Spectrophotometric multi-component analysisAbsorption spectroscopy is one of the most useful and widely used tools available to the analyte for quantitative analysis. The relation between the concentration of analyte and the amount of light absorbed is the basis of most analytical applications of molecular spectroscopy. This method of analysis is gaining importance due to simple, rapid, precise, highly accurate and less time consuming. Spectrophotometric multi-component analysis can be applied where the spectra of drugs overlaps. In such cases of overlapping spectra, simultaneous equation can be framed to obtain the concentration of individual component; otherwise multi-component analysis can be applied on any degree of spectral overlap provided that two or more spectra are not similar exactly. Some examples are listed in table-2.
The various spectroscopic techniques used for multi-component analysis are as follows
Simultaneous equation method (Vierodt’s method) 3
Two wavelength method3
The absorption ratio method3
Geometric correction method3
Absorption factor method (Absorption correction method) 3
Orthogonal polynomial method3
Area under curve method4
The methods deviated when overlapping of UV spectra of two drugs significantly and large difference in labeled strength5. e. g. Tizanidine HCl 3.0 mg and nimesulide 100.0 mg per tablet. The accuracy of the method depends upon nature of solvent, pH of solution, temperature, high electrolyte concentration and the presence of interfering substances.
High performance liquid chromatography (HPLC):This technique is based on the same method of separation as classical column chromatography. i.e. adsorption, partition, ion exchange and gel permeation but it differ from column chromatography, in that mobile phase is pumped through the packed column under high pressure. The technique is most widely used for all the analytical separation technique due to its sensitivity, its ready adaptability to accumulate quantitative determinations, its suitability for separating nonvolatile species or thermally fragile ones. In normal HPLC, polar solids such as silica gel; alumina (Al2O3) or porous glass beads and non-polar mobile phase such as heptane, octane or chloroform are used but if the opposite case holds, it is called as reversed phase HPLC. Some examples are listed in table-3 and 4
High performance thin layer chromatography (HPTLC):The principle is based on plane chromatography. The mobile phase normally is driven by capillary action. The prominent advantages of this technique includes possibilities of separating of up to 70 samples and standard simultaneously on a single plate leading to high throughout, low cost analogs and the ability to construct calibration curves from standard chromatography under the same condition as the sample. Analyzing a sample by use of multiple separation steps and static post chromatographic detection procedures with various universal and specific visualization regents that are possible because all the sample components are stored on the layer without the chance of loss. Some examples are listed in table-5.
Gas chromatography (GC):GC is one of the most extensively used separation technique in which separation is accomplished by partitioning solute between a mobile gas phase and stationary phase, either liquid or solid. The chief requirement is same degrees of stability at the temperature necessary to maintain the substance in gas state. Some examples are listed in table-6.
Validation of methods6:Validation by definition is an act of providing that any process, method, equipment, material, activity, system or analyst performs as expected under given set of conditions. When extended to an analytical procedure, depending upon the application it means that a method works reproducibility when carried out by a same or different person, in same or different laboratories, using different regent, different equipment etc. It will ensure commitment to quality of products and services. It builds a degree of confidence not only for the developer but also to the user.
Validation of analytical method should follow a well documented procedure beginning with the definition of the scope of the method and its validation criteria and including the compounds and matrices, desired detection and quantitation limits and any other important performance criteria. The scope of method should include different equipment and locations where the method will be run. The methods were validated in terms of linearity, accuracy, precision, specificity and reproducibility of sample applications. Analytical method validation has been performed according to ICH guidelines. Accuracy of the method is certain on the basis of recovery studies performed by the standard addition method. The formula used for calculating recovery of pure drug is
Percentage recovery = T - A X 100 / S
Where T = Total amount of drug estimated
A= Amount contributed by formulation
S = Amount of pure drug added.
Precision of analytical method is expressed as SD and RSD of series of measurement by replicate estimation of drug.
The stability indicating ability of the method has been investigated by deliberately degrading the sample preparation. The stress conditions applied are acidic (0.1 M HCl), alkalis (0.1M NaOH) and mild oxidizing condition (3% H2O2) for 24 hr at 50 C. Also heat (60C) and U.V. exposure for 24 hr will be carried out on the sample.
The linearity of the method was investigated by serially diluting the stock solutions of drugs and measured values.
Ruggedness studies has been carried out for different parameters i.e. days and analysts. The results shall be compared with the method.