GB/T 40267-2021 PDF in English
GB/T 40267-2021 (GB/T40267-2021, GBT 40267-2021, GBT40267-2021)
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Determination of Levodopa in plant derived products - High performance liquid chromatography
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Standards related to (historical): GB/T 40267-2021
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GB/T 40267-2021: PDF in English (GBT 40267-2021) GB/T 40267-2021
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 07.080
CCS A 40
Determination of Levodopa in Plant Derived Products
- High Performance Liquid Chromatography
ISSUED ON: MAY 21, 2021
IMPLEMENTED ON: DECEMBER 1, 2021
Issued by: State Administration for Market Regulation;
Standardization Administration of the People’s Republic of
China.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative References ... 4
3 Terms and Definitions ... 4
4 Principle ... 4
5 Reagents and Materials ... 4
6 Instruments and Equipment ... 6
7 Sample ... 6
8 Test Procedures ... 6
9 Test Data Processing ... 7
10 Precision ... 8
11 Others ... 8
Appendix A (informative) High Performance Liquid Chromatogram ... 9
Determination of Levodopa in Plant Derived Products
- High Performance Liquid Chromatography
1 Scope
This Standard describes the determination of levodopa content in plant derived
products through the high performance liquid chromatography.
This Standard is applicable to the determination of levodopa content in plant derived
dry samples, such as: Vicia faba L., Stizolobium cochinchinensis (Lour.) Tang et Wang
and Mucuna sempervirens Hemsl., etc.
2 Normative References
The content of the following documents constitutes indispensable clauses of this
document through normative references in the text. In terms of references with a
specified date, only versions with a specified date are applicable to this document. In
terms of references without a specified date, the latest version (including all the
modifications) is applicable to this document.
GB/T 6682 Water for Analytical Laboratory Use - Specification and Test Methods
3 Terms and Definitions
There are no terms and definitions that need to be defined in this document.
4 Principle
After the specimen is ultrasonically extracted with 2% (volume fraction) hydrochloric
acid solution, it is separated through reversed-phase chromatographic column and
detected by ultraviolet (or diode array) detector. It is qualitatively determined by
retention time and quantitatively determined by the external standard method.
5 Reagents and Materials
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.
6 Instruments and Equipment
6.1 High performance liquid chromatograph: equipped with ultraviolet detector (UV) or
diode array detector (DAD).
6.2 Analytical balance: with a division value of 0.01 mg and 0.0001 g.
6.3 Ultrasonic generator: with a power of 250 W and a working frequency of 40 kHz.
6.4 Centrifuge: with a rotating speed of not less than 8,000 g.
6.5 Acidity meter: with an accuracy of 0.01 pH unit.
6.6 Vortex mixer.
6.7 Electric pulverizer.
7 Sample
Take about 250 g samples of Vicia faba L., Stizolobium cochinchinensis (Lour.) Tang
et Wang and Mucuna sempervirens Hemsl.; use the electric pulverizer to smash them;
let them pass through a sieve with an inner diameter of 355 μm ± 13 μm. Mix the
prepared specimen and equally divide it into two parts. Put it into a clean specimen
container; seal and label it. Store it at room temperature. Keep it sealed and protected
from light.
8 Test Procedures
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
( 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 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.
9 Test Data Processing
The levodopa content in the specimen shall be calculated in accordance with Formula
(1):
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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