GB 31657.3-2022National food safety standards-Determination of cephalosporin residues in bee products-Liquid chromatography-tandem mass spectrometry (English PDF)

5.1 Reagents

5.1.1 Methanol (CH3OH). chromatographically pure.

5.1.2 Acetonitrile (CH3CN). chromatographically pure.

5.1.3 Formic acid (HCOOH). chromatographically pure.

5.1.4 Potassium dihydrogen phosphate (KH2PO4).

5.1.5 Sodium Hydroxide (NaOH).

5.2 Solution preparation

5.2.11 2.5mol/L sodium hydroxide solution. take 50g of sodium hydroxide, add water to dissolve and dilute to 500mL.

5.2.2 30% acetonitrile solution. Take 30mL of acetonitrile and dilute with water to 100mL.

5.2.3 0.05mol/L phosphate buffer solution (pH=8.5). Take 6.8g of potassium dihydrogen phosphate, dissolve it in water and dilute it to 1000mL, and use

2.5 mol/L sodium hydroxide solution to adjust the pH to 8.5.

5.2.4 0.1% formic acid solution. Take 1mL of formic acid, dissolve it in water and dilute to 1000mL.

5.2.5 0.1% formic acid solution-methanol. Take 95 mL of 0.1% formic acid solution, add 5 mL of methanol, and mix well.

5.3 Standards

Cefalexin, cephradine, cefazolin, cefoperazone, cefacetonitrile, cefapirin, deacetylcefapirin, cefaroning, cefquinone

oxime and cefotaxime, the content of which was >=95.0%, see Appendix A for specific information.

5.4 Preparation of standard solution

5.4.1 Standard stock solution. Take 10 mg of each standard product, weigh them accurately, add an appropriate amount of 30% acetonitrile solution to dissolve and dilute to a 25 mL volumetric flask,

It was prepared into a solution with a concentration of 400 µg/mL. It was stored in the dark below -18 degrees C, and the validity period was 1 month.

5.4.2 Mixed standard stock solution. Accurately pipette 0.25mL of each standard stock solution into a 10mL volumetric flask, dilute with 30% acetonitrile solution

To the mark, it was prepared into a mixed standard stock solution with a concentration of 10 µg/mL. Stored in the dark below -18°C, the validity period was 7 days.

5.4.3 Mixed standard working solution. Accurately pipette an appropriate amount of mixed standard stock solution, and dilute it with 0.1% formic acid solution-methanol to a concentration of

2.5µg/L, 5.0µg/L, 20µg/L, 100µg/L,.200µg/L and 500µg/L series mixed standard working solutions. Ready to use.

5.5 Materials

5.5.1 Solid-phase extraction cartridge. hydrophilic-lipophilic balanced solid-phase extraction column, 500mg/6mL, or equivalent.

5.5.2 Filter membrane. made of nylon, with a pore size of 0.22 µm or equivalent.

6.1 Liquid chromatography-tandem mass spectrometer. with electrospray ion source.

6.2 Analytical balance. Sensitivity 0.00001g and 0.01g.

6.3 Nitrogen blowing instrument.

6.4 Solid phase extraction device.

6.5 Vortex mixer.

6.6 Centrifuge. The maximum speed is 6000r/min or above.

6.7 Centrifuge tube. polypropylene plastic centrifuge tube, 10mL, 50mL.

6.8 pH meter.

7.1 Preparation of samples

Take an appropriate amount of fresh or refrigerated blank or test bee products, if there is no crystallization, stir it evenly, if there is crystallization, place it at no more than 60°C

Warm in a water bath, oscillate, stir evenly after the sample is completely melted, and cool to room temperature. Royal jelly and royal jelly freeze-dried powder are taken out from the frozen environment,

Samples were taken after stirring.

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

b) Take the uniform blank sample as the blank sample;

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

7.2 Storage of samples

Store below -18°C.

8.1 Extraction

8.1.1 Honey

Take 5g of the sample (accurate to +/-0.05g), put it in a 50mL centrifuge tube, add 25mL of 0.05mol/L phosphate buffer solution, dissolve the sample,

After vortex mixing, the pH was adjusted to 8.5 with 2.5 mol/L sodium hydroxide solution, and set aside.

8.1.2 Royal jelly and royal jelly freeze-dried powder

Take 2.5g (accurate to +/-0.05g) of royal jelly or 1.0g (accurate to +/-0.05g) of royal jelly freeze-dried powder in a 50mL centrifuge tube,

Add 25 mL of 0.05 mol/L phosphate buffer solution, vortex and mix well, adjust the pH to 8.5 with 2.5 mol/L sodium hydroxide solution, and

Centrifuge at 6000r/min for 5min, take the supernatant, and set aside.

8.2 Purification

Take the solid-phase extraction column, activate it with 5 mL of methanol and 10 mL of 0.05 mol/L phosphate buffer solution successively, take the spare solution, pass it through the column, and wait until the liquid level

When reaching the surface of the column bed, rinse with 3 mL of phosphate buffer solution and 2 mL of water in sequence, and discard all the effluent. Elute with 3 mL of acetonitrile,

Collect the eluate in a 10 mL centrifuge tube, dry the eluate in a 40°C water bath with nitrogen, add 0.1% formic acid solution-1.0 mL of methanol to dissolve, and

0.22 µm filter membrane for liquid chromatography-tandem mass spectrometry.

8.3 Preparation of matrix-matched standard curve

Take the blank sample and treat it according to 8.1 and 8.2 in turn, dry it with nitrogen in a water bath at 40°C, and add 1.0 mL of a series of mixed standard working solutions to dissolve it.

The solution residue was passed through a 0.22 µm filter membrane to prepare a series of bases of 2.5 µg/L, 5.0 µg/L, 20 µg/L, 100 µg/L,.200 µg/L and 500 µg/L

Mass matching standard working solution for liquid chromatography-tandem mass spectrometry determination. Take the peak area of the quantitative ion pair as the ordinate, and the concentration of the standard solution as the abscissa

Draw the standard curve. Find the regression equation and correlation coefficient.

8.4 Determination

8.4.1 Reference conditions for liquid chromatography

a) Chromatographic column. C18 chromatographic column (100mmx2.1mm, 1.7µm) or equivalent;

b) Mobile phase. A is 0.1% formic acid solution, B is methanol, and the gradient elution program is shown in Table 1;

c) Flow rate. 0.3mL/min;

d) Column temperature. 35°C;

e) Injection volume. 10 µL.

8.4.2 Reference conditions for mass spectrometry

a) Ion source. Electrospray (ESI) ion source;

b) Scanning mode. positive ion scanning;

c) Detection method. multiple reaction monitoring (MRM);

d) Capillary voltage..2000V;

e) RF lens voltage. 0.5V;

f) Ion source temperature. 150°C;

g) Desolvation temperature. 500°C;

h) Cone gas flow rate. 50L/h;

i) Desolvation gas flow rate. 1000L/h;

j) Secondary collision gas. argon;

k) See Table 2 for qualifier ion pairs, quantifier ion pairs, collision energy and cone voltage.

8.4.3 Assay

Take the sample solution and the matrix-matched standard solution for single-point or multi-point calibration, and use the external standard method to quantify the chromatographic peak area. The matrix-matched standard solution

The characteristic ion mass chromatographic peak area of the target drug in the solution and the sample solution should be within the linear range of the instrument detection.

The ratio of the retention time of the substance to the retention time of the substance to be tested in the matrix matching standard working solution, the deviation is within +/-2.5%, and the sample solution

Compared with the relative abundance of ions in the matrix-matched standard solution, if they meet the requirements in Table 3, it can be determined that there is a pair of ions in the sample.

The corresponding substances to be tested. See Appendix B for the MRM chromatogram of the standard solution.

8.5 Blank test

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

10.1 Sensitivity

In this method, the detection limits of cefoperazone, cefacetonitrile and cefazolin in honey, royal jelly and royal jelly freeze-dried powder are respectively

2.0 µg/kg, 4.0 µg/kg and 10 µg/kg, the limits of quantitation were 4.0 µg/kg, 8.0 µg/kg and 20 µg/kg respectively; other cephalosporins

The limits of detection for honey, royal jelly and royal jelly freeze-dried powder were 0.5 µg/kg, 1.0 µg/kg and 1.0 µg/kg, respectively.

2.5 µg/kg, the limits of quantitation were 1.0 µg/kg, 2.0 µg/kg and 5.0 µg/kg, respectively.

10.2 Accuracy

The recoveries of this method were 60%-120% at the spiked concentration levels of 1.0 µg/kg-40 µg/kg.

10.3 Precision

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