WS/T 684-2020Detection methods and evaluation requirements of antibacterial drugs in disinfectants and antibacterial agents (English PDF)

4.1 Principle

The antibacterial drugs in the sample are ultrasonically extracted with methanol or acetonitrile. After the extract is filtered, it is mixed with water and used by ultra-high performance liquid chromatography-tandem mass spectrometry.

Measured by instrument and quantified by internal standard method.

4.2 Reagents and materials

Unless otherwise specified, the reagents used are all chromatographically pure, and the experimental water is the first grade water specified in GB/T 6682.

4.2.1 Reagents

4.2.1.1 Acetonitrile (CH3CN).

4.2.1.2 Formic acid (HCOOH).

4.2.1.3 Methanol (CH3OH).

4.2.1.4 Sodium chloride (NaCl). analytically pure.

4.2.2 Reagent preparation

4.2.2.1 0.1% formic acid aqueous solution. take 1.0 mL of formic acid, dilute to 1000 mL with water, and mix.

4.2.2.2 Methanol aqueous solution. Take 800 mL of methanol, add.200 mL of water, and mix well.

4.2.3 Standard products

4.2.4 Standard solution preparation

4.2.4.1 Standard stock solution (about 1.0 mg/mL). accurately weigh tetracycline, oxytetracycline, chlortetracycline, minocycline, doxycycline, About 10.0 mg of erythromycin, cefradine, sulfadiazine, ofloxacin, norfloxacin, clindamycin, and chloramphenicol in a 10 mL volumetric flask, Dissolve it with methanol and make it to the mark, prepare a standard stock solution with a concentration of approximately 1.0 mg/mL, and store it in a frozen state at -20°C±2°C. The validity period is 3 months.

4.2.4.2 Mixed standard intermediate solution (10 µg/mL). accurately draw appropriate amounts of each single standard stock solution into a 25 mL volumetric flask, and use A Dilute with alcohol and make the volume up to the mark, prepare a mixed standard intermediate solution with a concentration of 10 µg/mL, store it in cold storage and avoid light, and the validity period is 1 month.

4.2.4.3 Internal standard compound standard stock solution (about 1.0 mg/mL). Weigh tetracycline-D6 and oxytetracycline-C22- respectively N2, minocycline-D6, doxycycline-D3, sulfadiazine-D6, cephalexin-D5, levofloxacin-D8, erythromycin-C-D3, Thiabendazole-D4, chloramphenicol-D5 about 10.0 mg in a 10 mL volumetric flask, dissolved in methanol and dilute to the mark, Prepare an internal standard stock solution with a concentration of about 1.0 mg/mL, at -20°C Frozen storage at ±2°C, valid for 6 months.

4.2.4.4 Standard intermediate solution of mixed internal standard compound. draw appropriate amount of single internal standard stock solution into 10 mL volumetric flask and dilute with methanol and dilute to the mark, prepare a 1 µg/mL mixed internal standard intermediate solution, store in refrigeration and avoid light, the validity period is 3 months.

4.2.4.5 Mixed standard solution. use methanol aqueous solution to prepare the standard intermediate solution of antibacterial drugs into a mixed standard series solution, such as 1 µg/L, 2 µg/L, 5 µg/L, 10 µg/L, 20 µg/L, 50 µg/L, 100 µg/L, the mixed internal standard compound concentration in each series of solutions is 10 µg/L, Ultra performance liquid chromatography-tandem mass spectrometer determination. Now equipped for temporary use.

4.2.5 Materials

Organic filter membrane. pore size is 0.22 µm.

4.3 Apparatus and equipment

4.3.1 Ultra high performance liquid chromatography-tandem mass spectrometer. charged electrospray ion source (ESI source).

4.3.2 Analytical balance. Sensitivity is 0.1 mg.

4.3.3 Centrifuge. speed >=9000 r/min.

4.3.4 Vortex mixer.

4.3.5 Ultrasonic cleaner.

4.4 Analysis steps

4.4.1 Sample preparation

4.4.1.1 Liquid dosage form

Weigh 0.2 g (accurate to 1 mg) of a well-mixed sample, place it in a 15 mL centrifuge tube, and add 10.0 µL of mixed internal standard compound standard Quasi-intermediate solution, add 3.0 mL methanol, vortex for 1 min to disperse the sample, ultrasonically extract for 10 min, add 0.15 g sodium chloride, vortex to mix, Centrifuge at 9000 r/min for 5 minutes, aspirate the supernatant, filter with a membrane, take 0.8 mL of filtrate into the sample bottle, add 0.2 mL of water, and mix well.

For ultra performance liquid chromatography-tandem mass spectrometry determination.

4.4.1.2 Other dosage forms

4.4.1.2.1 Gel dosage form

Weigh 0.2 g (accurate to 1 mg) of a well-mixed sample, place it in a 15 mL centrifuge tube, and add 10.0 µL of mixed internal standard compound standard Quasi-intermediate solution, add 3.0 mL acetonitrile, vortex for 1 min to disperse the sample, ultrasonically extract for 10 min, centrifuge at 9000 r/min for 5 min, and aspirate the supernatant Liquid, filter membrane, take 0.8 mL of filtrate into the sample bottle, then add 0.2 mL of water, mix well, for ultra performance liquid chromatography-tandem mass spectrometry determination.

4.4.1.2.2 Cream dosage form

Weigh 0.2 g (accurate to 1 mg) of a well-mixed sample, place it in a 15 mL centrifuge tube, and add 10.0 µL of mixed internal standard compound standard Quasi-intermediate solution, add 3.0 mL methanol, vortex for 1 min to disperse the sample, ultrasonically extract for 10 min, centrifuge at 9000 r/min for 5 min, and aspirate the supernatant Liquid, filter membrane, take 0.8 mL of filtrate into the sample bottle, add 0.2 mL of water, mix well, for ultra-high performance liquid chromatography-tandem mass spectrometry determination.

4.4.2 Sample determination

4.4.2.1 Reference conditions for ultra performance liquid chromatography

The reference conditions of ultra performance liquid chromatography are as follows.

a) Chromatographic column. C18 column (column length 100mm, inner diameter 2.1mm, particle diameter 1.8µm), or equivalent;

b) Mobile phase. Phase A is 0.1% formic acid aqueous solution, Phase B is acetonitrile;

c) Flow rate. 0.3 mL/min;

d) Column temperature. 40°C;

e) Injection volume. 5 µL;

f) Gradient elution procedure. see Appendix B.

4.4.2.2 Mass spectrometry reference conditions

The mass spectrometry reference conditions are as follows.

a) Ionization method. ESI, ESI-scan at the same time;

b) Ion source temperature. 550 °C;

c) Capillary voltage. ESI. 5.5 kV; ESI-. -4.5 kV;

d) Curtain air. 2.1 × 105 Pa;

e) Atomization gas. 4.5 × 105 Pa;

f) Auxiliary gas. 4.5 × 105 Pa;

g) Monitoring mode. multiple reaction monitoring (MRM) mode;

h) Other mass spectrometry parameters. see Appendix C.

4.4.2.3 Preparation of standard curve

The mixed standard series solutions were injected into the ultra performance liquid chromatography-tandem mass spectrometer in order from low to high concentration, and the corresponding peak surface was measured.

The ratio of the quantitative ion peak area of the measured component to the quantitative ion peak area of the internal standard compound is compared with the corresponding mass concentration to make a standard curve.

The sub-pair is qualitative, and the internal standard method is used for quantitative analysis. See Appendix D for the standard map.

4.4.2.4 Qualitative determination

Under the same test conditions, the retention time of the target compound chromatographic peak in the sample is compared with the retention time of the corresponding standard chromatographic peak.

The chemical range should be within ±2.5%, and the detected relative ion abundance should be consistent with the relative ion abundance in a standard solution of equivalent concentration.

The relative abundance ratio deviation should meet the requirements of Table 1.

Table 1 Maximum allowable deviation of relative ion abundance

Relative ion abundance/% >50 >20~<=50 >10~<=20 <=10 Allowable relative deviation/% ±20 ±25 ±30 ±50

4.4.2.5 Quantitative determination

This method uses internal standard method for quantification. The ordinate is the ratio of the area of the quantitative ion peak of the measured component to the area of the quantitative ion of the internal standard compound (fourth Cyclin and chlortetracycline use tetracycline-D6 as internal standard, oxytetracycline uses oxytetracycline-C22-N2 as internal standard, and minocycline uses minocycline-D6 as internal standard.

Doxycycline uses doxycycline-D3 as internal standard, erythromycin uses erythromycin-C-D3 as internal standard, cefradine uses cephalexin-D5 as internal standard, sulfa Pyrimidine uses sulfadiazine-D6 as internal standard, ofloxacin and norfloxacin uses levofloxacin-D8 as internal standard, clindamycin uses thiabendazole-D4 as internal standard Standard, chloramphenicol with chloramphenicol-D5 as the internal standard), and the abscissa is the mass concentration of the tested component in the sample.

4.4.2.6 Determination

Inject the sample solution into the ultra-high performance liquid chromatography-tandem mass spectrometer for retention time and two pairs of ions (characteristic ion pair/quantitative ion pair) the relative abundance of the corresponding chromatographic peak area is qualitative, and the mass concentration of the target compound in the sample solution is calculated according to the standard curve.

4.4.3 Blank test

Except that no sample is added, follow the steps in 4.4.1.

4.4.4 Calculation and expression of results

The content of the target compound in the sample is calculated according to formula (1).

4.4.5 Precision

The absolute difference between two independent determination results obtained under repeated conditions should not exceed 15% of the arithmetic mean. The recovery rate of this method and precision see Appendix E.

4.4.6 Detection limit and quantification limit

When the sampling volume is 0.2 g and the extract volume is 3.0 mL, the detection limit and quantification limit of this method are shown in Appendix F.