The document describes the development and validation of a reverse phase high performance liquid chromatography (RP-HPLC) method for the estimation of drugs in bulk and pharmaceutical dosage forms. It discusses the need for analytical methods in pharmaceutical analysis and introduces RP-HPLC as a commonly used technique. The document provides details of various studies done to develop and validate RP-HPLC methods for estimating specific drugs and drug combinations in research papers. It also provides chemical and pharmacological profiles of drugs that were estimated using RP-HPLC methods discussed in the literature review section. The aim is to develop a validated RP-HPLC method for estimation of a drug in bulk and marketed dosage form.

1. Development and validation of RP-HPLC
method for estimation of bulk dosage form as
marketed dosage form
DHANVANTHRI COLLEGE OF PHARMACEUTICAL SCIENCES
MAHABUBNAGAR, TELANGANA STATE-509002
February-2024 1
C POOJA
M.PHARMACY III SEMESTER
353122885011
[Department of Pharmaceutical Analysis]
2022-2024
Under the guidance of
K SUJATHA, M.Pharm
Assistant Professor

2. CONTENTS
CHAPTER NO TITLE
I INTRODUCTON & DRUG PROFILE
II LITERATURE REVIEW
III AIM, OBJECTIVES & PLAN OF WORK
IV
CONCLUSION
V BIBLIOGRAPHY

3. Introduction
» Herbal Products
» New process and reactions
» New molecules
» Active ingredients (Macro analysis)
» Residues (Microanalysis)
» Impurity Profiling
» Component of Interest in different matrices
3
Need of Analytical method

4. Analytical techniques
 Titrimetric & gravimetric
 Colorimetric & ultraviolet spectrophotometric
 Electrochemistry
 Fluorimetric
 Flame photometric & atomic absorption spectrometric
 Chromatography
4

5. High Performance Liquid Chromatography
(HPLC)
• HPLC is a separation technique based on a solid stationary phase
and a liquid mobile phase. Separations are achieved by partition,
adsorption, or ion-exchange processes, depending upon the type
of stationary phase used.
• Chiral
• Ion--exchange
• Ion--pair/affinity
• Normal phase
• Reversed phase
• Size exclusion
• The reversed-phase HPLC with UV detection is most commonly
used form of HPLC, is selected to illustrate the parameters of HPLC
method and validation.

6. • A FLOW SCHEME FOR HPLC

7. DRUG CATEGORY : Angiotensin receptor blockers
STRUCTURE :
IUPAC NAME : 2-(4{[4-methyl-6-(1-methyl-1H-1,3-benzodiazol-2-yl)-2-
propyl- 1H-1,3-benzodiazol-1-yl]methyl}phenyl)benzoic acid
PHYSICOCHEMICAL PROPERTIES :
Melting point : 261-263 °C
LogP : 3.2
Pka : 4.45
Density : 1.16
Colour : White
Solubility : Soluble in organic solvents
1.5 Drug Profile 8,9,10,11
1. INTRODUCTION
TELMISARTAN

8. Veeraswami et al (2019), Objective: The present paper describes a simple, accurate, and
precise reversed-phase high-performance liquid chromatography (HPLC) method for rapid
and simultaneous quantification of dolutegravir (DTG) and rilpivirine (RPV) in bulk and
pharmaceutical dosage form and rat plasma. Methods: The chromatographic separation was
achieved on Phenomenex C18 (150x4.6mm, 5μm). Mobile phase contained a mixture of
0.1% Ortho phosphoric acid and acetonitrile in the rato of 60:40 v/v, flow rate 1.0ml/min
and ultraviolet detection at 262nm. Results: The retention time of DTG and RPV was 4.35
min and 7.73 min, respectively. The proposed method shows a good linearity in the
concentration range of 10–150 μg/ml for DTG and 5–75 μg/ml for RPV under optimized
conditions. Precision and recovery study results are in between 98 and 102%. In the entire
robustness conditions, percentage relative standard deviation is <2.0%. Degradation has
minimum effect in stress condition and solutions are stable up to 24 h. DTG and RPV drugs
are release 98% at 2 h in rat body. Conclusion: This method is validated for different
analytical performance parameters like linearity. Precision, accuracy, limit of detection,
limit of quantification, robustness, and pharmacokinetic study were determined according
to the International Conference of Harmonization (ICH) Q2B guidelines. All the parameters
of validation were found in the acceptance range of ICH guidelines. The same method is
also applied for plasma samples study in bioanalytical work.
LITERATURE REVIEW

9. Swathi et al(2017), Objective: The objective and purpose of the analysis have sensibly assessed
by selecting of a rapid and sensitive RP-HPLC method for Entecavir in bulk and pharmaceutical
dosage form by using the most commonly employed C-18column with UV detection Methods:
In estimation by RP-HPLC method Agilent 1120 compact LC system with variable
programmable UV detector and Rheodyne injector with 20 μl fixed loop was used for the
chromatographic separation. The mode of operation was isocratic with the components of a
solution consisting of methanol: acetonitrile(70:30v/v) and triethanolamine (2-4drops)at the
flow rate of 1.2 ml/min and run time was 10 min. Forced degradation studies were conducted to
evaluate the stability and specificity of the method along with the validation parameters.
Results: Validation parameters of HPLC were found at a detection wavelength of 255 nm.
Linearity was observed with the concentration range (Beer’s law range) 20-100μg/ml with
R2Conclusion: The proposed RP-HPLC method was validated as per the ICH Q2B Guidelines,
and was found to be applicable for routine quantitative analysis of Entecavir by RP-HPLC using
UV detector in pharmaceutical dosage forms. The results of linearity, precision, accuracy and
specificity, were proved, that does not exceed certain specified limits. The method provides
selective quantification with no interference from other formulation excipients. The proposed
method was highly sensitive, reproducible, reliable, robust and specific. Therefore, this method
is a simple, analysis may actually be more desirable than a more complicated and time-
consuming process. The degradation studies at various stress conditions like thermal and
hydrolytic, drug gets degraded at a temperature of 80 °c and refluxing with water at 70 °c for
24hours. =0.9991. Robustness with detection wavelengths 253 and 257 nm with a flow rate of 1
ml/min and 1.4 ml/min showed good results. The retention time of the drug was 2.64 min and
assay showed 98.1%.

10. J. Sandya rani et al (2017),A simple, rapid, accurate, precise, specific and sensitive reverse phase-HPLC
method has been developed and validated for the simultaneous estimation of Sofosbuvir and Velpatasvir in
bulk drug and pharmaceutical dosage form. The chromatographic separation was performed on the Kromasil
C18 column (250mm×4.6mm, 5μm particle size), using a mobile phase of Buffer: Acetonitrile taken in the
ratio 45:55 v/v, at a flow rate of 1.0 ml/min at an ambient temperature of 30˚C with the detection wave length
at 260nm. The retention times of Sofosbuvir and Velpatasvir were 2.124 min and 3.334 min respectively. The
linearity was performed in the concentration range of 100-600 ppm, 25-150 ppm each of Sofosbuvir and
Velpatasvir with a correlation coefficient of 0.999 and 0.999 for Sofosbuvir and Velpatasvir respectively. The
percentage purity of Sofosbuvir and Velpatasvir was found to be 98.45% and 99.56% respectively. The
proposed method has been validated for specificity, linearity, range, accuracy, precision and robustness were
within the acceptance limit according to ICH Q2 (B) guidelines and the developed method can be employed
for routine quality control analysis in the bulk and combined pharmaceutical dosage form of Sofosbuvir and
Velpatasvir.
Jat R.K et al (2012), A simple, accurate rapid and precise RP-HPLC method has been developed
and validated for determ ination of simvastatin in bulk drug. The RP-HPLC separation was
achieved on Promosil C-18, (250 mm, 4.6 mm, 5μm) using mobile phase buffer: methanol ph 6.8
(96: 4 v/v) at flow rate of 1.0 ml/min at ambient temperature. The retention times were 9.546
min. for simvastatin. Calibration plots were linear over the concentration range 1-50μg/ml.
Quantification was achieved with photodiode array detection at 254 nm over the concentration
range of 1-50 μg/ml. The method was validated statistically and applied successfully for the
determination of simvastatin. Validation studies revealed that method is specific, rapid, reliable,
and reproducible. The high recovery and low relative standard deviation confirm the suitability of
the method for the routine determination of simvastatin in bulk drug.

11. 11
Chirag B. Pandya et al (2010), A simple, specific, accurate, and precise reverse phase high performance
liquid chromat ographic (RP-HPLC) met hod was developed and validat ed for the est imat ion of Rosuvast
at in Calcium (RC) in pharmaceut ical dosage forms. A Thermo hypersil reversed phase C- 18, 5 μm
column having 100 x 4.6 mm i.d. in gradient mode, with mobile phase containing HPLC grade Acetonitrile
: Potassium dihydrogen ort hophosphate (50 : 50 v / v, pH 3) was used. The flow rat e was 0.5 ml / min and
effluent s were monitored at 243 nm. Chromat ogram showed a main peak of RC at ret ent ion time was
3.333 ± 0.004 min. The method was validat ed for linearit y, accuracy, precision, limit of det ect ion, limit
of quantit at ion, robustness and ruggedness. The limit of det ect ion and limit of quantitation for estimation
of RC was found to be 0.14 μg / ml and 0.46 μg / ml, respectively. Recovery of RC was found to be in t he
range of 98.50-100.17 %. Proposed met hod was successfully applied for the quant it ative det ermination
of RC in pharmaceutical dosage forms.
Sandhya Donthula et al (2011), A simple, rapid, and precise RP-HPLC method for analysis of
Rosuvastatin calcium in bulk and its pharmaceutical formulations has been developed and
validated. The separation was achieved on Luna C18, 5μm 4.6 mm×250 mm column, using
mobile phase composition of buffer (pH 4.5): Acetonitrile: methanol (45:25:35), at a flow rate
of 1.0 ml/min at a detection wavelength of 248 nm. Rosuvastatin is eluted at retention time of
9.9 min. The method was validated for accuracy, precision, linearity, specificity and sensitivity
in accordance with ICH guidelines. Validation revealed the method is specific, rapid, accurate,
precise, reliable, and reproducible. Calibration plots were linear over the concentration ranges
25-75 μg mL−1. Limits of detection and limits of quantification were 3.5μg mL−1 and 10.5μg
mL−1, respectively for both the drugs. The high recovery and low coefficients of variation
confirm the suitability of the method for analysis of the rosuvastatin in formulations.

12. Nalini Kanta Sahoo et al (2014), A novel, simple and economic reverse phase high performance liquid
chromatography (RP-HPLC) method has been developed for the estimation of Simvastatin in bulk and tablet
dosage form with greater precision and accuracy. Separation was achieved on Develosil ODS HG-5 RP C18,
(150cmx4.6mm i.d. 5 m) column in isocratic mode with mobile phase consisting of acetonitrile :phosphate
buffer(pH 3.0) (85:15) with a flow rate of 1 mL/min. The detection was carried out at 236 nm. The retention
time of Simvastatin was found to be 5.84 min. The method was validated as per ICH guidelines.Linearity
was established for Simvastatin in the range 10 – 100 μg / ml with R2 value 0.99. The percentage recovery of
Simvastatin was found to be in the range 99.19-99.67 %. The high recovery and low relative standard
deviation confirm the suitability of the proposed method for the estimation of the drug in bulk and tablet
dosage forms. The LOD and LOQ were found to be 0.341 and 1.023 μg/ml respectively.Validation studies
demonstrated that the proposed RP-HPLC method is simple, specific, rapid, reliable and reproducible for the
determination of Simvastatin for Quality Control.
Mahfuza Maleque et al (2012), The present study was undertaken to develop a validated, rapid,
simple and economic HPLC method for estimating caffeine in pharmaceutical preparations.
Chromatographic determination was performed on a reversed phase C18 column (4.5 mm x 250
mm; 5 μm particle size) using a mixture of water and methanol (60:40) as mobile phase at a flow
rate of 1ml/min with UV detection at 272 nm. The method was validated for linearity, accuracy,
repeatability, precision, reproducibility, and specificity as per International ICH guidelines. The
method was also used in determination caffeine content in five commercial brands available in
Bangladeshi market. The method was linear in the range between 12 – 28 μg/ml, exhibited good
correlation coefficient (R2 = 0.9992) and good Accuracy study (97.35 %-100.02 Hence it can be
employed for routine analysis of caffeine both in bulk and commercial formulations and in
combination dosage form with paracetamol.

13. III. AIM, OBJECTIVES & PLAN OF WORK
AIM :The existing physicochemical methods are inadequate to meet the requirements; hence it
is proposed to improve the existing methods and to develop new methods for the estimation of
bulk in pharmaceutical dosage forms adapting different available analytical technique like
HPLC.
OBJECTIVES :The objectives of the proposed method is to develop simple and accurate
methods for the estimation of bulk by HPLC method in pharmaceutical dosage forms

14. The wide variety of equipment, columns, eluent and operational parameters involved makes
high performance liquid chromatography (HPLC) method development seem complex. The
process is influenced by the nature of the analytes and generally follows the following steps:
•step 1 - selection of the HPLC method and initial system
•step 2 - selection of initial conditions
•step 3 - selectivity optimization
•step 4 - system optimization
•step 5 - method validation.
PLAN OF WORK

15. Content of HPLC test procedure
• Any analytical procedure submitted should be described in sufficient
detail, includes:
• Preparation of mobile phase
• Chromatographic condition:
– Column: type (e.g., C18 or C8), dimension (length, inner diameter),
particle size (10μm, 5 μm)
– Detector: wavelength
– Injection volume
– column T
– flow rate,

16. Content of HPLC test procedure
• Elution procedure: isocratic or gradient elution
• Preparation of standards and samples
• Operation procedure: sequence of injections
• System suitability testing (SST) and criteria
• Calculations
• QOS 2.3.R.2 analytical procedures and validation summaries

17. Compendial methods
• When claim a compendial method, there should be no change in:
• The type of column i.e the stationary phases
• Detector wavelength
• Components in Mobile phase
• System suitability testing and criteria
• Adjustments to ratio of components in mobile phase, flow rate, column
temp, dimension of column, particle size (reduction only), may be
necessary to achieve the system suitability criteria. The allowable
variations for each parameter, see Int.Ph 1.14.4 or USP general chapter
<621>.

18. System suitability testing (SST)
• Precision:
– Assay: RSD ≤1% (API) or ≤ 2% (FPP), n ≥ 5
– Impurities: in general, RSD ≤ 5% at the limit level, up to 10% or higher
at LOQ, n ≥ 6
• Resolution (R): >2

19. System suitability testing (SST)
• Tailing factor/peak asymmetry: (≤ 2)

20. System suitability testing (SST)
• Number of theoretical plates (N): column
efficiency ≥ 2000
Gradient elution is one way to increase the N

21. System suitability testing (SST)
• A SST should contain:
• For Assay:
• precision + one or more other parameter

22. Linearity
• Rifampicine:
• y =31.312 x + 4.963
• Rifampicine Quinone:
• y = 26.198 x + 1.154
• RRF= 26.198 / 31.312
• =0.84

23. • To review:
• a) RRF calculation, and
• b) if RRF is properly used in the final calculation for % impurity
• If RRF within 0.8-1.2, correction may not be necessay
• Correction factor= 1/RRF, the reciprocal of the RRF
Linearity

24. Review points for HPLC method
• is the analytical procedure described in detail including all the parameters ?
• is SST well defined to ensure the consistency of system performance?
• The preparation of solutions:
– assay: concentration of reference standard should be close to the sample
solution

25. Validation – compendial methods
• Assay – API
– No validation generally required. Exception: specificity for major impurities not in
the monograph.
• Assay – FPP
-Specificity, accuracy and precision (repeatability).

26. Non-compendial methods
Full validation is required for purity, assay by HPLC :
• Specificity
• Linearity
• Accuracy
• Repeatability
• Intermediate precision
• LOD/LOQ (not required for assay, dissolution)
• Robustness (recommended)

27. CONCLUSION
Method development involves a series of sample steps; based on what is known about the
sample, a column and detector are chosen; the sample is dissolved, extracted, purified and
filtered as required; an eluent survey (isocratic or gradient) is run; the type of final separation
(isocratic or gradient) is determined from the survey; preliminary conditions are determined for
the final separation; retention efficiency and selectivity are optimized as required for the
purpose of the separation (quantitative, qualitative or preparation); the method should be
validated using ICH guidelines. The validated method and data can then be documented.

28. BIBILIOGRAPHY
28
1,Veeraswami B*, Naveen Vmk, development and validation of rp-hplc method for the estimation of dolutegravir and rilpivirine in bulk and
pharmaceutical dosage form and itsapplication to rat plasma, Asian J Pharm Clin Res, Vol 12, Issue 2, 2019, 267-271
2.Swathi p.1*, s. Vidyadhara2, r. L. C. Sasidhar3, k. Kalyan chakravarthi4. Method development and validation for the estimation of entecavir in
bulk And pharmaceutical dosage forms by rp-HPLC, International Journal of Current Pharmaceutical Research, Vol 9, Issue 5, 2017
3.J. Sandya rani, N. Devanna, A New RP-HPLC Method Development and Validation for Simultaneous Estimation of Sofosbuvir and Velpatasvir
in Pharmaceutical Dosage Form, International Journal of Engineering Technology Science and Research, Volume 4, Issue 11, November 2017
4,Jat R.K1, Sharma S2, Chhi pa RC1, Singh Rambir1, Alam Imran, development and validation of reversed phase hplc method for estimation of
simvastatin in pharmaceutical dosage form, J ournal of Drug Deli very & Therapeutics; 2012, 2(3): 121-124
5.Chirag B. Pandya* , K.P. Channabasavaraj, Jaydeep D. Chudasama, T.T. M ani, developm ent and validation of rp-hplc m ethod for
determination of Rosuvastatin calcium in bulk and pharm aceutical dosage form, International Journal of Pharmaceutical Sciences Review and
Research, Volume 5, Issue 1, November – Decem ber 2010
6.Sandhya Donthula*1, Meriga Kiran Kumar 1, G. Shiva Teja1, Y. Mohan Kumar1, J. Yasodha Krishna2 and D. Ramesh, A new validated RP-HPLC
method for determination of Rosuvastatin calcium in bulk and pharmaceutical dosage form Der Pharmacia Lettre, 2011, 3(3):350-356
7.Nalini Kanta Sahoo1*, Madhusmita Sahu1, P. Srinivasa Rao1, R.S.Vineela 1, J.N.V. Indira Devi1, N.Sandhya Rani1, Goutam Ghosh, alidation
of Assay Indicating Method Development of Simvastatin in Bulk and its Tablet Dosage form by RP-HPLC, Journal of Applied Pharmaceutical
Science Vol. 4 (01), pp. 117-122, January, 2014
8.Sharmin Reza Chowdhury, Mahfuza Maleque*, Mahbubul Hoque Shihan, Development and Validation of a Simple RP-HPLC Method for
Determination of Caffeine in Pharmaceutical Dosage Form, Asian J. Pharm. Ana. 2012; Vol. 2: Issue 1, Pg 01-04