The BP utilises a fluorescence assay to determine ethinyloestradiol in tablets. The tablets contain low dosages of the drug so that interference by excipients is likely to cause problems in UV/visible spectrophotometric measurements. The sample is measured using an excitation wavelength of 280 nm and measuring the emission at 320 nm. As was seen when the fluorescence spectrum of ethinyloestradiol was discussed earlier, the optimum excitation wavelength for ethinyloestradiol is 285 nm and the emission maximum is 310 nm. Thus, this assay as described brings out two important points that may have been either consciously or empirically adjusted for in the design of the assay:
(i) The use of a slightly shorter excitation wavelength reduces possible interference from Raman scatter, which may overlap with the fluorescence spectrum and is dependent on the wavelength of the exciting radiation, whereas the fluorescence maximum is not.
(ii) The intensity of Rayleigh and Tyndall scatter at shorter wavelengths is greater and thus the emission is observed at the slightly longer wavelength of 320 nm to reduce interference from this source.
After the fluorescence of the sample extract in methanol has been determined, 1 M sodium hydroxide solution is added to the sample solution and the fluorescence is determined again. The addition of sodium hydroxide removes the fluorescence by ionising the phenol group of the ethinyloestradiol and thus any residual fluorescence which is due to excipients can be subtracted from the reading. In the BP assay the ethinyloestradiol content of the tablet extract is determined by comparison with the fluorescence of a solution containing a known amount of ethinyloestradiol standard analysed using the same conditions.
Amethanolic extract from ethinyloestradiol tablets is measured using fluorescence spectrophotometry. A standard containing the pure drug is also measured under the same conditions. Calculate the content per tablet of the drug from the following data:
Weight of 20 tablets = 2.5673 g.
Weight of tablet powder taken for assay = 0.5257 g.
Volume of methanol extract of tablets = 50 ml.
Fluorescence reading of methanol extract of tablets = 64.1.
Fluorescence reading of sample after addition of 0.1 M NaOH = 3.5.
Concentration of standard solution of ethinyloestradiol = 4.85 /./g/ml.
Fluorescence reading of standard solution = 62.3.
Fluorescence reading of standard solution after addition of 0.1 M NaOH = 4.1.
Corrected reading for tablet extract = 64.1 - 3.5 = 60.6.
Corrected reading for standard = 62.3 - 4.1 = 58.2.
Amount of ethinyloestradiol in tablet extract = 60.2/58.2 x 4.85 = 5.02 jUg/ml.
Number of tablets in tablet powder analysed = 2.5673/0.5257= 4.884.
Content of ethinyloestradiol per tablet = 251/4.885 = 51.4 ¡jg.
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