GB 31658.25-2022National Food Safety Standard Determination of 10 Diuretic Drug Residues in Animal Foods by Liquid Chromatography-Tandem Mass Spectrometry (English PDF)

5.1 Reagents

5.1.1 Acetonitrile (CH3CN).

5.1.2 Methanol (CH3OH).

5.1.3 n-Hexane (C6H14).

5.1.4 Dimethyl sulfoxide (C2H6OS).

5.1.5 Ammonium acetate (CH3COONH4).

5.2 Solution preparation

5.2.1 90% acetonitrile aqueous solution. take 900mL of acetonitrile, add water to dilute to 1000mL.

5.2.2 50% acetonitrile aqueous solution. take 50mL of acetonitrile, add water to dilute to 100mL.

5.2.3 5mmol/L ammonium acetate buffer solution. Take 0.385g of ammonium acetate, dissolve it in water, and dilute to 1000mL.

5.2.4 90% acetonitrile methanol solution. Take 10mL of methanol, dilute to 100mL with acetonitrile, and mix well.

5.2.5 Acetonitrile saturated n-hexane solution. take the same volume of acetonitrile and n-hexane, put them in a separatory funnel, vibrate, let stand to separate layers, and take the upper layer

solution.

5.3 Standards

5.3.1 Standard products of diuretics. acetazolamide, 4-amino-6-chlorobenzene-1,3-disulfonamide, chlorothiazide, hydrochlorothiazide, chlorthalidone, furosemide, Bianfu

Thiazide, triamterene, spironolactone, canrenone, the purity of which is >=98.0%. Or the standard product certified by the state and awarded the standard material certificate. Standard product

See Table A.1 in Appendix A for information.

5.3.2 Isotopic internal standards. acetazolamide-D3, 4-amino-6-chlorobenzene-1,3-disulfonamide-15N2, chlorthalidone-D4, hydrochlorothiazide-13CD2,

Chlorthiazide-13C15N2, furosemide-D5, benfluthiazide-D5, triamterene-D5, canrenone-D6, all with a purity >= 98.0%.

Standards with certified reference materials. See Table A.1 in Appendix A for information on isotopic internal standards.

5.4 Preparation of standard solution

5.4.1 Standard stock solution (0.1 mg/mL). Weigh about 10 mg of each standard diuretic drug, weigh it accurately, dissolve in methanol and dilute to volume.

100mL brown volumetric flask, mix well, and prepare diuretic standard stock solution with a concentration of 0.1mg/mL. The standard triamterene should be used first

1mL dimethyl sulfoxide was dissolved, then diluted with methanol and adjusted to the mark. Stored at -20°C, the validity period was 6 months.

5.4.2 Internal standard stock solution (0.1 mg/mL). Weigh about 10 mg of each diuretic internal standard standard, accurately weighed, dissolve in methanol and make to volume.

into a 100mL brown volumetric flask, mix well, and prepare a diuretic internal standard standard stock solution with a concentration of 0.1mg/mL. Triamterene-D5 standard

The product needs to be dissolved in 1mL dimethyl sulfoxide first, then diluted with methanol and fixed to the mark. Stored at -20°C, the validity period is 6 months.

5.4.3 Mixed standard working solution. Accurately measure the standard stock solution (0.1 mg/mL) of chlorothiazide, hydrochlorothiazide, fenfluthiazide and canrenone.

1 mL, standard stock solution of chlorthalidone, acetazolamide, furosemide, spironolactone, 4-amino-6-chlorobenzene-1,3-disulfonamide, triamterene

(0.1mg/mL) each 2.5mL, in the same 100mL brown volumetric flask, dilute to the mark with methanol, and prepare the mixed standard working solution

The concentration of chlorothiazide, hydrochlorothiazide, benfluthiazide and canrenone was 1 µg/mL; chlorthalidone, acetazolamide, furosemide, spironolactone and 4-amino-6-

The concentrations of chlorobenzene-1,3-disulfonamide and triamterene were 2.5 µg/mL. Stored in the dark below 4 degrees C, the validity period was 1 month.

5.4.4 Internal standard working solution. Accurately measure 1 mL each of the internal standard stock solution (0.1 mg/mL), put it in a 100 mL brown volumetric flask, and dilute to volume with methanol.

To the mark, prepare the internal standard working solution with a concentration of 1 µg/mL, store it in the dark below 4°C, and have a validity period of 1 month.

5.4.5 Mixed standard series working solution

Accurately draw 10.0µL, 20.0µL, 50.0µL, 100µL,.200µL, 500µL of diuretic mixed standard working solution and internal standard working solution

200 µL of solution, respectively placed in 10mL brown volumetric flasks, and dilute to the mark with 50% acetonitrile aqueous solution. Among them, chlorothiazide, hydrochlorothiazide, Bian

The concentrations of fluthiazide and canrenone were 1.00ng/mL, 2.00ng/mL, 5.00ng/mL, 10.0ng/mL, 20.0ng/mL,

50.0ng/mL; the concentrations of chlorthalidone, acetazolamide, furosemide, spironolactone, 4-amino-6-chlorobenzene-1,3-disulfonamide and triamterene were

2.50ng/mL, 5.00ng/mL, 12.5ng/mL, 25.0ng/mL, 50.0ng/mL, 125ng/mL, internal standard in the standard series of working solutions

The concentration of the solution was 20.0 ng/mL, and it was prepared immediately after use.

5.5 Materials

5.5.1 Solid-phase extraction column. Pass-through reverse-phase mixed hydrophilic-lipophilic equilibrium copolymer solid-phase extraction column,.200mg/6mL, or equivalent performance.

5.5.2 Organic filter membrane. 0.22 µm.

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

6.2 Analytical balance. the sensitivity is 0.00001g and 0.01g respectively.

6.3 Nitrogen blowing concentrator.

6.4 Vortex mixer.

6.5 Ultrasonic generator.

6.6 Centrifuge. the speed is not lower than 8000r/min.

6.7 Solid phase extraction device.

6.8 Homogenizer.

7.1 Sample preparation

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

Take an appropriate amount of fresh or refrigerated blank or test eggs, shell them and mix them evenly.

Take an appropriate amount of fresh or refrigerated blank or test milk, and mix well.

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 Sample storage

Pork, beef, mutton, chicken, duck and egg samples were stored below -18°C, and milk samples were stored at 0°C to 4°C.

8.1 Extraction

8.1.1 Pork, beef, mutton, chicken, duck

Weigh 2 g of the sample (accurate to +/-0.05 g), put it in a 50 mL centrifuge tube, add 40.0 µL of internal standard working solution, add 90% acetonitrile water

8.0 mL of the solution was shaken vigorously on a vortex shaker for 2 min. Ultrasonic extraction was performed for 15 min, centrifuged at 8000 r/min for 5 min, and the supernatant was taken to centrifuge.

Add 8.0 mL of 90% acetonitrile aqueous solution to the residue and repeat the extraction once, and combine the supernatant. Add 5 mL of acetonitrile saturated n-hexane solution,

Vortex at low speed for 10 s, centrifuge at 8000 r/min for 3 min, remove the supernatant, and dilute to 20.0 mL with 90% acetonitrile aqueous solution for use.

8.1.2 Eggs, milk

Weigh 2 g of the sample (accurate to +/-0.02 g), add 40.0 µL of internal standard working solution to a 50 mL centrifuge tube, add 8.0 mL of acetonitrile, and

Vigorously oscillate on a vortex oscillator for 2 min. Ultrasonic extraction for 15 min, centrifugation at 8000 r/min for 5 min, and the supernatant was taken into a centrifuge tube. The residue was added to

Repeat the extraction once with 8.0 mL of acetonitrile, combine the supernatant, add 5 mL of acetonitrile saturated n-hexane solution, vortex at low speed for 10 s, 8000 r/min

Centrifuge for 3 min, remove the supernatant, and dilute to 20.0 mL with acetonitrile for later use.

8.2 Purification

Take 10.0mL of the reserve solution to pass through the solid-phase extraction column, pass through the solid-phase extraction column at a rate not greater than 1mL/min, receive the effluent sample solution, and

Elute with 1 mL of 90% acetonitrile methanol solution, receive all the eluate, and dry it with nitrogen at 40 °C. Dissolve it with 1.0 mL of 50% acetonitrile aqueous solution

The residue was vortexed and passed through a 0.22 µm filter membrane for determination by liquid chromatography-tandem mass spectrometer.

8.3 Determination

8.3.1 Reference conditions for liquid chromatography

a) Chromatographic column. C18 chromatographic column (100mmx2.1mm, 1.8µm), or equivalent;

b) Mobile phase. A is 5mmol/L ammonium acetate aqueous solution, B is acetonitrile, gradient elution conditions are shown in Table 1;

c) Flow rate. 0.3mL/min;

d) Column temperature. 35°C;

e) Injection volume. 5 µL.

8.3.2 Reference conditions for mass spectrometry

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

b) Scanning mode. positive ion mode for triamterene, spironolactone, canrenone; acetazolamide, 4-amino-6-chlorobenzene-1,3-disulfonamide, chloride

Thiazide, hydrochlorothiazide, chlorthalidone, furosemide, and Bianfluthiazide are in negative ion mode;

c) Detection method. multiple reaction monitoring;

d) Ionization voltage. positive mode 5500V, negative mode -4500V;

e) Temperature. 500°C;

f) Air curtain gas. 241kPa;

g) atomizing gas. 345kPa;

h) Auxiliary heating gas. 345kPa;

i) See Appendix B for qualifier ion pairs, quantifier ion pairs, declustering voltage and collision voltage.

8.3.3 Determination

8.3.3.1 Qualitative determination

Under the same experimental conditions, the deviation of the retention time of the substance to be tested in the sample from the corresponding retention time in the standard solution is within +/-2.5%;

And the relative ion abundance of the qualifier ions of the measured components in the sample is compared with the relative ion abundance of the corresponding qualifier ions in the standard solution with a similar concentration.

If the deviation does not exceed the range specified in Table 2, it can be determined that there is a corresponding analyte in the sample.

See Appendix C for the characteristic ion mass chromatograms of standard working solutions and internal standard working solutions of 10 diuretics.

8.3.3.2 Quantitative determination

Set the instrument conditions according to 8.3.1 and 8.3.2, take the sample solution and standard solution for single-point or multi-point calibration, and use the internal standard method to determine the peak area ratio.

Calculation. The peak area ratio of the 10 diuretics in the sample solution and the standard solution and the peak area of the corresponding internal standard should be within the linear range of the instrument detection.

The characteristic ion mass chromatograms of multiple reaction detection of 10 diuretics are shown in Appendix C.

8.4 Blank test

The blank sample was weighed, and the exact same measurement steps were used for parallel operation except that no drug was added.

10.1 Sensitivity

In this method, the detection limit of chlorothiazide, hydrochlorothiazide, benfluthiazide and canrenone was 1.00 µg/kg; the limit of quantification was 2.00 µg/kg. Acetyl

The detection limit of oxazolamide, 4-amino-6-chlorobenzene-1,3-disulfonamide, chlorthalidone, furosemide, spironolactone and triamterene was 2.50 µg/kg; the limit of quantification was

5.00 µg/kg.

10.2 Accuracy

In this method, when the concentration levels of chlorothiazide, hydrochlorothiazide, benfluthiazide and canrenone were added at 2 µg/kg~20 µg/kg, the recoveries were

60%~120%.

Acetazolamide, 4-amino-6-chlorobenzene-1,3-disulfonamide, chlorthalidone, furosemide, spironolactone, triamterene at 5 µg/kg~50 µg/kg

When the concentration level was added, the recovery rate was 60%~120%.

10.3 Precision

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