Analysis of Glyphosate and Aminomethyl Phosphonic Acid by Pre-column Derivatization-HPLC

Introduction

The analytical method for glyphosate and its metabolite, aminomethylphosphonic acid (AMPA), is specified in Appendix 12 of the “Methods for Testing Water Quality Management Target Parameters” (Notification No.1010001, October 10, 2003, Final Revision March 24, 2023). This method uses pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl), followed by high-performance liquid chromatography (HPLC) with fluorescence detection.

For water quality testing, the quantification limit is required to be one-hundredth of the target concentration, with a coefficient of variation of ≤20%. For glyphosate and AMPA; however, the specified quantification limit is 0.0005 mg/L, despite a target concentration of 2 mg/L.

This application note describes the analysis of glyphosate and AMPA using an Unifinepak C18 ODS column in accordance with the specified pre-column derivatization–HPLC method.

Experimental

Instruments

Pump:                          PU-4180*

Autosampler:              AS-4050

Column Oven:             CO-4060

Detector:                      FP-4025

*With option units

LC Conditions

Column:                        Unifinepak C18 (4.6 mm I.D. x 250 mm L, 5 µm)

Guard column:             Guard cartridge column (4.0 mm I.D. x 10 mm L, 5 µm)

Eluent A:                        0.05 M phosphate buffer (pH 2.5) /acetonitrile (75/25)

Eluent B:                        0.05 M phosphate buffer (pH 2.5) /acetonitrile (35/65)

Gradient:                       (A/B), 100/0 (0 min) → 0/100 (12 min)→ 100/0 (22.1 min); 1 cycle: 37 min

Flow rate:                      1.2 mL/min (0 to 12.1 min, 22.1 to 37 min), 1.5 mL/min (12.1 to 22.1 min)

Column temperature:  40ºC

Injection volume:          5 µL

Wavelength:                  Ex. 270 nm, Em. 315 nm, Gain x100

Standard:                       Mixed standard solutions of glyphosate and AMPA (0.0005, 0.002, 0.005, 0.002, 0.05, 0.1 mg/L each)

Keywords

Glyphosate, aminomethyl phosphate, aminomethyl phosphonic acid, AMPA, Unifinepak C18, water quality standards, water quality testing, water quality management target parameters, HPLC, 9-fluorenylmethyl chloroformate, FMOC-Cl, pre-column derivatization, fluorescence detector, fluorescence detection

Results

Figure 3-1 shows chromatograms of derivatized glyphosate and AMPA mixed standard solutions at concentrations ranging from 0.0005 to 0.1 mg/L. The calibration curves exhibited good linearity over this range, with correlation coefficients (r) of ≥0.9993 for both analytes (Figure 3-2).

Fig. 4 shows chromatograms obtained from six replicate measurements of a derivatized glyphosate and AMPA mixed standard solution containing 0.0005 mg/L of each analyte. Table 1 summarizes the repeatability of retention time and peak area. Both analytes exhibited good repeatability, with relative standard deviations (RSDs) of ≤0.1% for retention time and ≤7.3% for peak area.

Table 1. Retention time and peak area repeatability for derivatized glyphosate and AMPA (0.0005 mg/L each, n = 6)

Conclusion

Glyphosate and AMPA were successfully analyzed at the specified quantification limit of 0.0005 mg/L using pre-column derivatization–HPLC with fluorescence detection in accordance with the method for water quality management target parameters. Both analytes exhibited good calibration linearity over the concentration range of 0.0005 to 0.1 mg/L, with correlation coefficients (r) of ≥0.9993. Six replicate measurements at 0.0005 mg/L demonstrated good repeatability, with relative standard deviations of ≤0.1% for retention time and ≤7.3% for peak area. These results demonstrate the method’s suitability for determining glyphosate and AMPA at the specified quantification limit required by the Water Supply Law.