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Analytical Method Validation
Presented By -MR.MANOJ INGALE.
(Quality Assurance Techniques)
REGULATORY GUIDELINES
ICH-Q2R1/Q2A “Text on Validation of AnalyticalProcedure (1994)
ICH-Q2R1/Q2B “Validation of Analytical ProceduresMethodology (1995)
CDER “Reviewer Guidance: Validation ofChromatographic Method” (1994)
8/19/2015 2Analytical Method Validation
REGULATORY GUIDELINES
CDER “Submitting Samples and Analytical Data forMethod Validations” (1987)
CDER Draft “Analytical Procedures and MethodValidation” (2000)
CDER “Bioanalytical Method Validation for HumanStudies” (1999)
USP<1225> “Validation of Compendial Methods”
8/19/2015 3Analytical Method Validation
CONSIDERATIONS PRIOR TO METHOD VALIDATION
Suitability of InstrumentStatus of Qualification and Calibration
Suitability of MaterialsStatus of Reference Standards, Reagents, etc
Suitability of AnalystStatus of Training and Qualification Records
Suitability of DocumentationWritten analytical procedure and proper approved protocol withpre-established acceptance criteria.
8/19/2015 4Analytical Method Validation
TYPES OF ANALYTICAL PROCEDURES TO BE
VALIDATED1. Identification tests.
2. Quantitative tests for impurities content.
3. Limit tests for the control of impurities .
4. Quantitative tests of the active moiety in samples of drug
substance.
5. Bioanalytical methods.
6. Stability indicating methods.
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VALIDATION
Definition -
Validation of an analytical procedure is the process by which it
is established, by laboratory studies, that the performance
characteristics of the procedure meet the requirements for the
intended analytical applications.
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VALIDATIONCHARACTERISTICS
8/19/2015 7Analytical Method Validation
Accuracy Precision Range
Specificity Linearity
VALIDATIONCHARACTERISTICS
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Detection Limit
Quantitation Limit Robustness
Ruggedness System suitability
DATA ELEMENTS REQUIRED FOR ASSAY VALIDATION
Category I: Analytical methods for quantitation of majorcomponents of bulk drug substances
Category II: Analytical methods for determination ofimpurities in bulk drug substances
Category III: Analytical methods for determination ofperformance characteristics
Category IV: Identification tests.8/19/2015 Analytical Method Validation 9
DATA ELEMENTS REQUIRED FOR ASSAY VALIDATION
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ACCURACY
Definition-The accuracy of an analytical procedure is the closeness oftest results obtained by that procedure to the true value.
Determined by application of the analytical procedure to ananalyte of known purity (e.g. A Reference Standard).
Accuracy should be assessed using a minimum of ninedeterminations over a minimum of three concentrationlevels, covering the specified range.(i.e. Three concentrationsand three replicates of each concentration).
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ACCURACY
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Should be reported as:Percent recovery of known amount added or
the difference between the mean assay result and the acceptedvalue.
Assessment of accuracy –Evaluating the recovery of the analyte (percent recovery) acrossthe range of the assay, or evaluating the linearity of therelationship between estimated and actual concentrations.
ACCURACY DATA SET
AmountAdded (mg)
AmountFound (mg)
Percent Recovery
0.0 0.0 ---
50.2 50.4 100.5
79.6 80.1 100.6
99.9 100.7 100.8
120.2 119.8 99.7
150.4 149.7 99.5
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PRECISION
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Definition-It is the degree of agreement among individual test results
when the procedure is applied repeatedly to multiplesamplings of a homogeneous sample.
Precision may be a measure of either the degree ofreproducibility or of repeatability of the analytical procedureunder normal operating conditions.
PRECISION
Repeatability should be assessed using a minimum of nine
determinations covering the specified range for the procedure
(i.e., three concentrations and three replicates of each
concentration or using a minimum of six determinations at
100% of the test concentration).
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(PRECISION)1.REPEATABILITY
Precision Considered at 3 Levels
1. Repeatability- Express the precision under the same
operating conditions over a short interval of time. Also
referred to as Intra-assay precision.
Should be assessed using minimum of 9 determinations
( 3 concentrations/ 3 replicates) or
Minimum of 6 determinations at the 100% level.
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2.INTERMEDIATE PRECISION
Express within-laboratory variations.
Expressed in terms of standard deviation, relative standard
deviation (coefficient of variation) and confidence interval.
Studies should include varying days, analysts, equipment,
etc.
Depends on the circumstances under which the procedure is
intended to be used.8/19/2015 Analytical Method Validation 17
3.REPRODUCIBILITY
Ability reproduce data within the predefined precision
Determination: SD, RSD and confidence interval
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ACCURACY AND PRECISION
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Accurate & precise
Accurate & imprecise
Inaccurate &precise
Inaccurate & imprecise
SPECIFICITY
Definition -Its the ability to assess unequivocally the analyte in thepresence of components that may be expected to bepresent, such as impurities, degradation products, andmatrix components.
IUPAC, AOAC-I have preferred the term “selectivity,”reserving “specificity” for those procedures that arecompletely selective.
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SPECIFICITY
Specificity has the following implications:Identification Tests: Ensure the identity of the analyte.
Purity Tests: Ensure that all the analytical procedures performed allow an accurate statement of the content of impurities of an analyte (e.g. related substances test, heavy metals limit, organic volatile impurities).
Assays: Provide an exact result, which allows an accurate statement on the content or potency of the analyte in a sample.
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SPECIFICITY
Determination -If impurity or degradation product standards areunavailable, compare the test results of samples containingimpurities or degradation products to a second well-characterized procedure (e.g., a Pharmacopeial or othervalidated procedure).
The ICH documents state that when chromatographicprocedures are used, representative chromatograms should bepresented to demonstrate the degree of selectivity, and peaksshould be appropriately labeled.
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DETECTION LIMIT
Definition -It is the lowest amount of analyte in a sample that can bedetected, but not necessarily quantitated, under the statedexperimental conditions.
The detection limit is a characteristic of limit tests.
Its usually expressed as the concentration of analyte (e.g.,percentage, parts per billion) in the sample.
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DETECTION LIMIT
Determination -For non instrumental procedures -
• Generally determined by the analysis of samples with knownconcentrations of analyte and by establishing the minimumlevel at which the analyte can be reliably detected.For instrumental procedures -
• That exhibit background noise, which is to compare measuredsignals from samples with known low concentrations ofanalyte with those of blank samples.
• [Acceptable signal-to-noise ratios are 2:1 or 3:1.]
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QUANTITATION LIMIT
Definition-It is the lowest amount of analyte in a sample that canbe determined with acceptable precision and accuracyunder the stated experimental conditions.
The quantitation limit is a characteristic of quantitativeassays for low levels of compounds in sample matrices, suchas impurities in bulk drug substances and degradationproducts in finished pharmaceuticals.
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QUANTITATION LIMIT
Determination -For non instrumental procedures -
• Determined by the analysis of samples with knownconcentrations of analyte
For instrumental procedures -• the ICH documents describe a common approach, which is to
compare measured signals from samples with known lowconcentrations of analyte with those of blank samples.
[A typically acceptable signal-to-noise ratio is 10:1.]8/19/2015 Analytical Method Validation 26
LINEARITY
• Definition -Its ability to elicit test results that are directly, or by awell-defined mathematical transformation, proportional tothe concentration of analyte in samples within a givenrange.
• “linearity” refers to the linearity of the relationship ofconcentration and assay measurement.
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LINEARITY
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 > 0.95
Requires a minimum of 5 concentration levels
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RANGE
Definition -Its the interval between the upper and lower levels of
analyte (including these levels) that have been
demonstrated to be determined with a suitable level of
precision, accuracy, and linearity using the procedure as
written.
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RANGE
• For Drug Substance & Drug product Assay– 80 to 120% of test Concentration
• For Content Uniformity Assay– 70 to 130% of test Concentration
• For Dissolution Test Method– +/- 20% over entire Specification Range
• For Impurity Assays– From Reporting Level to 120% of Impurity
Specification for Impurity Assays– From Reporting Level to 120% of Assay Specification
for Impurity/Assay Methods8/19/2015 Analytical Method Validation 30
ROBUSTNESS
Definition –• It’s the measure of its capacity to remain unaffected by
small but deliberate variations in procedural parameterslisted in the procedure documentation and provides anindication of its suitability during normal usage.
Examples of typical variations are:
• Stability of analytical solutions
• Extraction time.
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ROBUSTNESS
In the case of LC, examples of typical
variations are:• - influence of variations of pH in a mobile phase.
• - influence of variations in mobile phase composition.
• - different columns (different lots and/or suppliers)
• - temperature.
• - flow rate.
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RUGGEDNESS
Degree of reproducibility of test results under a variety of
conditions
Different Laboratories
Different Analysts
Different Instruments
Different Reagents
Different Days
Expressed as %RSD8/19/2015 Analytical Method Validation 33
SYSTEM SUITABILITYICH
ICH
Definition: evaluation of equipment, electronic,
analytical operations and samples as a whole
Determination: repeatability, tailing factor (T), capacity
factor (k’), resolution (R), and theoretical Plates (N)
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SYSTEM SUITABILITYUSP
• USP 23 <621> :
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Parameters Recommendations
K In general k ≥ 2.0
R
R > 2, between the peak ofinterest and the closest potentialinterferences(degradant, internalStd, impurity, excipient etc)
T T ≤ 2
N In general N > 2000
Repeatability RSD ≤ 2.0% (n ≥ 5)
RESEARCH PAPERS1
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PURPOSE OF STUDY
• The stability-indicating LC assay method was developed andvalidated for quantitative determination of cefcapene pivoxilin the presence of degradation products formed during forceddegradation studies.
• The method was validated with regard to linearity, accuracy,precision, selectivity, and robustness.
• HPLC method was validated according to the InternationalConference on Harmonization Guidelines (ICH Q2B,validation of analytical procedures, methodology)
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SELECTIVITY
• The selectivity was examined for non-degraded and degradedsamples.
• The HPLC method for determination of cefcapene pivoxil wasfound selective in the presence of degradation products.
• The peak purity values were more than 98.79 % for cefcapenepivoxil at 270 nm, what proves that degradants were notinterfering with the mean peak.
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TABLE 1: RESULTS OF FORCED DEGRADATION STUDIES.
• Peak purity values in the range of 98.5–100 indicates a homogeneous.
• peak
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Stress conditions and time studies
Degradation(%) Peak purity
Acidic/0.5 mol L-1 HCl/363 K/240 min
56.4 100.00
Oxidizing/30 % H2O2/343 K/310 min
88.7 98.79
Thermal/373 K/28 days 9.4 100
Thermal/393 K/28 days 30.9 100Radiolytic/25 kGy 1.7 99.98
Radiolytic/400 kGy 10.8 99.15
LINEARITY
• Linearity was evaluated in the concentration range 20–240 mgL-1.
• The samples of each solution were injected three times andeach series comprised six experimental points.
• The calibration plots were linear in the followingconcentration range 20–240 mg L-1 (n = 6, r = 0.9992).
• Statistical analysis using Mandel’s fitting test confirmedlinearity of the calibration curves.
8/19/2015 Analytical Method Validation 40
PRECISION
• Precision of the assay was determined in relation torepeatability (intra-day) and intermediate precision(interday).
• six samples were determined during the same day for threeconcentrations of cefcapene pivoxil.
• The RSD values were 0.58 and 1.27 %, respectively,demonstrating that the method was precise.
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TABLE 2 :INTRA-DAY, INTER-DAY PRECISION (n = 6)
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ACCURACY AS RECOVERY TEST
• The accuracy of the method was determined by recovering cefcapene pivoxil from the placebo.
• The recovery test was performed at three levels 80, 100, and 120 %.
• Three samples were prepared for each recovery level.
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LOD AND LOQ :
• The LOD and LOQ parameters were determined from the
regression equation of cefcapene pivoxil.
• LOD = 3.3 Sy/a , LOQ = 10 Sy/a.
• where Sy is a standard error and a is the slope of the
corresponding calibration curve.
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ROBUSTNESS
8/19/2015 Analytical Method Validation 45
RESEARCH PAPERS2
8/19/2015 Analytical Method Validation 46
VALIDATION CHARACTERISTICS
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• Recovery studies and validation of the method(As per ICH Q2A Guidelines) :
• Recovery studies were carried out by adding 1, 2 and 3 mg ofpure drug to different samples of tablet powder containing theequivalent of 10 mg of drug.
• Percentage recovery was calculated from the amountobtained by recovery studies.
• Precision of the method was studied by carrying out intraday,interday analysis and expressed as % Relative Standard.
8/19/2015 Analytical Method Validation 48
RESULTS AND DISCUSSION
• In this proposed method, the determining conditions were established by varying one parameter at a time and keeping the other fixed by observing the effect produced on the absorbance of a complex.
• The parameters involves the maximum complex development viz. concentration of reagent, temperature and time required to yield complex of maximum sensitivity were optimized.
• The proposed method was found to be accurate, simple and rapid for routine analysis of cephalexin.
8/19/2015 Analytical Method Validation 49
RESEARCH PAPERS3
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• This method was based on the reaction of NBS with aromaticamines in an acidic medium to form a brominating coloredproduct.
• Cephalexin is let to react with a known excess of NBS inacidic media.
• This caused a proportional decrease in the concentration andabsorbance of formed color in the mixture by an increase inconcentration of cephalexin.
8/19/2015 Analytical Method Validation 51
STUDY OF INTERFERENCES BY COMMON EXCIPIENTS
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NameMAC*,μ
g/mLCephalexin,μg/mL Recovery
%TCR**
Added Found**
Glucose 50 15 15.20 101.33 3.33
Fructose 50 15 15.12 100.80 3.33
Sucrose 50 15 14.90 99.26 3.33
Starch 50 15 14.80 99.16 3.33
Ca2+ 100 15 15.10 100.66 6.67
CO32- 100 15 14.99 99.66 6.67
*Maximum allowable concentrations, **Mean of three replicate analyses, TCR: TolerableConcentration Ratio with no interferences (Interferent (μg/mL) / Cephalexin (μg/mL))
VALIDATION CHARACTERISTICS
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RESEARCH PAPERS4
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PURPOSE OF STUDY
An LC–MS/MS method was developed to measure SIM andits acid form (SIMA) in plasma and peripheral bloodmononuclear cells (PBMCs) obtained from patients.
Chromatographic analyte separation was carried out on areverse-phase column using 75:25 (% v/v)acetonitrile:ammonium acetate (0.1M, pH 5.0) mobile phase.
The assay was validated for specificity and sensitivity,linearity, precision and accuracy, extraction recovery, matrixeffect, and stability
8/19/2015 Analytical Method Validation 55
SPECIFICITY AND SENSITIVITY
• Assay specificity and sensitivity were conducted in eightdifferent lots of blank plasma that was either left blank orspiked with both analyte and IS.
• The lowest limit of quantitation (LLOQ) was assessed in thesame plasma lots used for specificity.
• The determination of LLOQ was based on the criteria that thedeviation of the measured concentrations should NMT 20%from the nominal concentration and that the signal to noiseratio be ≥5.
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LINEARITY
• Linearity was evaluated using plasma samples spiked withboth SIM and SIMA at concentration ranges 2.5–500 ng/mLand 5–500 ng/mL, respectively.
• The internal standard LOV, concentration was 50 ng/mL in allcalibration standards.
• Three calibration curves were prepared and analyzed byplotting area ratios of analyte to internal standard against theconcentration of each calibration standard.
8/19/2015 Analytical Method Validation 57
PRECISION AND ACCURACY
• The intra-day precision and accuracy was evaluated atthree different QC levels (low, medium and high) in eight replicates on the same day and in five replicates on three different days for inter-day precision and accuracy determination.
Acceptable deviation –Within 15% of the nominal concentration for accuracy .Within 15% relative standard deviation for precision
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PRECISION AND ACCURACY
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The results from intra- and inter-day precision and accuracy indicate that the method reproducibility is acceptable within the same day and on different days .
STABILITY
• The short term and long term stability of SIM and SIMA in plasma and cell lysate samples -
• Short term stability of SIM and SIMA was evaluated in plasma and cell lysate samples at 40C (ice-bath) for 6h.
• Long term stability of SIM and SIMA was evaluated by storing samples for a month at -800C.
• Interconversion can be reduced either at low temperature or when pH is adjusted between pH 4 and pH 5.tested the stability of both SIM and SIMA in working solution kept at -800C and they were found to be stable for at least one year .
8/19/2015 Analytical Method Validation 60
OUTSOURCING AGENCIES
Worldwide –Oxford labs.(USA , Europe)Applus laboratories.(china, Germany, France, Chile)India –India mart(Hyderabad ,Bangluru, Pune ,Ahmadabad) Pune-Operon strategies, Synapse labs,Bioanalytical tech.
8/19/2015 Analytical Method Validation 61
REFERENCES
Tamer A.Ahmeda, Jamie Horna, John Hayslipb, Markos Leggas, Validated LC–MS/MS method for simultaneous determination of SIM and its acid form in human plasma and cell lysate: Pharmacokinetic application, Journal of Pharmaceutical Analysis, 2(2012),403-411.
Przemysław Zalewski, Judyta Cielecka-Piontek et al., Stability-Indicating HPLC Method for the Determination of Cefcapene Pivoxil, Chromatographia (2013) 76:387–391.
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REFERENCES
A. Vyas, S.S. Shukla, R. Patel, R. Pandey,V. Jain, D. Singh and B.P. Nagori, Development and Validation of Spectrophotometric Method for Estimation of Cephalexin in Bulk and Tablet Dosage Forms, Oriental Journal of Chemistry,2011, Vol. 27, No. (1): Pg. 359-362.
Rebwar O. Hassan, Indirect Spectrophotometric Determination of Cephalexin in Pharmaceutical Formulations, Chemical Science Tran.sactions, 2013, 2(4), 1110-1117
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REFERENCES
www.fda.gov (Accessed on 11/412015)
www.ich.org (Accessed on 11/412015)
http://www.labcompliance.com/methods/meth_val.
htm#introduction. (Accessed on 11/412015)
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8/19/2015 Analytical Method Validation 65
Thank You For Your Attention