| ObjectiveTo establish a high performance liquid chromatography of detecting deoxidized VC and total VC in foods with a convenient,rapid,sensitive and accurate.Using this qualitative and quantitative method detected deoxidized and total VC in the foods such as beverages,fruits,vegetables and so on that abundant in VC.Researching the stability of VC when it is destroyed with different effects,to remind people eating foods that rich in VC reasonably in daily life and supply basement for eating scientifically.On the basis of previous study,an HPLC method using water-methanol as mobile phase to determine 5 kinds of water-soluble vitamins including VC,B1,B2,B3 and B6 was established. With this qualitative and quantitative method to analyse water-soluble vitamins in beverages and multivitamin tablets that rich in water-soluble vitamins accurately and rapidly.Method1.The condition of chromatographic analysis1.1 The determination of VCReversed phase high-performance liquid chromatography(RP-HPLC), Waters 515 Chromatograph of High-performance Liquid,Waters 2996 Diode Array Detector,Atlantis dC18column,and mobile phase 0.1%acidum aceticum water solution.The deoxidized VC was detected directly without deoxidizing sample and totle VC with DTT.To survey and evaluate total VC that deoxidized by DTT.Flow rate 1.0 ml/min;wave length of detection 245nm,sample size 10μL.Column temperature 30℃.1.2 The determination of water-soluble vitaminsReversed phase high-performance liquid chromatography(RP-HPLC), LC-10AVP Chromatograph of High-performance Liquid,UV Detector,Atlantis dC18column,and mobile phase 0.2%acidum aceticum water solution and methanol.Linear gradient elution is employed.Sample size 10μL.Column temperature 30℃.2.Deoxidize ability and stable time of reducing agent DTTAdjust power of hydrogen to 4.5～6 with standard solution of ascorbic acid. Then descend its concentration to 10%of stock solution by direct radiation of sunlight(used that day).Using the above oxidative ascorbic acid,reacting with the same volume of DTT in dark environment room temperature.Inject samples and detect with a certain time cell.3.Sample treatment3.1 BeveragesDilute beverages to a certain concentration with 0.01 mol/L HCl,then filter with 0.45μm filter membrane and inject samples to analyse.3.2 Fruits and vegetablesCut samples of fruits and vegetables into pieces,mix completely,weigh sufficient quantum samples,add in about 25mL 5 mmol/L KH2PO4 solutions, homogenize with super-speed disperser,then shift with fixed quantity to 50mL volumetric flask,metered volume with 5 mmol/L KH2PO4 water-solution,and then take 5.00mL to centrifuge at 4000rpm for 10min and the supernatant was stored at 4℃for analysis later.3.3 Vitamin tablet10 pills of Multivitamin tablets is grinded and sufficient quantum samples is weighed to 25mL volumetric flask,20mL of 0.01 mol/L HCl is added,sonic boom for 30min,then add to graduation line.Taking out 1mL from it,filter with 0.45μm filter membrane,then analysis.4.The determination of AA and total TAADilute sample solution of deoxidized ascorbic acid with 0.01 mol/L HCl to a certain concentration,then filter with 0.45μm filter membrane and inject samples to analysis.Dilute sample solution of total ascorbic acid with water to a certain concentration,adjust power of hydrogen to 4.5～6.0 before metered volume,shake equal,filter with 0.45μm filter membrane.Take filter liquor 200μL,adding in DTT at the same volume,vortex,react 20min in dark at room temperature,and then get out 10μL samples to inject and analysis.5.Accuracy and precision of the method5.1 Calibration curve:Prepare the standard curves of 5 kinds of vitamins,and observe their correlation coefficients and linear ranges;Dilute the minimum concentration of the standard series,then analyse,to obtain the detection limits of vitamins.5.2 Recovery and its precisionTwo concentration levels of standard solutions are employed to do recover experiments.To determine 5 times a day and continues for 5 days,calculate the recovery and its precision.6.Stability experiment6.1 Stability experiment at 4℃:prepare the standard solutions of various kinds of water-soluble vitamins and store at 4℃for 24h.6.2 The stability of VC:In various kinds of experiments conditions,to study the oxidized extent of deoxidize VC and the changes of total VC,approaching the effects of time,temperature,irradiation of ultraviolet radiation,alkaline condition,oxidant and cane sugar. Results:1.Sample treatment and the condition of chromatographic analysis:the method of sample treatment is simple;the retention time of detected component is short, the analysis can be over within 17 minutes,and the separation with the sample matrix is good at the chromatographic condition of this experiment.2.The deoxidized ability and stable times of reductant DTT:The sample matrix has no effect on deoxidized ability of DTT,the proportion of hydrogenising AA to DHAA are all exceeding 95%at the condition of this experiment;and keep stable in 2 hours.3.Evaluation of the accuracy and precision:①linear range of the standard curves are wide,r value 0.9982～0.9998,the detection limit of deoxidized ascorbic acid is 0.05μg/mL,totle VC is 0.1μg/mL;the other 4 kinds water-soluble vitamin is in 0.005μg/mL～0.05μg/mL②recovery rate of sample during 90.0%～108.7%;③intra-day RSD is 1.8%～4.8%,and inter-day RSD is 4.6%～8.9%;④the comparison between DTT reduction-high performance liquid chromatography and fluorometric method for detecting of total ascorbic acid:with matched-pairs T-test,t=-0.691(P＞0.50),according to the level ofα=0.05,the difference between the two methods has no statistically significant.4.The stability experiment:①5 kinds of water-soluble vitamins are stable in diluted hydrochloric acid at 4℃for long time;②When the temperature of heating is not to high(60℃),AA mainly transforms to DHAA,while at a higher temperature(100℃),AA mainly hydrolyze to 2,3-diketogulonic acid.And with the time of the heating prolong the proportion increased;the low temperature(4℃)has small effect.Ultraviolet,alkalinity environment and oxidation are all having an effect on the stability of ascorbic acid. ConclusionThis experiment method established for detecting deoxidized VC,total VC and 5 kinds of water-soluble vitamins simultaneouly with high performance liquid chromatography is convenient,rapid,and can be used for detecting many kinds of water-soluble vitamins that existence in foods and health care products;The food that rich in VC should avoid heating in high temperature,insolation in sunlight,treatment in alkaline condition and using oxidant;various kinds of water-soluble vitamins can be preserved at low temperature for long time.
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