The document discusses analytical and bioanalytical method validation. It defines validation as documented evidence providing a high degree of assurance that a specific process will produce a product meeting predetermined specifications. It describes the key parameters for analytical method validation including specificity, linearity, precision, accuracy, robustness, and validation reporting. For bioanalytical validation, it outlines the basic steps of selectivity, matrix effect, sensitivity, calibration/QC standards, recovery, dilution integrity, carryover, anticoagulant effect, and stability evaluation. The document emphasizes that validation ensures analytical methods and bioanalytical data support drug development and regulatory requirements.
Analytical method validation as per ich and usp shreyas B R
Analytical method validation is a process of documenting/ proving that an analytical method provides analytical data acceptable for the intended use.After the development of an analytical procedure, it is must important to assure that the procedure will consistently produce the intended a precise result with high degree of accuracy. The method should give a specific result that may not be affected by external matters. This creates a requirement to validate the analytical procedures. The validation procedures consists of some characteristics parameters that makes the method acceptable with addition of statistical tools.
Analytical method validation as per ich and usp shreyas B R
Analytical method validation is a process of documenting/ proving that an analytical method provides analytical data acceptable for the intended use.After the development of an analytical procedure, it is must important to assure that the procedure will consistently produce the intended a precise result with high degree of accuracy. The method should give a specific result that may not be affected by external matters. This creates a requirement to validate the analytical procedures. The validation procedures consists of some characteristics parameters that makes the method acceptable with addition of statistical tools.
In this slide contains Introduction, levels of cleaning, mechanism, sampling method of cleaning validation.
Presented by: P. VENKATESH (Department of pharmaceutical analysis).RIPER, anantapur
It is process of “Establishing documentary evidence that provide a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes”.
In the pharmaceutical industry, it is very important that in addition to final testing and compliance of products, it is also assured that the process will consistently produce the expected results.
Validation is action of proving in accordance with the principles of good manufacturing practices, that any procedure, process, equipment, material, activity or system actually leads to expected results.
Cleaning validation is documented evidence with a high degree assurance that one can consistently clean a system or a piece of equipment to predetermined and acceptable limits.
The primary regulatory concern driving the need for cleaning validation is cross contamination of the desired drug substance either by other API from previous batch runs or by residues from the cleaning agents used.
The prime purpose of validating a cleaning process is to ensure compliance with federal and other standard regulations
1. Cross contamination with active ingredients
Contamination of one batch of product with significant levels of residual active ingredients from previous batch cannot be tolerated.
In addition to the obvious problems posed by subjecting consumers or patients to unintended contaminants, potential clinically significant synergistic interactions between pharmacologically active chemicals are a real concern.
2. Contamination with unintended materials or compounds
While inert ingredients used in drug products are generally recognized as safe for human consumption, the routine use, maintenance and cleaning of equipment's provide the potential contamination with such items as equipment parts, lubricants and chemical cleaning agents3. Microbiological contamination
Maintenance , cleaning and storage conditions may provide adventitious microorganisms with the opportunity to proliferate within the processing equipment.
Analytical method validation, ICH Q2 guidelineAbhishek Soni
Analytical Method Validation, ICH Q2 Guideline.
General principles related to the analytical method validation.
Validation of analytical method as per International Council for Harmonisation(ICH) guidelines and the United States Pharmacopeia(USP).
Glossary.
Useful in understanding the terms :
Specificity
Linearity
Range
Accuracy
Precision
Detection limit
Quantitation limit
Robustness
Ruggedness
System suitability testing
University Institute of Pharmaceutical Sciences is a flag bearer of excellence in Pharmaceutical education and research in the country. Here is another initiative to make study material available to everyone worldwide. Based on the new PCI guidelines and syllabus here we have a presentation dealing with qualifications of HPLC which is the " High Performance Liquid Chromatography".
Thank you for reading.
Hope it was of help to you.
UIPS,PU team
Introduction, Regulatory requirements for validation, Role of FDA, Code of Federal regulation, Validation life cycle, Significance of validation, Types of validation, Process valiadation, Phases of process validation, Process capability design, Process Qualification, Validation maintainance phase
Types of Process validation, Examples
In this slide contains Introduction, levels of cleaning, mechanism, sampling method of cleaning validation.
Presented by: P. VENKATESH (Department of pharmaceutical analysis).RIPER, anantapur
It is process of “Establishing documentary evidence that provide a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes”.
In the pharmaceutical industry, it is very important that in addition to final testing and compliance of products, it is also assured that the process will consistently produce the expected results.
Validation is action of proving in accordance with the principles of good manufacturing practices, that any procedure, process, equipment, material, activity or system actually leads to expected results.
Cleaning validation is documented evidence with a high degree assurance that one can consistently clean a system or a piece of equipment to predetermined and acceptable limits.
The primary regulatory concern driving the need for cleaning validation is cross contamination of the desired drug substance either by other API from previous batch runs or by residues from the cleaning agents used.
The prime purpose of validating a cleaning process is to ensure compliance with federal and other standard regulations
1. Cross contamination with active ingredients
Contamination of one batch of product with significant levels of residual active ingredients from previous batch cannot be tolerated.
In addition to the obvious problems posed by subjecting consumers or patients to unintended contaminants, potential clinically significant synergistic interactions between pharmacologically active chemicals are a real concern.
2. Contamination with unintended materials or compounds
While inert ingredients used in drug products are generally recognized as safe for human consumption, the routine use, maintenance and cleaning of equipment's provide the potential contamination with such items as equipment parts, lubricants and chemical cleaning agents3. Microbiological contamination
Maintenance , cleaning and storage conditions may provide adventitious microorganisms with the opportunity to proliferate within the processing equipment.
Analytical method validation, ICH Q2 guidelineAbhishek Soni
Analytical Method Validation, ICH Q2 Guideline.
General principles related to the analytical method validation.
Validation of analytical method as per International Council for Harmonisation(ICH) guidelines and the United States Pharmacopeia(USP).
Glossary.
Useful in understanding the terms :
Specificity
Linearity
Range
Accuracy
Precision
Detection limit
Quantitation limit
Robustness
Ruggedness
System suitability testing
University Institute of Pharmaceutical Sciences is a flag bearer of excellence in Pharmaceutical education and research in the country. Here is another initiative to make study material available to everyone worldwide. Based on the new PCI guidelines and syllabus here we have a presentation dealing with qualifications of HPLC which is the " High Performance Liquid Chromatography".
Thank you for reading.
Hope it was of help to you.
UIPS,PU team
Introduction, Regulatory requirements for validation, Role of FDA, Code of Federal regulation, Validation life cycle, Significance of validation, Types of validation, Process valiadation, Phases of process validation, Process capability design, Process Qualification, Validation maintainance phase
Types of Process validation, Examples
Analytical method development and validation are one of the very imp aspects in Drug testing and approval process.Here I tried to explain the same with my experience.
Method validation is the process of documenting / proving that an analytical method provides analytical data acceptable for the intended use. A pharmaceutical drug product must meet all its specifications throughout its entire shelf-life. The performance of product characteristics throughout the shelf-life must be tested by analytical method for the product’s chemical, physiochemical, microbiological and biological characteristics. The method of analysis used must be validated. This is required to ensure the product’s safety and efficacy throughout all phases of its shelf-life. Validation is the process of establishing documentary evidence demonstrating that a procedure, process, or activity carried out in testing and then production maintains the desired level of compliance at all stages.
Analytical Method Validation is a process that is used to demonstrate the suitability of an analytical method for an intended purpose.Regulations and quality standards that have an impact on analytical laboratories require analytical methods to be validated.
Method validation for drug substances and drug product _remodified_2014Ramalingam Badmanaban
Method validation is the process of proving that an analytical method is acceptable for its intended purposes.
METHOD VALIDATION = ERROR ASSESSMENT
Method validation is the process of demonstrating that analytical procedures are suitable for their intended use and that they support the identity, strength, quality, purity and potency of the drug substances and drug products
Validation: Prior ConsiderationsSuitability of Instrument Status of Qualification and Calibration Suitability of Materials Status of Reference Standards, Reagents, Placebo Lots Suitability of Analyst Status of Training and Qualification Records Suitability of Documentation Written and approved standard test procedure and proper approved protocol with pre-established acceptance criteria
Compendial vs. Non-compendial Methods
Compendial methods-Verification
Regulatory analytical procedure in USP/NF
Non- compendial methods-Validation
Alternative analytical procedure proposed by the applicant for use instead of the regulatory analytical procedure
Chromatographic Methods
Demonstrate Resolution
Impurities/Degradants Available
Spike with impurities/degradants
Show resolution and a lack of interference
Impurities/Degradants Not Available
Stress SamplesFor assay, Stressed and Unstressed Samples should be compared.
Ability of an analytical method to measure the analyte free from interference due to other components.
Selectivity describes the ability of an analytical method to differentiate various substances in a sample
Original term used in USP
Also Preferred by IUPAC and AOAC
Also used to characterize chromatographic columns
Degree of Bias (Used in USP)
The difference in assay results between the two groups
the sample containing added impurities, degradation products, related chemical compounds, placebo ingredients
Selectivity: For impurity test, impurity profiles should be compared.
Temperature (50-60℃)
Humidity (70-80%)
Acid Hydrolysis (0.1 N HCl)
Base Hydrolysis (0.1 N NaOH)
Oxidation (3-30%)
Light (UV/Vis/Fl)
Intent is to create 10 to 30 % Degradation
Change in the analytical procedure, drug substance, drug product, the changes, may necessitate revalidation of the analytical procedures.
“The degree of revalidation depends on the nature of the change.”
“FDA intends to provide guidance in the future on post-approval changes in analytical procedures.”
By Visual Inspection of plot of signals vs. analyte concentration
By Appropriate statistical methods
Linear Regression (y = mx + b)
Correlation Coefficient, y-intercept (b), slope (m)
Acceptance criteria: Linear regression r2 > 0.999
Requires a minimum of 6 concentration levels
Normally derived from Linearity studies.
Established by confirming that the method provides acceptable degree of linearity, accuracy, and precision.
Specific range dependent upon intended application of the procedure.
Understanding of Analytical Method Validation Approach in Pharmaceutical Industry. Analytical method validation Verification is a wide chapter and a huge scope of applicability. In different types of methods, instrument, measurement approach all can effect the validation effort. However the basic fundamental will remains same, the parameters, acceptance criteria, functionality may vary depending upon the type of method, instrument etc.
This presentation include general introduction to validation of analytical method . analytical method validation include following points such as :
Introduction
Objective ,Types of analytical procedures to be validated,Validation parameters as per ICH and USP , cleaning validation , procedure , validation data, accuracy , range , precision, LOD, LOQ ,linearity, ruggedness , robustness
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
3. Validation is a documented evidence which high degree of
assurance that specific process will produce a product meeting it’s
pre determined specifications and quality attributes..
Drug substances and drug product manufacturers must perform
validations, it is very important that this understanding be shared
throughout the organization.
The term validation generally to cover the entire spectrum of
cGMP.
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4. ANALYTICAL METHOD VALIDATION
Validation of an analytical method is the process by which it is
established, by laboratory studies, that the performance
characteristics of the method meet the requirements for the
intended analytical applications.
The process to Confirm that the analytical procedure employed
for a specific test is suitable for intended use and that they
support the identity, quality, purity and potency of the drug
substances and drug products. 4
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6. CONCEPT OF REVALIDATION
When we make any changes inWhen we make any changes in
Analytical procedureAnalytical procedure
Drug substance (e.g. synthetic route)Drug substance (e.g. synthetic route)
Drug product (e.g. composition)Drug product (e.g. composition)
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7. PARAMETERSPARAMETERS
Specificity
LOD & LOQ (if applicable)
Linearity
Precision
i. System Suitability
ii. Method Repeatability
iii. Intermediate Precision (or) Ruggedness
iv. Method Reproducibility
Accuracy (or) Recovery
Robustness
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8. DEFINITION
SpecificitySpecificity of analytical method as its ability to measure
accurately an analyte in the presence of interference,
such as synthetic precursors, excipients, enantiomers
and known (or likely) degradation product that may be
expected to be present in the sample matrix.
1. SPECIFICITY
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9. 2. LIMIT OF DETECTION AND LIMIT OF
QUANTIFICATION (LOD & LOQ)
2. LIMIT OF DETECTION AND LIMIT OF
QUANTIFICATION (LOD & LOQ)
DEFINITIONDEFINITION
LOD:LOD: Lowest amount of analyte in a sample which can be detectedLowest amount of analyte in a sample which can be detected
but not necessarily quantitated, under the stated experimentalbut not necessarily quantitated, under the stated experimental
conditions (LOD)conditions (LOD)
LOQ:LOQ: Lowest amount of analyte in a sample which can beLowest amount of analyte in a sample which can be
quantitatively determined with suitable precision and accuracyquantitatively determined with suitable precision and accuracy
(LOQ)(LOQ)
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11. 3. LINEARITY
DEFINITIONDEFINITION
TheThe LinearityLinearity of an analytical procedure is its ability (within a given range) toof an analytical procedure is its ability (within a given range) to
obtain test results that are directly proportional to the concentration (amount) ofobtain test results that are directly proportional to the concentration (amount) of
analyte in the sampleanalyte in the sample
Range: The interval between the upper and lower level( Including these level)Range: The interval between the upper and lower level( Including these level)
that have been demonstrated to be determined with precision, accuracy andthat have been demonstrated to be determined with precision, accuracy and
linearity.linearity.
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12. 4. PRECISION
DEFINITIONDEFINITION
Closeness of agreement (degree of scatter) between a series ofCloseness of agreement (degree of scatter) between a series of
measurements obtained from multiple sampling of the samemeasurements obtained from multiple sampling of the same
homogenous sample under the prescribed conditions.homogenous sample under the prescribed conditions.
Precision may be considered at ….Precision may be considered at ….
1.1. System precision (System suitability)System precision (System suitability)
2.2. Method RepeatabilityMethod Repeatability
3.3. Intermediate precisionIntermediate precision
4.4. ReproducibilityReproducibility 12
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13. Repeatability:Repeatability: precision under same operating conditions (with-in a laboratory overprecision under same operating conditions (with-in a laboratory over
a short period of time using the same analyst with the same equipment)a short period of time using the same analyst with the same equipment)
Measurement / Injection repeatability (System Precision)Measurement / Injection repeatability (System Precision)
Method repeatability (Method Precision)Method repeatability (Method Precision)
Intermediate precisionIntermediate precision: precision under different laboratory conditions (within-: precision under different laboratory conditions (within-
laboratory variation, as on different days, or with different analysts, or equipmentslaboratory variation, as on different days, or with different analysts, or equipments
within the same laboratory)within the same laboratory)
ReproducibilityReproducibility: precision between laboratories / intermediate precision can be: precision between laboratories / intermediate precision can be
considered during the standardization of a procedure before it is submitted to theconsidered during the standardization of a procedure before it is submitted to the
pharmacopoeiapharmacopoeia
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14. 5. ACCURACY
DEFINITIONDEFINITION
The accuracy of an analytical procedure expresses the closeness ofThe accuracy of an analytical procedure expresses the closeness of
agreement between the value, which is accepted either as aagreement between the value, which is accepted either as a
conventional true value or an accepted reference value and the valueconventional true value or an accepted reference value and the value
foundfound
It is some times termed as truenessIt is some times termed as trueness
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15. 6. ROBUSTNESS
DEFINTIONDEFINTION
Measure of its capacity to remain unaffected by small, but deliberateMeasure of its capacity to remain unaffected by small, but deliberate
variations in method parameters and provides indication of its reliabilityvariations in method parameters and provides indication of its reliability
during its normal usageduring its normal usage
varying method parameters within a realistic range and the quantitativevarying method parameters within a realistic range and the quantitative
influence of the variables is determined, and, if the influence of theinfluence of the variables is determined, and, if the influence of the
parameter is within a previously specified tolerance, then, the parameter isparameter is within a previously specified tolerance, then, the parameter is
said to be within the method’s robustness range.said to be within the method’s robustness range.
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16. Typical variations include under validationTypical variations include under validation
programmeprogramme
Flow rateFlow rate
WavelengthWavelength
Mobile phase composition, generally, Organic compositionMobile phase composition, generally, Organic composition
TemperatureTemperature
pH of the mobile phasepH of the mobile phase
Stability of analytical solutionsStability of analytical solutions
Different columns ( Different lots and suppliers )Different columns ( Different lots and suppliers )
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17. VALIDATION REPORT
Generally method validation report should have
Objective and scope of the method
Molecule details (IUPAC name, CAS No. Molecular Formulae, Molecular
weight and its Molecular Structure etc.,)
Detailed list of chemicals, reagents, reference standards
Listing of equipment and its functional and performance requirements
Methodology followed
Validation data (parameter wise – procedure, results, conclusion etc.,)
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18. Instrument out puts, which should represent critical method parameters
Specificity and LOD – for Identification Test (Generally, photographs)
Selectivity / Specificity data (discriminating chromatogram, peak purity data,
blank and placebo chromatograms and stressed samples chromatograms)
Linearity graphs
Resolution and related system suitability chromatograms .
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20. INTRODUCTION OF BIO ANLAYTICAL METHOD
VALIDATION.
BASIC STEPS IN BIO ANALYTICAL METHOD
VALIDATION.
VALIDATION PARAMETERS.
CONCLUSION.
REFERENCE.
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21. INTRODUCTION
It involves the quantitative determination of drug and its
metabolites in biological fluids .
It plays a vital role in the evaluation & interpretation of the
bioavailability, bioequivalence, pharmacokinetic and
toxicokinetic study data.
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22. BASIC STEPS OF BIOANALYTICAL
PROCESS
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23. 1) SELECTIVITY
It is the ability of an analytical method to identify and
quantify the analyte in the presence of other
components in the sample.
LLOQ – It is the lowest amount of analyte in the sample
that can be quantified with accuracy and precision.
2) MATRIX EFFECT
It is the direct or indirect alteration or interference in
response due to the presence of unintended analytes in
the biological sample.
VALIDATION PARAMETERS
23
24. If MF = 1 ( No matrix effect)
If MF < 1 ( Suppression)
If MF > 1 ( Enhancement)
3) SENSITIVITY
Sensitivity test should be conducted to prove the
reproducibility for samples at LOQ.
ACCEPTANCE CRITERIA FOR LOQ :
For accuracy : ± 20%
For precision : ≤ 20%
For S/N ratio: 5: 1
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25. 4) CALIBRATION AND QC STANDARDS
Calibration standard is the biological matrix to which a known amount
of analyte is added.
QC standard is a spiked sample used to monitor the performance of a
bioanalytical method and is used to assess the integrity and validate
the results of unknown samples.
5) RECOVERY (OR) EXTRACTION EFFICIENCY
Determined by comparing the detector response of the analyte (or) IS
from an extracted sample to the detector response of the analyte from
an unextracted sample representing 100% recovery.
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26. 6) DILUTION INTEGRITY
Dilution of the study samples is performed when the
obtained concentration is exceeding the ULOQ, or when
there is less sample availability compared to the method
requirement.( can be tested on dilutions from 1:2 to 1:10
& dilution integrity will be evaluated at 1:2to 1:4)
ACCEPTANCE CRITERIA :
QC samples , Accuracy = ±15%
Precision = ≤15%
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27. 7) CARRYOVER
It is the appearance of an analyte signal in
blank sample peaks after the analysis of
samples with a high analyte concentration.
To evaluate carryover a blank sample will
be placed ULQ standard in a sequence.
It is insignificant when blank sample
response is ≤ 20% of LOQ sample.
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28. It is evaluated when different anticoagulants in plasma
are used in the preparation of the QC and Calibration
control standards.
Anticoagulant effect is nullified ,when QC standard
shows,
Accuracy = ±15% and Precision = ≤15%
9) STABILITY EVALUATION
The stability of analyte in the matrix during collection,
storage of samples should be assessed
8) ANTICOAGULANT EFFECT
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30. MATRIX STABILITY
AUTO SAMPLER STABILITY
It is evaluated to cover the anticipated run time for the
analytical batch and to handle the situations like system
malfunctioning.
ALL THE QC SAMPLE
CONC. ARE BACK
CALCULATED USING
CALIBRATION CURVE 30
31. It is performed to evaluate the stability of the samples,
which are kept on bench during the extraction process.
The anticipated time is usually 4 to 24 hrs
BENCH TOP STABILITY
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32. LONG TERM STABILITY
It is performed to demonstrate the stability of the
analyte in the matrix for longer duration of time.
ACCEPTENCE CRITERIA :
The relative means of back calculated
concentration (test/reference) for both levels must
be within 85-115%.
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33. FREEZE THAW STABILITY
It is performed , to evaluate the stability of the
analyte in the matrix after multiple cycles of freezing
and thawing.
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VIGNAN PHARMACY COLLEGE,VADLAMUDI.
34. CONCLUSION
Validation is done according to standard protocol and
used it always produce a product which meets its
predetermined specification and quality control.
Bioanalytical methods must be validated to objectively
demonstrate the fitness for their intended use.
Bioanalysis and the production of pharmacokinetic, toxicokinetic
and metabolic data plays a fundamental role in pharmaceutical
research and development involved in the drug discovery and
development process.
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35. REFERENCE
Bioanalytical Method Validation and Its
Pharmaceutical Application- A Review article
from Pharmaceutica Analytica
James agalloco,frederick J. Carleton. Validation
of pharmaceutical process, third edition, Page no
5.
The United State Pharmacopoeia 24; The National
Formulary 19; 2000: [1225] VALIDATION OF
COMPENDIAL METHODS.
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