Nilesh Dashrath Kamble presented a seminar on method development and validation in HPLC. The presentation discussed the steps involved in HPLC method development including column selection, mobile phase composition, pH range selection, and optimization of separation conditions. It also covered validation parameters such as accuracy, precision, specificity, limit of detection, and limit of quantification as per ICH guidelines. The presentation included an example method development for the simultaneous estimation of atorvastatin and telmisartan from a tablet formulation.

1. MIT-WPU | School of Pharmacy
WORLD’S FIRST UNIVERSITY
FOR LIFE TRANSFORMATION
Method development and Validation in HPLC
Name: Nilesh Dashrath Kamble
Class and Sem: F.Y. M Pharm Sem II
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2020-2021
Seminar on

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CONTENTS
Abstract
Introduction
Literature Survey
Conclusion
References

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ABSTRACT
Title: Method Development Of HPLC and Validation
High Pressure Liquid Chromatography (HPLC) is a column chromatography technique employing high
pressure pump to pass both mobile phase and sample mixture through the stationary phase column and
perform the efficient separation. The objective of this presentation is to review the method
development, optimization and validation. HPLC method development depends upon the physical and
chemical properties of an analyte drug, synthetic route, solubility, polarity, pH and pka values and
functional group activity, etc. as shown with the help of an example. Validation of HPLC method as per
ICH guideline gives information regarding various stages and knowing characteristic like accuracy,
specificity, linearity, limit of detection (LOD), limit of quantification (LOQ).
Name of Student: Nilesh D Kamble
Key words: High Pressure Liquid Chromatography (HPLC), Method validation, Method development.
Date:
10/06/2021
Time:16:30
Name of guide Dr. A R Chabukswar

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INTRODUCTION
High-performance liquid chromatography (HPLC) - is a technique is used to separate,
identify, and quantify each component in a mixture.
The main principle of separation is adsorption.
Types
--Adsorption - Ion exchange - Size exclusive
RP-HPLC
The stationary phase is nonpolar (hydrophobic) in nature, while the mobile phase is
polar(hydrophilic) in nature
•Non-polar stationary phase such as hydrocarbon and polar mobile phase such as water,
methanol or acetonitrile.

5. METHOD DEVELOPMENT
Steps involved in method development:-
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Information on
sample
Define separation
goals (Type of
Chromatography)
Special procedure
requirement &
pretreatment.
Detector selecting
and setting
Optimization
separation
conditions
Check for
problems or needs
of special
procedure
Recovery of
purified material
Quantitative
calibration /
Qualitative
method
Method validation
for release to
laboratories.

6. Column Selection
• Choosing the appropriate HPLC column is the most important requirement for HPLC analysis.
• The most commonly used HPLC column are C18 although C8, the octile bonded phases are also
used occassionally.
• The C18 are non polar columns but not as hydrophobic therefore retention time is higher,
whereas, hydrophobic compound is typically shorter, also they may show different selectivity than
C18, due to increased base silica exposure.
• There is unique selectivity which results in proton interaction of the bonded phase with electron
deficient functional groups of solute molecule.
• Following are the properties of column that needs to be selected :
• Column Dimensions
• Column Temperature.
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7. .
• The stationary phase material selection refers to the properties of the packing media that forms
the solid phase material in HPLC. Following consideration are included :
• 1) Particle shape : Varies from spherical to irregular shape. It have longer column life.
• 2)Particle size : Standard range of particle size =3 um to 15-20 um.
• 3) Surface area of particle : The high surface area of the particle shows high solute retention,
greater capacity and higher resolution.
• 4)Pore size : It is a average size of the pores present in porous packing particles, and is range of 60
Armstrong at the lower end upto 10,000 Armstrong at the higher end.
• 5) Bonding Type : Polymeric bonding and Monomeric bonding.
• 6) Carbon Load : Higher carbon load gives higher column capacities, greater resolution and longer
run time.
• 7) End Capping : It is the process of bonding short hydrocarbon chain to free silanols. It is
applicable only to reverse phase chromatography.
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8. Mobile Phase Composition
• Mobile phase composition (or solvent strength) plays an important role in RP-HPLC separation.
• Commonly used solvents in RP-HPLC have low UV cut off :
• These solvents are miscible with water.
• Mixture of acetonitrile and water is the best initial choice for the mobile phase during method
development.
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9. pH Range of Mobile Phase and Sample Mixture
• Selection of pH range for better resolution between two or more peak of an analyte depends
upon three main factors :
• Column efficiency.
• Selectivity.
• Retention time.
• Change in pH improve retention time and thus can lead to easy separation of ionizable analytes
from non-ionized forms.
• Change in the mobile phase pH can improve:
. Column efficiency because it alerts both the ionization of the analyte and the residual silanols.
. It also minimizes secondary interaction between analytes and the silica surface that lead to
poor peak shape.
. Optimum resolution is achieved by changing the pH of mobile phase.
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10. VALIDATION
The validation range is defined by IUPAC as the interval of analyte concentration within which the
method can be regarded as validated.
Various validation parameters includes :
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Accuracy
Precision
Specificity/
selectivity
Limit of
Detection
(LOD)
Limit of
Quantification
(LOQ).
Linearity Robustness
System
suitability

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LITERATURE SURVEY
1) Prajakta S Nawale et,al. reported simultaneous estimation of Telmisartan and
Metoprolol succinate by HPLC method from combined tablet dosage form. The mobile
phase consist of Methanol : Water and pH was adjusted to 5.5 by adding Trithylamine(
6:4:0.5 v/v) . The detection wavelength was 242 nm. Telmisartan and Metoprolol showed
R f values 0.55+_ 0.22 and 0.41+_0.02.
2) Krishna R Gupta et,al. reported simultaneous estimation of Atorvastatin and Atenolol
in pharmaceutical dosage by Reverse Phase HPLC. The mobile phase consists of phosphate
buffer and was 1 ml/min with detection wavelength at 238 nm. The retention time of
Atorvastatin was 2.1 min and Atenolol was 3.7 min.
3) Subhankar, et al. reported development and validation of RP-HPLC method for the
analysis of Telmisartan using C18 column. The mobile phase used was potassium
dihydrogen phosphate and acetonitrile (60:40% v/v) The flow rate was 1 ml/min. The
detection wavelength was monitored at 243 nm and the retention time was found to be
3.4 min with symmetric peaks.

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Example
Simultaneous estimation of telmisartan and metoprolol succinate by RP-HPTLC method
from combined tablet dosage form. The mobile phase consists of methanol: water:
triethylamine (6: 4:0.5 v/v) and the PH was adjusted to 5.5 by adding triethylamine. The
detection wavelength was 242nm. Telmisartan and metoprolol showed 𝑅 𝑓 values 0.55 ±
0.02 and 0.41 ± 0.02
- simultaneous estimation of atorvastatin and atenolol in pharmaceutical dosage by
reverse phase HPLC. The mobile phase consists of phosphate buffer and acetonitrile
(28:72%v/v) with PH adjusted to 4.58 with dilute orthophosphoric acid. Flow rate was
1ml/min with detection wavelength at 238nm. The retention time of atorvastatin was
2.1min and atenolol was 3.7min.

13. DRUG PROFILES
• ATORVASTATIN
• STRUCTURE:
• MOLECULAR FORMULA :C33H55FN2O5
• SOLUBILITY: soluble in methanol, ethanol
• CATEGORY: HMGcoA reductase inhibitor
• TELMISARTAN
• STRUCTURE:
• MOLECULAR FORMULA: C33H30N4O2
• SOLUBILITY : freely soluble in methanol, ethanol and butanol
• CATEGORY: anti- hypertensive CLASS : Angiotensin-II receptor type 1
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14. METHOD DEVELOPMENT
• wavelength – 245nm
• mobile phase :10mm phosphate buffer gave symmetrical peaks with good resolution., rf of
atorvastatin is 3.2 minutes and telmisartan is 4.6 minutes.
• optimization of separation conditions is based on the following factors : 1. effect of strength of
phosphate buffer 2. effect of ratio of mobile phase 3. effect of PH
• peaks water: acetonitrile 30:70 20mm phosphate buffer : acetonitrile(30:70)
• 10mm phosphate buffer: acetonitrile ( 30: 70)
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Analysis of formulation • preparation of standard solution: • aliquots of standard solution
contains 5 to 50microgram per ml of atorvastatin and telmisartan. • Preparation of sample
solution: • 10 mg of arbitel-av dissolved in mobile phase • final volume was made upto get
a working concentration of 20 microgram per ml each of atorvastatin and telmisartan.
Results: Retention for Atorvastatin & Telmisartan is 3.2 & 4.6 min
LOD - Atorvastatin & Telmisartan 0.20 & 0.11 mg/ml
LOQ - Atorvastatin & Telmisartan 0.32 & 0.39 mg/ml
The development method was found to be Robust.
A simple , specific, linear, precise and accurate RP-HPLC method has been developed and
validated for quantitative determination of atorvastatin and telmisartan in new tablet
formulation. The method is very simple and specific as both peaks are well separated from
its excipient peaks and with total runtime of 5 min, makes the developed method its
suitable for routine quality control analysis work.

16. • RP-HPLC method for routine analysis of Ketoconazole in formulations
• Verma Vikrant*, et al. -Stationary phase:Inertsil ODS-C18 column (150mm,4.6mm,5μm) . •Mobile
phase: Buffer (pH-4) and Methanol in the mixture of 30:70. •Wavelength:274nm. •. Injection
volume :20μl. •Flow rates: 1ml/min. 2.
• Olga Popovska, et al, -Stationary phase:LiChrospher®100 C-18 column (150 mm length x 4.6 mm
i.d., 5 µm particle size) •Mobile phase: Methanol and water (90:10 v/v) •pH :8.9 with phosphate
buffer. •Flow rate: 1.0 ml/min. 3.
• Rakesh Kumar Jat, et al. -Stationary phase:Promosil C-18, (250 mm, 4.6 mm, 5µm). •Mobile
phase: Water : acetonitrile : buffer ph 6.8 (51:45:4 v/v) . •Wavelength:238nm. •Flow rate: 1.0
ml/min. • Retention times: 2.713 min. •Concentration range: 1-50µg/ml. 8
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..

17. • Name : Ketoconazole
• Structure :
• Chemical formula : C26H28Cl2N4O4
• Solubility : Ketoconazole is soluble in organic solvents such as methanol, ethanol.
• Category : Anti-Infective Agents, Antifungal Agents.
Separation
Stationary Phase - the phase which remains fixed in the column. e.g. C18, C8. Mobile Phase - carries
the sample through the stationary phase as it moves through the column. e.g. methanol, ACN,OPA.
Injector Detector Column Solvents Mixer Pumps High Performance Liquid Chromatograph Waste
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DRUG PROFILE

18. .
• OPTIMIZED PEAK CONDITIONS
• Column: Phenomenex Luna C18, 100A, 5 µm,
250mmx4.6mm i.d. • Mobile phase:Acetonitrile :
Phosphate buffer = 68:32
• Wavelength:242 nm • Flow rate:1.0 ml/ min. •
Retention time:4.768. • Sampling System: Automatic •
Injection Volume:20µl • Temperature of Column:
Ambient • Run Time/ Stop Time:8.0minutes •
Concentration of Sample:10ppm
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CONCLUSION
Method development and validation are the key elements for any analytical process. It is
essential to establish the identity, purity, physical characteristic and potency of the
chemicals, pharmaceutical compounds and drugs. The selection of Column, buffer,
detector and another condition composition (organic and pH) plays a dramatic role on the
separation selectivity. Final optimization can be performed by changing the temperature
and flow rate as well as the type and concentration of mobile-phase modifiers. Validation
proves a scientific soundness of the process, measurements and characterization.
Optimized method is then validated with various parameters e.g. specificity, precision,
accuracy, detection limit, linearity, etc. as per ICH guidelines.

20. .
1) Rao BV, Sowjanya GN, Ajitha A, Rao Uma MV. A review on stability indicating HPLC
method development , World journal of pharmacy and pharmaceutical
sciences.2015;4(8):405-423.
2) Rajan HV. Development and validation of HPLC method- A Review International
Journal of current research in pharmacy. 2016;1(2):55-68.
3) Kumar V, Jain AD, Gill NS, Gupta G, Kumar S. An Overview on HPLC Method
Development, Optimization and Validation process for drug analysis. The
Pharmaceutical and Chemical Journal 2016;2(2):30-40.
4) Gupta V, Bharadwaj R, Kumar S. Development and Validation of HPLC method-a
review. International Research Journal of Pharmaceutical and Applied
Science.2017;2(4):17-25.
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REFERENCES

21. .
• 5) Snyder LR, Kirkland JJ, Glach JL, Practicle HPLC Method Development, John Wiley and Sons,
New York, 2018: 158-192.
• 6) HPLC- Chemiguide. June 8, 2021. www.chemguide.co.uk
• 7) http://www.scribd.com/doc/9508765/Physical-Properties-of-Drug.
• 8) Ranjit Singh. HPLC method development and validation. J Pharm Educ Res2018:4(1) :26-33.
• 9) Sabir AM, Molony M, Parminder SB, HPLC Method Development and Validation. A Review.
International research Journal of pharmacy 2018; 4(4) : 39-46.
• 10) Malviya R, Bansal V, Palo P and Sharma PK. High Performance Liquid Chromatography : A short
Review. Journal of Global Pharma Technology. 2019 ; 2(5): 22-23.
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