Importance Of Impurity Profiling 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, inevitably 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 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 in new drug substances can be addressed from two perspectives.

(1) The chemical aspect which includes classification and identification of impurities, report generation, listing of impurities in specifications and a brief discussion of analytical procedures.

(2) The safety aspect which includes specific guidance for quantifying impurities present substantially at lower levels in a drug substance used in clinical studies.

Impurity profiling is very important during the synthesis of drug substances and manufacture of dosage forms as it can provide crucial data regarding the toxicity, safety, various limits of detection, and limits of quantization, of several organic and inorganic impurities usually accompany with bulk drugs and finished products.

Sources of impurities..\impurity.pdf

1. Raw materials used in the manufacturing process.

2. Reagents and solvents used in .the process of manufacturing.

3. During storage of the product.

4. 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 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



Degradation products

Reagents, ligands and catalysts

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 (e.g., filter aids, charcoal)

Introduction on Development of analytical methods for the determination of related components in pharmaceutical compounds using chromatography technique4

Identification and quantification of impurities is a crucial task in pharmaceutical process development for quality and safety evaluation. Related components are the impurities in pharmaceuticals which are unwanted chemicals that remain with the Active Pharmaceutical Ingredients (APIs) or develop during stability testing or develop during formulation or up going of both API and formulated APIs to medicines. The presence of these unwanted chemicals even in small amounts may influence the efficacy and safety of the pharmaceutical products. Various analytical methodologies were employed for the determination of related components in pharmaceuticals.

Different analytical techniques for determination of impurities4

Different guidelines associated with relative 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;

A) Common names1

1. By-products

2. Degradation products

3. Interaction products

4. Penultimate intermediates

5. Related products

6. Transformation products



Process-related drug substance


- Starting material

- Intermediate

- By-product

- Impurity in starting material

Process-related drug substance

- Organic or inorganic

- Reagents, catalysts, etc.

Process-related drug substance or drug product

- Organic

-Degradation products

Process-related drug substance


- Excipient interaction

*Description * Description of impurity according to their sources

B) United State Pharmacopeia6

The United States Pharmacopoeia (USP) classifies impurities in various sections;

- Impurities in official articles

- Ordinary impurities

- Organic volatile impurities

C) ICH terminology

According to ICH guidelines, impurities in the drug substance formed

during the synthesis of chemical substance can be broadly classified under the following three categories;

- Organic Impurities (Process and Drug related)

- Inorganic Impurities

-The residual solvents

The different 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 or 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. Impurity profile is also the description of identified and unidentified impurities present in new drug substances. Impurity profiling is very important during the synthesis of drug substances and manufacture of dosage forms, as it can provide crucial data regarding the toxicity, safety, various limits of detection, and limits of quantization, of several organic and inorganic impurities, usually accompany with bulk drugs and finished products


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

Attachment 1: Thresholds for Degradation Products in New Drug Products

Reporting Thresholds

Maximum Daily Dose1 Threshold2,3

ï‚£1 g 0.1%

> 1 g 0.05%

Identification Thresholds

Maximum Daily Dose1 Threshold2,3

< 1 mg 1.0% or 5 µg TDI, whichever is lower

1 mg - 10 mg 0.5% or 20 µg TDI, whichever is lower

>10 mg - 2 g 0.2% or 2 mg TDI, whichever is lower

> 2 g 0.10%

Qualification Thresholds

Maximum Daily Dose1 Threshold2,3

< 10 mg 1.0% or 50 µg TDI, whichever is lower

10 mg - 100 mg 0.5% or 200µg TDI,whichever is lower

>100 mg - 2 g 0.2% or 3 mg TDI, whichever is lower

> 2 g 0.15%

Notes on Attachment 1

1. The amount of drug substance administered per day

2. 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.

3. Higher thresholds should be scientifically justified.

Forced degradation study8

Stability- indicating method is "a validated analytical procedure for the quantitation and detection of the changes that occurs with 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 active pharmaceutical ingredient (API).

Forced degradation or stress testing is performed for specificity where little information is available during development of stability-indicating methods, for potential degradation products. These studies provides information regarding degradation pathways and degradation products that could form during storage. 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 provides useful definitions and general comments about degradation studies. Various issues related to stress testing are mentioned in numerous guidance documents but not in the context of stress testing. For example, the available guidance discusses issues such as stereo chemical stability, degradation product identification thresholds, polymorphism and crystal forms, stability of (parenteral) combination products and mass balance but does not address these issues in the context of degradation studies.

Formal stability studies9

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

Long term and accelerated (intermediate) studies undertaken on primary and/or commitment batches according to a prescribed stability protocol to establish or confirm the re-test period of an API or the shelf life of a Finished Pharmaceutical Product (FPP).

Stress testing - forced degradation onActive Pharmaceutical Products (API)

Stress studies are performed to explain the intrinsic stability of the Active Pharmaceutical Ingredient (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


Drug substance Drug product

Solid Suspension Solid semisolid solution

Photolytic Acid/base hydrolysis Photolytic Photolytic Photolytic

Thermal Oxidative Oxidative Oxidative Oxidative

Thermal/humidity Thermal Thermal Thermal

ICH guidelines on stress testing11




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



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



400C,75%RH open storage **

3 months

50-600C, 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 science of obtaining, processing and communicating information about the composition and structure of matter. In other words, it 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, microbiology, nuclear science, electronics etc., and it deals with scientific and technical of measurement of compositional and constitutional features of the sample. The prime concern of analytical chemistry is the qualitative and quantitative analysis, this method provides information about the relative amount of one or more of these components.

Qualitative analysis is concerned with the description of chemical composition in terms of elements, compounds, or structural units, whereas quantitative analysis is concerned with the measurement of amount.

Analytical chemistry, once limited to the determination of chemical composition in terms of the relative amounts of elements or compounds in a sample, has been expanded to involve the spatial distribution of elements or compounds in a sample, the distinction between different crystalline forms of a given element or compound, the distinction between different chemical forms (such as the oxidation state of an element), the distinction between a component on the surface or in the interior of a particle, and the detection of single atoms on a surface. To permit these more detailed questions to be answered, as well as to improve the speed, accuracy, sensitivity, and selectivity of traditional analysis, a large variety 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-consuming process. Therefore, the developers want to maximize the profit from the drug by patenting the concerned molecule as well as its synthesis pathway. In a later stage a faster or cheaper manufacturing process can be developed and patented. Various regulatory authorities like ICH, USFDA, Canadian Drug and Health Agency and TGA (Therapeutics goods administration) are emphasizing on the purity requirements and the identification of impurities in API's. Qualification of the impurities is the process of acquiring and evaluating data that establishes biological safety of an individual impurity; thus, revealing the need and scope of impurity profiling of drugs in pharmaceutical research. 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 with TLC (Thin Layer Chromatography), HPLC (High Performance Liquid Chromatography), HPTLC (High Performance Thin layer Chromatography), AAS (Atomic Absorption Chromatography)etc. 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 drugs in pharmaceutical dosage form include.

Classical separation or wet analysis (Non instrumental)

Instrumental methods of Analysis

1. Spectral methods

2. Electrochemical methods

3. Chromatographic methods.

Chromatographic methods

Chromatography is a separation technique that is based on differing affinities of a mixture of solutes between at least two phases. The result is a physical separation of the mixture into its various components. The affinities or interactions can be classified in terms of a 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).

Concept of chromatography

In the following sections, individual chromatography techniques are discussed in relation to their usefulness as separation tools for drugs in different dosage forms.

Gas chromatography

Gas chromatography is one of the most extensively used tools for quantitative analysis of impurity. Gas chromatography is classified into Gas Liquid chromatography (GLC) and Gas Solid chromatography. In GLC, the stationary phase is a liquid that is coated into an inert solid support. The process is a form of partition chromatography, where the components of a drug mixture are separated based on the solute's vapor pressure (or B.P), solubility. In the GLC stationary phase was solid and the mobile phase was gas various liquid phases are chosen depending on the chemical nature of the drugs to be separated.

High performance liquid chromatography17-19

Reversed Phase High Performance Liquid Chromatography (RP-HPLC) is most commonly used to separate pharmaceutical compounds.

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 and size exclusion chromatography (gel permeation and gel filtration 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 octadecyl 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 octadecylsilane (ODS) or C18, C8, C4 etc. (in the order of increasing polarity of the stationary phase).

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

Most of the RI 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: This 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: This 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: This 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 to be reported in percent recovery as per the ICH document on validation methodology recommends, a minimum of nine determinations over a minimum of three concentration levels covering the specified range (for example, three concentrations with three replicates each).The assay of known added amount of analyte in the sample, or as the difference between the mean and the accepted true value, together with the confidence intervals. The RSD should not be ≥2 % and the replicated analysis will provide the analysis variation or how precise the test method is. The mean of the replicates, expressed as % label claim, to indicates the accuracy of the test method.


Precision is the measure of how close the data values are to each other for a number of measurements under the same analytical conditions. ICH has defined precision to contain three components: repeatability, intermediate precision and reproducibility. Precision is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of a homogenous sample.

Reproducibility expresses the precision between laboratories as in collaborative studies. Multiple laboratories are desirable but not always attainable because of the size of the firm.

The sensitivity or precision as measured by multiple injections of a homogeneous sample (prepared solution) indicates the performance of the HPLC instrument under the Chromatographic conditions.

The ICH documents recommend that repeatability should be assessed using a minimum of nine determinations covering the specified range for the procedure or a minimum of 6 determinations at 100 % of the test concentration.

For reproducibility calculate statistically valid estimates of standard deviation or relative standard deviation (coefficient of variation).

As part of methods validation, a minimum of 10 injections with an RSD of 11 % is recommended. With the methods for release and stability studies, an RSD of ≤ 1 % RSD for precision of the system suitability tests for at least five injections (n ≥ 5) for the active drug either in drug substance or drug product is desirable. For low level impurities, higher variations may be acceptable.

Repeatability should be assessed 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.

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 and non-instrumental methods detection limit is generally 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 it's detected based on the standard deviation of the response and the slope.

The detection limit and the method used for determining the detection limit should be presented. If detection limit is determined based on visual evaluation or based on signal to noise ratio, and Standard deviation of the response of the blank and based on the slope of the curve. The limit of detection varies with the detector used 0.16 % or 0.05 %. The detection limit for total impurity is not more than 0.2% and individual impurity not more than 0.10 %.

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 particularly 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. If the required precision of the method at the limit of quantitation has been specified, 5 or 6 samples with decreasing amounts of the analyte are injected six times.

For instrumental and non-instrumental methods, the quantitation limit is generally determined by the analysis of samples with known concentration of analyte and by establishing the minimum level at which the analyte can be determined with acceptable accuracy and precision. It's detected based on the standard deviation of the response and the slope, signal to noise ratio, and visual evaluation.

As per USP the detection limit and quantitation limit in terms of 2 .or 3, and 10 times noise level respectively, this concept is not very practical. Noise level on a detector during the method development phase may be different when samples are assayed on different detectors, etc.

As per ICH, limit should be subsequently validated by the analysis of a suitable number of samples known to be near or prepared at the quantitation limit vary with the detector used, like quantitation limit of 0.21 % or 0.07 %. The detection limit for total impurity is not more than 0.2 % and individual impurity not more than 0.10 %.

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.

These characteristics are determined by application of the procedure to a series of samples having analyte concentration spanning the claimed range of the procedure. 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, for example, by calculation of a regression line by the method of least squares.

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. In addition, an analysis of the deviation of the actual data points from the regression line may also be helpful for evaluating linearity.

As per ICH linearity establishment by a minimum of 5 concentrations normally used. Range for the assay of a drug substance or a finished (drug) product: normally from 80 to 120 % of the test concentration. The linearity range for examination depends on the purpose of the test method.

For example, the recommended range for an assay method for content would be NLT 20 % and the range.

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. Ruggedness is determined by the analysis of aliquots from homogeneous lots in different laboratories.

Determination of ruggedness is done 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

Influence of variations of pH in a mobile phase.

Influence of variations in mobile phase composition.

Different columns (different lots and/or suppliers).

Column temperature.

Mobile Phase flow rate.

System Suitability(8,21)

According to USP system suitability are an integral part of chromatographic methods.

System suitability verifies the resolution and reproducibility of the system are adequate for the analysis to be performed. One consequence of the evaluation of robustness and ruggedness should be that a series of system suitability parameters is established to ensure that the validity of the analytical method is maintained whenever used.