Understanding Bioanalytical Method Validation in a Regulatory Perspective

Dr. Ishaq B Mohammed M. Pharm., Ph.D., FAGE.
Professor, Department of Pharmaceutical Analysis,
Santhiram College of Pharmacy, Nandyal, Kurnool Dist. 518501. AP. India.
drbmdishaq@gmail.com
AICTE sponsored STTP program on
"Green Chemistry and advanced analytical techniques of Pharmaceuticals
and product development aspects as per the regulatory requirements“
11th Feb 2021
http://srcpnandyal.com/

Bioanalysis in the drug discovery and development
 The lead optimization/selection, confirmation, and testing process for
new drug candidates are well-defined as a series of activities.
 Broadly, these can be split into discovery, lead optimization, and
preclinical development, through to clinical evaluation (Phases I to IV).
Product life-stage management and bioequivalence
studies
 For marketed drugs, the application of PK and bioanalysis is
predominantly focused on the evaluation of line-extension products,
such as controlled-released formulations, and on the evaluation of
generic formulations.
 In a bioequivalence study, the aim is to demonstrate that two
formulations of an entity have the same bioavailability, to within certain
limits. Regulatory requirements (e.g. U.S. FDA, EMEA guidelines) for
such studies are explicit.

Overview of Bioanalytical Method Development & Validation

Bioanalytical method involves determination of concentration of
drugs or its metabolites or both in biological matrix such as plasma,
serum, urine etc.
A compound can often be measured by several methods and the
choice of analytical method involves many considerations, such as:
chemical properties of the analyte, concentrations levels, sample
matrix, cost of the analysis, speed of the analysis, quantitative or
qualitative measurement, precision required and necessary
equipment.
The analytical chain describes the process of method development
and includes sampling, sample preparation, separation, detection and
evaluation of the results.
Bioanalytical Method Development

The two most common biological samples used in analysis are:
Plasma/serum, to determine the
pharmacokinetic profile of the analyte and
thus the drug clearance, half-life, and
bioavailability.
 Urine, to determine the renal elimination
profile of the compound
The sample preparation process fulfills three major roles:
Removal of protein-related materials that may contaminate the
chromatography column
 Elimination of endogenous compounds, such as phospholipids that
are the major cause of ion suppression/enhancement in LC/MS
 Concentration to increase assay sensitivity

Common techniques for sample preparation are
• Protein precipitation
• Solid-phase extraction and
• Liquid/ liquid extraction
Protein precipitation

The changing landscape of chromatography
technology in bioanalysis

Reference Standards
•A highly characterized material (Drugs) suitable to test the identify,
strength, quality and purity of the substances for the pharmaceutical
and medicinal use .
•Generally available from USP, EP, BP and IP.
•Having purity of more than 99.5%.
Working Standards
•A drug susbtance of established quality and purity, which will be
characterized by comparing to the reference standard.
•Available from commercial source (Clearsynth).
Internal Standards
•A known concentration of a substance which will be added in a
specified quantities to every sample that is being analysed.
• Added to the analyte sample to check the variability due to analyte
loss during sample processing.

Internal Standards
Stable Isotope labeled IS
Structural related compound
(Non labeled)
Metronidazole
Metronidazole - D4 Ornidazole
Advantages:
Better accuracy and precision
Disadvantages:
More Expensive

Calibration Curve Standards (CC Std)
Relationship between instrumental response and drug standard within
intended quantification range.
Min. 6-8 non-zero standards along with blank and zero.
 Blank: Processed Matrix (Plasma or serum) without analyte.
Zero: Processed Matrix (Plasma or serum) with IS only.
How to decide range of CC
Cmax
Lower limit
(LLOQ or STD-1)
At least 5% of Cmax or
less
Upper limit
(ULOQ or STD-8)
1.5 to 2 times of Cmax
For ex: if Cmax of a drug is 50 ng/ml.
LLOQ: 1 or 5 ng/ml (1 ng/ml)
ULOQ : 100 ng/ml
Range of study is:
1 to 100 ng/ml

Quality Control Std
(QCs)
LLOQQC LQC MQC HQC
Half of LLOQ Three times of
LLOQ
Around 30-50%
of ULOQ
Atleast 75% of
ULOQ
0.5 ng/ml 3 ng/ml 50 ng/ml 85 ng/ml

CC & QC’s
STD-1 STD-2 STD-3 STD-4 STD-5 STD-6 STD-7 STD-8
LLOQ ULOQ
1 ng/ml 2 ng/ml 4ng/ml 10 ng/ml 20 ng/ml 30 ng/ml 70 ng/ml 100 ng/ml
LQC
3 ng/ml
LLOQ QC
0.5 ng/ml
MQC
50 ng/ml
HQC
85 ng/ml
Acceptance Criteria for CC STDs
Back calculated concentration of CC STDs should be in between± 15%
of nominal value except for LLOQ which will be ± 20% .
Atleast 75% of CC STDs should pass above criteria.
Area in blank at Rt of analyte should be <20% of area in LLOQ.
Area in blank at Rt of IS should be <5% of area in IS in LLOQ.

Acceptance Criteria for QC STDs
Atleast 67% of QCs should be ± 15% of nominal values.
 ≥ 50% of QCs at each level should be ± 15% of nominal values.
CC & QC should be prepared from separate stock solutions in same
matrix as the matrix of intended study sample.
Preparation of CC and QC STDs
1 mg/ml 10,00,000 ng/ml Stock solution
10,000 ng/ml Working STD
0.1 ml of stock is diluted to 10 ml with diluent.
1000 ng/ml Serial STD
1 ml of Working STD is diluted to 10 ml with diluent.
100 ng/ml STD – 8 in plasma
1 ml of Serial STD is diluted to 10 ml with Plasma.
N1 V1 = N2 V2

Definition of Bioanalytical Method 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.
Bio analytical Method Validation Parameters
Selectivity
Specificity
Carry over
Precision and Accuracy
Robustness and Ruggedness
Matrix effect
Recovery
Dilution integrity
Reinjection reproducibility
Stability

Selectivity
Is the extent to which the method can determine analyte of interest
and IS in the matrix without any interference from the matrix
components.
Purpose
To prove that our method can selectively determine our analyte of
interest and IS only.
To prove that our method is selective even if we use different plasma
lots.
Procedure
6 different lots of plasma (normal).
1 Hemolysed and 1 Lipolysed plasma lots.
 Prepared Blank and LLOQ (1ng/ml) from each plasma lot.
Acceptance criteria
Area in all blank samples
At Rt of analyte <20% of area of LLOQ.
At Rt of IS <5% of area of LLOQ.

Plasma Lot -1
Sample Area
Blank 10
LLOQ 1000
20% => 200
Pass
Plasma Lot -2
Sample Area
Blank 250
LLOQ 1000
20% => 200
Fail

Specificity
Ability of a method to assess the analyte of interest in the presence
of other components (eg: impurities, matrix components and other co-
medications).
Purpose
To prove that our method is specific to identify analyte of interest in
the presence of other plasma components or co-medicaiton is present
without any interferences.
Commonly used medication like paracetamol, Ibuprofen,
Chlorpheniramine, nicotine and caffeine.
Procedure
Spike blank plasma lots (6 Normal, 1 hemolysed and 1 lipemic) with
Cmax co-medication.
Prepare six LLOQ samples in each plasma lots.
Preapre six LQC samples and spike with Cmax co-medication.

IBU Analyte
Blank LLOQ LQC
Repeat all with all other plasma sample lots as well
Acceptance criteria
Accuracy of LQC samples should be within ± 15% of nominal value.

Carry over
Is the appearance of analyte signal in blank sample after the analysis
of sample with high analyte concentration.
Reason
Inappropriate sample dilution or diluent
Inappropriate mobile phase or mobile phase pH
Inappropriate washing duration.
Remedy
Extend washing duration.
Change mobile phase or diluent.
Procedure
Inject processed blank sample after High QC or ULOQ sample. Repeat
the procedure for six times.
Acceptance criteria

Precision and Accuracy
Precision
Closeness of agreement between series of measurements obtained
under prescribed conditions.
Precision =
Standard Deviation
Mean
X 100
Accuracy
Degree of closeness of determined value to the nominal or know value.
Accuracy=
Determined value
Nominal Value
X 100

Requirements
Freshly prepared CC and QC STDs
Atleast 3 runs required on atleast two different days.
Each Batch of PA contains: 1 Blank, 1 Zero, CC STDs (1-8), QC
(LLOQQC, LQC, MQC & HQC).
Atleast 5 samples per each QC level as per regulatory guidelines.
(inhouse: 6 samples).
Within Run P&A
Should be determined by analysing in a single run at minimum 5
samples of each QC at four QC levels.
Between Run P&A
Should be determined by analysing at least 3 runs at minimum of 5
samples of each QC at four QC levels at least for two different days.
Acceptance criteria for Calibration Standards
All CC STD (2 to 8) should be ±15% of nominal values.
 for STD-1 (LLOQ) should be ±20% of nominal values.
Atleast 75% and a minimum of 6 STD should meet above criteria.

Acceptance criteria for Blank & Zero Samples
Acceptance criteria for QC’s
Accuracy:
 The mean conc. of each level of QCs (L,M & H) should be within
±15% of nominal values.
For LLOQQC it should be within ±20% of nominal values.
Precision:
Co-efficient of Variance (%CV) of all QC’s (L, M &H) should not
exceed 15%
For LLOQQC it should not exceed 20% .
Atleast 67% of overall QC samples and 50% at each QC level should
comply with accuracy acceptance criteria in each run.

Example:
LLOQ QC: (0.500 ng/ml) – Nominal concentration
For LLOQQC acceptance criteria is ±20% of nominal concentration.
Therefore, 0.400 ng/ml to 0.600 ng/ml is allowed.
Sample No. LLOQQC Result
1 0.497 Pass
2 0.492 Pass
3 0.541 Pass
4 0.602 Fail
5 0.485 Pass
6 0.453 Pass
Mean 0.5117 --
STDEV 0.0525 --
%CV 10.26 % Pass (±20% )
%Recovery 102.33 % Pass (±20% )
%CV =
SD
Mean
X 100
%Accuracy =
Determined Value
Nominal Value
X 100
Atleast 50% of QC samples should pass.

Robustness & Ruggedness
Ability of an analytical method remain unaffected by small variations
in method parameters like column, instrument, analyst etc.
Evaluated by changing atleast one of the factors during one of the PA
batchs.
Acceptance criteria
As per P & A
Matrix Effect
Direct or indirect alteration of interferences in response due to the
presence of unintended analytes or other interfering substances in the
sample (Plasma, Blood and serum).
In simple – effect of matrix on the response of the method.
Matrix effect causes suppression or enhancement of ionization of
analyte, resulting in increase or decrease in analyte response.

Std solvent without matrix Set -1
A
A
A
A
A
A
H+
H+
H+ H+
H+
H+
H+X-
AH+
AH+
AH+
AH+
AH+
AH+
6 x [AH+]
M
A
A
A
M
A
H+
H+
M H+
H+
H+
H+X-
AH+
MH+
MH+
AH+
MH+
MH+
2 x [AH+]
How to Minimize
More selective extraction procedure.
Changing buffer and extraction solvent.
Use of stable labeled IS.
Std solvent with matrix – Set 2

Procedure
 8 different matrix lots (plasma)
Set – 1 & Set – 2 at LQC, MQC and HQC
Std solvent Spiked post extracted
Without plasma sample with matrix
6 Normal 1 Hemolysed 1 Lipemic
Matrix Factor =
Peak area in presence of matrix
Peak area in absence of matrix
If Matrix Factor is
 More than 1 => Ion Enhancement = > Increased response.
 Less than 1 => Ion Suppression = > Decreased response.
 1 => No Matrix Effect

 Matrix effect is calculated for both analyte and IS.
IS normalized matrix factor =
MF of analyte
MF of IS
Acceptance criteria
%CV of IS normalized MF from all lots should not be greater than
15%.
Recovery
Extraction efficiency of an analytical process reported as % of know
amount of analyte recovered through sample extraction.
Recovery need not be 100% always, but it must be consistent and
reproducible for analyte and IS.
Procedure
Determined at 3 levels – LQC, MQC and HQC
 6 replicates of extracted LQC, MQC and HQC.
6 replicates of post spiked LQC, MQC and HQC.

% Recovery =
Area of Extracted sample
Area in Post spiked sample
X 100
Acceptance criteria
%CV of calculated .
% recover should not be more than 15% at each QC level as well as
across all QC levels.
Dilution Integrity
This experiment is carried out to assess the ability of the method to
quantify the analyte and yield accurate and precise results after sample
dilution.
Range of CC: 0.500 to 100.00 ng/ml.
 while study, If any sample(s) goes beyond the range of CC then such
sample(s) will be diluted so as to fit in our CC range.

Procedure
Prepare six QC (Dilution QC) with concentration higher than ULOQ of
original concentration by at least two times
Eg: If ULOQ = 100ng/ml and CC Range: 0.5 to 100 ng/ml, dilution QC
shall be 200 ng/ml.
In order to fit in the range of our CC 200ng/ml will be diluted for 4
times to get 50 ng/ml.
Six replicates of 50ng/ml will be injected.
Acceptance criteria
%Accuracy and Precision (%CV) should be within 15%.
%CV =
SD
Mean
X 100
%Accuracy =
Determined Value
Nominal Value
X 100

Reinjection Reproducibility
To assess ability of the method to quantify analyte accurately and
precisely upon reinjection.
If any sample is reinjected we must able to get same result.
If any sample results goes wrong or any other error due to LCMS or
HPLC etc then under those circumstances that injection will be
reinjected.
Procedure
Inject one of the previously accepted P & A batch (P & A will be done
for 3 batches in two days, so any of these batches which has passed will
be reinjected and assessed for reinjection reproducibility)
Acceptance criteria
As per the P & A.

Stability
The chemical stability of an analyte in a given matrix under specific
conditions for given time intervals is assessed in several ways.
Pre-study stability evaluations should cover the expected sample
handling and storage conditions during the conduct of the study,
including conditions at the clinical site, during shipment, and at all
other secondary sites.
Drug stability in a biological fluid is a function of the storage
conditions, the physicochemical properties of the drug, the matrix,
and the container system.
Stability testing should evaluate the stability of the analytes during
sample collection and handling, after long-term (frozen at the
intended storage temperature) and short-term (bench top, room
temperature) storage, and after freeze and thaw cycles and the
analytical process.

Freeze and Thaw Stability
During freeze/thaw stability evaluations, the freezing and thawing of
stability samples should mimic the intended sample handling conditions
to be used during sample analysis. Stability should be assessed for a
minimum of three freeze-thaw cycles.
Bench-Top Stability
Bench top stability experiments should be designed and conducted to
cover the laboratory handling conditions that are expected for study
samples.
Long-Term Stability
The storage time in a long-term stability evaluation should equal or
exceed the time between the date of first sample collection and the
date of last sample analysis.

Stock Solution Stability
The stability of stock solutions of drug and internal standard should be
evaluated. When the stock solution exists in a different state (solution
vs. solid) or in a different buffer composition (generally the case for
macromolecules) from the certified reference standard, the stability
data on this stock solution should be generated to justify the duration
of stock solution storage stability.
Processed Sample Stability
The stability of processed samples, including the resident time in the
auto sampler, should be determined.
Procedure
6 Replicates of HQC and LQC will be injected after processing for
stability along with 6 replicates of freshly prepared samples.
Acceptance Criteria
% Mean Stability in all QCs should be ±15% of nominal values.
%Mean Stability =
Mean Determined stability
X 100
Mean Determined sample
Stability
X
Nominal Con. Of stability samp
Nominal Con. Of compari samp

Food and Drug Administration, FDA, Guidance for Industry: Bioanalytical Method
Validation, Rockville, MD: US Department of Health and Human Services, Food and Drug
Administration, Center for Drug Evaluation and Research, 2013.
Peters F.T., Review: Bioanalytical method validation – How, how much and why, Department
of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and
Toxicology, University of Saarland.
Pranay W, Bioanalytical Method Development –Determination of Drugs in Biological Fluids
2010.
Skoog DA, West DM, Holler FJ, Crouch SR. Fundamentals of Analytical chemistry. 18th ed.
Thomson Asia pvt Ltd. Chapter 1. The nature of analytical chemistry. 2004; 2-5 & 973-996.
ICH, Validation of analytical procedure, International conference on Harmonization, IFPMA,
Q2B Validation of Analytical Procedures: Methodology
References

Understanding Bioanalytical Method Validation in a Regulatory Perspective

Understanding Bioanalytical Method Validation in a Regulatory Perspective

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