Analytical

Method
Validation
Presented By -
MR.MANOJ INGALE.
(Quality Assurance Techniques)

SINHGA
D INSTI
TUT
NARHE E OF PHARMA
,PUNE - CY,
41
 REGULATORY GUIDELINES

 ICH-Q2R1/Q2A “Text on Validation of Analytical

Procedure (1994)

 ICH-Q2R1/Q2B “Validation of Analytical Procedures

Methodology (1995)

 CDER “Reviewer Guidance: Validation of

Chromatographic Method” (1994)

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 REGULATORY GUIDELINES
 CDER “Submitting Samples and Analytical Data for
Method Validations” (1987)

 CDER Draft “Analytical Procedures and Method

Validation” (2000)

 CDER “Bioanalytical Method Validation for Human

Studies” (1999)

 USP<1225> “Validation of Compendial Methods”

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 CONSIDERATIONS PRIOR TO
METHOD VALIDATION
 Suitability of Instrument
Status of Qualification and Calibration

 Suitability of Materials
Status of Reference Standards, Reagents, etc

 Suitability of Analyst
Status of Training and Qualification Records

 Suitability of Documentation
Written analytical procedure and proper approved protocol with
pre-established acceptance criteria.
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 TYPES OF ANALYTICAL
PROCEDURES TO BE
VALIDATED
1. 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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 VALIDATION
CHARACTERISTICS

Accuracy Precision Range

Specificity Linearity

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 VALIDATION
CHARACTERISTICS

Detection Quantitation
Robustness
Limit Limit

System
Ruggedness
suitability

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DATA ELEMENTS REQUIRED
FOR ASSAY VALIDATION

 Category I: Analytical methods for quantitation of major

components of bulk drug substances

 Category II: Analytical methods for determination of

impurities in bulk drug substances

 Category III: Analytical methods for determination of

performance characteristics

 Category IV: Identification tests.

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DATA ELEMENTS REQUIRED
FOR ASSAY VALIDATION

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 ACCURACY
 Definition-
 The accuracy of an analytical procedure is the closeness of
test results obtained by that procedure to the true value.

 Determined by application of the analytical procedure to an

analyte of known purity (e.g. A Reference Standard).

 Accuracy should be assessed using a minimum of nine

determinations over a minimum of three concentration
levels, covering the specified range.(i.e. Three concentrations
and three replicates of each concentration).
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 ACCURACY

Should be reported as:

Percent recovery of known amount added or
the difference between the mean assay result and the accepted
value.

Assessment of accuracy –
Evaluating the recovery of the analyte (percent recovery) across
the range of the assay, or evaluating the linearity of the
relationship between estimated and actual concentrations.

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 ACCURACY DATA SET

Amount Amount
Percent Recovery
Added (mg) Found (mg)

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

Definition-
It is the degree of agreement among individual test results
when the procedure is applied repeatedly to multiple
samplings of a homogeneous sample.

Precision may be a measure of either the degree of

reproducibility or of repeatability of the analytical
procedure under normal operating conditions.

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 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.
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 3.REPRODUCIBILITY

 Ability reproduce data within the predefined precision

 Determination: SD, RSD and confidence interval

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 ACCURACY AND PRECISION

Accurate & Accurate & Inaccurate & Inaccurate &

precise imprecise precise imprecise

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 SPECIFICITY

 Definition -
 Its the ability to assess unequivocally the analyte in the
presence of components that may be expected to be
present, such as impurities, degradation products, and
matrix components.

 IUPAC, AOAC-I have preferred the term “selectivity,”

reserving “specificity” for those procedures that are
completely 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 are
unavailable, compare the test results of samples containing
impurities or degradation products to a second well-
characterized procedure (e.g., a Pharmacopeial or other
validated procedure).

 The ICH documents state that when chromatographic

procedures are used, representative chromatograms should be
presented to demonstrate the degree of selectivity, and peaks
should be appropriately labeled.
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 DETECTION LIMIT
 Definition -
 It is the lowest amount of analyte in a sample that can be
detected, but not necessarily quantitated, under the stated
experimental 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 known
concentrations of analyte and by establishing the minimum
level at which the analyte can be reliably detected.
 For instrumental procedures -
• That exhibit background noise, which is to compare measured
signals from samples with known low concentrations of
analyte 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 can
be determined with acceptable precision and accuracy
under the stated experimental conditions.

 The quantitation limit is a characteristic of quantitative

assays for low levels of compounds in sample matrices, such
as impurities in bulk drug substances and degradation
products in finished pharmaceuticals.

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 QUANTITATION LIMIT
 Determination -
 For non instrumental procedures -
• Determined by the analysis of samples with known
concentrations of analyte

 For instrumental procedures -

• the ICH documents describe a common approach, which is to
compare measured signals from samples with known low
concentrations of analyte with those of blank samples.

 [A typically acceptable signal-to-noise ratio is 10:1.]

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 LINEARITY

• Definition -
Its ability to elicit test results that are directly, or by a
well-defined mathematical transformation, proportional to
the concentration of analyte in samples within a given
range.

• “linearity” refers to the linearity of the relationship of

concentration 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 Methods
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 ROBUSTNESS

 Definition –
• It’s the measure of its capacity to remain unaffected by
small but deliberate variations in procedural parameters
listed in the procedure documentation and provides an
indication 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 %RSD
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 SYSTEM SUITABILITY
ICH
 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 SUITABILITY
USP
• USP 23 <621> :
Parameters Recommendations
K In general k ≥ 2.0
R > 2, between the peak of in-
terest and the closest potential
R
interferences(degradant, internal
Std, impurity, excipient etc)
T T≤2
N In general N > 2000
Repeatability RSD ≤ 2.0% (n ≥ 5)
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 RESEARCH PAPERS
1

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 PURPOSE OF STUDY

• The stability-indicating LC assay method was developed and

validated for quantitative determination of cefcapene pivoxil
in the presence of degradation products formed during forced
degradation studies.
• The method was validated with regard to linearity, accuracy,
precision, selectivity, and robustness.
• HPLC method was validated according to the International
Conference on Harmonization Guidelines (ICH Q2B,
validation of analytical procedures, methodology)

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 SELECTIVITY

• The selectivity was examined for non-degraded and degraded

samples.

• The HPLC method for determination of cefcapene pivoxil was

found selective in the presence of degradation products.

• The peak purity values were more than 98.79 % for cefcapene
pivoxil at 270 nm, what proves that degradants were not
interfering 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.
•Stress
peak conditions and Degradation(%) Peak purity
time studies
Acidic/0.5 mol L-1 56.4 100.00
HCl/363 K/240 min
Oxidizing/30 % 88.7 98.79
H2O2/343 K/310 min
Thermal/373 K/28 days 9.4 100
Thermal/393 K/28 days 30.9 100
Radiolytic/25 kGy 1.7 99.98

Radiolytic/400 kGy 10.8 99.15

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 LINEARITY

• Linearity was evaluated in the concentration range 20–240 mg

L-1.
• The samples of each solution were injected three times and
each series comprised six experimental points.
• The calibration plots were linear in the following
concentration range 20–240 mg L-1 (n = 6, r = 0.9992).
• Statistical analysis using Mandel’s fitting test confirmed
linearity of the calibration curves.

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 PRECISION

• Precision of the assay was determined in relation to

repeatability (intra-day) and intermediate precision
(interday).

• six samples were determined during the same day for three
concentrations 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

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 RESEARCH PAPERS
2

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 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 of
pure drug to different samples of tablet powder containing the
equivalent of 10 mg of drug.
• Percentage recovery was calculated from the amount
obtained by recovery studies.
• Precision of the method was studied by carrying out intraday,
interday analysis and expressed as % Relative Standard.

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 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.

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 RESEARCH PAPERS
3

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• This method was based on the reaction of NBS with aromatic
amines in an acidic medium to form a brominating colored
product.

• Cephalexin is let to react with a known excess of NBS in

acidic media.

• This caused a proportional decrease in the concentration and

absorbance of formed color in the mixture by an increase in
concentration of cephalexin.
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 STUDY OF INTERFERENCES BY
COMMON EXCIPIENTS
MAC*,μ Cephalexin,μg/mL Recovery TCR**
Name g/mL %
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: Tolerable

Concentration Ratio with no interferences (Interferent (μg/mL) / Cephalexin (μg/mL))
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 VALIDATION
CHARACTERISTICS

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 RESEARCH PAPERS
4

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 PURPOSE OF STUDY

 An LC–MS/MS method was developed to measure SIM and

its acid form (SIMA) in plasma and peripheral blood
mononuclear cells (PBMCs) obtained from patients.

 Chromatographic analyte separation was carried out on a

reverse-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, matrix
effect, and stability
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SPECIFICITY AND SENSITIVITY

• Assay specificity and sensitivity were conducted in eight

different lots of blank plasma that was either left blank or
spiked with both analyte and IS.

• The lowest limit of quantitation (LLOQ) was assessed in the

same plasma lots used for specificity.

• The determination of LLOQ was based on the criteria that the

deviation of the measured concentrations should NMT 20%
from the nominal concentration and that the signal to noise
ratio be ≥5.
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 LINEARITY

• Linearity was evaluated using plasma samples spiked with

both SIM and SIMA at concentration ranges 2.5–500 ng/mL
and 5–500 ng/mL, respectively.

• The internal standard LOV, concentration was 50 ng/mL in all

calibration standards.

• Three calibration curves were prepared and analyzed by

plotting area ratios of analyte to internal standard against the
concentration of each calibration standard.

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 PRECISION AND ACCURACY

• The intra-day precision and accuracy was evaluated at

three 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

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 .

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 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 .

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 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.

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 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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Thank You For Your Attention

03/17/2023 Analytical Method Validation 65