This document describes the development and validation of an HPLC method for estimating drugs. It discusses the principles of HPLC, steps in method development including selecting the method, column, mobile phase and detector. Method validation parameters like accuracy, precision, specificity, linearity and robustness are also summarized. The document provides details on the optimization process and validation procedures to ensure the method is suitable for its intended use.
In this slide contains types of HPLC Columns, Plate theory and Van Deemter Equation.
Presented by : Malarvannan.M (Department of pharmaceutical analysis).
RIPER,anantpur.
In this slide contains types of HPLC Columns, Plate theory and Van Deemter Equation.
Presented by : Malarvannan.M (Department of pharmaceutical analysis).
RIPER,anantpur.
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.
The drug or drug combination may not be official in any pharmacopoeias.
A proper analytical procedure for the drug may not be available in the literature due to patent regulations.
Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients.
Analytical methods for the quantitation of the drug in biological fluids may not be available.
Analytical methods for a drug in combination with other drugs may not be available.
The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.
Ion pair chromatography for pharmacy studentsabhishek rai
Ion-PairChromatography
A GENERALISED OVERVIEW
Chromatography
HPLC
Reverse Phase Chromatography
Ion Pair Chromatography
Ion Pair Reagent
Mechanism of Ion Pair Chromatography
Ion Pair Wash Procedure
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR HIGH PRESSURE LIQUID CHROMAT...Dr. Ravi Sankar
GASSCHROMATOGRAPHY[GC], ADVANCED STUDY OF THE FOLLOWING AND THEIR APPLICATIONS, INTRODUCTION, THEORY, COLUMN OPERATION,INSTRUMENTATION AND DETECTION,APPLICATIONS AND ADVANTAGES OF GC,PRINCIPLE OF SEPARATION IN GC, HOW GC MECHINE WORKS? COLUMN, DETECTORS.
BY P.RAVISANKAR, VIGNAN PHARMACY COLLEGE, VADLAMUDI, GUNTUR, A.P, INDIA.
Bioanalytical Method Development and Validation..
Bioanalytical method validation include all the procedure that demonstrate that a particular method used for quantitative measurement of analyte in given biological matrix are reliable and reproducible for intended use.
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.
The drug or drug combination may not be official in any pharmacopoeias.
A proper analytical procedure for the drug may not be available in the literature due to patent regulations.
Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients.
Analytical methods for the quantitation of the drug in biological fluids may not be available.
Analytical methods for a drug in combination with other drugs may not be available.
The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.
Ion pair chromatography for pharmacy studentsabhishek rai
Ion-PairChromatography
A GENERALISED OVERVIEW
Chromatography
HPLC
Reverse Phase Chromatography
Ion Pair Chromatography
Ion Pair Reagent
Mechanism of Ion Pair Chromatography
Ion Pair Wash Procedure
HPLC[ HIGH PERPROMANCE LIQUID CHROMATOGRAPHY OR HIGH PRESSURE LIQUID CHROMAT...Dr. Ravi Sankar
GASSCHROMATOGRAPHY[GC], ADVANCED STUDY OF THE FOLLOWING AND THEIR APPLICATIONS, INTRODUCTION, THEORY, COLUMN OPERATION,INSTRUMENTATION AND DETECTION,APPLICATIONS AND ADVANTAGES OF GC,PRINCIPLE OF SEPARATION IN GC, HOW GC MECHINE WORKS? COLUMN, DETECTORS.
BY P.RAVISANKAR, VIGNAN PHARMACY COLLEGE, VADLAMUDI, GUNTUR, A.P, INDIA.
Bioanalytical Method Development and Validation..
Bioanalytical method validation include all the procedure that demonstrate that a particular method used for quantitative measurement of analyte in given biological matrix are reliable and reproducible for intended use.
Analytical method development and validation for simultaneous estimationProfessor Beubenz
Brief about analytical method development and validation
Subscribe to the YouTube Channel #Professor_Beubenz
https://www.youtube.com/channel/UC84jGf2iRN5VjwnQqi6qmXg?view_as=subscriber
Following are the list of attributes to be followed;
1.0 Objective:
The purpose of the study is to develop analytical method for determination of Assay & Related substances in new formulation product by HPLC using UV-Visible detector.
2.0 Introduction:
This document will help analyst to develop robust Analytical method for routine testing.
3.0 Scope:
This guideline will provide information about analytical method development to be carried out as per ICH Guidelines.
4.0 Physico-Chemical details of API:
Following items to be checked viz;
IUPAC name, Molecular formula, Chemical structure, Molecular weight, Solubility, Appearance, Melting Point, pH solubility, pKa, LogP, BCS Classification, Polymorphism, Isomerism, Density, Hygroscopicity, Impurities listed as per process either in DMF or official monograph
It was one of my presentation for my master's in pharmacy. It assisted me in better understanding the many pharmacy research fields as well as what to do before, during, and following a research project. I am hoping that it will also provide the readers a better understanding of the fascinating world of research.
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.
A Review HPLC Method Development and Validationijtsrd
Due to its very effective separations and often high detection sensitivity, HPLC is the most widely used separation method in contemporary pharmaceutical and biomedical analysis. The majority of medications in multiple component dosage forms can be examined using the HPLC method due to its many benefits, including speed, specificity, accuracy, precision, and ease of automation. The development and validation of HPLC procedures are crucial to novel discoveries, the creation of pharmaceutical medications, and numerous other investigations involving both humans and animals. To compare a defined characteristic of the drug substance or drug product to predetermined acceptance criteria for that characteristic, an analytical technique is designed. This review provides details on the numerous steps that go into developing and validating an HPLC technique. According to ICH Guidelines, validating an HPLC technique include testing for system appropriateness as well as accuracy, precision, specificity, linearity, range and limit of detection, limit of quantification, robustness, and other performance characteristics. Ajay Sanjay Salvi | Mohini S. Khamkar | Dr. Lahu D. Hingane "A Review: HPLC Method Development and Validation" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-7 | Issue-4, August 2023, URL: https://www.ijtsrd.com/papers/ijtsrd58595.pdf Paper Url:https://www.ijtsrd.com/pharmacy/other/58595/a-review-hplc-method-development-and-validation/ajay-sanjay-salvi
A brief review on development and validation of hplc method.adhirajain
the slides in the ppt gives a brief review on product development and its validation in HPLC method. Contents are with advantages, disadvantages, application , classification and methods for development.
Analytical Method Development by High Performance Liquid Chromatographyijtsrd
HPLC is the dominant separation technique in modern pharmaceutical and biomedical analysis because it results in highly efficient separations and in most cases provides high detection sensitivity. Most of the drugs in multi component dosage forms can be analyzed by HPLC method because of the several advantages like rapidity, specificity, accuracy, precision and ease of automation in this method. HPLC methods development and validation plays an important role in new discovery, development, manufacture of pharmaceutical drugs and various other studies related to humans and animals. This review gives information regarding various stages involved in development and validation of HPLC method. Validation of HPLC method as per ICH Guidelines covers all the performance characteristics of validation, like Accuracy, Precision, Specificity, Linearity, Range and Limit of detection, Limit of quantification, Robustness and system suitability testing. Tanmayi Kalamkar | Tejaswini Kande | Naziya Sayyad | Dipti Patil "Analytical Method Development by High Performance Liquid Chromatography" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-6 | Issue-4 , June 2022, URL: https://www.ijtsrd.com/papers/ijtsrd50177.pdf Paper URL: https://www.ijtsrd.com/pharmacy/analytical-chemistry/50177/analytical-method-development-by-high-performance-liquid-chromatography/tanmayi-kalamkar
Speaker at seminar "The Pharmaceutical quality system: ICH Q8/ICH Q9" - University of Parma, 18 May 2012.
Describing steps, tools, and approaches developed for application of QbD to manufacturing processes that have analogous application to the development and use of analytical methods.
Stability Indicating HPLC Method Development A Reviewijtsrd
High performance liquid chromatography HPLC is an essential analytical tool for evaluating drug stability. HPLC methods must be able to isolate, detect, and quantify drug related degradation products that may form during storage or production, and identify drug related impurities that may form during synthesis. .. This article describes strategies and challenges for designing HPLC methods to demonstrate drug stability. It will deepen our understanding of drugs and medicinal chemistry and demonstrate advances in stability that reflect an analytical approach. Several important chromatographic parameters were investigated to improve the detection of potentially related degradants. It is necessary to find suitable solvent and mobile phase samples that provide sufficient stability and compatibility with each component and potential impurities and degradants. This method should be carefully considered as it has the ability to distinguish between primary and secondary decomposers. The study of forced destruction of chemicals and new drugs is essential for the development and characterization of these immobilization methods. Practical guidance is provided at each stage of drug development to develop a forced disposal protocol and avoid common issues that might impede data interpretation. Suraj Nagwanshi | Smita Aher | Rishikesh Bachhav "Stability Indicating HPLC Method Development - A Review" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-5 , August 2021, URL: https://www.ijtsrd.com/papers/ijtsrd46310.pdf Paper URL: https://www.ijtsrd.com/pharmacy/other/46310/stability-indicating-hplc-method-development--a-review/suraj-nagwanshi
Method Validation - ICH /USP Validation, Linearity and Repeatability labgo
Prepared by : Santram Rajput (Technical Manager)
Validation of analytical procedures reinforce the reliability and suitability of a methodology for providing accurate and precise results. This slide show elaborates in detail about the need for method validation with examples, along with that it also covers the factors to be evaluated prior to validation. This slide show further touches upon the characteristics which are of significance in context of the validation procedure.
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.
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.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
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.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
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 .
HPLC Method Development & Method Validation (mr.s)
1. METHOD DEVELOPMENT AND METHOD VALIDATION
FOR THE ESTIMATION OF DRGS BY USING RP-
HPLC
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BY
@@@ Mr.S @@@
Pharmaceutical Analysis & Quality Assurance,
Vikas College Of Pharmacy,
Vissannapeta.
4. INTRODUCTION
High Performance Liquid Chromatography
(HPLC) is the most widely used of all analytical
separation techniques.
It is a technique in analytical chemistry used to
separate the components in a mixture , to identify
each component, and to quantify each component.
It relies on pumps to pass a pressurized liquid
solvent containing the sample mixture through a
column filled with a solid adsorbent material.
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5. Each component in the sample interacts
slightly differently with the adsorbent material ,
causing different flow rates for the different
components and leading to the separation of the
components as they flow out the column.
The development of HPLC from classical
column chromatography can be attributed to the
development of smaller particle is important.
They offer more surface area over the
conventional larger particle sizes.
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7. PRINCIPLE
“ The principle of separation in normal phase mode and
reversed phase mode is adsorption. When a mixture of
components are introduced into a HPLC column, they travel
according to their relative affinities towards the stationary
phase, more affinity towards stationary phase travels slower
and less affinity towards the stationary phase travels faster. No
two components have the same affinity towards the
stationary phase, the components are separated.”
In most of the cases in pharmaceutical industries –
RP-HPLC is used. Hence this gives detailing about
RP-HPLC.
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9. METHOD DEVELOPMENT
HPLC method development and validation
play important role in the discovery,
development and manufacture of
pharmaceutical products, agro chemicals.
HPLC method should be developed within
the GMP and GLP environments using the
protocols set out in ICH guide lines.
HPLC method development includes:
* Method Development
* Need for development
* Selection of suitable method
* Optimization
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10. STEPS INVOLVED IN METHOD DEVELOPMENT:-
Information on sample
Define separation goals
Special procedure requirement & pretreatment.
Detector selecting and setting
Optimization separation conditions
Check for problems or needs of special procedure
Recovery of purified material
Quantitative calibration / Qualitative method
Method validation for release to laboratories.
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11. NEED FOR DEVELOPING A NEW METHOD
Several reasons are available for the development of
a new method of analysis, they are :
There may not be a suitable method for a particular
analyte in the sample matrix.
Existing may be too erroneous.
Existing method may not provide adequate sensitivity.
Existing methods may be unreliable.
Existing methods are too expensive and time
consuming.
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12. SELECTION OF SUITABLE METHOD
Using the available literature and previous
methodology, the methods are adopted and
modified.
Sample preparation and instrument conditions
are adopted to make use of latest methods
and instrumentation.
If no previous methods exist for the analyte in
the literature , work from analogy to
investigate compounds that are similar in
structure and properties.
Usually a compound with analytical method
exists that is similar to the sample of interest.
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13. OPTIMIZATION
The principle of optimization in
the method development is to reduce the
cost, time and also to minimize the errors
which are arising in the development.
The process of optimization
includes:
* Selection of method
* Selection of mobile phase
* Selection of column
* Selection of detector
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14. SELECTION OF METHOD
The most commonly used
chromatographic methods are normal
phase, reverse phase, reverse phase ion
pair and ion exchange methods.
In the selection of method for the
organic compounds primarily reverse phase
method should be tried.
If not successful normal phase
should be tried, then reverse phase ion pair
method , ion exchange chromatographic
methods are at the end.
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15. SELECTION OF COLUMN
Columns being the heart of HPLC for
optimum separation method.
The selection of column involves the
following parameters:
* Column length
* Column diameter
* Column particle size
* Column particle shape
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16. SELECTION OF MOBILE PHASE
In reversed phase chromatography the
selection of mobile phase is very important for
the analysis of the drug .
The organic phase concentration
required for the mobile phase can be
estimated by “gradient elution method.”
Separation can be optimized by
changing the initial mobile phase composition
according to the chromatogram obtained from
preliminary run.
The pH of the mobile phase should not
alters the pH of the sample.
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17. SELECTION OF DETECTORS
Detectors are the eyes of the
chromatography system and measure the
compounds after separation of the column.
The must have certain characteristics
i.e, high Sensitivity, higher linear range,
application to most of the solutes,does not
contribute to band broadening, non-
destructive, faster response.
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19. METHOD VALIDATION
Validation Establish a 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.
Method validation is defined as the process of
proving (through scientific studies) analytical method
is acceptable for its intended use.
The validation of analytical procedures is directed to
the four most common types of analytical procedures:
Identification tests,
Quantitative tests for impurity content,
Limit tests for the control of impurities,
Quantitative tests of the active moiety in
samples.
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20. NEED OF METHOD VALIDATION
Before introduction of a new method in to
routine use.
Whenever condition change for which
method has been validation e.g. instrument
with different characteristics.
Whenever the method is changed and the
change is outside the scope of the original
method.
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21. DISCRIPTION OF METHOD
VALIDATION
PROCEDURES INVOLVED IN VALIDATION:
1.Accuracy.
2.Precision.
3.Specificity/selectivity.
4.Limit of Detection.
5.Limit of Quantification.
6.Linearity.
7.Robustness.
8.System suitability.
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22. ACCURACY
Accuracy The accuracy is the closeness of the test
results obtained by the method to he true value.
Accuracy should be established across its range.
Accuracy assessed using a minimum of 9 determinations
over a minimum of 3 concentration levels
The acceptance criterion for accuracy is the Relative
Standard Deviation (RSD) for all the recovery values
should not be more than 2%.
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23. PRECISION & REPEATABILITY
Precision : The precision of an analytical method is
the degree of agreement between a series of
measurements obtained from multiple sampling of the
same homogeneous sample. The RSD for all the
assays of sample preparations should not be more
than 2%.
Repeatability : Repeatability expresses the precision
under the same operating conditions over a short
interval of time. Repeatability is also termed intra-
assay precision . a minimum of 9 determinations
covering the specified range for the procedure ( e.g.,
3 concentrations/3 replicates each); or a minimum of
6 determinations at 100% of the test concentration.
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24. SPECIFICITY
Specificity/Selectivity: The ability to assess
unequivocally the sample in the presence of
components that may be expected to be present. –
Impurities – degrading agents – excipients ,
Specificity must be demonstrated for: –
Identification – Impurities Test – Assay Test.
The RSD for all the assays of sample preparations
should not be more than 2%.
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25. LINEARITY
Linearity: The linearity of an analytical procedure is
its ability to obtain test results that are directly
proportional to the concentration of the analyte in
the sample. Linearity is usually demonstrated by
the analysis of various concentrations of the analyte
(s) across the indented range and represented
graphically.
The relationship between the concentration (in %)
of drug in sample area of should be linear in the
specified range and the correlation should not be
less than 0.9.
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26. Robustness: The robustness of an analytical
procedure is a measure of its capacity to remain
unaffected by small but deliberate variations in
method parameters and provides an indication of its
reliability during use. The RSD for the samples
should not be more than 2%.
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27. System suitability: The simplest form of an HPLC
system suitability test involves a comparison of
chromatogram trace with a standard trace.
This allows a comparison of the peak shape, peak
width, baseline resolution. Parameters to be
calculated to provide a system suitability test report.
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Parameter Limit
Capacity factor K’>2
Injection Precision RSD < 1% for n ≥ 5
Resolution R>2
Tailing factor A≤2
Theoritical plates N>2000
28. PARAMETERS INVOLVED IN
VALIDATION
1.Mean. (Xi)
2.Standard deviation.(S.D)
3.Relative standard deviation.(RSD)
4.Correlation co-Efficient.(R)
5.Linear regression.
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29. VALIDATION PARAMETERS
MEAN :
The average result (ā) is calculated by
summing the individual results and dividing the sum
by the number (n) of individual values.
Xi= x1 + x2 + x3 ........ /n
Where,
x1 ,x2 ,x3...... = values of individual results
n = Number of individual results.
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30. STANDARD DEVIATION
It is the root mean square deviation of values from
their average
SD = [Σ (X- Xi) /n-1]1/2
Where,
Σ = sum of observations
Xi = mean
X = individual observation value
(X- Xi) = deviation of a value from mean
n =number of observations
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31. RELATIVE STANDARD DEVIATION
Relative Standard Deviation (RSD) is
defined as the standard deviation expressed as the
percentage of mean
RSD = (SD/XI) × 100
Where,
SD = Standard Deviation
XI = Mean
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32. CORRELATION CO-EFFECIENT
The correlation co-efficient (R) is used to
estimate the relationship of two random variables.
It provides a measure of the strength and
direction of the correlation varying from +1 to -1.
Positive values indicate the two variables are
positively correlated, meaning the two variables
vary in the same direction .
Negative values indicate that the two variables are
negatively correlated, meaning the two variables vary
in the contrary direction.
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33. Values close to +1 or -1 reveal the two variables
are highly related
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34. LINEAR REGRESSION
A regression is a statistical analysis
assessing the association between any two
variables. It is used to find the relationship
between two variables.
y = a + b x
where ,
a = Intercept
b = Slope
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35. CONCLUSION
Whenever the method is changed and the
change is outside the scope of the original
method.
If no previous methods exist for the analyte in
the literature , work from analogy to
investigate compounds that are similar in
structure and properties.
HPLC method should be developed within the
GMP and GLP environments using the
protocols set out in ICH guide lines.
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36. REFERENCES
Instrumental methods of chemical
analysis By B.K. Sharma (Pg:286-385)
Instrumental methods of Chemical
Analysis By Gurudeep R Chathwal
(Pg: 2.624-2.639)
WIKIPEDIA.
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