GB 29688-2013Determination of Chloramphenicol residues in milk by Liquid Chromatography-tandem Mass Spectrometric method (English PDF)

6.1 Preparation of the sample

An appropriate amount of fresh or frozen or blank test milk, mixed, and homogenized.

--- the test sample taken after homogenization, as the feed try.

--- blank sample taken after homogenization, as a blank sample.

--- blank sample taken after homogenization, adding a suitable concentration of the standard working solution, is added as a blank sample.

Save 6.2 sample

Or less at -20 °C.

Determination Step 7

7.1 Extract

Take Sample 10g ± 0.05g, in 50mL centrifuge tubes, add deuterated internal standard working solution 250 L of chloramphenicol, together with ethyl acetate 20mL, Oscillation 15min, 6000r/min centrifugal 10min, the ethyl acetate layer was in heart-shaped flask. Coupled with 20mL ethyl acetate extraction was repeated a Times, the two extracts were combined heart-shaped flask in a water bath at 45 °C rotary evaporated to dryness. The residue was dissolved with 4% sodium chloride solution 5mL, plus 5mL hexane oscillations 1min, standing layered hexane was discarded. Coupled with n-hexane 5mL, extraction was repeated once. Take subnatant spare.

7.2 Purification

C18 column and washed with 5mL methanol and 5mL water activation, taking stock solution through the column, controlling the flow rate of 1 drop/(3s ~ 4s), 5mL water leaching Washed, drained, washed with methanol 5mL, collecting eluate, blown dry with nitrogen at 50 °C. With 50% acetonitrile and the residue was dissolved 1.0mL, vortex mixed Uniform, membrane filtration, for liquid chromatography - tandem mass spectrometry.

7.3 Preparation of standard curve

The precise amount of 100µg/L of chloramphenicol standard working solution and 20µg/L of chloramphenicol deuterated amount of internal standard working solution, dilute with mobile phase Release, formulated at a concentration of chloramphenicol 0.10,0.25,0.50,1.0,2.0,5.0µg/L, chloramphenicol deuterated concentration 5µg/L series of standard solutions Solution for liquid chromatography - tandem mass spectrometer. A characteristic ion mass chromatogram peak area for the vertical axis, the standard solution at abscissa, standard curve line. Seeking regression equation and correlation coefficient.

7.4 Determination

7.4.1 LC Conditions

7.4.1.1 Column. C18 (150mm × 2.1mm, particle size 5µm), or equivalent person.

7.4.1.2 Column temperature. 30 °C.

7.4.1.3 flow rate. 0.2mL/min.

7.4.1.4 Injection volume. 20µL.

7.4.1.5 Run time. 8min.

7.4.1.6 Mobile phase. aqueous acetonitrile (50 50, volume ratio).

7.4.2 MS conditions

7.4.2.1 ionization mode. ESI.

7.4.2.2 Scan mode. negative ion scan.

7.4.2.3 Detection mode. multiple reaction monitoring.

7.4.2.4 ionization voltage. 2.8kV.

7.4.2.5 source temperature. 120 °C.

7.4.2.6 atomizing temperature. 350 °C.

7.4.2.7 Cone gas flow rate. 50L/h.

7.4.2.8 atomizing gas flow rate. 450L/h.

7.4.2.9 Data acquisition window. 8min.

7.4.2.10 dwell time. 0.3s.

7.4.2.11 qualitative and quantitative ion and the corresponding cone voltage and collision energy. see Table 1.

Table 1 chloramphenicol qualitative and quantitative Cone voltage of ion collision energy and

drug

Qualitative ion pair m/z Quantitative ion pair m/z Cone voltage Collision voltage Chloramphenicol (CAP) 321/151.6 321/256.8 321/151.6 30 Deuterated chloramphenicol (D5-CAP) 325.8/156.6 325.8/156.6 30 15

7.4.3 Assay

Take a sample solution and standard solutions corresponding, for single or multi-point calibration, according to the internal standard peak area ratio calculation. Sample solution and control solution Chloramphenicol broth and deuterated chloramphenicol response value should be within the linear range of the detection instrument. Ion in the sample solution and the relative abundance of labeled Ion standard solution relative abundance ratio in accordance with Table 2. Tissue white standard solution and sample solution was added and the total ion selected ion FIG flow Appendix A.

Table 2 sample ions in the solution relative abundance tolerance range

The relative abundance Tolerance > 50 ± 20 > 20 ~ 50 ± 25 > 10 ~ 20 ± 30 <=10 ± 50

7.5 Blank test

But without addition of the sample, the measurement step is performed in exactly the same operation in parallel.

8 and the result of the calculation expression Calculating A321/151.6/A325.8/156.6 peak area ratio, the standard calibration curve.

From the standard curve equation.

As/A'is = a × cs/c'is b (1) to obtain a and b, then c = c'is Ais-÷ b (2) Chloramphenicol residues in a sample according to formula (3) is calculated.

Where.

AS chloramphenicol --- the control solution peak area;

A'is --- internal control standard solution of chloramphenicol deuterated peak area;

--- CS control concentration deuterated internal standard solution of chloramphenicol, in units of nanograms per milliliter (ng/mL);

c'is --- chloramphenicol concentration control solution, in units of nanograms per milliliter (ng/mL);

C --- chloramphenicol test solution concentration unit nanograms per milliliter (ng/mL);

A --- chloramphenicol peak area of the sample;

Chloramphenicol deuterated standard peak area in the sample AIS ---;

X---- try feed for chloramphenicol residues in micrograms per kilogram (µg/kg);

V --- volume of the residue was dissolved in milliliters (mL);

m --- try supply feed mass in grams (g).

Note. The blank value should be subtracted from the results, expressed as the arithmetic mean of the measurement result measured parallel to three significant figures.

9.1 Sensitivity

This limit of detection is 0.01µg/kg, limit of quantification was 0.1µg/kg.

9.2 Accuracy

This method of adding 0.02µg/kg ~ 0.10µg/kg on recovery levels of 50% to 120%.

9.3 Precision

The relative standard deviation of the method <=17%, inter-assay relative standard deviation <=20%.

Appendix A

Chromatogram

Figure A.1 chloramphenicol standard solution and wherein the total ion chromatogram mass ion (0.25µg/L)

Figure A.2 wherein milk sample blank ion mass chromatogram

Figure A.2 (continued)

Figure A.3 milk sample blank add chloramphenicol total ion chromatogram and mass characteristic ions (0.02µg/kg)