GB 31658.19-2022National Food Safety Standard Determination of Atropine, Scopolamine, Anisodamine, Lidocaine, and Procaine Residues in Animal Foods-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 Dipotassium hydrogen phosphate (K2HPO4.3H2O).

5.1.6 phosphoric acid (H3PO4).

5.2 Solution preparation

5.2.11 0.1mol/L Potassium dihydrogen phosphate buffer solution. Take 13.6g of potassium dihydrogen phosphate, add 900mL of water to dissolve, adjust the pH to

4.0+/-0.05, dilute with water to 1000mL, and mix well.

5.2.2 0.2mol/L dipotassium hydrogen phosphate solution. Take 22.8g of dipotassium hydrogen phosphate, add water to dissolve and dilute to 500mL, and mix well.

5.2.3 5% methanol solution. Take 5mL of methanol, add water to dilute to 100mL, and mix well.

5.2.4 30% acetonitrile solution. take 30mL of acetonitrile, add water to dilute to 100mL, and mix well.

5.2.5 0.1% formic acid solution. take 0.5mL of formic acid, add water to dilute to 500mL, and mix well.

5.3 Standards

Atropine (Atropine, C17H23NO3, CAS number. 51-55-8), scopolamine (Scopolamine, C17H21NO4, CAS number. 51-

34-3), anisodamine (Anisodamine, C17H23NO4, CAS number. 55869-99-3), lidocaine (Lidocaine C14H22N2O, CAS

No.. 137-58-6), procaine (Procaine, C13H20N2O2, CAS No.. 59-46-1), the content is >=99%. The standard can also be the corresponding salt.

5.4 Preparation of standard solution

5.4.1 Standard stock solution. Take the appropriate amount of atropine, scopolamine, anisodamine, lidocaine and procaine

About 10 mg), accurately weighed, add methanol to dissolve and dilute to 10 mL volumetric flask, and prepare a standard concentration of 1 mg/mL

Stock solution. Stored at -18°C in the dark, valid for 6 months.

5.4.2 Mixed standard intermediate solution. Accurately measure 1mL of each standard stock solution, put it in a 100mL volumetric flask, dilute it to the mark with methanol, and prepare

The mixed standard working solution with a concentration of 10 µg/mL was stored at -18°C in the dark, and the validity period was 1 month.

5.4.3 series of standard working solutions. accurately measure an appropriate amount of mixed standard intermediate solution, dilute with 30% acetonitrile solution, and prepare the concentrations of

1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL and 100ng/mL series of standard solutions. Ready to prepare

Now in use.

5.5 Materials

1) The PEP-2 solid-phase extraction column listed here is for reference only and does not involve commercial purposes. Standard users are encouraged to try to use different manufacturers or models of solid-phase extraction columns.

5.5.1 PEP-2 solid phase extraction column 1). The filler is functionalized polystyrene/divinylbenzene, 60mg/3mL, or equivalent.

5.5.2 Microporous nylon filter membrane. 0.22 µm.

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

6.2 Analytical balance. Sensitivity 0.01g and 0.00001g.

6.3 Vortex mixer.

6.4 Vortex shaker.

6.5 High-speed refrigerated centrifuge. the speed can reach 10000r/min.

6.6 Solid phase extraction device.

6.7 Tissue homogenizer.

6.8 pH meter.

7.1 Preparation of samples

Take an appropriate amount of fresh or thawed blank or test tissue, mince it, and make it homogeneous.

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

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

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

7.2 Storage of samples

Store below -18°C.

8.1 Extraction

Take 2 g of the test material (accurate to +/-0.05 g) into a 50 mL centrifuge tube, and add 20 mL of 0.1 mol/L potassium dihydrogen phosphate buffer solution accurately.

(fat samples were heated in a 60°C water bath for 10min), vortexed for 1min, shaken for 10min, and centrifuged at 10000r/min for 10min at 4°C.

Take 10 mL of supernatant to another centrifuge tube, add 5 mL of 0.2 mol/L dipotassium hydrogen phosphate solution, mix well and set aside. If the solution is turbid,

Centrifuge at 10000r/min for 10min, and take the supernatant for later use.

8.2 Purification

The PEP-2 solid-phase extraction column was activated with methanol 3mL and water 3mL respectively, all the spare liquid was pipetted, passed through the column, water 3mL, 5% methanol solution

Rinse with 3mL, drain, and accurately pipette 2mL of 30% acetonitrile solution for eluting, drain, collect the eluate, pass through a microporous nylon filter membrane, and use it for liquid chromatography-series

Combined mass spectrometry.

8.3 Preparation of matrix-matched standard curve

Accurately pipette 100 µL each of the series of standard working solutions, dilute to 1 mL with the eluent of the blank sample after processing in steps 8.1-8.2, and prepare

A series of matrices with concentrations of 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/mL and 10ng/mL were prepared

Match the standard solution and pass through the microporous nylon filter membrane for liquid chromatography-tandem mass spectrometry.

The matrix matching standard solution concentration is the abscissa, the matrix matching standard curve is drawn, and the regression equation and correlation coefficient are calculated.

8.4 Determination

8.4.1 Reference conditions for liquid chromatography

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

b) Column temperature. 35°C;

c) Injection volume. 2 µL;

d) Flow rate. 0.3mL/min;

e) Mobile phase. A is 0.1% formic acid solution; B is methanol. The gradient elution program is shown in Table 1.

8.4.2 Reference conditions for mass spectrometry

a) Ion source. electrospray ion source;

b) Scanning mode. positive ion scanning;

c) Detection method. multiple reaction monitoring;

d) Spray voltage. 1.0kV;

e) Sheath gas. 30Arb;

f) Auxiliary gas. 5Arb;

g) Ion transfer tube temperature. 325°C;

h) Atomization temperature. 300°C;

i) Refer to Table 2 for the reference values of retention time, qualitative ion pair, quantitative ion pair, cone voltage and collision energy of the drug to be tested.

8.4.3.1 Qualitative determination

Under the same test conditions, the retention time of the drug peak of the analyte in the test solution was similar to that of the corresponding peak in the matrix-matched standard solution.

The deviation is within +/-2.5%, and the detected relative ion abundance should be equal to the relative ion abundance of the matrix matching standard solution with the same concentration.

The allowable deviation should meet the requirements of Table 3.

8.4.3.2 Quantitative determination

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

The response values of the analytes in the standard working solution and the sample solution should be within the linear range of the instrument detection.

See Appendix A for the characteristic ion mass chromatograms prepared with standard solutions.

8.5 Blank test

The blank sample was taken, except that no drug was added, and the same determination steps were used for determination.

10.1 Sensitivity

The detection limit of this method in the muscle and adipose tissue of pigs, cattle, and sheep was 0.2 µg/kg, and the limit of quantification was 0.5 µg/kg; in the kidneys of pigs, cattle, and sheep

The limit of detection in liver and liver tissues was 0.5 µg/kg, and the limit of quantification was 1.0 µg/kg.

10.2 Accuracy

The recovery rate of this method at the concentration level of 0.5 µg/kg~5 µg/kg in muscle and adipose tissue of pigs, cattle and sheep is 60%~

120%; the recovery rate was 60%~120% at the concentration level of 1.0µg/kg~10µg/kg in the kidney and liver tissues of pigs, cattle, and sheep.

10.3 Precision

In this method, the relative standard deviation within the batch was <=15%, and the relative standard deviation between batches was <=15%.