by Assay
Method A
This method is suitable for preparations which contain vitamin A as an ester and in a form directly soluble in cyclohexane.
Procedure
Dissolve an accurately weighed quantity of the substance under examination in sufficient cyclohexane to give a solution containing 9 to 15 Units of vitamin A per ml. Determine the wavelength of maximum absorption. Measure the absorbances of the solution against the cyclohexane at the wavelength given in Table 1. Calculate the absorbances at the wavelengths specified, as fractions relative to that at 328 nm. Calculate also the absorbance at 328 nm in terms of specific absorbance for the sample. If the wavelength of maximum absorption lies between 326
and 329 nm and the relative absorbances are within 0.02 of those in Table 1, calculate the vitamin A potency of the sample from the expression
A328(1 per cent, 1 cm) ×1900 = Vitamin A potency in Units per g.
| Table 1 | |
| Wavelength (nm) | Relative absorbance |
| 300 | 0.555 |
| 316 | 0.907 |
| 328 | 1.000 |
| 340 | 0.811 |
| 360 | 0.299 |
If the wavelength of maximum absorption lies between 326 nm and 329 nm but the relative absorbances are not within 0.02 of those in the table, calculate a corrected absorbance at 328 nm by applying the observed values to the equation
A328(corr.) = 3.52(2A328 – A316 – A340).
If the corrected absorbance lies within ± 3.0 per cent of the uncorrected absorbance, ignore the corrected absorbance and calculate the potency from the uncorrected absorbance. If the corrected absorbance lies outside –15 per cent to +3 per cent of the uncorrected absorbance or if the wavelength of maximum absorption does not lie between 326 nm and 329 nm, the substance under examination must be dealt with as described under Method B.
