GB 31656.4-2021National food safety standard - Determination of chlorpromazine residue in fishery products by liquid chromatography-tandem mass spectrometric method (English PDF)

Foreword

This document complies with the provisions of GB/T 1:1-2020 "Standardization Work Guidelines Part 1: Structure and Drafting Rules of Standardization Documents"

Drafting:

This document is published for the first time:

National food safety standards

Determination of chlorpromazine residues in aquatic products by liquid chromatography-tandem mass spectrometry

1 Scope

This document specifies the sample preparation and liquid chromatography-tandem mass spectrometry determination methods for the detection of chlorpromazine residues in aquatic products:

This document is applicable to the detection of chlorpromazine residues in edible tissues of fish, shrimp and crabs:

2 Normative reference documents

The contents of the following documents constitute essential provisions of this document through normative citations in the text: Among them, the cited documents with dates are:

Only the version corresponding to the date applies to this document; for undated referenced documents, the latest version (including all amendments) applies to this document:

document:

GB/T 6682 Specifications and test methods for water used in analytical laboratories

GB/T 30891-2014 Sampling specifications for aquatic products

3 Terms and definitions

There are no terms or definitions that need to be defined in this document:

4 Principles

The remaining chlorpromazine in the sample was extracted with acetonitrile, purified with a mixed cationic solid-phase extraction column, and measured by liquid chromatography-tandem mass spectrometry, using the internal standard method:

Quantitative:

5 Reagents and materials

The reagents used below are of analytical grade unless otherwise noted, and the water is first-grade water that complies with GB/T 6682:

5:1 Reagents

5:1:1 Acetonitrile (CH3CN): chromatographically pure:

5:1:2 Methanol (CH3OH): chromatographically pure:

5:1:3 Formic acid (HCOOH): chromatographically pure:

5:1:4 Ammonium acetate (NH4C2H3O2):

5:1:5 Ammonia (NH3.H2O):

5:2 Standard products

5:3 Solution preparation

5:3:1 5% ammoniated methanol solution: Take 5 mL of ammonia water and 95 mL of methanol, and mix well:

5:3:2 2mmol/L ammonium acetate solution: Take 0:154g of ammonium acetate, add 1mL of formic acid, dissolve with appropriate amount of water and dilute to 1000mL:

5:3:3 30% methanol-ammonium acetate solution: Take 30 mL of methanol and 70 mL of 2 mmol/L ammonium acetate solution, and mix well:

5:4 Preparation of standard solution

5:4:1 Chlorpromazine standard stock solution (100 µg/mL): Take about 11 mg of chlorpromazine hydrochloride standard, weigh it accurately, add an appropriate amount of methanol to dissolve and dilute to a 100 mL volumetric flask, shake well, and you get : Store in the dark below -18 degrees C, valid for 6 months:

5:4:2 Internal standard stock solution (100 µg/mL): Take about 11 mg of chlorpromazine hydrochloride-D6 standard, weigh it accurately, add an appropriate amount of methanol to dissolve and dilute to a 100 mL volumetric flask, shake well, and you get : Store in the dark below -18 degrees C, valid for 6 months:

5:4:3 Chlorpromazine standard intermediate solution (500ng/mL): Accurately measure an appropriate amount of chlorpromazine standard stock solution and dilute it with methanol to make a concentration of

500ng/mL chlorpromazine standard intermediate solution: Store in a dark place below -18°C and is valid for 3 months:

5:4:4 Internal standard working solution (200ng/mL): Accurately measure an appropriate amount of the internal standard stock solution, dilute it with methanol to prepare a chlorpromazine-D6 internal standard working solution with a concentration of:200ng/mL: Store in the dark below -18°C ,valid for 3 months:

5:5 Materials

5:5:1 Mixed cationic solid phase extraction column: 60mg/3mL, or equivalent:

5:5:2 Aqueous polysulfone ether needle filter: 0:22µm:

6 Instruments and equipment

6:1 Liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source:

6:2 Balance: sensitivity 0:01g and 0:00001g:

6:3 Homogenizer:

6:4 Centrifuge: 6000r/min:

6:5 Rotary evaporator:

6:6 Vortex mixer:

6:7 Ultrasonic cleaner:

7 Measurement steps

7:1 Preparation of specimens

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

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

b) Take the blank sample after homogenization as the blank sample;

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

7:2 Storage of specimens

Stored below -18 degrees C, the validity period is 3 months:

8 Measurement steps

8:1 Extraction

Take 5g of the sample (accurate to +/-0:05g) in a 50mL centrifuge tube, add 50µL of internal standard working solution, add 15mL of acetonitrile, homogenize for 1 minute, and ultrasonic

Sonicate for 10 min, centrifuge at 4000 r/min for 7 min, transfer the supernatant to another 50 mL centrifuge tube; take another 10 mL of acetonitrile and clean the homogenizer for 30 s, pour the washing liquid into the residue, vortex and mix for 2 min, ultrasonic for 10 min, 4000 r/min Centrifuge for 7 minutes, combine the supernatant, centrifuge at 6000r/min for 5 minutes, take the supernatant and set aside:

8:2 Purification

Take the solid phase extraction column, activate it with 5 mL each of methanol and water, pass the supernatant through the column at a speed of about 1 drop per second, and elute with 5 mL of methanol:

Discard the effluent and drain it: Elute with 3 mL of 5% ammoniated methanol solution: Collect the eluate and blow dry with nitrogen at 40°C: Add 30% methanol-ethyl alcohol accurately:

Dissolve the residue in 1 mL of ammonium acid solution and filter it with an aqueous needle filter into an injection vial for liquid chromatography-tandem mass spectrometry measurement:

8:3 Preparation of matrix standard curve

Accurately measure appropriate amounts of 500ng/mL chlorpromazine standard intermediate solution and:200ng/mL internal standard working solution, blow dry with nitrogen at 40°C, and use blank matrix

The solution was prepared into a series of matrices with an isotope internal standard concentration of 10ng/mL and chlorpromazine concentrations of 2:5ng/mL, 5:0ng/mL, 25:0ng/mL, 100:0ng/mL and 250:0ng/mL: The standard working solution is used for liquid chromatography-tandem mass spectrometry: The standard curve is drawn using the ratio of the characteristic ion mass chromatographic peak area of chlorpromazine to the characteristic ion mass chromatographic peak area of the isotope internal standard as the ordinate and the corresponding concentration as the abscissa: Regression equation and correlation coefficient:

8:4 Determination

8:4:1 Liquid chromatography conditions

a) Chromatographic column: C18 column, 150mmx2:1mm, 5µm, or equivalent performance;

b) Flow rate: 0:2mL/min;

c) Column temperature: 25 degrees C;

d) Injection volume: 25µL;

e) Mobile phase: A is methanol, B is 2mmol/L ammonium acetate, and the gradient elution conditions are shown in Table 1:

8:4:2 Mass spectrometry conditions

a) Ion source: electrospray ion source;

b) Scanning method: positive ion scanning;

c) Detection method: multiple reaction monitoring;

d) Spray voltage: 4500V;

e) Ion transmission capillary temperature: 300 degrees C;

f) In-source collision-induced dissociation voltage: 8V;

g) Atomizing gas flow rate: 12:3L/h;

h) Auxiliary air flow rate: 1:7L/h;

i) The reference values of precursor ions, product ions and collision energy are shown in Table 2:

8:4:3 Determination method

Inject equal volumes of sample solution and matrix standard working solution, perform single-point or multi-point calibration, and calculate using the chromatographic peak area and internal standard method:

The response values of chlorpromazine in the matrix standard solution and the sample solution should be within the linear range of the instrument detection:

The retention time and relative abundance of characteristic ions are compared qualitatively with the retention time and relative abundance of characteristic ions of a matrix standard solution of similar concentration:

The retention time deviation of the analyte in the sample solution and the standard substance is within +/-2:5%; the relative abundance deviation of the characteristic ions meets the requirements of Table 3, then

It can be determined that the corresponding analyte exists in the sample: Under the above chromatography-mass spectrometry conditions, the characteristic ion chromatograms of chlorpromazine and internal standard standard solution

See Appendix A:

8:5 Blank test

Take a blank sample and perform parallel operations using the same measurement steps except that no standard solution is added:

9 Calculation and presentation of results

10 Method sensitivity, accuracy and precision

10:1 Sensitivity

The detection limit of this method is 0:5 µg/kg, and the quantitation limit is 1:0 µg/kg:

10:2 Accuracy

In this method, when chlorpromazine is added at a concentration range of 1 µg/kg to 50 µg/kg, the recovery rate is 70% to 110%:

10:3 Precision

The intra-batch relative standard deviation of this method is <=15%, and the inter-batch relative standard deviation is <=15%: