High-throughput analysis of neonicotinoids and metabolites in urine by LC-MS

Protocol Steps

1. 1
   Add internal standard and methanol

   Pipette 10 µL of internal‑standard (IS) solution and 10 µL of 50 % methanol in water into each 100 µL urine aliquot.

2. 2
   Precipitate proteins and centrifuge

   Add 600 µL acetonitrile to each tube, mix, then centrifuge at 4 °C, 2000 × g for 1 min.

3. 3
   Prepare SPE plate and load sample

   Pre‑wash an ISOLUTE® HYDRO DME+ 400 mg plate with 100 µL acetonitrile and centrifuge at 4 °C, 1000 × g for 1 min; load the supernatant onto the plate and centrifuge again at 4 °C, 1000 × g for 1 min.

4. 4
   Evaporate eluate to dryness

   Place the plate in a centrifugal vacuum concentrator coupled to the TurboVap 96‑well system and evaporate at 45 °C until completely dry.

5. 5
   Reconstitute residues

   Add 200 µL of 5 % methanol in 0.1 % formic acid containing 10 mM ammonium acetate to each well and mix for 30 seconds.

6. 6
   Inject sample into LC‑MS/MS

   Transfer 10 µL of the reconstituted eluate to the autosampler vial and inject 10 µL onto the LC‑MS/MS system.

7. 7
   Configure liquid chromatography

   Use an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 µm, 100 Å). Mobile phase A: 0.1 % formic acid in 10 mM ammonium acetate. Mobile phase B: methanol. Set flow rate to 0.4 mL/min, column oven at 50 °C, autosampler at 4 °C. Gradient program: 0.0 min – 0 % B, 0.0 min – 1.955 % B; 0.0 min – 5.0 % B; 5.0 min – 5.0 % B (load to MS); 19.0 min – 19.0 % B (switch to waste); 24.5 min – 24.5 % B (end). Injection volume 10 µL. Needle wash solvent methanol:water = 7:3. Total LC run time 24.5 min per sample; overall analysis time estimated 26.5 min per sample.

8. 8
   Set MS source conditions

   Operate the Triple Quad™ 6500 in positive electrospray mode with the following source settings: ion‑spray voltage 5500 V, heating gas temperature 450 °C, nebulizer gas 50 psi, heating gas 60 psi, curtain gas 20 psi, collision gas pressure 11 psi.

9. 9
   Program MRM transitions

   Enter the MRM transitions for each analyte and its internal standard as listed in Table 3 (target ion m/z, qualifier ion m/z, declustering potential, collision energy, dwell time, retention time). Example entries: ACE – 223.2→223.3 (DP 22 V, CE 6 V, dwell 50 ms, RT 5.0 min); ACE‑IS – 229.0→232.2 (DP –50 V, CE –30 V, dwell 30 ms, RT 5.0 min); … (include all compounds and ISs exactly as shown).

10. 10
    Re‑analyse out‑of‑range samples

    If a sample’s measured concentration falls outside the calibration range (see Table 5), dilute the sample further (appropriate dilution factor) and repeat the entire preparation and injection steps.

11. 11
    Prepare fortified QC urine

    Pool maternal urine, then spike to achieve the following target concentrations (ng/mL): ACE 0.5, TCP 0.5, SUL‑A 0.5, SUL‑B 0.5, FLN 10, THX 2.0, DIN 5.0, CLO 5.0, IMI 5.0, NIT 5.0, dm‑ACE 5.0, TCP‑amid 1.0, dm‑CLO 20, IMI‑OF 100. Process QC samples exactly as unknowns, with five replicates per analytical sequence.

Materials & Reagents

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