An Introduction To Analytical Method

Development For Pharmaceutical

Formulations
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Rashmin B. Patel

Analytical methods development and validation play important roles

in the discovery, development, and manufacture of
pharmaceuticals.

Pharmaceutical products formulated with more than one drug, typically referred to as

combination products, are intended to meet previously unmet patients need by combining the

therapeutic effects of two or more drugs in one product. These combination products can

present daunting challenges to the analytical chemist responsible for the development and

validation of analytical methods. This presentation will discuss the development and validation

of analytical method (Spectrophotometric, High performance liquid chromatography (HPLC), &

High performance thin layer chromatography (HPTLC)) for drug products containing more than

one active ingredient. The official test methods that result from these processes are used by

quality control laboratories to ensure the identity, purity, potency, and performance of drug

products.

Introduction
The number of drugs introduced into the market is increasing every year. These drugs may be

either new entities or partial structural modification of the existing one. Very often there is a

time lag from the date of introduction of a drug into the market to the date of its inclusion in

pharmacopoeias. This happens because of the possible uncertainties in the continuous and

wider usage of these drugs, reports of new toxicities (resulting in their withdrawal from the

market), development of patient resistance and introduction of better drugs by competitors.

Under these conditions, standards and analytical procedures for these drugs may not be

available in the pharmacopoeias. It becomes necessary, therefore to develop newer analytical

methods for such drugs.

Basic criteria for new method development of drug analysis:

• The drug or drug combination may not be official in any pharmacopoeias,

• A proper analytical procedure for the drug may not be available in the literature due to

patent regulations,
• Analytical methods may not be available for the drug in the form of a formulation due

to the interference caused by the formulation excipients,

• Analytical methods for the quantitation of the drug in biological fluids may not be

available,
• Analytical methods for a drug in combination with other drugs may not be available,

• The existing analytical procedures may require expensive reagents and solvents. It

may also involve cumbersome extraction and separation procedures and these may

not be reliable.

Introduction To Spectrophotometric Methods Of Analysis For Drugs In Combination2

Simultaneous estimation of drug combination is generally done by separation using

chromatographic methods like HPLC, GC and HPTLC etc. These methods are accurate and

precise with good reproducibility, but the cost of analysis is quite high owing to expensive

instrumentation, reagent and expertise. Hence it is worthwhile to develop simpler and cost

effective method for simultaneous estimation of drugs for routine analysis of formulation.

Spectrophotometric analysis fulfils such requirement where the simultaneous estimation of the

drug combination can be done with similar effectiveness as that of chromatographic methods.

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

samples 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.

If the formula of the samples is known, the identity and concentration of the interfering

substance are known and the extent of interference in the assay may be determined.

A number of modifications to the simple spectrophotometric procedure are available to the

analyst, which may eliminate certain sources of interference and permit the accurate

determination of all of the absorbing components. Each modification of the basic procedure

may be applied if certain criteria are satisfied.

The basis of all the spectrophotometric techniques for multicomponent samples is the property

that at all wavelengths:

• the absorbance of a solution is the sum of absorbance of the individual components

or
• the measured absorbance is the difference between the total absorbance of the

solution in the sample cell and that of the solution in the reference cell.

There are various spectrophotometric methods are available which can be used for the

analysis of a combination samples. Following methods can be used

• Simultaneous equation method

• Derivative spectrophotometric method

• Absorbance ratio method ( Q-Absorbance method)

• Difference spectrophotometry

• Solvent extraction method

Simultaneous Equation Method2

If a sample contains two absorbing drugs (X and Y) each of which absorbs at the lmax of the

other (as shown in figure 1. λ1 andλ2), it may be possible to determine both drugs by

the technique of simultaneous equation (Vierodt’s method) provided that certain criteria

apply.

The informations required are:

• the absorptivities of X at λ1 and λ2, ax1 and ax2 respectively

• the absorptivities of Y at λ1 andλ2, ay1 and ay2 respectively

• the absorbance of the diluted sample at λ1 and λ2, A1 and A2 respectively.

Let Cx and Cy be the concentration of X and Y respectively in the diluted samples.

Two equations are constructed based upon the fact that at λ1 andλ2, the absorbance of the

mixture is the sum of the individual absorbance of X and Y.

At λ1

A1 = ax1bCx + ay1bCy ……………. (1)

Atλ2

A2 = ax2bCx + ay2bCy ………. (2)

For measurements in 1 cm cells, b =1.

Rearrange equation (2)

Cy = (A2 - ax2 Cx) / ay2

Substituting for Cy in eq. (1) and rearranging gives

Cx = (A2 ay1 - A1 ay2) / (ax2 ay1 - ax1 ay2)

Cy = (A1 ax2 - A2 ax1) / (ax2 ay1 - ax1 ay2)

Fig. 1: The overlain spectra of substance X and Y, showing the wavelength for the

assay of X and Y in admixture by the method of simultaneous equation.

Criteria for obtaining maximum precision have been suggested by Glenn3. According to him

absorbance ratio place limits on the relative concentrations of the components of the mixture.

(A2/A1) / (ax2/ax1) and (ay2/ay1)/ (A2/A1)

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 λmax of the

two components are reasonably dissimilar. An additional criterion is that the two components

do not interact chemically, thereby negating the initial assumption that the total absorbance is

the sum of the individual absorbance. The additive of the absorbance should always be

confirmed in the development of a new application of this technique.

Simultaneous equation method using Matrices and Cramer's Rule can be explained as follows:
Consider a binary mixture of component X and Y for which the absorption spectra of individual

components and mixture are shown in figure 1.

-1 is the λmax of component X

-2 is the λmax of component Y

the total absorbance of a solution at a given wavelength is equal to the sum of the absorbance

of the individual components at the wavelength. Thus the absorbance of mixture at the

wavelength 1 and 2 may be expressed as follows:

At λ1

A1 = ax1bCx + ay1bCy ………………… (1)

At λ2

A2 = ax2bCx + ay2bCy ………………. (2)

Such equation can be solved using matrices.

From equation (1) and (2),

A1 = kx1 Cx + ky1Cy …………. (3)

A2 = kx2 Cx + ky2Cy ………………. (4)

Where k = a x b

Let A, be a column matrix with 'i' elements [i, is the number of wavelength at which

measurements are done; here two wavelength 1 and 2 are taken in to consideration, so i=2].

Let C, be a column matrix with 'j' elements [j, is the number of components, in this case X and

Y are present, so j = 2]. Let k, be a matrix with i x j values so that it has number of rows

equals to number of wavelength and number of columns equal to number of components ( in

this case it has two rows and two columns). Hence we have

A=kxC …………. (5)

Since the number of wavelength equal to number of components, the equation (5) has a

unique solution.

C = k-1 x A ………….(6)

However, it will be faster to solve the equation (3) and (4) by means of cramer's rule. And

unknown concentration Cj of component j is found by replacing' j column of matrix A. The

determinant of the new matrix is divided by determinant of 'k' matrix.

Cx = (A1 ky2 - A2 ky1) / (kx1 ky2 - kx2 ky1) …………. (7)

Cy = (kx1 A2 - kx2 A1) / ( kx1 ky2 - kx2 ky1) ………….. (8)

Therefore

Cx = (A1 ay2 - A2 ay1) / ( ax1 ay2 - ax2 ay1) ……………(9)

Cy = (ax1 A2 - ax2 A1) / ( ax1 ay2 - ax2 ay1) …………..(10)

In British Pharmacopoeia the assay of quinine-related alkaloids and Cinchonine related

alkaloids are based on this technique.

Following drugs have been reported to be estimated simultaneously by the simultaneous

equation method.

- Estimation of Gliclazide and Metformin hydrochloride in combined dosage forms4.

- Estimation of Losartan potassium and Hydrochlorthiazide in tablets5.

- Estimation of Salbutamol and Theophylline from tablets6.

- Estimation of Amlodipine besylate and Enalepril maleate from tablets7.

Drugs with large difference in the content in the combined dosage form have been estimated

simultaneously by Simultaneous equation method by standard addition technique. In which

known amount of pure drug have been added to the sample drugs.

-Estimation of Ibuprofen and Pseudoephedrine hydrochloride from tablets8.

-Estimation of Salbutamol and Theophylline from tablets6.

Q-Absorbance Method (Absorbance Ratio Method)2

Q-Absorbance method depends on the property that, for a substance which obeys Beer's law

at all wavelength, the ratio of absorbances at any two wavelengths is a constant value
independent of concentration or pathlength. For example, two different dilution 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 a mixture by the absorbance ratio method,

absorbances are measured at two wavelengths. One being the λmax of one of the component

(λ2) and the other being a wavelength of equal absorptivities of the two components (As

shown in figure 2) i.e. an isoabsorptive point9.

Two equations are constructed as described for the method of simultaneous equation. Their

treatment is somewhat different, however, and uses the relationship ax1 = ay1 at λ1. Assume b

= 1cm

A1 = ax1Cx + ax1Cy ……………….. (1)

A2 / A1 = (ax2Cx + ay2Cy) / (ax1Cx + ax1Cy)

Divide each term by Cx + Cy and let Fx = Cx / (Cx + Cy) and Fy = Cy / (Cx + Cy) i.e. Fx and Fy

are the fraction of X and Y respectively in the mixture:

A2 / A1 = (ax2 Fx + ay2Fy) / (ax1Fx + ax1Fy)

But Fy = 1 - Fx

A2 / A1 = (ax2 Fx - Fx ay2 + ay2) / ax1

A2 / A1 = (ax2 Fx)/ ax1 - (Fx ay2)/ ay1 + (ay2) / ay1

Fig. 2: Wavelength for the assay of substances X and Y in admixture by the method

of absorbance ratio method.

Let QX = ax2 / ax1, QY = ay2/ ay1, and QM = A2 / A1

QM = Fx (QX - QY) + QY

Fx = (QM - QY) / (QX - QY) …………. (2)

Above equation gives fraction, rather than the concentration of X in the mixture in terms of

absorbance ratios. As these are independent of concentrations, only approximate, rater than
accurate, dilutions of X and Y and the sample mixture are required to determine QX,QY, and QM

respectively.

For absolute concentration of X and Y, eq. (1) is rearranged

A1 = ax1 (Cx +CY)

Cx +Cy = A1 / ax1

From equation (2)

Cx / (Cx +Cy) = (QM - QY) / (QX - QY)

Cx / (A1 / ax1) = (QM - QY) / (QX - QY)

Cx = (QM - QY) A1 / (QX - QY) ax1

Above equation gives the concentration of X in terms of absorbance ratios, the absorbance of

mixture and the absorptivities of the compounds at the isoabsorptive wavelength.

The British Pharmacopoeia suggests the use of this method as the identification test for

Cyanocobalamin10.

Following drugs have been reported to be estimated simultaneously by the Q- Absorbance

method.

- Estimation of Rifampicin and Isoniazide in pharmaceutical dosage forms11.

- Estimation of Spiranolactone and hydroflumethiazide12.

- Estimation of Nalidixic acid and Metronidazole from tablets13.

- Estimation of Noscapine, Chlorpheniramine Maleate and Ephedrine hydrochloride from

tablets14.

Derivative Spectroscopy 2

For the purpose of spectral analysis in order to relate chemical structure to electronic

transitions, and for analytical situations in which mixture contribute interfering absorption, a

method of manipulating the spectral data is called derivative spectroscopy15.

Derivative spectrophotometry involves the conversions of a normal spectrum to its first,

second or higher derivative spectrum. (As shown in figure 3). In the context of derivative

spectrophotometry, the normal absorption spectrum is referred to as the fundamental, zero

order, or D0 spectrum.

The first derivative D1 spectrum is a plot of the rate of change of absorbance with wavelength

against wavelength i.e. a plot of the slope of the fundamental spectrum against wavelength or

a plot of dA/dλ vs. λ. . The maximum positive and maximum negative slope respectively in the

D spectrum correspond with a maximum and a minimum respectively in the D1 spectrum. The

λmax in D spectrum is a wavelength of zero slope and gives dA/dλ = 0 in the D1 spectrum.
The second derivative D2 spectrum is a plot of the curvature of the D spectrum against

wavelength or a plot of d2A/ dλ2 vs. λ. The maximum negative curvature in the D spectrum

gives a minimum in the D2 spectrum, and the maximum positive curvature in the D spectrum

gives two small maxima called satellite bands in the D2 spectrum. The wavelength of

maximum slope and zero curvature in the D spectrum correspond with cross-over points in the

D2 spectrum.

These spectral transformations confer two principal advantages on derivative

spectrophotometry. Firstly, an eve order spectrum is of narrower spectral bandwidth than its

fundamental spectrum. A derivative spectrum therefore shows better resolution of

overlapping bands than the fundamental spectrum and may permit the accurate determination

of the λmax of the individual bands. Secondly, derivative spectrophotometry discriminates in

favour of substances of narrow spectral bandwidth against broad bandwidth substances. All

the amplitudes in the derivative spectrum are proportional to the concentration of the analyte,

provided that Beer's law is obeyed by the fundamental spectrum.

Fig. 3: (b) First, (c) Second, (d) Third and (e)fourth derivative Spectrum of (a)

Gaussian peak.

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. For quantitative

purposes, second and fourth derivative spectra are the most frequently employed derivative

orders.

Derivative spectra may be generated by any of three techniques. The earliest derivative

spectra were obtained by modification of the optical system. Spectrophotometers with dual
monochromator set a small wavelength interval (Δλ, typically 1-3nm) apart, or with the facility

to oscillate the wavelength over a small range, are required. In either case the photodetector

generates a signal with amplitude proportional to the slope of the spectrum over the

wavelength interval. Instruments of this type are expensive and are essentially restricted to

the recording of first derivative spectra only.

The second technique to generate derivative spectra is electronic differentiation of the

spectrophotometer analog signal. Resistance capacitance (RC) modules may be incorporated

in series between the spectrophotometer and recorder to provide differentiation of the

absorbance, not with respect to wavelength, but with respect to time, thereby producing the

signal dA/dt. If the wavelength scan rate is constant (dλ/dt = C), the derivative with respect

to wavelength is given by

dA/ dλ = (dA/dt) / (dλ /dt) = (dA/dt)(1/C)

Derivative spectra obtained by RC modules are highly dependent on instrumental parameters,

in particular the scan speed and the time constant. It is essential, therefore, to employ a

standard solution of the analyte to calibrate the measured value the instrumental conditions

selected.

The third technique is based upon microcomputer differentiation. Microcomputers incorporated

into or interfaced with the spectrophotometer may be programmed to provide derivative

spectra during or after the scan, to measure derivative amplitudes between specified

wavelengths and to calculate concentrations and associated statistics from the measured

amplitude.

For the estimation of drugs in combinations, simultaneous use of derivative spectroscopy

along with simultaneous equation has been reported in the literature16.

Following drugs have been reported to be estimated simultaneously by the Derivative

spectroscopy method.

-Estimation of Propranolol and Hydrochlorthiazide17.

-Estimation of Phenylpropanolamine, chlorpheniramine and Bromhexine18.

-Estimation of Naphazoline hydrochloride and Chlorpheniramine maleate19.

Drugs with large difference in the content in the combined dosage form have been estimated

simultaneously by Simultaneous equation method by standard addition technique. In which

known amount of pure drug have been added to the sample drugs.

-Estimation of Ibuprofen and Pseudoephedrine hydrochloride from tablets20.

-Estimation of Salbutamol and Theophylline from tablets21.

Solvent Extraction Method 2

In solvent extraction method quantitation of individual drugs in combinations has been

performed by separation of individual drugs based on their selective solubility followed by

spectrophotometric measurement22.

If the interference from the other absorbing substances is large, it may be possible to separate

the absorbing interferent from the analyte by solvent extraction procedure. These are

particularly appropriate for acidic or basic drugs whose state of ionisation determines their

solvent partitioning behavior. The judicious choice of pH of the aqueous medium may effect

the complete separation of the interferents from the analyte, the concentration of which may

be obtained by a simple measurement of absorbance of the extract containing the analyte.

Following drugs have been reported to be estimated by the Solvent extraction method.

-Estimation of Probenecid and Ampicillin from tablets22.

-Estimation of Probenecid and Cephalexine from tablets22.

-Estimation of Caffeine from Aspirin and Caffeine tablets23.

-Estimation of Paracetamol and Diclofenac sodium from tablets24.

Introduction To Hplc Methods Of Analysis For Drugs In Combination 25 - 27

Most of the drugs in multicomponent dosage forms can be analyzed by HPLC method because

of the several advantages like rapidity, specificity, accuracy, precision and ease of automation

in this method. HPLC method eliminates tedious extraction and isolation procedures. Some of

the advantages are:

§ speed (analysis can be accomplished in 20 minutes or less),

§ greater sensitivity (various detectors can be employed),

§ improved resolution (wide variety of stationary phases),

§ reusable columns (expensive columns but can be used for many analysis),

§ ideal for the substances of low volatility,

§ easy sample recovery, handling and maintenance,

§ instrumentation tends itself to automation and quantitation (less time and less labour),

§ precise and reproducible,

§ calculations are done by integrator itself,

§ suitable for preparative liquid chromatography on a much larger scale.

There are different modes of separation in HPLC. They are normal phase mode, reversed

phase mode, reverse phase ion pair chromatography, affinity chromatography and size

exclusion chromatography.

In the normal phase mode, the stationary phase is polar and the mobile phase is nonpolar in

nature. In this technique, nonpolar compounds travel faster and are eluted first. This is
because of the lower affinity between the nonpolar compounds and the stationary phase. Polar

compounds are retained for longer times because of their higher affinity with the stationary

phase. These compounds, therefore take more times to elute. Normal phase mode of

separation is therefore, not generally used for pharmaceutical applications because most of

the drug molecules are polar in nature and hence take longer time to elute.

Reversed phase mode is the most popular mode for analytical and preparative separations of

compound of interest in chemical, biological, pharmaceutical, food and biomedical sciences. In

this mode, the stationary phase is nonpolar hydrophobic packing with octyl or octa decyl

functional group bonded to silica gel and the mobile phase is polar solvent. An aqueous mobile

phase allows the use of secondary solute chemical equilibrium (such as ionization control, ion

suppression, ion pairing and complexation) to control retention and selectivity. The polar

compound gets eluted first in this mode and nonpolar compounds are retained for longer time.

As most of the drugs and pharmaceuticals are polar in nature, they are not retained for longer

times and hence elute faster. The different columns used are octa decyl silane (ODS) or C18,

C8, C4, etc., (in the order of increasing polarity of the stationary phase).

In ion exchange chromatography, the stationary phase contains ionic groups like NR3+ or SO3- ,

which interact with the ionic groups of the sample molecules. This is suitable for the

separation of charged molecules only. Changing the pH and salt concentration can modulate

the retention.

Ion pair chromatography may be used for the separation of ionic compounds and this method

can also substitute for ion exchange chromatography. Strong acidic and basic compounds may

be separated by reversed phase mode by forming ion pairs (coulumbic association species

formed between two ions of opposite electric charge) with suitable counter ions. This

technique is referred to as reversed phase ion pair chromatography or soap chromatography.

Affinity chromatography uses highly specific biochemical interactions for separation. The

stationary phase contains specific groups of molecules which can adsorb the sample if certain

steric and charge related conditions are satisfied. This technique can be used to isolate

proteins, enzymes as well as antibodies from complex mixtures.

Size exclusion chromatography separates molecules according to their molecular mass.

Largest molecules are eluted first and the smallest molecules last. This method is generally

used when a mixture contains compounds with a molecular mass difference of at least 10%.

This mode can be further subdivided into gel permeation chromatography (with organic

solvents) and gel filtration chromatography (with aqueous solvents).

A schematic diagram of HPLC equipment is given in Fig.4.28

Fig. 4: A schematic diagram of HPLC equipment.

Various components of HPLC are: 29-33

§A solvent delivery system, including pump,

§Sample injection system,

§A chromatographic column,

§A detector,

§A strip chart recorder,

§Data handling device and microprocessor control.

a) Solvent delivery system:

A mobile phase is pumped under pressure from one or several reservoir and flows through the

column at a constant rate. For normal phase separation eluting power increases with

increasing polarity of the solvent but for reversed phase separation, eluting power decreases

with increasing polarity.

A degasser is needed to remove dissolved air and other gases from the solvent. Special grades

of solvents are available for HPLC and these have been purified carefully in order to remove

absorbing impurities and particulate matter to prevent these particles from damaging the

pumping or injection system or clogging the column.

Pumps:

The pump is one of the most important component of HPLC, since its performance directly

affects retention time, reproducibility and detector sensitivity.

Three main types of pumps are used in HPLC to propel the liquid mobile phase through the

system.

1. Displacement pump: It produces a flow that tends to independent of viscosity and back

pressure and also output is pulse free. But it possesses limited capacity (250 ml).

2. Reciprocating pump: It has small internal volume (35 to 400 µl), their high output pressure

(upto 10,000 psi) and their constant flow rates. But it produces a pulsed flow.

3. Pneumatic or constant pressure pump: They are pulse free; suffer from limited capacity as

well as a dependence of flow rate on solvent viscosity and column back pressure. They are

limited to pressure less than 2000 psi.

(b) Sample injection system:

Insertion of the sample onto the pressurized column must be as a narrow plug so that the

peak broadening attributable to this step is negligible. The injection system itself should have

no dead (void) volume.

There are three important ways of introducing the sample into injection port.

· Loop injection: In which, a fixed amount of volume is introduced by making use of

fixed volume loop injector.

· Valve injection: In which, a variable volume is introduced by making use of an injection

valve.

· On column injection: In which, a variable volume is introduced by means of a syringe

through a septum.

(c) Chromatographic column:

The column is usually made up of heavy glass or stainless steel tubing to withstand high

pressure. The columns are usually 10-30 cm long and 4-10 mm inside diameter containing

stationary phase at particle diameter of 25 µm or less.

Columns with an internal diameter of 5 mm give good results because of compromise between

efficiency, sample capacity, and the amount of packing and solvent required.

Column packing:

The packing used in modern HPLC consist of small, rigid particles having a narrow particle size

distribution. There are three main types of column packing in HPLC.

1. Porous, polymeric beds: Porous, polymeric beds based on styrene divinyl benzene co-

polymers used doe ion exchange and size exclusion chromatography. For analytical purpose
these have now been replaced by silica based, packing which are more efficient and more

stable.

2. Porous layer beds: Consisting of a thin shell (1-3 µm) of silica or modified silica on an

spherical inert core (e.g. Glass). After the development of totally porous micro particulate

packings, these have not been used in HPLC.

3. Totally Porous silica particles (dia. <10 µm): These packing have widely been used for

analytical HPLC in recent years. Particles of diameter >20 µm are usually dry packed. While

particles of diameter <20 µm are slurry packed in which particles are suspended on a suitable

solvent and the slurry so obtained is driven into the column under pressure.

(d) Detectors:

The function of the detector in HPLC is to monitor the mobile phase as it merges from the

column. Detectors are usually of two types:

1. Bulk property detectors: It compares overall changes in a physical property of the mobile

phase with and without an eluting solute. e.g. refractive index, dielectric constant or density.

2. Solute property detectors: It responds to a physical property of the solute which is not

exhibited by the pure mobile phase. e.g. UV absorbance, fluorescence or diffusion current.

Such detectors are about 1000 times more sensitive giving a detectable signal for a few

nanograms of sample.

Quantitative Analysis In HPLC

Three methods are generally used for quantitative analysis. They are the external standard

method, the internal standard method and the standard addition method.

1. External standard method: The external standard method involves the use of a single

standard or up to three solutions. The peak area or the height of the sample and the standard

use are compared directly. One can also use the slope of the calibration curve based on

standard that contain known concentrations of the compound of interest.

2. Internal standard method: A widely used technique of quantitation involves the addition

of an internal standard to compensate for various analytical errors. In this approach, a known

compound of a fixed concentration is added to the known amount of samples to give separate

peaks in the chromatograms to compensate for the losses of the compounds of interest during

sample pretreatment steps. Any loss of the component of interest will be accompanied by the

loss of an equivalent fraction of the internal standard. The accuracy of this approach obviously
dependence on the structural equivalence of the compounds of interest and the internal

standard.

The requirements for an internal standard are:

a. It must have a completely resolved peak with no interferences,

b. It must elute close to the compound of interest,

c. It must behave equivalent to the compound of interest for analysis like pretreatments,

derivative formations, etc.,

d. It must be added at a concentration that will produce a peak area or peak height ratio of

about unity with the compound,

e. It must not be present in the original sample,

f. It must be stable, unreactive with sample components, column packing and the mobile

phase and

g. It is desirable that this compound is commercially available in high purity.

The internal standard should be added to the sample prior to sample preparation procedure

and homogenized with it. To be able to recalculate the concentration of a sample component

in the original sample, we have to demonstrate first the response factor. The response factor

(RF) is the ratio of peak areas of sample component (Ax) and the internal standard (AISTD)

obtained by injecting the same quantity. It can be calculated by using the formula,

RF = Ax / AISTD

On the basis of the response factor and strength of the internal standard (NISTD), the amount

of the analyte in the original sample can be calculated using the

formula,

X =AS / RF * AISTD X NISTD

The calculations described above can be used after proving the linearity of the calibration

curve for the internal standard and the analytical reference standard of the compound of

interest. When more than one component is to be analyzed from the sample, the response

factor of each component should be determined in the calculations using similar formula.

3. Standard addition method: In the standard addition method, a known amount of the

standard compound is added to the sample solution to be estimated. This method is suitable if

sufficient amount of the sample is available and is more realistic in the sense that it allows

calibration in the presence of excipients or other components.

As gradient elution can be a source of additional error in quantitative analysis. It is also

necessary to keep the flow rate and the mobile phase composition constant. The sample

should be dissolved in the mobile phase. If the solvent used in the preparing the sample

solution and the mobile phase are not identical, the analysis can become less accurate and it

is also possible that the detector response is more dependent on the sample.
Design And Development And Of Separation Method

Methods for analyzing drugs in multicomponent dosage forms can be developed, provided one

has knowledge about the nature of the sample, namely, its molecular weight, polarity, ionic

character and the solubility parameter. An exact recipe for HPLC, however, cannot be provided

because method development involves considerable trial and error procedures. The most

difficult problem usually is where to start, what type of column is worth trying with what kind

of mobile phase. In general one begins with reversed phase chromatography, when the

compounds are hydrophilic in nature with many polar groups and are water soluble.

The organic phase concentration required for the mobile phase can be estimated by gradient

elution method. For aqueous sample mixtures, the best way to start is with gradient reversed

phase chromatography. Gradient can be started with 5-10% organic phase in the mobile

phase and the organic phase concentration (methanol or acetonitrile) can be increased up to

100% within 30-45min. Separation can then be optimized by changing the initial mobile phase

composition and the slope of the gradient according to the chromatogram obtained from the

preliminary run. The initial mobile phase composition can be estimated on the basis of where

the compounds of interest were eluted, namely, at what mobile phase composition.

Changing the polarity of mobile phase can alter elution of drug molecules. The elution strength

of a mobile phase depends upon its polarity, the stronger the polarity, higher is the elution.

Ionic samples (acidic or basic) can be separated, if they are present in undissociated form.

Dissociation of ionic samples may be suppressed by the proper selection of pH.

The pH of the mobile phase has to be selected in such a way that the compounds are not

ionized. If the retention times are too short, the decrease of the organic phase concentration

in the mobile phase can be in steps of 5%. If the retention times are too long, an increase of

the organic phase concentration is needed.

In UV detection, good analytical results are obtained only when the wavelength is selected

carefully. This requires knowledge of the UV spectra of the individual components present in

the sample. If analyte standards are available, their UV spectra can be measured prior to

HPLC method development.

The molar absorbance at the detection wavelength is also an important parameter. When

peaks are not detected in the chromatograms, it is possible that the sample quantity is not

enough for the detection. An injection of volume of 20 µl from a solution of 1 mg/ml

concentration normally provides good signals for UV active compounds around 220 nm. Even if

the compounds exhibit higher lmax, they absorb strongly at lower wavelength. It is not always

necessary to detect compounds at their maximum absorbance. It is, however, advantageous

to avoid the detection at the sloppy part of the UV spectrum for precise quantitation. When
acceptable peaks are detected on the chromatogram, the investigation of the peak shapes can

help further method development.

The addition of peak modifiers to the mobile phase can affect the separation of ionic samples.

For examples, the retention of the basic compounds can be influenced by the addition of small

amounts of triethylamine (a peak modifier) to the mobile phase. Similarly for acidic

compounds small amounts of acids such as acetic acid can be used. This can lead to useful

changes in selectivity.

When tailing or fronting is observed, it means that the mobile phase is not totally compatible

with the solutes. In most case the pH is not properly selected and hence partial dissociation or

protonation takes place. When the peak shape does not improve by lower (1-2) or higher (8-

9) pH, then ion-pair chromatography can be used. For acidic compounds, cationic ion pair

molecules at higher pH and for basic compounds, anionic ion-pair molecules at lower pH can

be used. For amphoteric solutes or a mixture of acidic and basic compounds, ion-pair

chromatography is the method of choice.

The low solubility of the sample in the mobile phase can also cause bad peak shapes. It is

always advisable to use the same solvents for the preparation of sample solution as the mobile

phase to avoid precipitation of the compounds in the column or injector.

Optimization can be started only after a reasonable chromatogram has been obtained. A

reasonable chromatogram means that more or less symmetrical peaks on the chromatogram

detect all the compounds. By sight change of the mobile phase composition, the position of

the peaks can be predicted within the range of investigated changes. An optimized

chromatogram is the one in which all the peaks are symmetrical and are well separated in less

run time.

The peak resolution can be increased by using a more efficient column (column with higher

theoretical plate number, N) which can be achieved by using a column of smaller particle size,

or a longer column. These factors, however, will increase the analysis time. Flow rate does not

influence resolution, but it has a strong effect on the analysis time.

System Suitability Tests For Chromatographic Methods 36

System suitability is the checking of a system to ensure system performance before or during

the analysis of unknowns. Parameters such as plate count, tailing factors, resolution and

reproducibility (%RSD retention time and area for six repetitions) are determined and

compared against the specifications set for the method. These parameters are measured

during the analysis of a system suitability "sample" that is a mixture of main components and
expected by-products. Lists of the terms to be measured and their recommended limits

obtained from the analysis of the system suitability sample are given below.

Definition

The purpose of the system suitability test is to ensure that the complete testing system

(including instrument, reagents, columns, analysts) is suitable for the intended application.

The USP Chromatography General Chapter states:

"System suitability tests are an integral part of gas and liquid chromatographic methods. They

are used to verify that the resolution and reproducibility of the chromatographic system are

adequate for the analysis to be done. The tests are based on the concept that the equipment,

electronics, analytical operations and samples to be analyzed constitute an integral system

that can be evaluated as such."

Evolution of System Suitability

Similar to the analytical method development, the system suitability test strategy should be

revised as the analysts develop more experience with the assay. In general, consistency of

system performance (e.g., replicate injections of the standard) and chromatographic suitability

(e.g. tailing factor, column efficiency and resolution of the critical pair) are the main

components of system suitability.

Early Stage of Method Development

During the early stage of the method development process some of the more sophisticated

system suitability tests may not be practical due to the lack of experience with the method. In

this stage, usually a more "generic" approach is used. For example, evaluation of the tailing

factor to check chromatographic suitability, and replicate injections of the system suitability

solution to check injection precision may be sufficient for an HPLC impurities assay.

In the early method development, it may be useful to perform some additional system

suitability tests to evaluate the system performances under different method conditions. This

information will help to develop an appropriate system suitability test strategy in the future.

As The Method Matures

As more experience is acquired for this method, a more sophisticated system suitability test

may be necessary. For HPLC impurities method intended to be stability indicating, a critical
pair for resolution determination should be identified. The critical pair is defined as the two

peaks with the least resolution in the chromatographic separation. Generally, a minimum

resolution limit is defined for the critical pair to ensure that the separations of all other

impurities are acceptable. All critical factors that will significantly impact the method

performance will need to be identified. Therefore, if the resolution test results exceed the

acceptance limit, the critical factors can be adjusted to optimize the system performance. If %

organic in the mobile phase has a significant impact on the resolution of the critical pair,

organic composition in the mobile phase can be adjusted within a predetermined range to

achieve the acceptable resolution. Therefore, system suitability strategy not only consists of

the tests and limits, but also the approach used to optimize system performance when the

original test result exceeds the limit. In addition, if the method demands high method

sensitivity (e.g. to analyze very low impurity levels), a detector sensitivity solution may be

required to demonstrate suitable signal-to-noise from the HPLC system. These system

suitability tests, combined with the typical replicate injections of the standard solution, may be

used to demonstrate the system suitability for this method.

Long Term System Suitability Strategy

During the final stage of method development, there is a need to define the long-term

strategy for system suitability requirements, and the practicalities for all laboratories using

this method. If the system suitability test involves the use of any reference sample (i.e.

isolated and characterized impurity), the laboratory needs to have enough supply of this

reference sample to complete the system suitability test. However, maintaining the supply of

this reference sample in the long term is usually not an easy task. If the reference sample is a

degradation product of the drug substance, it is desirable to generate the reference sample in-

situ by artificially degrading the drug substance in order to streamline the method. Therefore,

extensive investigations must be done to evaluate the best approach to generate the

reference sample, and to identify the critical factors needed to ensure that the degradation

process is reproducible.

How to Set Limits

Numerous approaches can be used to set limits for system suitability tests. This depends on

the experience with the method, material available and personal preference. During method

development, it may be useful to perform some system suitability tests with no acceptance

limit. Firstly, it is premature to set any limit during the very early stage of method
development. Secondly, since experimental conditions will be varied intentionally during

method development, collecting system suitability data in these experiments will help the

analyst to evaluate the impact of results generated under different method conditions. This

information will be used to set appropriate system suitability limits in the future.

Default Values from Regulatory Guidelines

There are numerous guidelines which detail the expected limits for typical chromatographic

methods. In the current FDA guidelines on "Validation of Chromatographic Methods" , the

following acceptance limits are proposed as initial criteria:

These suggested limits may be used as a reference to set up the initial system suitability

criteria in the early method development process.

Method Validation Results

Making use of the method validation results is yet another approach. During the robustness

testing of method validation, critical method parameters such as mobile phase composition,

column temperature are varied to mimic the day-to-day variability. Therefore, the system

suitability results from these robustness experiments should reflect the expected range for the

system suitability results. As a result, the limits for system suitability tests can be determined

from these experiments. This is a very effective approach since the required system suitability

results can be generated during method validation, and no special study is required. However,

these results only reflect the expected performance of the system, but not necessarily the

minimum "performance standard" for acceptable results. For example, the minimum resolution

of the critical pair from method validation may be 3.5; however, a resolution of 2.0 may still

be acceptable as long as they are baseline resolved, and all other chromatographic parameters

remain acceptable.

Simulated Conditions

Ideally the analyst should observe the results from a "deteriorating" system and determine the

situations under which the results are no longer acceptable. One way to simulate the

deterioration of the system is to use an old or artificially degraded column in the analysis.

Typically, a column can be degraded artificially by numerous injections or heating at extreme

pH conditions. These old columns will provide the information about the changes

System Suitability Parameters and Recommendations

Parameter Recommendation
Capacity Factor (k’) The peak should be well-resolved from other peaks and the

void volume, generally k’>2.0

Repeatability RSD </= 1% for N >/= 5 is desirable.

Relative retention Not essential as long as the resolution is stated.

Resolution (Rs) Rs of > 2 between the peak of interest and the closest eluting

potential interferent (impurity, excipient, degradation product, internal standard, etc.

Tailing Factor (T) T of </= 2

Theoretical Plates (N) In general should be > 2000

If the results are adversely affected by the changes in column performance (e.g. unacceptable

precision of results due to overlapping peaks), the system suitability results from these

experiments will help to determine the limits for system suitability criteria.

This approach facilitates the investigation of the worst case scenario, which reflects minimum

performance standard used to ensure that the chromatography is not adversely affected.

The parameters that are affected by the changes in chromatographic conditions are:

§ Resolution (Rs),

§ Capacity factor (k’),

§ Selectivity (a),

§ Column efficiency (N) and

§ Peak asymmetry factor (As).

1. Resolution (Rs): Resolution is the parameter describing the separation power of the

complete chromatographic system relative to the particular components of the mixture.

The resolution, Rs, of two neighboring peaks is defined as the ratio of the distance between

two peak maxima. It is the difference between the retention times of two solutes divided by

their average peak width. For baseline separation, the ideal value of Rs is 1.5. It is calculated

by using the formula,

Fig. 5: Resolution between two peaks.

where, Rt1 and Rt2 are the retention times of components 1 and 2 and
W1 and W2 are peak width of components 1 and 2.

There are three fundamental parameters that influence the resolution of a chromatographic

separation:

·capacity factor (k')

·selectivity (α)

·column efficiency (N)

These parameters provide you with different means to achieve better resolution, as well as

defining different problem sources

2. Capacity Factor (k’): Capacity factor is the ratio of the reduced retention volume to the

dead volume. Capacity factor, k’, is defined as the ratio of the number of molecules of solute

in the stationary phase to the number of molecules of the same in the mobile phase. Capacity

factor is a measure of how well the sample molecule is retained by a column during an

isocratic separation. The ideal value of k’ ranges from 2-10. Capacity factor can be determined

by using the formula,

Fig. 6: Retention Factor

Where, tR = retention volume at the apex of the peak (solute) and

t0 = void volume of the system.

Capacity Factor (k') changes are typically due to:

· Variations in mobile phase composition

· Changes in column surface chemistry (due to aging)

· Changes in operating temperature.

In most chromatography modes, capacity factor (k') changes by 10 percent for a temperature

change of 5 C.

Adjusting Capacity Factor (k')

Good isocratic methods usually have a capacity factor (k') in the range of 2 to 10 (typically

between 2 and 5). Lower values may give inadequate resolution. Higher values are usually

associated with excessively brood peaks and unacceptably long run times.

If the analytes fall outside their specified windows run the initial column test protocol to

compare the results obtained with a new column.

Capacity Factor (k') values are sensitive to:

· solvent strength

· composition

· purity

· temperature

· column chemistry

· sample

If the shift in Capacity Factor (k') value is observed with both analytes and the column test

solution, the problem is most likely due to change in the column, temperature or mobile phase

composition. This is particularly true if the shift occurred gradually over a series of runs. If,

however the test mixture runs as expected, the problem is most likely sample-related.

3. Selectivity(a): The selectivity (or separation factor), a, is a measure of relative retention of

two components in a mixture. Selectivity is the ratio of the capacity factors of both peaks, and

the ratio of its adjusted retention times. Selectivity represents the separation power of

particular adsorbent to the mixture of these particular components.

This parameter is independent of the column efficiency; it only depends on the nature of the

components, eluent type, and eluent composition, and adsorbent surface chemistry. In

general, if the selectivity of two components is equal to 1, then there is no way to separate

them by improving the column efficiency.

The ideal value of a is 2. It can be calculated by using formula,

a= V2 – V1 / V1 – V0 = k1’/ k2’

Where, V0 = the void volume of the column,

V1 and V2 =the retention volumes of the second and the first peak

respectively.
Fig. 7: Selectivity

Adjusting selectivity (α)

When troubleshooting changes in Selectivity (α), the approach is similar to the approach used

to troubleshoot changes in Capacity Factor (k').

When Selectivity (α) is affected, the corrective action depends on whether the problem is

mobile phase or column related.

Be sure to compare results obtained with the test solution to those observed when the column

was new. Use these results to distinguish column changes from problems with mobile phase

or other operating parameters.

Selectivity (α) values are sensitive to:

·changes in mobile phase composition (pH ionic strength)

·purity

·temperature

4. Column Efficiency/ Band broadening: Efficiency, N, of a column is measured by the number

of theoretical plates per meter. It is a measure of band spreading of a peak. Similar the band

spread, higher is the number of theoretical plates, indicating good column and system

performance. Columns with N ranging from 5,000 to 100,000 plates/meter are ideal for a good

system. Efficiency is calculated by using the formula,

Fig. 8: Number of Theoretical Plates

Where, tR is the retention time and

W is the peak width.

A decline in measured efficiency may be due to:

·age and history of the column

· extra column band broadening (such as due to malfunctioning injector or improper tubing

ID)

· inappropriate detector settings (for example, time constant)

· change in flow rate and solvent viscosity.

You can recognize problems in your separation due to a loss of column efficiency when the

width and/or shape of all peaks are affected.

If the measured efficiency has degraded, either the column has degraded, or system

bandbroadening has increased. At this point, check system bandspreading against established

benchmarks.

Methods of measuring column efficiency (N)

Methods used for the measurement and calculation of column include (in order to sensitivity to

abnormal peak shape):

· Asymmetry-based (Most sensitive to tailing or fronting)

· 5 sigma

· 4 sigma

· Tangent

· 3 sigma

· ½ height

· 2 sigma (infection) (Least sensitive to tailing or fronting)

Choose the method that best suits your operating requirements. It is critical that the same

method always be used and executed reproducibly.

Figure above illustrates the use of the different peak widths of a Gaussian peak for the

calculation of column efficiency (N).

When measuring Column Efficiency, use test conditions identical to those used in the

established benchmark performance (such as test sample, flow rate, mobile phase composition

and so on). Measure the column efficiency against the established performance.

5. Peak asymmetry factor (Tf): Peak asymmetry factor, Tf, can be used as a criterion of column

performance. The peak half width, b, of a peak at 10% of the peak height, divided by the

corresponding front half width, a, gives the asymmetry factor.

Fig. 9A: Asymmetric Factor

For a well packed column, an asymmetry factor of 0.9 to 1.1 should be achievable.

Fig. 9B: Asymmetric Factor

Introduction To Hptlc Methods Of Analysis For Drugs In Combination37

HPTLC (High Performance Thin Layer Chromatography) is a well known and versatile

separation method which shows a lot of advantages in comparison to other separation

techniques.
Layer of Sorbent Ø 100µm

Efficiency Ø High due to smaller particle size generated

Separations Ø3 - 5 cm

Analysis Time ØShorter migration distance and the analysis time is greatly
reduced
Solid support ØWide choice of stationary phases like silica gel for normal
phase and C8 , C18 for reversed phase modes

Development chamber ØNew type that require less amount of mobile phase

Sample spotting ØAuto sampler

Scanning ØUse of UV/ Visible/ Fluorescence scanner scans the entire

chromatogram qualitatively and quantitatively and the scanner
is an advanced type of densitometer

Features of HPTLC

1. Simultaneous processing of sample and standard - better analytical precision and

accuracy less need for Internal Standard

2. Several analysts work simultaneously

3. Lower analysis time and less cost per analysis

4. Low maintenance cost

5. Simple sample preparation - handle samples of divergent nature

6. No prior treatment for solvents like filtration and degassing

7. Low mobile phase consumption per sample

8. No interference from previous analysis - fresh stationary and mobile phases for each

analysis - no contamination

9. Visual detection possible - open system

10. Non UV absorbing compounds detected by post-chromatographic derivatization

Steps involved in HPTLC

1. Selection of chromatographic layer

2. Sample and standard preparation

3. Layer pre-washing

4. Layer pre-conditioning

5. Application of sample and standard

6. Chromatographic development

7. Detection of spots

8. Scanning

9. Documentation of chromatic plate

Selection of chromatographic layer

- Precoated plates - different support materials - different Sorbents available

- 80% of analysis - silica gel GF · Basic substances, alkaloids and steroids Aluminum oxide

- Amino acids, dipeptides, sugars and alkaloids - cellulose

- Non-polar substances, fatty acids, carotenoids, cholesterol - RP2, RP8 and RP18

- Preservatives, barbiturates, analgesic and phenothiazines- Hybrid plates-RPWF254s

Sample and Standard Preparation

- To avoid interference from impurities and water vapours.

- Low signal to noise ratio - Straight base line- Improvement of LOD

- Solvents used are Methanol, Chloroform: Methanol (1:1), Ethyl acetate: Methanol (1:1), - --

Chloroform: Methanol: Ammonia (90:!0:1), Methylene chloride : Methanol (1:1), 1%

Ammonia or 1% Acetic acid

- Dry the plates and store in dust free atmosphere

Activation of pre-coated plates

- Freshly open box of plates do not require activation

- Plates exposed to high humidity or kept on hand for long time to be activated

- By placing in an oven at 110-120ºc for 30’ prior to spotting

- Aluminum sheets should be kept in between two glass plates and placing in oven at 110-

120ºc for 15 minutes.

Application of sample and standard

- Usual concentration range is 0.1-1µg / µl

- Above this causes poor separation

- Linomat IV (automatic applicator) - nitrogen gas sprays sample and standard from syringe o-

n TLC plates as bands

- Band wise application - better separation - high response to densitometer

Selection of mobile phase

- Trial and error

- one’s own experience and Literature

Normal phase

- Stationary phase is polar

- Mobile phase is non polar

- Non-polar compounds eluted first because of lower affinity with stationary phase

- Polar compounds retained because of higher affinity with the stationary phase

Reversed phase

- Stationary phase is non polar

- Mobile phase is polar

- Polar compounds eluted first because of lower affinity with stationary phase

- Non-Polar compounds retained because of higher affinity with the stationary phase

- 3 - 4 component mobile phase should be avoided

- Multi component mobile phase once used not recommended for further use and solvent

composition is expressed by volumes (v/v) and sum of volumes is usually 100

- Twin trough chambers are used only 10 -15 ml of mobile phase is required

- Components of mobile phase should be mixed introduced into the twin - trough chamber

Pre- conditioning (Chamber saturation)

- Un- saturated chamber causes high Rf values

- Saturated chamber by lining with filter paper for 30 minutes prior to development - uniform

distribution of solvent vapours - less solvent for the sample to travel - lower Rf values.

Chromatographic development and drying

- After development, remove the plate and mobile phase is removed from the plate - to avoid

contamination of lab atmosphere

- Dry in vacuum desiccator - avoid hair drier - essential oil components may evaporate

Detection and visualization

- Detection under UV light is first choice - non destructive

- Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm

(long wave length)

- Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used -

silica gel GF

- Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1%

iodine solution

- When individual component does not respond to UV - derivatisation required for detection
Quantification

- Sample and standard should be chromatographed on same plate - after development

chromatogram is scanned

- Camag TLC scanner III scan the chromatogram in reflectance or in transmittance mode by

absorbance or by fluorescent mode - scanning speed is selectable up to 100 mm/s - spectra

recording is fast - 36 tracks with up to 100 peak windows can be evaluated

- Calibration of single and multiple levels with linear or non-linear regressions are possible

when target values are to be verified such as stability testing and dissolution profile single

level calibration is suitable

- Statistics such as RSD or CI report automatically

- Concentration of analyte in the sample is calculated by considering the sample initially taken

and dilution factors.

Fig. 10: Schematic procedure for HPTLC 38

HPTLC Method design and development

Set the analytical objective first that may be quantification or qualitative identification or

separation of two components/multicomponent mixtures or optimization of analysis time

before starting HPTLC. Method for analyzing drugs in multicomponent dosage forms by HPTLC

demands primary knowledge about the nature of the sample, namely, structure, polarity,

volatility, stability and the solubility parameter. An exact recipe for HPTLC, however, also

same like HPLC cannot be provided because method development involves considerable trial

and error procedures. The most difficult problem usually is where to start, with what kind of

mobile phase.

Selection of stationary phase is quite easy that is to start with silica gel which is reasonable

and nearly suits all kind of drugs. Mobile phase optimization is carried out by using three level

techniques. First level involves use of neat solvents and then by finding some such solvents

which can have average separation power for the desired drugs. Second level involves

decreasing or increasing solvent strength using hexane or water for respective purposes.

Third level involves trying of mixtures instead of neat solvents from the selected solvents of

first and second level which can further be optimized by the use of modifier like acids or

bases.

Analytes are detected using fluorescence mode or absorbance mode. But if the analytes are

not detected perfectly than it need change of stationary phase or mobile phase or need the

help of pre or post chromatographic derivatization.

Optimization can be started only after a reasonable chromatogram which can be done by slight

change in mobile phase composition. This leads to a reasonable chromatogram which has all

the desired peaks in symmetry and well separated.

Parameters that are affected by the changes in chromatographic conditions are:

·Retention factor (Rf) and

·Peak purity

1. Retention factor (Rf): Retention factor (Rf) is defined as the amount of separation due to

the solvent migration through the sorbent layer as shown in the formula. It depends on time

of development and velocity coefficient or solvent front velocity.

Migration distance of substance

Rf = --------------------------------------------------------

Migration distance of solvent front from origin

2. Peak purity: The null hypothesis “these spectra are identical” can in this case (purity) with

two sided significance. During the purity test the spectrum taken at the first peak slope is

correlated with the spectrum of peak maximum [r(s,m)] and the correlation of the spectra

taken at the peak maximum with the one from the down slope or peak end [r(m,e)] which is

used as a reference spectra for statistical calculation. An error probability of 1% only be

rejected if the test value is greater than or equal to 2.576.

Validation Of Analytical Method 39-41

Validation is an act of proving that any procedure, process, equipment, material, activity or

system performs as expected under given set of conditions and also give the required

accuracy, precision, sensitivity, ruggedness, etc.

When extended to an analytical procedure, depending upon the application, it means that a

method works reproducibly, when carried out by same or different persons, in same or

different laboratories, using different reagents, different equipments, etc.

The various validation parameters are:

§accuracy,

§precision(repeatability and reproducibility),

§linearity and range,

§limit of detection(LOD)/ limit of quantitation(LOQ),

§selectivity/ specificity,

§robustness/ ruggedness and

§Stability and system suitability studies.

Advantages of Analytical method Validation:-

§The biggest advantage of method validation is that it builds a degree of confidence, not only

for the developer but also to the user.

§Although the validation exercise may appear costly and time consuming, it results

inexpensive, eliminates frustrating repetitions and leads to better time management in the

end.

§Minor changes in the conditions such as reagent supplier or grade, analytical setup are

unavoidable due to obvious reasons but the method validation absorbs the shock of such

conditions and pays for more than invested on the process.

Analytical method validation: The Regulatory Perspective

In the US, there was no mention of the word validation in the cGMP’s of 1971, and precision

and accuracy were stated as laboratory controls. It was only in the cGMP guideline of March

1979 that the need for validation was implied. It was done in two sections: (1) Section

211.165, where the word ‘validation’ was used and (2) section 211.194, in which the proof of

suitability, accuracy and reliability was made compulsory for regulatory submissions. Another

guidance on validation of chromatographic methods was released by CDRE on 1st Nov. 1994.

The WHO published a guidelines 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 for adoption in the US, Japan and EC , has

issued two guidelines under the titles-‘ Text on validation of Analytical procedures(Q2A) and

validation of Analytical procedure Methodology (Q2B)’.

Among the pharmacopoeias, USP XXII 1225 (1995) carries a section which describes

requirements of validation of compendial methods. The British Pharmacopoeia includes the

definition of method validation in 15 latest editions, but the term is completely missing form

the Indian Pharmacopoeia. (1996).

Key parameters of the Analytical method validation:-

It is important for one to understand the parameters or characteristics involved in the

validation process. The various Performance parameters, which are addressed in a validation

exercise, are grouped as follows.

(1) Accuracy: -

The accuracy of an analytical method may be defined as the closeness of the test results

obtained by the method to the true value. It is the measure of the exactness of the analytical

method developed. Accuracy may often express as percent recovery by the assay of a known

amount of analyte added.

Accuracy may be determined by applying the method to samples or mixtures of excipients to

which known amount of analyte have been added both above and below the normal levels

expected in the samples. Accuracy is then calculated from the test results as the percentage of

the analyte recovered by the assay. Dosage form assays commonly provide accuracy within 3-

5% of the true value.

The ICH documents recommend that accuracy should be assessed using a minimum of nine

determinations over a minimum of three concentration levels, covering the specified range

(i.e. three concentrations and three replicated of each concentration).

(2) Precision: -

The precision of an analytical method is the degree of agreement among individual test results

when the method is applied repeatedly to multiple samplings of homogenous samples. This is

usually expressed as the standard deviation or the relative standard deviation (coefficient of

variation). Precision is a measure of the degree of reproducibility or of the repeatability of the

analytical method under normal operating circumstances.

Repeatability involves analysis of replicates by the analyst using the same equipment and

method and conducting the precision study over short period of time while reproducibility

involves precision study at

§ Different Occasions,

§ Different Laboratories,

§ Different Batch of Reagent,

§ Different Analysts,

§ Different Equipments.

Determination of Repeatability:- Repeatability can be defined as the precision of the procedure

when repeated by same analyst under the same operating conditions (same reagents,

equipments, settings and laboratory) over a short interval of time.

It is normally expected that at least six replicates be carried out and a table showing each

individual result provided from which the mean, standard deviation and co-efficient of

variation should be calculated for set of n value. The RSD values are important for showing

degree of variation expected when the analytical procedure is repeated several time in a

standard situation. (RSD below 1% for built drugs, RSD below 2% for assays in finished

product).

The ICH documents recommend that repeatability should be assessed using a minimum of

nine determinations covering the specified range for the procedure (i.e. three concentrations

and three replicates of each concentration or using a minimum of six determinations at 100%

of the test concentration).

Determination of reproducibility:- Reproducibility means the precision of the procedure when it

is carried out under different conditions-usually in different laboratories-on separate,

putatively identical samples taken from the same homogenous batch of material.
Comparisions of results obtained by different analysts, by the use of different equipments, or

by carrying out the analysis at different times can also provide valuable information.

(3) Linearity and range:-

The linearity of an analytical method is its ability to elicit test results that are directly (or by a

well defined mathematical transformation) proportional to the analyte concentration in

samples within a given range. Linearity usually expressed in terms of the variance around the

slope of regression line calculated according to an established mathematical relationship from

test results obtained by the analysis of samples with varying concentrations of analyte.

The linear range of detectability that obeys Beer’s law is dependent on the compound analyzed

and the detector used. The working sample concentration and samples tested for accuracy

should be in the linear range. The claim that the method is linear is to be justified with

additional mention of zero intercept by processing data by linear least square regression. Data

is processed by linear least square regression declaring the regression co-efficient and b of the

linear equation y= ax + b together with the correlation coefficient of determination r. For the

method to be linear the r value should be close to1.

The range of an analytical method is the interval between the upper and lower levels of the

analyte (including these levels) that have been demonstrated to be determined with precision,

accuracy and linearity using the method as written.

(4) Limit of Detection and limit of Quantitation:-

Limit of detection:- The limit of detection is the parameter of limit tests. It is the lowest level

of analyte that can be detected, but not necessarily determined in a quantitative fashion,

using a specific method under the required experimental conditions. The limit test thus merely

substantiates that the analyte concentration is above or below a certain level.

The determination of the limit of detection of instrumental procedures is carried out by

determining the signal-to-noise ratio by comparing test results from the samples with known

concentration of analyte with those of blank samples and establishing the minimum level at

which the analyte can be reliably detected. A signal-to-noise ratio of 2:1 or 3:1 is generally

accepted.

The signal-to-noise ratio is determined by dividing the base peak by the standard deviation of

all data points below a set threshold. Limit of detection is calculated by taking the

concentration of the peak of interest divided by three times the signal-to-noise ratio.
For spectroscopic techniques or other methods that rely upon a calibration curve for

quantitative measurements, the IUPAC approach employs the standard deviation of the

intercept (Sa) which may be related to LOD and the slope of the calibration curve, b, by

LOD = 3 Sa / b

Limit of quantitation:- Limit of quantitation is a parameter of quantitative assays for low levels

of compounds in sample matrices such as impurities in bulk drugs and degradation products in

finished pharmaceuticals. The limit of quantitation is the lowest concentration of analyte in a

sample that may be determined with acceptable accuracy and precision when the required

procedure is applied.

It is measured by analyzing samples containing known quantities of the analyte and

determining the lowest level at which acceptable degrees of accuracy and precision are

attainable. Where the final assessment is based on an instrumental reading, the magnitude of

background response by analyzing a number of blank samples and calculating the standard

deviation of this response. The standard deviation multiplied by a factor (usually 10) provides

an estimate of the limit of quantitation. In many cases, the limit of quantitation is

approximately twice the limit of detection.

(5) Selectivity and Specificity:-

The selectivity of an analytical method is its ability to measure accurately and specifically the

analyte of interest in the presence of components that may be expected to be present in the

sample matrix.

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

and detect the analyte qualitatively the method is called selective. On the other hand, if the

method determines or measures quantitatively the component of interest in the sample matrix

without separation, it is said to be specific.

Hence one basic difference in the selectivity and specificity is that, while the former is

restricted to qualitative detection of the components of a sample, the latter means

quantitative measurement of one or more analyte.

Selectivity may be expressed in terms of the bias of the assay results obtained when the

procedure is applied to the analyte in the presence of expected levels of other components,

compared the results obtained when the procedure is applied to the analyte in the presence of

expected levels of other components, compared to the results obtained on the same analyte

without added substances. When the other components are all known and available, selectivity

may be determined by comparing the test results obtained on the analyte with and without

the addition of the potentially interfering materials. When such components are either
unidentified or unavailable, a measure of selectivity can often be obtained by determining the

recovery of a standard addition of pure analyte to a material containing a constant level of the

other components.

(6) Robustness and Ruggedness:-

Robustness:- The robustness of an analytical method is a measure of its capacity to remain

unaffected by small but deliberate variation in method parameters and provides an indication

of its reliability during normal usage. The determination of robustness requires that methods

characteristic are assessed when one or more operating parameter varied.

Ruggedness:- The ruggedness of an analytical method is the degree of reproducibility of test

results obtained by the analysis of the same samples under a variety of normal test

conditions such as different laboratories, different analysts, using operational and

environmental conditions that may differ but are still within the specified parameters of the

assay. The testing of ruggedness is normally suggested when the method is to be used in

more than one laboratory. Ruggedness is normally expressed as the lack of the influence on

the test results of operational and environmental variables of the analytical method.

For the determination of ruggedness, the degree of reproducibility of test result is determined

as function of the assay variable. This reproducibility may be compared to the precision of the

assay under normal condition to obtain a measure of the ruggedness of the analytical method.

(7) Stability and System suitability tests:-

Stability of the sample, standard and reagents is required for a reasonable time to generate

reproducible and reliable results. For example, 24 hour stability is desired for solutions and

reagents that need to be prepared for each analysis.

System suitability test provide the added assurance that on a specific occasion the method is

giving, accurate and precise results. System suitability test are run every time a method is

used either before or during analysis. The results of each system suitability test are compared

with defined acceptance criteria and if they pass, the method is deemed satisfactory on that

occasion. The nature of the test and the acceptance criteria will be based upon data generated

during method development optimization and validation experiments.

Data Elements Required For Assay Validation 34, 35

There are various analytical methods used for the examination of pharmaceutical materials.

Not all the characteristics referred above will need to be considered in all cases. Analytical

methods may be broadly classified as Per WHO as follows:

Class A: Tests designed to establish identity, whether of bulk drug substances or of a

particular ingredient in a finished dosage form.

Class B: Methods designed to detect and quantitative impurities in a bulk drug substance or

finished dosage form.

Class C: Methods used to determine quantitatively the concentration of a bulk drug substance

or of a major ingredient in a finished dosage form.

Class D: Methods used to assess the characteristic of finished dosage forms, such as

dissolution profiles and content uniformity.

Table: Characteristic that should be considered for different types of analytical

procedure:
Class A Class B Class C Class D

Quantitative Limit tests

tests

Accuracy X X X

Precision X X X

Robustness X X X X X

Linearity and X X X
range

Selectivity X X X X X

Limit of X X
Detection

Limit of X
Quantitation

Where, X indicates the tests to be performed.

As per USP:

Category I: Analytical methods for quantitation of major components of bulk drug substances

or active ingredients including preservatives in finished pharmaceutical products.

Category II: Analytical methods for determination of impurities in bulk drugs or for

determination of degradation compounds in finished pharmaceutical products.

Category III: Analytical methods for determination of performance characteristics (e.g.

dissolution, drug release).

Category IV: Identification tests.

Table: Data Elements Required for Assay Validation:

Analytical Assay Assay Category II Assay Category Assay Category
Performance Category I III IV
Characteristics Quantitative Limit tests
tests

Accuracy X X May be May be

Precision X X X

Specificity X X X May be X

Limit of Detection X May be

Limit of Quantitation X May be

Linearity X X May be

Range X X May be May be

Where, X indicates the tests to be performed.

Conclusion:

The efficient development and validation of analytical methods are critical elements in the

development of pharmaceuticals. Success in these areas can be attributed to several

important factors, which in turn will contribute to regulatory compliance. Experience is one of

these factors both the experience level of the individual scientists and the collective

experience level of the development and validation department.

Reference:

1.Swarbrick James., and Boylan James.C., Encyclopedia of pharmaceutical technology, Volume

I, Marcel Dekker Inc., New York, (1998), 217 - 224.

2.Beckett,A.H., Stenlake J.B., Practical Pharmaceutical Chemistry, (1997), 4th edition, Part 2,

CBS Publishers and distributors, 275-337.

3.Glenn A.L., Journal of pharmacy and pharmacology, (1960), 12,598-608.

4.Jain H.K., Agrawal R.K., Indian Journal of pharmaceutical sciences, (2002), 64(1), 88-71.

5.Shankar M.B., Mehta F.A., Bhatt K.K., Mehta R.S., and Geetha M., Indian Journal of

pharmaceutical sciences, 65(2), 167-170.

6. Singhvi I., Chaturvedi S.C., Indian drugs, (1998), 35(7), 421-426.

7.Dhake A.S., Kasture V.S., and Syed M.R., Indian drugs, (2002), 39(1), 14-17.

8. Singhvi I., Chaturvedi S.C., Indian drugs, (1998), 35(4), 234-238.

9.Pernarowski M., Knevel A.M., Christian J.E.., Journal of Pharmaceutical Science, (1960), 50,

943-946.

10. British Pharmacopoeia (1993), Her Majesty's stationary office, Volume II, 180-190.

11. Kakde R.B., Kasture A.V., and Vadodkar S.G., Indian Journal of Pharmaceutical sciences,

(2002), 64(1), 24-27.

12. Dyade G.K., Sharma A.K., Indian drugs, (2001), 38(2), 75-78.

13. Jadhav G.P., More H.N., Mahadik K.R., Indian drugs, (1998), 35(8), 475-480.

14. Gangwal S., Trivedi P., Indian drugs, (1998), 35(5), 291-295.

15. Connors K.A., A textbook of pharmaceutical Analysis, (1999), 3rd edition, John wiley and

sons, 221-224.

16. Panda S.K., Sharma A.K., and Sahu L.K., Indian Journal of Pharmaceutical sciences,

(2002), 64(6), 540-544.

17. Jain S.K., Jain Deepti., Tiwari M., and Chaturvedi S.C. , Indian Journal of Pharmaceutical

sciences, (2002), 64(3), 267-270.

18. Panda S.K., Sharma A.K., and Sahu L.K., Indian Journal of Pharmaceutical sciences,

(2002), 64(6), 540-544.

19. Kelani K.M., Journal of AOAC International, (1998), 81(6), 1128-1134.

20. Singhvi I., Chaturvedi S.C., Indian drugs, (1998), 35(4), 234-238.

21. Singhvi I., Chaturvedi S.C., Indian drugs, (1998), 35(7), 421-426.

22. Prasad P.B.N., Rao A.C.S., Kumar Y., and Mathur S.C., Singhvi I., Chaturvedi S.C., Indian

drugs, (1995), 32(9), 451-453.

23. British Pharmacopoeia (1993), Her Majesty's stationary office, Volume II, 780.

24. Sethi P.D., Qualitativie Analysis of drugs in Pharmaceutical Formulations, (1997), 3rd

edition, 182-184.

25. Swarbrick James., and Boylan James.C., Encyclopedia of pharmaceutical technology,

Volume I, Marcel Dekker Inc., New York, (1998), 217 - 224.

26. Lindsay Sandy., HPLC by open learning, John wiley and sons, London , (1991), 30-45.

27. Lough W.J., Wainer I.W.W., HPLC fundamental principles and practices, Blackie Academic

and professional, (1991), 52-67.

28. Meyer Veronica. R., Practical High Performance Liquid Chromatography, 2nd edition, John

wiley and sons, London , (1993), 26, 27, 40, 222, 246, and 258.

29. http://hplc.chem.shu.edu/NEW/HPLC_Book

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(1991), 217-235.
32. Willard Hobart. H., Merritt L.L., Dean John.A., Instrumental Methods of Analysis, 7th

edition, CBS Publishers, 580-610.

33. Sharma B.K., Instrumental Methods of Chemical Analysis, 20th edition, GOEL Publishing

House, (2001), 54-83.

34. Yu Joseph. G., Kruszynska Yolanta. T., and Mulfors MIm. I. , Diabetes, (1999), 48(12),

2414-2421.

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Pharmaceutical Science., 2003., 65(6).,530-531.

36. www.waters.com

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38. Sethi P. D.; HPTLC – High Performance Thin Layer Chromatography; CBS Publisher, New

Delhi ; First Edition, 1996; 4.

39. Validation of Analytical Procedures: Methodology, ICH Harmonised Tripartite Guidelines,

(1996), 1-8.

40. United States Pharmacopoeia and National Formulary, (24th) Asian Edition, The United

States Pharmacopoeia Convention Inc., U.S.A., 2149-2152.

41. Quality Assurance of Pharmaceuticals, (A compendium of guidelines and related

materials), (1997), Volume I, WHO, Geneva , 119-124.

About Authors:

Rashmin B. Patel

Mr. Rashmin B. Patel is currently working as a lecturer, department of Pharmaceutical

Chemistry, A R College of Pharmacy, Vallabh Vidyanagar, Gujarat; India. He completed his

B.Pharm and M.Pharm from A R College of Pharmacy, Vallabh Vidyanagar, Gujarat; India. His

areas of interest in research includes New Drug Delivery systems & Pharmaceutical Analysis.
Mrunali R. Patel

Mrs. Mrunali R. Patel is currently working as a lecturer, Department of Pharmaceutics &

Pharmaceutical Technology, Indukaka Ipcowala College of Pharmacy, New Vallabh Vidyanagar,

Gujarat; India. She completed her B.Pharm and M.Pharm from A R College of Pharmacy,

Vallabh Vidyanagar, Gujarat; India. Her area of interest in research includes New Drug

Delivery systems and Pharmaceutical Analysis.

Dear Rashmin and Mrunali...

Submitted by eswar on Mon, 08/25/2008 - 17:31.

It taken lot of time to go through your article but I felt very happy to know the things

presented in a pictural way... Its really good.

Go on keeping such useful blogs beneficial to All visitors of Pharmainfo.net.

eswar :-)

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reply to eswar

Submitted by Rashmin on Tue, 08/26/2008 - 04:18.

Dear Eswar

thank you for taking interest in my article and your comment regarding it.

Rashmin

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Analytical Method Development : Good representation

Submitted by lucky_pharmacist on Thu, 07/24/2008 - 17:18.

Dear Sir, you have really made good effort in expalining the Analytical Method Development

for Pharmaceuticals I will ask all my friends to visit this article to gain good knowledge about

this topic.

In the Table: Data Elements Required for Assay I was not able to understand the term maybe,

is it offically mentioned or you have edited it for ease.

I will sugest you to ask the Pharmainfo.net team to align the text more properly so that your

work looks even more better.

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thank you very much for your

Submitted by Rashmin on Sat, 08/09/2008 - 16:16.

thank you very much for your compliments and feedback to my article.

i 've already forwarded your request to editor.

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Nice article

Submitted by sailajabyrisetty on Tue, 07/22/2008 - 17:32.

Very good information. I think it should be promoted to book chapters section .

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Reply to your comment

Submitted by Rashmin on Sat, 08/09/2008 - 15:41.

thank you very much for your encouraging feedback to my article.

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