Bioanalytical Aspect

in BE Study

Yantirta Indra Kurniawan

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 Competencies & Compliance in BA/BE

Competent Competent
Clinical Analytical
Resources Resources

Subjects / Biological Bio-analytical

Healthy Volunteers Procedures
Samples

Good Clinical Practice (ICH- Good Laboratory Practice

GCP E6) (GLP) – ISO/IEC 17025
Ethics Committee Approval
Comply to Helsinki Declaration
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 Guideline in Bioanalytical
 Guidelineon Bioanalytical Method
Validation, EMeA (European
Medicaines Agency), 21 July 2011
 Bioanalytical
Method Validation,
Guidance for Industry, US-FDA, May
2018

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 Bioanalytical Method Validation
• Selectivity
• Lower Limit of Quantification (LLOQ)
• Calibration Curve
• Accuracy, precision, and recovery
• Carry over
• Stability
• Short term
• Long term
• Freeze and Thaw
• Post preparative
• Reinjection (if necessary)
• Matrix effect (for MS detector only)
• Cross over of Plasma anti coagulant (if necessary)

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 Selectivity
• Each of 6 blank should be tested for interference, and selectivity
should be ensured at the lower limit of quantification (LLOQ).
%Interference of Analyte and IS should be < 20% and < 5% to
LLOQ, respectively
• If method of simultaneous determination of multiple analytes,
must demonstrate that analytes do not interfere with each other

Example – peak interference

Analyte of interest

Interfering analytes

Equilab Presentation 5
 Lower Limit of Quantification (LLOQ)
• The lower limit of quantification (LLOQ) is the lowest
concentration of analyte in a sample which can be
quantified reliably, with acceptable accuracy and
precision
• < 1/20 Cmax (less than 5% of Cmax)
• Normally 5-fold blank noise
• within- run accuracy and between-run accuracy
• within- run precision and between-run precision
• N = 5 or N > 5 for between
• < 20% nominal value

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Lower Limit of Quantification
(LLOQ)

LLOQ

Noise

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 The calibration curve (1)
• The relationship between instrument response and known
concentrations of the analyte.
• The calibration curve should be prepared in the same
biological matrix with known concentrations of the analyte

 Define concentration range (LLOQ/ULOQ)

 Minimum of 6 calibration standards excluding blank/zero sample)
 Simple relationship without blank and zero
 Calibration curve parameters and back-calculated values reported
 Back calculated concentration: accuracy < 15% (except LLOQ)
 > 75% should comply, with a minimum of 6 standards
 Minimum N=3 curves

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 The calibration curve (2)
 criteria not met: reject calibration standard and re-evaluated
standard curve
 recommended using freshly prepared curves during validation

continuous &
reproducible

Equilab Presentation 9
 Accuracy

levels: LLOQ, LQC, MQC, HQC 4

.obtained vs. nominal conc

1 run/within-accuracy between-accuracy
N=5 3 runs/minimum 2 days
<15%,
except LLQC (<20%)
 Precision

closeness of repeated values

expressed as CV: S.D./mean

same QC samples used as for determination of

accuracy
 Precision

levels: LLOQ, LQC, MQC, HQC 4

.obtained vs. nominal conc

1 run/within-precision between-precision
N=5 3 runs/minimum 2 days
<15%,
except LLQC (<20%)
 Recovery
Recovery need not be 100%, but recovery of an
analyte and Internal standard (IS) should be
consistent and reproducible

Extracted samples at Low, Medium, and High

QC concentrations versus extracts of blanks
spiked with the analyte post extraction (US-FDA
2018)

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 Stability

!Stability assessed prior sample analysis

Required data freshly prepared cal.curve

Stock /working solutions
 Freeze and thaw stability
 Short term stability /
bench top stability
 Long term stability <15% nominal
 Post preparative stability
If applicable:
Reinjection stability

freeze/thaw: > 12h

 Long term stability:

bracketing approach applicable

study samples only for investigational issues

multi-analyte: should not interfere

results available before report is issued

Stock/working solutions:

bracketing approach applicable

stable-isotope labelled IS!!

 Matrix effect
the direct or indirect alteration or interference in
response due to the presence of unintended analytes
(for analysis) or other interfering substances in the
sample
applicable to mass spectrometric methods

6 lots of (individual) blank matrix; not pooled ;

<6 lots may be acceptable (rare matrix) but matrix
effects still be investigated

recommended haemolysed and hyperlipidaemic

and special populations if applicable
 Cross over of Plasma Anticoagulant
In case anticoagulant of plasma used in study
samples is different with plasma used in
method validation, cross over test should be
performed

Cross over effect is investigated by perform the

selectivity, accuracy, and precision test using 2
different plasma anticoagulants

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 QC Samples in Method Validation

 QC Samples :
 LLOQ QC
 Low QC : 3x LLOQ
 Mid QC : 30-50% of ULOQ
 High QC : > 75% of LLOQ
 For Accuracy and precision Runs :
Four QCs, including LLOQ, low, mid, and high from at least
five replicates in at least three runs
 For Other Validation Runs (US-FDA 2018):
Low, Mid, and High QCs in duplicates

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Bioanalysis

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 Challenges in Bioanalysis
Reference Standard
QC Samples
Audit Trail & Data Integrity
Incurred Sample Reanalysis (ISR)
Reanalysis of Sample

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 Reference Standard
• Reference standards used during the validation and study
sample analysis should be obtained from an authentic and
traceable source :
• Compendial standard
• Commercial available standard
• Characterised standards prepared in-house or by an external
non-commercial organisation
• A certificate of analysis (CoA) is required to ensure purity and
provide information on storage conditions, expiration date and
batch number of the reference standard
• When mass-spectrometry (MS) detection is used in the
bioanalytical method, a stable isotope-labelled IS is
recommended to be used whenever possible

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 QC Samples (1)
• QC Solution should be prepared separately from stock
solution
• Distribution and position of QC Samples in analytical
run should be divided over the run
• At least 2 QC sample levels should fall within the range
of concentrations measured in study samples 
Additional QC should be applied if samples only
disperse at one area of calibration range
• The overall (mean) accuracy and precision of the QC
samples of all accepted runs should be calculated at each
concentration level and reported in the analytical report. In
case the overall mean accuracy and precision exceeds
15%, this should lead to additional investigations justifying
this deviation. In the case of bioequivalence trials it may
result in the rejection of the data
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QC Samples (2)

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QC Samples (3)

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Audit Trail & Data Integrity (1)

gap should be
explained

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 Audit Trail & Data Integrity (2)
• Electronic Audit Trail should be provided for every
batch of Analytical Run

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 Audit Trail & Data Integrity (3)
• Manual integration can be captured in Electronic Audit Trail
automatically  Reason should be explained

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Incurred Sample Reanalysis (1)
Used to measure the reproducibility of study
sample concentrations
The total number of ISR samples should be
depending on the total number of samples
(EMA : 10% for total samples <1000 or 5% for
total samples > 1000, US-FDA : The first 1000
samples = 10%, 5% of the remaining samples)
Samples should cover the entire PK profile
(Cmax concentrations and elimination
concentration)
 Incurred Sample Reanalysis (2)
• ISR should be performed in separate runs at
different days (after all study samples
completed or throughout the conduct of study)
• Two-thirds (67%) of the repeated sample results
should be within 20% of the mean of 2 values

• Large differences between results may indicate

analytical issues and should be investigated

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 Reanalysis of Sample
• Rejection of an analytical run because the run did not fulfil the
acceptance criteria with regard to accuracy of the calibration
standards and/or the QC samples
• Internal standard response significantly different from the
response for the calibration standard and QC samples, if
such criteria have been pre-defined in a SOP, e.g. -50% to
+50%
• Improper sample injection or malfunction of equipment,
• The obtained concentration is above the ULOQ or below the
run’s LLOQ, in runs where the lowest standard sample has been
rejected from a calibration curve, resulting in a higher LLOQ
compared with other runs
• Identification of quantifiable analyte levels in pre-dose samples
or placebo sample
• Poor chromatography
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Thank you
yantirta.kurniawan@
equilab-int.com

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