Pharmaceutical analysis may be defined as the application of analytical procedures used to determine the purity, safety and quality of drugs and chemicals. More recently it also deals with biological samples in support of biopharmaceutical and pharmacokinetic studies. Some of the medicinal products are still being assayed by the time-tested procedures of gravimetric and titrimetric technique. A wide diversity in the type of analytical technique has been characteristic of assay method for pharmaceuticals. The following analytical techniques have been employed for estimation of different components in formulation. Pharmaceutical analysis includes both qualitative and quantitative analysis.
Qualitative analysis:Deals with identification of the substance.
Quantitative analysis: Deals with the determination of how much of the constituent is present.
Some of the key components in quality control which have a great impact of the quality control of the final product are:
Raw material inspection
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In process inspection
The instrumental technique can be categorized into five techniques such as
The spectral methods are based on the nature of absorption or emission of electromagnetic radiation by the system being analyzed.
UV - Visible spectroscopy
Fluorescence and Phosphorescence spectroscopy
X ray radiation technique
Nuclear magnetic resonance
Electron spin resonance
The electro chemical techniques are based on interdependence of Electro chemical properties and composition of system.
High performance thin layer chromatography
Super critical chromatography
High performance liquid chromatography
Analytical method development is required for
Active ingredients (Macro analysis)
Component of Interest in different matrices
New process and reactions
Sample transport and storage
Information to customer
Sample collection and sampling
Steps in analytical cycle
Among the various spectrophotometric methods available, the technique of ultraviolet-visible spectroscopy is the most common method used in the analytical techniques used in the field of pharmacy.It deals with the determination of the amount of UV (190-380 nm) or visible (380-800 nm) radiation absorbed by a substance in a solution.
When a beam of monochromatic light passed through a transparent cell containing a solution of an absorbing substance, reduction of the intensity of the light may occur.
A compound or drug possess conjugated double bond, absorbs UV radiation at a specific wavelength and this character of drug is specific for a fixed solvent system. The drugs are quantitatively analyzed by ultraviolet analytical method which is governed by Beer Lambert's law, which is represented as
A = abc
Where, A = absorbance,
a = absorptivity,
b = path length,
c = concentration
ASSAY OF SUBSTANCES IN MULTICOMPONENT SAMPLE
For multi-component analysis of the compounds many methods are followed. They are
ï€ Simultaneous equation method (Vierodt's method)
ï€ Absorbance ratio method
ï€ Geometric correction method
ï€ Absorption factor method (absorption correction method)
ï€ Orthogonal polynomial method
ï€ Difference spectrophotometry
ï€ Derivative spectrophotometry
The essential components of spectrometers
Source of electromagnetic radiation
Moderate to high selectivity
Wide applicability to both organic and inorganic systems
Easy and convenience of data acquisition
SIMULTANEOUS EQUATION METHOD
Concentration of several components present in the same mixture can be determined by solving a set of simultaneous equation even if their spectra overlap. If beer's law is followed these equations are linear.
If a sample contains two absorbing drugs (X and Y) each of which absorbs at the Î»max of the other, it may be possible to determine both drugs by the technique of simultaneous equation (Vierodt's method)
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The absorptivity of X at Î»1 and Î», ax1 and ax2 respectively.
The absorptivity of Y at Î»1 and Î», ay1 and ay2 respectively.
The absorbance of the diluted sample at Î»1 and Î»2, A1 and A2, respectively.
Let Cx and Cy be the concentration of X and Y, respectively in the diluted sample.
Two equations are constructed based upon the fact that at Î»1 and Î»2, the absorbance of the mixture is the sum of the individual absorbance of X and Y.
At Î»1 A1 = ax1bcx + ay1bcy
At Î»2 A2 = ax2bcx + ay2bcy
Cx = (A2ay1 - A1ay2) / (ax2ay1 - ax1ay2)
Cy = (A2ax2 - A1ax1) / (ax2ay1 - ax1ay2)
Using the above equations, concentration of individual components in a mixture can be determined by simple calculation.
HIGH PERFORMANCE THIN LAYER CHROMATOGRAOHY
HPTLC is one of the most widely used methods for both qualitative and quantitative analysis. Steps involved in HPTLC method development can be summarized as follows.
Selection of chromatographic layer
Pre coated plates with different support materials and different sorbents are available. Of these silica gel is far most important sorbent.
The sample preparation procedure is to dissolve the dosage form with complete recovery of intact compound of interest and minimum of matrix with a suitable concentration of analytes for direct application on the HPTLC plate.
Layer pre treatments
Prior to chromatography it is a common practice to prepare the layer by any or all of the following steps; washing, activation, conditioning and equilibrium so as to avoid problems like irregular and drifting densitometric baselines, ghost peaks and reduced sample detectability.
Application of the sample
Use of automatic application devices is recommended for quantitative analysis. Preferably samples are applied as bands because it ensures better separation because of the rectangular area in which the compounds are present on the plate.
Mobile phase optimization
A solvent of correct strength for a single development separation will migrate the sample into the Rf range of 0.2 to 0.8. Mobile phase should chose taking into consideration chemical properties of analytes and sorbent layer.
If the tank is saturated prior to development, solvent vapour soon get uniformly distributed throughout the chamber, hence less solvent shall be required to travel at a particular distance, resulting in the lower Rf values.
Development and detection
Ascending, descending, two dimensional, horizontal, multiple over run gradient, radial and anti-radial are most common methods of development. Detection may be done manually or instrumentally; instrumental detection is recommended for quantitative analysis.
Criteria for selection of most suitable wave length
The light intensity re-emitted by chromatographic zones is usually lower than the sorbent layer around it. Therefore, absorption spectra of a compound can be directly determined on HPTLC plate itself in comparison to substance free portion of sorbent layer.
Scanning is mostly carried out at a wavelength of maximum absorption because the difference between, absorption by the chromatographic zone and the blank area of sorbent layer around it is the largest, background being least.
In case of complex formulations, the analyst should explore the possibility of selecting a single wavelength at which the entire chromatogram could be scanned referred to as "most suitable wavelength".
While selecting single wavelength, the interest of minor components in the formulation needs special consideration.
Advantage of HPTLC
Ability to analyse several samples
Visual chromatogram and simplicity
Multiple sample handling
Low running and maintenance cost
Disposable layer can be used
Quantification of crude drug
Simultaneous analysis of samples
Automatic sample application
Small quantity of mobile phase sufficient
ANALYSIS OF BIOLOGICAL SAMPLES
Bioanalytical methods employed for the quantitative determination of drugs and their metabolites in biological samples plays a significant role in the evaluation and interpretation of bioavailability, bioequivalence and pharmacokinetic study data. These studies generally support regulatory findings.
In order to determine the optimum dose it is necessary to know in detail the pharmacokinetics of drug and its metabolites. Today such requirements are put upon all newly introduced drugs and this is the reason why so much interest is focused on the topic.
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The most widely employed bioanalytical techniques include but not limited to conventional chromatography methods (such as HPLC, HPTLC and GC), mass spectrometry based methods (such as LC-MS, LC-MSMS, GC-MS) and ligand bind assays.
The biological samples generally used are plasma, serum, urine and saliva. However in most of the cases plasma is the sample of choice. This is due to the fact that the analyte concentration in plasma can be used reliably to interpret the analyte concentration in the site of action.
Sample preparation involves separation of analyte from the matrix. Sample preparation step largely depends on the nature of sample and type of method employed for estimation. For chromatographic techniques commonly employed methods are
Liquid-liquid extraction: Hydrophobic drugs in unionized form can be extracted into an immiscible organic solvent.
Protein precipitation: Protein is denatured by precipitation an its drug binding ability is destroyed, resulting in the releasing of the drug into filtrate.
Solid phase extraction: Analytes can be separated from interference by a specific interaction with a solid phase cartridge.
The parameters essential to ensure the acceptability of the performance of a bio-analytical method is accuracy, precision, selectivity, sensitivity, reproducibility and stability. The stability of the analyte in the biological matrix at indented storage and operating conditions should be established. The matrix based standard curve should consist of a minimum of five standard points, excluding blanks, using single or replicate samples, and should cover the expected range of concentrations.
High Performance Liquid Chromatography
High performance liquid chromatography is a convenient separation technique used for wide types of samples, with exceptional resolving power, speed and Nano molecular detection levels. This technique is based on the same modes of separation as that of classical chromatography i.e. adsorption, partition, ion exchange and gel permeation, but it differs from column chromatography in the fact that the mobile phase is passed through the packed column under high pressure.
According to the phases involved, HPLC can be classified in to several types which are as follows:
Normal phase chromatography
Reverse phase chromatography
b) Liquid- solid chromatography or adsorption HPLC
c) Liquid- liquid chromatography or partition HPLC
d) Ion exchange chromatography
e) Size exclusion or gel permeation
f) Ion- pair HPLC
g) Affinity HPLC
Parameters Used in Chromatographic Characterization
The retention of a drug with a given packing material and eluent can be expressed as retention time or retention volume, but both of this are dependent on flow rate, column length and column diameter. The retention is best described as a column capacity ratio (k), which is independent of these factors. The column capacity ratio of a compound (A) is defined as
kA = VA - Vo = tA - to
VA = Elution volume of A
Vo = Elution volume of a. non retained compound (void volume)
The distance between any two adjacent peaks in a multi peak chromatogram is referred to as Resolution 'R.' and is calculated as
R = 2(t2 - t1)
W1 + W2
t1, and t2 are the retention times for the latest and the earliest eluting peak and W, and W2, are the peak width at baseline.
R>1 = Components completely separated
R<1 = Components overlap.
Capacity Factor (k')
The retention of the analyte expressed as the number of void volumes of the system, needed for the peak to elute is called the capacity factor. The expression for k is
k' = tr - to
tr retention time
to = void volume
Theoretical Plates (N)
The number of theoretical plates generated on a column is a measure of its performance. The definition of N is
N = 5.54 tr
tr = retention time
tw1/2 = is the peak width at half height
'N' may also be calculated from the width along the baseline of the peak. This is accomplished by extending tangents from the two peak inflection points through the baseline.
Separation factor (Î±)
This parameter is used to quantify the separation between adjacent peaks. Ideally, the peaks should not overlap, that is they should be baseline-resolved. This condition is met for peaks of similar size when a > 1.15. The separation factor is calculated as follows
Î± = K'2
The subscripts refer to the order of elution.
Î± is always >1
The asymmetry is a tool for quickly determining how much if any, of an eluting peak profile deviates in shape from a normal distribution. The subscript Y refers to the percentage of peakheight at which the asymmetry is determined.
Eg: A10(determined at 10% peak height)
Peak asymmetry is given as:
b = The distance between the perpendicular connecting the baseline to peak maximum and the latest eluting portion of the curve.
a = The distance between the perpendicular connecting the baseline to the peak maximum and the earliest eluting .portion of the curve.
Quantification involves the measurement of peak height or peak area. To determine the concentration of a compound, the peak area or height is plotted Vs the concentration of the substance. For well resolved. peaks, both peak height and area are proportional to the concentration. Three different calibration methods used in quantitative analysis is external standard, internal standard and the standard addition method.
RETENTION IN RP-HPLC
The reverse phase chromatography is based upon its polarity and experimental conditions such as mobile phase, column and temperature.
Mobile Phase effects
Retention (compound K value) can be preferably adjusted by changing mobile phase composition or solvent strength. In RPC, retention is less for stronger, less polar mobile phase. Solvent strength depends on the choice of organic solvent and its concentration in the mobile phase. A retention range of 0.5 < k < 20 are allowable for samples to be separated using isocratic condition but 1 < k < 10 is generally preferred.
a) Choice of organic phase (%B)
A mobile phase of 100% ACN is a stronger polar solvent, which may result in (k < 0.2), so a weaker mobile phase is required to retain the compound. This can be attained by decreasing the percentage of ACN which in turn increases the retention time. When organic phase is decreased by 10% the k value increases 3 times approximately.
b) Mobile-phase strength
Mobile phase strength in RPC depends upon both % B and the type of organic solvent. RPC solvent strength varies as water (weakest) < methanol < acetonitrile < ethanol <tetrahydrofuran< propanol <methylenechloride (strongest). Solvent strength increases as solvent polarity decreases.
Column and Temperature effect
An increase in temperature by 1Â°C will usually decrease values of k by 2% fornon-ionic compounds, but it is not mostly used in RPC. For very hydrophobic samples it can be useful to operate at higher temperatures with a very strong mobile phase and very weak column.
SELECTIVITY IN RP-HPLC
Once overall sample retention is adjusted (0.5 < k < 20), it is necessary to change the band spacing or selectivity (Î±) of different bands. Three main variables used in RPC to change selectivity for neutral samples are mobile phase, column type and temperature. A small change in ' Î± ' is adequate for separating many samples.
Solvent- strength selectivity
The best sample resolution will occur for a %B value where both pairs have the same resolution peak spacing can be explored while %B is varied for optimum sample retention (0.5 < k < 20). The use of solvent strength selectivity is limited mainly by the retention range of the sample.
b) Solvent Type selectivity
A change in organic solvent type is often used to change peak spacing and improve resolution. The selection of RPC solvents for this purpose is guided by solvent properties that are believed to affect selectivity, acidity, basicity and diploarity.
c) Column Type selectivity
A change in column type can produce useful changes in selectivity and overall sample retention. Retention is greater on the stronger C8 and phenyl column Vs the weaker cyano column.
d) Temperature Selectivity
Values of k decreases at higher temperature for the RPC separation of neutral compounds. This is less effective for non-ionic compounds.
FORCED DEGRADATION STUDY
Stability testing forms an important part of drug product development. The purpose of stability testing is to provide evidence on how the qualities of the drug substance or drug product varies with time under the influence of variety of environmental factors such as temperature, humidity and enables recommendation storage conditions. The two main aspects of drug product that play an important role in shelf life determination are assay of active drug and degradents generated during the stability study. The assay of drug in stability test sample needs to be determined using stability indicating method, as recommended by ICH guidelines and USP.
The ICH guidelines entitled " stability testing for new drug substance and products"(Q1A) requires that stress testing carried out to elucidate the inherent stability characteristics of active substance. It suggest that the testing should include the effect of temperature, humidity where appropriate, oxidative photolysis and susceptibility to hydrolysis.
ANALYTICAL METHOD VALIDATION
The word "Validation" means "Assessment" of validity or action of proving effectiveness'.
The steps involved in method development and validation largely depend upon types of method being developed. However the following steps are common to most of analytical methods.Analytical data are used to screen potential drug candidates, aid in development of drug synthesis, support formulation studies, monitor the stability of bulk pharmaceuticals and formulated products and test final products of release. The process of method development and validation has a direct impact on the quality of these data.
A well-developed method should be easy to validate. A method should be developed with a goal to rapidly test preclinical samples. The extent of method validation are defined and characterized as follows.
a) Full validation
A full validation is important when developing a method for the first time.
b) Partial validation
Partial validations are modifications of already validated methods.
c) Cross validation
Cross validation is a comparison of validation parameters when two or more methods are used to generate data within same study or across different studies.
A common weakness in development and validation of methods is that the methods are not robust enough. As the method will be run by several groups during its progression from development to validation, the method must be robust. This means that the method should provide reliable data, both on a wide range of equipment and in the hands of several chemists. If robustness is not built into methods early in development, then the result most likely will be loss of efficiency during routine quality control testing and a lengthy and complicated validation process as well. Another challenge encountered early in the development of methods intended to support stability studies is ensuring that the method is stability indicating. This process is typically achieved by conducting forced-degradation studies. The design and execution of these studies requires thorough knowledge of the product being tested as well as a good understanding of the analysis technique.
A well-developed method should be easy to validate. A method should be developed with the goal torapidly test preclinical samples, formulation prototypes and commercial samples. For validation, the developed method is subjected to the following studies.
It is the ability of the method to accurately measure the analyte response in the presence of all potential sample components. The response of the analyte in test mixtures containing the analyte and all potential sample components (placebo formulation, synthesis intermediates, excipients, degradation products, process impurities etc.) is compared with the response of a solution containing only the analyte.
A linearity study verifies that the sample solutions are in a concentration range where the analyte response is linearly proportional to concentration. Acceptability of linearity data is often judged by examining the correlation coefficient and y-intercept of the linear regression line for the response versus concentration plot.
The range of ananalytical method is the concentration over which acceptable accuracy, linearity, and precision are obtained in practice, the range is determined using data from the linearity and accuracy studies.
The accuracy of a method is the closeness of the measured value to the true value for the sample. Accuracy is usually determined in four ways:
ï· It can be assessed by analyzing. A sample of known concentration and comparing the measured value to the true value.
ï· The second approach is to compare test results from the new method with the results from an existing alternate method that is known to be accurate.
ï· The third approach, which is the most widely used recovery study, is performed by spiking analyte in blank matrices. For assay methods, spiked samples are prepared in triplicate at three levels over a range of 50-150% of the target concentration.
ï· The fourth approach is the technique of Standard additions, which can also be used to determine recovery of spiked analyte.
The precision of an analytical method is the amount of scatter in the results obtained from multiple analyses of a homogenous sample. It is determined at three levels.
It is obtained when analysis is carried out in one laboratory by one operator using one piece of equipment over relatively short time span for at least 5 or 6 determinations of three different matrices at 2 or 3 different concentrations.
It represents the precision obtained between laboratories. The objective is to verify that the method will provide tile same results in different laboratories. It is determined by analyzing aliquots from homogenous lots in different laboratories with different analysts with the specified parameters of the method.
Limit of detection (LOD)
The detection limit is the lowest analyte concentration that produces a response detectable above the noise level of the system, typically, three times the noise level.
S/N = 3/1
Limit of Quantitation (LOQ)
The quantitation limit is the lowest level of analyte that can be accurately and precisely measured. It is calculated as the, analyte concentration that gives S/N = 10/1.
The robustness of a method is its ability to remain unaffected by small changes in parameters such as % organic content and pH of mobile phase, buffer concentration, and temperature and injection volume.
Degree of reproducibility of test results obtained by analyzing the same sample under variety of normal test conditions such as different analytes, instruments, days, reagents, column and TLC plates.
BENEFITS OF VALIDATION
Minimize rejection and reworking
Minimize utility costs
Reduce testing requirements
More rapid and reliable start-up new equipment
Easier maintenance of equipment