Importance Of Impurity Determination In Drug Substance

Published Date: 02 Nov 2017

Bulk drugs or drug substances, is known to be of products that are having the biological activity to treat certain disease in human beings. These drug substances are formulated to made available for the human consumption. So it is essential to determine the purity and safety of the bulk drug substances before using them in different formulations. Good manufacturing practices (GMP) are most inherent and provide valuable information and guidance for the selection of manufacturing process [1]. Establishment of impurities and fixing the specified limits for the drug substance are some of the important steps to be carried out during the manufacturing of the bulk drugs. The United States Pharmacopeia (USP), National Formulary (NF), British Pharmacopeia (BP) and European Pharmacopeia (EP) are well known as standards for potency and purity of drugs. The third International Conference on Harmonization (ICH) conducted in Japan, in 1995 released new guidelines on impurities in new drug products [2]. The main objective of the guidelines is to provide the quantification, reporting and qualification of impurities in key starting materials, in new drug substances and in new drug products.

An impurity as defined by the ICH guidelines is "Any component of the medicinal product which is not the chemical entity defined as the active substance or an Excipients in the product", while the definition of impurity profile is same as "A description of the impurities present in the medicinal product [3]. Regulatory filings require specifying all the potential impurities with the specifications, generated during the manufacturing process.

1.1.1 Classification of Impurities:

Impurities are broadly classified as Organic impurities (Process-and drug related), Inorganic impurities and Residual solvents. Organic impurities are generally originated during the manufacturing, storage and handling of the drug substance. Organic impurities include starting materials, by-products, intermediates, degradation products, reagents, ligands and catalysts. Inorganic impurities can be reagents, ligands and catalysts, heavy metals or other residual metals, inorganic salts, filter aids, charcoal etc. Residual solvents are normally organic or inorganic liquids usually used during the manufacturing process. Since toxicity of these is generally known, the selection of appropriate Controls can be accomplished easily. For a given drug substances the possible impurities can be readily derived from the knowledge of the raw materials used in the process of manufacturing and the stability of the product. To these must be added impurities, which may arise from physical contamination or inadequate storage conditions [4].

It is very mandatory from the regulatory bodies like Food drug and Administration (FDA) point of view, to characterize an impurity if the determinations indicate that the impurity content is greater than 0.1%. For Characterization of an impurity different Hyphenated methods such as Gas chromatography, or liquid chromatography, Mass spectrometry or the number of other chromatographic-spectroscopic combination techniques can be used [5]. Highly sophisticated instrumentation, gas chromatography or HPLC are the modern tools in the identification of minor components (drugs, impurities, degradation products, metabolites) in various matrices. NMR Spectroscopy and Mass spectroscopy, which involve complete structure elucidation, are very expensive. However the basic requirements for impurity profiling using HPLC techniques, is that all the components should be spectrophotometrically active and its spectrum differs sufficiently from that of the main components (Parent drugs ) and from other small components [6].

It is very essential to perform forced degradations studies (may be oxidation or hydrolysis), in the initial stages of the method development to understand the degradation pathways and possible degradants. The degradation products can be listed and summarized as a reference library of degradation products for routine use during an impurity profiling and trace the nature and structure of the impurity [7].

The separation and quantification of impurities by chromatographic methods are one of the widely used techniques for the control of the impurities present in the drugs. Establishing the Specifications for finished products are some important steps to be carried out during the manufacturing of the bulk drugs. To characterize impurities, it is often necessary to perform several analytical experimental sequence injections to obtain the necessary MS and MS/MS data.

1.2 Chromatographic techniques for separation and Quantification of Impurities:

Chromatography is a separation technique where the mixture of the sample getting separated between two phases. Solutes are separated by a dynamic differential migration process in a system consisting of two or more phases, one of which is moving mobile phase and other is the fixed stationery phase. Chromatography is classified into four categories based on the separation process involved and the forces between the moving mobile phase and stationery phase.

Adsorption chromatography: This technique is widely used for the analysis of polar non ionic compounds.

Ion Exchange Chromatography: This technique is very much useful for the ionic compounds, separations is achieved based on the principle of Ion exchange. This is further subdivided as, Anion Exchange, wherein the sample to be analyzed is an anion, bonded phase has positive charge and Cation Exchange, where the sample to be analyzed is a Cation; bonded phase has negative charge.

Partition Chromatography: This technique is based on the principle of relative solubility of the analytes in the mobile phase and stationery phase. Again it is sub-classified into two types depending on the polarity of stationery Phases, Normal Phase: where in the mobile phase is non polar organic mixture, Stationery phase is more Polar than the mobile Phase. Reverse phase: Mobile phase is Polar Organic mixture: stationery phase is less Polar than the mobile phase.

Size Exclusion Chromatography: This technique separation is based on the size of the analyte. Porous matrix is used as the Stationery phase.

1.2.1 High performance liquid chromatography (HPLC) and related techniques in separation and quantification of impurities in pharmaceutical compounds:

It is confronting activity for the scientists to separate the impurities like process related impurities, un-reacted substance and degradation products with good resolution, precision and accuracy at levels in the higher concentration of drug substances. Chromatography is widely used for the separation of compounds based on the functional groups present in the atom, polarity of the compounds, solubility nature of the substances, pKa value of the compound and its absorbance at selected wavelength. Chromatography in general and high performance liquid chromatography (HPLC) in particular, plays an important role as a separation/detection technique in pharmaceutical analysis. The compounds to be determined are generally dissolved in a suitable solvent and injected into the chromatographic system, separated under normal room temperature condition and then detected by various detection techniques. It is widely used for separation, identification and determination of both sample and complex present in the raw materials, intermediates, bulk drugs and their drug substances and drug product in the pharmaceutical sector [8]. Thin layer chromatography (TLC) is a powerful technique for rapid screening of unknown materials in bulk drugs [9]. Gas liquid chromatography (GLC) has a significant role in the analysis of pharmaceutical products.

High performance Liquid Chromatography (HPLC), which was introduced in 1960’s is the most common and sensitive of the several chromatographic techniques employed in the impurity profiling of pharmaceuticals. Organic impurities in bulk drug substances at levels of 0.1% area or even less can be detected by HPLC. HPLC system mainly consists of four main parts namely, the solvent Reservoir, Pump, Column, and the detector. The Pumping systems deliver metered amounts of mobile phase from the solvent reservoirs to the column through high-pressure tubing and fittings. Advanced technologies in the chromatographic field introduced pumping systems consisting of one or more computer-controlled metering pumps that can be programmed to vary the ratio of mobile phase components, as is required for gradient chromatography, or to mix isocratic mobile phases.

Sensitivity of the HPLC detection system plays a major role, in detection and quantification of the impurities at trace levels [10]. The evaluation of HPLC profile of bulk drug substances and pharmaceutical dosage forms using different Liquid chromatographic systems and the selection of sensitive detection system is the focus of current research in the field of impurity profiling of pharmaceutical compounds.

1.2.1.1 Commonly used Detectors in HPLC:

In general the HPLC detectors are classified in to two main types, selective and universal. The selective (specific) detectors gives the response based on the different typical physiochemical or chemical properties of the samples investigated (UV, fluorescence etc.,). Whereas the universal (non-specific) detectors gives a response for all peaks eluted from the column independent of their chemical structure (refractive index, evaporative light scattering, etc.).

Ultraviolet (UV) absorbance detector:

UV detectors are the most popular and are commercially available for HPLC use in a different type of configurations.

Variable wavelength detector (VWD):

Monochromator starts with a continuum UV source, such as deuterium lamp, producing a broad band of radiation from 190 to 800 nm. The light is then scattered off a grating, which separates the light into a spectrum of its various wavelengths. As the grating is placed on a movable platform, it allows the user to choose any single wavelength from the spectrum.

Photodiode array detector (PDA):

The photodiode array detector takes the UV detector one step further than the variable wavelength detector by allowing the user access to all the wavelengths simultaneously. This is accomplished by stating with a continuum source as in the VWD and passing the entire spectrum of light through the detector cell. The light is then bounced off a grating as in the VWD. In this case the grating does not move and the single detector is replaced with an array of multitude of individual detectors (photodiodes). These detectors are arranged on a single chip referred to as photodiode array. The advantage of this detector is scanning of the entire UV spectrum, establishing the peak purity and compound confirmation.

Fluorimetric detectors: These type of detectors are useful for the detection of compounds that are inherently fluorescent or that can be converted to fluorescent derivatives either by chemical transformation of the compound or by coupling with fluorescent reagents at specific functional groups. If Derivitazation is required, it can be done prior to chromatographic separation or, alternatively, the reagent can be introduced into the mobile phase just prior to its entering the detector

Potentiometric, Voltammetric, or Polarographic electrochemical detectors are useful for the quantitation of species that can be oxidized or reduced at a working electrode. These detectors are selective, sensitive, and reliable, but require conducting mobile phases free of dissolved oxygen and reducible metal ions.

Refractive index (RI) detector:

Refractive index detector is sensitive to the minor refractive index changes that occur when the analyte concentration changes in the column effluent. The detector is universal because the RI of a solution changes if the temperature, density or concentration changes. Therefore, if any analyte passes through the detector cell, the changes in RI will be referred as a chromatographic peak.

Evaporative Light scattering detector (ELSD):

ELSD detector is a universal detector and more sensitive than RI detector. Useful for the separation of non Chromophoric molecules with the volatile mobile phases and has the sensitivity in the range of 0.2 ng µl-1.

1.3 Importance of Stability indicating methods in pharmaceutical analysis:

Stability indicating nature of the chromatographic method indicates its suitability for the testing of stability samples, holding time study samples, sample under transit and samples stored for long period without appropriate storage condition and within appropriate storage condition. Before the introduction of liquid chromatographic techniques, in 1950 only quantitative or semi-quantitative method and procedures were used in pharmaceutical studies. Establishing the credible expiration dates for pharmaceutical products by kinetic and predictive studies is now accepted worldwide. The most important factors needs to be considered for the quality and safety of pharmaceutical product is stability at different storage condition in different atmospheres. Regulatory authorities require stability testing at different storage conditions like real time and accelerated condition to understand how the quality of an API (active pharmaceutical ingredient) or a drug product changes with such conditions like with time under the influence of various environmental factors such as Moisure, humidity, heat, light and pressure. The data obtained during these stability studies defines storage conditions and expiry date / retest date/shelf life, the selection of proper formulations and protective packaging, and is required for regulatory documentation. Forced degradation, or stress testing, is carried out by altering the conditions deliberately than those used for accelerated stability testing, they are performed during the initial drug development process, involving potential drug to degrade under a variety of conditions like, acid and base hydrolysis, peroxide oxidation, photo stability, and thermal to understand resulting byproducts and pathways that are necessary to develop stability indicating methods.

Stability of pharmaceutical product may be defined as the capability of particular product, in a specific/ container/closure system, to remain within its physical, chemical, microbiological, therapeutic, and toxicological specification.

According to FDA guidelines "Stability is defined as the capacity of a drug substance or drug product to remain within established specifications to maintain its identity, strength, quality, and purity throughout the retest or expiration dating periods.

1.3.1. Techniques employed for Stability-indicating method

Various analytical techniques such as titrimetric, spectrophotometric and chromatographic techniques are in place for the analysis of stability samples. Based on the available technology in current scenario there so many sophisticated techniques are used for the stability studies, based on the nature of the compound and storage conditions. The analytical methods which are reports till date literature are depicted below:

Table1.3.T1: Techniques employed, stability-indicating method [31]

S.No.

Principle

Technique

1.

Electrometric

Titrimetric, Polarographic

2.

Spectrophotometric

Ultra violet (UV)/Visible (VIS), Fluorescence, Nuclear magnetic resonance (NMR)

3.

Chromatographic

Thin layer chromatography (TLC), High performance thin layer chromatography (HPTLC), Gas chromatography (GC),

High performance liquid chromatography (HPLC), Capillary electrophoresis (CE)

4.

Hyphenated

techniques

Gas chromatography–Mass spectrometry (GC-MS), Liquid chromatography– Mass spectrometry (LC-MS), LC-MS-MS,

Liquid chromatography–Nuclear magnetic resonance (LC-NMR), Capillary electrophoresis–Mass spectrometry (CE-MS)

Titrimetric and spectrophotometric methods are simple and cost effective but they are not enough insightful as that of chromatographic method and these techniques have the limitations with respect to specificity. When compared to these methods chromatographic methods are sensitive, accurate and can detect traces amount of degradation product/impurities as well as capable of separating multiple components in a single run. In chromatographic methods, HPLC has been very widely employed. It has gained popularity in stability studies due to its high-resolution capacity, sensitivity and specificity. Non-volatile, thermally unstable or polar/ionic compounds can also be analyzed by this technique.

1.4Analytical Method development:

Modern active pharmaceutical ingredients are complex in nature and it contains various number of functional groups with different characteristics. To ensure the quality of the final product or API it is essential to have good chromatographic method to identity and quantity the impurities, starting materials, intermediates, impurities and degradation products. To separate these mixtures into individual components prior to quantitative analysis, chromatographic techniques comprise of methods for separating complex mixtures that depends on the differential affinities of the solute between two immiscible phases. The relative affinity of the solutes for each of phases must be reversible to ensure that mass transfer occurs during chromatographic separation.

Nowadays reverse phase liquid chromatographic methods are the best in choice for for small molecular weight pharmaceutical moieties in stability indicating and stability specific methods. Non chromatographic and spectroscopic techniques such as titrimetry, atomic absorption, UV Spectrophotometry and IR spectroscopy, though they are precise, are not considered as specific and stability indicating, and as such not suitable for stability assessment application.

Based on the solubility and nature of the substances method development shall be initiated by selecting primary conditions. Based on the outcome of the different experimental conditions to achieve good resolution and symmetry of the peak the chromatographic conditions shall be adjusted to meet the objective. For example reduction of the runtime of components in a liquid chromatographic method can be achieved by means of increasing the concentration of the organic phase in mobile phase or adjusting the pH of the buffer or increasing the flow rate or increasing the temperature of the column. By doing so we need to compromise the resolution between the components. The adjustment should be in such a way to optimize the appropriate symmetry and resolution. Reverse phase HPLC consists of non polar stationery phase and moderately polar mobile phase. High performance RPLC columns are efficient, stable, and reproducible. Depending on the number of active component to be separated, the more complex the separation the more gradient elution would be advantageous over isocratic modes.

Analytical stability indicating method developments involves various Practical steps like, Collection of information on physiochemical properties of the analytes, Preliminary separations with known impurities, Stress or forced degradation studies, Separation studies on stressed samples, Optimization and final method development, Identification and characterization of degradation products for selective Stability indicating methods (SIM).

1.4.1 Factors influencing the Separation in HPLC:

Chromatographic separation can be influenced by any one or a combination of different factors that include solvent composition, type of column stationary phase, and mobile phase buffers and pH. The column is the heart of HPLC separation process. It is very essential to have stable HPLC column for the development of rugged, reproducible analytical method. Different types of column packing materials are available for the HPLC. Silica based reverse phase pickings are made by covalently bonding an organosilane or by depositing a polymeric organic layer on the support surface. Column polarity depends on the polarity of the bound functional groups, which range from relatively nonpolar octadecyl silane to very polar nitrile groups. The concentration of the organic stationery phase or Percent Carbon for bonded phase packing is a rough guide to the level of retention provided by a particular column. The surface area of the bonded phase is a one of the important factor for the retention. Larger the surface area the greater is retention (k).Retention tends to increase with percent carbon in separation involving hydrophobic interactions. Sample retention marginally increases for bonded phases of greater length (C18>C8>C3>C1) but it can be controlled only to a limited extent by changing the bonded phase Stability of the bonded phase columns are also one important factor to be considered in method development. Once the desired separation is achieved, column characteristics should remain unchanged for as long as possible. Column stability is dependent on the type of silica supports, bonded phases and also on mobile phase pH, type of buffer and organic modifier used.

Most of the Acidic compounds can be well retained and separated at Lower pH (pH < 3) but some separation are performed at higher pH because, Compounds of interest are unstable at low pH or needed selectivity is not available at low pH or protonated basic compounds are too poorly retained at low pH. High pH conditions are favorable for separating basic compounds, as they are in a free, non-ionic state. Also the unreacted silanol groups are totally ionized at high pH, creating less interactive surface than at intermediate pH where silanol groups can only be partially ionized.

The retention and selectivity due to difference in bonded Phase columns with the same functionality (i.e. C18, C8 etc) are affected by factors like, difference in silica Support, Choice of silane, monofunctinal or polyfunctinal,Completeness of bonding, partially or fully reacted, Presence or absence of end capping, bonding Chemistry, Support surface area etc.

The mobile phase composition plays a significant role in chromatographic performance and the resolution of compounds in the mixture being chromatographed. Change in composition has a much greater effect on the capacity factor k, than temperature. In partition chromatography, the partition coefficient, and hence the separation, can be changed by addition of another component to the mobile phase. In Ion-exchange chromatography, pH and ionic strength, as well as changes in the composition of the mobile phase, affect capacity factors. So depending on the chromatographic techniques, it is very important to consider each and every above discussed point, while developing new LC analytical methods.

1.4.2 Forced degradation studies (Stress testing):

Forced degradation studies of active pharmaceutical ingredients, drug products or any other substance is useful in understanding the intrinsic stability of the molecule, identify the likely degradation product, establish the degradation pathways and hence to validate the stability indicating power of the analytical method used. Forced degradation studies are different from accelerated stability studies, students should not confused. Forced degration studies are carried out in extreme conditions such as acidic, basic, photolytic, oxidative and thermal conditions to temperature, whereas accelerated stability studies requires testing at high relative humidity and temperature i.e 40°C/75% RH, and serves to gener ate the data which is useful in predicting the changes that might occur in the drug substance or product during normal storage conditions.

As per ICH guidelines Stress testing [11] is likely to be carried out on a single batch of the drug substance The study should include the effect of temperatures (in 10ºC increments (e.g., 50ºC, 60ºC, etc.) above that for accelerated testing), humidity (e.g.,75% RH or greater) where appropriate, oxidation, and photolysis on the drug substance. The testing should also evaluate the susceptibility of the drug substance to hydrolysis across a wide range of pH values when in solution or suspension. Photo stability testing should be an integral part of stress studies.

The primary goal of forced degradation studies should be to generate the degradation samples realistic of those formed during manufacturing, handling and normal ICH storage conditions of the Active pharmaceutical ingredients and drug product. Overstressing may destroy the relevant primary degradants and lead to unrealistic secondary degradants. Under stressing may fail to produce realistic degradant. As a General rule, the stress testing experiments to be designed in such a way that, about 10-20 % degradation is achieved under different stress conditions. Forced degradation studies may help facilitate pharmaceutical development as well as in area such as formulation development, manufacturing and packaging, in which knowledge of chemical behavior can be used to improve a drug product.

The API shall be subjected to various forced degradation conditions to effect partial degradation of the drug preferably in 20-80% range [12]. The forced degradation studies provide information about the conditions in which the drug is unstable, so that measures can be taken during the manufacturing of API’s or the formulation of drug products to avoid any potential instability. The stability samples were prepared by dissolving the API in diluent, aqueous hydrochloric acid, aqueous sodium hydroxide or aqueous hydrogen peroxide solution at a concentration of sample preparation for routine analysis, separately. After the degradation, these samples shall be collected, filtered using syringe filters and injected in to HPLC system.

Solutions for neutral degradation studies were prepared in diluent at a concentration of principle concentration by taking appropriate amount of API in suitable mL of diluent solution, at room temperature and heated in a water bath at the selected temperature for 2h. The mixture was then allowed to cool at room temperature, filtered using syringe filters and analyzed.

Solutions for acid degradation studies were prepared in 1N hydrochloric acid at a concentration of principle concentration, at room temperature by dissolving appropriate amount of API in suitable mL of 1N hydrochloric acid solution in diluent to get a concentration of sample concentration. The resultant solution was heated in a water bath at selected temperature for 2h. The mixture was then allowed to cool at room temperature, filtered using syringe filters and analyzed.

Solutions for base degradation studies were prepared in 1N sodium hydroxide at a concentration of principle concentration, at room temperature by dissolving appropriate amount of API in suitable mL of 1N sodium hydroxide solution in diluent to get a concentration of sample concentration. The resultant solution was heated in a water bath at selected temperature for 2h. The mixture was then allowed to cool at room temperature, filtered using syringe filters and analyzed.

Solutions for oxidation studies were prepared in 50% hydrogen peroxide solution at a concentration of principle concentration, at room temperature by dissolving appropriate amount of API in suitable mL of 50% hydrogen peroxide in diluent to get a concentration of sample concentration. The resultant solution was heated in a water bath at selected temperature for 2h. The mixture was then allowed to cool at room temperature, filtered using syringe filters and analyzed.

For Photo-stability studies, API in powder form was directly exposed to short wavelength light (254nm) for 4 days. The degraded sample were withdrawn at appropriate time and subjected to analysis.

For Temperature stress studies, API in powder form was exposed to dry heat in an oven for 4 days. The degraded sample were withdrawn at appropriate time and subjected to analysis.

1.5 HPLC Method validation:

Analytical test method validation is conducted to assure that the analytical methodology is repeatable, accurate, linear, specific, and robust over the specified range that an analyte will be analyzed. Method validation provides an assurance of reliability during normal use, and in sometimes defined as "the process of providing documented evidence that the method does what it is intended to do".

Analytical methods need to be validated or revalidated, before their introduction into routine use; or whenever the conditions change for which the method has been validated (e.g,an instrument with different characteristics or samples with a different matrix); and whenever the method is changed and the change is outside the original scope of the method.

The Food and drugs administration has projected adding section 211.222 on method validation to the current Good Manufacturing Practices (cGMP) regulations. As per International Conference on Harmonization (ICH) guidelines [13] the parameters like System suitability test (SST), Selectivity, Precision, Limit of Detection (LOD), Limit of Quantitation (LOQ) , Linearity, Accuracy, Ruggedness, Robustness, Solution and Mobile phase stability should be studied during the method validation. Validation parameters carried out for different analytical methods were presented in the individual chapters.

During the method validation the scope and objective method needs to be considered and based on the scope parameters selection needs to be performed. All the materials used for the validation study should be of appropriate suitability and purity. The objective of the method and its validation criterion should be distinct early in the process. The complete information regarding the Number of analytes, expected concentration levels, required detection and quantitation limits, the Sample matrix, required precision and accuracy range, should be available before starting the validation experiment.

1.5.1 System suitability test:

System suitability test (SST) is an integral part of method validation. Before performing any method validation experiments we should establish that the HPLC system and procedure are capable of providing data of acceptable quality. These tests are used to verify that the resolution and repeatability of the system are adequate for the analysis to be performed. System suitability tests are based on the concept that the equipment, electronics, analytical operations and samples constitute an integral system that can be evaluated as a whole. System suitability is used to ensure system performance before or during the analysis of unknowns. Parameters such as tailing factor, resolution, theoretical plates count and relative retention time are determined and compared against the specifications set for the method. The following system suitability parameters will be generally evaluated for LC methods.

Number of Theoretical plates (N) :

The column efficiency (N) also known as theoretical plate count is a measure of the efficiency and related to the band spreading of a peak. The smaller the band spread, the higher the number of theoretical plates, which indicates good column and system performance. The measurement of column efficiency is actually the total efficiency for the Liquid chromatographic system and the column combined.

N= 16(tr/tw) 2 =L/H

tr = Retention time of the chromatographic peak

tw= Peak width of the chromatographic peak

L= Length of the column

H= Height equivalent to the theoretical plate.

USP Resolution (Rs) :

USP Resolution is a measure of separation of the two peaks. Most simply, it is a function of how far apart the peaks are relative to how broad they are. For accurate quantification of the closely eluting chromatographic peaks, a USP resolution  1.5 is desirable.

Rs = V2-V1

½ (W1+W2)

V1 & V2 are the retention times of the peaks 1 and 2

W1 & W2 are the widths of the peaks 1 and 2

USP tailing factor (T) :

USP tailing factor is a measure of symmetry of the chromatographic peak. T= Wx/2f

Wx = Width of the peak determined at either at 5% or 10% from the baseline of the peak height.

f = Distance between peak maximum and peak from at Wx

Relative retention time or selectivity () :

Relative retention is a measure of the relative location of the two chromatographic peaks,  = k2/k1

k1 & k2 are capacity factor values of chromatographic peaks 1 and 2.

1.5.2 Specificity:

Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities. The specificity of the developed HPLC method is carried out in the presence of its impurities. Stress degradation studies were performed for drug substance to provide an indication of the stability indicating property and specificity of the proposed method. If the method lacks the selectivity parameter, method accuracy, precision, and linearity all are seriously compromised. So during method development, one of the first steps is to ensure the selectivity. Selectivity of the method can be assessed by spiking the known interference impurities and by conducting sample forced degradation studies.

1.5.3 Precision:

Precision is the measure of the degree of repeatability of an analytical method under normal operation and is normally expressed as the percent relative standard deviation (RSD) for a statistically significant number of samples. According to the ICH, Precision of the method can be expressed as three different types: repeatability, intermediate precision and reproducibility.

Repeatability is the precision of a method under the same operating conditions over a short period of time. Intermediate precision is the agreement of complete measurements when the same method is applied many times within the same laboratory. The objective of intermediate precision in validation is to ensure that in the same laboratory the method will provide the same results once the development phase is over. Reproducibility examines the precision between laboratories and is often determined in Interlaboratory studies or method transfer experiments. Precision is the measure of the degree of agreement among test results when the method is applied repeatedly to multiple samplings of a homogeneous sample.

1.5.4 Limit of detection (LOD):

There are two types of the methods are available for establishing the LOD.

Method 1: S/N ratio method:

The limit of detection (LOD) is defined as the lowest concentration of an analyte in a sample that can be detected but not necessarily quantified. LOD is a limit test that specifies whether or not an analyte is above or below a certain value. It is expressed as a concentration at a specified signal-to-noise ratio of usually 3:1.

Method 2: Slope method:

The LOD is determined by measuring the standard deviation of the response and slope. The LOD for API and Impurities were determined by injecting a series of dilute solutions with known concentrations.

The limit of detection (LOD) may be expressed as:

3.3 ∞

LOD = _________________

S

where ∞ = the standard deviation of the response

S = the slope of the calibration curve

1.5.5 Limit of Quantification (LOQ):

Method 1: S/N ratio method:

The limit of quantification (LOQ) is defined as the lowest concentration of an analyte in a sample that can be quantified with acceptable precision and accuracy under the stated operated conditions of the method. The LOQ is often based on a certain signal to noise ration usually 10:1.The LOD and LOQ values determined during method validation are affected by the separation conditions: columns, reagents and especially instrumentation and data systems.

Method 2: Slope method:

The LOQ is determined by measuring the standard deviation of the response and slope. The LOQ for API and Impurities were determined by injecting a series of dilute solutions with known concentrations.

The limit of quantification (LOQ) may be expressed as:

10 ∞

LOD = _________________

S

where ∞ = the standard deviation of the response

S = the slope of the calibration curve

1.5.6 Linearity:

Linearity is defined as the "ability of the method to elicit test results that are directly proportional to analyte concentration within a given range". Linearity can be assessed by performing single measurements at several analyte concentrations. The Linearity of the method for all the related substances was determined by analyzing dilute solution of API and its related substances at different concentration levels of LOQ-150 % of each in triplicate. The correlation coefficient was calculated for each substance. The expected concentration range, the data are then processed using a linear least square regression analysis. The resulting graph provides the desired information on slope, intercept and correlation coefficient. A linearity coefficient above 0.980 for drug substance and 0.970 for impurities is generally acceptable.

A typical linearity equation for HPLC method is as follows

Y = mX+C where

m= Slope of the calibration curve

C = Y-intercept of the calibration curve

Y = Response or peak area of the analyte of interest

X = Concentration or amount of the analyte

1.5.7 Accuracy:

Accuracy is defined as "the closeness of the test results obtained by the analytical method to the true value". In the case of the assay of a drug substance, accuracy can be determined by application of the analytical procedure to an analyte of known purity. Accuracy can be established by comparing the results of the method with results from an established reference method. This approach assumes that the uncertainty of the reference method is known. Secondly, accuracy can be assessed by analyzing a sample with known concentrations, for example, a certified reference material, and comparing the measured value with the true value as supplied with the material.

The accuracy of the method for all the related substances was determined by analyzing API sample solutions spiked with all the related substances at four different concentration levels of LOQ-150 % of each in triplicate. LOQ-150% of impurities solutions were prepared considering impurity limit as 100%. LOQ is the concentration obtained in LOQ study. The percentage of recoveries for the impurities was calculated by injecting the standard solution for each level.

1.5.8 Ruggedness:

Method ruggedness is defined as ‘the reproducibility of the results when the method is performed under actual use conditions’. This includes different analysts, laboratories, columns, instruments, sources of reagents and so on.

1.5.9 Robustness:

The robustness of a method can be defined as "ability of the method to remain unaffected by small deliberate variations in method parameters and provides an indication of its reliability during normal usage". This includes flow rate, pH, mobile phase composition, ionic strength, column temperature etc. These method parameters may be evaluated one factor at a time or simultaneously as part of a factual experiment.

1.5.10 Solution and mobile phase stability:

Solution stability is one of the important parameter to be studied during the validation. Analytical laboratories utilize auto samplers with overnight runs and the sample will be in solution for hours in the laboratory environment before the test procedure is completed. This is of serious concern especially for drugs that can undergo degradation by hydrolysis, photolysis and adhesion to glassware. Data to support the sample solution stability under normal laboratory conditions for the duration of the test procedure, e.g., 24 or 48 h should be generated. In exceptional cases where multiple days needed for sample preparation or solution storage an appropriate stability time can be selected. Mobile phase stability can also be assessed by using the same mobile phase during the study and by estimating the stability of the freshly prepared sample solutions at each study interval.

1.6 Scope and objectives of the present research study:

The main objective of the study is to develop simple, specific, sensitive, cost effective, time effective methods for the selected compounds N-methyl fluoxetine oxalate, Etoricoxib, Prasugrel hydrochloride and Pioglitazone hydrochloride. To attain so, it is required to have high scientifically sound analytical techniques to separate and quantify the impurities present in the drug intermediate and substance. I have selected an analytical tool "Liquid chromatography (LC) / High performance liquid chromatography (HPLC)" to separate and quantify the impurities.

Regulatory agencies insists development of specific and sensitive analytical methods for assurance of quality, safety and efficacy of drugs. This is very essential because of their use not only as health care products but also life saving substances. The development of new analytical methods becomes essential whenever there is a development of a new drug, whenever there are continuous changes in manufacturing processes for existing drugs and also whenever new threshold limits for individual and total impurities of drugs by regulatory authorities are being set. Keeping this in view, an attempt is made in the present investigation to develop new analytical methods for some of the important drugs and pharmaceutical compounds.

Upon thorough literature survey it is revealed the absence of the stability indicating validated liquid chromatographic methods for the quantification and determination of selected process related impurities and degradation impurities formed during the manufacture, handling and storage of N-methyl fluoxetine oxalate, Etoricoxib, Prasugrel hydrochloride and Pioglitazone hydrochloride in drug substance and pharmaceutical preparations. Keeping in this in mind, cost effective, simple and sensitive liquid chromatographic methods were developed, optimized and validated for the determination of the purity and related substances. All the methods developed were validation as per the ICH Q2 Validation of analytical procedures guideline.

The compounds under study are mentioned below

1. Compound name : Etoricoxib

Etoricoxib : 5-Chloro-2-(6-methyl pyridin-3-yl) 3-(4-methyl sulfonyl phenyl) pyridine.

Molecular Formula : C18H15ClN2O2S

Molecular Weight : 358.84 g.mol-1

Therapeutic activity:  Non-Steroidal Anti-Inflammatory 

2. Compound name : N-Methyl Fluoxetine Oxalate

N-Methyl Fluoxetine oxalate: Dimethyl-3[3-phenyl-3-(4-trifluoromethyl-phenoxy)-propyl]-amine oxalic acid slat.

Molecular Formula : C18H20F3NO.C2H2O4

Molecular Weight : 413 g.mol-1

Therapeutic activity: Drug substance intermediate (Antidepressant)

3. Compound name: Prasugrel Hydrochloride

Prasugrel Hydrochloride 2-[2-(Acetyloxy)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone hydrochloride

Molecular Formula : C20H21ClFNO3S

Molecular Weight : 409.9 g.mol-1

Therapeutic activity:  Platelet Inhibitor

4. Compound name: Pioglitazone Hydrochloride

Pioglitazone Hydrochloride [5-[[4-[2-(5-Ethyl-2-pyridinyl)ethoxy]phenyl]methyl]-2,4-] thiazolidinedione hydrochloride

Molecular Formula : C19H20N2O3S•HCl 

Molecular Weight : 392.9 g.mol-1

Therapeutic activity: Antidiabetic