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

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. 1 andλ2), it may be possible to determine both drugs by the technique of simultaneous equation (Vierodts method) provided that certain criteria apply. The informations required are:

the absorptivities of X at 1 and 2, ax1 and ax2 respectively the absorptivities of Y at 1 and2, ay1 and ay2 respectively The absorbance of the diluted sample at 1 and 2, 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 1 and2, the absorbance of the mixture is the sum of the individual absorbance of X and Y. At 1 A1 = ax1bCx + ay1bCy At 2 A2 = ax2bCx + ay2bCy . (2)
.

(1)

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 max 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 max of component X -2 is the max 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 1 A1 = ax1bCx + ay1bCy At 2 A2 = ax2bCx + ay2bCy
. (1)

(2)

Such equation can be solved using matrices. From equation (1) and (2), A1 = kx1 Cx + ky1Cy A2 = kx2 Cx + ky2Cy 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) . (3)
.

(4)

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 Cramers 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 = Cy = (A1 ay2 - A2 ay1) / ( ax1 ay2 - ax2 ay1) (ax1 A2 - ax2 A1) / ( ax1 ay2 - ax2 ay1) (9) ..(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 particular advantage 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 Layer of Sorbent Efficiency Separations HPTLC 100m High due to smaller particle size generated 3 - 5 cm Shorter migration distance Analysis Time and the analysis time is greatly reduced Wide choice of stationary Solid support phases like silica gel for normal phase and C8 , C18 for reversed phase modes Silica gel , Alumina & Kiesulguhr Slower TLC 250m Less 10 - 15 cm

Development chamber Sample spotting

New type that require less amount of mobile phase Auto sampler Use of UV/ Visible/ Fluorescence scanner scans

More amount Manual spotting

Scanning

the entire chromatogram qualitatively and quantitatively densitometer

Not possible

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

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 110-120c for 30 prior to spotting. Aluminum sheets should be kept in between two glass plates and placing in oven at 110-120c for 15 minutes. Application of sample and standard Usual concentration range is 0.1-1g / l 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 - Ones 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 -15 ml 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 camera transmits 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 Dimensions(Length ID) Column Particle Size Stationary Phase Solvents A and B %B Buffer (compound, pH, concentration) Mobile Phase Additives(e.g., amine modifiers, ion-pair reagents) Flow-rate Temperature Volume Sample Size Weight < 100 g Do not use initially 1.5-2.0 ml/min 35 45C < 25 l Preferred initial choice 15 0.46 cm 5m C8 or C18 Buffer-Acetonitrile 80-100% 10 - 25mM Phosphate Buffer 2.0 < pH < 3.0

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 UVvisible detection.

Table-20. Detector options Detector Analytes Solvent Requirements UV-grade nonUV absorbing solvents UV-grade nonUV absorbing solvents Cannot run mobile phase gradients Comments Has a degree of selectivity and is useful for many HPLC applications Highly selective and sensitive. Often used to analyze derivatized compounds Virtually a universal detector but has limited sensitivity

UV-visible

Any with chromophores

Fluorescence

Fluorescent compounds Compounds with a different RI to the mobile phase Readily oxidized or reduced compounds, especially biological samples Virtually all compounds

Refractive Index (RI)

Electrochemical

Mobile phase must be conducting

Very selective and sensitive

Evaporative Light Scattering (ELSD)

Must use volatile solvents and volatile buffers

Mass Spectrometer (MS)

Broad range of compounds

Must use volatile solvents and volatile buffers

A universal detector which is highly sensitive. Not selective Highly sensitive and is a powerful 2nd dimensional analytical tool. Many modes available. Needs trained operators

c) The Best Column Length Many 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 best separation 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 columns 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 40C 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 is neutral, 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 TFA Phosphate,pK1 H2PO4 Phosphate,pK2 HPO42Phosphate,pK3 PO43Citrate, pK1 C3H5O(COOH)2(COO)1Citrate, pK2 C3H5O(COOH)1(COO)2Citrate, pK3 C3H5O(COO)3Carbonate, pK1 HCO31Carbonate, pK2 CO32Formate Acetate Ammonia Borate TEA PKa (25C) 0.3 2.1 7.2 12.3 3.1 4.7 6.4 6.1 10.3 3.8 4.8 9.3 9.2 10.8 1.1-3.1 6.2-8.2 11.3-13.3 2.1-4.1 3.7-5.7 4.4-6.4 5.1-7.1 9.3-11.3 2.8-4.8 3.8-5.8 8.3-10.3 8.2-10.2 9.8-11.8 Maximum Buffer Range UV Cut-off (nm) 210 < 200 < 200 < 200 230 230 230 < 200 > 200 210 210 200 N/A < 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 in which 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 method 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. analytical

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 TESTING Type of analytical procedure Characteristics IDENTIFICATION FOR IMPURITIES Quantitative Limit Accuracy Precision Repeatability Interm.Precision Reproducibility Specificity (3) Detection Limit Quantitation Limit Linearity Range + + +(1) - (2) + + + + + + + +(1) - (2) +(4) + + + ASSAY -dissolution (measurement only) -content/potency +

- 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 more than 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 Expression/calculation: Correlation coefficient, y-intercept, slope of regression line, residual sum of squares. Acceptance criteria: 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. Expression/calculation: Correlation coefficient, y-intercept, slope of regression line, residual sum of squares. Acceptance criteria: 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

Expression/calculation: 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,

% Re covery
Where,

( a b) a b 100

a Amount of drug present in sample b Amount of standard added to the sample Acceptance criteria: 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. Expression/calculation: Standard deviation, % RSD and confidence interval Acceptance criteria: 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 /s s = Slope of calibration curve = 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 Expression/calculation: 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.

Acceptance criteria: 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 /s

s = Slope of calibration curve = 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.

Acceptance criteria: 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 in method 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 adequately controlled and a precautionary statement included in the method documentation. Expression/calculation: Effect of these changed parameters on system suitability parameters. Acceptance criteria: 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.

Expression/calculation: % 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 5C3C 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; TA % Response = 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.

Acceptance criteria: 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 determined and compared against the specifications set for the method. Table- 23. System Suitability Parameters and their recommended limits Parameter Capacity Factor (K) Repeatability Relative Retention Recommendation The peak should be well-resolved from other peaks and the void volume, generally K > 2 RSD 1% ; N 5 is desirable Not essential as the resolution is stated. Rs of > 2 between the peak of interest and the closest Resolution(Rs) eluting potential interferent (impurity, excipients, degradation product, internal standard, etc.) Tailing Factor(T) Theoretical Plates(N) T2 In general should be > 2000.

Table -24. Characteristics to be validated in HPLC Characteristics Accuracy/trueness Precision Repeatability Intermediate Precision Specificity / Selectivity Detection Limit Quantitation Limit Linearity Range Stability Acceptance Criteria Recovery 98-102% (individual) RSD < 2% RSD < 2% RSD < 2% No interference S/N > 2 or 3 S/N > 10 Correlation coefficient r2 > 0.999 80 120 % > 24 h or >12 h