Method Development And Validation By Hplc Pdf
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Download the PDF version. Analytical method development, validation, and transfer are key elements of any pharmaceutical development program. This technical brief will focus on development and validation activities applicable to drug products.
- HPLC Method Development and Validation for Pharmaceutical Analysis
- A Review on Method development and validation by HPLC
- HPLC METHOD DEVELOPMENT AND VALIDATION: AN OVERVIEW
A reliable, selective and sensitive stability-indicating RP-HPLC assay was established for the quantitation of bromazepam BMZ and one of the degradant and stated potential impurities; 2- 2-aminobromobenzoyl pyridine ABP. Read More.
HPLC Method Development and Validation for Pharmaceutical Analysis
ABSTRACT: Bioanalytical method development is the process of creating a procedure to enable a compound of interest to be identified and quantified in a biological matrix. A compound can often be measured by several methods and the choice of analytical method involves many considerations. Analysis of drugs and their metabolites in a biological matrix is carried out using different extraction techniques like liquid-liquid extraction, solid phase extraction SPE and protein precipitation from these extraction methods samples are spiked with calibration reference standards and using quality control QC samples.
These methods and choice of analytical method describes the process of method development and includes sampling, sample preparation, separation, detection and evaluation of the results.
The developed process is then validated. These bioanalytical validations play a significant role in evaluation and interpretation of bioavailability, bioequivalence, pharmacokinetic, and toxicokinetic studies. In which different parameters like accuracy, precision, selectivity, sensitivity, reproducibility, and stability are performed. The official test methods that result from these processes are used by quality control laboratories to ensure the identity, purity, potency and performance of drug products 1 and includes all the procedures demonstrating particular method used for quantitative measurement of analytes in a given biological matrix, such as blood, plasma, serum, or urine, reliable and reproducible for the intended use 2, 3.
The recent studies show that sample throughput is an important part in Bioanalytical method development involving an efficient preparation 4. The analysis thus carried out must be verified for its alleged purpose and must be validated.
An investigation should be performed during each step to determine whether the external environment, matrix or procedural variables can affect the estimation of analyte in the matrix from the time of collection up to the time of analysis 5.
Recent progress in methods development has been largely a result of improvements in analytical instrumentation. This is true especially for chromatographs and detectors. Isocratic and gradient high-performance liquid chromatography HPLC have evolved as the primary techniques for the analysis of non-volatile active pharmaceutical ingredients and impurities.
The emphasis on the identification of analytes and impurities has led to the increased use of hyphenated techniques such as liquid chromatography-mass spectrometry LC-MS and liquid chromatography-nuclear magnetic resonance spectroscopy LC-NMR 1.
So, in the development stage, decisions regarding choice of column, mobile phase, detectors, and method of quantitation must be addressed. Method development is a complex process that involves a number of steps 8 , which are as follows:. Collection of physicochemical properties of the analytes and the related compounds are essential for the development of the analytical method.
Same molecule with different isotopes like deuterium, C 13 and N 15 will be a better alternative for internal standards. Setting the initial method conditions include diluent selection based on the solubility of the drug, drug metabolites and internal standard and compatibility with analytical method.
The lowest concentration to be quantified shall be assessed using aqueous solutions during this phase. Run time and resolution between the peaks should be taken care during this phase. Before going to analyze a method in biological matrix, first check the analytical method in aqueous standards. Prepare aqueous calibration curve standards, at least with four concentrations, including the highest and lowest.
Concentration of the highest standard shall be based on C max and lowest standard shall be tentatively fixed based on the preliminary studies.
Make injections of each calibration curve standard and find the correlation coefficient. Correlation co-efficient r should not be less than 0. If required, adjust the mobile phase, mass spectral parameters if applicable and chromatographic conditions such as mobile phase constituents, buffer strength, ratio, pH, flow rate, wavelength, column, column oven temperature etc.
When the instrumental method is concluded with aqueous standards, prepare matrix sample. Liquid-liquid extraction is useful for separating analytes from interferences by partitioning the sample between two immiscible liquids or phases.
One phase in LLE often is aqueous and second phase an organic solvent. More hydrophilic compounds prefer the polar aqueous phase; whereas more hydrophobic compounds will be found mainly in the organic solvents.
Analyte extracted into the organic phase are easily recovered by evaporation of the solvent, while analytes extracted in to the aqueous phase can often be injected directly on to a reversed-phase column.
The technique is simple, rapid and has relatively small cost factor per sample when compared to others. The extraction containing drug can be evaporated to dryness and the residue reconstituted in a smaller volume of an appropriate solvent preferably mobile phase. Solid phase extraction is the most important technique used in sample pretreatment for HPLC. SPE occur between a solid phase and a liquid phase.
It is easier to obtain a higher recovery of analyte. SPE employs a small plastic disposable column or cartridge, often the barrel of a medical syringe packed with 0. When the analyte is strongly retained, interferences are eluted or washed from the cartridge so as to minimize their presence in the final analyte fraction.
The analyte is then eluted in a small volume with strong elution solvent, collected, and either. In the opposite case, where analyte is weakly retained, interferences are strongly held on the cartridge and the analyte is collected for the further treatment. This can be carried out by using the suitable organic solvents which has good solubility of the analyte and protein precipitating properties. Acetonitrile is the first choice of solvent for protein precipitation due to its complete precipitation of proteins and methanol is the second choice of organic precipitant provided the solubility of the analyte in these solvents.
After protein precipitation the supernatant obtained can be injected directly in to the HPLC or it can be evaporated and reconstituted with the mobile phase and further cleanup of the sample can be carried out by using micro centrifuge at very high speed. Preliminary evaluation of lower limit of quantification to be done after fixing the sample processing technique. The wash volume and washing pattern of auto injector needle to be evaluated to avoid carryover of previous injections to next injections.
When sensitivity of the drug is more, prefer protein precipitation and check for recovery, precision and interferences. When sensitivity of the drug is less, prefer liquid-liquid extraction and check for recovery, precision and interferences. When the recovery and reproducibility is less in liquid-liquid extraction, prefer solid phase extraction for better sensitivity, recovery, precision and low interferences. Checking the developed bioanalytical method with matrix samples for accuracy, precision and recovery is essential before finalizing the method for pre-validation.
Minimum three aliquots each of Higher Quality Control HQC and Lower Quality Control LQC and Lower Limit of Quantification LLOQ matrix samples are analysed with one set of extracted calibration curve standards including matrix blank and zero standard blank with only internal standard and the results shall be compared for recovery with aqueous quality control samples of equivalent concentration.
The method is accepted if it meets the criteria of accuracy, precision and recovery. If needed, the method shall be considered for modification. When the method is evaluated to be reliable, prepare a brief procedure with the details of sample preparation, instrumental conditions and method conditions, to proceed for pre-validation. Selectivity, Accuracy, Precision, Recovery parameters should be evaluated in Pre-validation stage.
In reverse phase mode, the mobile phase is comparatively more polar than the stationary phase. For the separation of polar or moderately polar compounds, the suitable mode is reverse phase.
The nature of the analyte is the primary factory in the selection of mode of separation. Selection of the column is the first and the most important step in method development, because the column is the heart of separation process. The optimum length of the column required for a particular separation is dictated by the number of theoretical plates needed to give the desired resolution.
The most common column lengths used in regular analytical HPLC are 10, Most analytical HPLC columns have internal diameters i. It is generally considered that spherical forms give superior column packing properties to the non spherical forms.
As the particle size decreases the surface area for coating increases. Generally high specific surface area will increase the retention of solutes by increasing the capacity factor. For example: A compound A is assayed in three different columns of size, different particle size and different internal diameter. As shown in the figure.
Reverse phase mode of chromatography facilitates a wide range of columns like dimethylsilane C2 , butylsilane C4 , octyl silane C8 , octadecyl silane C18 , cyanopropyl CN , nitro, amino etc 11, The primary objective in selection and optimization of mobile phase is to achieve optimum separation of all the individual impurities and degradants from each other and analyte peak.
The following are the parameters to be considered during selection and optimization of mobile phase. Buffer and its strength play an important role in deciding the peak symmetries and separations. The retention time depends on molar strength of buffer. Molar strength is proportional to retention time. In order to achieve better separation the strength of the buffer can be increased. Another important component is the influence of the pH since this can change the hydrophobicity of the analyte.
For this reason most methods use a buffering agent, such as sodium phosphate, to control the pH. The buffers serve multiple purposes: they control pH, neutralize the charge on any residual exposed silica on the stationary phase and act as ion pairing agents to neutralize charge on the analyte. Ammonium formate is commonly added in mass spectrometry to improve detection of certain analytes by the formation of ammonium adducts.
A volatile organic acid such as acetic acid, or most commonly formic acid, is often added to the mobile phase if mass spectrometry is used to analyze the column eluent. Trifluoroacetic acid is used infrequently in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but can be effective in improving retention of analytes such as carboxylic acids in applications utilizing other detectors, as it is one of the strongest organic acids.
The effects of acids and buffers vary by application but generally improve the chromatography. A different concentration of buffer was chosen to achieve required separations. It is important to maintain the pH of mobile phase in the range of 2.
As Siloxane linkages are cleaved below pH 2 and at above pH 8 silica dissolves. Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability and consistency of analytical results; it is an integral part of any good analytical practice. Owing to the importance of method validation in the whole field of analytical chemistry, a number of guidance documents on this subject have been issued by various international organizations and conferences Partial validations are modifications of already validated bio analytical methods.
Partial validation can range from as little as one intra-assay accuracy and precision determination to a nearly full validation. Typical bio analytical method changes that fall into this category include, but are not limited to:. Cross-validation is a comparison of validation parameters when two or more bio analytical methods are used to generate data within the same study or across different studies.
An example of cross-validation would be a situation where an original validated bio analytical method serves as the reference and the revised bio analytical method is the comparator.
The comparisons should be done both ways. When sample analyses within a single study are conducted at more than one site or more than one laboratory, cross-validation with spiked matrix standards and subject samples should be conducted at each site or laboratory to establish inter laboratory reliability.
Selectivity is evaluated by injecting extracted blank plasma and comparing with the response of extracted LLOQ samples processed with internal standard. Sensitivity is measured using Lower Limit of Quantification LLOQ is the lowest concentration of the standard curve that can be measured with acceptable accuracy and precision. The LLOQ should be established using at least five samples independent of standards and determining the coefficient of variation and appropriate confidence interval.
The LLOQ should serve the lowest concentration on the standard curve and should not be confused with limit of detection and low QC sample.
A Review on Method development and validation by HPLC
ABSTRACT: Bioanalytical method development is the process of creating a procedure to enable a compound of interest to be identified and quantified in a biological matrix. A compound can often be measured by several methods and the choice of analytical method involves many considerations. Analysis of drugs and their metabolites in a biological matrix is carried out using different extraction techniques like liquid-liquid extraction, solid phase extraction SPE and protein precipitation from these extraction methods samples are spiked with calibration reference standards and using quality control QC samples. These methods and choice of analytical method describes the process of method development and includes sampling, sample preparation, separation, detection and evaluation of the results. The developed process is then validated. These bioanalytical validations play a significant role in evaluation and interpretation of bioavailability, bioequivalence, pharmacokinetic, and toxicokinetic studies.
Rasul Jan, Sultan Shah, and M. Naeem Khan. Received March 7, Accepted June 3, A reversed phase high performance liquid chromatographic method has been developed for the simultaneous determination of cefaclor and ceftriaxone cephalosporin antibiotic. The developed method has been validated and applied to mixtures of the commercial formulation and spiked human plasma.
HPLC METHOD DEVELOPMENT AND VALIDATION: AN OVERVIEW
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