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)

Analytical Method Validation 2

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”

Analytical Method Validation 3
 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.
Analytical Method Validation 4
 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.

Analytical Method Validation 5

 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.

Analytical Method Validation 6

 VALIDATION
CHARACTERISTICS

Accuracy Precision Range

Specificity Linearity

Analytical Method Validation 7

 VALIDATION
CHARACTERISTICS

Detection Quantitation
Robustness
Limit Limit

System
Ruggedness
suitability

Analytical Method Validation 8

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.

Analytical Method Validation 9
DATA ELEMENTS REQUIRED
FOR ASSAY VALIDATION

Analytical Method Validation 10

 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).
Analytical Method Validation 11
 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.

Analytical Method Validation 12

 ACCURACY DATA SET

Amount Amount
Percent Recovery
Added Found (mg)
(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

Analytical Method Validation 13

 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.

Analytical Method Validation 14

 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).

Analytical Method Validation 15

 (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.

Analytical Method Validation 16

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.
Analytical Method Validation 17
 3.REPRODUCIBILITY

 Ability reproduce data within the predefined precision

 Determination: SD, RSD and confidence interval

Analytical Method Validation 18

ACCURACY AND PRECISION

Accurate & Accurate & Inaccurate & Inaccurate &

precise imprecise precise imprecise

Analytical Method Validation 19

 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.

Analytical Method Validation 20

 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.
Analytical Method Validation 21
 SPECIFICITY
 Determination -
 If impurity degradation product standards are
or
unavailable, compare the test results of samples containing
impurities or degradation products to a second well-
characterized procedure a Pharmacopeial or other
(e.g., validated procedure).

 The ICH documents that when chromatographic

procedures
state are used, representative chromatograms should be
presented to demonstrate the degree of selectivity, and peaks
should be appropriately labeled.
8/19/2015 Analytical Method Validation 22
 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.

Analytical Method Validation 23

 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.]

Analytical Method Validation 24

 QUANTITATION LIMIT

 Definition-
It is the lowest amount of analyte in a sample that can
be determined with acceptableprecision 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.

Analytical Method Validation 25

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

Analytical Method Validation 26
 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.

Analytical Method Validation 27

 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

Analytical Method Validation 28

 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.

Analytical Method Validation 29

 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
Analytical Method Validation 30
 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.

Analytical Method Validation 31

 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.
Analytical Method Validation 32
• - flow rate.
 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
Analytical Method Validation 33
 SYSTEM SUITABILITY
ICH
 ICH
 Definition: evaluation of electronic,
equipment, analytical operations and
 samples as a whole
Determination: repeatability, tailing factor (T), capacity
factor (k’), resolution (R), and theoretical Plates (N)

Analytical Method Validation 34

 SYSTEM SUITABILITY
USP
• USP 23 <621> :
Parameters Recommendations
K In general k ≥ 2.0
R > 2, between the peak of
interest 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)
Analytical Method Validation 35
RESEARCH PAPERS
1

Analytical Method Validation 36

 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)

Analytical Method Validation 37

 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.

Analytical Method Validation 38

 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

Analytical Method Validation 39
 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.

Analytical Method Validation 40

 PRECISION

• Precision the assay was determined in relation to

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

Analytical Method Validation 41

TABLE 2 :INTRA-DAY, INTER-
DAY PRECISION (n = 6)

Analytical Method Validation 42

 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.

Analytical Method Validation 43

 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.

Analytical Method Validation 44

ROBUSTNESS

Analytical Method Validation 45

RESEARCH PAPERS
2

Analytical Method Validation 46

 VALIDATION
CHARACTERISTICS

Analytical Method Validation 47

• 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.

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.

Analytical Method Validation 49

RESEARCH PAPERS
3

Analytical Method Validation 50

• 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.
Analytical Method Validation 51
 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))
Analytical Method Validation 52
 VALIDATION
CHARACTERISTICS

Analytical Method Validation 53

RESEARCH PAPERS
4

Analytical Method Validation 54

 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
Analytical Method Validation 55
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.
Analytical Method Validation 56
 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.
Analytical Method Validation 57
 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

Analytical Method Validation 58

 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 .

Analytical Method Validation 59

 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 .
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.
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.

Analytical Method Validation 62

 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

Analytical Method Validation 63

 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)

Analytical Method Validation 64

Thank You For Your Attention

Analytical Method Validation 65