various uv spectrophotometric simultaneous estimation methods

7/28/2019 Various UV Spectrophotometric Simultaneous Estimation Methods

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Various UV spectrophotometric Simultaneous

estimation methods

Submitted by Anonymous on Tue, 04/04/2006 - 07:00

Prof. S. Saraf

Modern medicines for human use are required to standards which relate to their quality, safety and

efficacy (quantity of the active ingredient). The evaluation of safety and efficacy and their maintenance

in practice is dependent upon the existence of adequate methods for quality control of the product. The

standard of purity must, therefore, be strictly defined in such a way as to ensure that successive batches

are consistent in composition, irrespective of whether they come from the same or different

manufactures.

The multicomponent formulations have gained a lot of importance now a day due to greater patient

acceptability, increased potency and decreased side effects. The quantitative analysis of such

multicomponent formulations is very important. One of the quantitative procedures for

multicomponent formulations is the simultaneous spectrophotometric method, which utilizes the

measurement of intensity of electromagnetic radiation emitted or absorbed by the analyte. This review

contains the various simultaneous estimation methods which are employed for the quantitative

estimation of multicomponent formulations. The spectrophotometer has become a useful instrument

for drug analysis. Now it is the instrument of choice in conducting quantitative estimation of colored and

colorless solutions. The spectrophotometers are based on the principle of over determination, where

the number of observation wavelengths may exceed the number of components present. The

spectrophotometers have an inbuilt microprocessor for spectral data processing. The instrument

computes accurate results within minimal time. The concentrations of each of the component in the

mixtures are printed through an inbuilt system. This article also covers the method validation which

ensures that the selective method will give reproducible and reliable results adequate for intended

purpose. It is therefore necessary to defies precisely both the condition in which the procedures are to

be used and the purpose for which it is intended.

INTRODUCTION

The quantitative estimation is the method to determine how much of each constituent is in the

sample1-2. Estimation of a given drug or medicine in the dosage forms needs the quantitative analysis ofthat drug or medicinal in it. The first quantitative analyses were gravimetric, made possible by the

invention of a precise balance. It was soon found that carefully calibrated glassware made considerable

saving of time through the volumetric measurement of gravimetrically standardized solutions.


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In contrast to above two techniques namely gravimetric and volumetric analysis, methods of drug

analysis today utilize rather sophisticated instruments involving simple physico-chemical properties.

In instrumental analysis, a physical property of a drug is utilized to determine its chemical composition.

A study of the physical properties of drug molecules is a prerequisite for product formulation and often

leads to a better understanding of the inter-relationship between molecular structure and drug action.These properties may be thought of as either additive or constitutive such as mass is an additive

property. Many physical properties are constitutive and yet have some measure of additivity such as

specific gravity, surface tension and viscosity. Molar refraction of a compound is the sum of the

refraction of the atoms and groups making up the compound. The arrangements of atoms in each group

are different however and so the refractive index of two molecules will be different; that is the

individual groups in two different molecules contribute different amounts to the overall refraction of the

molecules. In the instrumental analysis some physical properties of molecules such as absorption of

radiation, scattering of radiation, Raman Effect, emission of radiation, rotation of the plane of the

polarized light and diffraction phenomenon are involving interaction with the radiant energy. Physical

properties encompass specific relations between the molecules and well defined forms of energy e.g.Half-cell potential, current voltage, electric conductivity, dielectric constant, heat of reaction, thermal

conductivity or other yardsticks of measurements. By carefully associating specific physical properties

with the chemical nature of closely related molecules. Conclusions can be drawn that (1) Describe the

spatial arrangement of drug molecules (2) Provide evidence for the relative chemical or physical

behavior of a molecule and (3) Suggest methods for quantitative and qualitative analysis of a particular

pharmaceutical agents3,4.

Analytical methods, in a broad sense, can be classified into chemical methods and instrumental

methods. Chemical methods are defined as those that depend on chemical operations in combination

with the manipulation of simple instruments. In general, the measurement of mass, i.e. gravimetric andof volume, i.e., volumetric analysis falls in this class. An instrumental method encompasses the use of

more complicated instrumentation based on analytical methods:

Although in recent years, spectrophotometric methods are extensively used, but it would be wrong to

conclude that instrumental methods have totally replaced chemical methods. In fact, chemical steps are

often an integral part of an instrumental method. The sampling, dissolution, change in oxidation state,

removal of excess reagent, pH adjustment, addition of complexing agent, precipitation, concentration

and the removal of interferences are the various chemical steps which are part of an instrumental

method. In recent years HPLC5 (High Performance Liquid Chromatography) is extensively used, because

HPLC is not limited by sample volatility or thermal stability. HPLC is able to separate macromolecules

and ionic species, labile natural products, polymeric material and a wide variety of other high molecular

weight poly-functional group because of the relatively high pressure necessary to perform this type of

chromatography; a more elaborate experimental setup is required.

Because of the high cost of the instrument and costly analytical process, small-scale industries cannot

afford to procure and use HPLC. So, in spite of other advantages of HPLC, we often select the

spectrophotometric method of analysis. The variation of the color of a system with change in


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concentration of some component forms the basis of what the chemists commonly term as colorimetric

analysis. The color is usually due to the formation of a colored compound by the addition of an

appropriate reagent, or it may be inherent in the constituent itself. Colorimetry is concerned with the

determination of the concentration of a substance by measurement of the relative absorption of light

with respect to a known concentration of the substance. Colorimetric determinations are usually made

with a simple instrument termed a colorimeter.

In spectrophotometric analysis a source of radiation is used that extend into the ultraviolet region of the

spectrum. From this, definite wavelengths of radiation are chosen possessing a bandwidth of less than 1

nm. This process necessitates the use of a more complicated and consequently more expensive

instrument. All atoms and molecules are capable of absorbing energy in accordance with certain

restrictions; these limitations depend upon the structure of the substance. Energy may be furnished in

the form of electromagnetic radiation (light). The kind and amount of radiation absorbed by a molecule

depend upon the structure of the molecule, the amount of radiation absorbed also depend upon the

number of molecules interacting with the radiation. The study of these dependencies is called

absorption spectroscopy.

THEORY OF SPECTROPHOTOMETRY AND COLORIMETRY

Wavelength and Energy:

Absorption and emission of radiant energy by molecules and atoms is the basis for optical spectroscopy.

By interpretation of these data both qualitative and quantitative information can be obtained.

Qualitatively, the positions of the absorption and emission lines or bands, which occur in the

electromagnetic spectrum, indicate the presence of a specific substance. Quantitatively, the intensities

of the some absorption and emission lines or bands for the unknown and standards are measured. The

concentrations of the unknown is then determined from these data6,7.

The absorption and the emission of energy in the electro-magnetic spectrum occur in discrete packets of

photons. The relation between the energy of a photon and the frequency appropriate for the

description of its propagation is

E = hv

Where E = Energy in ergs

v = Represents frequency in cycles per second

h = Plank's constant (6.6256 x 10-27 erg-sec)


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The data obtained from a spectroscopic measurement are in the form of a plot of radiant absorbed or

emitted as a function of position in the electromagnetic spectrum. This is known as a spectrum and the

position of absorption or emission is measured in units of energy, wavelength or frequency8.

Beer-Lamberts law:

Colorimetry is the determination of the light absorbing capacity of a system. A quantitative

determination is therefore, carried out by subjecting a colored solution to those wavelengths of visible

energy which are absorbed by that solution. UV and visible absorption bands are due to electronic

transitions in the region of 200 nm to 780 nm. In case of organic molecules, the electronic transitions

could be ascribed to a s, p or n electron transition from the ground state to an excited state (s*, p* or

n*).There are four types of absorption bands that occur due to the electronic transition of a

molecule9,10:

R - Bands: n p*, in compounds with C=O or NO2 group

k - Bands: p p*, in conjugated systems.

b - Bands (Benzenoid bands): Due to aromatic and heteroaromatic systems

E - Bands (ethylenic bands): In aromatic systems.

When light (monochromatic or heterogeneous) falls upon a homogeneous medium, a portion of the

incident light is reflected, a portion is absorbed within the medium and the remainder is transmitted. If

the intensity of the incident light is expressed by I, that of the absorbed light by Ia, that of the

transmitted light by It, and that of the reflected light by Ir, then:

I = Ia + It + Ir .......................... (1)

Credit for investigating the change of absorption of light with the thickness of the medium is frequently

given to Lambert; Beer later applied similar experiments to solutions of different concentrations and


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published his results. The two separate laws governing absorption are usually known as Lambert's law

and Beer's law. In the form they are referred to as the

Beer-Lambert law. Mathematically, the radiation-concentration and radiation-path-length relation can

be expressed by11

...................... (2)

The more familiar equation used in spectrometry

log (I/I) = cl .........................(3)

Where I is the intensity of the incident energy

I is the intensity of the emergent energy

c is the concentration

l is the thickness of the absorber (in cm)

and is the molar absorbtivity for concentration in moles/L

, which is encountered less frequently in the literature, represents a concentration of 1% w/v and 1 cmcell thickness and is used primarily in the investigation of those substances of unknown or undetermined

molecular weight. A typical UV absorption spectrum, shown in fig. 1, is the result of plotting wavelength

v/s absorbtivity, max is denoted by lmax.


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Fig. 1.1: A representative Beer-Lambert law plot

PRINCIPLE OF QUANTITATIVE SPECTROPHOTOMETRIC ASSAY OF MEDICINAL SUBSTANCES12:

The assay of an absorbing substance may be quickly carried out by preparing a solution in a transparent

solvent and measuring its absorbance at a suitable wavelength. The concentration of the absorbing

substance calculated from the measured absorbance using one of three principal procedures.

Use of a standard absorbtivity value:

This procedure is adopted by official compendia, e.g. British Pharmacopoeia, for substances such as

methyl testosterone that has reasonably broad absorption variation of instrumental parameters e.g. slit

width, scan speed.

Use of a calibration graph:

In this procedure the absorbances of a number (typically 4-6) of standard solutions of the reference

substance at concentrations encompassing the sample concentrations are measured and a calibration

graph is constructed. The concentration of the analyte in the sample solution is read from the graph as

the concentration corresponding to the absorbance of the solution.

Single or double point standardization:

The single-point procedure involves the measurement of the absorbance of a sample solution and of a

standard solution of the reference substance. The standard and sample solutions are prepared in a

similar manner. Ideally, the concentration of the standard solution should be close to that of the sample

solution. A 'two-point bracketing' standardization is therefore required to determine the concentration

of the sample solutions. The concentration of one of the standard solutions is greater than that of the

sample while the other standard solution has a lower concentration than the sample.


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DIFFERENT SPECTROPHOTOMETRIC SIMULTANEOUS ESTIMATION METHODS FOR MULTICOMPONENT

SAMPLES13

The spectrophotometric assay of drugs rarely involves the measurement of absorbance of sample

containing only one absorbing component. The pharmaceutical analyst frequently encounters the

situation where the concentration of one or more substances is required in samples known to contain

other absorbing substances, which potentially interfere in the assay.

The basis of all the spectrophotometric techniques for multi-component samples is the property that all

wavelengths:

(a)The absorbance of a solution is the sum of absorbances of the individual components; or

(bThe measured absorbance is the difference between the total absorbance of the solution in the

sample cell and that of the solution in the reference (blank) cell.

In multi-component formulations the concentration of the absorbing substance is calculated from the

measured absorbance using one of the following procedures:

(a)Assay as a single-component sample: The concentration of a component in a sample which contains

other absorbing substances may be determined by a simple spectrophotometric measurement of

absorbance, provided that the other components have a sufficient small absorbance at the wavelength

of measurement.

(b) Assay using absorbance corrected for interference: If the identity, concentration and absorbtivity ofthe absorbing interferents are known, it is possible to calculate their contribution to the total

absorbance of a mixture.


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(c) Simultaneous equation method: If a sample contains two absorbing drugs (X and Y) each of which

absorbs at the lmax of the other, it may be possible to determine both drugs by the technique of

simultaneous equations (Vierodt's method).

Where:

a) The absorptivity of X at 1 and 2, ax1 and ax2 respectively.

b) The absorptivity of Y at 1 and 2, ay1 and ay2 respectively.

c) The absorbances of the diluted sample at 1 and 2, A1 and A2 respectively.

Criteria for obtaining maximum precision, based upon absorbance ratios that place limits on the relative

concentrations of the components of the mixture.

The criteria are that the ratios

should lie outside the range 0.1- 2.0 for the precise determination of Y and X respectively. These criteria

are satisfied only when the max of the two components are reasonably dissimilar. An additional

criterion is that the two components do not interact chemically.

To reduce the random errors during measurements, sometimes instead of carrying out analysis of two

components at two wavelengths, it is carried out at 3 or 4 wavelengths. The equations will no longer

have a unique solution but the best solution can be find out by the least square criterion.


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(d) Absorbance ratio method14: The absorbance ratio method is a modification of the simultaneous

equations procedure. It depends on the property that, for a substance, which obeys Beer's law at all

wavelengths. Q-analysis is based on the relationship between absorbance ratio value of a binary mixture

and relative concentrations of such a mixture. The ratio of two absorbance determined on the samesolution at two different wavelengths is constant. This constant was termed as Hufners Quotient or Q-

value which is independent of concentration and solution thickness e.g. two different dilutions of the

same substances give the same absorbance ratio A1/ A2. in the USP this ratio is referred to as a Q value.

In the quantitative assay of two components in admixture by the absorbance ratio method, absorbance

are measured at two wavelengths, one being the max of one of the components ( 2) and the other

being a wavelength of equal absorptivity of the two components ( 1), an iso-absorptive point.

Cx = Qm Qy / Qx Qy . A1/ ax1

Equation gives the concentration of X in terms of absorbance ratios, the absorbance of the mixture and

the absorptivity of the compounds at the iso-absorptive wavelengths. Accurate dilutions of the sample

solution and of the standard solutions of X and Y are necessary for the accurate measurement of A1 and

A2 respectively.

(e) Geometric correction method: A number of mathematical correction procedures have been

developed which reduce or eliminate the background irrelevant absorption that may be present insamples of biological origin. The simplest of this procedure is the three point geometric procedure,

which may be applied if the irrelevant absorption is linear at the three wavelengths selected. If the

wavelengths 1, 2 and 3 are selected to that the background absorbances B1, B2 and B3 are linear,

then the corrected absorbance D of the drug may be calculated from the three absorbances A1, A2 and

A3of the sample solution at 1, 2 and 3 respectively as follows

Let vD and wD be the absorbance of the drug alone in the sample solution at 1 and 3 respectively, i.e.

v and w are the absorbance ratios vD/D and wD/D respectively.

B1 = A1 vD, B2 = A2D and B3 = A3wD

Let y and z be the wavelengths intervals ( 2 1) and ( 3- 2) respectively


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D= y(A2 -A3) + z(A2 A1) / y (1-w) + z(1-v)

This is a general equation which may be applied in any situation where A1, A2 and A3 of the sample, thewavelength intervals y and z and the absorbance ratio v and w are known.

(f) Orthogonal polynomial method15: The technique of orthogonal polynomials is another

mathematical correction procedure, which involves more complex calculations than the three-point

correction procedure. The basis of the method is that an absorption spectrum may be represented in

terms of orthogonal functions as follows

A( ) = p P ( ) p1 P1 ( ) p2 P2 ( ) .. pn Pn ( )

Where A denotes the absorbance at wavelength belonging to a set of n1 equally spaced wavelengths

at which the orthogonal polynomials, P ( ) , P1 ( ), P2 ( ) .. Pn ( ) are each defined.

The accuracy of the orthogonal functions procedure depends on the correct choice of the polynomial

order and the set of the wavelengths. Usually, quadratic or cubic polynomials are selected depending on

the shape of the absorption spectra of the drug and the irrelevant absorption. The set of the

wavelengths is defined by the number of wavelengths, the interval and the mean wavelength of the set

( m). Approximately linear irrelevant absorption is normally eliminated using six to eight wavelengths,

although many more up to 20, wavelengths may be required if the irrelevant absorption contains high-

frequency components. The wavelengths interval and m are best obtained from a convulated

absorption curve. This is a plot of the absorptivity coefficient for a specified order of polynomial, a

specified number of wavelengths and a specified wavelengths interval against the m of the set of

wavelengths. The optimum set of wavelengths corresponds with a maximum or minimum in the

convoluted curve of the analyte and with a coefficient of zero in the convoluted curve of the irrelevant

absorption. In favorable circumstances the concentration of an absorbing drug in admixture withanother may be calculated if the correct choice of polynomial parameters is made, thereby eliminating

the contribution of the drug from the polynomial of the mixture.

(g)Difference spectrophotometry16-20: Difference spectroscopy provides a sensitive method for

detecting small changes in the environment of a chromophore or it can be used to demonstrate


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ionization of a chromophore leading to identification and quantitation of various components in a

mixture. The selectivity and accuracy of spectrophotometric analysis of samples containing absorbing

interferents may be markedly improved by the technique of difference spectrophotometry. The

essential feature of a difference spectrophotometric assay is that the measured value is the difference

absorbance ( A) between two equimolar solutions of the analyte in different forms which exhibit

different spectral characteristics.

The criteria for applying difference spectrophotometry to the assay of a substance in the presence of

other absorbing substances are that:

A) Reproducible changes may be induced in the spectrum of the analyte by the addition of one or more

reagents.

B) The absorbance of the interfering substances is not altered by the reagents.

The simplest and most commonly employed technique for altering the spectral properties of the analyte

properties of the analyte is the adjustment of the pH by means of aqueous solutions of acid, alkali or

buffers. The ultraviolet-visible absorption spectra of many substances containing ionisable functional

groups e.g. phenols, aromatic carboxylic acids and amines, are dependent on the state of ionization of

the functional groups and consequently on the pH of the solution.

If the individual absorbances, Aalk and Aacid are proportional to the concentration of the analyte and

path length, the A also obeys the Beer-Lambert law and a modified equation may be derived

A = abc

Where a is the difference absorptivity of the substance at the wavelength of measurement.

If one or more other absorbing substances is present in the sample which at the analytical absorbance

Ax in the alkaline and acidic solutions, its interference in the spectrophotometric measurement is

eliminated


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A = (Aalk Ax) (Aacid + Ax)

The selectivity of the A procedure depends on the correct choice of the pH values to induce the

spectral change of the analyte without altering the absorbance of the interfering components of the

sample. The use of 0.1M sodium hydroxide and 0.1M hydrochloric acid to induce the A of the analyte

is convenient and satisfactory when the irrelevant absorption arises from pH-insenstive substances.

Unwanted absorption from pH-sensitive components of the sample may also be eliminated if the pKa

values of the analyte and interferents differ by more than 4.

(h) Derivative spectrophotometry: Direct spectrophotometric determination of multicomponent

formulation is often complicated by interference from formulation matrix and spectral overlapping; such

interferences can be treated in many ways like solving two simultaneous equations, using absorbance

ratios at certain wavelengths, but still may give erroneous results21. Other approaches include PH

induced differential least squares22 and orthogonal function methods23. Also the compensation

technique can be used to detect and eliminate unwanted or irrelevant absorption. Derivative

spectrophotometry is a useful means of resolving two overlapping spectra and eliminating matrix

interferences or interferences due to an indistinct shoulder on side of an absorption band24. Derivative

spectrophotometry involves the conversion of a normal spectrum to its first, second or higher derivative

spectrum. In the context of derivative spectrophotometry, the normal absorption spectrum is referred

to as the fundamental, zeroth order or D spectrum. The absorbance of a sample is differentiated with

respect to wavelength to generate first, second or higher order derivative

*A+ = f ( ): zero order

*dA/d + = f ( ): first order

*d2A/d 2+ = f ( ): second order

The first derivative spectrum of an absorption band is characterized by a maximum, a minimum, and a

cross-over point at the max of the absorption band. The second derivative spectrum is characterized

by two satellite maxima and an inverted band of which the minimum corresponds to the max of the

fundamental band.


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The spectral transformation confer two principal advantages on derivative spectrophotometry, firstly an

even order spectrum is of narrower spectral bandwidth than its fundamental spectrum, secondly

derivative spectrophotometry discriminates in favor of substances of narrow spectral bandwidth

substances. This is because the derivative amplitude.

The enhanced resolution and bandwidth discrimination increases with increasing derivative order.

However it is also found that the concomitant increase in electronic noise inherent in the generation of

the higher order spectra, and the consequent reduction of the signal to noise ratio, place serious

practical limitations on the higher order spectra.

The important features of derivative technique include enhanced information content, discrimination

against back ground noise and greater selectivity in quantitative analysis. It can be used for detection

and determination of impurities in drugs, chemicals and also in food additives and industrial wastes25.

(i)Least square approximation26-28: Occasionally one finds it advisable to admit that experimental

measurements are not as accurate as might be desired, but are subject to random errors. An answer

with higher probable accuracy can be obtained if excess experimental information is applied. Instead of

carrying out analysis of two components at two wavelengths, it is carried out at three or four

wavelengths. If it is carried out at three wavelengths the problem becomes the solution of three

equations in two unknowns. This can not be carried out by any other method. The best solution is theleast square criterion, which is found by multiplying by transponse of the absorptivity matrix. This now

gives two equations in two unknowns, such that the solution to these two equations is also the optimum

solution to the three original equations. The method yields a higher precision of determinations for

systems whose absorption spectra are very similar. With increasing diversity of the absorption curves,

the efficiency of the method of measurements taken at a large number of wavelengths i.e. the method

of an over-determined system of linear equations, decreases and for systems with highly diversified

curves it may even deteriorate the precision of the determination.

All the foregoing methods of calculating the content of individual component in a multicomponent

analysis fail to use the entire information capacity of the spectrophotometric method of analysis. Only

the method that stores the whole spectra of standard substance in the computer memory and uses the

algorithm matching the absorption spectrum of the sample with the spectrum obtained mathematically

by adding up the individual spectra of components makes a full use of the information load of the

spectrophotometric method. This is also the operating principle of advanced design uv-vis

spectrophotometers equipped with multicomponent analysis program.


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SELECTION PARAMETERS OF AN ANALYTICAL METHOD

Once the problem is defined the following important factors are considered in choosing the analyticalmethod. These are concentration range, required accuracy and sensitivity, selectivity time requirements

and cost of analysis.

Concentration Range:

The ability to match the method to the optimum sample size is usually gained through experience and

awareness of the different methods.

Sensitivity, as it applied to an analytical method, corresponds to the minimum concentration or lowest

concentration of a substance that is detectable with a specified reliability. It is often expressed

numerically as a detection limit or sensitivity. Different analytical methods will provide different

sensitivities and the one chosen will depend on the sensitivity that is required to solve a particular

problem. Accuracy refers to the correctness of the result achieved by the analytical method.

Selectivity:

Selectivity is an indication of the preference that a particular method shows for one substance over

another.

Time and Cost:

Time and cost often go hand in hand usually are a reflection of the equipment, personnel and space

required to complete a determination.

ANALYTICAL METHOD VALIDATION


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Regulatory perspective: In the US, there was no mention of the word validation in the cGMPs of 1971,

and precision and accuracy were stated as laboratory controls. It was only in the cGMP guidelines of

March 28, 1979, that the need for validation was implied. It was done in two sections:

i)Section 211.165, it was the word validation was used and

ii)Section 211.194, in which the proof of suitability,

Accuracy and reliability was made compulsory for regulatory submission. Subsequently a guideline was

issued on 1st February, 1987, for submitting samples and analytical data for method validation. Theworld health organization (WHO) published a guideline under the title validation of analytical

procedures used in the examination of pharmaceutical materials. It appeared in the 32nd report of the

WHO expert committee on specifications for pharmaceutical preparations which was published in 1992.

The international conference on harmonization (ICH) which has been on the forefront of developing the

harmonized tripartite guidelines under the titles text on validation of analytical procedures (Q2A) and

Validation of analytical procedures: methodology (Q2B). on 1st march,1999, the FDA published a final

guideline on the validation of analytical procedures. The contents of this guideline were prepared under

the auspices of the technical requirements for registration of pharmaceuticals for human use. Accordingto section 501 of the federal food, drugs and cosmetics act, assays and specifications in monographs of

the USP and the NF constitutes legal standards. As a result every analytical method should be validated

according to the current pharmacopoeial standards.

The ability to provide timely, accurate, and reliable data is central to the role of analytical chemists and

is especially true in the discovery, development, and manufacture of pharmaceuticals. Analytical data

are used to screen potential drug candidates, aid in the development of drug syntheses, support

formulation studies, monitor the stability of bulk pharmaceuticals and formulated products, and testfinal products for release. The quality of analytical data is a key factor in the success of a drug

development program. The process of method development and validation has a direct impact on the

quality of these data.


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Although a thorough validation cannot rule out all potential problems, the process of method

development and validation should address the most common ones. Examples of typical problems that

can be minimized or avoided are synthesis impurities that co elute with the analyte peak in an HPLC

assay; a particular type of column that no longer produces the separation needed because the supplier

of the column has changed the manufacturing process; an assay method that is transferred to a second

laboratory where they are unable to achieve the same detection limit; and a quality assurance audit of avalidation report that finds no documentation on how the method was performed during the validation.

Problems increase as additional people, laboratories, and equipment are used to perform the method.

When the method is used in the developer's laboratory, a small adjustment can usually be made to

make the method work, but the flexibility to change it is lost once the method is transferred to other

laboratories or used for official product testing. This is especially true in the pharmaceutical industry,

where methods are submitted to regulatory agencies and changes may require formal approval before

they can be implemented for official testing. The best way to minimize method problems is to perform

adequate validation experiments during development.

Method Validation

Method validation is the process of proving that an analytical method is acceptable for its intended

purpose. For pharmaceutical methods, guidelines from the United States Pharmacopeia (USP) 29,

International Conference on Harmonization (ICH) 30, and the Food and Drug Administration (FDA) 31,32

provide a framework for performing such validations. In general, methods for regulatory submission

must include studies on specificity, linearity, accuracy, precision, range, detection limit, quantitation

limit, and robustness. Although there is general agreement about what type of studies should be done,

there is great diversity in how they are performed 33. The literature contains diverse approaches to

performing validations 34-35. Validation requirements are continually changing and vary widely,

depending on the type of drug being tested, the stage of drug development, and the regulatory group

that will review the drug application. In the early stages of drug development, it is usually not necessary

to perform all of the various validation studies. Many researchers focus on specificity, linearity,

accuracy, and precision studies for drugs in the preclinical through Phase II (preliminary efficacy) stages.

The remaining studies are performed when the drug reaches the Phase III (efficacy) stage of

development and has a higher probability of becoming a marketed product. The process of validating a

method cannot be separated from the actual development of the method conditions, because the

developer will not know whether the method conditions are acceptable until validation studies are

performed. The development and validation of a new analytical method may therefore be an iterative

process. Results of validation studies may indicate that a change in the procedure is necessary, which

may then require revalidation. During each validation study, key method parameters are determined

and then used for all subsequent validation steps. To minimize repetitious studies and ensure that the


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validation data are generated under conditions equivalent to the final procedure, we recommend the

following sequence of studies.

Establish minimum criteria:

The first step in the method development and validation cycle should be to set minimum requirements,

which are essentially acceptance specifications for the method. A complete list of criteria should be

agreed on by the developer and the end users before the method is developed so that expectations are

clear. For example, is it critical that method precision (RSD) be 2%? Does the method need to be

accurate to within 2% of the target concentration? During the actual studies and in the final validation

report, these criteria will allow clear judgment about the acceptability of the analytical method. The

statistics generated for making comparisons are similar to what analysts will generate later in the

routine use of the method and therefore can serve as a tool for evaluating later questionable data. Morerigorous statistical evaluation techniques are available and should be used in some instances, but these

may not allow as direct a comparison for method troubleshooting during routine use.

Specificity:

Specificity is the ability of the method to accurately measure the analyte response in the presence of all

potential sample components. The response of the analyte in test mixtures containing the analyte and

all potential sample components (placebo formulation, synthesis intermediates, excipients, degradation

products, process impurities, etc.) is compared with the response of a solution containing only the

analyte. If an analytical procedure is able to separate and resolve the various components of a mixture

and detect the analyte quantitatively, the method is called selective. If the method determines or

measures quantitatively the compound of interest in the sample matrix without separation it is said to

be specific. Measuring a methods specificity is extremely important during the validation of non-

chromatographic methods because they do not contain a separation step that ensures non-interference

from Excipients. They rely on intrinsic differences in chemical or physical properties to ensure their

ability to accurately determine the concentration of analyte in complex sample mixture.

Linearity:

A linearity study verifies that the sample solutions are in a concentration range where analyte response

is linearly proportional to concentration. For assay methods, this study is generally performed by


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preparing standard solutions at five concentration levels, from 50 to 150% of the target analyte

concentration; at least six replicates per concentration must be used. The 50 to 150% range for this

study is wider than what is required by the FDA guidelines. In the final method procedure, a tighter

range of three standards is generally used, such as 80, 100, and 120% of target; and in some instances, a

single standard concentration is used. Validating over a wider range provides confidence that the

routine standard levels are well removed from nonlinear response concentrations, that the methodcovers a wide enough range to incorporate the limits of content uniformity testing, and that it allows

quantitation of crude samples in support of process development. For impurity methods, linearity is

determined by preparing standard solutions at five concentration levels over a range such as 0.05-2.5

wt%. Acceptability of linearity data is often judged by examining the correlation coefficient and y-

intercept of the linear regression line for the response versus concentration plot. A correlation

coefficient of > 0.999 is generally considered as evidence of acceptable fit of the data to the regression

line. The y-intercept should be less than a few percent of the response obtained for the analyte at the

target level. Although these are very practical ways of evaluating linearity data, they are not true

measures of linearity 36-37. These parameters, by themselves, can be misleading and should not be

used without a visual examination of the response versus concentration plot.

Accuracy:

The accuracy of a method is the closeness of the measured value to the true value for the sample.

Accuracy is usually determined in one of four ways. First, accuracy can be assessed by analyzing a

sample of known concentration and comparing the measured value to the true value. National Institute

of Standards and Technology (NIST) reference standards are often used; however, such a well-characterized sample is usually not available for new drug-related analytes. The second approach is to

compare test results from the new method with results from an existing alternate method that is known

to be accurate. Again, for pharmaceutical studies, such an alternate method is usually not available. The

third and fourth approaches are based on the recovery of known amounts of analyte spiked into sample

matrix. The third approach, which is the most widely used recovery study, is performed by spiking

analyte in blank matrices. For assay methods, spiked samples are prepared in triplicate at three levels

over a range of 50--150% of the target concentration. If potential impurities have been isolated, they

should be added to the matrix to mimic impure samples. For impurity methods, spiked samples are

prepared in triplicate at three levels over a range that covers the expected impurity content of the

sample, such as 0.1--2.5 wt%. The analyte levels in the spiked samples should be determined using thesame quantitation procedure as will be used in the final method procedure (i.e., same number and

levels of standards, same number of sample and standard injections, etc.). The percent recovery should

then be calculated. The fourth approach is the technique of standard additions, which can also be used

to determine recovery of spiked analyte. This approach is used if it is not possible to prepare a blank

sample matrix without the presence of the analyte. This can occur, for example, with lyophilized

material, in which the speciation in the lyophilized material is significantly different when the analyte is


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absent. An example of an accuracy criteria for an assay method is that the mean recovery will be 100 +

2% at each concentration over the range of 80--120% of the target concentration. For an impurity

method, the mean recovery will be within 0.1% absolute of the theoretical concentration or 10%

relative, whichever is greater, for impurities in the range of 0.1--2.5 wt%.

Determine the range:

The range of an analytical method is the concentration interval over which acceptable accuracy,

linearity, and precision are obtained. In practice, the range is determined using data from the linearity

and accuracy studies. The following minimum specified ranges should be considered:

1) For the assay of an active substance or a finished product normally from 80-120% of the testconcentration.

2) For the determination of an impurity; from reporting level of an impurity to 120% of the specification.

3) For content uniformity; covering minimum range is from 70-130%of the test concentration, unless a

wider more range based on the nature of the dosage form.

4)For dissolution testing + 20% over the specified range.

5) If assay and purity are performed together as one test and only a 100 % standard is used, linearity

should cover the range from reporting level of the impurities to 120% of the assay specification.

Determine precision:

The precision of an analytical method is the amount of scatter in the results obtained from multiple

analyses of a homogeneous sample. To be meaningful, the precision study must be performed using the

exact sample and standard preparation procedures that will be used in the final method.


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The first type of precision study is instrument precision or repeatability. In this a method when reported

by the same analyst, same test method and under same set of laboratory conditions within a short

interval of time, also known as intra-assay precision.

Second type of precision is reproducibility; the measure of test methods variability when carried out by

different analysts in different laboratories using different equipments, reagents and laboratory settings

and on different days. It is assessed by means of an inter-laboratory crossover studies. Reproducibility

should be considered in case of the standardization of an analytical procedure, for instance, for inclusion

of procedures in pharmacopoeias.

Scope:

Once these validation studies are complete, the method developers should be confident in the ability of

the method to provide good quantitation in their own laboratories. This result may be sufficient for

many methods, especially in the early phases of drug development. The remaining studies should

provide greater assurance that the method will work well in other laboratories, where different

operators, instruments, and reagents are involved and where it will be used over much longer periods of

time. This is a good time to begin accumulating data for two or more system suitability criteria, which

are required prior to routine use of the method to ensure that it is performing appropriately.

Detection limit:

The detection limit of a method is the lowest analyte concentration that produces a response detectable

above the noise level of the system, typically, three times the noise level. It is a limit test where

concentrations below this may not be detected while concentrations above this limit are certainly

detected in analysis. The detection limit should be estimated early in the method development-

validation process and should be repeated using the specific wording of the final procedure if anychanges have been made. It is important to test the method detection limit on different instruments,

such as those used in the different laboratories to which the method will be transferred. An example of

a detection limit criteria is that, at the 0.05% level, an impurity will have S/N = 3.

Quantitation limit:


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The quantitation limit is the lowest level of analyte that can be accurately and precisely measured. This

limit is required only for impurity methods and is determined by reducing the analyte concentration

until a level is reached where the precision of the method is unacceptable. If not determined

experimentally, the quantitation limit is often calculated as the analyte concentration that gives S/N =10. An example of quantitation limit criteria is that the limit will be defined as the lowest concentration

level for which an RSD 20% is obtained when an intra-assay precision study is performed.

Stability:

During the earlier validation studies, the method developer gained some information on the stability of

reagents, standards, and sample solutions. For routine testing in which many samples are prepared and

analyzed each day, it is often essential that solutions be stable enough to allow for delays such as

instrument breakdowns or overnight analyses. At this point, the limits of stability should be tested.

Samples and standards should be tested over at least a 48-h period, and quantitation of components

should be determined by comparison to freshly prepared standards. If the solutions are not stable over

48 h, storage conditions or additives should be identified that can improve stability. An example of

stability criteria for assay methods is that sample and standard solutions and the mobile phase will be

stable for 48 h under defined storage conditions. Acceptable stability is 2% change in standard or sample

response, relative to freshly prepared standards. The mobile phase is considered to have acceptable

stability if aged mobile phase produces equivalent chromatography (capacity factors, resolution, or

tailing factor) and assay results are within 2% of the value obtained with fresh mobile phase. In case ofuv the absorption must be same.

Robustness:

The robustness of a method is its ability to remain unaffected by small changes in parameters such as

percent organic content, pH of the solvent, buffer concentration and temperature. These method

parameters may be evaluated one factor at a time or simultaneously as part of a factorial experiment

38.

The evaluation of the robustness should be considered during the development phase. Often such

testing is not performed as a part of the official method validation during the transfer of the method to

another laboratory.


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CONCLUSION

We can conclude that the simultaneous spectrophotometric methods for quantitative estimation ofpharmaceuticals are fast, less time consuming, reproducible and highly sensitive even microgram of

compound can be measured. Performing a thorough method validation can be a tedious process, but

the quality of data generated with the method is directly linked to the quality of this process. Time

constraints often do not allow for sufficient method validations. Many researchers have experienced the

consequences of invalid methods and realized that the amount of time and resources required to solve

problems discovered later exceeds what would have been expended initially if the validation studies had

been performed properly.

REFERENCES

Vasudevan, R. and Mathai, I. M., Ind. J. Chem., 1972, 10, 175.

Vasudevan, R., Subramanian, P. S. and Mathai, I. M., J. Ind. Chem. Soc., 1984, 61, 395.

Kampli, S. R., Nandibewoor, S. T. and Raju, J. R., Ind. J. Chem, 1990, 29, 908.

Hanumantha, R. K. and Bhaganwanth, R. M., J. Ind. Chem. Soc., 1991, 68,160.

Amjad, Z. and Mc Auley, A.M., J. Am. Chem. Soc., 1977, 99, 304.

Jwo, J. J. and Noyes, R. M., J. Am. Chem. Soc., 1975, 97, 5422.

Amjad, Z. and Mc Auley, A., J. Am. Chem. Soc., 1977, 99, 82.

Richardson, W.H., Oxidation in organic Chemistry K .B. Wiberg, Academic Press, New York Part-A 1965,

245.

Richardson, W.H., Oxidation in organic Chemistry K .B. Wiberg, Academic Press, New York Part-A 1965,

262.

Sethuram, B. and Mushamad, S. S., Acta Chim. Acad. Sci., 1965, 46, 115.

Guelbautt, G. C. and Mc Curdy W. R., J. Phys. Chem., 1963, 67, 283.

Mehrotra, R. N. and Ghos, S., Ind. J. Chem., 1976, 14, 663.

Khanna, P. K. and Krishna, B., Proc. Natl. Acad. Sci., 1977, 12, 478.

Pernarowski, M., Knevel, A. M., and Christian, J.E., J. Pharm. Sci., 1960, 50, 943.

Glenn, A.L., J. Pharm. Pharmacol., 1963, 15, 123.


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Wahbi, A. M. and Farghaly, A. M., J. Pharm. Pharmcol. 1970, 22, 848.

Kartal, M. and Erk, N., J. Pharm. Biomed. Anal., 1999, 19, 477-485.

Erk, N., J. Pharm. Biomed. Anal., 1999, 20, 155-167.

Skoog, A.D. and West M.D., Principles of instrumental analysis, Saunders golden, Japan, 3rd ed., 1985,212-213.

OHaver, D.Y., J. Assoc. of Anal. Chem., 1983, 66(6), 1450.

Porro, T. J., Anal. Chem., 1972, 44(4), 93.

Allen, G.C. and Mc Mecking, R. F., Anal. Chem., 1975, 47, 2124.

Shibata, S., Furukawa, M. and Goto, K., Anal. Chim. Acta., 1973, 65, 49.

Shiga, T., Shiga, K. and Kuroda, M., Anal. Biochem., 1971, 44, 291.

OHaver, T. C. and Green, G. L., Anal. Chem. 1976, 48(2), 313.

Nraxin, Z. and Liming, L., Analyst, 1991, 116(9), 919-922.

Thomas, M., Ultraviolet and visible spectroscopy, John willey and sons ltd. U.K., 2nd ed., 1996, 131-132.

Glenn, A .L., J. Pharm. Pharmcol., 1963,15, 123.

United State Pharmacopeia, 23rd ed., United States Pharmacopeial Convention, Inc., 1994,1982-84.

International Conference on Harmonisation, Draft Guideline on Validation of Analytical Procedures:

Definitions and Terminology, Federal Register, Volume 60, March 1, 1995, 11260.

Reviewer Guidance, Validation of Chromatographic Methods, Center for Drug Evaluation and Research,

Food and Drug Administration, 1994.

Guideline for Submitting Samples and Analytical Data for Methods Validation, Food and Drug

Administration, 1987.

Clarke, G. S., J. Pharm. Biomed. Anal., 1994, 12, 643.

34. Inman, E. L., Frischman, J. K., Jimenez, P. J., Winkel, G. D., Persinger, M. L. and Rutherford, B. S., J.

Chromatogr. Sci., 1987, 25, 252.

Wilson, T. D., J. Pharm. Biomed. Anal., 1990, 8, 389.

Hokanson, G. C., Pharm. Technol.,1994, 18, 118.

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Dorschel, C. A., Ekmanis, J. L., Oberholtzer, J. E., Warren, F. V. and Bidlingmeyer, B. A., Anal. Chem.,

1989, 61, 951.

Gopal Garg, S. J. Daharwal, Swarnlata Saraf and S. Saraf*

Institute of Pharmacy, Pt. Ravi Shankar Shukla University,Raipur (C.G.), 492010, INDIA

Corresponding Author E-mail: [email protected]

Mr. Gopal Garg has nearly 2 years of research and teaching experience. He is a hard working researcher.

Mr. Garg did his masters degree from Dept. of Pharmacy, Dr. H. S. Gour University, SAGAR. He has over

7 publications to his credit published in international and national journals. His research interest extends

from Analytical technique to phytochemical estimation. Presently, he is working as a Lecturer at

Institute of pharmacy Pt. Ravishankar Shukla University, Raipur, (C.G.)

Mr. S.J. Daharwal has nearly 15 years of research and teaching experience. He is a hard working

researcher . Mr . Daharwal did his masters degree from Dept. of Pharmacy, of Nagpur University. He

has over 12 publications to his credit published in international and national journals. His research

interest extends from analytical methods, Drug synthesis and computer added drug designing.

Presently, he is working as a Lecturer at Institute of Pharmacy Pt. Ravishankar Shukla University, Raipur,

(C.G.)

Dr. (Mrs). Swarnlata Saraf has nearly 14 years of research and teaching experience. She is a leading

scientist and well-known in the field of herbal Cosmetics. Dr. (Mrs.) Saraf did her doctoral research at

the Dept. of Pharmacy, Dr. H. S. Gour University, SAGAR. She has over 40 publications to her credit

published in international and national journals. She is an active member of IPA ,APTI and ISTE. Her


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research interest extends from Herbal Cosmetics to transdermal drug delivery (Iontiphoresis), New Drug

Delivery Systems for biological therapeutic agents. She has Co-authored 1 books, (in press). Presently

She is working as a Reader at Institute of pharmacy Pt. Ravishankar Shukla University, Raipur, (C.G.).

Email: [email protected]

Prof. S. Saraf *has nearly 17 years of research and teaching experience at U.G. and P.G. level. He is a

leading scientist and well-known academician . Prof. Saraf did his doctoral research at the Dept. of

Pharmacy, Dr. H. S. Gour University, SAGAR. He has over 50 research publications to his credit published

in international and national journals. He has delivered invited lectures and chaired many sessions in

several National Conferences and Symposia in India. His research interest extends from Herbal

Cosmetics to Herbal drug standardization Modern analytical techniques, New Drug Delivery Systems

with biotechnology bias. He has authored 1 books, in press. Presently, he is Professor and Director

Institute of pharmacy and Dean, Faculty of Technology, Pt. Ravishankar Shukla University , Raipur ,

(C.G.). E-mail: [email protected]

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various uv spectrophotometric simultaneous estimation methods

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