Validation of HPLC method for
analysis of plasma drug
concentration
Muhammad Usman
[email protected]
HPLC
• High Performance Liquid Chromatography
(HPLC) is one of the most widely used
techniques for identification, quantification
and purification of mixtures of organic
compounds.
• Components of a mixture are partitioned
between an adsorbent (the stationary
phase) and a solvent (the mobile phase).
Analytical method
• An analytical technique is a method that is
used to determine the concentration of a
chemical compound or chemical element.
Method Validation
• Validation of analytical procedures is the
process of determining the suitability of a given
methodology for providing useful analytical data.
• Method validation is the process of
demonstrating that analytical procedures are
suitable for their intended use and that they
support the identity, strength, quality, purity
and potency of the drug substances and drug
products
Method Validation
• Method validation is primarily concerned with:
identification of the sources of potential errors
quantification of the potential errors in the method
• It describes the performance characteristics of an assay in
mathematical and quantifiable terms
Examples of Methods That Require
Validation
• Chromatographic Methods - HPLC, GC, TLC, GC/MS, etc.
• Spectrophotometric Methods – UV/VIS, IR, NMR,
• Capillary Electrophoresis Methods - Zone, Isoelectric
Focusing
• Particle Size Analysis Methods - Laser, Microscopic, Sieving
etc.
Guidelines
Validation
Parameters
» Selectivity
» Calibration Curve
» Lower Limit of Quantification (LLOQ)
» Carry Over
» Accuracy and Precision
» Stability
Muhammad Usman 24-May-21
Selectivity
• Protocol:
o At least 6 individual sources of the appropriate blank matrix
o Individually analyzed and evaluated for interference
o Response should less than 20% of the lower limit of
quantification
Selectivity
Protocol:
oAt least 6 individual sources of the appropriate blank matrix
oIndividually analyzed and evaluated for interference
oResponse should less than 20% of the lower limit of
quantification
600
Blank
BlankPlasma
Plasma
500
Vancomycin
Response (mV)
Vancomycin
400
Response (mV)
Blank Plasma
Meropenem
Blank Plasma
Vancomycin
Meropenem
300 Imipenem
Imipenem
Serum Sample
200
100
0
6 7 8 9 10 11 12
Time (min)
Calibration curves
• Protocol:
• A minimum of six calibration concentration levels should be
used, in addition to the blank sample and a zero sample.
• Each calibration standard can be analyzed in replicate.
• The calibration curve parameters should be reported (slope
and intercept in case of linear fit).
Standard Calibration Curve
600000
500000
y = 9155.2x + 1181.4
R² = 0.9998
Peak Area (mAu)
400000
300000
200000
100000
0
0 10 20 30 40 50 60
Conc. (mg/L)
Calibration curves
• Back Calculations
• The back calculated concentrations of the calibration
standards should be within ±15% of the nominal value, except
for the LLOQ for which it should be within ±20%.
• At least 75% of the calibration standards, with a minimum
of six calibration standard levels, must fulfil this criterion.
Back calculated concentrations of calibration standards
Level Mean (± S.D) Mean %age
n=5 Conc. found found C.V %
(mg/L)
60 mg/L
60.1 ± 0.14 100.3 % 0.23 %
30 mg/L
29.6 ± 0.30 98.2 % 1.03 %
10 mg/L
10.5 ± 0.19 104.2 % 1.81 %
1 mg/L
0.94 ± 0.05 98.8 % 4.90 %
0.5 mg/L
0.43 ± 0.06 94.5 % 13.7 %
0.25 mg/L
0.15 ± 0.05 78.6 % 25.1 %
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 an acceptable accuracy and precision.
LLOQ = 0.5 mg/L
Accuracy & Precision
• Accuracy:
The accuracy of an analytical method describes the
closeness of the determined value obtained by the
method to the nominal concentration of the analyte
(expressed in percentage)
• Precision:
The precision of the analytical method describes the
closeness of repeated individual measures of analyte.
Precision is expressed as the coefficient of variation (CV).
i) Within run Accuracy and Precision
ii) Between run Accuracy and Precision
With-in run Accuracy and Precision:
• Protocol:
• Within-run accuracy and precision should be determined by analyzing
in a single run a minimum of 5 samples per level at a minimum of 4
concentration levels which are covering the calibration curve range:
i) Lower Limit of Quantification (LLOQ)
ii) Low Quality Control (LQC) within three times the LLOQ
iii) Medium Quality Control (MQC) around 50% of the calibration curve range
iv) High Quality Control (HQC) at least at 75% of the upper calibration curve range.
• For accuracy The mean concentration should be within ±15% of the
nominal values for the QC samples, except for the LLOQ which should be
within 20% of the nominal value.
• For Precision The within-run CV value should not exceed 15% for the QC
samples, except for the LLOQ which should not exceed 20%.
Samples
• Calibration • Quality Control
standard Solutions samples
60 mg/L 50 mg/L (HQC)
30 mg/L 25 mg/L (MQC)
10 mg/L
2 mg/L (LQC)
5 mg/L
0.5 mg/L (LLOQ)
1 mg/L
0.5 mg/L
Results
Mean (± S.D) Mean
Level Conc found %age found C.V %
n=5 (mg/L)
50 mg/L
(HQC)
25 mg/L
(MQC)
2 mg/L
(LQC)
0.5 mg/L
(LLOQ)
Between run Accuracy and
Precision
• Protocol: For the validation of the between-run accuracy,
LLOQ, low, medium and high QC samples from at
least three runs analyzed on at least two different
days should be evaluated.
• For accuracy The mean concentration should be within
±15% of the nominal values for the QC samples,
except for the LLOQ which should be within 20% of the
nominal value.
• For precision The between-run CV value should not
exceed 15% for the QC samples, except for the LLOQ
which should not exceed 20%.
Stability
• Freeze and Thaw stability
• Long term stability
• Stability of processed sample under storage conditions
• On-instrument stability of processed sample
Freeze and Thaw stability
• Protocol:
• The QC samples are stored and frozen in the freezer at the
intended temperature and thereafter thawed at room or
processing temperature.
• After complete thawing, samples are refrozen again
applying the same conditions.
• At each cycle, samples should be frozen for at least 12
hours before they are thawed.
• The number of cycles in the freeze-thaw stability should
equal or exceed that of the freeze/thaw cycles of study
samples.
Freeze and Thaw stability
• The QC samples are analyzed against a
calibration curve, obtained from freshly
spiked calibration standards, and the
obtained concentrations are compared to the
nominal concentrations.
• The mean concentration at each level should
be within ±15% of the nominal concentration.
Freeze and Thaw stability
Samples
• 50 mg/L (HQC) 3ml
• 0.5 mg/L (LQC) 3ml
Storage Temperature:
• -20ºC
Five runs after four freeze and thaw cycles
Results
Level Mean ±S.D Mean
n=5 Conc. found %age found C.V %
(mg/L)
50 mg/L
(HQC)
2 mg/L
(LQC)
Stability of processed sample under
storage conditions
• HQC (50 mg/L) and LQC (2 mg/L) are
prepared and stored in refrigerator at 2-8ºC
after extraction.
• After 24 hours these samples are injected to
HPLC and concentration is calculated from
calibration curve drawn through freshly
prepared calibration standard solutions.
Level Mean ±S.D Mean
n=5 Conc. found %age found C.V %
(mg/L)
50 mg/L
(HQC)
2 mg/L
(LQC)
Short term and long term stability
• HQC (50 mg/L) and LQC (2 mg/L) are prepared and freezed at -
20ºC.
• For short term stability the samples are thawed at room
temperature and extraction is performed.
• For long term stability the samples are thawed after 3 months or 6
months and extraction is performed
• The concentration is quantified through freshly prepared
calibration curve.
• The mean concentration at each level should be within ±15% of
the nominal concentration and CV% should be ≤ 15%.
