Validated Analytical Methods For Determination Of Active Ingredients From Bulk Drugs And Pharmaceutical Dosage Forms

Mr. Vitthal V. Chopade

Validation of Anaflytical Method

Method validation has received considerable attention in literature and from industrial committees and regulatory agencies.

The International Conference on Harmonization (ICH) of technical requirements for the registration of pharmaceuticals for human use has developed a consensus text on the validation of analytical procedures. The document includes definition of different validation parameters. The United States Environmental Protection agency (US EPA), Resource Conservation and Recovery Act (RCRA). The American Association of Official Analytical Chemist (AOAC), United States Environmental Protection Agency (USP) and other scientific organizations provide methods that are validated through multi-laboratory studies¹.

The United States Food and Drug Administration (US FDA) has proposed guidelines on submitting sample and analytical data for methods validation. The United States Pharmacopoeia (USP) has published specific guidelines for method validation and compound evaluation².

The objective of validation of analytical procedures is to demonstrate that it is suitable for its intended purpose. The discussion of the validation of analytical procedures is directed to the four most common types³

• Identification tests.
• Quantitative tests for impurities content.
• Limit tests for the control of impurities.
• Quantitative tests of the active moiety in samples of drug substance or drug product or other selected components in the drug product.

 Methods need to be validation and revalidation⁴

• Before their introduction into routine use. 
• Whenever the condition change for which the method has been validated e.g. instrument with different characteristics.
• Whenever the method is changed and the changes are outside the original scope of the method.

Analytical Procedure

Analytical procedure refers to the way of performing the analysis. It should describe in detail the steps necessary to perform each analytical test. This may include sample, the reference standard and the reagent preparation, use of the apparatus, generation of the calibration curve, use of the formula for the calculation, etc.

Specificity

Specificity is the ability to access unequivocally the analyte in presence of components which may be expected to present. Typically these might include impurities, degradents, matrix, etc.

Accuracy

The accuracy of an 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.

Precision

The precision of an analytical procedure expresses the closeness of agreement between a series of measurement obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. It may be considered at three levels: repeatability, intermediate precision and reproducibility.

Repeatability

Repeatability expresses the precision under the same operating conditions over a small interval of time. Repeatability is also termed intra-assay precision.

Intermediate Precision

 Intermediate precision expresses within-laboratories variation: different days, different  equipment, etc.

Reproducibility

Reproducibility expresses the precision between laboratories.

Detection Limit

The detection limit of an individual analytical procedure is the lowest amount of analyte in the sample that can be detected but not quantified as an exact value.

Quantitation Limit

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in the sample that can be quantitatively determined with precision and accuracy. Quantitation limit is a parameter for quantitatively assay of low level of compounds in sample matrices and is used particularly for the determination of impurities and/or degradation products.

Linearity

The linearity of an analytical procedure is the ability to obtain test results that are directly proportional to the concentration of an analyte in the sample.

Range

The range of an analytical procedure is the interval between the upper and lower concentration of an analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable precision, accuracy and linearity.

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 ability during normal range.

High Performance Thin Layer Chromatography

Chromatography is a powerful separation technique that finds application to all branches of science ⁶. Chromatography is a separation technique whereby the components of a mixture may be separated by allowing the sample to be transported through packed bed of material by fluid mobile phase ^7-8.  Out of almost 700 pharmaceutical formulations that are documented in the USP almost 43% of the procedures are those documented by thin layer chromatography.

The significant popularity of HPTLC in the analytical testing of pharmaceutical, bulk drugs and herbal lends its fame to the attributes ^9-10 

1. High speed quantitative analysis.

2. Flexibility to analyze different samples concurrently.

3. Low running cost making the analysis more cost efficient.

4. The sample clean up especially for this technique is minimal.

5. HPTLC provides accuracy, precision and sensitivity comparable to HPLC.

6. Qualitative, quantitative and preparative analysis with the same system is possible.

7. HPTLC provides positive identification as well as visualization of the separated fraction of  sample component. 

8.Pre and post derivatization of sample bands for enhanced stability and visualization is  possible.

9. The cost for maintenance as well as methods development is comparatively low.                                               

10.Preparative work for large sample amounts is a positive advantage of HPTLC.

Steps involves in the development of HPTLC method

High Perfomance Liquid Chromatography ¹¹

Chromatography is a method in which the components of a mixture are separated on an adsorbent column in a flowing system. The separation occurs because, under an optimum set of conditions, each component in a mixture will interact with the two phases differently relative to the other components in the mixture. Chromatography is described and measured in terms of four major concepts: capacity, efficiency, selectivity, and resolution. The capacity and selectivity of column are variables that are controlled, to some extent by the chromatography, to obtain the best possible separation; the efficiency of the chromatographic system must be optimized in order to minimize band broadening. The column should have capacity to retain the solutes and it should have the appropriate selectivity to resolve the analytes of interest.

Steps involved in the development of HPLC method ¹²

Literature survey

Here a detailed account of all analytical methods existing for the drug is made to avoid duplication of the method developed. Details about the structure of the drugs and their physicochemical properties are also collected.

Selection of chromatographic mode

 A variety of chromatographic modes have been developed on the basis of mechanism of retention and operation introduced. The key chromatographic modes are normal phase, reversed phase, ion exchange, size exclusion, and affinity chromatography.

Normal phase chromatography and its mechanism

The term normal phase is used to denote a chromatographic system in which a polar stationary phase is employed and a less polar mobile phase is used for elution of analytes.

In the normal phase mode, neutral solutes in solution are separated on the basis of their polarity: the more polar the solute, the greater is its retention on the column.

Mechanism of retention

Two models have been developed to describe the adsorption process. The first model known as the competition model, assumes that the entire surface of the stationary phase is covered by the mobile phase molecules and that adsorption occurs as a result of competition for the adsorption sites between the solute molecule and the mobile phase molecules. The solvent interaction model

on the other hand suggests that a bilayer of solvent molecule is formed around the stationary phase particles. In the latter model, retention results from interaction of the solute molecule with the secondary layer of adsorbed mobile phase molecules.

Reversed phase chromatography and its mechanism

It is most widely used chromatographic mode and used to separate neutral molecules in solution on the basis of their hydrophobicity. It involves the use of a non-polar stationary phase and a polar mobile phase. As a result, a decrease in the polarity of the mobile phase results in a decrease in solute retention. Modern reversed phase chromatography typically refers to the use of chemically bonded stationary phase, where a functional group is bonded to silica.

Mechanism of retention

Two main theories, the so called solvophobic and partitioning theories have been developed to explain the separation mechanism. In solvophobic theory the stationary phase is thought to behave more like a solid than a liquid, and retention is considered to be related primarily to hydrophobic interaction between the solutes and the mobile phase (solvophobic effects), because of the solvophobic effects, the solutes bind to the surface of the stationary phase, there by reducing the surface area for the retention of analyte exposed to the mobile phase. Solutes are retained more as a result of solvophobic interactions with the mobile phase than through specific interaction with the stationary phase. In partitiong theory, the stationary phase plays a more important role in the retention process. The solute is thought to be fully embedded in the stationary phase chains, rather than adsorbed on the surface and therefore is considered to be partitioned between the mobile phase and liquid like stationary phase.

Ion Exchange Chromatography¹³

Ion Exchange Chromatography, species are separated on the basis of difference in electric charge. The primary mechanism of retention is the electrostatic attraction of ionic solutes in solution to fixed ions of opposite charge on the stationary phase support. The stationary phase or ion exchanger is classified as an anion exchange material when the fixed ion carries a positive charge and as a cation exchanger when the fixed ion carries a negative charge.

Mechanism of retention

As an ionic solute passes through the column, it distributes itself between the mobile phase and stationary phase by exchanging with the couter ions associated with the stationary phase. As the electro neutrality of the solution must be maintained during the ion exchange process, the exchange is stoichiometric: a single monovalent solute ion displaces a single monovalent counter ion. Separation occurs as a consequence of difference in the size, charge density, and structure of the different ionic solutes.

Size Exclusion Chromatography ^14-17

Size exclusion chromatography is a convenient and highly predictable method for separating simple mixtures whose components are sufficiently different in molecular weight. For small molecules, a size difference of more than about 10 % is required for acceptable resolution: for macromolecules a two fold difference in molecular weight is necessary.

Mechanism of retention

Stationary phase in SEC is a highly porous substrate whose pores are penetrated best by small solute molecules. Because higher solutes molecules are unable to enter as deeply into the pores, they will travel further down the column in the same time. The largest molecules, which are totally excluded from the pores, are eluted first from the column. Because the solvent molecules are usually the smallest, they are normally the last to be eluted. The rest of the solute molecules are eluted between these two extremes, at a time dependent on their ability to penetrate into the pores.

Affinity chromatography

Affinity chromatography may be applied to the separation of any biological entity that is capable of forming a dissociable complex with another species. It is based on the ability of biological macromolecules to recognize and bind to other molecules, often in a highly specific manner.

Mechanism of retention

An affinity ligand that is specific to only one type of biological molecule is covalently bonded to an inert support material. A sample containg mixture of biomolecules is applied to the head of the column. As the sample is washed through the column only those species that are complementary to the affinity ligand molecules are adsorbed; the other components are eluted without retention. The adsorbed species are eluted after the rest of the sample by changing the composition of the mobile phase.

Selection of stationary phase

The simplest criteria for selection of stationary phase is like have affinity for like. Matching the polarity of sample and stationary phase and using mobile phase of different polarity, achieves a successful separation.

Selection of mobile phase

Reversed phase bonded packing when used in conjunction with highly polar solvents is the ideal approach and universal system for liquid chromatography. Similarly polar sample will need a non polar mobile phase. In liquid chromatography mobile phase may be either single liquid or combination of liquids, which are compatible with the sample, column and instrument.

Selection of suitable detector

When the entire component absorbs electromagnetic radiations the most suitable detector is a variable wavelength UV detector. Refractometer is the only universal detector but it suffers from lack of sensitivity and unable to cope with the solvent programming.

Steps involved in Quantitative Analysis

After resolution is accomplished an appropriate method for quantitative analysis is to be set up, for multicomponent analysis it is essential that good resolution for all component is obtained. 

Sampling and sample preparation

The sample should be homogeneous. Sample must be completely soluble and solvent used to dissolve the sample should be the initial mobile phase or any solvent miscible with the mobile phase.

Chromatographic separation

After achieving resolution with a pre- optimized solvent system to obtain reproducible results following criteria must be satisfied.

a. Monitoring flow rate.

b. Keeping the composition intact.

c. Solvent system must be covered before storage.

d. Monitoring column temperature.

Detection

The response obtained from a given detector will vary according to the nature of solutes and the chromatographer must calibrate by running standards at several levels to determine the response factor. With a UV detector the response is related to both concentration and molecular extinction coefficient of the component at the wavelength of detection.

Measurement and calibration

The various approaches used for quantitative analysis:

• Peak height method.
• Peak area method.
• Use of internal standards.
• Use of external standards.

In the peak area method a mixed standard solution containing known quantities of all the components of the mixture is chromatographed several times. The mean of the peak area of each component is calculated and is used to calibrate the detector response. The sample is also chromatographed several times and mean peak area for each component is correlated with peak area of tile standard and the amount of each component present in the mixture is calculated.

Role of temperature

Although temperature is less significant in liquid chromatography it deserves consideration as a variable to be optimized for difficult separation. Since increasing temperature reduces carrier viscosity and increases diffusion rate, increasing temperature can increase column efficiency. 

References:

1. International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use, Validation of analytical procedures, ICH-Q2A, Geneva 1995.

2. US FDA Technical Review Guide: Validation of Chromatographic Methods, Center for Drug Evaluation and Research (CDER), Rockville , MD , 1993.

3. International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use, Validation of analytical procedures: Methodology, ICH-Q2B, Geneva 1996.

4. General Chapter 1225, Validation of compendial methods , United States Pharmacopeia XXIII, National Formulary, XVIII, Rockville, MD, The United States Pharmacopeial Convention, Inc, 1995, 1710–1612.

5.Pikering, W. F., Modern Analytical Chemistry, Macel Dekker Inc, New york , 1971, 265.

6.Skoog, D. A., West, D. M., Holler, F. J., Analytical Chemistry- An Introduction, Saunders College Publishing, Philadelphia , 1996 Edn. 7^th, 5-11. 

7. Szepesi, M. Gazdag and K. Mihalyfi, Selection of HPLC methods in pharmaceutical analysis -III method validation, www.labcompliance.com Page 21 J.Chromatogr. 464, 265-278.

8.Zaltkis. A., Kaiser. R. E., HPTLC- High Performance Thin Layer Chromatography, 1977, 619-625A.

9.Sethi, P. D., Quantitative Analysis of Drugs in Pharmaceutical Formulations, Unique Publisher, 1997, 11.

10.J.M.Green, A Practical Guide to Analytical Method Validation, Anal.Chem. News & Features, May 1, 1996, 305A/309A 14. B. Renger, H.Jehle, M.Fischer and w. Funk, Validation of analytical procedures in pharmaceutical analytical chemistry: HPTLC assay of theophylline in an effervescent tablet, J.Planar Chrom., 8, July/Aug 1995, 269-278.

11.M. Huber, Applications of diode-array detection in HPLC, Waldbronn , Germany , Agilent Technologies, 1989, publ. number 12-5953-2330.

12.Ewing, G. W., Instrumental methods of Chemical Analysis, Mc Graw Hill International Book Co, London, Ed. 4^th , 1981, 1, 7.

13.Braithwalte, K. A., Smith, F. J., Chromatographic Methods, Ed. 4^th, 1992, 124-129

14.Swel, P. A., Clarke, B., Chromatographic Separation, Analytical Chemistry, 1991, 170, 184-187.

15.Weston. A., Brown. R., HPLC and CE Principles and Practices, 1997, 24-52, 233-235.

16.Singh. S., Bakshi. M., Guidance on the Conduct of Stress Tests to Determine the Inherent Stability of Drugs, Pharmaceutical technology, Asia Sep/Oct. 2000.

17.Overholser, B. R., Michael, B., Sowinski, K. M., Determination of Gatifloxacin in human serum and urine by high-performance liquid chromatography with ultraviolet detection. J. Chromatography., B. 2003, 798, 167-173.

About Authors:

Mr. Vitthal V. Chopade
Correspondence Address
Lcturer in Pharmaceutics Modern College of Pharmacy, Yemuna Nagar Nigdi, Pune-44. He has done his M. Pharm in Quality Assurance from Nagpur University. He is a Life member of APTI. He has published and presented several research and review articles in national level and International level. E- Mail: vi_research@rediffmail.com Phone No. 9923277395

Mr. Sameer Lakade
Lcturer in Pharmaceutics Modern College of Pharmacy, Yemuna Nagar Nigdi, Pune. E- Mail: Sameer_patil97@rediffmail.com

Miss. Suvarna J. Kshirsagar
Lcturer in Pharmaceutics Modern College of Pharmacy, Yemuna Nagar Nigdi, Pune. E-mail: - suvarnajk_26d@yahoo.co.in

Prof.Satish A. Polshettiwar
Working as Lecturer at MAEER’s, Maharashtra Institute of Pharmacy, MIT Campus, Pune. He has done his M.Pharm in Quality Assurance from Nagpur University. He is a Life member of APTI. He has published and presented several research articles in national level and International level
E.Mail: contact_psatish@yahoo.co.in, Cell No. 09422842838

Prof.Manish S. Wani
Working as Lecturer at MAEER’s Maharashtra Institute of Pharmacy, MIT campus, Pune. He has done his M.Pharm in Pharmaceutics from Pune University. He has also done his MBA from Pune University