Agilent 1200 Series HPLC System. HPLC Column: Phenomenex. ®. Luna. ®. Phenyl-Hexyl (150 à ... Rudolph von Abele, and Barbara Schumacher for helpful.
Development of Quantitative LC-MS/MS Method for Glyphosate Residue Analysis Ganesh S. Moorthy, Karen M. L’Empereur, Ann B. Orth , and Julie E. Eble Critical Path Services, LLC, 3070 McCann Farm Drive, Suite 112, Garnet Valley, PA 19060
Abstract A sensitive and specific reversed-phase liquid chromatography coupled electrospray ionization tandem mass spectrometry (LC-MS/MS) method was developed for quantitative analysis of glyphosate in soybean seed and corn grain. Utilizing liquid-liquid extraction followed by protein precipitation and LC-MS/MS analysis, we demonstrate good recovery for glyphosate in soybean seed and corn grain. This method is simple, specific, and efficient.
Figure 1: Structure of Glyphosate
Experimental (continued) Instruments: Agilent 1200 Series HPLC System HPLC Column: Phenomenex Luna Phenyl-Hexyl (150 4.6 mm, 3 m) Mobile Phases: A: 0.8 % Formic Acid in water B: Methanol Applied Biosystems API 4000 triple quadrupole mass spectrometer with a TurboIonSpray ionization source.
Time (min)
Flow Rate (mL/min)
%A
%B
0.0
0.750
95
5
3.5
0.750
55
45
3.6
0.750
5
95
*
Introduction Glyphosate (N-phosphonomethyl glycine) is a widely used herbicide.1 It has a broad spectrum of activity with little harmful effects on mammals. Glyphosate’s high efficacy, low toxicity, and low cost have led to its widespread utilization in many crops. Due to its low toxicity, the maximum residue levels (MRLs) for glyphosate established around the world are generally greater than those set for other pesticides (MRL of 0.2 mg/kg in soybean).2 Glyphosate’s involatility, high water solubility, and absence of chromophores make it challenging to analyze without derivatization using GC or HPLC with traditional detectors. However, it can readily be analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The majority of published MS/MS methods involve derivatization, and only recently a method for direct quantitation of glyphosate by LC-MS/MS was reported.3 In the current study, we present a sensitive method for quantitation of glyphosate in agricultural samples.
Experimental Materials: Glyphosate and 2-13C, 15N-glyphosate (internal standard) were received from Cambridge Isotope Laboratories, Inc. The stock solutions of glyphosate (0.5 mg/mL) were prepared in 50/50 (v/v) methanol/water. A calibration standard curve for glyphosate was prepared at concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 25, 50, and 100 ng/mL with internal standard (final concentration: 5 ng/mL).
Results Representative chromatograms, calibration standard curve . and summary of recovery for glyphosate in soybean seed and corn grain is presented below.
Figure 2: Representative Chromatogram of Glyphosate (0.5 ng/mL, LOQ) and Internal Standard (5 ng/mL) Glyphosate (MRM: 17088)
HPLC Conditions: Injection Volume: 25 L
*
Discussion
5.0
0.750
5
95
5.1
0.750
95
5
10.0
0.750
95
5
Internal Standard 2-13C, 15N-Glyphosate (MRM: 17290)
MS/MS Conditions: Polarity and Ionization: Positive ESI Analyte
Parent (m/z) Product (m/z) Dwell Time (msec)
Glyphosate 2-13C,
15N-Glyphosate
170
88
200
172
90
200
Polarity and Ionization: Positive ESI CAD: 6 GS2: 40 DP: 28 CUR: 25 TEM: 500 CE: 13 GS1: 35 IS: 4500
EP: 5 CXP: 10
Fortifications: Fortification of soybean seed (dried) and corn grain were performed at 0.05 mg/kg and 0.5 mg/kg as triplicates. Soybean seed and corn grain were extracted with methylene chloride and protein precipitated with methanol as described previously3. The extract was diluted 1:5 or 1:50 with water or 0.02 M phosphoric acid solution with addition of internal standard (5 ng/mL) and filtered. The samples were analyzed by LC-MS/MS and recoveries calculated for each fortification level (Table 1). Blended Soybean Seed or Corn Grain (0.5 g)
Shake for 60 min Centrifuge at 3000 rpm, 15 min + CH2Cl2 (1.25 mL) + Water (5 mL) + Glyphosate (0.05 or 0.5 mg/kg)
Figure 3: Calibration Standard Curve for Glyphosate
LC-MS/MS Analysis 1:5 or 1:50 dilution + Internal standard Filter (Acrodisc 0.2 M) Centrifuge, 3000 rpm, 15 min + 0.5 mL MeOH, vortex Transfer 0.5 mL of aqueous layer
Glyphosate ionizes in both positive and negative ion mode; however, positive electrospray ionization mode has better sensitivity. Unlike the recently published method3, use of stable labeled glyphosate as internal standard in the present study is valuable in addressing matrix effects. During method development, a variety of HPLC columns (Zorbax Eclipse XDB-C8 or -C18, Zorbax SB-Aq, and Luna phenyl-hexyl) were evaluated. The Luna phenyl-hexyl column gave better chromatographic peak shape and sensitivity under the present LC conditions. A Zorbax Eclipse XDB-C18 RRHT column provided much shorter run time (1.5 min) and excellent sensitivity under isocratic conditions; however, this method was not selected due to the potential for matrix effects resulting from limited separation. In the present study, we have utilized liquid-liquid extraction and protein precipitation for processing samples. Minimal matrix effects were seen at 1:50 dilution. However, ion counts were significantly suppressed with 1:5 dilutions (0.05 mg/kg). The extent of ion suppression was severe with soybean seed compared to corn grain fortification extracts. Future studies will evaluate the utility of solid phase extraction in minimizing matrix effects during residue analysis of various agricultural samples.
Conclusions • A specific and sensitive LC-MS/MS method was developed for analysis of glyphosate (LOQ was 0.5 ng/mL and LOD was 0.1 ng/mL). • The method was successfully employed for quantitation of glyphosate in fortified soybean seed and corn grain. • Acceptable recoveries were seen at both 0.05 and 0.5 mg/kg levels. Ion suppression due to matrix effects were compensated by using stable labeled glyphosate as internal standard.
References
Table 1: Summary of Fortification Recovery for Glyphosate in Soybean Seed and Corn Grain (n = 3) Matrix
Fortification Level (mg/kg)
Soybean Seed Corn Grain
0.05 0.50 0.05 0.50
Recovery, Mean SD 90 14 100 9 78 11 92 5
[1] Glyphosate: a once-in-a-century herbicide. Duke SO, Powles SB. Pest Manag Sci. 2008 Apr;64(4):319–25. [2] Codex Alimentarius Commission Maximum Residue Limits for Pesticides—CAC/MRL, 1 (2001). [3] An alternative and fast method for determination of glyphosate and aminomethylphosphonic acid (AMPA) residues in soybean using liquid chromatography coupled with tandem mass spectrometry. Martins-Júnior HA, Lebre DT, Wang AY, Pires MA, Bustillos OV. Rapid Commun Mass Spectrom. 2009 Apr;23(7):1029–34.
Acknowledgements We wish to thank Spencer Drummond and Elizabeth Cherkauskas for technical support and James McVicker, Rudolph von Abele, and Barbara Schumacher for helpful discussions.
