion ratio range, which is the average for standards of the same sequence. 

We determined the creatinine concentration in urine using Jaffé's reaction method to adjust the Gly and AMPA urinary concentrations. Performance criteria for the proposed method were generated considering SANTE 12682/2019⁶ and the Bioanalytical Method Validation Guidance for Industry.⁷ The average recovery ranged from 84% to 113% for Gly and from 95% to 117% for AMPA. For both analytes, the RSD was lower than 13%, indicating good precision. The lower limit of quantification (LLOQ) was 0.1 μg/L for both analytes (Table 2). 

For quantification purposes, a 6-point, matrix-matched calibration curve and ILIS were employed. The linear dynamic range (LDR) was established from 0.1 to 10 μg/L and the accuracy of all calibrators was in the range of ±20% (Figure 3). A series of quality control samples was also run and prepared by spiking blank urine with standard solutions to levels of 0.1, 1 and 10 μg/L. Likewise, a reagent blank (water) and a blank sample were included in each batch to check for process contamination. The method successfully fulfilled the requirements of the German External Quality Assessment Scheme (G-EQUAS) intercomparison program, round 66, in 2020.

Conclusion

This was the first work to assess the levels of Gly and AMPA in urine from and risk of Spanish breastfeeding mothers. In this study, the pesticide Gly and its main metabolite AMPA were analyzed in urine samples of Spanish lactating mothers with good sensitivity and linearity. Ruiz P et al. report that glyphosate and AMPA had detection frequencies of 54% and 60%, with means of 0.12 μg/L and 0.14 μg/L, respectively. ⁵ Their studied population had similar levels of both analytes to those reported in other parts of the world. They detected Gly and AMPA in the urine of different groups within their population, including vulnerable groups, similar to the breastfeeding mothers included in the present study. Given the prevalence of these compounds and their potential effects on the human body, it would be important to include these two compounds in the scope of analyzed substances in human biomonitoring programs.

References
 

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6. SANTE, (2019) Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed, SANTE/12682/2019
   
   
7. FDA, US, (2018) US Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research and Center for Veterinary Medicine, Bioanalytical Method Validation Guidance for Industry