Edition: BP 2025 (Ph. Eur. 11.6 update)
DEFINITION
Natural Vitamin A Ester Concentrate consists of a natural ester or a mixture of natural esters of retinol or of a solution of the ester or mixture of esters in Arachis Oil or other suitable vegetable oil. It contains in 1 g not less than 485,000 IU of vitamin A and not less than 97.0% of the number of IU of vitamin A stated on the label. It may contain a suitable antioxidant or mixture of antioxidants.
CHARACTERISTICS
A yellow oil or a mixture of oil and crystalline material which yields a homogeneous yellow oil on warming.
Solubility
Practically insoluble in water; soluble or partly soluble in ethanol (96%); miscible with ether and with petroleum spirit.
IDENTIFICATION
A. Dissolve a quantity containing 10 IU in a mixture of 100 parts of absolute ethanol and 1 part of hydrochloric acid. The light absorption, Appendix II B, of the solution immediately after preparation exhibits a single maximum at 326 nm. Heat the solution in a water bath for 30 seconds and cool rapidly. The light absorption in the range 300 to 400 nm exhibits a low maximum or inflection at 332 nm and sharp maxima at 348, 367 and 389 nm.
B. Dissolve a quantity containing 30 IU in 1 mL of chloroform and add 10 mL of antimony trichloride solution. A transient bright blue colour is produced immediately.
TESTS
Acid value
Not more than 2.0, Appendix X B.
Peroxide value
Place 1 g in a boiling tube (20 cm × 2.5 cm) and dissolve in 20 mL of a mixture of 2 volumes of glacial acetic acid and 1 volume of ethanol-free chloroform. Add 1 g of finely powdered potassium iodide and pass a rapid current of oxygen-free nitrogen through the mixture for 1 minute. Stopper the tube loosely, partly immerse in boiling water for 30 seconds and then in water at 80° for 2 minutes, tighten the stopper and cool rapidly.
Transfer the contents to a flask containing 25 mL of a freshly prepared 1% w/v solution of potassium iodide, rinse the tube with a further 25 mL of the potassium iodide solution, shake the combined solution and rinsings and titrate with 0.01M sodium thiosulfate VS. Repeat the procedure without the concentrate. The difference between the titrations does not exceed 1.4 mL.
Retinol
Carry out the method for descending paper chromatography, Appendix III E, using a mixture of 70 volumes of 1,4-dioxan, 15 volumes of methanol and 15 volumes of water containing 1% w/v of butylated hydroxyanisole in the bottom of the tank and as the mobile phase. Saturate the paper with a 10% w/v solution of liquid paraffin in petroleum spirit (boiling range, 40° to 60°) and dry without the aid of heat. Apply separately to the impregnated paper 5 µL and 10 µL of each of the following freshly prepared solutions. For solution (1) dissolve sufficient of the concentrate in petroleum spirit (boiling range, 40° to 60°) to yield a solution containing 16,150 to 17,850 IU per mL. For solution (2) mix a quantity of the concentrate containing not less than 500 IU of vitamin A and not more than 1 g of fat with 30 mL of absolute ethanol and 3 mL of a 50% w/w solution of potassium hydroxide. Boil gently under a reflux condenser in a current of oxygen-free nitrogen for 30 minutes, cool rapidly and add 30 mL of water. Transfer the hydrolysate to a separating funnel using three 50 mL quantities of ether and extract the vitamin A by shaking for 1 minute. After complete separation discard the aqueous layer and wash the extract with four 50 mL quantities of water, mixing very cautiously during the first two washes to avoid the formation of emulsions. Evaporate the separated extract to about 5 mL and remove the remaining solvent in a current of oxygen-free nitrogen without the application of heat. Dissolve the residue in sufficient petroleum spirit (boiling range, 40° to 60°) to produce a solution of retinol containing about 340 IU per mL.
Develop until the solvent front approaches the bottom of the paper. Examine the dried paper under ultraviolet light (365 nm). The fluorescence of any spot corresponding to retinol in the chromatograms obtained with solution (1) is not more intense than that of the spot in the corresponding chromatogram obtained with solution (2).
ASSAY
Carry out the test as rapidly as possible, avoiding exposure to actinic light and air, oxidising agents, oxidation catalysts (e.g. copper and iron) and acids.
Examine by ultraviolet absorption spectrophotometry, Appendix II B (Method A). If method A is found not to be valid, examine by liquid chromatography, Appendix III D (Method B).
Method A
Test solution To a quantity of the substance being examined containing 50,000 IU in a round-bottomed flask, add 3 mL of a freshly prepared 50% w/w solution of potassium hydroxide and 30 mL of absolute ethanol. Boil under a reflux condenser in a current of nitrogen for 30 minutes. Cool rapidly and add 30 mL of water. Extract with four 50 mL quantities of ether discarding the lower layer after complete separation. Wash the combined upper layers with four 50 mL quantities of water and evaporate to dryness under a gentle current of nitrogen at a temperature not exceeding 30° or in a rotary evaporator at a temperature not exceeding 30° under reduced pressure (water ejector). Dissolve the residue in sufficient propan-2-ol to give an expected concentration of vitamin A equivalent to 10 to 15 IU per mL.
Measure the absorbances, Appendix II B, of the solution at 300 nm, 310 nm, 325 nm and 334 nm and at the wavelength of maximum absorption with a suitable spectrophotometer in 1-cm specially matched cells, using propan-2-ol as the compensation liquid.
Calculate the content of vitamin A, as all-trans-retinol, in IU per gram from the expression:
where A325 = absorbance at 325 nm,
m = mass of the substance to be examined in grams,
V = total volume of solution containing 10 IU to 15 IU of vitamin A per mL,
1830 = conversion factor for the specific absorbance of all-trans-retinol in IU.
The above expression can be used only if A325 has a value of not greater than A325, corr / 0.970 where A325, corr is the corrected absorbance at 325 nm and is given by the equation:
A325, corr = 6.815A325 – 2.555A310 – 4.260A334
where A designates the absorbance at the wavelength indicated by the subscript.
If A325 has a value greater than A325, corr / 0.970, calculate the content of vitamin A from the expression:
The assay is not valid unless:
(a) the wavelength of maximum absorption lies between 323 nm and 327 nm and
(b) the absorbance at 300 nm relative to that at 325 nm is at most 0.73.
Method B
Carry out the method for liquid chromatography, Appendix III D, using the following solutions. Prepare solution (1) as follows. To a quantity of the substance being examined containing 50,000 IU in a round- bottomed flask, add 5 mL of a freshly prepared 10% w/v solution of ascorbic acid and 10 mL of a freshly prepared 80% w/v solution of potassium hydroxide and 100 mL of ethanol (96%). Boil under a reflux condenser on a water bath for 15 minutes. Add 100 mL of a 1% w/v solution of sodium chloride and cool. Transfer the solution to a 500 mL separating funnel rinsing the round-bottomed flask with about 75 mL of a 1% w/v solution of sodium chloride and then with 150 mL of a mixture of equal volumes of petroleum spirit (boiling range, 40° to 60°) and ether. Shake for 1 minute. When the layers have separated completely, discard the lower layer and wash the upper layer, first with 50 mL of a 3% w/v solution of potassium hydroxide in a 10% v/v solution of ethanol (96%) and then with three 50 mL quantities of a 1% w/v solution of sodium chloride. Filter the upper layer through 5 g of anhydrous sodium sulfate on a fast filter paper into a 250 mL flask suitable for a rotary evaporator. Wash the funnel with 10 mL of fresh extraction mixture, filter and combine the upper layers. Distil them at a temperature not exceeding 30° under reduced pressure (water ejector) and fill with nitrogen when evaporation is completed. Alternatively evaporate the solvent under a gentle current of nitrogen at a temperature not exceeding 30°. Dissolve the residue in propan-2-ol, transfer to a 25 mL volumetric flask and dilute to 25 mL with propan-2-ol. Gentle heating in an ultrasonic bath may be required. For solution (2) prepare a solution of retinyl acetate EPCRS in propan-2-ol R1 so that 1 mL contains about 1000 IU of all-trans-retinol.
The exact concentration of solution (2) is assessed by ultraviolet absorption spectrophotometry, Appendix II
B. Dilute solution (2) with propan-2-ol R1 to a presumed concentration of 10 IU per mL to 15 IU per mL and measure the absorbance at 326 nm in matched 1-cm cells using propan-2-ol R1 as the compensation liquid.
Calculate the content of vitamin A in IU per mL of solution (2) from the following expression, taking into account the assigned content of retinyl acetate EPCRS:
where A326 = absorbance at 326 nm,
V2 = volume of the diluted solution,
V1 = volume of solution (2) used,
1900 = conversion factor for the specific absorbance of retinyl acetate EPCRS in IU.
For solution (3) proceed as described for solution (1) but use 2 mL of solution (2) in place of the substance being examined.
The exact concentration of solution (3) is assessed by ultraviolet absorption spectrophotometry, Appendix II
B. Dilute solution (3) with propan-2-ol R1 to a presumed concentration of 10 IU per mL to 15 IU per mL of all- trans-retinol and measure the absorbance at 325 nm in matched 1 cm cells using propan-2-ol R1 as the compensation liquid.
Calculate the content of all-trans-retinol in IU per millilitre of solution (3) from the expression:
where A325 = absorbance at 325 nm,
V3 = volume of the diluted solution,
V4 = volume of solution (3) used,
1830 = conversion factor for the specific absorbance of all-trans-retinol in IU.
The chromatographic procedure may be carried out using (a) a stainless steel column (25 cm × 4.6 mm) packed with octadecylsilyl silica gel for chromatography (5 µm to 10 µm), (b) as mobile phase at a flow rate of 1 mL per minute a mixture of 3 volumes of water and 97 volumes of methanol, (c) a detection wavelength of 325 nm, (d) a 10 µL loop injector and (e) an electronic integrator.
Inject in triplicate solution (1) and solution (3). The retention time of all-trans-retinol is 5 minute ± 1 minute.
The assay is not valid unless (a) the chromatogram obtained with solution (1) shows a peak corresponding to that of all-trans-retinol in the chromatogram obtained with solution (3), (b) when using the method of standard additions to solution (1) there is greater than 95% recovery of the added retinyl acetate EPCRS, and (c) the recovery of all-trans-retinol in solution (3) as assessed by direct absorption spectrophotometry is greater than 95%.
Calculate the content of vitamin A using the following expression:
where A1 = area of the peak corresponding to all-trans-retinol in the chromatogram obtained with solution (1),
A2 = area of the peak corresponding to all-trans-retinol in the chromatogram obtained with solution (3),
C = concentration of retinyl acetate EPCRS in solution (2) as assessed prior to the saponification in International Units per mL (1000 IU
per mL),
V = volume of solution (2) treated,
m = mass of the substance being examined in solution (1).
STORAGE
Vitamin A Ester Concentrate (Natural) should be kept in an airtight container, protected from light and stored at a temperature of 8° to 15°. Once the container has been opened its contents should be used as soon as possible; any part of the contents not used at once should be protected by an atmosphere of an inert gas.
LABELLING
The label states (1) the number of IU (Units) of vitamin A per g; (2) the name of the ester or esters; (3) the name and proportion of the principal excipients; (4) the method of restoring the solution if partial crystallisation has occurred.
