UPLC-MS/MS Determination of Paraquat and Diquat in Potato and Wheat Using the CORTECS UPLC HILIC Column

In this application, we demonstrate a UPLC-MS/MS method using a CORTECS HILIC Column with significantly improved retention and resolution of both analytes compared with other current HILIC methods.

Benefits

• The CORTECS UPLC HILIC Column gives improved retention and resolution of paraquat and diquat compared with other current HILIC methods
  
• The CORTECS UPLC HILIC is compatible with the fast screening method adopted for use in the EU
• The CORTECS UPLC HILIC separation requires no ion-pairing reagents and provides superior sensitivity for LC-MS analysis

Diquat and paraquat are doubly charged quaternary ammonium herbicides (Figure 1). They have been and still are extensively used worldwide for  controlling both crop and aquatic weeds. The US EPA tolerances for diquat are 100 ppb (µg/kg) in potato and 20 ppb in wheat. The US EPA tolerances for paraquat are 500 ppb in potato and 1100 ppb in wheat Diquat and paraquat are ionic substances that are poorly retained on C₁₈ or other reversed phase LC columns. Ion-paring reagents can be employed to enhance reversed-phase retention but this approach usually results in significant ion-suppression if  mass-spectrometry is used for detection and quantitation (LC-MS). An alternative methodology, Hydrophilic Interaction Liquid Chromatography (HILIC) has a distinct advantage for  LC-MS determination of diquat and paraquat; ionization efficiency is improved because no ion-pairing reagents are required. In this application, we demonstrate a UPLC-MS/MS method using a CORTECS HILIC Column with significantly improved retention and resolution of both analytes compared with other current HILIC methods.

Sample preparation

Note: Polypropylene containers should be used for sample collection and for all sample preparation steps. Polypropylene auto sampler vials (part number 186002642) are recommended for UPLC analysis.

Sample of fresh potato and processed whole wheat flour were prepared using a procedures described in a recently published method.¹ This methodology, available on the web,² is recommended by the EU for screening commodities for paraquat and diquat residues. 

Potato Analysis. Samples (10 g) were weighed into 50 mL centrifuge tubes. Fortified samples were prepared by spiking aqueous standards into the pre-weighed sample followed by 30 minute equilibration. 10 mL of extraction solution (50:50 methanol/0.1 M HCl in water) was then added and the samples were shaken by hand for 2 minutes. The samples were then heated at 80 °C for  15 minutes, allowed to cool and then centrifuged at 4000 rpm (rcf 3250x g) for 4 minutes. An aliquot of the supernatant was filtered using a 45 micron PTFE syringe filter. A 400 µL portion of the filtered sample was diluted to 1.0 mL with acetonitrile prior to UPLC-MS analysis.

Flour Analysis. Samples (4 g) were weighed into 50 mL centrifuge tubes. Fortified samples were prepared by spiking aqueous standards into the pre-weighed sample followed by 30 minute equilibration. 10 mL of reagent water was then added to the samples, the samples were vortexed and allowed to equilibrate for 15 minutes. The samples were then extracted and processed in the same manner as the potato samples discussed above.

Table 1 summarizes the MRM transitions and  LC-MS parameters used for this study.

Figure 2 shows typical UPLC-MS/MS extracted ion chromatograms obtained from the analysis of a  sample of whole wheat flour fortified to 10 ppb of each analyte prior to all sample preparation steps. The ion-chromatogram obtained for potato was similar.

The CORTECS UPLC HILIC separation was shown to be suitable for use with the fast screening method adopted for use in the EU. Prior to UPLC-MS/MS analysis the extracted sample was simply filtered and diluted. The CORTECS UPLC HILIC method is isocratic (50:50 acetonitrile/buffer). Therefore, the sample extract need only be diluted 1:1 with acetonitrile to achieve good chromatographic performance. The limit of quantitation (LOQ) was below 10 ng/g for diquat in potato and wheat flour. The LOQ for paraquat was below 10 ppb for potato and about 10 ppb for wheat flour.

• Baseline separation of diquat and paraquat was achieved using the CORTECS HILIC Column with total analysis time of 4 minutes
  
• Detection limits at or below 10 ppb were achieved for determination of both quats in fresh potato and in wheat flour

1. Kohlberg, D.I.S., Mack, D., Anastassiades, M., Hetmanski, M.T., Fussell, R.J., Meijer, T., and Mol, H.G.J., Anal. Bioanal. Chem, 404, 2465-2474 (2012).
   
2. Anastassiades, M, Kolberg, D.I., Mack, D., Wildgrube C., Sigalova, I., Roux, D., and Fügel, D., “Quick Method for the Analysis of Residues of numerous Highly Polar Pesticides in Foods of Plant Origin involving Simultaneous Extraction with Methanol and LC-MS/MS Determination”, EU Reference Laboratory for pesticides requiring single Residue Methods (EURL-SRM), Version 7 (Dec 2012).