In this study, an analytical method for the simultaneous determination of the novel insecticide flupyradifurone and its two metabolites in a variety of traditional Chinese herbal medicines was developed for the first time using high-performance liquid chromatography-tandem mass spectrometry. A simple and efficient method using dispersive solid-phase extraction was employed for the pretreatment of the samples. Several extractions and cleanup strategies were evaluated. The recoveries (
Flupyradifurone (IUPAC name, 4-((6-chloro-3-pyridylmethyl) (2,2-difluoroethyl)amino)furan-2(5H)-one, referred to as FPO in this paper) is a novel butenolide insecticide developed by Bayer with systemic action and low mammalian toxicity. FPO is used to control a variety of sucking pests in agricultural and horticultural crops and is extremely effective against pests resistant to neonicotinoid insecticides. FPO is the first nicotinic acetylcholine receptor (nAChR) insecticide containing the stemofoline-derived (natural compound) butenolide pharmacophore [
Traditional Chinese herbal medicines (TCHMs) play a very important role in the Chinese medical system. Many varieties of TCHM are employed, most of which are currently artificially planted. To ensure the quality and yield of TCHM crops, pesticides are widely used in their cultivation to protect them from pests and diseases. However, not only does the excessive or incorrect use of these chemicals cause environmental pollution, but the resulting pesticide residues in TCHMs may also affect the health of consumers. Additionally, with the increasing popularity of TCHMs worldwide, many organizations, including the Food and Agriculture Organization (FAO) and the European Union (EU), have set maximum residue limits (MRLs) for some pesticides in TCHMs and related products.
In studies used to set MRLs, the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method developed by the US Department of Agriculture (USDA) in 2003 is widely used [
To the best of our knowledge, only Li et al. [
Flupyradifurone (purity > 99.5%), difluoroacetic acid (DFA, purity > 98.0%), and 6-chloronicotinic acid (6-CNA, purity > 99.2%) were purchased from Chem Service (West Chester, PA, USA).
HPLC-grade methanol and acetonitrile were purchased from MREDA (Beijing, China). Formic acid (FA, purity > 99.0%) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water was prepared using a Milli-Q system (Bedford, MA, USA). The sorbents primary-secondary amine (PSA), octadecyl silica (C18), graphitized carbon black (GCB), and polar enhanced polymer-2 (PEP-2) were purchased from Agela Technologies (Tianjin, China). Sodium chloride (NaCl) and anhydrous magnesium sulfate (MgSO4) were obtained from Beijing Chemical Reagent Company (Beijing, China).
Stock solutions containing 1,000 mg/L FPO, 6-CNA, or DFA were prepared in methanol. The stock standards were placed in 10 mL amber bottles and stored in a refrigerator at 4 ± 3°C. Under these storage conditions, the stock standards were replaced after three months.
Samples of four TCHMs (
An Agilent 1260 HPLC (Santa Clara, CA, USA) equipped with an Agilent ZORBAX RRHD Eclipse Plus C18 column (3.0 × 100 mm, 1.8
The HPLC system was coupled to an Agilent 6470 triple quadrupole mass spectrometer (Santa Clara, CA, USA) equipped with an electrospray ion source (ESI). The ionization of FPO and its metabolites was performed in the positive and negative ionization modes. The following source parameters were used: Nebulizer pressure: 30 psi; capillary and nozzle voltage for positive and negative modes: 4.0 kV and 0.5 kV, respectively; desolvation (drying gas) and sheath gas flow rates: 7 and 8 L/min, respectively; desolvation and source temperatures: 330 and 300°C, respectively.
Based on the SANTE/11813/2017 guidelines [
The quantitation of FPO was performed using the external standard method. Because of the influence of matrix effects (ME) on quantitation during LC-MS analysis [
The MS/MS parameters of the three compounds were investigated in both ESI+ and ESI− modes. The results showed that DFA and 6-CNA had higher responses in the negative mode, while FPO showed a higher response in the positive mode.
The MS/MS parameters that could affect the intensity of the ion transitions, including the fragmentor voltage and collision energy (CE), were optimized by ramping the values of each parameter over a certain range and selecting the value that yielded the highest signal response. The selected ion pairs and optimized MS/MS parameters are listed in Table
The mobile phase for the chromatographic separation was optimized to achieve sufficient peak separation with high sensitivity and for the switch between the negative and positive ESI modes. Gradient elution on the C18 column with methanol-water and acetonitrile-water as the mobile phase was used to separate the three target compounds. The retention time of FPO was shorter when the proportion of methanol or acetonitrile in the mobile phase was increased, and the retention of FPO was affected when the proportion of methanol or acetonitrile in the mobile phase was less than 20%. The proportions of phases A and B had little effect on 6-CNA and almost no effect on DFA. The separation of the three compounds met the requirements for positive and negative mode switching when the mobile phase was acetonitrile : water = 1 : 4 (v/v). However, the solvent effect affected the peak shapes of DFA and 6-CNA. Therefore, the addition of different proportions of water to the injection solutions to eliminate the solvent effect was evaluated. The results (Figure
HPLC-MS/MS chromatograms of DFA (1.0 mg/L, peak 1), 6-CNA (1.0 mg/L, peak 2), and flupyradifurone (0.1 mg/L, peak 3) on C18 column and the same elution procedure under different injection solutions conditions. ((a) Acetonitrile: water = 1 : 0 (v/v), (b) acetonitrile: water = 4 : 1 (v/v), (c) acetonitrile: water = 1 : 1 (v/v), (d) acetonitrile: water = 1 : 4 (v/v)).
In addition, the effect of the addition of 0.1% formic acid, 5 mmol ammonium acetate, or both on the sensitivity of the three compounds was studied. The results (Figure
HPLC-MS/MS chromatograms of DFA (1.0 mg/L), 6-CNA (1.0 mg/L) and flupyradifurone (0.1 mg/L) on C18 column and the same elution procedure under different mobile phase conditions. ((a) 5 mmol ammonium acetate aqueous solution/acetonitrile, (b) 0.1% formic acid and 5 mmol ammonium acetate aqueous solution/acetonitrile, (c) 0.1% formic acid aqueous solution/acetonitrile, (d) water/acetonitrile).
The improved QuEChERS method (with the addition of 10 mL of water) has been recommended for the determination of pesticide residues in dried samples. However, the recoveries of DFA using this method were less than 10%, and no significant change was observed after adjusting the pH. This indicated that the partitioning of DFA into acetonitrile was difficult because of its high solubility in water. To solve this problem, different amounts (1, 2, or 5 g) of NaCl were added to the extraction solution. The efficiency of the solvent extraction process can be improved by salt [
Recovery of flupyradifurone, DFA, and 6-CNA for the improved method under different amounts of sodium chloride condition (
Recovery of flupyradifurone, DFA, and 6-CNA for the method using different concentrations of formic acid (
The choice of a suitable purification step is very important to obtain accurate results using LC–MS/MS and GC–MS/MS. Matrix extracts often contain many polar organic coextracts (including organic acids and aliphatic acid) and nonpolar organic coextracts (such as fats and pigments). These organic coextracts will enhance the matrix effect, increase the background in the mass spectrum, and pollute the ion source [
In this paper, the purification effects of several common sorbents on DFA, 6-CNA, and FPO in dried samples of
Recovery and ME of flupyradifurone, DFA, and 6-CNA for the method using different sorbents (
The recoveries of the three compounds in all the matrices only reached greater than 70% when C18 was used. However, the matrix effects for
Recovery and ME of flupyradifurone, DFA, and 6-CNA for the method under different combinations of sorbent conditions (
The calibration data, ME, and LOQs of FPO, DFA, and 6-CNA in the different matrices are listed in Table
Calibration information of target compounds in the range of 0.01–1 mg/L in different matrices.
Compound | Matrix | Calibration equation | ME (%) | LOQ ( | |
---|---|---|---|---|---|
DFA | Acetonitrile | 0.9999 | — | — | |
0.9998 | −17.9 | 100 | |||
1 | −45.0 | 100 | |||
0.9997 | −58.4 | 100 | |||
0.9998 | −50.6 | 100 | |||
6-CNA | Acetonitrile | 0.9998 | — | — | |
0.9998 | −20.0 | 100 | |||
0.9999 | 12.3 | 100 | |||
0.9998 | 20.0 | 100 | |||
0.9999 | 15.8 | 100 | |||
FPO | Acetonitrile | 0.9999 | — | — | |
0.9998 | −23.3 | 10 | |||
0.9998 | 8.5 | 10 | |||
0.9998 | −4.2 | 10 | |||
0.9999 | −1.1 | 10 |
The mean recoveries (
To confirm the applicability of the developed and validated method, the method was applied for the determination of FPO, DFA, and 6-CNA in 10 commercial TCHMs (
A modified version of the original QuEChERS method was established for the determination of FPO and its two metabolites (DFA and 6-CNA) in dried samples of TCHMs. The three target compounds were successfully separated on a C18 column. The samples were extracted using acetonitrile containing formic acid and cleaned up using different combinations of sorbents. This method reduced the extraction of hydrophilic organic coextracts in the extraction process and caused the hydrophobic organic coextracts to precipitate when the extraction solution was diluted to prepare the injection solutions, which reduced the interference with the target compounds. In the validation of the method, satisfactory linearity, repeatability, intermediate precision, and accuracy were obtained. The recoveries of the method were in the 70 to 120% range. The method precision in terms of repeatability and intermediate precision was adequate, with RSD values of <20%. These results demonstrated that the developed method is rapid and reliable for monitoring FPO and its two metabolites (DFA and 6-CNA) in TCHMs.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This study was financially supported by the Pesticide Residue Foundation of the Ministry of Agriculture and Rural Affairs of the People’s Republic in China (grant no. 2019RS01).
Table S1: chemical information of flupyradifurone and its metabolites. Table S2: optimized MRM conditions for analysis of flupyradifurone, DFA, and 6-CNA. Table S3: mean recoveries and RSD for target compounds from different matrices at three spiked levels.