GB 23200.23-2016Food safety national standard -- Determination of duliphenol residues in food by liquid chromatography-mass spectrometry / mass spectrometry (English PDF)

4.1 Reagents

4.1.1 acetonitrile (CH3CN), high performance liquid chromatography grade.

4.1.2 Methanol (CH3OH), high performance liquid chromatography grade.

4.1.3 Cyclohexane (C6H12).

4.1.4 Ethyl acetate (C4H8O2).

4.1.5 Sodium chloride (NaCl).

4.2 Solution preparation

4.2.1 Ethyl acetate-cyclohexane (1 1, volume ratio). Equal volume of ethyl acetate and cyclohexane are miscible.

4.3 Standards

4.3.1 Diphenols standard. Molecular formula, C10H12N2O5; CAS 88-85-7; purity >= 99%.

4.4 Standard solution preparation

4.4.1 duliphenolic standard stock solution. accurately weighed the amount of Diphenols standard, with methanol prepared into 100 µg/mL standard stock solution,-18 °C below the storage. The

4.4.2 Dulu phenolic standard working solution. accurately absorb the appropriate amount of levophenol standard stock solution, diluted with methanol to the required concentration, -18 °C the following storage.

4.5 Materials

4.5.1 0.22 µm organic filter.

6.1 Preparation of the sample

6.1.1 Fruits, vegetables, nuts

Fruit, nuts. take the sample about 500 g, crushed with a pulverizer, into a clean container as a sample, sealed and do a good job.

6.1.2 Grain, tea

Take about 500 g of the sample and crush it all through a 20 mesh sieve into a clean container as a sample, seal and mark it.

Warm preservation.

6.1.3 Meat products

Take a representative sample of about 500 g, crushed with a pulverizer, into a clean container as a sample, sealed and labeled.

6.1.4 Honey

Take a representative sample of about 500 g, the amorphous sample will be forced to stir evenly, there are crystallization of the sample can be sealed after the sample cap, Placed in a water bath of not more than 60 °C, until the sample all dissolved and stir well, quickly cooled to room temperature. The prepared sample is packed in a clean container Sealed and identified.

6.1.5 Milk

Take a representative milk or milk powder sample, mix well and mark it.

Note. The above sample sampling site according to GB 2763 Appendix A implementation.

6.2 Save

Fruits, vegetables, nuts, meat products, milk and other samples in -18 °C below the frozen preservation; grain, tea, honey and other samples in the room Keep the temperature away from light.

During the sample preparation operation, it is necessary to prevent the sample from being contaminated or the change in the content of the residue.

7.1 Extraction

Approximately 2.5 g of the sample of the tea leaves and about 5 g (accurate to 0.01 g) of the other substrate samples in a 50 mL centrifuge tube (3 mL Water, so that the sample is fully wetted), adding 25 mL of acetonitrile and 5 g of sodium chloride to 10 000 r/min homogenization 2 min, centrifuged at 4000 r/min for 10 min, the supernatant was transferred to a 25 mL colorimetric tube and fixed to 25 mL with acetonitrile.

7.2 Purification

7.2.1 Gel Permeation Chromatographic Conditions

A) Purification column S-X3 Bio-Beads filler, particle size 38 µm -75 µm,.200 mm × 22 mm (inner diameter), or equivalent;

B) mobile phase. ethyl acetate-cyclohexane, flow rate. 5 mL/min;

C) Injection volume. 5 mL;

D) Purification procedure. 0 min-10.5 min The eluent was discarded and the eluate was collected at 10.5 min-15 min.

7.2.2 Purification process

Accurately remove 5 mL of the above set of liquid, water bath at 45 °C nitrogen flow to near dry, constant volume to 10 mL, take 5 mL of ethyl acetate -the alkane solution was purified by gel permeation chromatography under the conditions of 6.2.1. The collected eluate was concentrated on a 45 ° C water bath to dryness and accurately added Into 1 mL of methanol dissolved residue, dissolved solution over 0.22 µm organic filter for liquid chromatography-mass spectrometry/mass spectrometry.

7.3 Determination

7.3.1 Liquid Chromatographic Reference Conditions

A) Column. C18 150 mm × 2.1 mm (inner diameter), particle size 5 µm, or equivalent;

B) Column temperature. 30 ° C;

C) Injection volume. 5 µL.

D) mobile phase gradient and flow rate in Table 1.

Table 1 Liquid Chromatography Gradient Elution Conditions

Time (min)

0.1% acetic acid is water soluble liquid(%) Acetonitrile (%) Flow rate (µL/min) 0.0 30 70.200 4.0 30 70.200 5.0 10 90.200 6.0 10 90.200 GB 23200.24-2016 6.1 30 70.200 8.0 30 70.200

7.3.2 Mass spectrometry reference conditions

A) ion source. electrospray ion source (ESI);

B) scanning mode. negative ion scanning mode;

C) See Appendix A for other reference mass spectrometry conditions.

7.3.3 Quantitative determination

According to the content of the sample in the sample solution, select the standard response value similar to the working fluid. Standard working solution and the analyte in the sample solution the values shall be within the linear range of the instrument. The standard working solution and sample solution volume measurement. Under the above chromatographic conditions the reference retention time of levophenol is about 4.55 min. The standard multi-reaction monitoring chromatogram of dinomyol is shown in Appendix B.

7.3.4 Qualitative determination

When the sample is measured, if the mass retention time of the detected mass is consistent with that of the standard sample, and the sample spectrum after subtracting the background , The relative abundance of the qualitative ions is close to the concentration of the matrix under the same conditions obtained under the standard solution spectrum, the error does not exceed the table Set the range, you can determine the existence of the corresponding sample in the sample.

Table 2 The maximum allowable error of relative ion abundance when qualitative confirmation

Relative abundance (base) 50% 20% to 50% 10% to 20% <=10% Allowable relative deviation ± 20% ± 25% ± 30% ± 50%

7.4 Blank experiment

In addition to the sample, according to the above determination steps.

10.1 Quantitation limits

The quantitative limit of diclofenac in tea was 10 µg/kg, and the other substrates were 5 µg/kg.

10.2 Recovery rate

For different substrates, the recovery rates for the three different additive levels are given in Appendix C.

GB 23200.24-2016

Appendix A

(Informative)

Reference mass spectrometry conditions 1) Reference mass spectrometry conditions.

A) sheath gas pressure. 137.9 kPa (20 psi);

B) Auxiliary gas pressure. 206.85 kPa (30 psi);

C) Negative ion mode Electrospray voltage (IS). -3.200 V;

D) Capillary temperature. 320 ° C;

E) Source induced dissociation voltage. 10 V;

F) Q1, Q3 resolution. Q1 is 0.4, Q3 is 0.7;

G) collision gas. high purity argon;

H) Collision gas pressure. 1.5 mTorr.

I) Other mass spectral parameters are shown in Table A.1.

Table A.1 Mass Spectral Acquisition Parameters and Retention Time

Analyte retention time/min acquisition window/min ion pair (m/z) collision energy/eV Digoxin 4.55 3 ~ 6 239.02/194.05a 239.02/193.02 A for the quantitative ion pair, for different mass spectrometry equipment, instrument parameters may be different, before the determination of the mass spectrometry parameters should be optimized to the best.

1) Non-commercial declaration. The reference mass spectrometry conditions listed in Appendix B are performed on a Thermo TSQ Quantum Ultra AM-type LC/MS.

The instrument type is for reference only and does not involve commercial purposes, and the standard user is encouraged to try different manufacturers or models.

GB 23200.24-2016

Appendix B

(Informative)

The multi-reaction monitoring quality chromatogram of dinol Diphenols Reference Substance Multi-Reaction Monitoring Mass Chromatogram, see Figure B.1.

Figure B.1 Diphenols Reference Substance Multi-Reaction Monitoring Quality Chromatogram

239.02/193.02

239.02/194.05

GB 23200.24-2016

Appendix C

(Informative)

Sample concentration and recovery of the experimental data

Table C.1 Experimental data on the concentration and recovery of the sample

Substrate addition level (µg/kg) Recovery% apple 5 76.4 ~ 94.8 10 72.2 ~ 85.6 50 86.9 ~ 100 Cabbage 5 72.7 to 92.4 10 79.8 to 98.4 50 84.3 ~ 100 Chestnut 5 77.2 ~ 95.6 10 75.4 to 94.9 50 79.6 ~ 95.6 beef 5 77.3 ~ 94.2 10 79.2 ~ 94.7 50 80.0 to 97.7 honey 5 76.3 ~ 91.4 10 77.5 ~ 94.2 50 79.5 ~ 95.0 wheat 5 74.9 ~ 96.5 10 78.2 ~ 94.1 50 81.1 to 99.8 chicken 5 77.4 ~ 91.0 10 76.1 to 97.4 50 82.8 ~ 94.8 5 81.1 to 91.3 10 79.2 ~ 101 50 82.5 to 99.3 Soybeans 5 76.2 ~ 97.8 10 85.8 ~ 102 50 86.8 ~ 100 milk 5 71.8 ~ 100 10 74.2 ~ 94.1 50 86.8 ~ 101 tea 10 87.4 ~ 102 50 84.7 ~ 101 100 89.9 ~ 100 GB 23200.24-2016 Appendix D (Normative appendix) Laboratory repeatability requirements

Table D.1 Laboratory repeatability requirements

Measured component content Mg/kg Precision <=0.001 36 > 0.01 > 1 14 GB 23200.24-2016 Appendix E (Normative appendix) Inter-laboratory reproducibility requirements

Table E.1 Inter-laboratory reproducibility requirements

Measured component content Mg/kg Precision <=0.001 54 > 0.01 > 1 19