noopur pharmacist project

1. Analytical method development and validation
for simultaneous estimation of
Antihypercholesterolemic oral formulation by
HPLC
Guided By : Mrs. Dhabarde Mam
Presented By : N. R. Gaikwad

2. Sr.
No.
Content Page
No.
1. Introduction 3
2. Literature Survey 10
3. Aim and Objective 13
4. Plan of work 14
5. References 16

3. Introduction
Analytical chemistry is the analysis of separation, quantification
and chemical additives identification of herbal and synthetic
materials constituted with one or more compounds or factors.
Analytical chemistry is separated into two predominant classes, a
qualitative evaluation that is to say the identification with regard
to the chemical additives exists in the sample, whereas
quantitative evaluation estimates the amount of positive detail or
compound within the substance, i.e. the sample[1].

4. Introduction
• CHROMATOGRAPHY
Chromatography is a technique in which solutes are resolved
by differential rates of elution as they pass through a
chromatographic column. Chromatography is essentially a
group of techniques for the separation of the compounds of
mixtures by their continuous distribution between two phases,
one of which moves relative to the other.[2]

5. Introduction
• High Performance Liquid Chromatography
HPLC is the fastest growing analytical technique for the
analysis of the drugs. Its simplicity, high specificity and wide
range of sensitivity make it ideal for an analysis of many drugs
in both dosage forms and biological fluids. Today it is widely
accepted separation technique for both sample analysis and
purification in variety of areas. The successful use of liquid
chromatography requires the right combination of a variety of
operating conditions such as the type of,
⮚ Column packing.
⮚ Mobile phase and its flow rate.
⮚ Column length and diameter.
⮚ Column temperature and sample size.

6. Introduction
• General uses of HPLC
1. Separation of wide variety of compounds, organic, inorganic
and biological compounds, polymers, chiral compounds,
thermally liable compounds and small ions to macro molecules.
2. Analysis of impurities.
3. Analysis of both volatile and nonvolatile compounds.
4. Determination of neutral ionic or zwitter ionic molecules.
5. Isolation and purification of compounds.
6. Ultra trace to preparative and process scale separations.
7. Qualitative and quantitative method.[3]

7. Introduction
• Most common HPLC detectors
⮚ UV-Visible absorbance detector (UV-VIS)
⮚ Photo-diode array detector (PDA)
⮚ Fluorescence detector
⮚ Electrochemical (ECD)
⮚ Refractive Index (RI)
⮚ Mass detectors (MS)
⮚ Conductometric detector

8. Method Validation
According to ICH guidelines [VALIDATION OF ANALYTICAL
PROCEDURES: TEXT AND METHODOLOGY Q2(R1)][5]
There are following parameter taken into consideration :
1. Analytical Procedure
2. Specificity
3. Accuracy
4. Precision
5. Detection limit
6. Quantitation limit
7. Linearity
8. Range
9. Robustness

9. Introduction of Disease
Hypercholesterolemia : Hypercholesterolemia is a lipid disorder in which
your low-density lipoprotein (LDL), or bad cholesterol, is too high.
High cholesterol can limit blood flow, increasing the risk of a heart attack
or stroke. It's detected by a blood test.
Providers classify all of these as high levels of low-density lipoprotein
cholesterol:
• 190 mg/dL or higher without other risk factors.
• Higher than 160 mg/dL with another major risk factor.
• Above 130 mg/dL with two risk factors.
Depending on your risk of a cardiovascular event, your target LDL-C level
can range from 116 mg/dL to less than 70 mg/dL.[6]

10. Causes
Hypercholesterolemia causes include:
• Your genes (pure or familial hypercholesterolemia).
• A diet that includes a lot of saturated and/or trans fats.
• A lack of exercise.
• Tobacco products.
• Obstructive liver disease.
• Diabetes.
• Hypothyroidism.
Risk Factors includes :
• Age
• High blood pressure
• Family history of premature atherosclerotic heart disease.
• Diabetes.
• Low levels of HDL

11. Hypercholesterolemia can lead to cardiovascular issues, such as:
• Stroke
• Coronary artery disease
• Peripheral artery disease
Treatment:
• Exercising more.
• Staying at a healthy weight.
• Eating foods low in saturated fat.
• Lowering your stress level.
• Taking cholesterol-lowering medications.
• Avoiding tobacco products.
• Controlling high blood pressure and blood sugar.

12. Literature Survey
Rameshwar Gholve, Sanjay Pekamwar, Sailesh Wadher and
Tukaram Kalyankar (2021) has developed and validated for
simultaneous estimation of telmisartan and rosuvastatin calcium in
bulk and in tablet dosage form. . The RP-HPLC elution was
carried out at 242.0 nm using column Oyster ODS3 (150×4.6 mm,
5 µm) isocratically, and a mobile phase containing 10 mM
phosphate bufer with 1.1 g octane-1-sulfonic acid sodium salt
having pH 2.5 (adjusted with 5% OPA) and acetonitrile, with a
proportion of 500:500, v/v was pumped through the column
maintained at ambient (about 25 °C) temperature with 1.0 mL/min
fow rate. The proposed method was validated according to ICH
Q2 (R1) guideline. The developed stability-indicating method is
linear in studied concentration range as well as precise, accurate,
specifc, and robust. [7]

13. Literature Survey
Uma Sai Teja Yarra and Sowjanya Gummadi (2021) et al, has
performed chromatographic separation for simultaneous
estimation of Bempedoic acid and Ezetimibe in the
pharmaceutical formulation with Waters Acquity C18 [50×2.1
mm, 1.7 μ] column using methanol: acetonitrile: water [50: 30:
20, by volume] as mobile phase pumped at a fow rate 0.5
mL/min. The separated analytes were detected at 260 nm using
UV detector. The separation of Bempedoic acid (BA) and
Ezetimibe (EZ) was done at a retention time of 1.827 min. and
3.577 min. respectively. The developed method was found to be
simple, sensitive, accurate, precise, robust, rapid and yet stability
indicating.[8]

14. Literature Survey
Mula. Anusha Reddy, C. Parthiban, M. Sudhakar (2022) et
al, has reported the method developed for the simultaneous
estimation of the Bempedoic acid and ezetimibe in
Pharmaceutical Dosage Form . Chromatogram was run through
Agilent 150 x 4.6 mm, 5mm. Mobile phase containing
Acetonitrile: KH2 in the ratio 55:45 was pumped through column
at a flow rate of 0.9 ml/min. Retention time of Bempedoic Acid
and Ezetimibe were found to be 2.252 min and 2.987. % RSD of
the Bempedoic Acid and Ezetimibe were and found to be 0.5 and
0.7 respectively. Because retention times and run times were
reduced, the method developed was simple and cost-effective,
and it can be used in regular quality control tests in industries.[9]

15. Literature Survey
Simran patel , Devanshi upadhyay, Prof. Mitali Dalwadi, Dr.
Umesh Upadhyay (2022) et al, has developed a reverse phase
high performance liquid chromatography (RP-HPLC) method for
the determination of bempedoic acid, ezetimibe and atorvastatin
in a synthetic mixture. The chromatographic sepration was
achieved isocratically on C 18 (250 mm 4.6 mm),5 µm at
ⅹ
ambient temperature using Potessium dihydrogen phosphate,
methanol and acetonitrile (30:60:10) as a mobile phase at flow
rate of 1 ml/min and UV detection at 262 nm. Retention time for
bempedoic acid, ezetimibe and atorvastatin were 3.76, 5.49 and
6.85 min respectively. % Recovery for Bempedoic Acid (BEM)
was found to be in range of 99.90-103.46 %, for), Ezetimibe
(EZT) it was found to be range of 97.02 -102.43 % and
Atorvastatin (ATR) it was found to be range of 97.97 -102.17.
[10]

16. Literature Survey
Pimpale Awdhut and Kakde Rajendra (2020) et al, ha
reported the reversed-phase HPLC method for the simultaneous
estimation of rosuvastatin calcium and fenofibrate in combined
bulk and tablet formulation. The chromatographic separation was
performed on reverse phase Princeton (C18) (250 mm x 4.6 mm,
5µ) column with mobile phase as a mixture of water and
acetonitrile in the ratio (40:60) v/v at the flow rate 1.0 ml/min.
Detection was carried out at wavelength 240 nm. The retention
time under the optimized condition of Rosuvastatin calcium and
Fenofibrate was found to be 2.485 & 3.905 minutes respectively.
The percentage recovery was found to be 99.66-100.37% and
99.13-100.44% for rosuvastatin calcium and fenofibrate
respectively.[10]

17. Literature Survey
Vadthya Rajashekar, K.Rajeswar Dutt, N.Ramathilagam A simple
reversed-phase high-performance liquid chromatographic (RP-
HPLC) method has been developed and validated for simultaneous
determination of Rosuvastatin and Ezetimibe in pharmaceutical
tablet dosage form. Chromatographic analysis was performed on a
Symmetry X-terra C8 (4.6mm x 100mm, 5 m)column at ambient

temperature with a mixture of ortho phosphoric acid buffer and
Acetonitrile in the ratio 40:60 v/v as mobile phase, at a flow rate of
1.0 mL min-1 . UV detection was performed at 237 nm.. The
retention times of Rosuvastatin and Ezetimibe were 2.490 and
3.173 min, respectively. The accuracy of the proposed method was
determined by recovery studies and found to be 99.59% to
100.70%. The method was validated according to the ICH
guidelines.[11]

18. Aim and objectives
• Aim:-
Analytical method development and validation for simultaneous
estimation of Antihypercholesterolemic oral formulation by
HPLC.
• Objectives:-
 To develop assay method for estimation of oral formulation by
using HPLC.
 To develop a precise, accurate and simple validation method for
the estimation of drug in combined dosage form using HPLC.
 Study the stability of oral formulation.

19. PLAN OF WORK
Estimation of oral formulation will be done by following method
 UV Spectrophotometric Method
 HPLC ( High Performance liquid Chromatography) Method
A] Literature Survey
B] Selection Of Drug Molecule
C] Method Development of Proposed Method
  Wavelength selection and it's Detection
 Selection of mobile phase
 Preparation of Standard Calibration Curve
 Estimation of Drug in Marketed formulation by proposed method

20. PLAN OF WORK
D] Validation of Proposed Method
 Similarity factor
 System suitability
 Specificity
Identification
Interference
 Linearity and
 Accuracy
 Precision
System precision
Method precision
Intermediate precision
 Stability of analytical solution
 Filter compatibility
 Robustness
 LOD and LOQ

21. References
1.Ravisankar P, Navya CN, Pravallika D, Sri DN. A review on step-by-
step analytical method validation. IOSR J Pharm 2015;5:7-19.
2.McMurry J (2011). Organic chemistry: with biological applications
(2nd ed.). Belmont, CA: Brooks/Cole. pp. 395. ISBN 9780495391470.
3.Sharma BK. Organic Spectroscopy. 6th edition. New Delhi: Goel
Publishing House.
2002; P.75.
4.Hdicostas E, Wendawiak B.W. Koch M. Quality Assurance in
Analytical Chemistry Training and Teaching. 1stedition. New Delhi:
Springer Pvt Ltd. International Springer
Group. 2004; P. 204-219.

22. 5.VALIDATION OF ANALYTICAL PROCEDURES:
TEXT AND METHODOLOGY Q2(R1).
6.https://my.clevelandclinic.org/health/
diseases/23921-hypercholesterolemia
7.Gholve et al. Futur J Pharm Sci (2021) 7:224
https://doi.o8.