Materials and Chemicals
The analytical standards of 20 neonicotinoids and their metabolites (Fig 1), including IMI (≥99.8%), CYC (≥92.7%), THX (≥99.0%), CLO (≥99.8%), and FLO (≥99.0%), were purchased from Dr. Ehrenstorfer Gmbh (Germany); 5-OH-IMI (≥99.7%), DN (≥99.0%), UF (≥99.0%), and TFNG (≥99.7%) from A ChemTek (USA); 6-CHL (≥99.0%), ACE (≥99.2%), DM-ACE (≥99.5%), THA (≥99.4%), IMIT (≥98.0%), DM-CLO (≥98.2%), and DNT (≥99.9%) from Tan-Mo Technology (China); SUL (≥99.0%) and TFNA-AM (≥99.8%) from CATO Research Chemicals (USA); IM-1-4 (≥98.0%) AltaScientific (China). Sorbents, such as Neutral alumina (Alumina-N) was obtained from Kermel (China); Graphitized carbon black (GCB), C18 (ODS), Primary and secondary amine (PSA), and aminopropyl (-NH2) from Biocomma (China); Silica mesoporous SBA-15 (Pore size 6-13 nm), and reduced graphene [email protected]3O4 ([email protected]3O4) from XFNANO (China); Multi-walled carbon nanotubes (MWCNT) (≥99%) from Tanfeng Tech (China); Captiva EMR-Lipid from Agilent (USA). HPLC grade formic acid, acetonitrile, acetic acid, methanol, and ethyl acetate were acquired from Merk (USA). Deionized water was produced using a Milli-Q purification system (Millipore, USA). Infant foods, including vegetable & fruit cookies, grain rice cereals, and vegetable purees, were sourced from a local supermarket.
Individual standard solutions of 20 neonicotinoids and their metabolites were prepared by separately dissolving the technical grade materials in methanol. Mixed standard solutions containing each target compound for this study were prepared in a mixture of appropriate amounts of the individual stock solutions with 10% aqueous acetonitrile (containing 0.1% formic acid). A series of working solutions (mixed standard solutions and matrix-matched standard solutions) were prepared at the concentration of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 150 ug L-1. All the stock and working standard solutions were stored at -20 ℃ until further use.
The representative sample's aliquot (4.0 g) was initially weighed and transferred into a 50 mL poly-propylene centrifuge tube. Then, it was dissolved with 4 mL of deionized water and extracted with 20 mL of acetonitrile/ethyl acetate acidified with 0.1% acetic acid (50/50, v/v). Each extraction process should be vortexed for 30 s and ultrasonicated for 10 min and then centrifuged at 5000 rpm for 10 min. Subsequently, 10 mL of the upper extract was collected, transferred into a 15 mL glass tube, and concentrated to dryness under a gentle stream of nitrogen. The residues were then redissolved with 1 mL of 10% aqueous acetonitrile (containing 0.1% formic acid), and treated with 40 mg PSA, 30 mg [email protected]3O4 powder, vortexed 1 min. Take the upper transparent layer to pass through a 0.22 um nylon membrane for HPLC-MS/MS analysis.
A 2040C HPLC (Shimadzu, Japan) coupled with an 8045 Triple Quadrupole mass spectrometer (Shimadzu, Japan) was used for sample analysis. Chromatographic separation was performed at 30 ℃ on an InertSustain AQ-C18 column (2.1 mm × 100 mm, 3.0 um, Shimadzu, Japan), in which the mobile phase consisted of 5 mM ammonium acetate and 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The following gradient program was developed by applying 5 uL injection volume and 0.3 mL/min flow rate. Starting with 0-10% B in 0.2 min, 10-35% B in 2.8 min, 35-75% B in 4 min, re-equilibration at 10% B for 0.5 min, and held at 10 % B in 4.5 min.
The MS/MS detection was performed in multiple reaction monitoring (MRM) mode with positive ESI. Data collection was monitored using the LabSolution Insight software (5.91), and the optimized source parameters were as follows:
• interface voltage of 4 kV
• desolvation line temperature of 250 ℃
• heat block temperature of 400 ℃
• nebulizer gas (nitrogen) flow at 3 L/min
• drying gas (air) flow at 10 L/min
• heating gas (nitrogen) flow at 10 L/min