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The determination methods of vitamin D mainly include colorimetry, ultraviolet spectrophotometry, gas chromatography, liquid chromatography and thin layer chromatography
.
The official methods selected by AOAC are colorimetry and high performance liquid chromatography
1.
Principle
After the sample is saponified, the unsaponifiable matter is extracted with benzene.
After the benzene is distilled off, the first stage fractionation HPLC is used to fractionate the vitamin D component to remove most of the interfering substances
.
The obtained vitamin D component was used in the second stage analytical HPLC to obtain a sample chromatogram, which was compared with the chromatogram of the vitamin D standard product obtained under the same operating conditions for quantification
2.
Operation steps
(1) Sample saponification and extraction of unsaponifiable matter.
Weigh 1~10g of the crushed sample (with vitamin D content below 2IU) and place it in a saponification bottle, add 40mL 10% pyrogallic acid- ethanol solution and 10mL 0.
9g/ mL KOH solution, equipped with a reflux device, saponified in a boiling water bath for 30 minutes, then cooled to room temperature with flowing cold water, accurately added 100 mL of benzene, stoppered, vigorously shaken for 15 seconds, and then transferred to a 200-250 mL separatory funnel (such as Precipitates remain in the flask.
At this time, there is no need to wash the saponification bottle with benzene), add 50 mL of 1mol/L KOH solution, shake and mix, and let it stand, discarding the water layer
.
Add 50 mL of 0.
(2) Vitamin D fractionation.
Accurately pipette 80mL of the above benzene solution into a round-bottomed flask, evaporate the benzene under reduced pressure at a temperature below 40℃, and accurately add 5mL n-hexane to the resulting residue to dissolve it, and take 4.
5mL into the round bottom flask.
In a 10mL test tube with a stopper, the solvent is evaporated under reduced pressure, and 500uL of acetonitrile - methanol (1:1) solution is accurately added to the residue to dissolve it
.
Accurately draw 200uL of the solution and inject it into the chromatographic column for fractionation
(3) Quantitative decompression of vitamin D.
The solvent in the vitamin D component was distilled under reduced pressure.
The residue was dissolved in 200 uL of 0.
4% isopropanone- n-hexane solution, and 100 uL of it was injected into the analytical column to obtain a sample chromatogram
.
Take 1 mL of the vitamin D standard solution and follow the steps (1) to (3) above to obtain a standard vitamin D chromatogram
.
3.
Result calculation
Where x-the content of vitamin D, IU/100g
h sa ——The peak height or area of vitamin D in the sample chromatogram
h st ——The peak height or area of vitamin D in the chromatogram of the standard solution
c——Concentration of vitamin D standard solution, IU/mL
m——sample mass, g
4.
Tips
(1) The aliquoted vitamin D should be measured after replacing the column within half a day.
If it exceeds half a day, it should be sealed with inert gas for cryopreservation, and then the column should be changed for measurement
.
This method does not distinguish between vitamin D 2 and vitamin D 3.
(2) When washing the unsaponifiable matter extracted from benzene with water, in order to prevent the loss in water due to the formation of colloidal particles, wash with 1mol/L KOH solution, 0.
(3) Pyogallic acid is added as an antioxidant