Simultaneous Detection Of Retinal And α-tocopherol From Food By Using High Performance Liquid Phase Chromatography

Simultaneous detection of retinal andα-tocopherol from food by using high performance liquid phase chromatographyIntroduceRetinol and Tocopherol are needed lipo-soluble vitamins for body keeping natural body metabolize. As a result of these two vitamins are unstable in outside surrounding, they are liable to light and oxygen, and oxidized and destroyed., there are much affecting factors, so finding a determination method to determine these two vitamins is a difficulty in inspection field. Now, the standard method cost much more time and the method is fussy, possoblely led to lipo-soluble vitamins losing, and the recovery is too low. We set up a Simultaneous detection of retinal andα-tocopherol from food by using high performance liquid phase chromatogra-phy method which can quickspeed, delicacy, accuracy in the text. And its sample handling is easy, handy, we detection of retinal andα-tocopherol from baby food and healthy food selling in Anshan by using this method, obtaining good results.Material and method一,Material(一) Mostly instrument and reagentAgilent 1100 HPLC, DAD, chromatogram column: Supelco C 18, 15cm×2.1mm, 5μm(Agilent)ultrasonic launder, N-EVAP TM 112 N₂ blowing instrument, LXJâ…¡centrifugal machine, 0.45μm organic needle cylinder filtration filter, ultrapure water system(China Beijing), retinal andα-tocopherol standard sample,＞99ï¼…, (Sigama)(二) Sample sourceRandom baby-food and healthy-food sample selling in Anshan 二,Methodfood samples were added in anhydrous alcohol, potassium hydroxide and pyro-gallic acid. The mixture was shaken and saponified at 80℃for 30 minutes. The lipo-soluble vitamins were extracted by petroleum ether. The organic layer was dried by gas of N₂, dissolved by mobile phase, and analyzed by HPLC. The HPLC system consisted of Supleco C 18 column(10cm×2.1mm, 5×μm), mobile phase of methanol: water(93:7), flow rate:0.4ml/min, column temperature 40℃, wavelength of scan from 210nm to 370nm, DAD detector at 325nm of retinal and 294nm ofα-tocopherol.Experiment results一,Retinol and Tocopherol determination relativity studyRetinol's linearity is good in 1.0～100.0mg/L, r=0.9998; and Tocopherol's linearity is good in 0.10～10.0mg/L, r=0.9991.二,Retinol and Tocopherol determination reclaiming rate experimentRetinol and Tocopherol determination reclaiming rate＞96ï¼…using the method, effect is good.三,Retinol and Tocopherol precision determinationUsing one sample in the precision determination, repeat five times,effect is good.å››,High performance liquid phase chromatography-DAD comparing with standard methodWeighting Wondersun milk powder five lot, determine using the method and standard method GB/T5009.82-2003, the results have no significance difference.五,Using high performance liquid phase chromatography -DAD evaluate sample selling in Anshan.Sampling baby-food and healthy-food in Anshan 30 lot, determine using the method and standard method GB/T5009.82-2003, judge by standard and sample marker, Va and Ve's eligibility rate is 100ï¼…. Discussion一,Chromatogram condition selectionUsing methanol, methanol: water, methanol: acetic acid: water verified NH4Ac liquor and methanol in varied proportion, proved by experiments, under methanol: water (93:7), are separated on base line, in 0.2ml/min～2.0ml/min, change velocity of flow, in 0.2ml/min～0.8ml/min, lipo-soluble vitamins peak time is appropriate, peak is good, and has low disturbtion. Scanning with ultraviolet spectrum instrument, Va at 325nm, Ve at 294nm, have biggest absorb. DAD's ultraviolet spectrum approved it. Ensuring DAD wavelength containing it, we use wavelength in 210～370nm. We use sampling at 5μl, result is good.二,Sample pre-deal condition selectionCompare with verified saponification and concentration, we choose 1:1KOH liquor 20ml, high temperature got high saponification effect, so we choose 80℃water bath,30min to get whole saponification effect. Compare with verified antioxidant such as BHA, hydroquinone, pyro-gallic acid, we found that adding 16g/L pyrogallic acid liquor, can protect effectively lipo-soluble vitamins to be un- destroyed.三,Retinol and Tocopherol determination relativity studyRetinol's linearity is good in 1.0～100.0mg/L, r=0.9998; and Tocopherol's linearity is good in 0.10～10.0mg/L, r=0.9991。四,Retinol and Tocopherol precision determination and reclaiming rate experimentUsing one sample in the precision determination, repeat five times, effect is good.Retinol and Tocopherol determination reclaiming rate＞96% using the method, effect is good.五,High performance liquid phase chromatography-DAD comparing with standard methodWeighting Wondersun milk powder five lot, determine using the method and standard method GB/T5009.82-2003, the results have no significance difference.å…­,Using high performance liquid phase chromatography -DAD evaluate sample selling in Anshan.Sampling baby-food and healthy-food in Anshan 30 lot, determine using the method and standard method GB/T5009.82-2003, judge by standard and sample marker, Va and Ve's eligibility rate is 100ï¼….ConclusionUsing high performance liquid phase chromatography-DAD, sample dealing is easier, the method is simpler, speeder, veracity, and stability are higher, reclaiming rate is higher, compare with standard method, saving determination cost, so the method suits spread and actualize.