GB 29698-2013Determination of 17β-Estradiol, Estriol and Ethinylestradiol Residues in Milk and Milk Products by Gas Chromatography-Mass Spectrometric method (English PDF)

6.1 Preparation of the sample

An appropriate amount of fresh or frozen blank or test sample, 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

Save 2 °C ~ 8 °C.

Determination Step 7

7.1 Preparation of standard curve

The precise amount of 10mg/L working standard solutions amount, diluted with methanol, and formulated to a concentration of 10,50,100,200,500 1000µg/L series of working standard solution, blown dry with nitrogen at 40 °C water bath, treated according to the derivatization step, process gas chromatography - mass spectrometry. With Measured 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 Liquid Sample

Sample Weigh 10g ± 0.05g, in 50mL centrifuge tube, add acetonitrile 5mL, ethyl acetate 15mL, vortexed 3min, 8000r/min centrifugal 5min, the supernatant was collected in another 50mL centrifuge tube, the residue was extracted repeatedly once, twice combined supernatants at at 40 °C rotary evaporated to near dryness, dissolved in 1mol/L sodium hydroxide solution three times 6mL, 50mL transferred to another centrifuge tube, add 20mL hexane vortexed 1min, 8000r/min centrifugal 3min, extract the lower layer was collected, adjusted to pH with 5mol/L hydrochloric acid solution to 5.0 to 5.2, the standby.

7.2.2 Solid samples

Sample Weigh 10g ± 0.05g, in 50mL centrifuge tubes, ethyl acetate was added 15mL, vortexed 3min, 8000r/min centrifugal 5min, supernatant was collected in another 50mL centrifuge tube, the residue was extracted repeatedly once, twice supernatants were combined, rotary evaporated to 40 deg.] C water bath Near dryness, dissolved in 1mol/L sodium hydroxide solution 6mL three times, transferred to another centrifuge tube 50mL, 20mL hexane plus vortex transducer Swing 1min, 8000r/min centrifugal 3min, lower layer was collected extracts were washed with 5mol/L hydrochloric acid solution was adjusted to pH 5.0 to 5.2, the standby.

7.3 Purification

C18 column and washed with 5mL methanol and 5mL water activated, taking stock solution through the column, 5mL water rinsed, drained, eluting with methanol 5mL, the eluate was collected, dried in a water bath at 40 °C nitrogen. The residue was dissolved with n-hexane to 95% ethyl acetate 5mL, through with hexane 5mL the activated silica gel, n-hexane was added 5mL rinsed, drained, and then 5mL of 70% ethyl acetate-hexane, collecting eluate, Water bath at 40 °C blown dry with nitrogen.

7.4 Derivatization

The residue was added toluene and 100µL of each derivatization reagent were dissolved, mixed, sealing, derived 60min, cooled, gas phase in an oven at 80 °C Chromatography - Mass Spectrometry.

7.5 Determination

7.5.1 Chromatographic conditions

7.5.1.1 column HP-5MS capillary column (30m × 0.25mm, film thickness 0.25µm), or equivalent person.

7.5.1.2 The carrier gas was Helium, constant flow 1.0mL/min.

7.5.1.3 injector temperature 220 °C.

7.5.1.4 Injection volume. 1µL, splitless.

7.5.1.5 column starting temperature of 100 deg.] C (holding 1min), at a heating rate of 20 °C/min was raised to 200 °C (holding 3min), and then to Heating rate 20 °C/min was raised to 260 °C (holding 5min), and then at a heating rate of 20 °C/min was raised to 280 deg.] C (holding 5min).

7.5.2 MS conditions

7.5.2.1 ion source (EI) temperature.200 °C.

7.5.2.2 EM Voltage. tuning voltage is higher than 200V.

7.5.2.3 electron energy. 70eV.

7.5.2.4 GC/MS transfer line temperature. 280 °C.

7.5.2.5 Quadrupole temperature. 160 °C.

7.5.2.6 selected ion monitoring (SIM) .( m/z) 232,285,326,416 (17beta- estradiol); 311,345,414,504 (estriol); 285, 300,425,440 (ethinyl estradiol).

7.5.3 Assay

7.5.3.1 Qualitative determination

By retention time of sample chromatogram with retention times of the respective standards, wherein each of the ion peaks and the corresponding concentration of the standard Wherein each of the ion peaks qualitative contrast. Sample and standard deviation of the relative retention time of not more than 5%; with a sample wherein ions Relative abundance consistent with the abundance of an equivalent concentration of a mixed standard solution, the relative abundance of the deviation does not exceed specified in Table 1, the sample can be stored is determined the respective analyte.

The maximum allowable error relative ion abundances% Table 1 Qualitative diagnosis Relative abundance > 50 20 ~ 50 10 ~ 20 <=10 Tolerance ± 10 ± 15 ± 20 ± 50 Quantitative determination 7.5.3.2 Take appropriate sample solution and standard solutions appropriate to do single-point or multi-point calibration, by external standard method, in order to quantify the peak area, sample and standard solutions Liquid 17beta- estradiol, estriol and ethinyl estradiol response value shall be within the linear range of the detection instrument. In the chromatographic conditions, the standard Solution, add blank sample and blank sample chromatogram and mass spectrum given in Appendix A.

7.6 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 the sample of 17beta- estradiol, estriol remaining amount, calculated as ethinyl estradiol formula (1).

A × cS × V

AS × m

Where.

--- for X-try feed corresponding 17beta- estradiol, estriol, ethinylestradiol residues micrograms per kilogram (µg/kg);

A --- Sample peak area corresponding 17beta- estradiol, estriol and ethinyl estradiol; and cS --- standard solution corresponding 17beta- estradiol, estriol and ethinyl estradiol concentration, in micrograms per liter (µg/L);

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

--- the AS standard solution corresponding 17beta- estradiol, estriol and ethinyl estradiol peak area;

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 method detection limit of 0.5µg/kg, the limit of quantitation was 1.0µg/kg.

9.2 Accuracy

This method of adding 1µg/kg ~ 10µg/kg on recovery levels of 60% to 120%.

9.3 Precision

The relative standard deviation of the method <=15%, inter-assay relative standard deviation <=20%.

Appendix A

Chromatogram

Description.

ES --- 17beta- estradiol derivatives;

--- the EES ethinyl estradiol derivative;

EST --- estriol derivatives.

FIG A.1 17beta- estradiol, ethinylestradiol, estriol chromatogram of the standard solution (100µg/L)

Figure A.2 milk blank sample chromatogram

Description.

ES --- 17beta- estradiol derivatives;

--- the EES ethinyl estradiol derivative;

EST --- estriol derivatives.

Figure A.3 blank add milk 17beta- estradiol, ethinyl estradiol and estriol sample chromatogram (1µg/kg)