Bioanalytical validation.pptx

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©2023 Waters Corporation COMPANY CONFIDENTIAL
Understanding Bioanalytical method validation in a regulatory
Perspective

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Overview of Bioanalytical Method development & Validation

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Two most common biological samples used in analysis

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common techniques for sample preparation
 Protein precipitation
 Solid phase extraction
 Liquid / liquid extraction

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Drug reference standard
• A drug reference standard or pharmaceutical reference standard is a highly characterized material
suitable to test the identity, strength, quality and purity of substances for pharmaceutical use and
medicinal products.
• Generally available from USP IP BP EP
• Having purity more than 99.5%
Drug Working standard
• A drug substance of established quality and purity as shown by comparison to the reference
standards.
• Available from commercial source like Clearsynth.
internal standard
• An internal standard is a substance added in a known amount to standards, samples, and blanks during
an analysis.
• Added to the analyte sample to check the variability due to analyte loss during the sample processing.

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internal standard
internal standard
Stable isotope labeled IS
Structural related compound
(non labeled)
Metronidazole d4
Metronidazole Ornidazole
Advantages:
Better accuracy and prison
Disadvantages:
More expensive

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Calibration curve standards (CC Std.)
 Relation between instrumental response and the drug standard within intended quantification range.
 Minimum 6 to 8 standards.
 Blank: processed matrix (plasma or serum) without analyte.
 Zero: processed matrix (plasma or serum) with IS only.
How to decide the range
Cmax
Lower limit
LLOQ or STD-1
Upper limit
ULOQ or STD-8
At least 5% of Cmax or less
At least 1.5 to 2.0 times of Cmax
For example, If Cmax of drug is 50 ng/mL
LLOQ: 1 to 2.5 ng/mL (1.0 ng/mL)
ULOQ: 100 ng/mL
Range of the study is 1.0 to 100 ng/mL

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QUALITY CONTROL STANDEREDS
LLOQQC LQC MQC HQC
Quality control standards
Half of LLOQ
0.5 ng/mL
Three times of LLOQ
3.0 ng/mL
Around 30 to 50% of ULOQ
50.0 ng/mL
At least 75% of ULOQ
85.0 ng/mL

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CC & QC’S
STD-1 STD-2 STD-3 STD-4 STD-5 STD-6 STD-7 STD-8
CC & QC’S
LLOQ
1.0 ng/mL
LLOQC
0.5 ng/mL
2.0 ng/mL 4.0 ng/mL 10.0 ng/mL 20.0 ng/mL 30.0 ng/mL 70.0 ng/mL 100.0 ng/mL
LQC
3.0 ng/mL
MQC
50.0 ng/mL
HQC
85.0 ng/mL
ULOQ
Acceptance criteria for cc STD’s
 At least 75% CC STD’s 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 less than <5% of area in IS in
LLOQ.
Acceptance criteria for QC STD’s
 At least 67% QC should be ± 15% of nominal
value.
 ≥ 50% of QC at each level should be 15% of
nominal value.
 CC and QC should be prepared from the
separate stock solution.

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Bioanalytical method validation parameters
 Selectivity
 Specificity
 Carryover
 Prison and Accuracy
 Robustness Ruggedness
 Matrix effect
 Recovery
 Dilution integrity
 Reinjection reproducibility
 Stability

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Selectivity
 Is the extent to which the method can determine the analyte of interest and IS in the matrix without any interference
from the matrix component.
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
 Six different lots of plasma ( Normal )
 One Hemolysed and one Lipolysed plasma lot
 Prepare Blank and (1.0 ng/mL) LLOQ samples in each plasma lots.
Acceptance criteria
 Area in all blank samples at the RT of analyte <20% area of LLOQ & at the RT of IS <5% area of LLOQ.

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Specificity
 Ability of method to assess the analyte of interest in presence of other components ( eg.impurities,matrix components &
other comedication.
Purpose
 To prove that our method is specific to identify the analyte of interest in the presence of other plasma components or
any comedication is present without any interference.
 Commonly used medication are paracetamol, ibuprofen, chlorpheniramine, Nicotine & caffeine.
Procedure
 Spike plasma lots six normal One Hemolysed and one Lipolysed plasma lot Cmax of comedication
 Prepare six LLOQ samples in each plasma lots
 Prepare six LQC samples and spike with Cmax of comedication
Acceptance criteria
 Accuracy of LQC should be within ±15% nominal value.

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Carry over
 Is the appearance of analyte signal in Blank 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 the washing duration.
 Change the mobile phase or diluent.
Acceptance criteria
 Accuracy of LQC should be within ±15% nominal value.
Procedure
 Inject the processed blank sample after higher concentration QC repeat the procedure for six times.

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Precision and Accuracy
 Freshly prepared CC and QC standard’s
 At least two runs are required in two days
 Each batch of PA contains : 1 Blank, 1 Zero, CC STD’S (1-8) QC (LLOQQC,LQC,MQC,HQC)
Acceptance criteria for CC
 All CC STD (2-8) should be ±15% nominal value.
 For STD-1 (LLOQ) should be ±20% nominal value.
 At least 75% of and minimum of 6 STD. should meet above criteria.
Acceptance criteria for QC
 Accuracy : The mean concentration of each level of QC ( L,M & H) should be within ±15% nominal value.
 For LLOQQC it should be within ± 20% nominal value.
 At least 67% of overall QC & 50 % at each QC level should meet acceptance criteria.

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Robustness & Ruggedness
 Ability of analytical method remains unaffected by the small variations in the method parameters like
column,analyst,instrument.
 Evaluated by changing at least one of the factors during one of the P&A batch.
Acceptance criteria
 As per P&A
Matrix Effect
 Effect of the matrix on the method.
 Matrix effects can be observed either as a loss in response (ion suppression) or as an increase in response (ion enhancement)
 Can be minimized by selective extraction procedure changing extraction solvents and buffers.
 Matrix Factor (MF) = Peak response in presence of matrix ions / Peak response in absence of matrix ions

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Procedure to check matrix effect
 Eight different matrix lot
Six normal one Haemolysed one lipemic
Set 1 set 2
Standard solvent without plasma At LQC,MQC,HQC Spiked post extracted sample with matrix
Matrix factor =
𝑃𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝑖𝑛 𝑝𝑟𝑒𝑠𝑒𝑛𝑠𝑒 𝑜𝑓 𝑚𝑎𝑡𝑟𝑖𝑥
𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝑖𝑛 𝑎𝑏𝑠𝑒𝑛𝑐𝑒 𝑜𝑓 𝑚𝑎𝑡𝑟𝑖𝑥
If matrix effect is =>1 ion enhancement
If matrix effect is =<1 ion Suppression
If matrix factor is =1 No matrix effect

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Recovery
 Extraction efficacy of an analytical process reported as % of known amount of analyte recovered through sample
extraction.
 Recovery need not to be 100% always but it should be consistent and reproducible for analyte and IS
 Determined at three levels LQC,MQC&HQC
 6 Replicates of Extracted LQC,MQC&HQC
 6 Replicates of Post spiked LQC,MQC&HQC
Procedure
Acceptance criteria for QC
Precision (%RSD) of overall % Recovery should be within 15% and % Recovery should be ≥ at individual quality control level
(i.e. LQC, MQC, HQC).
%Recovary =
𝑎𝑟𝑒𝑎 𝑜𝑓 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒
𝑎𝑟𝑒𝑎 𝑖𝑛 𝑝𝑜𝑠𝑡 𝑠𝑝𝑖𝑘𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒
x 100

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Dilution Integrity
 This experiment is carried out to assess the ability of method to quantify analyte and yield accurate and precise result
even after sample dilution
Procedure
 Prepare the six QC (Dilution QC) with concentration higher than ULOQ of the original concentration by at least two times;
 Eg: If ULOQ is 100ng/mL & cc range is 0.5 to 100ng/mL Dilution QC should be 200ng/mL
 In order to fit in the range of our CC 200ng/mL will be diluted for 4 times to get 50ng/mL
 Six replicates of 50ng/mL will be injected
Acceptance criteria
 % Accuracy & Precision should (%CV) should be within 15%.

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Reinjection reproducibility
 To access the ability of method to quantify analyte accurately and precisely upon reinjection.
 If any sample is reinjected, we must get same result.
 If any sample results goes wrong or any other error of LCMS or HPLC etc. then under those circumstances, then that
injection will be reinjected.
Procedure
 Inject previously accepted P&A batch
Acceptance criteria
 As per P&A

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Stability
 The chemical stability of analyte in a given matrix under specific condition in given time intervals is assessed in several
ways.
Freeze and thaw stability
 During the freeze/thaw stability evaluation the freezing and thawing of stability should mimic the intended sample handling
conditions to be used during sample analysis. Stability should be accessed for minimum of 3 freeze thaw cycle.
Bench top Stability
 Bench top stability experiments should be designed and conducted to cover the laboratory handling conditions that are
expected for the study samples.
Long term stability
 Storage time should exceed the time between the date of first sample collection and the date of last sample analysis.

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