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Analytical chemistry is one of a branch of chemistry that determines the nature and identity of a substance and its composition. In the before time there were only four accepted branches of chemistry, organic chemistry, inorganic chemistry, physical chemistry and biochemistry. During that time, analysis was considered to be a service to the other four branches. Its importance grew, and in the process, fascinated techniques and skills from all other four branches so by the 1950s, analytical chemistry was finally acknowledged as a branch of chemistry in its own right. There are fundamentally two types of analysis, qualitative analysis and quantitative analysis. The qualitative analysis identifies the nature of substance, and if it is mixture, the nature of the components present, whereas, the quantitative analysis determines the elemental composition of the substance and/or the quantitative distribution of each component.
Pharmaceutical analysis deals with the analysis of a pharmaceutical(s). It is generally recognized that pharmaceutical is a chemical entity of therapeutic interest. A more suitable term for pharmaceutical is active pharmaceutical ingredient (API) or active ingredient.
Pharmaceutical analysts in research and development (R&D) of pharma industry plays a very widespread role in new drug development and follow up activities to assure that, a new drug product meets the reputable standards, its stability, and continued to meet the professed quality throughout its shelf life.
The different activity of R&D includes drug development, (synthesis and manufacture), formulation, clinical trials, evaluation and finally launching i.e. finished products. Closely coupled with these processes are regulatory and quality assurance functions.
Before submitting the drug product for approval to the regulatory authorities, assuring that all batches of drug products comply with specific standards, utilization of approved ingredients and production methods. It becomes the responsibility of pharmaceutical analysts in quality control (QC), quality assurance (QA) department. The methods are generally developed in an analytical R&D and transferred to QC department, or other departments.
Quality Assurance and Quality Control plays a central role in determining the safety and efficacy of medicines. A highly specific and sensitive analytical technique holds the key to Design, Development, Standardization and quality control of medicinal products. They are equally important in pharmacokinetics and in drug metabolism studies, both of which are fundamental to the assessment of bioavailability and the duration of clinical response.
The pharmaceutical analysts play, a major role in assuring the individuality, safety, efficacy and quality of the drug product. Safety and efficacy studies entail that drug substance and drug product meet two critical requirements
Established identity & purity
Established bioavailability &dissolution Â.
Until 1920 all the methods based upon volume and mass like volumetric and gravimetric methods have come to be known as classical or chemical method of analysis.
After that there is a radical change in pharma analysis field due to the introduction of highly sensitive instrumental methods. In instrumental methods a physical property of a substance is measured to determine its chemical composition. These methods may be used by the analytical chemist to save time with increased accuracy in the method.
A convenient classification of analytical procedure is based on the theoretical principles involved in them. Accordingly quantitative analytical method may be divided into the following categories
Spectral (optical) methods
Advantages of instrumental methods over chemical methods:
More accurate & precise
Can be used for the analysis of complex mixtures
Can be used for multi component analysis
Small quantity of sample required
Widely accepted in research and quality control
Useful in invivo & invitro studies of drugs and their metabolites
Employed to design new drug molecules.
Spectroscopy is the division of science dealing with the study of interaction of electromagnetic radiation with matter resulting in the absorption or emission of energy in discrete amount called quanta. It is one of the most powerful tools available for the study of atomic and molecular structure and is used in the analysis of a wide range of samples.
Among the various spectrophotometric methods available, the technique of Ultraviolet-Visible spectroscopy is one of the most frequently employed in pharmaceutical analysis. It involves the measurement of the amount of ultraviolet (190-380 nm) or visible (380-800 nm) radiation absorbed by a substance in a solution.
Important characteristics of spectrophotometer methods include.
Moderate to high selectivity
Wide applicability to both organic and inorganic systems
Easy and convenience of data acquisition
Single component analysis may be done by the use of standard absorptive value, calibration graph and single or double point standardization methods. In standard absorptive value method, the use of standard A (1%, 1Cm) or E values are used in order to determine its absorptivity. It is advantageous in situations where it is difficult or expensive to obtain a sample of the reference substance. In calibration graph method, the absorbances of a number of standard solutions of the reference substance at concentrations encompassing the sample concentrations are measured and a calibration graph is constructed. The amount of the analyte in the sample solution can be easily read from the graph as the concentration equivalent to the absorbance of the solution.
The single point standardization procedure involves the measurement of the absorbance of a sample solution and of a standard solution of the reference substance. The concentration of the substances in the sample is calculated from the proportional relationship that exists between absorbance and concentration.
Atest x Cstd
Where Ctest and Cstd are the concentrations in the sample and standard
solutions respectively and Atest and Astd are the absorbances of the sample and standard solutions respectively.
This method is the preferred method of assay of substances that obey Beer's law and for which a reference standard of adequate purity is available. In multicomponent formulations, the presence of two or more drugs in a formulation give rise to interference components which mutually interfere with each other in their estimation. For the simultaneous estimation of drugs in such formulations many techniques have been applied.
Simultaneous equation method
Absorbance ratio method
Multicomponent mode of analysis.
Simultaneous Equation Method
In this method sample containing two absorbing drugs (X and Y) each of which absorbing at the ï¬max of the other may be determined. In this method, two equations are constructed based upon the fact that at ï¬1 and ï¬2 the absorbance of the mixture is the sum of the individual absorbances of X and Y. The two equations are
A1 = ax1 C1 + ay1 C2
A2 = ax2 C1 + ay2 C2
Where ax1 and ax2 = absorptivity values of compound X at
ï¬Â1 and ï¬2 respectively
ay1 and ay2 = absorptivity values of compound Y at
ï¬Â1 and ï¬2 respectively
A1 and A2 = absorbances of diluted sample at ï¬1
and ï¬2 respectively
Absorbance ratio method
This method depends on the property of a substance which obeys Beer's Law at all wavelength the ratio of absorbance of any two wavelengths is a constant value independent of concentration or path length. The two different dilutions of the same sample give the same absorbance ratio. This ratio is referred to as a Q value.
Derivative Spectroscopic Method
For the rationale of spectral analysis in order to relate chemical structure to electronic transition for analytical situations in which mixture contribute interfering absorption, as method of manipulating the spectral data called derivative spectroscopy. In this technique spectra are obtained by plotting the first or higher order derivation of absorbance or transmittance with respect to wavelength versus the wavelength. Often these plots reveal a spectral detail which is lost in an ordinary spectrum. In addition, concentration measurements of an analyte in the presence of their interference can sometimes be made easily or more accurately.
Chemical derivatization method
In the chemical derivatization, spectrophotometric assays are based on the conversion of the analyte by a chemical reagent to a derivative that has different spectral properties, indirect method. This methods is employed if the analyte absorbs weekly in the UV-region, a more sensitive method of assay is obtained by converting the substance to a derivative with a more intensely absorbing chromophore.
The interference absorption from irrelevant species may be avoided by converting the analyte to its derivative which absorbs in the visible region, where irrelevant absorption is negligible. Indirect spectrophotometric procedures are also used to advance the selectivity of the assay of an ultraviolet absorbing substance in a sample that contains other uv-absorbing components.
Multi-Component mode of analysis
Mixtures containing more than two absorbing species can be analyzed by this method. The total absorbance at a given wavelength is equivalent to the sum of the absorbance of the individual components present in the solution. This relationship make possible the quantitative determination of the individual constitutes of a mixtures even if their spectral overlap, the absorption spectra for the components are determined. The analytical wave lengths are selected such that the difference in absorption by the components is maximum. The next step in the analysis is to make Beer's Law plot using solutions of the pure substances for each component at respective wavelength. All quantities being evaluated at the same wavelength should be linear.
Computer software programmes can handle multi-components mixture and deviation from Beer's Law. In this type of analysis it is possible to determine the concentration of each component of a mixture containing multicomponents.
In multicomponent mode of analysis, five mixed standards and sample solutions were prepared and scanned using sampling points of the drugs between 200-400 nm in the multicomponent mode of spectrophotometer. The spectral data obtained from these scans are used to determine the concentration of the drugs present in the given sample formulation.
Chromatography is a group of technique for the separation of the compounds of mixtures by their continuous distribution between two phases. One is stationary phase and the other is mobile phase.
The different types of chromatography
Ion Exchange chromatography
Gel permeation chromatography
Advances in technology have resulted in wide range of techniques varying in complexity, separating ability, sensitivity and cost.
The modern instrumental techniques of Gas-liquid chromatography and high performance liquid chromatography provide excellent separation and allow the accurate measurement of very low concentrations of a wide variety of substances in complex mixtures.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
High Performance Liquid Chromatography is unquestionably the most widely used analytical separation technique. HPLC has been rapidly developed with the introduction of new pumping methods, more reliable columns and a variety of detectors has made. HPLC is one of the commonly used analytical techniques. HPLC can also be automated which involve automated sampling, separation, detection, recording and calculation and printing of results. Due to its high selectivity, specificity and sensitivity achieved by HPLC methods are mainly used for the analysis of most drugs.
HPLC is one of the most versatile instruments used in the field of pharmaceutical analysis. It provides the following features.
High resolving power
Continuous monitoring of the column effluent
Accurate quantitative measurement
Repetitive and reproducible analysis using the same column
Automation of the analytical procedure and data handling
In HPLC, the analyst has a wide choice of Chromatographic separation methodologies from normal to reverse phase and a whole range of mobile phases using isocratic (or) gradient elution techniques.
Various detectors are also available for HPLC like electrochemical detectors, refractive index detectors, fluorescence detectors, radiochemical detectors, mass-sensitive detectors, UV detectors.
Separation method developed for analysis of compounds ranged from nanograms (or) even micrograms levels are transferred to larger columns at higher flow rates for preparative chromatography for isolation of materials. This has resulted in the extensive use of preparation HPLC in the clean up and isolation of compounds from synthetic and biological samples.
HPLC methods can be classified based on separation modes as follows:
Liquid -Solid chromatography
Liquid- Liquid Chromatography
Bonded - phase chromatography
Ion - Exchange chromatography
Size - Exclusion chromatography
Reverse - Phase high performance liquid chromatography
Reverse Phase Ion - Pair chromatography
Liquid - Solid Chromatography
In this separation mode the stationary phase is a solid that retains solutes by adsorption. Usually silica or alumina is utilized as the adsorbent with relatively non polar solvents such as hexane as the mobile phase. This type of chromatography is known as normal phase adsorption. Where non polymer beads are used as stationary phase with relatively polar solvents such as water, acetonitrile, methanol as mobile phase, the separation mode can be referred to as reverse phase adsorption.
Liquid - Liquid Chromatography
In this mode, the stationary phase is a liquid the separation occurs due to solutes partitioning between two liquid phases. In normal-phase partition chromatography the stationary phase is polar and the mobile phase is non-polar. If the stationary phase is non polar liquid and the mobile phase is polar it is reversed phase partition chromatography. The method suffers from disadvantage due to some solubility of stationary phase in the mobile phase hence precautions must be taken to limit the dissolution of the stationary phase.
The salient feature of this chromatographic mode is bonding of an organic substrate to a silica-based support material. The resultant stationary phase is stable. If the bonded-phase is nonpolar and the mobile phase is relatively polar, the chromatographic mode is reversed phase bonded-phase chromatography.
In Ion-exchange chromatography, a charged stationary phase is employed containing oppositely charged counter-ions, choice are available to exchange with solute ions of the same charge in the mobile phase. The technique is known as cation-exchange or anion-exchange chromatography, depending on whether the solutes to be exchanged are positively or negatively charged.
Size - Exclusion Chromatography
In this chromatographic mode the stationary phase contains polymeric resin matrix or silica-based support material of controlled pore size, where the predominant mechanism of separation is based on the effective size of solutes in the mobile phase. This type of chromatography is suitable for solutes with molecular weight of 2000 Daltons or more and is also useful for preliminary investigation of unknown samples.
REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
Reversed-phase chromatography refers to the use of a polar eluent with a nonpolar stationary phase in contrast to normal phase chromatography, where a polar stationary phase is employed with a non polar mobile phase.
Reverse phase chromatography is widely in use due to the following advantages.
Many compounds such as biologically active substances, have limited solubility in the nonpolar mobile phase that are employed in normal-phase chromatography. Ionic or highly polar compounds have high rate of adsorption on straight silica or alumina columns and therefore can elute as tailing peaks.
Column deactivation from polar modifiers is a problem in liquid-solid chromatography which frequently can lead to irreproducibility in chromatographic systems. Long re-equilibration times during gradient elution are common in adsorption chromatography. The use of reversed phase chromatography with bonded-phase columns is advantageous because of the short re-equilibration time required. Ionic compounds can be chromatographed by using ion-exchange chromatography. This mode of chromatography is tedious because precise control of variables such as pH and ionic strength is required for reproducible chromatography.
As implied by the name, the order of elution of solutes from a reverse phase column would be the reverse of that of a normal phase column. The interactions involved in reverse phase chromatography are quite different from those of the normal phase although the predominant process is one of partition chromatography in which components are separated according to their relative partition co-efficient between the mobile and stationary phases. Highly lipophilic non polar solutes will be better retained by the stationary phase and hence have longer retention time. In contrast polar molecules will be less retained.
In reverse-phase liquid chromatography the stationary phase is prepared by chemically bonding a relatively non-polar group on to the stationary phase support. The most frequent non-polar group on to the stationary phase support is octadecaylsilane (ODS or C18) which gives a highly lipophilic stationary phases. Less lipophilic stationary phases are produced when octylsilance (C8, C2, Phenyl or cyanopropyl) - bonded phases are used. The shorter silanol chain bonded phases (C2 and C8) are often most appropriate when highly lipophlic solutes are to be separated which would be highly retained on C18 column. The mobile phase in RP-HPLC is polar, generally consisting of water and a water-miscible organic solvent, such as methanol or acetonitrile. In bonded phases the silanol groups of the silica support are reacted with alkyl groups which probably form a monolayer over the surface of the silica and such monomeric phases provide a high separation efficiency since mass transfer between the solid and mobile phases should be rapid when the sample mixture contains components having a wide range of polarity, the peak capacity of the separation column often has to be increased by using gradient elution. In reversed phase mode, solvent gradients are generated by a continuous decrease in the polarity of the eluent during the separation.
Reverse-Phase Ion-Pair Chromatography
The solvents generally employed in reversed phase chromatography consist of water and an organic modifier for neutral compounds. For basic and acidic substances an aqueous buffer system plus an organic modifier is employed. The typical buffers consist of acetate and phosphate salts at a pH value that favours the formation of the unionized form of a solute. For basics that are completely ionized at the mobile phase, pH mixed mechanisms such as ion-pairing and ion exchange which leads to tailing peaks could occur. Ion-pairing chromatography, which is a type of reversed phase chromatography employing secondary equilibrium, is an excellent alternative for the chromatography of basic solutes.
The simplicity and reproducibility of reversed phase chromatography on bonded phases makes this method particularly attractive in quantitative analysis of analytical chemistry.
Quantitation Methods in HPLC:
Peak height or peak area measurements only provide a response in terms of detector signal. This response must be related to the concentration or mass of the compound of interest. To accomplish this, some type of calibration must be performed.
The four primary techniques for quantitation are
Normalized peak area method
External Standard method
Internal Standard method
Method of Standard addition
1. Normalized peak area method:
The area percent of any individual peak is referred to the normalized peak area. This technique is widely used to estimate the relative amounts of small impurities or degradation compounds in a purified material and in this method; the response factor for each component is identified.
2. External Standard method:
This method includes injection of both standard and unknown and the unknown is determined graphically from a calibration plot or numerically using response factors.
A response factor (Rf) can be determined for each standard as follows
Rf = Standard Area (Peak height)
The external standard approach is preferred for most samples in HPLC that do not require extensive sample preparation. For good quantitation using external standards, the chromatographic conditions must remain constant during the separation of all standards and samples. External standards are often used to ensure that the total chromatographic system is performing properly and can provide reliable results.
3. Internal Standard method:
The compound, different from the analyte, one that is well resolved in the separation is called internal standard. The internal standard should be chosen to mimic the behavior of the sample compound.Response Factor is used to determine the concentration of a sample component in the original sample. The response factor is the ratio of peak areas of sample component (Ax) and the internal standard (AISTD). It can be calculated using the formula,
Rf = Ax
On the basis of strength of the internal standard (NISTD) and the response factor, the amount of the analyte in the original sample can be calculated using the formula,
Requirements for a proper internal standard include:
Well resolved from the compound of interest and other peaks
Similar retention (K) to the analyte
Should not be in the original sample
Should mimic the analyte in any sample preparation steps
Does not have to be chemically similar to the sample
Commercially available in high purity
Stable and should not react with sample or mobile phase
Should have a similar detector response to the analyte for the concentration used.
4. Method of Standard addition:
The method of standard addition can be used to provide a calibration plot for quantitative analysis. It is most often used in trace analysis. An important aspect of the method of standard addition is that the response prior to spiking additional analytes should be high enough to provide a reasonable S/N ratio (>10), otherwise the result will have poor precision.
Optimization of Chromatographic Condition
After obtaining a reasonable chromatogram only the optimization procedure can be started. A reasonable chromatogram means that all the compounds are detected by more or less symmetrical peaks on the chromatogram. By a slight change of the mobile phase composition, the shifting of the peaks can be expected. From a few experimental measurements, the position of the peaks can be predicted within the range of investigated changes. The chromatogram in which all the peaks are well resolved and symmetrical in less run time is called optimized chromatogram.
By using a more efficient column with higher theoretical plate number (N), the peak resolution can be increased.
The parameters that are affected by the changes in chromatographic conditions are,
Capacity factor (k'),
Column efficiency (N) and
Peak asymmetry factor (As).
i) Resolution (Rs)
The resolution, Rs, of two nearest peaks is defined by the ratio of the distance between the two peak maxima. It is the difference between the retention times of two components divided by their average peak width. For baseline separation, the ideal value of Rs is 1.5. It is calculated by using the formula,
Where, Rt1 and Rt2 are the retention times of components 1 and 2 and
W1 and W2 are peak widths of components 1 and 2.
ii) Capacity factor (k')
Capacity factor, k', is defined as the ratio of the number of components of solute in the stationary phase to the number of components of the same in the mobile phase. Capacity factor is used to explain how well the sample molecule is retained by a column or TLC plate during an isocratic separation. The ideal value of k' ranges from 2-10. Capacity factor can be determined by using the formula,
Where, V1 = retention volume at the apex of the peak (solute) and
V0 = void volume of the system.
The values of k' of individual bands increase or decrease with changes in solvent strength. In reversed phase HPLC, solvent strength increases with the increase in the volume of organic phase in the water / organic mobile phase. Typically an increase in percentage of the organic phase by 10 % by volume will decrease k' of the bands by a factor of 2-3.
iii) Selectivity (ï¡)
The selectivity (or separation factor), ï¡, is a measure of relative retention of two components in a mixture. The ideal value of selectivity is 2. It can be calculated by using the formula,
Where, V0 is the void volume of the column and V2 and V1 are the retention volumes of the second and the first peak respectively.
iv) Column efficiency (N)
Efficiency, N, of a column is calculated by the number of theoretical plates per meter. It is a measure of band spreading of a peak. Smaller the band spread, higher is the number of theoretical plates, indicating good column and system performance. Columns with N ranging from 2000 to 100,000 plates/meter are ideal for a good system. Efficiency is calculated by using the formula,
N = ,
Where, Rt is the retention time and W is the peak width.
v) Peak asymmetry factor (As)
Peak asymmetry factor, As, can be used as a criterion of column performance. The peak half width, b, of a peak at 10 % of the peak height, divided by the corresponding front half width, a, gives the asymmetry factor.
For a well packed column, an asymmetry factor of 0.9 to 1.1 should be achievable.
VALIDATION OF THE HPLC METHOD
Validation is a procedure of establishing documented proof, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics.
Method validation is the procedure of demonstrating that analytical procedures are appropriate for their intended use and that they support the identity, quality, purity, and potency of the drug substances and drug products. Simply, method validation is the process of proving that an analytical method is acceptable for its intended purpose. A successful Validation guarantees that both the technical and regulatory objectives of the analytical methods have been fulfilled. The transfer of a method is best accomplished by a systematic method validation process. The real goal of validation process is to confront the method and determine limits of allowed variability for the conditions needed to run the method.
Type of analytical procedures to be validated.
Validation of analytical measures is focussed to the four most common types of analytical procedures.
Quantitative test for impurities content.
Limit test for the control of impurities.
Quantitative test of the active moiety in samples of drug substance on drug product on other selected components in the drug product.
In our method of validation, we are following last type.
Assay procedures are intended to measure the analyst present in given sample, assay represent a quantitative measurement of the major component(s) in the drug sample.
Objective of validation
The most important objective of validation is to form a basis for written procedure for production and process control which are designed to assure that the drug products have the identity, strength, quality and purity they purport or are represented to possess quality, safety and efficacy must be designed to build into the product. Each step of the manufacturing process must be controlled to maximize the possibility that the finished products meet all quality and design specification.
Benefits of Validation:
Produces quality products
Helps in process improvement technology transfer, related product validation, failure investigation, and increased employee awareness.
Cost reduction by increasing efficacy, few reject, longer equipment life, production of cost effective products
Helps in optimization of process or method.
Regulatory affairs-produces approved products and increased ability to export.
VALIDATION AS DEFINED BY DIFFERENT AGENCIES
1. USFDA: According to this "Validation is the process of establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes.
2. WHO: Defines Validation as an action of providing any procedure, process, equipment, material, activity or system actually leads to the expected results.
3.EUROPEAN COMMITTEE: Defines Validation as an action of providing in accordance with the principles of GMP that any procedure. process, material, activity or system actually lead to expected results.
This process consists of establishment of the performance characteristics and the limitations of the method.
Method performance parameters are determined using equipment that is :
1. With in specification
2. Working correctly
3. Adequately calibrated
Method validation is required when:
1. A new method is been developed
2. Revision established method
3. When established methods are used in different laboratories and different analysts etc.
4. Comparison of methods
5. When quality control indicates method changes.
Performance characteristics examined when carrying out method validation are.
1. Accuracy / Precision
2. Repeatability / Reproducibility
3. Linearity / Range
4. Limit of detection (LOD)/ Limit of quantification (LOQ)
5. Selectivity / Specificity
6. Robustness / Ruggedness
Significance of Method Validation:
The quality of analytical data is a key factor in the success of a drug development programme. The process of method development and validation has a direct impact on the quality of these data.
To trust the method.
Analytical validation is a very important feature of any package of information submitted to international regulatory agencies in support of new product marketing or clinical trials applications. A thorough method development can almost rule out all potential problems, at the same time, a thorough validation programme can address the most common ones and provide assurance to the intended purpose (can be used with 100% confidence). In other words, a thorough validation can fulfil all the technical and regulatory objectives. A direct consequence and most significant out come from any method validation exercise is 'the development of meaningful specifications can be predicted upon the use of validated analytical procedures that can assess changes in a drug substance or drug product during its life time.
Analytical characteristics listed below may not be applicable to every test procedure or every particular material. It will mostly depend on the purpose for which the procedure is required, however, these following aspects of validation should be given due importance.
CHARACTERISTICS THAT SHOULD BE CONSIDERED FOR DIFFERENT TYPES OF ANALYTICAL PROCEDURE:
(As per WHO guidelines)
Linearity and Range
Limit of Detection
Limit of Quantification
The accuracy of an analytical procedure expresses the closeness of agreement between the value, which is accepted either as a conventional true value or an accepted reference value and the value found.
In a method with high accuracy, a sample is analyzed and the measured value should ideally be identical to the true value. Accuracy is represented and determined by recovery experiments. The usual range being 10% above or below the expected range of claim. The % recovery was calculated using the formula,
a - Amount of drug present in sample
b - Amount of standard added to the sample
The ICH guidelines recommended to take minimum of 3 concentration levels covering the specified range and 3 replicates of each concentration are analyzed (totally 3* 3=9 determinations.)
Precision is the measure of how data values are close to each other for a number of measurements under similar analytical conditions.
Precision may be considered at three levels according to ICH guidelines.
It expresses the precision under same operating conditions (with-in a laboratory over a short period of time using the same analyst with the same equipment)
Measurement / Injection repeatability (System Precision)
Method repeatability (Method Precision)
It expresses the precision under different laboratory conditions (within-laboratory variation, as on different days, or with different analysts, or equipments within the same laboratory).
It expresses the precision between laboratories and is often determined in collaborative studies or method transfer experiments.
The Linearity of an analytical procedure is its ability (within a given range) to obtain test results, which are directly proportional to the concentration (amount) of analyte in the sample.
The linearity is determined from 50% of the ICH reporting level to 150 % of the proposed shelf life specifications of the related substance as a minimum.
The range of an analytical procedure is the interval between the upper and lower of analyte, which is studied.
The range of an analytical procedure was the concentration interval over which acceptable accuracy, precision and linearity were obtained. In practice, the range was determined using data from the linearity and accuracy studies. Assuming that acceptable linearity and accuracy (recovery) results were obtained as described earlier. The only remaining factor to be evaluated was precision. To confirm the 'range' of any analytical procedure, linearity studies alone are not sufficient, and accuracy at each concentration (minimum three concentration levels covering lower and upper levels) should be proved.
Specificity is the ability to assess unequivocally the respective analyte in the presence of other components, which may be expected to be present in the sample.
Three methods were employed for demonstrating specificity. In the first method, the conditions of HPLC method developed, namely, percent of organic solvent in mobile phase, ionic strength, pH of the mobile phase, flow rate etc. were changed and the presence of additional peaks, if any, was observed.
The second method involves the peak purity test method using diode array detector. The diode array spectrum and diode array first derivative spectrum of the standard and sample drug peaks were recorded and compared.
The third method is based on measurement of the absorbance ratio of the drug peaks at two different wavelengths.
Limit of Detection (LOD) and Limit of Quantification (LOQ):
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be detected but not necessarily quantified as an exact value. Detection limit corresponds to the concentration that will give a signal-to-noise ratio of 3:1.
Measurement is based on:
1. Signal to noise ratio
2. Visual evaluation (relevant chromatogram acceptable)
3. The standard deviation of the response and the slope.
ï³ = Standard deviation of the response.
S = Slope of the calibration curve of the analyte
The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample that can be determined quantitatively with suitable precision and accuracy. Quantitation limit is the concentration of related substance in the sample that will give a signal-to-noise ratio of 10:1.
Based on the standard deviation of the response and the quantitation limit QL may expressed as
ï³ = Standard deviation of the response.
S = Slope of the calibration curve of the analyte
Both LOD and LOQ are affected by the separation conditions.
The reproducibility of the results when the method is performed under actual use conditions.
This includes different analysts, laboratories, columns, instruments, sources of reagents, chemicals, solvents and so on.
It is the measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides indication of its reliability during its normal usage. This is to verify that the method performance is not affected by typical changes in normal experiments.
System Suitability Studies:
System Suitability Studies were carried out as specified in the United States Pharmacoepia (USP). These parameters include column efficiency, resolution, peak asymmetry factor, capacity factor and percent coefficient of variation of peak area or height on respective injections.