This method was designed to simultaneously extract ciprofloxacin, enrofloxacin, marbofloxacin, danofloxacin and sarafloxacin residues in milk, using norfloxacin as an internal standard (taken from Journal of Chromatography A 2004, 1034, 25-32).
Pretreatment (defatting & protein removal): In a 50mL polypropylene centrifuge tube, add 5g of pasteurized whole milk and fortify with FQs. Place centrifuge tube on a vortex mixer for 30 s and then let stand for at 4°C in the dark for 30 min.
Add 2.5mL of trichloroacetic acid (20% in methanol) and shake sample for 30s. Centrifuge at 13,800 x g for 30min at 10°C ((Eppendorf-5804R).
Add 12.5mL 50mM, pH 9 ammonium chloride buffer to the sample and centrifuge again at 13,800 x g for 30min at 10°C.
Remove the clear supernatant with a pipette and keep it for SPE.
Solid phase extraction clean up using strata X:
To a 200mg strata X cartridge,
Condition: 4mL methanol
Equilibrate: 4mL 50mM ammonium chloride buffer, pH 9
Load: pretreated sample
Wash I: 3mL 5% methanol in water
Wash II: 3mL 5% methanol in 50mM ammonium chloride buffer, pH 9
Dry cartridge on full vac for 5 min
Elution: 2x 2 mL 4% orthophosphoric acid in methanol
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