Introduction On Development Of Methods For Related Components Biology Essay

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The substances that occur naturally or during production of a chemicals or commercial drug product are defined as impurities. During production or synthesis, impurities may be purposely, accidentally, predictably or incidentally added into the substance.

Impurity Profiling1

The intentions of impurity profiling are to detect, structure elucidate, identify and quantify the organic and inorganic impurities, as well as residual solvents in bulk drugs and pharmaceutical formulations. The core activity in modern drug analysis is for the characterization of the stability and quality of bulk drugs and pharmaceutical formulations.

Importance of Impurity Profiling2

Impurities that are present in excess of 0.1 % when compared with the concentration of the active pharmaceutical ingredient (API), should be identified and quantified by selective methods. The predicted structures of the impurities are synthesized and proved for their structures by spectroscopic methods. The structure of these impurities in the bulk drug helps in altering the reaction condition and to minimize the quantity of impurity to an acceptable level. Isolation, identification and quantification of impurities help us in various ways, to obtain a pure substance with less toxicity and safety in drug therapy. Quantitative determination of these impurities could be used as a method for the quality control and validation of drug substances. Regulatory authorities such as International Conference on Harmonization (ICH), Food and Drug Administration (US FDA), Current Good Manufacturing Practice (CGMP), Therapeutics goods administration (TGA) and Ministry of corporate affairs (MCA), insist on the impurity profiling of drugs.

Impurities present in new drug substances are addressed under two aspects.

The chemical aspect includes identification and classification of impurities, generation of report, listing of impurities in specifications and explanation of analytical procedures in brief.

The safety aspect includes specific guidance for the quantification of impurities present significantly at lower levels in a drug substance that are used in clinical studies.

Impurity profiling is significant during the synthesis of drug substances (API) and manufacture of dosage since it provides crucial data concerning the safety, toxicity, LOD, LOQ of several organic and inorganic impurities that are possibly to be present in bulk drugs and finished products.

Sources of impurities

Raw materials used in the manufacturing process.

Reagents and solvents used in the process of manufacturing.

During storage of the product.

Containers used in packaging.

Classification of impurities3

Impurities can be classified into the following categories:

Organic impurities (process and drug-related)

Inorganic impurities

Residual solvents

Organic impurities

Organic impurities can arise during the manufacturing process and/or storage of the new drug substance. They can be identified or unidentified, volatile or nonvolatile.

Organic impurities includes the following

Starting materials


Intermediates product

Degradation products

Isomers, reagents, ligands and catalysts

Inorganic impurities

Inorganic impurities occur during manufacturing process. They are normally known and identified. In organic impurities includes the following

Reagents, ligands and catalysts

Heavy metals or other residual metals

Inorganic salts

Other materials from filter aids and charcoal

Residual solvents

Residual solvents are volatile organic chemicals employed in the manufacturing process of bulk drug and formulation as solvent for dissolving chemical substance in synthesis or purification of the API or for dissolving coating substances like polymers. Solvents that are toxic should be avoided in the production.

Residual solvents are classified into 3 classes as per the ICH guidelines with respect to the human health. They are

Class I: pyridine benzene, toluene, carbon tetrachloride, methylene chloride, methanol.

Class II: N, N-dimethyl formamide, acetonitrile. 

Class III: ethanol, acetic acid, acetone

Introduction on Development of methods for related components 4

Identification and quantification of impurities is essential during pharmaceutical process development for the evaluation of quality and safety. In pharmaceuticals related substances/impurities are the unwanted chemicals that remain with the API or develop during the stability testing studies or formulation or upon aging of both API and formulated products. Safety and efficacy of the pharmaceutical products are influenced by the presence of these impurities (or) unwanted chemicals in the smaller quantities. Different types of analytical methodologies were used for the determination of related substances/components in various pharmaceutical substances.

Different analytical techniques employed for determination of impurities4

Different guidelines associated with related substances/impurities5

PhRMA Position Paper: PhRMA GTI Task Force in 2005- Muller L et al, A Rationale for determining, testing and controlling specific impurities in pharmaceuticals that possess potential for genotoxicity. Regulatory Toxicology and Pharmacology 2006; 44, 198- 211.

CHMP guideline on the limits of genotoxic impurities. CPMP/SWP5199/02EMEA/ CHMP/QWP/ 251344/2006: became effective on January 1, 2007.

Questions and answers on the CHMP guideline on the limits of genotoxic impurities. EMEA/ CHMP/SWP/431994/2007, Rev. 3, Sep 2010.

FDA draft guidance for industry. Genotoxic and carcinogenic impurities in drug substances and products, recommended approaches. Center for drug evaluation and research:

Australian regulatory guidelines for prescription medicines Appendix 18:Impurities in active pharmaceutical ingredients and finished products, June 2004.Impurities have been named differently or classified as per the ICH as follows;

Common names1


Degraded products

Interaction products

Penultimate intermediates

Related products

Transformation products

Description of impurities according to their sources

Types of Impurity

Source of Impurity

Process-related drug substance


Starting material



Impurity in starting material

Process-related drug substance

Organic or inorganic

Reagents, catalysts, etc.

Process-related drug substance or drug product


Degradation products

Process-related drug substance


Excipient interaction

United State Pharmacopoeia6

Classification of impurities according to United States Pharmacopoeia (USP) is as follows-

Impurities in official articles

Ordinary impurities

Organic volatile impurities

ICH terminology

According to ICH guidelines, impurities in the drug substance that are formed during the synthesis of chemical substance are broadly classified under the following three categories;

Organic Impurities (Process and Drug related)

Inorganic Impurities

The residual solvents

Various pharmacopoeias, such as the Indian Pharmacopoeia (IP), British Pharmacopoeia (BP), Japanese Pharmacopoeia and United States Pharmacopoeia (USP) publish the impurity level that can be present in API's and in pharmaceutical formulations. The ICH of technical requirements for registration of pharmaceuticals for human use has also published guidelines for validation of methods for analyzing impurities in new drug substances, products, residual solvents and microbiological impurities.

History of impurity guidelines7


ICH Q3A/B (R) issued in 2002

Lower thresholds may be appropriate for unusually toxic impurities

Lacks specific guidance on how to address mutagenic/carcinogenic impurities

Increased awareness and regulatory scrutiny on residual levels of genotoxic impurities in API and drug products

EMEA issues draft guidance, stressing avoidance vs. acceptance of a low limit


EMEA updates draft guidance and introduces the Threshold of toxicological concern(TTC) limit (1.5 µg/day) for drugs


PhRMA Publication (Muller et al., 2006). "A rationale for determining, testing and controlling specific impurities in pharmaceuticals that possess potential for genotoxicity".

Introduces concept of the 'staged TTC' for clinical trial materials


CHMP Guideline on the Limits of Genotoxic Impurities effective January 2007.

CHMP Q&A document generated based on industry questions and EMA answers


FDA Draft Guidance for Industry. Genotoxic and Carcinogenic Impurities in Drug Substances and Products: Recommended Approaches.

EMA letter requesting evaluation of sulfonate esters in all marketed products


November 2009 -Concept paper issued and ICH M7 topic agreed

September 2010 -CHMP Q&A document updated

November 2010 -First ICH EWG M7 Meeting in Fukuoka Japan

Limits of impurities1

Thresholds for Degradation Products in New Drug Products Reporting Thresholds

Maximum Daily Dose1

% Threshold2,3

£1 g


> 1 g


Identification of Thresholds

Maximum Daily Dose in mg1

% Threshold2,3

< 1

1.0 or 5 µg TDI, whichever is lower

1 - 10

0.5 or 20 µg TDI, whichever is lower

> 10 - 2000

0.2 or 2 mg TDI, whichever is lower

> 2000


Qualification Thresholds

Maximum Daily Dose in mg1

% Threshold2,3

< 10

1.0 or 50 µg TDI, whichever is lower

10 - 100

0.5 or 200µg TDI, whichever is lower

> 100 - 2000

0.2 or 3 mg TDI, whichever is lower

> 2000


Notes on Attachment1

The amount of drug substance administered per day

Thresholds for degradation products are expressed either as a percentage of the drug substance or as total daily intake (TDI) of the degradation product. Lower thresholds can be appropriate if the degradation product is unusually toxic.

Higher thresholds should be scientifically justified.

Forced degradation study8

Stability- indicating method is a validated analytical procedure for the Identification and quantitation of the changes that occurs with time to time in the physico-chemical properties of the drug substance and drug product.

A stability-indicating method accurately measures the interference of the degradation products, excipients, process impurities or other potential impurities with the drug substance and drug product.

Forced degradation or stress testing is performed for determining the specificity of the developed stability-indicating method, for potential degradation products. These studies provide information regarding degradation pathways and degradation products that could form during storage of the drug/drug products. Forced degradation studies are useful during the pharmaceutical development in various areas such as formulation development, manufacturing and packaging in which information regarding chemical behavior is used to obtain improved drug product. The various regulatory guidelines provide useful definitions and general comments about degradation studies. Various guidelines discuss the stress testing related issues such as stereo chemical stability, identification of degradation product thresholds, polymorphism and the possible crystal forms, stability of combination products, incompatibility studies in parenteral products and mass balance.

Formal stability studies9

Long term and accelerated (intermediate) studies were performed to confirm the re-test period of an Active Pharmaceutical Ingredient or the shelf life of a finished Pharmaceutical Product.Stability studies were performed on primary and/or commitment batches according to the stability protocol.

Stress testing - forced degradation on Active Pharmaceutical Products

Stress studies are performed to explain the intrinsic stability of the API. These studies are performed under more severe conditions than those used for accelerated testing.

Stress testing - forced degradation on FinishedPharmaceutical Products (FPP)

Stress studies are performed to estimate the effect of severe conditions on the FPPs. These studies includes photo stability testing as per ( ICHQ1B) and compatibility testing on APIs with each other in FPPs of multi drug combinatios and APIs with excipients during formulation development.

Reasons for conducting forced degradation studies10

Forced degradation studies are carried out for the following reasons:

Development and validation of stability-indicating methodology

Determination of degradation pathways of drug substances and drug products

Discernment of degradation products in formulations that are related to drug substances versus those that are related to non-drug substances (e.g., excipients).

Structure elucidation of degradation products.

Determination of the intrinsic stability of a drug substance molecule.

Different forced degradation conditions used for drug substances and drug products10

ICH guidelines on stress testing11


Title and Reference


Stability Testing of New Drug Substances and Products (the parent guideline)


Photo stability Testing of New Drug Substances and Products


Validation of Analytical Procedures: Methodology


Impurities in New Drug Substances

Stress Testing of API in Solution11

Storage Conditions

Testing Period

pH ±2,Room temperature

2 weeks

pH ±7, Room temperature

2 weeks

pH 10-12,Room temperature

2 weeks

H2O2 0.1-2% at neutral pH, room temperature

24 Hrs

Storage conditions given or 5-15% degradation, whatever comes first.

Stress testing of FPPS in solid state11

Storage Conditions

Testing Period*

40 °C,75% RH open storage **

3 months

50-60 °C, ambient RH, open storage

3 months

Photo stability according to ICH

According to ICH

*3 months or 5-15% degradation whatever comes first

**For API 1 API 2 or API excipients or FPP without packing material,

typically a thin layer of material is spread on petridish.

Open storage is recommended if possible.

Analytical chemistry13

Analytical chemistry is the art and science of determining what matter is and how much of it exists. Analytical chemistry derives its principles from various branches of science like, physics, chemistry, nuclear science, microbiology, electronics etc., and it deals with scientific and technical measurement of compositional and constitutional features of the sample. The major concern of analytical chemistry is to perform qualitative and quantitative analysis. Qualitative analysis deals with the identification and description of chemical composition in terms of elements, compounds, structural units and quantitative analysis is to measure the amount of individual substance present in a chemical composition.

Analytical chemistry, once limited to the determine the chemical composition in terms of the relative amounts of elements or compounds in a chemical substance and has been expanded to involve the spatial distribution of elements or compounds in a chemical substance, the distinction between different crystalline forms of a given element.To improve the speed, accuracy, sensitivity, and selectivity of traditional analysis, a large number of physical measurements are used. These are based on Spectrophotometric, electro photometric, chromatographic, chemical and nuclear principles.

The development of a new drug substance is an expensive and time-taking process. Therefore, the developers maximize the pro¬t from the drug by patenting the concerned molecule as well as the pathway of synthesis. In a later stage a faster or cheaper manufacturing process can be developed and patented.

Identification of impurities is done by variety of chromatographic and spectroscopic techniques, either alone or in combination with other techniques. There are different methods for detecting and characterizing impurities. They are

Thin Layer Chromatography (TLC)

High Performance Liquid Chromatography (HPLC)

High Performance Thin layer Chromatography (HPTLC)

Gas Liquid Chromatography (GLC)

Atomic Absorption Chromatography (AAS)

Conventional Liquid Chromatography, particularly, HPLC has been exploited widely in field of impurity Profiling. Impurity profiling is now gaining critical attention from regulatory authorities. The instrumental employed commonly for the analysis are spectrophotometry, GLC, HPLC, HPTLC etc. These methods are based upon the measurement of specific and nonspecific physical properties of the substances.

Estimation of drugs in pharmaceutical dosage form 14-16

Analytical methods for the estimation of drugs in pharmaceutical dosage form include.

Non instrumental methods of Analysis

Titrimetric methods

Instrumental methods of Analysis

Spectral methods

Electrochemical methods

Chromatographic methods.

Chromatographic methods

Chromatography is a separation technique based on differing affinities of a mixture of solutes between two different phases which results in physical separation of the mixture into its various components. The affinities or interactions are classified in terms of solute adhering to the surface of a polar solid (adsorption), a solute dissolving in a liquid (partition), and a solute passing through or impeded by a porous substance based on its molecular size (exclusion).

High performance liquid chromatography17-19

High Performance Liquid Chromatography (HPLC) is one of the modes of chromatography and is one of the most used analytical techniques.

In this technique the components are first dissolved in a solvent, and then forced to flow through analytical column under high pressure. In the column, the mixture separates into its components. The amount of resolution is important, and is dependent upon the extent of interaction between the solute components (Mobile phase) and the stationary phase. The stationary phase is defined as the immobile packing material in the column. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and it has the ability to easily separate a wide variety of chemical mixtures.

Modes of separation by HPLC

There are different modes of separation in HPLC. They are

Normal phase mode

Reverse phase mode

Reverse phase ion pair chromatography

Ion exchange chromatography

Affinity chromatography

Size exclusion chromatography

In normal phase mode, the nature of stationary phase is polar and the mobile phase is non-polar. In this technique, non-polar compounds travel faster and are eluted first because of lower affinity between the non-polar compounds and the polar stationary phase. Polar compounds are retained for longer times and take more time to elute because of their higher affinity with the stationary phase. Normal phase mode of separation is, therefore, not generally used for pharmaceutical applications because most of the drug molecules are polar in nature and hence take longer time to elute. The silica structure is saturated with silanol groups at the end. These -OH groups are statistically distributed over the whole of the surface. The silanol groups represent the active sites (very polar) in the stationary phase. This forms a weak type of bond with any molecule in the vicinity when any of the following interactions are present.

Dipole-induced dipole


Hydrogen bonding

Complex bonding

Reverse phase mode is the most popular mode for analytical and preparative separations of compounds of interest in chemical, biological, pharmaceutical, food, biomedical sciences and etc. In this mode, the stationary phase is non-polar hydrophobic packing with octyl or octa decyl functional group bonded to silica gel and the mobile phase is a polar solvent. An aqueous mobile phase allows the use of secondary solute chemical equilibrium (such as ionization control, ion suppression, ion pairing and complexation) to control retention and selectivity. The polar compound gets eluted first in this mode and non-polar compounds are retained for longer time. As most of the drugs and pharmaceuticals are polar in nature, they are not retained for longer times and hence elute faster. The different columns used are octa decyl silane (ODS) or C18, C8, C4 etc. (in the order of increasing polarity of the stationary phase).

Estimation of relative impurities in pharmaceutical dosage forms by HPLC20-22

Most of the relative impurities in bulk drug and pharmaceutical dosage form can be analyzed by HPLC method because of several advantages like rapidity, repeatability, reproducibility, specificity, accuracy, precision, ease of automation, eliminates tedious extraction and isolation procedures. Some of the advantages are:

Speed (analysis can be accomplished in 20 min or less)

Greater sensitivity (various detectors can be employed)

Improved resolution ( wide variety of stationary phases

Reusable columns (expensive columns but can be used for many samples)

Ideal for the substances of low volatility

Easy sample recovery, handling and maintenance

Instrumentation lends itself to automation and quantization (Less time and less labor)

Precise and reproducible

Calculations are done by integrator itself and

Suitable for preparative liquid chromatography on a much large scale.


Validation is defined as follows by different agencies

Food and Drug administration (FDA): Establishing documentation evidence, which provides a high degree of assurance that specific process, will consistently produce a product meeting its predetermined specification and quality attributes.

World Health Organization (WHO): Action of providing that any procedure, process, equipment, material, activity, or system actually leads to the expected results.

European Committee (EC): Action of providing in accordance with the principles of good manufacturing practice, that any procedure, process, equipment material, activity or system actually lead to the expected results. In brief validation is a key process for effective Quality Assurance.

Types of Validation

Prospective validation is performed for all new equipments, products, and processes. It is a proactive approach of documenting the design, specifications and performance before the system is operational. This is the most defendable type of validation.

Concurrent Validation is performed in two instances, i.e., for existing equipment, verification of proper installation along with specific operational tests is done. In case of an existing, infrequently made product, data is gathered from at least three successful trials.

Retrospective validation is establishing documented evidence that the process is performed satisfactory and consistently over time, based on review and analysis of historical data. The source of such data is production and QA/QC records. The issues to be addressed here are changes to equipment, process, specifications, and other relevant changes in the past.

Phases of Validation

Design qualification (DQ): Documented verification of the design of equipment and manufacturing facilities.

Installation qualification (IQ): documented verification of equipment or system design and adherence to manufacturer's recommendations.

Operational qualification (OQ): Documented verification of equipment or system performance in the target operating ranges.

Process performance qualification (PQ): documented verification that equipment or systems operate as expected under routine production conditions. The operation is reproducible, reliable and in a state of control.

Process / Product validation: Validation is establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its pre-determined specifications and quality attributes.

Analytical method validation

Analytical monitoring of a pharmaceutical product or of specific ingredients within the product is necessary to ensure its safety efficacy throughout all phases of its shelf life. Such monitoring is in accordance with the specifications elaborated during product development.

Analytical validation is the corner stone of process validation without a proven measurement system it is impossible to confirm whether the manufacturing process has done what it purports to do. All new methods developed are validated.

Steps followed for validation procedures

Proposed protocols or parameters for validations are established.

Experimental studies are conducted.

Analytical results are evaluated.

Statistical evaluation is carried out.

Report is prepared documenting all the results.

Objectives & parameters of validation

The objective of validation of an analytical procedure is to demonstrate that method is suitable for its intended purpose. According to ICH, typical analytical performance characteristics that should be considered in the validation of the types of methods are:








The parameters which are recommended by International Committee of harmonization to be validated for different types of assays are shown in following table. 



Identification tests are intended to ensure the identity of an analyte in a sample. This is normally achieved by comparison of a property of the sample to that of a reference standard.


Impurities quantitation is intended to accurately reflect the purity characteristic of the sample. Different validation characteristics are required for a quantitative test than for a limit test.

Accuracy, Precision, Specificity, Detection limit, Quantitation limit, Linearity Range.

Impurities Limit is intended to reflect the purity characteristics of the sample.


Detection limit

Content / Potency, Dissolution is intended to measure the analyte present in a given sample. A quantitative measurement of the major component (s) in the drug substance.

Accuracy , Precision, Specificity, Linearity, Range

Validation parameters


The accuracy of an analytical procedure as the closeness of agreement between the conventional true value or an accepted reference value and the value found. Accuracy can also be described as the extent to which test results generated by the method and the true value agree.

Accuracy is reported in percent recovery by performing a minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range test method is. The mean of the replicates, expressed as % label claim, to indicates the accuracy of the test method.


Precision is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of a homogenous sample. According to ICH precision should contain repeatability, intermediate precision and reproducibility

Reproducibility expresses the precision between laboratories as in collaborative studies. The sensitivity or precision indicates the performance of the HPLC instrument under the Chromatographic conditions.

Standard deviation or relative standard deviation (coefficient of variation) calculation is done for reproducibility.

Repeatability is performed using a minimum of 9 determinations covering the specified range for the procedure (e.g.3 concentrations/ 3 replicates each) or a minimum of 6 determinations at 100 % of the test concentration.


Specificity should be performed during the validation of identification tests, the determination of impurities and the assay. The specificity procedure depends on the proposed objective of the analytical procedure.


Identification tests should be able to distinguish the compounds of closely related structures which are likely to be present. The discrimination of a procedure may be confirmed by obtaining positive results (perhaps by comparison with a known reference material) from samples containing the analyte, coupled with negative results from samples which do not contain the analyte.

Assay and Impurity Test(s)

For chromatographic procedures, representative chromatograms should be used to demonstrate specificity, and individual components should be appropriately labeled.

For critical separations, specificity can be demonstrated by the resolution of the two components which elute closest to each other. For nonspecific assay , other supporting analytical procedures should be used to demonstrate overall specificity.

If Impurities are available the assay should involve the separation of analyte in the presence of impurities and/or excipients, which is done by spiking pure substances (drug substance or drug product) with impurities and/or excipients, indicating that the assay result is unaffected even in the presence of these materials.

If impurity or degradation product standards are unavailable, specificity may be established by comparing the test results of samples containing impurities or degradation products to a second well-characterized procedure.

Peak purity test is performed to show that there is no interference with the sample analyte peak, which indicates that the analyte chromatographic peak is pure. This is usually performed by using Photo diode array, mass spectrometry.

Limit of Detection

ICH defines the "detection limit of an individual analytical procedure as the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value."

For instrumental methods detection limit is determined by the analysis of samples with known concentration of analyte and by establishing the minimum level at which the analyte can be reliably detected. For impurities detection limit is established based on the standard deviation of the response and the slope.

The detection limit and the method used should be presented whether detection limit is determined based on visual evaluation or based on signal to noise ratio, and/or Standard deviation of the response in the blank chromatogram and based on the slope of the curve.

Limit of Quantitation

ICH defines the "limit of quantitation (LOQ) of an individual analytical procedure as the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy".

The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices and for the determination of impurities or degradation products. The quantitation limit is generally determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be quantified with acceptable accuracy and precision. The detection is based on the standard deviation of the response and the slope, signal to noise ratio, and visual evaluation.

Linearity and Range

Linearity of an analytical method is its ability to produce results that are directly, proportional to the concentration of analyte in samples.

The range of the procedure is an expression of the lowest and highest levels of analyte that have been demonstrated to be determinable with acceptable precision, accuracy, and linearity.

When the relationship between response and concentration is not linear, standardization may be providing by means of a calibration curve.

Linearity is evaluated by a plot of signals as a function of analyte concentration. If there is a linear relationship, test results should be evaluated by appropriate statistical methods.

The correlation coefficient, y-intercept, slope of the regression line and residual sum of squares should be submitted. A plot of the data should be included

As per ICH linearity is established by using a minimum of 5 concentrations. For the assay of a drug substance or a finished product range normally from 80 to 120 % of the test concentration. The linearity range for examination depends on the purpose of the test method.

For an assay impurities combination method based on area % (for impurities) would be +20 % of target concentration down to the limit of quantitation of the drug substance or impurity. Under most circumstances, regression coefficient (r) is ≥ 0.999. Intercept and slope should be indicated.


Ruggedness is "a measure of the reproducibility of test results under normal, expected operational conditions from laboratory to laboratory and from analyst to analyst."

Determination of ruggedness is performed by analysis of aliquots from homogenous lots in different laboratories, by different analysts, using operational and environmental conditions that may differ but are still within the specified parameters of the assay. Degree of reproducibility of test results is then determined as a function of the assay variables.


Robustness of an analytical method is "measure of its capacity to remain unaffectedly small but deliberate variations in method parameters and provides an indication of its reliability during normal usage."

Typical Variations are

pH of mobile phase.

Mobile phase composition.

Anlytical columns (different lots and/or suppliers).

Column temperature.

Mobile Phase flow rate.

System Suitability8,21

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. System suitability test parameters to be established for a particular procedure depend on the type of procedure being validated. System suitability of the method was performed by calculating the chromatographic parameters namely, column efficiency, resolution, peak asymmetry factor and capacity factor on the repetitive injection of standard solutions using the following formula.

Capacity factor (k) is a measure of how well the sample molecule is retained by a column during an isocratic separation. The ideal value of (K) ranges from 2-10.

Capacity Factor = vt-v0/v0

where, vt is the retention volume at the apex of the peak (solute) and v0 is the void volume of the system

Resolution factor (Rs) is the difference between the retention times of two solutes divides by their average peak width. The ideal valve of (Rs) is

Resolution (Rs) = Rt2-Rt1/0.5 (W1+W2)

Where Rt1 and Rt2 are the retention times of components 1 and 2 and W1 and W2are peak widths of components 1and 2 respectively.

Efficiency (N) of the column is measured by number of theoretical plates per meter. Column with N ranging from 5,000 to 1,00,000 plates/meter are ideal for a good separation.

Column efficiency (N) = 16Ã-Rt2/w2

Where, Rt is the retention time and W is the peak width.

Peak Asymmetry Factor can be used as a criterion of column performance.

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

Peak asymmetry factor (As) = b/a

Where a and b are the distances on either side of the peak mid-point.