In general, when a radiation is made to pass through a layer of a solution containing an absorbing pharmaceutical substance, a portion of the radiation is absorbed by it, whereas the intensity of the radiation emerging from the solution is always found to be less than the intensity of the radiation entering it, Therefore, the quantum of the absorption is designated in terms of the extinction E, that is represented by the following expression :
E = log 10 (Io/I)
Io = Intensity of radiation passing into the absorbing layer, and
I = Intensity of radiation passing out of the absorbing layer.
Extinction is solely dependent upon the following two factors, namely :
(a) Concentration of the absorbing substance present in the solution, and
(b) Thickness of the absorbing layer taken for measurement.
Bearing in mind the ease in calculations and also the convenience of reference, the extinction of a 1-cm layer of a 1% w/v solution is usually recommended in most of the official compendia (i.e., USP ; BP ; EP : IP :) for many pharmaceutical substances and is evaluated by the following expression :
E (1% ; 1-cm) = E/cl
c = Concentration of the absorbing substance represented as a percentage (w/v) ; and
l = Thickness of the absorbing layer (cm).
It is however, pertinent to mention here that most pure pharmaceutical substances possess a characteristic value of E (1% ; 1-cm) at a specific wavelength in a given spectroscopic-grade solvent (UVASOL(R)-Merck). This particular property is the basis for most assay methods included in pharmacopoeia that are absolutely free from interfering materials, besides being utilized for identifying substances.
In all other instances, the recommended tests specified in pharmacopoeia and prescribed assay methods normally call for comparison against Reference Substances (RS) to ensure measurements under conditions identical for the substance under examination and the reference substance.
In actual practice, where a test or an assay recommends the usage of a Reference Substance, the spectrophotometric measurements are always performed first with the solution prepared from the Reference Substance by the directions provided in the specific monograph and then with the corresponding solution prepared from the substance under examination. Nevertheless, the second measurement must be done immediately after the first, by employing the same cell and the same instrumental parameters.
Any appropriate spectrophotometer capable for measuring both in the ultra-violet (UV) and visible range of the spectrum must essentially consist of an optical system that should produce monochromatic light in the range 190-780 nm and a suitable device for measuring the extinction (E) precisely and accurately.
Besides, the two empty cuvettes (or cells) normally employed for the solution under examination and the reference substance (RS) should have exactly the same spectral features and characteristics. Importantly, when a double bond recording instrument is being employed the solvent cell is always placed in the reference beam.
The pharmaceutical substance under examination is usually dissolved in a spectroscopic grade UVASOL(R) SOLVENT. Particular care must be taken to employ solvents free from contaminants absorbing in the specific spectral region being used. In measuring the extinction of a solution at a given wavelength, the extinction of the solvent cell and its contents must not exceed 0.4 and should be preferably less than 0.2 when measured with reference to air at the same wavelength. Particularly, the solvent in the solvent cell should always be of the same purity, grade and batch as that employed to prepare the respective solution and above all it must be free from fluorescence at the wavelength of measurement.
Ethyl alcohol, methyl alcohol and cyclohexane (UVASOL(R)-Grade) employed as solvents shall have an extinction, measured in a 1 cm cell at 240 nm with reference to water (spectroscopic grade), not exceeding 0.10.
(a) Unless otherwise prescribed, measure the extinction (E) or the absorbance (A), at the prescribed wavelength using a path-length of 1 cm at 25 ± 1°C (IP) and at 20 ± 1°C (BP). All the measure-ments are normally performed with reference to the solvent used to prepare the solution being examined, unless otherwise indicated in the individual monograph.
(b) In the case of an assay or a limit test where the extinction forms the basis for a quantitative determination, a manually scanning instrument is employed invariably. In tests for identification, a recording instrument is always preferred ; besides, the concentration of the solution and the path-length are specifically monitored. In case, the laid down conditions are not suitable for a particular instrument, the thickness of the solution (i.e., path-length) may be varied without altering the concentration of the solution,
(c) Each assay of a pharmaceutical substance by UV-method specifies a wavelength at which maximum absorption takes place which implies the maximum occurring either precisely at or in the vicinity of the given wave length,
(d) Pharmaceutical assays (i.e., quantitative determinations) are normally performed at wavelength above 235 nm,
(e) In case, the measurements are specifically to be carried out at a wavelength between the range 190-210 nm, the following extra and special precautions must be adhered to rigidly, namely :
(i) Purging the cell compartments with N2,
(ii) Making use of only spectroscopic grade solvents e.g., UVASOL(R) (Merck), and
(iii) Making use of cells that are absolutely transparent in the region 190-210 nm.
(f) The requirements for light absorption in the official compendia invariably apply to the dried, anhydrous, or solvent free material in all such monographs in which standards for loss on drying, water or solvent content are provided.
A few typical examples for the assay of pharmaceutical substances by UV-spectrophotometric method are described below :
Materials Required : Amoxycillin trihydrate : 0.17 g ; 100-ml volumetric flask ; 2 ; buffer solution pH 9.0 (Solution : I : Boric acid and Potassium Chloride (0.2 M)-Dissolve 12.366 g of Boric acid and 14.911 g of KCl in DW and dilute with water to 1000 ml ; Solution : II NaOH (0.2 N) : Dissolve 8.0 g of NaOH in CO2-free DW to produce 1000 ml ; Now, transfer 50 ml of solution I into a 200-ml volumetric flask and add to it 20.8 ml of solution II, then add sufficient DW to make up the volume to 200 ml) : 10 ml ; acetic anhydride-dioxan solution (add 1 ml of acetic anhydride to 50-ml of dioxan) : 1.0 ml ; imidazole-mercury reagent (dissolve 8.25 g of recrystallized imidazole in 60 ml of DW and add 10 ml of 5N HCl. Stir the solution magnetically and, add dropwise, 10 ml of a 0.27% w/v solution of Hg2Cl2. Adjust the pH to 6.8 ± 0.05 with 5 N HCl (about 4.0 ml is needed) and add sufficient DW to produce 100 ml) : 10.0 ml ;
Procedure : Weigh accurately about 0.17 g of amoxycillin trihydrate and dissolve in sufficient DW to produce 500 ml. Now, transfer 10 ml of this solution into a 100 ml volumetric flask, add 10 ml of buffer solution pH 9.0 followed by 1 ml of acetic anhydride-dioxan solution, allow to stand for 5 minutes, and add sufficient water to produce 100 ml. Pipette 2 ml of the resulting solution into each of the two stoppered tubes. To tube 1 add 10 ml of imidazole-mercury reagent, mix, stopper the tube and immerse it in a water-bath previously maintained at 60 °C for exactly 25 minutes, with occasional swirling. Remove the tube from the water-bath and cool rapidly to 20 °C (Solution-1). To tube 2 add 10 ml of DW and mix thoroughly (Solution-2). Immediately, measure the extinctions of Solutions 1 and 2 at the maximum at about 325 nm, as detailed above, employing as the blank a mixture of 2 ml of DW and 10 ml of imidazole-mercury reagent for Solu-tion-1 and simply DW for Solution-2.
Calculations : The content of C16H19N3O5S may be calculated from the difference between the extinctions of Solution-1 and that of Solution-2 and from the difference obtained by repeating the operation using 0.17 g of amoxycillin trihydrate (RS), instead of the sample being examined and the declared content of C16H19N3O5S in the amoxycillin trihydrate (RS).
Cognate Assays : Ampicillin can also be assayed by employing the above method using 0.15 g of the sample.
Theory : Folic acid (I) undergoes cleavage by reduction with Zn-Hg in acidic medium to yield p-aminobenzoylglutamic acid (II). The primary aromatic amino group present in the latter is subsequently diazotized in the usual manner and coupled in acidic solution with N-(1-naphthyl)-ethylenediamine hydro-chloride in the absence of light (caution). The colour thus produced has a maximum absorption at 550 nm and the extinction (E) is consequently compared with a calibration curve obtained from p-aminobenzoic acid (PABA) that has been duly diazotized and coupled exactly in the same fashion as the p-aminobenzoylglutamic acid.
The reaction involved is expressed by the following equation :
Note : In order to ensure that the extinctions recorded exclusively refer to folic acid (I), and also that they do not necessarily include a contribution from a free-primary-amino-aromatic-moiety obtained from a decomposition product, a blank estimation is always performed with the unreduced solution and an appropriate correction is applied. The colour thus corresponds to a definite quantity of C16H19O6N7. Thus, we have :
C7H7O2N = C19H19O6N7
Materials Required : Folic acid : 0.05 g : 0.1 N NaOH : 100 ml ; 2 N HCl : 30 ml ; Zn-powder : 0.5 g ; sodium nitrite solution (0.1% w/v in DW) : 5 ml ; ammonium sulphamate (0.5% w/v in DW : 5 ml ; N-(1-naphthyl) ethylene-diamine hydrochloride solution (0.1 % w/v in DW) : 5 ml ;
Procedure : Accurately weigh about 0.5 g, dissolve in 50 ml of 0.1 N NaOH and add sufficient 0.1 N NaOH to produce 100 ml (Solution-1). To 3 ml add 20 ml of 2 N HCl and dilute to 100 ml with DW. To 50 ml of this solution, add 0.5 g of zinc powder, allow to stand in a dark place for 20 minutes with intermittent shaking and filter, Dilute 10 ml of the filtrate to 25 ml with DW, add 5 ml of 2N HCl and 5 ml of a 0.1% solution of sodium nitrite, mix and allow to stand for 2 minutes. Add 5 ml of a 0.5% w/v solution of ammonium sulphamate, mix and allow to stand for 2 minutes. Now, add carefully 5 ml of a 0.1% solution of N-(1-naphthyl) ethylene diamine hydrochloride, mix thoroughly and allow to stand for 10 minutes. Add sufficient DW to produce 50 ml and measure the extinction of the resulting solution at about 550 nm, as discussed earlier, using as blank a solution prepared exactly in a similar manner but employing 25 ml of DW and beginning the procedure at ‘‘add 5 ml of 2 N HCl...’’
To a further portion of 30 ml of solution-1, add 20 ml of 2N HCl and sufficient DW to produce 100 ml. Mix 10 ml of this solution with 15 ml of DW and repeat the operations stated above beginning the procedure at ‘‘add 5 ml of 2 N HCl ...’’
Finally, substract 1/10th of the extinction of the unreduced solution from that of the reduced solution and from the result thus obtained calculate the amount of C19H19O6N7, using the result obtained by repeating the operation using folic acid (RS) instead of the substance being examined and the declared content of C19H19O6N7 in folic acid (RS).
Theory : First and foremost the active ingredient i.e., glyceryl trinitrate is extracted completely from the tables by shaking with glacial acetic acid. To an aliquot of the resulting acetic acid solution an excess of phenoldisulphonic acid is added to produce a yellow colour which is subsequently intensified by adding an excess of ammonia. The following reactions take place :
The standard substance in this assay is KNO3, which conforms to the nitric acid released by acidolysis in the test solution.
Materials Required : Glyceryl trinitrate tablets : 20 ; glacial acetic acid (90% v/v) : 5 ml ; phenoldisulphonic acid solution (heat 3 g of phenol with 20 ml of sulphuric acid on a water-bath for 6 hours, and transfer the resulting liquid to a stoppered vessel) : 2 ml ; strong ammonia solution ; 20 ml ; potassium nitrate (previously dried at 105 °C) : 1 g ;
Procedure : Weigh and powder 20 tablets. Now, weigh accurately a quantity of the powder equiva-lent to 0.5 mg of glyceryl trinitrate, add 5 ml of glacial acetic acid, shake thoroughly for 1 hour and then centrifuge. To 2 ml of the supernatant liquid add 2 ml of phenoldisulphonic acid solution and allow to stand for 15 minutes. Add 8 ml of DW, make alkaline with strong ammonia solution, cool to about 20 °C, dilute to 20 ml with DW and filter. Finally, measure the extinction of a 1-cm layer of the filtrate at 405 nm, as described earlier, employing as blank 2 ml of glacial acetic acid, treated exactly in a similar fashion, begin-ning at ‘‘add 2 ml of phenoldisulphonic acid solution .........’’.
Dissolve 133.5 mg of potassium nitrate, in sufficient DW to produce 100 ml ; to 10 ml add sufficient glacial acetic acid to produce 100 ml. Taking 2 ml of this solution, just repeat the assay beginning the procedure at ‘‘add 2 ml of phenoldisulphonic acid solution......’’
The content of C3H5N3O9 may be calculated from the values of the extinctions thus obtained. Each ml of the potassium nitrate solution is equivalent to 0.1 mg of C3H5N3O9.
Cognate Assays : The following two pharmaceutical products, namely : Pentaerythritol tetranitrate Tablets and Diluted Isosorbide dinitrate are assayed by using a solution of phenoldisulphonic acid as detailed below :
Theory : The assay of stilbonesterol is exclusively based upon photochemical reactions whereby the trans-isomer firstly gets converted into its corresponding cis-isomer (Geometrical Isomerism) and then followed by intramolecular rearrangement therby causing ring closure as expressed in the equations :
The highly conjugated diketo system obtained as a result of irradiation of the stilbosterol solution placed in a closed spectrophotometer cell for a duration of 10 minutes and exposed to a 15-watt short-wave ultraviolet lamp. Ultimately the extinction is duly measured at 418 nm and compared with stilboesterol (RS) treated exactly in the same manner.
Materials Required : Stilbosterol : 20 mg ; ethyl alcohol (absolute) : 250 ml ; dipotassium hydrogen phosphate solution (dissolve 1 g in 55 ml of DW) : 25 ml ;
Procedure : Weigh accurately about 20 mg of stilbosterol in sufficient ethyl alcohol to produce 100 ml ; and dilute 10 ml of this solution to 100 ml with ethyl alcohol. To 25 ml of the resulting solution add 25 ml of dispotassium hydrogen phosphate solution, transfer a portion of the mixture to a 1-cm closed quartz cell, place the cell 10 cm from a 15 watt short-wave UV-lamp, and subject it to irradiation for 10 minutes. Now, measure the extinction of the irradiated solution at the maximum at about 418 nm as described earlier.
Calculations : Calculate the content of C18H20O2 from the extinction obtained by repeating the op-eration with stilbosterol (RS).
The ultra-violet absorption characteristics of a number of official pharmaceutical substances have been duly provided in Table 21.2.