estimation of multi-component formulations

Spectrophotometric multi-component analysis

Absorption 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

Concentration of several components present in the same mixture can be determined by solving a set of simultaneous equation even if their spectra overlap. If Beer’s law is followed, these equations are linier.

• Two wavelength method³

The method can be used to calculate the concentration of component of interest found in a mixture containing it along some unwanted interfering component. The absorption different between two points on the mixture spectra is directly proportional to the concentration of the component to be determined irrespective of the interfering component.

• The absorption ratio method³

The absorbance ratio method is a modification of the simultaneous equation procedure. It depends on the property that for a substance, which obeys Beer’s law at all wavelength, the ratio of absorbance at any two wavelengths is constant value independent of concentration or path length. e.g. Two dilutions of the same substance give the same absorbance ratio A₁ / A₂. In the USP, this ratio is referred to as Q value. In the quantitative assay of two components in admixture by the absorbance ratio method, absorbances are measured at two wavelengths. One being the λ _max of one of the components (λ₂) and the other being a wavelength of equal absorptivity of the two components (λ₁), i.e., an iso-absorptive point.  

• Geometric correction method³

A number of the mathematical correction procedures have been developed which reduce or eliminate the background irrelevant absorption that may be present in the samples of biological origin. The simplest of this procedure is the three-point geometric procedure, which may be applied if the irrelevant absorption is linier at the three wavelengths selected. This procedure is simply algebraic calculations of what the baseline technique in infrared spectrophotometry dose graphically.

• Absorption factor method (Absorption correction method) ³

It is further modification of simultaneous equation method. Quantitative determination of one drug is carried out by E (1%, 1 cm) value and quantitation of another drug is carried out by subtraction absorption due to interfering drug using absorption factors.

• Orthogonal polynomial method³

The technique of orthogonal polynomials is another mathematical correction procedure, which involves complex calculation than the three-point correction procedure. The basis of the method is that an absorption spectrum may be represented in terms of orthogonal functions.

• Difference spectrophotometry³

Difference spectrophotometry provides a sensitive method for detecting small changes in the environment of a chromophore or it can be used to demonstrate ionization of a chromophore leading to identification and quantitation of various components in mixture. The essential feature of difference spectrophotometric assay is that the measured value is the difference absorbance (∆A) between two equimolar solutions of the analyte in different chemical forms, which exhibits different spectral characteristics.

• Derivative spectrophotometry³

Derivative spectrophotometry is useful means of resolving two overlapping spectra and eliminating matrix interference due to an indistinct shoulder on side of an absorption bands. It involves conversion of normal spectrum [A= f (λ)]to its first [dA/ dλ = f (λ)], second [d²A/ dλ² = f (λ)]and higher derivatives spectra where the amplitude in the derivative spectrum is proportional to the concentration of the analyte provided that Beer’s law is obeyed by the fundamental spectrum.

• Area under curve method⁴

In this method, the absorptivity values (ε₁ and ε₂) of each of the two drugs were determined at the selected wavelength range. Total area under curve of a mixture at wavelength range is equal to the sum of area under the individual component at that wavelength range. This method is applicable when the λ _max of the two components are reasonably dissimilar, the two components do not interact chemically and both the component must be soluble in same solvent.
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.

Biopharmaceutical Classification Of Drugs

Biopharmaceutical Classification System:

The biopharmaceutical classification system was developed primarily in the context of immediate release (IR) solid oral dosage forms. It is the scientific framework for classifying drug substances based on their aqueous solubility and intestinal permeability (2). It is a drug development tool that allows estimation of the contributions of three major factors, dissolution, solubility and intestinal permeability that affect oral drug absorption from immediate release solid oral dosage forms. The interest in this classification system is largely because of its application in early drug development and then in the management of product change through its life cycle. It was first introduced into regulatory decision-making process in the guidance document on Immediate Release Solid Oral Dosage Forms: Scale Up And Post Approval Changes (3).

Classification:

According to BCS, drug substances are classified as (Figure 3):

Class I : High Solubility – High Permeability

Class II : Low Solubility – High Permeability

Class III: High Solubility – Low Permeability

Class IV: Low Solubility – Low Permeability

Combined with the dissolution, the BCS takes into account the three major factors governing bioavailability viz. dissolution, solubility and permeability.

This classification is associated with drug dissolution and absorption model, which identifies the key parameters controlling drug absorption as a set of dimensionless numbers viz.

Absorption number, defined as the ratio of the mean residence time to mean absorption time.
Dissolution number, defined as the ratio of mean residence time to mean dissolution time.
Dose number, defined as the mass divided by the product of uptake volume (250 ml) and solubility of drug (4).

Class I drugs exhibit a high absorption number and a high dissolution number. The rate limiting step is drug dissolution and if dissolution is very rapid then gastric emptying rate becomes the rate determining step. e.g. Metoprolol, Diltiazem, Verapamil, Propranolol.

Class II drugs have a high absorption number but a low dissolution number. In vivo drug dissolution is then a rate limiting step for absorption except at a very high dose number. The absorption for class II drugs is usually slower than class II and occurs over a longer period of time. In vitro- In vivo correlation (IVIVC) is usually excepted for class I and class II drugs. e.g. Phenytoin, Danazol, Ketoconazole, Mefenamic acid, Nifedinpine.

For Class III drugs, permeability is rate limiting step for drug absorption. These drugs exhibit a high variation in the rate and extent of drug absorption. Since the dissolution is rapid, the variation is attributable to alteration of physiology and membrane permeability rather than the dosage form factors. e.g. Cimetidine, Acyclovir, Neomycin B, Captopril.

Class IV drugs exhibit a lot of problems for effective oral administration. Fortunately, extreme examples of class IV compounds are the exception rather than the rule and are rarely developed and reach the market. Nevertheless a number of class IV drugs do exist. e.g. Taxol.

Applications of BCS in oral drug delivery technology (5):

Once the solubility and permeability characteristics of the drug are known it becomes an easy task for the research scientist to decide upon which drug delivery technology to follow or develop.

The major challenge in development of drug delivery system for class I drugs is to achieve a target release profile associated with a particular pharmcokinetic and/or pharmacodynamic profile. Formulation approaches include both control of release rate and certain physicochemical properties of drugs like pH-solubility profile of drug.

The systems that are developed for class II drugs are based on micronisation, lyophilization, addition of surfactants, formulation as emulsions and microemulsions systems, use of complexing agents like cyclodextrins.

Class III drugs require the technologies that address to fundamental limitations of absolute or regional permeability. Peptides and proteins constitute the part of class III and the technologies handling such materials are on rise now days.

Class IV drugs present a major challenge for development of drug delivery system and the route of choice for administering such drugs is parenteral with the formulation containing solubility enhancers.

Conclusion:

The in vivo performance of the drug depends upon its solubility and permeability. The biopharmaceutical classification system is the guiding tool for the prediction of in vivo performance of the drug substance and development of drug delivery system to suit that performance. The knowledge of the biopharmaceutical class of the drug substance is also essential for biowaivers thereby reducing the cost both in terms of money and time.

References:

1. Draft Guidance for Industry, Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate Release Solid Oral Dosage Forms containing certain Active Moieties/ Active Ingredients based on a Biopharmaceutic Classification System, February 1999, CDER/FDA.

2. Amidon G.L., Lennernas H., Shah V.P., Crison J.R.A., A Theoretical Basis For a Biopharmaceutic Drug Classification: The Correlation of In Vitro Drug Product Dissolution and In Vivo Bioavailability. Pharm. Res. 12: 413-420 (1995).

3. Guidance for Industry, Immediate Release Solid Oral Dosage Forms: Scale Up and Post Approval Changes, November 1995, CDER/FDA.

4. Medicamento Generico from website http://www.anvisa.go/.

5. Devane J., Oral drug delivery technology: addressing the solubility/ permeability paradigm, Pharm. Technol. 68-74, November 1998.