GB 31656.14-2022National food safety standard - Determination of 27 sex hormones residues in fishery products by liquid chromatography-tandem mass spectrometric (English PDF)

5.1 Reagents

5.1.1 Methanol (CH3OH). chromatographically pure.

5.1.2 n-Hexane (C6H14). chromatographically pure.

5.1.3 Methyl tert-butyl ether (C5H12O). chromatographically pure.

5.1.4 Ethyl acetate (C4H8O2). chromatographically pure.

5.1.5 Glacial acetic acid (CH3COOH).

5.1.6 Sodium acetate trihydrate (C2H3NaO2.3H2O).

5.1.7 Ammonia (NH3.H2O).

5.2 Solution preparation

5.2.1 Acetic acid-sodium acetate buffer solution. Take 21.5g of sodium acetate trihydrate and 2.6mL of glacial acetic acid, add appropriate amount of water to dissolve and dilute to 500mL,

mix well.

5.2.2.0.02% ammonia water. Take 100 µL of ammonia water in 500 mL of water, mix well, and prepare before use.

5.2.3 Ethyl acetate-methyl tert-butyl ether solution. Mix equal volumes of ethyl acetate and methyl tert-butyl ether.

5.3 Standards

5.3.1 Sex hormones. diethylstilbestrol, diethylstilbestrol, estrone, hexestrol, estradiol, estriol, ethinylestradiol, estradiol benzoate, trenbolone,

Nandrolone, Androstenedione, Boldenone, Testosterone, Norethindrone, Meandrosterone, Methyltestosterone, Stanozolol, Nandrolone Phenylpropionate, Testosterone Propionate, Progesterone, 21alpha-hydroxy

Progesterone, 17alpha-hydroxyprogesterone, medroxyprogesterone, megestrol acetate, chlormadinone acetate, medroxyprogesterone acetate and levonorgestrel. content >= 97%,

See Appendix A for details.

5.3.2 Internal standard. diethylstilbestrol-D8, estrone-D2, estradiol-13C2, methyltestosterone-D3, progesterone-D9, levonorgestrel-D6 and medroxyprogesterone-D3.

Content >= 97%, see Appendix A for details.

5.4 Preparation of standard solution

5.4.1 Standard stock solution. Take 10mg of sex hormone standard products, weigh them accurately, dissolve them in appropriate amount of methanol and dilute to 100mL.

Color volumetric flasks were used to prepare standard stock solutions with a concentration of 100 µg/mL. Stored below -18°C, the validity period was 1 year.

5.4.2 Mixed standard intermediate solution. Accurately measure the appropriate amount of each standard stock solution, put it in a 100mL brown volumetric flask, dilute it with methanol to prepare hexadiene

Diethylstilbestrol, Diethylstilbestrol, Estrone, Trenbolone, Nandrolone, Androstenedione, Boldenone, Testosterone, Meandrosterone, Methyltestosterone, Nandrolone Phenylpropionate, Testosterone Propionate,

The concentrations of progesterone, 21alpha-hydroxyprogesterone, 17alpha-hydroxyprogesterone, medroxyprogesterone, megestrol acetate, chlormadinone acetate and medroxyprogesterone acetate were all

1 µg/mL; the concentrations of estradiol, levonorgestrel, norethindrone, estradiol benzoate and stanozolol were all 2 µg/mL; estriol and

The concentration of ethinyl estradiol was 4 µg/mL, and the mixed standard intermediate solution was stored below -18 degrees C, and the validity period was 6 months.

5.4.3 Mixed standard working solution. Accurately measure 10mL mixed standard intermediate solution, put it in a 50mL brown volumetric flask, dilute it to the mark with methanol,

Diethylstilbestrol, Diethylstilbestrol, Estrone, Trenbolone, Nandrolone, Androstenedione, Boldenone, Testosterone, Meandrosterone, Methyltestosterone, Phenylpropionate

Nandrolone, testosterone propionate, progesterone, 21alpha-hydroxyprogesterone, 17alpha-hydroxyprogesterone, medroxyprogesterone, megestrol acetate, chlormadinone acetate, and medroxyprogesterone acetate

The concentrations of progesterone were all 0.2 µg/mL; the concentrations of hexestrol, estradiol, levonorgestrel, norethindrone, estradiol benzoate and stanozolol were all

0.4 µg/mL; estriol and ethinyl estradiol concentrations are both 0.8 µg/mL mixed standard working solution. Store below -18 degrees C, valid for 3

month.

5.4.4 Internal standard standard stock solution. Take 10 mg of each isotope internal standard, accurately weigh it, dissolve it in an appropriate amount of methanol and set the volume to 50 mL.

Brown volumetric flask, prepared as an internal standard single-standard stock solution with a concentration of.200 µg/mL, stored below -18°C, and valid for 1 year.

5.4.5 Mixed internal standard working solution. Accurately measure an appropriate amount of internal standard stock solution, put it in a 100mL brown volumetric flask, dilute it to the mark with methanol,

The concentration of estradiol-13C2 was prepared to be 2 µg/mL; diethylstilbestrol-D8, estrone-D2, methyltestosterone-D3, progesterone-D9, medroxyprogesterone-D3 and levonyl

Norgestimate-D6 concentration was 1 µg/mL mixed internal standard working solution. Stored below -18 degrees C, valid for 6 months.

5.5 Materials

5.5.1 C18 solid phase extraction column. 500mg/3mL, or equivalent.

5.5.2 Microporous nylon filter membrane. 0.22 µm.

6.2 Analytical balance. Sensitivity 0.00001g and 0.01g.

6.3 High speed centrifuge. 10000r/min.

6.4 Vortex mixer.

6.5 Homogenizer.

6.6 Rotary evaporator.

6.7 Solid phase extraction device.

6.8 Pear-shaped bottle. 100mL.

6.9 Ultrasonic cleaner.

6.10 Plastic centrifuge tubes. 10mL and 50mL.

7.1 Preparation of samples

Prepare samples according to the requirements of Appendix B of GB/T 30891-2014.

a) Take the homogeneous test sample as the test sample;

b) Take a homogeneous blank sample as a blank sample;

c) Take a homogeneous blank sample, add a standard solution of appropriate concentration, and add a sample as a blank.

7.2 Storage of samples

Store below -18°C.

8.1 Extraction

Take 5 g of the test material (accurate to +/-0.05 g), and add 30 µL of mixed internal standard working solution and acetic acid-sodium acetate buffer to a 50 mL centrifuge tube.

Flush 10mL, vortex 30s, add ethyl acetate-methyl tert-butyl ether solution 10mL, vortex 1min, ultrasonic 10min, vortex mix 1min,

Centrifuge at 8000r/min for 8min, take the upper organic phase into a 100mL pear-shaped bottle; repeat the extraction once with 10mL of ethyl acetate, organic

The phases were combined into a pear-shaped flask, and evaporated to dryness in a water bath at 35°C. For samples with oil residue after evaporation, use nitrogen or air to remove the remaining oil

Blow off the ethyl acetate in the fat. Add 7 mL of n-hexane to dissolve the residue and set aside.

8.2 Purification

Take a C18 solid-phase extraction column and activate it with 5 mL of methanol and 5 mL of n-hexane in turn. Take the spare liquid and pass it through the column, controlling the flow rate to not exceed 1 drop/s;

Rinse with 10mL of hexane and blow dry; elute with 10mL of methanol, collect the eluate, and evaporate it in a water bath at 40°C to dryness. Add methanol accurately

0.85mL, vortex mixed for 1min, then accurately added 0.15mL of water, mixed for 30s, 8000r/min, centrifuged for 6min, the supernatant was washed with 0.22µm

Nylon filter membrane was used for liquid chromatography-tandem mass spectrometry determination.

8.3 Preparation of matrix matching standard working curve

Take 10 µL, 25 µL, 50 µL, 100 µL, 250 µL and 500 µL of mixed standard working solutions in 5 mL glass centrifuge tubes respectively,

Add 30 µL of mixed internal standard working solution, blow dry, add 1.0 mL of matrix blank solution obtained through steps 7.1 and 7.2, mix for 1 min, and prepare

The hormone concentration was 2ng/mL ~ 400ng/mL series matrix matching standard working solution, measured on the machine. Characteristic ion chromatography of standard substance

The ratio of the peak area of the peak to the peak area of the characteristic ion chromatographic peak of the internal standard is the ordinate, and the corresponding concentration is the abscissa, and the matrix standard working curve is drawn.

Line. Find the regression equation and correlation coefficient.

8.4 Determination

8.4.1 Reference conditions for liquid chromatography

a) Chromatographic column. XBridgeShield RP18 column (4.6mmx150mm, 5µm), or equivalent;

b) Flow rate. 0.4mL/min;

c) Injection volume. 20 µL;

d) Column temperature. 25°C;

e) Mobile phase A is methanol; mobile phase B is 0.02% ammonia water, and the gradient elution program is shown in Table 1.

8.4.2 Reference conditions for mass spectrometry

a) Ion source. ESI;

b) Spray voltage. 3.0kV;

c) Ion transfer tube temperature. 350°C;

d) Sheath gas flow rate. 13.3L/min;

e) Auxiliary gas flow rate. 3.3L/min;

f) Collision air pressure. 0.1995Pa;

g) Detection method. multiple reaction monitoring (MRM), 27 kinds of hormones and internal standard multiple reaction monitoring parent ion, product ion, collision energy and scanning

The method is shown in Table 2.

8.5 Determination method

8.5.1 Qualitative determination

Under the same test conditions, the difference between the retention time of hormone drugs in the test solution and the retention time of hormone drugs in the standard working solution

The ratio deviation is within +/-2.5%; and the relative abundance of the detected ions should be equal to the relative abundance of the ions in the calibration standard solution with the same concentration.

The allowable deviation should meet the requirements in Table 3.

8.5.2 Quantitative determination

Take the sample solution and matrix matching standard working solution for single-point or multi-point calibration, and quantify according to the internal standard method. Matrix matching standard working solution and sample

The response values of the target substances in the solution should be within the linear range of instrument detection. Under the above conditions of chromatography-mass spectrometry, the characteristic ion mass of the standard solution

For chromatograms see Appendix B.

8.6 Blank test

Take the blank sample, except that no drug is added, the same measurement steps are used for parallel operation.

10.1 Sensitivity

Diethylstilbestrol, Diethylstilbestrol, Estrone, Trenbolone, Nandrolone, Androstenedione, Boldenone, Testosterone, Meandrosterone, Methyltestosterone, Phenylpropionate

Nandrolone, testosterone propionate, progesterone, 21alpha-hydroxyprogesterone, 17alpha-hydroxyprogesterone, medroxyprogesterone, megestrol acetate, chlormadinone acetate, and medroxyprogesterone

The detection limit of progesterone was 0.5 µg/kg, and the limit of quantification was 1.0 µg/kg; hexestrol, estradiol, levonorgestrel, norethindrone, estradiol benzoate and

The detection limit of stanozolol was 1.0 µg/kg, and the limit of quantification was 2.0 µg/kg; the detection limit of estriol and ethinyl estradiol was 2.0 µg/kg, and the limit of quantification was

4.0 µg/kg.

10.2 Accuracy

Diethylstilbestrol, Diethylstilbestrol, Estrone, Trenbolone, Nandrolone, Androstenedione, Boldenone, Testosterone, Meandrosterone, Methyltestosterone, Phenylpropionate

Nandrolone, testosterone propionate, progesterone, 21alpha-hydroxyprogesterone, 17alpha-hydroxyprogesterone, medroxyprogesterone, megestrol acetate, chlormadinone acetate, and medroxyprogesterone

The recovery rate of progesterone was 60%~120% within the concentration range of 1.0µg/kg~20µg/kg; hexestrol, estradiol, levonorgestrel, acetylene

The recoveries of nordone, estradiol benzoate and stanozolol were 50%-120% in the concentration range of 2.0 µg/kg-40 µg/kg; estriol and

The recoveries of ethinyl estradiol were 60%~120% in the concentration range of 4.0µg/kg~80µg/kg.

10.3 Precision

The intra-assay relative standard deviation of this method was <=20%, and the inter-assay relative standard deviation was <=20%.