Introduction

The analytical method for glyphosate and its metabolite, aminomethylphosphonic acid (AMPA), is outlined in Appendix 12 to the “Methods for Testing Water Quality Management Target Parameters” (Notification No. 1010001, October 10, 2003, Final Revision March 24, 2023). It specifies the use of a derivatization-HPLC method. This analytical method involves derivatizing glyphosate and AMPA in a water sample using 9-fluorenylmethyl chloroacetate (FMOC-Cl) and detecting the resulting compound by fluorescence using an HPLC system. For water quality testing, the quantification limit must be 1/100th of the target concentration, and the coefficient of variation should be ≤20 %. However, for glyphosate and AMPA, while the target concentration is set at 2 mg/L, the quantification limit is 0.0005 mg/L.

In this application note, we present the results of glyphosate and AMPA measurements using an Unifinepak C18 ODS column based on this testing method.

LC-4000 series HPLC system

Experimental

Instruments
Pump:  PU-4180*
Autosampler:  AS-4050
Column oven:  CO-4060
Detector:  FP-4025
*with option units

Conditions
Column:   Unifinepak C18  (4.6 mmI.D. x 250 mmL, 5 µm)
Guard column:  Guard cartridge column (4.0 mmI.D. x 10 mmL, 5 µm)
Eluent A:   0.05 mol/L phosphate buffer (pH 2.5) /acetonitrile (75/25)
Eluent B:  0.05 mol/L phosphate buffer (pH 2.5) /acetonitrile (35/65)
Gradient:  (A/B) , 100/0 (0 min) -> 0/100 (12 min) -> 100/0 (22.1 min) , 1cycle; 37 min
Flow rate:  1.2 mL/min (0～12.1 min, 22.1～37 min), 1.5 mL/min (12.1～22.1 min)
Column temp.:   40 ºC
Wavelength:   Ex. 270 nm, Em. 315 nm, Gain x 100
Inj. volume:   5 µL
Standard:  Mixed standard solutions of glyphosate and AMPA (0.0005, 0.002, 0.005, 0.002, 0.05, 0.1 mg/L each)

Procedure for derivatization of glyphosate and AMPA

Reaction formulas for glyphosate and AMPA with FMOC-Cl

Keywords

Glyphosate, Aminomethyl phosphate (AMPA), Water quality standards, Water quality management target, FMOC-Cl, Pre-column derivatization, Unifinepak C18, Fluorescence detector

Results

Figure 1-1 shows chromatograms of mixed standard solutions of glyphosate and AMPA with concentrations of 0.0005 to 0.1 mg/L. As shown in Figure 1-2, the calibration curves exhibited good linearity, with a correlation coefficient (r) of ≥ 0.9993.

Fig. 1-1   Chromatograms of mixed standard solutions of derivatized glyphosate and AMPA (0.0005, 0.002, 0.005, 0.02, 0.05, 0.1 mg/L)
(A): Full time range, (B): Enlargement of the retention time range of 6.0-12.0 min for low-concentration solutions (0.0005, 0.002, 0.005 mg/L)
1: Derivatized glyphosate, 2: Derivatized AMPA

Fig. 1-2 Calibration curve for derivatized glyphosate and AMPA

Fig. 2 shows chromatograms for derivatized glyphosate and AMPA (n = 6) obtained by repeat measurements of 0.0005 mg/L standard solutions, and Table 1 shows the repeatability of the retention times and the peak areas. For all glyphosate and AMPA, good results were obtained, with a repeatability of ≤0.1 % for retention time and ≤7.3 % for peak area.

Fig.2   Chromatograms for derivatized glyphosate and AMPA (0.0005 mg/L, n = 6)
1: Derivatized glyphosate, 2: Derivatized AMPA

Table 1   Repeatability of retention time and peak area for derivatized glyphosate and AMPA (0.0005 mg/L, n = 6)

Conclusion

Measurements of glyphosate and AMPA using pre-column derivatization-HPLC based on water quality management target parameters showed that concentrations as low as 0.0005 mg/L could be detected, which is required by the Water Supply Law. A calibration curve was produced by measuring derivatized mixed standard solutions with concentrations of 0.0005, 0.002, 0.005, 0.02, 0.05, and 0.1 mg/L, and it was confirmed that the method ensures good linearity. For repeat measurements at the limit of quantification of 0.0005 mg/L, good repeatability for both the retention time and peak area was obtained.