GB 29685-2013Determination of Lincomycin, Clindamycin and Spectinomycin residues in animal derived food by Gas Chromatography Mass Spectrometric method (English PDF)

6.1 Preparation of the sample

6.1.1 Pork, beef and chicken muscle, liver and kidney tissue

Fresh or thawed take appropriate blank or test tissue, minced, 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.

6.1.2 Milk and eggs

An appropriate amount of fresh or frozen test blank or milk and eggs, 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

The following tissue samples stored at -20 °C. Eggs and milk sample or less 2 °C ~ 8 °C stored.

Determination Step 7

7.1 Preparation of matrix-matched standard curve

Were the precise amount of 100µg/mL lincomycin, clindamycin and spectinomycin appropriate amount of working standard solution, diluted with methanol and formulated into concentrated 50,100,200,400,800,1600 degrees and 3200µg/L of a mixed series of standard solution from each 1.0 mL, extracted separately dissolved, Purifying and drying the residue of the sample blank, thoroughly mixed, dried at room temperature, was added N, O- bis (trimethylsilyl) trifluoroacetamide and 500µL

100 L acetonitrile, vortex mixed for 1min, sealed at 75 deg.] C incubator IH derivatization reaction, dry nitrogen at room temperature, was added n-hexane solution 0.5mL the residue solution was vortexed, gas chromatography - mass spectrometry. To measure peak area for the vertical axis, corresponding to the concentration of standard solution as abscissa, the standard curve. Seeking regression equation and correlation coefficient.

7.2 Extract

7.2.1 Muscle and kidney tissue

Sample Weigh 5g ± 0.05g, polytetrafluoroethylene in 50mL centrifuge tube, the extract was added 10mL, shaking 5min, 5000r/min Centrifugal 10min, supernatant to another centrifuge tube, extract the residue was added 10mL, extraction was repeated once, the two supernatants were combined, plus 3% trichloroacetic acid solution 5mL, mix, 8000r/min centrifugal 10min, the supernatant was 2mol/L potassium hydroxide solution adjusted to pH 5.8 ± 0.2, sodium dodecyl sulfate solution was added 2mL, vortexed, allowed to stand for 15min, standby.

7.2.2 Milk and eggs organization

Sample Weigh 5g ± 0.05g, polytetrafluoroethylene in 50mL centrifuge tube, the extract was added 10mL, shaking 5min, 5000r/min Centrifugal 10min, supernatant to another centrifuge tube, extract the residue was added 10mL, extraction was repeated once, the two supernatants were combined, add n Hexane 5mL, mixing, 5000r/min centrifugal 15min, the lower layer were washed with 2mol/L potassium hydroxide solution adjusted to pH 5.8 ± 0.2, add Sodium dodecyl sulfate solution 2mL, vortexed, allowed to stand for 15min, standby.

7.3 Purification

HLB 3mL column successively with methanol, water and sodium dodecyl sulfate 3mL 3mL activation buffer, taking stock solution through the column, rinsed with water Twice 3 mL, eluted with 4mL of methanol, collect the eluate, at blowing nitrogen at 40 °C to nearly dry, dry at room temperature, was added N, O- bis (C Butyldimethylsilyl) trifluoroacetamide and acetonitrile 500µL 100 L, vortex mixed for 1min, sealed, derived from the reaction 75 deg.] C incubator IH, chamber Temperature nitrogen blow, the residue was dissolved with n-hexane 0.5mL, vortexed, gas chromatography - mass spectrometry.

7.4 Determination

7.4.1 Chromatographic conditions

7.4.1.1 Column. capillary column Rtx-5 (5% phenyl methyl polysiloxane, 30m × 0.25mm), or equivalent person.

7.4.1.2 Inlet temperature. 280 °C.

7.4.1.3 Injection mode. splitless.

7.4.1.4 Injection volume. 1µL.

7.4.1.5 modes. constant current.

7.4.1.6 Carrier gas. helium (99.999%).

7.4.1.7 column flow rate. 1mL/min.

Table 1 7.4.1.8 oven ramp program.

Table 1 Column temperature heating procedure

Starting temperature Termination temperature Heating rate °C/min Hold Time min total time min 2.0 2.0 19.5

7.4.2 MS conditions

7.4.2.1 Ion source temperature.200 °C.

7.4.2.2 Interface Temperature. 280 °C.

7.4.2.3 Mass collected (the SIM). lincomycin and clindamycin scanning ion. 126 (quantitative ion), 127,73.

Spectinomycin scanning ion. 145 (quantitative ion), 171,187,201.

7.4.3 Assay

7.4.3.1 Qualitative determination

By retention time and the retention time of the standard sample chromatogram, wherein each of the ion peaks corresponding standard concentration for each chromatographic in contrast to the qualitative characteristics of ion peaks. The relative abundance of the sample ions characteristic; relative deviation of retention time with the standard sample is not more than 5% Relative abundance consistent with a considerable degree of mixed standard solution concentration, relative abundance does not exceed a predetermined deviation Table 2, it can be determined present in the sample with the object to be measured.

The maximum allowable deviation relative ion abundance qualitative determination of Table 2 the relative ion abundances > 50% > 20-50% of > 10% to 20% <=10% Permissible relative deviation ± 10% ± 15% ± 20% ± 50% Ratio for the sample and standard solutions try quantitative ion area of single-point calibration for quantification.

7.4.3.2 Quantitative determination

Take a sample and standard solutions, by external standard method, in order to quantify the peak area of the standard solution and sample solution lincomycin, clindamycin, and large Spectinomycin response value should be within the linear range of the detection instrument. In the above-described chromatography - mass spectrometry under conditions, lincomycin, clindamycin and mildew Grand Su added standard solution and blank sample solution ion mass chromatogram characterized in Appendix A.

7.5 Blank test

But without addition of the sample, the same steps employed in parallel operation.

Calculation and Expression of Results 8 Single-point calibration.

cSA

Matrix-matched standard curve calibrated by. AS = acS b, to obtain a and b, then c = Ab (2) the specimens lincomycin, clindamycin residues and spectinomycin (3) is calculated according to the formula.

Where.

C --- try to supply feed solution corresponding lincomycin, clindamycin, or spectinomycin concentration, in micrograms per liter (µg/L);

cS --- respective standard solution lincomycin, clindamycin, or spectinomycin concentration, in micrograms per liter (µg/L);

A --- try to supply feed solution of the peak area of the corresponding derivative of lincomycin, clindamycin, or spectinomycin;

Peak area of the AS derivatives --- respective standard solution lincomycin, clindamycin, or spectinomycin;

--- for the respective X-try feed lincomycin, clindamycin residues or spectinomycin micrograms per kilogram (µg/kg);

The residue was dissolved V --- volume 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

The detection limit of the method, lincomycin and clindamycin 15µg/kg, spectinomycin as 25µg/kg.

This method Lin quantitative limits and clindamycin in muscle, milk and eggs is 20µg/kg, the quantitative limits for the kidney 50µg/kg; spectinomycin limit of quantitation was 50µg/kg.

9.2 Accuracy

This method clindamycin 20µg/kg ~ 200µg/kg, in the lincomycin at 20µg/kg ~ 3000µg/kg and spectinomycin 50µg/kg ~ 4000µg/kg the levels recoveries of 70% to 110%.

9.3 Precision

<=15% relative standard deviation in this method and inter - batch relative standard deviation of <=20%.

Appendix A

Ion chromatogram

Figure A.1 ion chromatogram of the standard solution derivative

(Lincomycin and clindamycin 100µg/L, spectinomycin 250µg/L)

Figure A.2 in pig muscle ion chromatogram of blank sample

Figure A.3 blank in pig muscle added lincomycin, clindamycin and spectinomycin ion chromatogram of the sample

(Lincomycin and clindamycin 20µg/kg, spectinomycin 50µg/kg)