GB/T 40267-2021Determination of Levodopa in plant derived products—High performance liquid chromatography (English PDF)

5.1 Reagents

Unless it is otherwise specified, the reagents used by this Method shall be analytically pure. The water shall be Grade-1 water specified in GB/T 6682.

Microporous membrane (aqueous phase): with a pore size of 0.45 µm.

8.1 Specimen Processing

Weigh-take 1 g of specimen (accurate to 0.001 g) into a 50 mL centrifuge tube with stopper. Add 10 mL of 2% (volume fraction) hydrochloric acid solution. After vortex mixing, ultrasonically extract it for 30 min; every 15 min, shake it once. Then, at 8,000 (x g), centrifuge for 5 min; transfer the supernatant to a 25 mL volumetric flask. Use 10 mL of 2% (volume fraction) hydrochloric acid solution to re-extract the residue.

Combine the two extracts. Then, use 2% (volume fraction) hydrochloric acid solution to reach a constant volume; shake it well. Let it pass through a 0.45 µm microporous membrane; reserve it for later test.

8.2 Determination

8.2.1 Liquid chromatography reference conditions

The liquid chromatography reference conditions are shown below:

a) Chromatographic column: C18 chromatographic column, 4.6 mm x 250 mm, particle size 5 µm, or others with equivalent performance;

b) Mobile phase: methanol-10 mmol/L dipotassium hydrogen phosphate solution = 5:95;

c) Flow rate: 0.7 mL/min;

d) Column temperature: 20 °C;

e) Injection volume: 10 µL;

f) Detection wavelength: 280 nm.

8.2.2 Drawing of standard working curve

In accordance with the levodopa content in the specimen, select the standard working solution with an appropriate range of mass concentration. From low concentration to high concentration, successively inject specimen for analysis. Take the chromatographic peak area of levodopa as the y-coordinate; take the mass concentration of the solution as the x-coordinate; draw the standard working curve.

The chromatogram of the standard working solution is shown in Figure A.1 of Appendix A.

8.2.3 Sample determination

Inject the specimen solution into the high performance liquid chromatograph. Conduct qualitative determination by the retention time; record the peak area. In accordance with the standard working curve, obtain the mass concentration of levodopa in the specimen solution. The mass concentration of levodopa in the specimen solution shall be within the range of mass concentration of the standard working curve. Samples that exceed the upper limit of the mass concentration of the standard working curve shall be diluted and injected for analysis. The chromatogram of a typical specimen solution is shown in Figure A.2.

8.3 Parallel Test

In accordance with the stipulations of 8.1 and 8.2, conduct two parallel determinations on the same specimen.