Vitamin A

Name : WINDA A ENGKESA

JITRO K WELLEM
Vitamin A (C20H30O)
which has a scientific
name or latin name is
Retinol is one of fat-
soluble vitamins or
oil. Vitamin A is
stable against heat,
acid and acting but is
very easily oxidized
by water and will be
damaged at high
temperatures
 The benefits of vitamin A in the body is able
to function in the process of vision, in this
process vitamin A serves as retinal (retinete)
which is a component of vision. In addition to
serving as the process of vision, vitamin A
can also keep the cornea of ​the eye to always
be healthy.
 Metabolically, vitamin A plays a role in
stimulating the synthesis of corticosteroids,
in the process of hydroxylation of
pregnenolone into progesterone, spurring the
change of mevalonate into squalen which is
then converted to cholesterol and as a carrier
of membrane glycoprotein synthesis.
1. Identification of Vitmin A (Pulvis) according to
European Pharmacopoeia p. 2685
Thin-layer chromatography (TLC)

 Test Solution

Put into a 20 ml glass-stoppered test tube to make a

solution equivalent to 17,000 IU of vitamin A. Add ± 20
mg bromelains R, 2 ml water and 150 μl 2-propanol R,
heat and rotate slowly for 2 to 5 minutes above the
waterbath at a temperature of 60 C to 65 C. Cool to
below 30 C and add 5 ml 2-propanol R containing 1 g /
l butylhydroxytoluene R. Shake firmly for 1 minute, let
stand a few minutes then take the supernatant solution
 Standard Solution

Prepare 10 mg / ml of retinol esters CRS

solution (3.3 IU each per microlitre) in 2-
propanol R containing 1 g / l of
butylhydroxytoluene R. Totolkan 3 μl each
solution on the plate. Develop to 15 cm using a
mixture of ether motion phases: cyclohexane
(20:80). Leave it in the open air and check
under UV light 254 nm.
 Procedure

Totolkan 3 μl of solution. Develop up to 2/3

plates using a mixture of mobile phase up to
2/3 plates using a mixture of ether motion
phases: cyclohexane (20:80). Place the plate in
the open air allow it to dry and then inspected
under 254 nm uv. A valid identification when a
chromatogram obtained with standard solution
exhibits a single stain of the ester. The order
of elution from the bottom up is retinol
acetate, retinol propionate and retinol
palimate. The composition of the ester is
determined according to the suitability of the
stains or the major stain of the test solution
obtained from the standard solution
2. . Determination of Vitamin A (Pulvis) Levels
according to European Pharmacopoeia p. 2686
Liquid Chromatography

 Test solution (a)

Provide 50 ml measuring flask, weigh accurately

0.1% and equivalent to 120,000 IU of vitamin A.
Add 20 mg - 30 mg butylhydroxytoluene R, 2.0 ml
aquaest and 0.15 ml 2-propanol R. Heat in water
bat with temperature 60 C - 65 C for 2-5 minutes.
Cool to a temperature below 30 C and add 20 ml
of 0.1 M tetrabutylammonium hydroxide in 2-
propanol. Dissolve for 5 minutes using ultrasonic
pad. Dilute to 50 ml with 2-propanol and
homogenize. Residue can cause a turbid solution.
 Test solution (b)
Add 20 mg-30 mg of butylhydroxytoluene R in
a 50 ml flask. Add 5 ml 2-propanol R, 5 ml test
solution (a) and dilute to 50 ml with 2-propanol.
Homogenize slowly to prevent air bubbles. Filter
first before it is injected.

 Comparative Solution (a)

Enter into a 50 ml volumetric tube of 120 mg
retinol acetate with a heavy insurance of 0.1
percent and then dissolve it with 5 ml of pentaine.
Add 20-30 mg of butylhydroxyltoluene and 20 ml
of 0.1 M tetrabutylamine hydroxide in 2-
propaneol. For 5 minutes place in ultrasonic bath,
then dilute to 50 ml with 2-propanol.
Comparative Solution (b)
Enter 20-30 mg of butylhydroxyltolene into a 50
ml volumetric tube, add 5 ml 2-propanol, 5 ml test
solution (a) and dilute to 50 ml with 2-propanol.
Homogenize carefully to avoid the formation of air
bubbles.

 Chromatographic Implementation Procedure:

- Using stainless steel columns of 0.125 m long

and 4 mm in diameter that have been coated by
octadecyl silica gel (5 μl)

- The mobile phase consists of a mixture of water:

methanol (5: 95)
- The spectrophotometer detector is installed at
325 nm
- An injector loop
 Procedure

Inject a volume amount of the reference solution

(b) in order to obtain uptake between 0.5 to 1.0 at
325 nm. Perform with six injection times and the
standard deviation of the solution is not greater
than 1%.

 Calculate the content of vitamin A by using the

following formula:

 A1 x C x m2

 A2 x m1
 Information:

A1 = The corresponding peak area for all retinol in

the chromatographic file in Test solution (b)

A2 = The corresponding peak area for all retinol in

the chromatographic file in standard solution (b)

C = concentration of retinol acetate with unit UI /

g.

m1 = the period of material tested in the test

solution (a), in milligrams

m2 = retinol acetate period in the standard

solution (a), in milligrams
 To determine the exact concentration of retinol
acetate can be done by UV spectrophotometry.
Dissolve 25 - 100 mg of the substance in 5 ml of
pentane and volume it with 2-Propanol to obtain
10-15 IU / ml concentration.

 Check the maximum absorbance of the solution

at wavelengths between 325 and 327 nm. And
measured up to 300 nm, 326 nm, 350 nm and
370 nm. Calculate the ratio of Aλ / A326 for each
wavelength.

 The result will not exceed 0.593 at 300 nm,

0.537 at 350 nm and 0.142 at 370 nm. Then
calculate the content of vitamin A in units of IU /
g, using the formula;
 A326 x V x 1900

 100 x m

 Information :

 A326 = absorbance at 326 nm

 m = the mass of the substance tested, in grams

 V = total volume tested diluted 10-15 IU / ml

 1900 = factor to change the specific absorbance

of the retinol esters into International Units per
gram
3.Analisys TLC VITAMIN A (Form of Oil)
Vitamin A (form of oil) is made from a
synthetic retinol ester or a liquid with fatty oils
of the corresponding fruits. This concentrate
should use a stabilizer like antioxidants.
Vitamin A contains no more than 500,000 IU /
g and for the concentrate contains not less
than 95.0% and not more than 110.0% as
indicated on the label.
 Pemerian
Oily yellow, brownish yellow liquid, practically
insoluble in water, soluble or partially soluble in
ethanol, and soluble in organic solvents Crystal
form can occur in a very concentrated solution.

 a. Identification of Vitamin A (Forms of oil)

according to European Pharmacopoeia p. 2684
Thin layer chromatography

 Inspection with TLC, using TLC silica gel F254 R

plate.
 Test solution.
Prepare a solution containing 3.3 IU / μl of
vitamin A in cyclohexane P containing 1 g / l
butylhydroxytoluene P.

Comparative Solution
Prepare 10 mg / ml of a CRS retinol ester
solution (ie 3.3 IU / μl ester respectively) in a
P-cyclohexane containing 1 g / l of
butylhydroxytoluene P.
 Procedure

Strip each 3 μl solution over the plate. Eluate

immediately no more than 15 cm using a mixture
of 20 parts of P ether and 80 parts of cyclohexane
P. Dry the plate and look under 254 nm Ultraviolet
light. The identification is invalid unless the
chromatogram results with the comparison
solution show a separate stain of the same ester.
Elementation results from the bottom up are:
retinol assetate, retinol propionate and retinol
palmitate. The composition of the test solution
shows the corresponding primary stain or stain
indicated by the comparison solution.
 PH test

 acidic pH: not more than 2.0, set in 2.0 g.

alkaline pH: not more than 10.0
 . Determination of Vitamin A (Form of Oil)
according to European Pharmacopoeia p.
2685

Loading the assay immediately if possible,

avoid lighting from strong light and air,
oxidizing agents, oxidation catalysts (eg
copper, iron), acid and old heating; use fresh
solvents. If crystals are formed, homogenize
the material at temperatures above 65oC, but
do not heat up for too long. Assignment of
Content is performed in accordance with
Method A. Assessment The content does not
show valid results, use Method B.
 Method A

 Testing using UV-VIS Spectrophotometry

Dissolve 25-100 g of the substance in 5 ml of pentane P

and dilute with 2-propanol LP to obtain concentration of 10-
15 IU / ml. Check the maximum absorbance of the solution
at wavelengths between 325-327nm of its uptake at a
wavelength of 300nm, 326nm, 350nm, 370nm. Repeat
readings at each wavelength and grab the average rating.
Calculate the A / A326 ratio for each wavelength. Calculate
the A / A326 ratio for each wavelength. If the ratio does not
exceed: 0.593 at 300 nm, 0.537 at 350 nm, 0.142 at 370
nm, calculate the vitamin A content in IU / g, where: A326 =
absorption at 326 nm, m = the weight of the test materials
in grams, V = total volume of the test solution ie 10 IU / ml
dilution up to 15 IU / ml, 1900 = conversion factor of
specific uptake of retinol ester into IU / g. If one or more of
the A / A326 ratios exceeds the given value, or if the
maximum absorption wavelength does not lie between 325
nm and 327 nm, use Method B.
 Method B

 Testing using Liquid Chromatography

 Test solution (a)

 Put into a 50 ml volumetric flask, a number of

test materials, using a scaler with an accuracy of
0.1%, and the equivalent of 120,000 IU of vitamin
A and dissolve immediately in 5 ml of pentane P.
Add 20 mg to 30 mg of butylhydroxytoluene P
and 20 ml tetrabutylammonium hydroxide 0 , 1
M in 2-propanol. Stir gently for 5 minutes (using
the appropriate ultrasonic). Dilute to 50.0 ml with
2-propanol P then homogenize carefully to avoid
foaming.
 Test solution (b)

Input 20 mg to 30 mg of butylhydroxytoluene P into a 50

ml volumetric flask, add 5 ml 2-propanol P, 5.0 ml test
solution (a) and dilute to 50.0 ml with 2-propanol P.
Homogenize carefully to avoid foaming.

 Comparative solution (a)

Put into a 50 ml volumetric flask of 120 mg retinol acetate
CRS, using a scaler with an accuracy of 0.1% and then make
it as test solution (a).

 Comparative solution (b)

Add 20 mg to 30 mg of butylhydroxytoluene P into a 50
ml volumetric flask, add 5 ml 2-propanol P, 5.0 ml
comparative solution (a) and dilute to 50.0 ml with 2-
propanol P. Homogenize carefully to avoid foaming.
 Assignment is not eligible except:

 The chromatogram results from the comparative solution

(b) show the main peak corresponding to all retinol esters,
retention time of all retinol esters for 3 min.

 No peak is similar to retinol acetate derivative on

chromatogram results from comparative solution (b) at 6
minutes retention time. A suitable volume injection of
comparative solution (b) at a command indicated in a 0.5
to 1.0 uptake range at 325 nm and a chromatogram
record showing peak height equal to vitamin A of not less
than 50% of the full scale of the recorded. Make total
injection 6 times. The relative standard deviation resulting
from comparative solution (b) is not greater than 1%.

 Injection the same volume of the test solution (b) and

record the chromatogram in the same way. Calculate the
content of vitamin A by using the following formula:
A1 x C x m2
A2 x m1

 Information :

A1 = area of ​peak equal to all retinol esters in chromatogram yield
with test solution (b).

A2 = area of ​peak equal to all retinol esters in chromatogram yield

with comparative solution (b).

C = concentration of CRS retinol acetate in IU / g, determined by

method A; the A / A326 uptake ratio must be met.

m1 = mass of the material tested in the test solution (a), in mg.

m2 = mass of CRS retinol acetate in comparative solution (a), in mg.

 Storage

Stored in an airtight container, fully loaded containers,

protected from light. A container that can be opened,
the contents are used as soon as possible; as soon as
some of its contents are not used it will be protected
by an atmosphere of inert gas.

Vitamin A (Mixture / Emulsions

Vit A concentration as a solution or emulsion is a liquid

form (water commonly used as a solvent) of a Vit A
ester and a suitable solvent. Vit A contains not less
than 100,000 UI / g and its activity is not less than
95.0% and not more than 115.0% of the amount
indicated on the label.

Such concentrations may be added to suitable carriers

as antimicrobials and antioxidants.
 Pemerian

In liquid form of liquid yellow or light yellow

and thick. At high concentrations as in the
mixture can be solid at low temperatures or
into gel form. A mixture of 1 g in 10 ml of
water previously heated at a temperature of
500C was then cooled to 200C, to be uniform,
sufficiently dense and yellow dispersion.
Thank you