GB 31659.3-2022National Food Safety Standard Determination of Cephalosporin Residues in Milk and Milk Powder by 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 n-Hexane (C6H14).

5.1.5 Potassium dihydrogen phosphate (KH2PO4).

5.1.6 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 and dilute it with water to 1000mL.

5.2.5 0.1% formic acid solution-methanol (95.5). take 95mL of 0.1% formic acid solution and 5mL of methanol, and mix well.

5.3 Standards

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

Oxime and cefotaxime standard products, the content of which is >=95%, see Appendix A for details.

5.4 Preparation of standard solution

5.4.1 Standard stock solution. take 10 mg of each standard product, weigh them precisely, dissolve them in an appropriate amount with 30% acetonitrile solution and dilute to 25 mL.

A volumetric flask was prepared into a standard stock solution with a concentration of 400 µg/mL. It was stored at -18°C in the dark, 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, and dilute with 30% acetonitrile solution.

The solution was released to the mark, and prepared into a mixed standard stock solution with a concentration of 10 µg/mL. Stored in the dark at -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 (95.5) 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 and prepare.

5.5 Materials

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

5.5.2 Syringe filter. 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 tube. polypropylene plastic centrifuge tube, 10mL, 50mL.

6.7 pH meter.

7.1 Preparation of samples

Take an appropriate amount of fresh or thawed blank or test sample, and homogenize.

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

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

c) Take the homogenized 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

Take 5g (accurate to +/-0.05g) of milk or goat milk sample or 0.5g (accurate to +/-0.01g) of milk powder sample, put it in a 50mL centrifuge tube, add phosphorus

20mL salt buffer solution, vortexed and mixed for 30s, adjusted to pH 8.5 with 2.5mol/L sodium hydroxide solution, set aside.

8.2 Purification

Take the solid-phase extraction column and activate it with 5 mL of methanol and 10 mL of phosphate buffer solution in turn. Take the spare solution and pass it through the column until the liquid level reaches the surface of the column bed.

Then rinse with 3 mL of phosphate buffer solution and 2 mL of water in sequence, discard all the effluent, elute with 3 mL of acetonitrile, and collect the eluate

Add 3 mL of n-hexane to a 10 mL centrifuge tube, vortex and mix for 1 min, let stand for 5 min, discard the upper n-hexane layer, take the acetonitrile layer in 40 °C water

Blow dry in a nitrogen bath, add 1.0 mL of 0.1% formic acid solution-methanol (95.5) to dissolve, pass through a 0.22 µm filter membrane, and use for liquid chromatography-tandem mass spectrometry determination.

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

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

Draw a 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 (100mmx2.0mm, 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, if it exceeds the linear range

range, the matrix matching standard solution and the test solution should be diluted accordingly and re-measured. The retention time of the substance to be tested in the test solution is different from that of the matrix

Match the ratio of the retention time of the substance to be tested in the standard working solution, the deviation is within +/-2.5%, and the relative abundance of ions in the sample solution is consistent with that of the matrix.

Compared with the relative abundance of ions in the matching standard solution, if it meets the requirements of Table 3, it can be determined that the corresponding substance to be tested exists in the sample. The standard solution

See Appendix B for the liquid multiple reaction monitoring chromatogram.

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 milk and milk powder were 2.0 µg/kg and 20 µg/kg, respectively.

The limits of detection were 4.0 µg/kg and 40 µg/kg respectively; the detection limits of other cephalosporins and deacetylcefapirin in milk and milk powder were

The limits of quantitation were 1.0 µg/kg and 10 µg/kg respectively.

10.2 Accuracy

The recoveries of this method were 60%-120% at the spiked concentration levels of 1.0 µg/kg-200 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%.