Highly polar pesticide multi-residue analysis in food safety by LC-MS/MS

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  • Published: May 1, 2015
  • Categories: Base Peak
thumbnail image: Highly polar pesticide multi-residue analysis in food safety by LC-MS/MS

David R. Baker, Shimadzu, Manchester, UK
Mikaël Levi, Shimadzu, Marne-La-Vallée, France
Eric Capodanno, Phytocontrol, Nimes, France


Food safety laboratories involved in pesticide residue monitoring utilise multi-residue LC-MS/MS methods for the quantification of an ever increasing list of target pesticides. However, the determination of several highly polar pesticides is extremely challenging due to their low mass, amphoteric character and lack of chemical groups for detection by LC/MS/MS. For this reason, single residue methods or small group specific methods are often used to analyse these compounds, in some cases including the use of pre- or post-column derivatisation. However, the application of single residue methods requires considerable laboratory resources relative to the number of compounds analysed; hence very few laboratories actually target these compounds on a regular basis. Consequently, there is a clear need for simple and fast LCMS methods that are capable of analysing many highly polar pesticides in a single run and achieving the required regulatory reporting limits.

The aim of this study was to develop a fast, sensitive and simple methodology for a range of challenging highly polar pesticides that require single-residue methods, by as few multi-residue LC-MS/MS runs as possible and without the need for derivatisation. Several different analytical columns and mobile phases were evaluated in this study, in addition to assessing the MS/MS parameters.

Figure 1. Target analyte structures.


Sample extracts were provided by Phytocontrol and extracted according to the EURL-SRM QuPPe methodology.1 Briefly, apple samples (10 g) were prepared by chopping up the sample, freezing, homogenizing with dry ice, adding 1% formic acid in methanol solution (10 mL) and centrifuging (4000 RPM).

Preliminary investigations involved the testing of several different analytical columns: SIELC Obselisc R (150 x 2.1mm, 5µm); Hypercarb PGC (100 x 2.1mm, 5µm); SeQuant ZiC-HILIC (100 x 2.1mm, 3.5µm), SeQuant ZIC-cHILIC (100 x 2.1mm, 3.5µm), Scherzo SM-C18 (50 x 2, 3 µm), Scherzo SW-C18 (50 x 2, 3 µm), Fortis Phenyl (100 x 2.1mm, 5µm), Luna Phenyl-Hexyl (100 x 2.1mm, 3µm), and Restek IBD (150 x 2.1mm, 3µm). These columns were tested with several different mobile phase additives including acetic acid, formic acid, ammonium formate, ammonium acetate and ammonium hydroxide (depending on appropriate conditions for each column and the progression of results). Reversed phase, HILIC, and mixed mode chromatography were tested depending on the column suitability for each mode. Finally, two methods were developed, the LC method conditions are listed in Table 1 and LCMS-8050 conditions listed in Table 2.

Linearity was evaluated by spiking sample extracts at the following levels: 0.005, 0.01, 0.02, 0.05, 0.1 and 0.2 mg/kg. Deuterated internal standards were used for calibration; Table 3 lists the internal standard used for each target analyte. All calibration points were analysed in duplicate. Plastic vials were used for analysis to prevent adsorption to glass surfaces (e.g. paraquat, diquat and glyphosate)1 (Figure 1).

Table 1
Table 1. UHPLC conditions for method 1 and method 2.


Table 2
Table 2. LCMS-8050 parameters.


Following evaluation of several different analytical columns, mobile phases and ma

ss spectrometer settings, two methods were developed for a range of highly polar pesticides that typically require single residue methods to analyse. Three MRM transitions were acquired for each analyte, with the exception of two transitions for kasugamycine. Linearity was evaluated for all compounds in the range 0.005 mg/kg – 0.2 mg/kg (5 – 200 ppb) in apple matrix. Thereby ensuring that the sensitivity of the method is below the European Union (EU) maximum residue limit (MRL) for all of the target analytes in this study.2

A ZIC-HILIC column, a zwitterionic stationary phase covalently attached to porous silica, was utilised in method 1 to analyse the following; amitrole, chlormequat, daminozide, diquat, kasugamycine, mepiquat, paraquat and trimesium. All analytes in method 1 displayed excellent linearity with R2 > 0.998 as listed in Table 3. A chromatogram of the target analytes in apple at 0.05mg/kg is shown in Figure 2, with examples calibration curves displayed in Figure 3.

A Hypercarb PGC (porous graphitic carbon), which behaves as a strongly retentive alkyl-bonded silica gel, was used in method 2 to analyse the following; glufosinate, glyphosate, ethephon, fosethyl aluminium, maleic hydrazide, perchlorate, ETU, PTU, and nicotine. All analytes in method 2 displayed excellent linearity with R2 > 0.999. A chromatogram of the target analytes in apple at 0.05mg/kg is shown in Figure 4, with examples calibration curves displayed in Figure 5.

Figure 2. Target analytes at 0.05mg/kg in apple matrix using using a ZIC-HILIC based separation (LC Method 1).


Figure 3
Figure 3. Calibration curves for paraquat, mepiquat, trimesium and diquat using a ZIC-HILIC based separation (LC Method 1).


Figure 4
Figure 4. Target analytes at 0.05mg/kg in apple matrix using a Hypercarb PGC based separation (LC Method 2).


Figure 5
Figure 5. Calibration curves for glyphosate, gluphosinate, maleic hydrazide
and fosethyl using Hypercarb PGC based separation (LC Method 2).


Table 3
Table 3. Target analyte summary.



Two LC-MS/MS methods were developed for the measurement of a range of highly polar pesticides in their underivatised state using the LCMS-8050 triple quadrupole mass spectrometer. The developed multi-residue methods offer significant time savings in comparison to single residue methods typically used for analyse of these analytes. All compounds were quantified in the range 0.005 – 0.2 mg/kg with correlation coefficients greater than 0.997. The excellent sensitivity achieved, which is most cases is far below the EU MRL, offers the opportunity to dilute sample extracts prior to LC-MS/MS injection in order to reduce matrix effects.


  1. Reference Laboratory for pesticides requiring Single Residue Methods (EURL-SRM). 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 (QuPPe-Method). 2012. Version 7.
  2. Commission Regulation (EC). 2005. No 396/2005 of the European Parliament and of the Council, maximum residue levels of pesticides in or on food and feed of plant and animal origin. Official Journal of the European Union, L 70: 1-16

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www.shimadzu.eu, shimadzu@shimadzu.eu

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