Introduction Analysis and Validation

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CHAPTER 4

INTRODUCTION TO METHOD OF ANALYSIS AND METHOD

VALIDATION

Contents

4.1 UV- VIS SPETROPHOTOMETRIC METHOD FOR ANALYSIS OF DRUG

COMPONENTS

4.2 HPTLC METHOD FOR ANALYSIS OF DRUG COMPONENTS

4.2.1 Introduction to HPTLC

4.2.2 Steps involved in HPLC

4.3 RP-HPLC METHOD FOR ANALYSIS OF DRUG COMPONENTS

4.4 VALIDATION OF ANALYTICAL METHODS

4.4.1 Linearity

4.4.2 Precision

4.4.3 Range

4.4.4 Accuracy

4.4.5 Specificity and Selectivity

4.4.6 Limit of detection (LOD) and Limit of Quantification

4.4.7 Ruggedness

4.4.8 Robustness



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.

Development and validation of analytical method (Spectrophotometric, High

performance liquid chromatography (HPLC), & High performance thin layer

chromatography (HPTLC)) is carried out for drug products containing more than one

active ingredient

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 quantification 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 UV-VIS Spectrophotometric Methods of

Analysis for Drugs in Combination

Ultraviolet-visible spectroscopy or ultraviolet – visible spectroscopy (UV-Vis)

involves the spectroscopy of photons in the UV-visible region. This means it uses

light in the visible and adjacent (near ultraviolet (UV) and near infrared (NIR)

ranges. The absorption in the visible ranges directly affect the colour of the

chemicals involved .In the region of the electromagnetic spectrum ,molecules

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undergo electronic transitions .this technique is complementary to fluorescence

spectroscopy ,in that fluorescence deals with transitions from the excited state to

the ground state ,while absorption measures transitions from the ground state to

the excited state .

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

Absorbance ratio method

Geometric correction method



Orthogonal polynomial method

Least square approximation

Dual wavelength spectrometry

Assay as a single – component sample

Assay using absorbance corrected for interference

Simultaneous Equation Method

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 information’s 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)

At λ2

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 = k x1 Cx + k y1Cy …………. (3)

A2 = k x2 Cx + k y2Cy ………………. (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 = k x C …………. (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 k y2 - A2 k y1) / (k x1 k y2 - k x2 k y1) …………. (7)

Cy = (k x1 A2 - k x2 A1) / ( k x1 k y2 - k x2 k y1) ………….. (8)

Therefore

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

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



4.2. HPTLC METHOD FOR ANALYSIS OF DRUG

COMPONENTS[15, 16]

4.2.1 Principles of thin-layer chromatography:

Thin-layer chromatography (TLC), also known as planar chromatography (PC), is one

of the oldest methods in analytical chemistry still in use.

In TLC, the different components of the sample are separated by their interaction with

the stationary phase (bonded to the glass, aluminium, or plastic support) and the liquid

mobile phase that moves along the stationary phase.

TLC is a fast, simple, and low-cost method suitable for any laboratory. A particularadvantage is that it allows the analysis of many samples simultaneously. In contrast to

liquid chromatography (LC), TLC offers separation without or at least with minimal sample preparation. Also, the plates are disposable, and there is no memory effect,

such as may occur in LC. TLC is also an off-line method: sample application,

separation, and detection take place in different processes. Because of its off-line

character, TLC allows the use of a number of detection methods and appropriate

derivatization reagents in sequence, which improves the reliability of the detection

Table 18. Difference between HPTLC and TLC:-

Parameters HPTLC TLC

Layer of Sorbent 100µm 250µm

EfficiencyHigh due to smaller particle

size generatedLess

Separations 3 - 5 cm 10 - 15 cm

Analysis Time

Shorter migration distance

and the analysis time is

greatly reduced

Slower

Solid support

Wide choice of stationary

phases like silica gel for

normal phase and C8 , C18

for reversed phase modes

Silica gel ,

Alumina &

Kiesulguhr



4.2.2. Features of HPTLC:

Simultaneous processing of sample and standard - better analytical precision and

accuracy less need for Internal Standard

Several analysts work simultaneously

Lower analysis time and less cost per analysis

Low maintenance cost

Simple sample preparation - handle samples of divergent nature

No prior treatment for solvents like filtration and degassing

Low mobile phase consumption per sample

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

each analysis - no contamination

Visual detection possible - open system

Non UV absorbing compounds detected by post-chromatographic derivatization

4.2.3. 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

Development

chamber

New type that require less

amount of mobile phaseMore amount

Sample spotting Auto sampler Manual spotting

Scanning

Use of UV/ Visible/ Fluorescence scanner scans

the entire chromatogram

qualitatively and

quantitatively densitometer

Not possible



9. Documentation of chromatic plate

Selection of chromatographic layer

· Precoated plates with different support materials and different Sorbents are available

· 80% of analysis is done on silica gel GF. Activation of pre-coated plates

Plates exposed to high humidity or kept on hand for long time requires activation.

Activation of pre-coated plates is done by placing them 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

· automatic applicators are available wherein nitrogen gas sprays sample and standard

from syringe on TLC plates as bands



· Band wise application provides better separation and shows high response to

densitometer

Selection of mobile phase

- Trial and error

- One’s own experience and Literature

- When the mobile phase is polar, polar compounds would be eluted first because of

lower affinity with stationary phase

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

for chromatographic development twin trough chambers are used where only 10 -15ml of mobile phase is required

- Components of mobile phase should be mixed thoroughly and then introduced into

the twin trough chamber

Pre- conditioning (Chamber saturation)

· Unsaturated chamber may lead to high Rf values

· Saturation of chamber is done by lining with filter paper for 30 minutes prior to

development.

. This allows uniform distribution of solvent vapors in the chamber so less solvent is

required for the sample to travel.

Chromatographic development and drying

After development, plate is removed from the chamber and mobile phase is removed

from the plate. Drying can be done either at room temperature or at alleviated

temperatures if solvents like water or acids are used.

Detection(Quantification)

1) Densitometry

Densiometry is a mean of measuring the concentration of the chromatographic zones

on the developed HPTLC layer with damaging the separated substance.



There are three possible scanning modes ,single beam,single wavelength and double

beam combined in to a single beam .the single beam fortmat is most popular ,as the

beam of electromagnetic radiation hits the electromatographic layer, some passes into

and through the layer whilst the remainder is reflected back from the surface

reflectance occurs due to the opaqueness of the layer .this reflected radiation is

measured by the photomultiplier unit or photoelectric cell in the instrument .

The spectro densiometric scanner scan separate tracks and wavelength produces vast

amount data .these data includes peak heights and areas ,and position of zones (start,

middle and end ) for every resolved component on every chromatographic track on

the HPTLC plate .A baseline adjustment is applied so that all peaks can be accurately

integrated ready for possible quantification .

2) Video imaging and densiometry

The developed chromatogram is illuminated from above with visible ,254 nm (UV) or

366nm (UV) light ,depending on the radiation required to visualize the analytes

.Illumination from below the plate can often improve the brightness of the image with

the plate suitably lit, an image acquisition device,usually CCD (charged coupled

device) camera with zoom attachment is positioned vertically above .the CCD cameratransmits a digital signal to a computer and video printer.

4.3 RP-HPLC METHOD FOR ANALYSIS OF DRUG COMPONENTS

High Performance Liquid Chromatography is unquestionably the most widely used

analytical separation technique. HPLC has been rapidly developed with the

introduction of new pumping methods, more reliable columns and a variety of

detectors has made. HPLC is one of the commonly used analytical techniques.

HPLC can also be automated which involve automated sampling, separation,

detection, recording and calculation and printing of results. Due to its high

selectivity, specificity and sensitivity achieved by HPLC methods are mainly used

for the analysis of most drugs.

HPLC is one of the most versatile instruments used in the field of pharmaceutical

analysis. It provides the following features.



High resolving power

Speedy separation

Continuous monitoring of the column effluent

Accurate quantitative measurement

Repetitive and reproducible analysis using the same column

Automation of the analytical procedure and data handling

In HPLC, the analyst has a wide choice of Chromatographic separation methodologies

from normal to reverse phase and a whole range of mobile phases using isocratic (or)

gradient elution techniques. Various detectors are also available for HPLC like

electrochemical detectors, refractive index detectors, fluorescence detectors,

radiochemical detectors, mass-sensitive detectors, UV detectors.

4.3.1 Characteristics of HPLC method

Efficient, highly selective, widely applicable.

Only small sample required.

May be non-destructive of sample.

Readily adapted to quantitative analysis.

High resolving power.

Speed of separation.

4.3.2 STRATEGY FOR METHOD DEVELOPMENT IN HPLC:

Everyday many chromatographers face the need to develop a high-performance liquid

chromatography (HPLC) separation.37

Method development and optimization in

liquid chromatography is still an attractive field of research for theoreticians

(researchers) and attracts also a lot of interest from practical analysts. Complex

mixtures or samples required systematic method development involving accurate

modelling of the retention behaviour of the analyte. Among all, the liquid

chromatographic methods, the reversed phase systems based on modified silica offers

the highest probability of successful results. However, a large number of (system)

variables (parameters) affect the selectivity and the resolution.



HPLC method development follows a series of steps which are summarized as below

Information on a sample, define separation goals

Need for special HPLC procedure, sample pretreatment, etc.?

Choose detector and detector settings

Choose LC method; preliminary run; estimate best separation conditions

Optimize separation conditions

Check for problems or requirement for special procedure

Recover purified material Quantitative calibration Qualitative method

Validate method for release to routine laboratory

Fig.2: Steps in HPLC method development



Table 19. Preferred Experimental Conditions for the Initial HPLC Separation

Separation Variable Preferred initial choice

Column

Dimensions(Length × ID) 15 × 0.46 cm

Particle Size 5µm

Stationary Phase C8 or C18

Mobile Phase

Solvents A and B Buffer-Acetonitrile

% B 80-100%

Buffer

(compound, pH, concentration)

10 - 25mM Phosphate Buffer

2.0 < pH

< 3.0

Additives(e.g., amine modifiers,

ion-pair reagents)Do not use initially

Flow-rate 1.5-2.0 ml/min

Temperature 35 – 45°C

Sample Size

Volume

< 25 µl

Weight

< 100 µg

4.3.3. Getting Started on Method Development

“Best column, best mobile phase, best detection wavelength, efforts in separation

can make a world of difference while developing HPLC method for routine

analysis. Determining the ideal combination of these factors assures faster

delivery of desired results – a validated method of separation. ”

a) The Best Mobile Phase

In reverse-phase chromatography, the mobile phase is more polar than the stationary

phase. Mobile phase in these systems is usually mixtures of two or more individual

solvents with or without additives or organic solvent modifiers. The usual approach

is to choose what appears to be the most appropriate column, and then to design a

mobile phase that will optimize the retention and selectivity of the system.

Separations in these systems are considered to be due to different degrees of

hydrophobicity of the solutes. The polarity of organic modifier and its proportion

control the rate of elution of the components in the mobile phase. The rate of elution



is increased by reducing the polarity. The simple alteration of composition of the

mobile phase or of the flow rate allows the rate of the elution of the solutes to be

adjusted to an optimum value and permits the separation of a wide range of the

chemical types. First isocratic run followed by gradient run is preferred.

Since the mobile phase governs solute-stationary phase interaction, its choice is

critical.

Practical considerations dictate that it should not degrade the equipment or the

column packing. For this reason, strong acids, bases and halide solutions should

be avoided.

Chemical purity of solvents is an important factor. Since large volumes of solvent

are pumped through the column, trace impurities can easily concentrate in

column and eventually be detrimental to the results. Spectro or HPLC grade

solvents are recommended.

Volatility should be considered if sample recovery is required.

Viscosity should be less than 0.5 centipoises, otherwise higher pump pressures

are required and mass transfer between solvent and stationary phase will be

reduced.

LC/MS-only volatile buffers.

b) The Best Detector

The next consideration should be the choice of detector. There is little use in

running a separation if detector one uses cannot “see” all the components of

interest, or conversely, if it “sees” too much. UV-visible detectors are the most

popular as they can detect a broad range of compounds and have a fair degree of

selectivity for some analytes. Unfortunately UV-visible detectors are not

universal detectors so it is worthwhile to look at the chemical structure of the

analyte to see if it has suitable chromophores, such as aromatic rings, for UV-

visible detection.



Table-20. Detector options

Detector AnalytesSolvent

RequirementsComments

UV-visibleAny with

chromophores

UV-grade non-UV absorbing

solvents

Has a degree of

selectivity and is useful

for many HPLC

applications

FluorescenceFluorescent

compounds

UV-grade non-

UV absorbing

solvents

Highly selective and

sensitive. Often used to

analyze derivatized

compounds

Refractive Index

(RI)

Compounds with

a different RI to

the mobile phase

Cannot run

mobile phase

gradients

Virtually a universal

detector but has limited

sensitivity

Electrochemical

Readily oxidized

or reduced

compounds,

especially

biological

samples

Mobile phase

must be

conducting

Very selective and

sensitive

Evaporative Light

Scattering (ELSD)

Virtually all

compounds

Must use volatile

solvents and

volatile buffers

A universal detector

which is highly

sensitive. Not selective

Mass

Spectrometer

(MS)

Broad range of

compounds

Must use volatile

solvents and

volatile buffers

Highly sensitive and is a

powerful 2nd

dimensional analytical

tool. Many modes

available. Needs trained

operators

c) The Best Column LengthMany chromatographers make the mistake of simply using what is available. Often this

is a 250 × 4.6mm C18 column. These columns are able to resolve a wide variety of

compounds (due to their selectivity and high plate counts) and are common to most

laboratories. While many reverse phase separations can be carried out on such column,

its high resolving capabilities are often unnecessary, as illustrated in



Figure 2. Method development can be streamlined by starting with shorter columns; 150,

100 or even 50mm long. This is simply because they have proportionally shorter run

times.

Fig.3: Effect of Column length

d) The Best Stationary Phase

Selecting an appropriate stationary phase can also help to improve the efficiency

of method development. For example, a C8 phase (reversed phase) can provide a

further time saving over a C18, as it does not retain analytes as strongly as the

C18 phase. For normal phase applications, cyano (nitrile) phases are most

versatile.

e) The Best Internal Diameter

By selecting a shorter column with an appropriate phase, run times can be

minimized so that an elution order and an optimum mobile phase can be



quickly determined. It can also be advantageous to consider the column

internal diameter. Many laboratories use 4.6mm ID columns as a standard, but

it is worth considering the use 4.6mm ID columns as an alternative. These

require only 75% of the solvent flow that a 4.6mm column uses. This

translates to a 25% solvent saving over the life of the column and can be even

more significant if a routine method is developed for such a column.

f) Gradient Programming

The fastest and easiest way to develop a method is to use a mobile phase

gradient. Always start with a weak solvent strength and move to a higher

solvent strength. To begin, use a very fast gradient (e.g.10 minutes) and then

modify the starting and finishing mobile phases to achieve a suitable separation.

Of course the choice of solvents and buffers may need to be modified during

method development. (Different HPLC instruments will give different results

for the same gradient, so if a method is to be validated for use by several

different laboratories, isocratic methods are recommended). Optimizing the

mobile phase for an analysis will help to improve the separation. A number of

factors depend upon the solvents chosen.

g) Retention

Analytes may be too strongly retained (producing long run times). If this occurs,

the solvent strength should be increased. In reverse phase analysis this means a

higher % of organic solvent in the mobile phase.

h) Poor Separation

Analytes often co-elute with each other or impurities. To overcome this, the

analysis should be run at both higher and lower solvent strengths so the bestseparation conditions may be determined. Varying solvents may help - try

methanol instead of acetonitrile for reversed phase analysis. Using buffers and

modifying the pH (within the column’s recommended pH range) can also assist

the separation. When the optimum conditions have been achieved, improving

the resolution is often just a case of changing to a longer column and/or one

with a smaller particle size to increase the column efficiency. (For reversed

phase analysis, having started with a 100mm C8 column there is also the option



of trying C18 columns to get better resolution. The important point is having

used a short column for this stage of the development a lot of time was saved).

i) Peak Shape

This is often a problem, especially for basic compounds analyzed by reversed

phase HPLC. To minimize any potential problems always use a high purity

silica phase such as Wakosil II. These modern phases are very highly

deactivated so secondary interactions with the support are minimal. Buffers can

be used effectively to give sharp peaks. If peak shape remains a problem, use an

organic modifier such as triethylamine, although this should not be necessary

with modern phases like Wakosil. One point often forgotten is the effect of

temperature changes on a separation. To maximize the reproducibility of a

method, it is best to use a column heater to control the temperature of the

separation. A temperature of 35 – 40°C is recommended.

j) Buffer selection

In reverse phase HPLC, the retention of analytes is related to their

hydrophobicity. The more hydrophobic the analyte, the longer it is retained.

When an analyte is ionized, it becomes less hydrophobic and, therefore, it

retention decreases. When separating mixtures containing acid and/or bases by

reversed phase HPLC, it is necessary to control the pH of mobile phase using

appropriate buffer in order to achieve reproducible results.

When separating acids and bases a buffered mobile phase is recommended to

maintain consistent retention and selectivity. A buffered mobile phase, by

definition, resists changes in pH so that the analytes and silica will be

consistently ionized, resulting in reproducible chromatography. If the sample isneutral, buffers or additives are generally not required in the mobile phase.

Acids or bases usually require the addition of a buffer to the mobile phase. For

basic or cationic samples, “less acidic” reverse-phase columns are

recommended and amine additives for the mobile phase may be beneficial.

Optimum buffering capacity occurs at a pH equal to the pKa of the buffer.

Beyond that, buffering capacity will be inadequate.



Buffers play an additional role in the reproducibility of a separation. The buffer

salts reduce peak tailing for basic compounds by effectively masking silanols.

They also reduce potential ion-exchange interactions with unprotonated silanols

(Figure 3). To be most effective, a buffer concentration range of 10 - 50 mM is

recommended for most basic compounds.

Fig.-4: Peak Tailing Interaction

Table21. Commonly used Buffers for reversed phase HPLC

Buffer PKa

(25°C)Maximum

Buffer RangeUV Cut-off (nm)

TFA 0.3 - 210

Phosphate,pK

1 H2PO4 2.1 1.1-3.1 < 200

Phosphate,pK

2 HPO42-

7.2 6.2-8.2 < 200

Phosphate,pK

3 PO43-

12.3 11.3-13.3 < 200

Citrate, pK

1

C3H5O(COOH)2(COO)1-

3.1 2.1-4.1 230

Citrate, pK

2

C3H5O(COOH)1(COO)2-

4.7 3.7-5.7 230

Citrate, pK

3

C3H

5O(COO)

3-

6.4 4.4-6.4 230

Carbonate, pK

1

HCO31-

6.1 5.1-7.1 < 200

Carbonate, pK

2

CO32-

10.3 9.3-11.3 > 200

Formate 3.8 2.8-4.8 210

Acetate 4.8 3.8-5.8 210

Ammonia 9.3 8.3-10.3 200

Borate 9.2 8.2-10.2 N/A

TEA 10.8 9.8-11.8 < 200



k) Selection of pH

The pH range most often used for reversed-phase HPLC is 1 - 8 and can be

divided into low pH (1 - 4) and intermediate pH (4 - 8) ranges. Each range has a

number of advantages. Low pH has the advantage of creating an environment inwhich peak tailing is minimized and method ruggedness is, maximized. For this

reason, operating at low pH is recommended.

At a mobile phase pH greater than 7, dissolution of silica can severely shorten

the lifetime of columns packed with silica-based stationary phases.

The pKa value (acid dissociation [ionization] constant) for a compound is the

pH at which equal concentrations of the acidic and basic forms of the molecule

are present in aqueous solutions. Analytes may sometimes appear as broad or

tailing peaks when the mobile phase pH is at, or near, their pKa values. A more

rugged mobile phase pH will be at least 1 pH unit different from the analyte

pKa. This shifts the equilibrium so that 99% of the sample will be in one form.

The result is consistent chromatography.

Dramatic changes in the retention and selectivity (peak spacing) of basic and

acidic compounds can occur when the pH of the mobile phase is changed. This

is often a result of different interactions between the column and the analytes

when the ionization of these compounds changes. It is important to evaluate

these changes when a method is developed in order to select the mobile phase

pH that provides the most reproducible results.

4.4. ANALYTICAL MEHOD VALIDATION TERMINOLOGY:

[6-9]

“Doing thorough method validation can be tedious, but the consequences of

not doing it right are wasted time, money, and resources.”

4.4.1 Definition:

Validation is a process of establishing documented evidence, which provides a

high degree of assurance that a specific activity will consistently produce a



desired result or product meeting its predetermined specifications and quality

characteristics.

Method validation is the process of demonstrating that analytical procedures are

suitable for their intended use and that they support the identity, quality, purity,

and potency of the drug substances and drug products. Simply, method validation

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

purpose. A successful Validation guarantees that both the technical and

regulatory objectives of the analytical methods have been fulfilled. The transfer

of a method is best accomplished by a systematic method validation process. The

real goal of validation process is to challenge the method and determine limits of

allowed variability for the conditions needed to run the method.

4.4.2 Objective of validation

The objective of validation of analytical procedure is to demonstrate that it is

suitable for its intended purpose. Validation is documented evidence, which

provide a high degree of assurance for specific method. Any developed method

may be influenced by variables like different elapsed assay times, different days,

reagents lots, instruments, equipments, environmental conditions like

temperature, etc so it is expected that after the method has been developed and

before it is communicated or transferred from one lab to the other, it is properly

validated and the result of validity tests reported.

Two steps are required to evaluate an analytical method.

1) First determine the classification of the method.

2) The second step is to consider the characteristics of the analyticalmethod

For analytical method validation of pharmaceuticals, guidelines from the

International Conference on Harmonization (ICH), United States Food and Drug

Administration (US FDA), American Association of Official Analytical Chemists

(AOAC)United States Pharmacopoeia (USP), and International Union of Pure

and Applied Chemists (IUPAC) provide a framework for performing such

validations in a more efficient and productive manner50.



The primary objective of validation is to form a basis for written procedure for

production and process control which are designed to assure that the drug

products have the identity, strength, quality and purity they purport or are

represented to possess quality, safety and efficacy must be designed to build into

the product. Each step of the manufacturing process must be controlled to

maximize the probability that the finished products meet all quality and design

specification.

4.4.3 Data Elements Required for Assay Validation

Both the USP and ICH recognize that is it not always necessary to evaluate every

analytical performance parameter. The type of method and its intended use

dictates which parameters needed to be investigated, as illustrated in Table 4

Table-22. ICH Validation Guideline

Type of analytical

procedure

Characteristics

IDENTIFICATION

TESTING

FOR

IMPURITIES

Quantitative

Limit

ASSAY

-dissolution

(measurement only)

-content/potency

Accuracy

Precision

Repeatability

Interm.Precision

Reproducibility

Specificity (3)

Detection Limit

Quantitation Limit

Linearity

Range

-

-

-

-

+

-

-

-

-

+ -

+ -

+(1) -

- (2) -

+ +

- +

+ -

+ -

+ -

+

+

+(1)

- (2)

+(4)

-

-

+

+

- Signifies that this characteristic is not normally evaluated.

+ Signifies that this characteristic is normally evaluated.

1. Intermediate precision is not needed in some case, when

reproducibility is checked.

2. May be needed in some cases.



3. Lack of specificity of one analytical procedure could be

compensated by other supporting analytical procedure(s).

4. May not be needed in some cases.

The different parameters of analytical method development are discussed below

as per ICH guideline:-

1) Specificity:

Specificity is the ability to assess unequivocally the analyte in the presence of

components which may be expected to be present. Typically these might include

impurities, degradants, matrix, etc.

Method:

When the impurities are available: Spiking of pure substance (drug

substance or drug product) with appropriate levels of impurities/excipients

and demonstrate the result is unaffected.

When the impurities are not available: Comparing the test results of sample

containing impurities or degradation product to second well-characterized

procedure. These comparisons should include sample under relevant stress

condition.

In chromatographic method: Peak purity test to be done by diode array and

mass spectrometry.

Expression/calculation:

Proof of discrimination of analyte in the presence of impurities. e.g. for

chromatography chromatogram should be submitted.

Peak purity test helps in demonstrating that the peak is not attributable to

more than one component.

For assay two results should be compared and for impurity tests two

profiles should be compared.

Acceptance criteria:

a) Interference from blank, placebo and impurities:

There should not be any interference from blank, placebo and impurities

peak with the main peaks.



Peak purity factor for the main peaks in standard preparation, unspiked

sample preparation and spiked sample preparation with known

impurities should be equal to or more than 995.

Assay difference of spiked and unspiked samples should not be morethan 2.0% absolute.

b) Interference from degradation products by stress study:

Degradation impurities in all degraded API preparations and sample

preparations should be separated from the main peak.

Peak purity factor for the main peaks in all unstressed and degraded API

and sample preparations should be equal to or more than 995.

2) Linearity:

The linearity of an analytical procedure is its ability (within given range) to

obtain test results, which are directly proportional to the concentration

(amount) of analyte in the sample.

Method:

Drug (different dilution) and/or separately weighed synthetic mixture.

Measurement of response and plot response vs. concentration of analyte and

demonstration of linearity by

Visual inspection of plot

Appropriate statistical methods

Recommendation:

Minimum of 5 concentrations are recommended


Correlation coefficient, y-intercept, slope of regression line, residual sum of

squares.


The correlation co-efficient (r) value should not be less than 0.995 over the

working range.

3) Range:



The range of analytical procedure is the interval between the upper and

lower concentration (amounts) of analyte in the sample (including these

concentrations) for which it has been demonstrated that the analytical

procedure has a suitable level of precision, accuracy and linearity.

Method:

Drug (different dilution) and/or separately weighed synthetic mixture.

Measurement of response and plot response vs. concentration of analyte and

demonstration of linearity by

Visual inspection of plot

Appropriate statistical methods

Recommendation:

Assay of drug/finished product: 80 – 120% of test concentration.

For content uniformity: 70 – 130% of test concentration.

For dissolution testing: ± 20% over specified range.

For impurity: from reporting level to 120% of specification.


Correlation coefficient, y-intercept, slope of regression line, residual sum of

squares.


Not specified

4) Accuracy:

The accuracy of analytical procedure expresses the closeness of agreement

between the value which is accepted either as a conventional true value or

an accepted reference value and the value found. This is sometimes termed

trueness.

Method:

Application of procedure to analyze synthetic mixture of known purity.

Comparison of result with already established procedure.

Accuracy may be inferred once precision, linearity and specificity have

been established.



Recommendation:

Minimum of nine determinations

Low concentration of range × 3 replicates

Medium concentration of range × 3 replicates

High concentration of range × 3 replicates


Percent recovery by the assay of known added amount of analyte

Mean – Accepted true value with confidence interval

The % recovery was calculated using the formula,

100

)(covRe%

b

abaery

Where,

a – Amount of drug present in sample

b – Amount of standard added to the sample


Individual and mean % recovery at each level should be 98.0% to 102.0%.

5) Precision:

The precision of an analytical procedure expresses the closeness of agreement

(degree of scatter) between the series of measurements obtained from multiple

sampling of the same homogeneous sample under the prescribed conditions.

Method:

Determination of % relative standard deviation (RSD) of response of

multiple aliquots.

Recommendation:

a) Repeatability (Same operating condition over short interval of time):



Minimum of nine determinations

Low concentration of range × 3 replicates

Medium concentration of range × 3 replicates

High concentration of range × 3 replicates

(Or)

At target concentration × 6 determinations

Acceptance Criteria:

RSD for assay of six determinations should not be more than 2.0%.

b) Intermediate precision (within laboratory variation):

Different Days

Different Analysts

Different Equipment etc.


Standard deviation, % RSD and confidence interval


RSD for assay of six determinations should not be more than 2.0%.

Difference between the mean assay value obtained in the intermediate

precision study and method precision study should not be more than 2.0%

absolute.

6) Detection Limit:

The detection limit of an individual analytical procedure is the lowest

amount of analyte in a sample, which can be detected but not necessarily

quantitated under stated experimental conditions.

Method:

1. By visual evaluation

2. Based on S/N ratio

Applicable to procedure, which exhibit baseline noise.

Actual lowest concentration of analyte detected in compared with

blank response

3. Based on S.D. of response and slope



LOD = 3.3 σ/s

s = Slope of calibration curve

σ = S.D. of response; can be obtained by

Standard deviation of blank response

Residual standard deviation of the regression line

Standard deviation of the y-intercept of the regression line

Sy/x i.e. standard error of estimate


If based on visual examination or S/N ratio – relevant chromatogram is to

be presented.

If by calculation/extrapolation – estimate is validated by analysis of suitable

no. of samples known to be near or prepared at detection limit.


S/N ratio > 3 or 2:1; not specified in other cases.

7) Quantitation Limit:

The quantitation limit of an individual analytical procedure is defined as the

lowest amount of analyte in a sample, which can be quantitatively determined

with suitable precision and accuracy.

Method:

1. By visual evaluation

2. Based on S/N ratio

Applicable to procedure, which exhibit baseline noise.

Actual lowest concentration of analyte detected in compared with

blank response

3. Based on S.D. of response and slope

LOQ = 10 σ/s



s = Slope of calibration curve

σ = S.D. of response; can be obtained by

Standard deviation of blank response

Residual standard deviation of the regression line

Standard deviation of the y-intercept of the regression line

Sy/x i.e. standard error of estimate

Recommendation:

Limit should be validated by analysis of suitable no. of samples known to

be near or prepared at quantitation limit.Expression/calculation:

Limits of quantitation and method used for determining should be

presented.

Expresses as analyte concentration.


S/N ratio > 10:1; not specified in other cases.

8) Robustness:

The robustness of an analytical procedure is a measure of its capacity to

remain unaffected by small, but deliberate variations in method parameters

and provides an indication of its reliability during normal usage.

Method:

It should show the reliability of an analysis with respect to deliberate variations inmethod parameters.

In case of liquid chromatography, examples of typical variations are

Influence of variations of pH in a mobile phase,

Influence of variations in mobile phase composition,

Different columns (different lots and/or suppliers),

Temperature,

Flow rate.



Recommendation:

Robustness should be considered early in the development of a method.

If the results of a method or other measurements are susceptible to

variations in method parameters, these parameters should be adequatelycontrolled and a precautionary statement included in the method

documentation.


Effect of these changed parameters on system suitability parameters.


System suitability criteria should meet as per test procedure.

The difference between assay value of sample analyzed as per test

procedure and analyzed by applying proposed changes should not be more

than 2.0% absolute.

9) Ruggedness:

The ruggedness of an analytical method is the degree of reproducibility of test

results obtained by analysis of the same samples under a variety of conditions.

Method:

Analysis of aliquots of homogenous lots in different laboratories by

different analysts under different operational and environmental

conditions.


% RSD

Note: In the guideline on definitions and terminology, the ICH did not address

ruggedness specifically. This apparent omission is really a matter of semantics,

however, as ICH chose instead to cover the topic of ruggedness as part of

precision, as discussed previously.



10) Solution stability:

Prepare standard and sample as per test procedure and determine initial assay

value. Store the standard and sample preparation up to 48 hours at room

temperature. Determine the assay of sample preparation after 24 hours and 48

hours storage against freshly prepared standard and determine % response of

standard preparation after 24 hours and 48 hours storage against initial standard

response. The assay value of sample and % response of standard calculated after

24 hours and 48 hours storage should be compared with the initial value and

recorded.

If the stability of solution fails to the acceptance criteria at 24 hour interval at

room temperature, repeat the experiment and injecting after standing for 2, 4, 8,

12, and 18 hours at room temperature.

If the stability of solution is found to be less than 24 hours at room temperature,

then establish the solution stability at 5°C±3°C as per the above procedure.

Calculation: Calculate results as follows:

Standard preparation stability: Calculate the % response of the Standard

preparation after specified period using the formula;

% Response =TA

SA× 100

Where,

TA = the peak area of standard preparation after standing for specified period,

SA = the initial peak area of standard preparation subjected for solution stability,

Sample preparation stability: Calculate the % Assay of the Sample preparation

after specified period as per the test procedure against freshly prepared standard.

Calculate the difference of the result obtained after each interval against initial

result.




The difference in the response of standard preparation should not be more

than ± 2.0% from the initial value at any time interval.

The absolute difference in the assay value of sample should not be more

than ± 2.0% from the initial value at each time point.

11) System Suitability Testing:

The system has to be tested for its suitability for the intended purpose. System

suitability testing is an integral part of many analytical procedures. 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.

Numerous approaches may be used to set the limits for system suitability tests.

This depends on experience with the method, material available and personal

preference. Parameters such as plate count, tailing factors, resolution and

reproducibility (% RSD retention time and area for six repetitions) are determinedand compared against the specifications set for the method.

Table- 23. System Suitability Parameters and their recommended limits

Parameter Recommendation

Capacity Factor (K’) The peak should be well-resolved from other peaks and

the void volume, generally K’ > 2

Repeatability RSD ≤ 1% ; N ≥ 5 is desirable

Relative Retention Not essential as the resolution is stated.

Resolution(Rs)

Rs of > 2 between the peak of interest and the closest

eluting potential interferent (impurity, excipients,

degradation product, internal standard, etc.)

Tailing Factor(T) T ≤ 2

Theoretical Plates(N) In general should be > 2000.



Table -24. Characteristics to be validated in HPLC

Characteristics Acceptance Criteria

Accuracy/trueness Recovery 98-102% (individual)

Precision RSD < 2%

Repeatability RSD < 2%

Intermediate Precision RSD < 2%

Specificity / Selectivity No interference

Detection Limit S/N > 2 or 3

Quantitation Limit S/N > 10

Linearity Correlation coefficient r2

> 0.999

Range 80 – 120 %

Stability > 24 h or >12 h

Documents

Introduction Analysis and Validation