A.1 General requirements
Unless otherwise stated, the analysis using only the reagents were of analytical grade and recognized GB / T 6682-2008 specified water. Analysis in the standard titration solution, impurities measured by standard solution, preparations and products, while other requirements not specified, according to GB T 601, GB / T 602, the provisions of the preparation / GB / T 603 used. The test used in the preparation of the solution does not indicate what kind of solvent used, refer to the aqueous solution. A.2 Identification Test
A.2.1 weighed amount of sample, the sample solution prepared 13.3mg/mL, 3mL sample was weighed, placed in a 15mL tube, prepared at concentrations latest 3mL of 100 mg / mL solution of catechol, mixed uniform. Then add 6mL sulfuric acid, and mix. The tube is heated in the flame slowly 30s, was dark pink or red wine.
A.2.2 D-mannitol in the determination of the test, the peak retention time and peak chromatogram of the standard solution of the sample liquid chromatographic retention time consistent with FIG.
A.3 D-mannitol Determination A.3.1 A.3.1.1 reagents and materials sorbitol standards. A.3.1.2 D-mannitol standard. A.3.2 instruments and equipment
HPLC: equipped with a differential refractive index detector, or other equivalent detector. A.3.3 reference chromatographic conditions
A.3.3.1 Column: 10cm × 7.8mm column packing L34 (U.S. Bio-Rad Laboratories), or equivalent thereof; or other equivalent column.
A.3.3.2 mobile phase: degassed water. A.3.3.3 column temperature: (50 ± 2) ℃, constant temperature. A.3.3.4 detector temperature: 35 ℃. A.3.3.5 mobile phase flow rate: approximately 0.7mL/min. A.3.3.6 injection volume: about 10μL.
A.3.3.7 relative retention time of sorbitol is 0.6, the relative retention time of D-mannitol is 1.0. A.3.4 analysis step
A.3.4.1 Preparation of the solution separated
Weighed amount of standard sorbitol and D-mannitol standard, were prepared at a concentration of 4.8mg / g of sorbitol solution and D-mannitol was added.
A.3.4.2 Preparation of the standard solution
Weighed amount of D-mannitol standard, formulated at a concentration of 4.8mg / g of D-mannitol standard solution. A.3.4.3 Preparation of test solution
Weighed amount of sample, prepared at a concentration of 5 mg / g of the sample solution. System suitability test A.3.4.4
A.3.4.4.1 adaptability 1
Solution for chromatography separation, sorbitol and D-mannitol separation R ≥ 2.0. A.3.4.4.1 adaptability 2
Chromatographic analysis of the standard solution, the results of repeated injections of the relative standard deviation of the detected ≤ 2.0%.
A.3.4.5 Determination
In A.3.3 reference chromatographic conditions, respectively, the standard solution and the sample solution was measured, the injection volume was 10 μL, repeat injections once, calculate its main peak response average.
A.3.5 calculated results
The content of D-mannitol by the formula X1 (A.1) calculated: () () w
rrCCXSUUS – × × = 10010000 1 …………………………… (A.1)
The formula:
X1 – sample D-mannitol content,%;
CS – concentration of the standard units of milligrams per gram (mg / g); CU – the concentration of the sample solution, in milligrams per gram (mg / g); rU – the main sample liquid chromatogram peak response mean; rS – standard solution chromatogram main peak response mean;
w – Loss on drying the sample measured in units of grams per hundred grams (g/100g). The results parallel arithmetic mean of the measurement results shall prevail.
A.4 pH measurement
Weighed amount of sample, the preparation of the mass fraction of 10% of the sample solution, the solvent is carbon dioxide-free water and then measured with a pH meter pH.
A.5 Determination of reducing sugars
3.3g sample was accurately weighed, was added 25mL of water in the sample was slowly dissolved by heating. The solution was cooled, added to 20mL of test solution and an alkaline copper citrate few glass beads. The solution was heated, 4min The solution began to boil and keep boiling 3min. After rapid cooling, dilute acetic acid test solution and 100mL 20.0mL0.025mol / L iodine titration [Note: The dilution 0.05mol / L iodine titration solution 1:1 with water]. Shaking side solution, the mixed solution was added 25mL (6mL hydrochloric acid and 94mL of water). When the precipitate was dissolved with 0.05mol / L sodium thiosulfate solution [NOTE: 0.1mol / L sodium thiosulfate solution diluted with water 1:1] titration of excess iodine. At the endpoint, adding 2mL starch test solution, as the display agent. 0.05mol / L sodium thiosulfate solution required for the titration volume ≥ 12.8mL.