Vitamin A
EUROPEAN PHARMACOPOEIA 5.0
Acidity or alkalinity. To 25 ml of solution S add 0.1 ml
of phenolphthalein solution R and to another 25 ml add
0.05 ml of methyl orange solution R. Neither solution is
pink.
Foreign fibres. Examined under a microscope, it is seen to
consist exclusively of viscose fibres, except that occasionally
a few isolated foreign fibres may be present.
Fluorescence. Examine a layer about 5 mm in thickness
under ultraviolet light at 365 nm. It displays only a slight
brownish-violet fluorescence. It shows no intense blue
fluorescence, apart from that which may be shown by a few
isolated fibres.
Absorbency
Apparatus. A dry cylindrical copper-wire basket 8.0 cm high
and 5.0 cm in diameter. The wire of which the basket is
constructed is about 0.4 mm in diameter, the mesh is 1.5 cm
to 2.0 cm wide and the mass of the basket is 2.7 ± 0.3 g.
Sinking time. Not more than 10 s. Weigh the basket
to the nearest centigram (m1). Take a total of 5.00 g in
approximately equal quantities from 5 different places in
the product to be examined, place loosely in the basket and
weigh the filled basket to the nearest centigram (m2). Fill
a beaker 11 cm to 12 cm in diameter to a depth of 10 cm
with water at about 20 °C. Hold the basket horizontally
and drop it from a height of about 10 mm into the water.
Measure with a stopwatch the time taken for the basket to
sink below the surface of the water. Calculate the result as
the average of 3 tests.
Water-holding capacity. Not less than 18.0 g of water per
gram. After the sinking time has been measured, remove
the basket from the water, allow it to drain for exactly 30 s
suspended in a horizontal position over the beaker, transfer
it to a tared beaker (m3) and weigh to the nearest centigram
(m4). Calculate the water-holding capacity per gram of
absorbent viscose wadding using the following expression :
400 ml of the filtrate (corresponding to 4/5 of the mass of
the sample taken) and dry the residue to constant mass at
100 °C to 105 °C.
Hydrogen sulphide. To 10 ml of solution S add 1.9 ml of
water R, 0.15 ml of dilute acetic acid R and 1 ml of lead
acetate solution R. After 2 min, the solution is not more
intensely coloured than a reference solution prepared at the
same time using 0.15 ml of dilute acetic acid R, 1.2 ml of
thioacetamide reagent R, 1.7 ml of lead standard solution
(10 ppm Pb) R and 10 ml of solution S.
Loss on drying (2.2.32). Not more than 13.0 per cent,
determined on 5.000 g by drying in an oven at 100 °C to
105 °C.
Sulphated ash (2.4.14). Not more than 0.45 per cent for
the lustrous variety and not more than 1.7 per cent for the
matt variety. Introduce 5.00 g into a previously heated and
cooled, tared crucible. Heat cautiously over a naked flame
and then carefully to dull redness at 600 °C. Allow to cool,
add a few drops of dilute sulphuric acid R, then heat and
incinerate until all the black particles have disappeared.
Allow to cool. Add a few drops of ammonium carbonate
solution R. Evaporate and incinerate carefully, allow to cool
and weigh again. Repeat the incineration for periods of
5 min to constant mass.
Calculate the result as the average of 3 tests.
Ether-soluble substances. Not more than 0.30 per cent. In
an extraction apparatus, extract 5.00 g with ether R for 4 h
at a rate of at least 4 extractions per hour. Evaporate the
ether extract and dry the residue to constant mass at 100 °C
to 105 °C.
Extractable colouring matter. In a narrow percolator,
slowly extract 10.0 g with alcohol R until 50 ml of extract is
obtained. The liquid obtained is not more intensely coloured
(2.2.2, Method II) than reference solution Y5, GY6 or a
reference solution prepared as follows : to 3.0 ml of blue
primary solution add 7.0 ml of hydrochloric acid (10 g/l HCl)
and dilute 0.5 ml of this solution to 10.0 ml with hydrochloric
acid (10 g/l HCl).
Surface-active substances. Introduce the 10 ml portion of
solution S reserved before filtration into a 25 ml graduated
ground-glass-stoppered cylinder with an external diameter
of 20 mm and a wall thickness of not greater than 1.5 mm,
previously rinsed 3 times with sulphuric acid R and then
with water R. Shake vigorously 30 times in 10 s, allow to
stand for 1 min and repeat the shaking. After 5 min, any
foam present does not cover the entire surface of the liquid.
Water-soluble substances. Not more than 0.70 per cent. Boil
5.00 g in 500 ml of water R for 30 min, stirring frequently.
Replace the water lost by evaporation. Decant the liquid,
squeeze the residual liquid carefully from the sample with
a glass rod and mix. Filter the liquid whilst hot. Evaporate
Substance
2682
STORAGE
Store in a dust-proof package in a dry place.
01/2005:0217
VITAMIN A
Vitaminum A
R
Molecular
formula
Mr
R=H
C20H30O
286.5
CO-CH3
C22H32O2
328.5
all-(E)-retinol propionate
CO-C2H5
C23H34O2
342.5
all-(E)-retinol palmitate
CO-C15H31
C36H60O2
524.9
all-(E)-retinol
all-(E)-retinol acetate
DEFINITION
Vitamin A refers to a number of substances of very similar
structure (including (Z)-isomers) found in animal tissues and
possessing similar activity. The principal and biologically
most active substance is all-(E)-retinol (all-(E)-3,7-dimethyl9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraen-1-ol ;
C20H30O). Vitamin A is generally used in the form of esters
such as the acetate, propionate and palmitate.
Synthetic retinol ester refers to an ester of synthetic retinol
(acetate, propionate or palmitate) or a mixture of synthetic
retinol esters.
The activity of vitamin A is expressed in retinol equivalents
(R.E.) 1 mg R.E. corresponds to the activity of 1 mg of
all-(E)-retinol. The activity of the other retinol esters is
calculated using the stoichiometry, so that 1 mg R.E. of
vitamin A corresponds to the activity of :
— 1.147 mg of all-(E)-retinol acetate,
— 1.195 mg of all-(E)-retinol propionate,
— 1.832 mg of all-(E)-retinol palmitate.
See the information section on general monographs (cover pages)
Vitamin A
EUROPEAN PHARMACOPOEIA 5.0
International units (IU) are also used. 1 IU of vitamin A is
equivalent to the activity of 0.300 µg of all-(E)-retinol. The
activity of the other retinol esters is calculated using the
stoichiometry, so that 1 IU of vitamin A is equivalent to the
activity of :
— 0.344 µg of all-(E)-retinol acetate,
— 0.359 µg of all-(E)-retinol propionate,
— 0.550 µg of all-(E)-retinol palmitate,
1 mg of retinol equivalent is equivalent to 3333 IU.
CHARACTERS
Retinol acetate occurs as pale-yellow crystals (melting point
about 60 °C). Once melted retinol acetate tends to yield a
supercooled melt.
Retinol propionate occurs as a reddish-brown oily liquid.
Retinol palmitate is a fat-like, light yellow solid or a yellow
oily liquid, if melted (melting point about 26 °C).
All retinol esters are practically insoluble in water, soluble or
partly soluble in ethanol and miscible with organic solvents.
Vitamin A and its esters are very sensitive to the action of
air, oxidising agents, acids, light and heat.
Carry out the assay and all tests as rapidly as possible,
avoiding exposure to actinic light and air, oxidising agents,
oxidation catalysts (e.g. copper, iron), acids and heat ; use
freshly prepared solutions.
IDENTIFICATION
A. Examine by thin-layer chromatography (2.2.27), using a
TLC silica gel F254 plate R.
Test solution. Prepare a solution containing about 3.3 IU
of vitamin A per microlitre in cyclohexane R containing
1 g/l of butylhydroxytoluene R.
Reference solution. Prepare a 10 mg/ml solution
of retinol esters CRS (i.e. 3.3 IU of each ester per
microlitre) in cyclohexane R containing 1 g/l of
butylhydroxytoluene R.
Apply to the plate 3 µl of each solution. Develop
immediately over 2/3 of the plate using a mixture of
20 volumes of ether R and 80 volumes of cyclohexane R.
Allow the plate to dry in air and examine in ultraviolet
light at 254 nm. The identification is not valid unless
the chromatogram obtained with the reference solution
shows the individual spots of the corresponding esters.
The elution order from bottom to top is : retinol acetate,
retinol propionate and retinol palmitate. The composition
of the test solution is confirmed by the correspondence
of the principal spot or spots with those obtained with
the reference solution.
B. It complies with the test for related substances.
no or only traces of the retinol ester are seen. Any spot
corresponding to retinol in the chromatogram obtained with
the test solution is not more intense than the spot in the
chromatogram obtained with the reference solution (1.0 per
cent).
Related substances. Examine by ultraviolet absorption
spectrophotometry (2.2.25). Determine the absorption
maximum of the solution for “Activity”. The solution shows
an absorption maximum at 325 nm to 327 nm. Measure the
absorbances at 300 nm, 350 nm and 370 nm and calculate
the ratio Aλ/A326 for each wavelength. None of the ratios
Aλ/A326 exceed 0.593 at 300 nm, 0.537 at 350 nm, 0.142 at
370 nm.
ACTIVITY
The activity of the substance is determined in order to be
taken into account for the production of concentrates.
Examine by ultraviolet absorption spectrophotometry
(2.2.25). Dissolve 25 mg to 100 mg, weighed with an
accuracy of 0.1 per cent, in 5 ml of pentane R and dilute with
2-propanol R1 to a presumed concentration of 10 IU/ml to
15 IU/ml. This solution is also used for the test for related
substances.
Determine the absorbance of the maximum at 326 nm.
Calculate the activity of vitamin A in International Units per
gram from the expression :
A326 =
m
=
V
=
absorbance at 326 nm,
1900 =
factor to convert the specific absorbance of esters
of retinol into International Units per gram.
mass of the substance to be examined, in grams,
total volume to which the substance to be
examined is diluted to give 10 IU/ml to 15 IU/ml,
STORAGE
Store in an airtight, well-filled container, protected from
light.
Once the container has been opened, its contents are to be
used as soon as possible ; any part of the contents not used
at once should be protected by an atmosphere of inert gas.
LABELLING
The label states :
— the number of International Units per gram,
— the name of the ester or esters.
IMPURITIES
TESTS
Retinol. Examine by thin-layer chromatography (2.2.27),
using a TLC silica gel F254 plate R.
Test solution. Prepare a solution in cyclohexane R, stabilised
with a 1 g/l solution of butylhydroxytoluene R, containing
about 330 IU of vitamin A per microlitre.
Reference solution. Shake 1 ml of the test solution with 20 ml
of 0.1 M tetrabutylammonium hydroxide in 2-propanol for
2 min and dilute to 100 ml with cyclohexane R, stabilised
with a 1 g/l solution of butylhydroxytoluene R.
Apply to the plate 3 µl of each solution. Develop immediately
over a path of 15 cm using a mixture of 20 volumes of
ether R and 80 volumes of cyclohexane R. Allow the plate
to dry in air and examine in ultraviolet light at 254 nm. In
the chromatogram obtained with the reference solution
A. R = H, CO-CH3 : kitols (Diels-Alder dimers of vitamin A),
General Notices (1) apply to all monographs and other texts
2683
Vitamin A concentrate (oily form), synthetic
EUROPEAN PHARMACOPOEIA 5.0
ASSAY
Carry out the assay as rapidly as possible, avoiding
exposure to actinic light and air, oxidising agents, oxidation
catalysts (e.g. copper, iron), acids and prolonged heat ; use
freshly prepared solutions. If partial crystallisation has
occurred, homogenise the material at a temperature of
B. (3E,5E,7E)-3,7-dimethyl-9-[(1Z)-2,6,6-trimethylcyclohex-2- about 65 °C, but avoid prolonged heating.
enylidene)nona-1,3,5,7-tetraene (anhydro-vitamin A),
Carry out the assay according to Method A. If the assay is
not shown to be valid, use Method B.
Method A. Examine by ultraviolet absorption
spectrophotometry (2.2.25). Dissolve 25 mg to 100 mg,
weighed with an accuracy of 0.1 per cent, in 5 ml of
pentane R and dilute with 2-propanol R1 to a presumed
concentration of 10 IU/ml to 15 IU/ml.
C. (3E,5E,7E)-3,7-dimethyl-9-[(1Z)-2,6,6-trimethylcyclohex-2- Verify that the absorption maximum of the solution lies
enylidene)nona-3,5,7-trien-1-ol (retro-vitamin A),
between 325 nm and 327 nm and measure the absorbances
at 300 nm, 326 nm, 350 nm and 370 nm. Repeat the readings
D. oxidation products of vitamin A.
at each wavelength and take the mean values. Calculate the
ratio Aλ/A326 for each wavelength.
01/2005:0219 If the ratios do not exceed : 0.593 at 300 nm, 0.537 at
350 nm, 0.142 at 370 nm, calculate the content of vitamin A
in International Units per gram from the expression :
VITAMIN A CONCENTRATE (OILY
FORM), SYNTHETIC
Vitaminum A densatum oleosum
DEFINITION
Synthetic vitamin A concentrate (oily form) is prepared
from synthetic retinol ester (0217) as is or by dilution with
a suitable vegetable fatty oil. The concentrate may contain
suitable stabilisers such as antioxidants.
The declared content of vitamin A is not less than
500 000 IU/g and the concentrate contains not less than
95.0 per cent and not more than 110.0 per cent of the
content stated on the label.
CHARACTERS
A yellow or brownish-yellow, oily liquid, practically insoluble
in water, soluble or partly soluble in ethanol, miscible with
organic solvents. Partial crystallisation may occur in highly
concentrated solutions.
IDENTIFICATION
Examine by thin-layer chromatography (2.2.27), using a TLC
silica gel F254 plate R.
Test solution. Prepare a solution containing about 3.3 IU of
vitamin A per microlitre in cyclohexane R containing 1 g/l
of butylhydroxytoluene R.
Reference solution. Prepare a 10 mg/ml solution of retinol
esters CRS (i.e. 3.3 IU of each ester per microlitre) in
cyclohexane R containing 1 g/l of butylhydroxytoluene R.
Apply to the plate 3 µl of each solution. Develop immediately
over a path of 15 cm using a mixture of 20 volumes of
ether R and 80 volumes of cyclohexane R. Allow the plate
to dry in air and examine in ultraviolet light at 254 nm. The
identification is not valid unless the chromatogram obtained
with the reference solution shows the individual spots of the
corresponding esters. The elution order from bottom to top
is : retinol acetate, retinol propionate and retinol palmitate.
The composition of the test solution is confirmed by the
correspondence of the principal spot or spots with those
obtained with the reference solution.
TESTS
Acid value (2.5.1). Not more than 2.0, determined on 2.0 g.
Peroxide value (2.5.5). Not more than 10.0.
2684
A326 =
m
=
V
=
absorbance at 326 nm,
1900 =
factor to convert the specific absorbance of esters
of retinol into International Units per gram.
mass of the preparation to be examined, in grams,
total volume to which the preparation to be
examined is diluted to give 10 IU/ml to 15 IU/ml,
If one or more of the ratios Aλ/A326 exceeds the values given,
or if the wavelength of the absorption maximum does not lie
between 325 nm and 327 nm, use Method B.
Method B. Examine by liquid chromatography (2.2.29).
Test solution (a). Introduce into a 50 ml volumetric flask, an
amount of the preparation to be examined, weighed with an
accuracy of 0.1 per cent and equivalent to about 120 000 IU
of vitamin A and dissolve immediately in 5 ml of pentane R.
Add 20 mg to 30 mg of butylhydroxytoluene R and 20 ml of
0.1 M tetrabutylammonium hydroxide in 2-propanol. Swirl
gently for 5 min (an ultrasonic bath is recommended). Dilute
to 50.0 ml with 2-propanol R and homogenise carefully to
avoid air-bubbles.
Test solution (b). Introduce 20 mg to 30 mg of
butylhydroxytoluene R into a 50 ml volumetric flask, add
5 ml of 2-propanol R, 5.0 ml of test solution (a) and dilute to
50.0 ml with 2-propanol R. Homogenise carefully to avoid
air-bubbles.
Reference solution (a). Introduce into a 50 ml volumetric
flask about 120 mg of retinol acetate CRS, weighed with an
accuracy of 0.1 per cent and proceed as described for test
solution (a).
Reference solution (b). Introduce 20 mg to 30 mg of
butylhydroxytoluene R into a 50 ml volumetric flask, add
5 ml of 2-propanol R, 5.0 ml of reference solution (a) and
dilute to 50.0 ml with 2-propanol R. Homogenise carefully
to avoid air-bubbles.
The chromatographic procedure may be carried out using :
— a stainless steel column 0.125 m long and 4 mm in
internal diameter packed with octadecylsilyl silica gel for
chromatography R (5 µm),
— as mobile phase at a flow rate of 1 ml/min a mixture of
5 volumes of water R and 95 volumes of methanol R,
See the information section on general monographs (cover pages)