This standard specifies the HPLC determination method for thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid, nicotinamide and caffeine in health foods. This standard is applicable to the HPLC determination method for thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid, nicotinamide and caffeine as dietary supplement additives added to tablets, capsules, oral liquids, beverages and other sample types. GB/T 5009.197-2003 Determination of thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid, nicotinamide and caffeine in health foods GB/T5009.197-2003 Standard download decompression password: www.bzxz.net

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National Standard of the People's Republic of China
GB/T5009.197--2003
Determination of thiamine hydrochloride,pyridoxine hydrochloride,niacin,niacinamide and caffeine in health foods
Determination of thiamine hydrochloride,pyridoxine hydrochloride,niacin,niacinamide and caffeine in health foods2003-08-11Promulgated
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
2004-01-01Implementation
This standard is proposed and managed by the Ministry of Health of the People's Republic of China. This standard is drafted by the Food Hygiene Supervision and Inspection Institute of the Ministry of Health. The main drafters of this standard are Yang Dajin, Fang Congrong and Wang Zhutian. GB/T5009.197—2003
GB/T5009.197—2003
Thiamin hydrochloride, pyridoxine hydrochloride, niacin, niacinamide and other B vitamins are important nutrients that are indispensable to the human body and play an important auxiliary role in the prevention and treatment of various diseases. At present, B vitamins and caffeine have been added to health foods as effective ingredients. Based on the vitamin detection method of Taisho Pharmaceutical Co., Ltd. in Japan and combined with the specific instrumental analysis conditions in my country, this method has been developed to determine the content of B vitamins and caffeine such as thiamin hydrochloride, pyridoxine hydrochloride, niacin, niacinamide and other B vitamins added to health foods such as tablets, capsules, oral liquids and beverages by high performance liquid chromatography. 558
1 Scope
Determination of thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid, nicotinamide and caffeine in health foods
GB/T5009.197—2003
This standard specifies the HPLC determination method for thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid, nicotinamide and caffeine in health foods.
This standard is applicable to the HPLC determination method for thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid, nicotinamide and caffeine added as dietary supplements in tablets, capsules, oral liquids, beverages and other sample types. The detection limits of this method are: thiamine hydrochloride 0.02ug, pyridoxine hydrochloride 0.02μg, nicotinic acid 0.10μg, nicotinamide 0.06ug, caffeine 0.05μg. Linear range: Thiamine hydrochloride 0.500ug/ml~50.0μg/ml, pyridoxine hydrochloride 0.500#g/mL~50.0μg/ml, nicotinic acid 1.00μg/ml~100μg/ml; nicotinamide 1.00μg/ml~100μg/mL; caffeine 5.00±g/mL~500μg/mL.
2 Principle
Use methanol decahydrate decaphosphoric acid = 100+400+0.5 to extract and dilute the crushed and mixed tablets, capsules or liquid samples, and perform qualitative and quantitative detection according to the external standard method of HPLC UV detector. 3 Reagents
Unless otherwise specified, the water used in the analysis is double distilled water. 3.1 Methanol: high-grade pure.
3.2 Ethylene: chromatographic pure.
3.3 Phosphoric acid: analytical pure.
Sodium dodecyl sulfate: Reagent for high performance liquid chromatography. 3.5 Sodium 1-decanesulfonate: Reagent for high performance liquid chromatography. 3.6 Standard stock solution A: Accurately weigh 0.0500g of thiamine hydrochloride and pyridoxine hydrochloride standards, which have been deducted from the water content, dissolve in water and make up to 50mL.
3.7 Standard stock solution B: Accurately weigh 0.0400g of nicotinamide and nicotinic acid standards, which have been deducted from the water content, dissolve in water and make up to 20mL.
3.8 Mixed standard solution: Accurately weigh 0.0500g of caffeine dried at 80℃ for 4h, accurately add 5mL of standard stock solution A, 10mL of standard stock solution B, and then add methanol + water + phosphoric acid = 100+400+0.5 mixed solution to 20mL. 4 Instruments
High performance liquid chromatograph: with UV detector. 4.2 Ultrasonic cleaner.
4.3 Centrifuge.
5 Analysis steps
5.1 Sample treatment
5.1.1 Grind or mix more than 20 tablets or capsules, weigh quantitative samples in a test tube (accurate to 0.001g), add methyl alcohol + water + phosphoric acid = 100 + 400 + 0.5 mixed solution, so that the concentration is about 0.25mg of thiamine hydrochloride and pyridoxine hydrochloride, 1tmg of nicotinic acid and nicotinamide per milliliter. After ultrasonic extraction for 5 minutes, centrifuge at 3000r/min for 5 minutes, and filter the supernatant through a 0.45um filter membrane before injection.
5.1.2 Liquid samples are directly diluted with a mixed solution of methanol + water + phosphoric acid = 100 + 400 + 0.5, and after sufficient mixing, filtered through a 0.45μm filter membrane before injection.
5.2 Chromatographic conditions
5.2.1 Chromatographic column: TSKCs4.6mmX150mm, 5μm. 5.2.2 Column temperature: room temperature.
5.2.3 Detection wavelength: 260nm for thiamine hydrochloride; 280nm for caffeine, nicotinic acid, nicotinamide, and pyridoxine hydrochloride. 5.2.4 Mobile phase: Thiamine hydrochloride: sodium lauryl sulfate solution (5g530mL) + acetonitrile + phosphoric acid = 530 + 470 + 1 Caffeine, nicotinic acid, nicotinamide, pyridoxine hydrochloride: sodium 1-decanesulfonate solution (1.22g→850mL) + acetonitrile + phosphoric acid = 850 + 150 + 1. 5.2.5 Flow rate: mL/min.
5.2.6 Injection volume: 10μl.
5.3 Chromatogram
-Thiamine hydrochloride.
Figure 1 Chromatogram of thiamine hydrochloride
-Caffeine,
-Nicotinic acid:
-Nicotinamide;
4--Pyridoxine hydrochloride.
Figure 2 Chromatograms of caffeine, nicotinic acid, nicotinamide, hydrochloric acid, and pyridoxine. Under the chromatographic conditions, the retention time of thiamine hydrochloride is 14.305min, the retention time of pyridoxine hydrochloride is 18.512min, the retention time of nicotinic acid is 8.728min, the retention time of nicotinamide is 9.341min, and the retention time of caffeine is 4.986min. The concentrations are thiamine hydrochloride 8.80μg/ml., pyridoxine hydrochloride 9.60μg/ml, nicotinic acid 23.8μg/mL, nicotinamide 25.0μg/ml, and caffeine 50.4μg/mL. 6 Calculation of results
x=hxcxVx100
hzXm×1000
Wherein:
X--the content of a single component in the sample, in milligrams per hundred grams (or milligrams per hundred milliliters) [mg/100g(mL)]hi——sample peak height or peak area;
-standard solution concentration, in micrograms per milliliter (μg/ml);560
-sample fixed volume, in milliliters (mL);h2--standard solution peak height or peak area,
m——sample volume, in grams (or milliliters) [g(or mL)]. The calculation result shall retain two significant figures.
Precision
GB/T5009.197-2003
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 561
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