Determination of Quinolones Residues in Milk

Application range

This method specifies the determination of quinolones residues in milk by HPLC.

This standard applies to the determination of residues of dafloxacin, enfloxacin and cyprofloxacin in milk. (This test sample is milk).

Reference standard: GB 29692-2013 Determination of quinolones in Milk HPLC

Extraction steps

Accurately weigh 1.0g milk sample in 15mL centrifuge tube, then add 5.0mL acetonitrile-10% trichloroacetic acid solution. Mix well and centrifuge at 8000r/min with ultrasound for 5min. Take the supernatant to use.

Purification steps

SPE column: Welchrom^® BRP column, specification: 60mg/3mL.

Activation: 3mL methanol, 3mL water, discard;

Sample loading: all the supernatant, discard;

Washing: 3mL10% methanol aqueous solution, drain and discard;

Elution: 3mL methanol, drain and discard;

Blow the collected eluent to dry with nitrogen at 60 ℃, then dissolve the residue with 500 μL acetonitrile-citric acid/ammonium acetate buffer. Mix well with vortex, and centrifuge at 8000r/min for 10min. Take the supernatant to filter through 0.22μm filter membrane to be tested by machine.

Chromatographic conditions

Column: Ultisil^® XB-C18,4.6 × 250mm, 5μm.

Flow rate: 1.5mL/min

Column temperature: 50 ℃

Injecton volume: 20 μL

Fluorescent detector

Time/min	Ex/nm	Em/nm
0	278	465
23.4	312	366
32.0	278	465

Elution program

Time/min	Acetonitrile/%	Citric acid / ammonium acetate Buffer solution /%
0.01	8	92
30	55	45
32	8	92
35	8	92

Chromatograph or spike recovery rate

Fig 1. Chromatogram of darfloxacin 10ng/mL, enfloxacin, ciprofloxacin 50ng/mL

Peak name	Rt	Area	Height	Area	Tailng factor	Plates
_	min	mV*min	mV	%	EP	EP
Ciprofloxacin	9.860	359804	50002	19.108	1.113	42506
Danofloxacin	10.561	738438	101616	39.217	1.118	48636
Enrofloxacin	11.354	784732	105225	41.675	1.123	53569

Fig 2. Blank chromatogram of milk sample

Fig 3. Chromatogram of milk sample floloxacin spiked at 5 μg/kg, enfloxacin, cyloprofloxacin spiked at 25 μg/kg

Tab 1. Spike recovery rate

Substance	Spike level	Average recovery rate	RSD
–	μg/kg	%	%（n=2）
Ciprofloxacin	5	78.47	0.79
Danofloxacin	25	75.36	0.26
Enrofloxacin	25	79.01	2.88

Ordering Information

P/N	Product	Specification
00522-20009	SPE column	Welchrom® BRP, 60mg/3mL, 50pk
00837-05002	15mL centrifuge tube with screw mouth and conical bottom	Disposable centrifuge tube, flat cap, conical bottom, RCF 12000xg, 15 mL, 50/ pk without sterilization
00824-31001	Welch SPE instrument	12 ports square cylinder
00821-32291	Caps and septa	Pre-slit red PTFE/white Silicone septa, 9mm blue short screw-thread polypropylene cap, 6mm centre hole, 100/pk
00821-40927	Vials	2ml wide opening short screw-thread vial with write-on spot, clear, 11.6×32mm, 100/pk
00201-31043	HPLC column	Ultisil® XB-C18, 4.6×250mm,5μm
00802-02201	Syringe filter	Import NY, 13mm×0.22μm, 100pk

If you have any problem or require further information, please contact info@welchmat.com.