Determination of Vitamin E by HPLC

Determination of Vitamin E by HPLC1.Introduction1.1 Chemical molecular formula3H 33Vitamin E 35231O H C Mol.Wt: 472.75 1.2 Properties of vitamin ECharacters:Vitamin E is a fat soluble vitamin that is essential for the maintenance of normal metabolism and function of the body.Vitamin E is light yellow to yellow or yellow green clear viscous liquid; it is almost odorless;once it is illuminated, its color turns deep;it is solified in nature;it melts at about 25℃.Solubility:Vitamin E is soluble in alcohol, acetone, ether, or vegetable oil,but not in water.Absorbance index:Take this product, weigh it precisely, add ethanol to make it dissolved and diluted into quantitative solution 0.1mg/mL; according to the UV Visspectrophotometry, the absorption coefficient () is 41.0 ~ 45.5 at 284nmwavelength.1.3 Method of the determination from the CP2015Method:Determination of gas chromatographic method (general rules 0521). Chromatographic conditions and system suitability method:Place silicone (OV-17) as the stationary liquid, the coating concentration of packed column 2%, or place 100% methyl polysiloxane as stationary liquid capillary column; control column temperature at 265℃. The number of theoretical plates by vitamin E peak is not less than 500 (packed column) or 5000 (Mao Xiguanzhu); the separation between vitamin E peak and internal standard material peak should meet the%1cm 1Erequirements.Determination of correction factor:Take n-dotriacontane properly, add n-hexane to dissolve and dilute it into 0.1mg/mL; consider it as the internal standard solution. Take another vitamin E about 20mg, weighing accurately, put into the brown stoppered bottle.Add internal standard solution 10ml precisely, plug, shake to dissolve;consider it as the reference solution. Take 1 ~ 3μL, injected into gas chromatography,calculate the correction factor. Determination method:Take the product about 20mg,weighing precisely, put into the brown stoppered bottle, adding internal standard solution 10ml precisely, plug, shake to dissolve;consider it as the test solution; inject 1 ~ 3μL into gas chromatography, determine and calculate. 1.4 Method of the determination from the USP36-NF31Internal standard solution: 10 mg/mL of squalane in cyclohexaneSystem suitability solution: 0.1 mg/mL each of USP Al-pha Tocopherol RS and USP Alpha Tocopheryl Acetate RS in cyclohexaneStandard solution: 10 mg/mL of USP Alpha Tocopherol RS in Internal standard solutionSample solution: 10 mg/mL of Vitamin E in Internal standard solution Chromatographic system(See Chromatography<621>,System Suitability.)Mode: GCDetector: Flame ionizationColumn:0.25mm*30m fused silica capillary,bonded with a 0.25μm film of phase G2 Temperature:Column: 280℃Injection port: 290℃Detector: 290℃Carrier gas:HeliumFlow rate:1 mL/minSplit ratio:100:1Injection size:1μLSystem suitabilitySamples:System suitability solution and Standard solutionSuitability requirementsResolution:NLT 3.5 between alpha tocopherol and alpha tocopheryl acetate, System suitability solutionRelative standard deviation:NMT 2.0% for ratios of alpha tocopherol to internal standard peak responses from replicate injections, Standard solution2.Method and Materials2.1Instrument & AapparatusChromatograph:Agilent 1200 (N2000 chromatographic work station)Column: C18(ODS); 4.6×150mm,dp5µmMicroinjector:100μL2.2 ReagentsMethanol(023060401,Cinc High Purity Solvents (Shanghai)Co.,Ltd.)Acetone(1503043870,Nanjing Chemical Industrial Park)Vitamin E soft capsules(140624,Zhejiang Medicine Co.,Ltd. Xinchang Pharmaceutical Factor,100mg/grain,H20003539)Standard vitamin E(National Institute for the Control of Pharmaceutical and Biological Products,99.24%)2.3 ConditionMobile phase: MeOH(methanol)Stationary phase: ODS(C18)Flow rate: 1.5 mL/minWavelength: 284nmColumn temperature: Room temperatureInjection volume: 20µL（60 ~ 80µL）2.4 Sample preparationSample solution:Take one Vitamin E soft capsule,peel off the capsule,pour the contains into 10mL volumetric flask,dissolved into acetone,constant volume to 10mL. Take 1mL,diluted in the 50mL volumetric flask with methanol,shake todissolve;consider it as sample solution.Standard solution:0.2mg/mL3.ResultThe chart of standard solution’s chromatographic peak:The chart of sample solution’s chromatographic peak:4.CaculationContent(%)=C R ×A X ×Dilute multipleA R ×M×100%R C : concentration of standard solution R A : area of vitamin E standard solution X A : area of vitamin E sample solutionM : labeled amount of vitamin E%67.93%1001007521675010706818/2.0%100(%)=⨯⨯⨯⨯⨯=⨯=⨯⨯⨯mgmLmL mg Content MA ipleDilutemult A C R X R5.Disscusion5.1 The resolution(R) is much more than 1.5.The separation result is very good.Hovever,at 284nm the UV absorption of acetone is too much. 5.2 Due to standard solution ’s long existence,the first peak is not acute. 5.3 Comparison between this method and the method from pharmacopoeiaReferences[1]Chinese Pharmacopoeia Commission..Chinese Pharmacopoeia(second part).Beijing:Chemical Industry Press,2015.[2]United States Pharmacopoeia Convention..United States Pharmacopoeia(Official Monographs / Vitamin).Chemical Industry Press,2013.。