Analytical Method Validation [PHA4132]
Development and Validation
of RP-HPLC Method For The
Estimation of Lansoprazole In
Tablet Dosage Form
Kumar et al., 2010
Table of Contents
01 02
Analytical Method AMV Data Output
Development (AMD)
03 04
Challenges Troubleshooting
05
References
01 Analytical Method Development (AMD)
● Use for routine determination of Lansoprazole in tablet dosage
form and its pharmaceutical dosage form (API chemical analysis).
● RP-HPLC method:
○ Separation using mobile phase consist of Disodium
Hydrogen Phosphate Buffer (pH 3) : Acetonitrile [30:70]
○ Column → Phenomenex Luna C8 column with flow rate of 1.0
mL/min using UV detection at 285 nm.
○ Analysis results will validated statistically.
Michellaine (Kumar et al., 2010)
02 AMV Data Output
Linearity and Range Accuracy
● Accuracy was determined at five
● A calibration curve was constructed concentrations and each of the solutions
from five non-zero samples covering was analyzed and the percentage
the total range of 40 - 60 µg/ml. The standard deviation was determined.
peak area was plotted versus the
concentration. ● % RSD: <2
● Linearity regression coefficient : 0.992 ● Acceptance criteria: <2
● Linearity acceptance criteria: ≥ 0.990
● Range: 40-60 µg/ml.
Qastalia (Ravisankar et al., 2021; ICH.,2005)
02 AMV Data Output
System Suitability
● The system suitability was performed by injecting the pre-made sample
solution into the injection port and a chromatogram was recorded. The
injections were repeated 6 times and peak areas were recorded.
● Theoretical plates (N): 4798
● Tailing Factor (T): 1.164
● Acceptance criteria:
- Theoretical plates (N): >2000
- Tailing factor (T): < 2
Qastalia (Panchumarthy & Ravi, 2015)
02 AMV Data Output
Precision Intra day
● % RSD: 0.16
● Precision for repeatability (intra day)
and intermediate (inter-day) was done ● Acceptance criteria: <2
by taking 5 Levels of concentration in
triplicate
● 40, 45, 50, 55, and 60
Inter-day
microgram/milliliter ● % RSD: 0.27
● Acceptance criteria: <2
Rachel (Ravisankar et al., 2021; ICH.,2005)
02 AMV Data Output
Robustness [Link]
● % RSD: 1.29
● Robustness was determined by
varying the mobile phase flow rate and ● Acceptance criteria: <2
the ratio in the mobile phase
● Flow rate ratio example
[Link]
0.8, 1, 1.2 ml/min
● % RSD: 1.23
● Mobile phase ratio example
● Acceptance criteria: <2
phosphate buffer : acetonotrile
28:72, 30:70, 32:68
Rachel (Ravisankar et al., 2021; ICH.,2005)
03 Challenges
Rapid Absorption & Metabolism
Implementation of Method
● Cmax → 1.7 hours
Only for routine analysis of 2
● Metabolism in the liver
yields 2 plasma derivatives components in formulation
Challenges
Interference of
Plasma Protein Excipients
97% of Lansoprazole is Minimize interference from
bound to plasma proteins excipients to enhance method
specificity and accuracy
Kim Jung Yeu
03 Limitations
Sample Size Limited Concentration Range
A larger sample size could provide The concentration range for
more robust results regarding the the assay of Lansoprazole is
accuracy and precision of the specified as 40-60 μg/ml.
method.
Limitations
Single Method Specificity Concerns
Validation A broader exploration of
The study focuses on a single potential interference from
method for the estimation of different excipients is not
Lansoprazole in tablet dosage form. provided.
Kim Jung Yeu
04 Troubleshooting
Column Stability Instrument Calibration
Regular column maintenance or
Calibrate detectors, pumps, and
replacement may be necessary
other components according to
(cases of column
the recommended schedule.
degradation/contamination).
Trouble-
Shooting
Mobile Phase Sample Preparation
Compatibility Consistency
Mobile phase should be properly Inconsistent sample preparation
stored (maintain temperature and can lead to variability in results.
humidity).
Kim Jung Yeu
04 Troubleshooting
Robustness Metabolite Separation
● Deliberate variation in mobile ● Modification of
phase flow rate and ratio chromatographic
● Assessing the method's conditions to enhance the
robustness by monitoring the separation of Lansoprazole
recovery of the drug under
and its metabolites
different conditions
● Ensuring specificity in
detection to focus on the
Can also be for:
Retention Time Optimization → parent compound
Adjustment of mobile phase
composition, flow rate, or column
temperature to optimize retention
time.
Kim Jung Yeu (Panchumarthy & Ravi, 2015)
05 References
HPLC Troubleshooting Guide. HPLC troubleshooting guide. (n.d.).
[Link]
hemistry/small-molecule-hplc/hplc-troubleshooting-guide
Kumar, S. M., Kumar, D. S., Rajkumar, T., Kumar, E. U., Geetha, A.
S., & Diwedi, D. (2010). Development and Validation
of RP-HPLC Method for the Estimation of Lansoprazole in
Tablet Dosage Form , 2(6), 291–295. Retrieved from
[Link]
[Link]
ICH (Q2A) Validation of analytical Procedures: Text and Methodology. Geneva:
International Conference on Harmonization, International Conference on
Harmonization; 2005.
Ravisankar, Panchumarthy & Sankar, Ravi. (2015). A Review on Step-by-Step Analytical
Method Validation. 7-19.
Ravisankar, P & Pentyala, Abhinav & Sai, Ch & Hemasri, P & Babu, Puttagunta & Sankar,
Ravi. (2021). VALIDATION CHARACTERISTICS AND STATISTICS IN ANALYTICAL METHOD
DEVELOPMENT. High Technology Letters. 27. 76-88.
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