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84 | P a g e International Standard Serial Number (ISSN): 2319-8141

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International Journal of Universal Pharmacy and Bio Sciences 4(1): January-February 2015

INTERNATIONAL JOURNAL OF UNIVERSAL

PHARMACY AND BIO SCIENCES IMPACT FACTOR 2.093*** ICV 5.13*** Pharmaceutical Sciences RESEARCH ARTICLE……!!!

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF

EZETIMIBE AND FENOFIBRATE COMBINATION BY USING RP-HPLC

METHOD

Jabeen Farhana, Wajeed Khan, Vinayak K Durgakar, Asiya Begum

Azad college of Pharmacy,Moinabad.

KEYWORDS:

Ezetimibe, Fenofibrate,

RP-HPLC.

For Correspondence:

Jabeen Farhana*

Address:

Azad college of

pharmacy, Moinabad.

Email:

[email protected]

ABSTRACT

A simple, Accurate, precise method was developed for the

simultaneous estimation of the Ezetimibe and Fenofibrate in tablet

dosage form. Chromatogram was run through Kromasil (250 mm

x4.6 mm, 5µ). Mobile phase containing Buffer and Acetonitrile in

the ratio of 60: 40 was pumped through column at a flow rate of

1ml/min. Buffer used in this method was 0.01M KH2PO4 with pH

3.80. Temperature was maintained at 30°C. Optimized wavelength

for Ezetimibe and Fenofibrate was 230nm. Retention time of

Ezetimibe and Fenofibrate were found to be 3.0min and 2.3 min.

%RSD of the Ezetimibe and Fenofibrate were and found to be 0.13

and 0.19 respectively. % Recovery was Obtained as 99.9% and

100.03% for Ezetimibe and Fenofibrate respectively. LOD, LOQ

values are obtained from regression equations of Ezetimibe and

Fenofibrate were 0.39, 1.18 and 0.48, 1.44 respectively. Regression

equation of Ezetimibe is y = 42418x + 5004 and of Fenofibrate is y

= 18765x + 2704.

mailto:[email protected]



INTRODUCTION:

Quantitative chemical analysis is an important tool to assure that the raw material used and the

intermediate products meet the required specifications. Drug analysis is the base for the

determination of the product. Every year numbers of drugs are introduced into the market. Also

quality is important in every product or service but it is vital in medicines as it involves life. Very

often, there is a time lag from the date of introduction of a drug into the market to the date of its

inclusion in pharmacopoeias. This happens because of the possible uncertainties in the

continuous and wider usage of these drugs, report of new toxicities, and development of patient

resistance and introduction of better drugs by the competitors. Under these conditions, standard

and analytical procedures for these drugs may not be available in Pharmacopoeias.

Quality control is a concept, which strives to produce a perfect product by series of measures

designed to prevent and eliminate errors at different stage of production. The decision to release

or reject a product is based on one or more type of control action. With the growth of

pharmaceutical industry during last several years, there has been rapid progress in the field of

pharmaceutical analysis involving complex instrumentation. Providing simple analytical

procedure for complex formulation is a matter of most importance.

DRUG PROFILE:

Drug profile of Ezetimibe:

Ezetimibe is a drug that lowers plasma cholesterol levels. It acts by decreasing cholesterol

absorption in the small intestine. It may be used alone (marketed as Zetia or Ezetrol), when other

cholesterol-lowering medications are not tolerated, or together with statins (e.g.,

Ezetimibe/simvastatin, marketed as Vytorin and Inegy) when statins alone do not control

cholesterol. Ezetimibe is generally administered orally in a 10mg tablet taken once a day with or

without food.

Ezetimibe decreases cholesterol levels, but has not been shown to improve outcomes in

cardiovascular disease patients by decreasing atherosclerotic or vascular events compared to

placebo. Ezetimibe is endorsed in the Canadian Lipid Guidelines and is considered a well-

tolerated option for an add-on agent to statin, to help patients achieve their LDL (or so-called

"bad cholesterol") targets. [1] Ezetimibe is the only add-on to statin therapy that has

successfully shown cardiovascular benefit when combined with statin, but has not been proven

to have an incremental benefit compared to statins alone. [2] Britain's NICE statement,



published in 2007, endorses its use for monotherapy if statins are not tolerated or as add-on

therapy.

Fig 2.1 Ezetimibe

Ezetimibe reduces blood cholesterol by inhibiting the absorption of cholesterol by the small

intestine. In a 2-week clinical study in 18 hypercholesterolemic patients, ZETIA inhibited

intestinal cholesterol absorption by 54%, compared with placebo. ZETIA had no clinically

meaningful effect on the plasma concentrations of the fat-soluble vitamins A, D, and E (in a

study of 113 patients), and did not impair adrenocorticalsteroid hormone production (in a study

of 118 patients).

The cholesterol content of the liver is derived predominantly from three sources. The liver can

synthesize cholesterol, take up cholesterol from the blood from circulating lipoproteins, or take

up cholesterol absorbed by the small intestine. Intestinal cholesterol is derived primarily from

cholesterol secreted in the bile and from dietary cholesterol.

Ezetimibe has a mechanism of action that differs from those of other classes of cholesterol-

reducing compounds (statins, bile acid sequestrants [resins], fibric acid derivatives, and plant

stanols). The molecular target of ezetimibe has been shown to be the sterol transporter,

Niemann-Pick C1-Like 1 (NPC1L1), which is involved in the intestinal uptake of cholesterol

and phytosterols.

Ezetimibe does not inhibit cholesterol synthesis in the liver, or increase bile acid excretion.

Instead, Ezetimibe localizes at the brush border of the small intestine and inhibits the absorption

of cholesterol, leading to a decrease in the delivery of intestinal cholesterol to the liver. This

causes a reduction of hepatic cholesterol stores and an increase in clearance of cholesterol from

the blood; this distinct mechanism is complementary to that of statins and of Fenofibrate.

Pharmacodynamics:

Clinical studies have demonstrated that elevated levels of total-C, LDL-C and Apo B, the major

protein constituent of LDL, promote human atherosclerosis. In addition, decreased levels

of HDL-C are associated with the development of atherosclerosis. Epidemiologic studies have

http://www.rxlist.com/cholesterol/article.htm

http://www.rxlist.com/script/main/art.asp?articlekey=2101














established that cardiovascular morbidity and mortality vary directly with the level of total-C

and LDL-C and inversely with the level of HDL-C. Like LDL, cholesterol-enriched

triglyceride-rich lipoproteins, including very-low density lipoproteins (VLDL), intermediate-

density lipoproteins (IDL), and remnants, can also promote atherosclerosis. The independent

effect of raising HDL-C or lowering TG on the risk of coronary and cardiovascular morbidity

and mortality has not been determined.

ZETIA reduces total-C, LDL-C, Apo B, non-HDL-C, and TG, and increases HDL-C in patients

with hyperlipidemia. Administration of ZETIA with a statin is effective in improving serum

total-C, LDL-C, Apo B, non-HDL-C, TG, and HDL-C beyond either treatment alone.

Administration of ZETIA with Fenofibrate is effective in improving serum total-C, LDL-C,

Apo B, and non-HDL-C in patients with mixed hyperlipidemia as compared to either treatment

alone. The effects of Ezetimibe given either alone or in addition to a statin or Fenofibrate on

cardiovascular morbidity and mortality have not been established.

Pharmacokinetics:

Absorption

After oral administration, Ezetimibe is absorbed and extensively conjugated to a

pharmacologically active phenolic glucuronide (ezetimibe-glucuronide). After a single 10-mg

dose of ZETIA to fasted adults, mean Ezetimibe peak plasma concentrations (Cmax) of 3.4 to

5.5 ng/mL were attained within 4 to 12 hours (Tmax). Ezetimibe-glucuronide mean Cmax

values of 45 to 71 ng/mL were achieved between 1 and 2 hours (Tmax). There was no

substantial deviation from dose proportionality between 5 and 20 mg. The absolute

bioavailability of Ezetimibe cannot be determined, as the compound is virtually insoluble in

aqueous media suitable for injection.

Effect of Food on Oral Absorption:

Concomitant food administration (high-fat or non-fat meals) had no effect on the extent of

absorption of Ezetimibe when administered as ZETIA 10-mg tablets. The Cmax value of

Ezetimibe was increased by 38% with consumption of high-fat meals. ZETIA can be

administered with or without food.

Distribution:

Ezetimibe and ezetimibe-glucuronide are highly bound ( > 90%) to human plasma proteins.





Metabolism and Excretion

Ezetimibe is primarily metabolized in the small intestine and liver via glucuronide conjugation

(a phase II reaction) with subsequent biliary and renal excretion. Minimal

oxidative metabolism (a phase I reaction) has been observed in all species evaluated.

In humans, Ezetimibe is rapidly metabolized to ezetimibe-glucuronide. Ezetimibe and

Ezetimibe-glucuronide are the major drug-derived compounds detected in plasma, constituting

approximately 10 to 20% and 80 to 90% of the total drug in plasma, respectively. Both

Ezetimibe and ezetimibe-glucuronide are eliminated from plasma with a half-life of

approximately 22 hours for both Ezetimibe and ezetimibe-glucuronide. Plasma concentration-

time profiles exhibit multiple peaks, suggesting enterohepatic recycling.

Following oral administration of 14C-ezetimibe (20 mg) to human subjects, total Ezetimibe

(Ezetimibe + ezetimibe-glucuronide) accounted for approximately 93% of the total radioactivity

in plasma. After 48 hours, there were no detectable levels of radioactivity in the plasma.

Approximately 78% and 11% of the administered radioactivity were recovered in the faces and

urine, respectively, over a 10-day collection period. Ezetimibe was the major component in

faces and accounted for 69% of the administered dose, while ezetimibe-glucuronide was the

major component in urine and accounted for 9% of the administered dose.

Table 1 Physical properties of Ezetimibe

S.NO PARAMETER EZETIMIBE

1 Molecular formula

C24H21F2NO3

2 Structural Formula

(3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-

fluorophenyl)-3-hydroxypropyl]-4-(4-

hydroxyphenyl)azetidin-2-one

2 Molecular weight 409.4 g/mol

3 Categories

Anticholesteremic Agents

Cholesterol Absorption Inhibitors

4 Solubility

Ezetimibe is a white, crystalline powder that is

freely to very soluble in ethanol, methanol, and

acetone and practically insoluble in water

5 Half life 22 hours



http://en.wikipedia.org/wiki/Carbon

http://en.wikipedia.org/wiki/Hydrogen

http://en.wikipedia.org/wiki/Fluorine

http://en.wikipedia.org/wiki/Nitrogen





Drug profile of Fenofibrate:

Fenofibrate is a drug of the fibrate class. Fenofibrate was developed by Groupe Fournier SA,

before it was acquired in 2005 by Solvay Pharmaceutical, a business unit owned by the Belgian

corporation, Solvay S.A. In 2009 Solvay Pharmaceutical was acquired by Abbott Laboratories. It

is mainly used to reduce cholesterol levels in patients at risk of cardiovascular disease. Like other

fibrates, it reduces both low-density lipoprotein (LDL) and very low density lipoprotein (VLDL)

levels, as well as increasing high-density lipoprotein (HDL) levels and reducing triglycerides

level. It also appears to have a beneficial effect on the insulin resistance featured by the

metabolic syndrome. It is used alone or in conjunction with statins in the treatment of

hypercholesterolemia and hypertriglyceridemia.

Fig 1 Fenofibrate

Mechanism of Action

The active moiety of TRICOR is fenofibric acid. The pharmacological effects of fenofibric acid

in both animals and humans have been extensively studied through oral administration of

Fenofibrate.

The lipid-modifying effects of fenofibric acid seen in clinical practice have been explained in

vivo in transgenic mice and in vitro in human hepatocyte cultures by the activation of

peroxisome proliferator activated receptor α (PPARα). Through this mechanism, fenofibrate

increases lipolysis and elimination of triglyceride-rich particles from plasma by activating

lipoprotein lipase and reducing production of apoprotein C-III (an inhibitor of lipoprotein lipase

activity).

The resulting decrease in TG produces an alteration in the size and composition of LDL from

small, dense particles (which are thought to be atherogenic due to their susceptibility to

oxidation), to large buoyant particles. These larger particles have a greater affinity

http://en.wikipedia.org/wiki/Medication

http://en.wikipedia.org/wiki/Fibrate

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http://en.wikipedia.org/wiki/Business

http://en.wikipedia.org/wiki/Belgium

http://en.wikipedia.org/wiki/Corporation

http://en.wikipedia.org/wiki/Solvay_%28company%29

http://en.wikipedia.org/wiki/Abbott_Laboratories

http://en.wikipedia.org/wiki/Cholesterol

http://en.wikipedia.org/wiki/Cardiovascular_disease

http://en.wikipedia.org/wiki/Low-density_lipoprotein

http://en.wikipedia.org/wiki/Very_low_density_lipoprotein

http://en.wikipedia.org/wiki/High-density_lipoprotein

http://en.wikipedia.org/wiki/Triglycerides

http://en.wikipedia.org/wiki/Insulin_resistance

http://en.wikipedia.org/wiki/Metabolic_syndrome

http://en.wikipedia.org/wiki/Statin

http://en.wikipedia.org/wiki/Hypercholesterolemia

http://en.wikipedia.org/wiki/Hypertriglyceridemia



for cholesterolreceptors and are catabolized rapidly. Activation of PPARα also induces an

increase in the synthesis of apolipoproteins A-I, A-II and HDL-cholesterol.

Fenofibrate also reduces serum uric acid levels in hyperuricemic and normal individuals by

increasing the urinary excretion of uric acid.

Pharmacodynamics

A variety of clinical studies have demonstrated that elevated levels of total-C, LDL-C, and apo

B, an LDL membrane complex, are associated with human atherosclerosis. Similarly, decreased

levels of HDL-C and its transport complex, apolipoprotein A (apo AI and apo AII) are

associated with the development of atherosclerosis. Epidemiologic investigations have

established that cardiovascular morbidity and mortality vary directly with the level of total-C,

LDL-C, and TG, and inversely with the level of HDL-C. The independent effect of raising

HDL-C or lowering triglycerides (TG) on the risk of cardiovascular morbidity and mortality has

not been determined.

Fenofibric acid, the active metabolite of fenofibrate, produces reductions in total

cholesterol, LDL cholesterol, apolipoprotein B, total triglycerides and triglyceride rich

lipoprotein (VLDL) in treated patients. In addition, treatment with fenofibrate results in

increases in high density lipoprotein (HDL) and apolipoproteins apoAI and apoAII.

Pharmacokinetics

Plasma concentrations of fenofibric acid after administration of three 48 mg or one 145 mg

tablets are equivalent under fed conditions to one 200 mg micronized fenofibrate capsule.

Fenofibrate is a pro-drug of the active chemical moiety fenofibric acid. Fenofibrate is converted

by ester hydrolysis in the body to fenofibric acid which is the active constituent measurable in

the circulation.

Absorption

The absolute bioavailability of fenofibrate cannot be determined as the compound is virtually

insoluble in aqueous media suitable for injection. However, fenofibrate is well absorbed from

the gastrointestinal tract. Following oral administration in healthy volunteers, approximately

60% of a single dose of radiolabelled fenofibrate appeared in urine, primarily as fenofibric acid

and its glucuronate conjugate, and 25% was excreted in the feces. Peak plasma levels of

fenofibric acid occur within 6 to 8 hours after administration.

http://www.rxlist.com/cholesterol/article.htm






Exposure to fenofibric acid in plasma, as measured by Cmax and AUC, is not significantly

different when a single 145 mg dose of fenofibrate is administered under fasting or nonfasting

conditions.

Distribution

Upon multiple dosing of fenofibrate, fenofibric acid steady state is achieved within 9 days.

Plasma concentrations of fenofibric acid at steady state are approximately double of those

following a single dose. Serum protein binding was approximately 99% in normal and

hyperlipidemic subjects.

Metabolism

Following oral administration, fenofibrate is rapidly hydrolyzed by esterases to the active

metabolite, fenofibric acid; no unchanged fenofibrate is detected in plasma.

Fenofibric acid is primarily conjugated with glucuronic acid and then excreted in urine. A small

amount of fenofibric acid is reduced at the carbonyl moiety to a benzhydrol metabolite which is,

in turn, conjugated with glucuronic acid and excreted in urine.

In vivo metabolism data indicate that neither fenofibrate nor fenofibric acid undergo oxidative

metabolism (e.g., cytochrome P450) to a significant extent.

Elimination

After absorption, fenofibrate is mainly excreted in the urine in the form of metabolites,

primarily fenofibric acid and fenofibric acid glucuronide. After administration of radiolabelled

fenofibrate, approximately 60% of the dose appeared in the urine and 25% was excreted in the

feces.





Table 1.2 Physical properties of Ezetimibe

S.NO PARAMETER EZETIMIBE

1 Molecular formula

C20H21O4Cl

2 Structural Formula

propan-2-yl 2-{4-[(4-

chlorophenyl)carbonyl]phenoxy}-2-

methylpropanoate

2 Molecular weight 360.831 g/mol

3 Categories

Antilipemic Agents

Fribic Acid Derivatives

4 Solubility

Fenofibrate is soluble in organic solvents such as ethanol, DMSO, and dimethyl formamide. It is insoluble in water.

5 Half life

20 hours

Materials and reagents used

S.No. Name Grade Make

1 Ezetimibe - -

2 Fenofibrate - -

3 Ezetimibe and Fenofibrate SR

Tablets

- -

4 Potassium dihydrogen phosphate HPLC Merck

5 Ammonium Acetate HPLC Merck

6 Ortho phosphoric acid HPLC Merck

7 Glacial Acetic acid HPLC Merck

8 Acetonitrile gradient HPLC Rankem

9 Methanol HPLC Merck

10 Tetrahydrofuran HPLC Merck

11 Triethylamine HPLC Merck

12 Water HPLC Milli Q (purification system)



Table 1.3: Instruments used

S.No. Name of Instrument 1 Waters HPLC with PDA Detector

2 Empower-2 Software 3 Rankem Pipette 4 Metrohm pH meter

5 Metrohm Electronic Balance FX -400

6 Elma S 300H Ultra sonicator

7 Hermle Z 323 centrifuge

8 PG UV- Visible Spectrophotometer- T60

9 Special bandwidth Quartz 2mm and 10mm

Method Development:

Selection of Chromatographic technique:

The selection of the method depends on the physico-chemical properties of the drug substance

i.e. on the nature of the sample, molecular weight and solubility.

As the drug is moderately polar, the reverse phase chromatographic technique was selected from

the knowledge of properties of the compound and reverse phase chromatographic conditions

needs to be optimized.

Preparation of buffer:

Diluted Ortho phosphoric Acid Solution:

Diluted 10 mL of the ortho phosphoric acid into 50 mL with water and mixed well.

0.01M Potassium dihydrogen phosphate buffer with pH 3.80:

Accurately weighed 1.36gm of Potassium dihydrogen ortho phosphate in a 1000ml of

Volumetric flask add about 900ml of milli-Q water added and degas to sonicate and finally make

up the volume with water and pH adjusted to 3.8 with dil. OPA.

Diluent Preparation:

Used Methanol.

Standard Preparation:

Standard preparation for Ezetimibe:

Accurately Weighed and transferred 10 mg of Ezetimibe working Standards into 50 ml clean

dry volumetric flask, add 3/4 th of diluent , sonicated for 5 minutes and make up to the final

volume with diluent.



Standard preparation for Fenofibrate:

Accurately Weighed and transferred 29 mg of Fenofibrate working Standards into 10 ml clean



Standard Solution Final:

From the above stock solutions pipetted 1ml of the each solution into a 10ml volumetric flask

and made up to 10ml with diluent and mixed well.

Sample Preparation:

Weighed 10 tablets and calculated the average weight of each tablet.

Then the weight equivalent to 5 tablets was transferred into a 50 mL volumetric flask, 35mL of

diluent added and sonicated for 25 min with intermediate shaking, allowed to cool at room

temperature and made up to volume with diluent.

Centrifuged a portion of above solution at 5000 rpm, in a centrifuge tube with cap, for about 10

minutes and filtered the solution.

From the filtered solution pipetted 1ml of the solution into a 50 ml volumetric flask and made up

to 50ml with diluent.

Linearity:

Linearity solutions are prepared such that 0.25ml, 0.5ml, 0.75ml, 1ml, 1.25ml, 1.5ml from the

Stock solutions of Ezetimibe and Fenofibrate are taken in to 6 different volumetric flasks and

diluted to 10ml with diluents to get 5ppm, 10ppm, 15ppm, 20ppm, 25ppm, 30ppm of Ezetimibe

and 72.5ppm, 145ppm, 217.5ppm, 290ppm, 362.5ppm, 435ppm of Fenofibrate.

Precision:














Sample Preparation:









Accuracy:












Sample preparation for Recovery:

Test preparation at 50% Level:

Weighed 10 tablets and calculated the average weight of each tablet and crushed the tablets.

Then powder weight of 750mg was transferred into a 25 mL volumetric flask, 20mL of diluent

added and sonicated for 25 min, further the volume made up with diluent. Centrifuged a portion



of above solution at 5000 rpm, in a centrifuge tube with cap, for about 10 minutes and filtered

the solution.

From the filtered solution 1ml was pipetted out into a 10 ml volumetric flask and made up to

10ml with diluent and mixed well.






the solution.


10ml with diluent.






the solution.


10ml with diluent.

TEST METHOD

Diluted Orthophosphoric Acid Solution:

Diluted 10 mL of the orthophosphoric acid into 50 mL with water and mixed well.

0.01M Potassium dihydrogen phosphate buffer with pH 3.80:

Accurately weighed 1.36gm of Potassium dihydrogen ortho phosphate in a 1000ml of

Volumetric flask add about 900ml of milli-Q water added and degas to sonicate and finally make

up the volume with water and pH adjusted to 3.8 with dil. OPA.

Preparation of Mobile phase:

Mix 600 volume of pH 3.80 buffer and 400 volume of Acetonitrile and degas.

Preparation of Diluent:

100% Methanol.



Preparation of Blank: Diluent is used as Blank.

Standard Preparation:












Sample Preparation:









Procedure:

Inject 10µL Test preparation (in duplicate), record the chromatogram and measure the Peak

response.

System suitability:

Inject 10µL of blank and standard solution (five times) record the chromatograms and measure

the peaks response.

The tailing factor for the Ezetimibe and Fenofibrate peak should be not more than 2.0 from the

chromatogram of standard solution.

The Plate count for Ezetimibe and Fenofibrate peak should be not less than 3000 from the

chromatogram of standard solution.



The Relative standard deviation of Ezetimibe and Fenofibrate peak area from five replicate

injections of standard solution should be not more than 2.0%.

Calculations:

A x Ws x 1 x 50 x 50 x P x 100

Assay of Ezetimibe = --------------------------------------------

as % of Label claim S x 50 x 10 x N x 1 x 100 x LC

A x Ws x 1 x 50 x 50 x P x 100

Assay of Fenofibrate = --------------------------------------------

as % of Label claim S x 10 x 10 x N x 1 x 100 x LC

A = Average Peak area of Ezetimibe and Fenofibrate from test preparation,

S = Average peak area of Ezetimibe and Fenofibrate from Standard preparation.

Ws = Weight of Ezetimibe and Fenofibrate working/reference standard taken for

standard preparation in ‘mg’

P = Potency of Ezetimibe and Fenofibrate sodium Standard in % on as is basis.

L C = Labeled amount of Ezetimibe and Fenofibrate, in mg, per tablet.

N = Number of tablets taken for test preparation

RESULTS AND DISCUSSIONS

1. System suitability: All the system suitability parameters are within range and

satisfactory as per ICH guidelines.

Fig : 1.2Chromatogram of blank.



Fig : 1.3 Typical chromatogram of Ezetimibe and Fenofibrate

2. Linearity: Six Linear concentrations of Ezetimibe (5ppm to 30ppm) and Fenofibrate

(72.5ppm to 435ppm) are prepare and Injected. Regression equation of the the Ezetimibe

and Fenofibrate are found to be, y = 42418x + 5004 and y = 18765x + 2704. And

regression co-efficient was 0.999.

Table: 1.5Calibration data of RC by RP-HPLC method.

S.no

Concentration

Ezetimibe

(µg/ml

)

Responce

(

m

V

)

Concentration

Fenofibrate

Responce

1 0 0 0 0

2 5 215556 72.5 1330216

3 10 430154 145 2752063

4 15 636958 217.5 4056127

5 20 869696 290 5493558

6 25 1079556 362.5 6858560

7 30 1256985 435 8098366



Fig : 1.4 Calibration curve of Ezetimibe

Fig : 1.5Calibration curve of Fenofibrate



Fig : 1.6 Linearity 25% Chromatogram of Ezetimibe and Fenofibrate

Fig : 1.7 Linearity 50% Chromatogram Ezetimibe and Fenofibrate





Fig : 2.0Linearity 125% Chromatogram Ezetimibe and Fenofibrate

Fig : 2.1Linearity 150% Chromatogram Ezetimibe and Fenofibrate



3. Precision:

Intraday precision (Repeatability): Intraday Precision was performed and % RSD for

Ezetimibe and Fenofibrate were found to be 0.13% and 0.19% respectively.

Table: 1.4 Repeatability results Ezetimibe and Fenofibrate.

Sr. No. Ezetimibe Fenofibrate

1 864985 5507140

2 863536 5508008

3 864958 5533177

4 863268 5523937

5 862154 5520321

6 864154 5508560

Mean 863843 5516857

Std. Dev. 1088.274 10677.39

%RSD 0.13 0.193541

*Average of six determinations

Fig : 2.2. Repeatability Chromatogram of Ezetimibe and Fenofibrate

Intermediate precision: Intraday Precision was performed and % RSD for Ezetimibeand

Fenofibrate were found to be 0.47% and 0.06% respectively.



Table: 1.4Intermediate precision results for RC by RP-HPLC.

Sr. No. Ezetimibe Fenofibrate

1 866279 5499784

2 866653 5505251

3 869438 5505100

4 860312 5503928

5 860288 5498009

Mean 864594 5502414

Std. Dev. 4105.49 3311.959

%RSD 0.47 0.060191

Fig : 2.3 Inter Day precision Chromatogram of Ezetimibe and Fenofibrate

4. Accuracy:

Accuracy: Three concentrations 50%, 100%, 150%, were injected in a triplicate manner and

amount Recovered and % Recovery were displayed in Table 6.5.



Table: 1.5 Accuracy results of RC by RP-HPLC

Sample

Amount added

(µg/ml)

Amount

Recovered

(µg/ml)

Recovery

(%)

% RSD

Ezetimibe

10

9.98

99.9

0.19

20

20.04

100.201

0.59

30

29.88

99.61

0.55

Fenofibrate

145

145.29

100.2

0.47

290

289.68

99.89

0.13

435

434.9

100.00

0.16

Fig : 2.5 Accuracy 50% Chromatogram of Ezetimibe and Fenofibrate

Fig : 2.6 Accuracy 100% Chromatogram of Ezetimibe and Fenofibrate



Fig : 2.7Accuracy 150% Chromatogram of Ezetimibe and Fenofibrate

1. LOD: Limit of ditection was calculated by intercept method and LOD for Ezetimibeand

Fenofibrate wre found to be 0.39 and 0.48 respectively.

Fig : 2.8LOD Chromatogram of Ezetimibe and Fenofibrate

2. LOQ: Limit of Quantification was calculated by intercept method and LOQ for

Ezetimibeand Fenofibrate wre found to be 1.18 and 1.44 respectively.

Fig : 2.9 LOQ Chromatogram of Ezetimibe and Fenofibrate.



3. Robustness: Small deliberate changes in the method like Flow rate, mobile phase ratio

and temperature are made but there were no recognized change in the result and are

within range as per ICH guideline.

Table 1.6 Robustness data of Ezetimibe and Fenofibrate

S.NO

Robustness condition

Ezetimibe

%RSD

Fenofibrate

%RSD

1

Flow minus

0.8

0.11

2

Flow Plus

0.76

0.85

3

Mobile phase minus

0.85

1.3

4

Mobile phase Plus

0.9

1.1

5

Temperature minus

0.3

0.4

6

Temperature Plus

0.5

0.1

Typical chromatograms of the Robustness parameter as below

2.6 Flow minus of Ezetimibe and Fenofibrate



2.7 Flow plus of Ezetimibe and Fenofibrate

2.8 Mobile phase minus of Ezetimibe and Fenofibrate



2.9 Mobile phase Plus of Ezetimibe and Fenofibrate

2.9 Temperature Minus of Ezetimibe and Fenofibrate



3.0Temperature Plus of Ezetimibe and Fenofibrate

Assay: Standard preparations are made from the API and Sample Preparations are from

Formulation. Both sample and standards are injected six homogeneous samples. Drug in the

formulation was estimated by taking the standard as the reference. The Average %Assay was

calculated and found to be 99.94 and 99.995 for Ezetimibe and Fenofibrate respectively.

Table 1.6 Assay of Tablet

S. No. Ezetimibe

%Assay

Fenofibrate

%Assay

1 100.0702 99.81923

2 99.90257 99.83496

3 100.0671 100.2912

4 99.87156 100.1237

5 99.74268 100.0581

6 99.97406 99.84496

AVG 99.94 99.995

STDEV 0.1259 0.194

%RSD 0.13 0.194



3.1Assay of Tablet

SUMMARY

Table: 1.7 Summary tables of Ezetimibe and Fenofibrate

Parameters Ezetimibe Fenofibrate

alibration range (mcg / ml) 5 -30 ppm 72.5 – 435 ppm

Optimized wavelength 245 nm 245 nm

Mobile phase

(Acetonitrile : Buffer)

Buffer:Acetonitrile

(60:40)

Buffer:Acetonitrile

(60:40)

Retention time 3.0 2.1

Regression equation (Y*) y = 42418x + 5004. y = 18765x + 2704.

Correlation coefficient(r2)

0.999 0.999

Precision (% RSD*) 0.13

0.19

Limit of Detection (mcg / ml) 0.39 0.48

Limit of Quantitation (mcg / ml) 1.18 1.44

CONCLUSION

A simple, Accurate, precise method was developed for the simultaneous estimation of the

Ezetimibe and Fenofibrate in Tablet dosage form. Retention times of Ezetimibe and Fenofibrate

were found to be 3.0min and 2.3 min. %RSD of the Ezetimibe and Fenofibrate were and found to

be 0.13 and 0.19 respectively. %Recover was Obtained as 99.9 and 100.031 for Ezetimibe and

Fenofibrate respectively. LOD, LOQ values are obtained from regression equations of Ezetimibe

and Fenofibrate were 0.39, 1.18 and 0.48, 1.44 respectively. Regression equation of Ezetimibe is

y = 42418x + 5004, and of Fenofibrate is y = 18765x + 2704. Regression co-efficient was 0.999.



Retention times are decreased and that run time was decreased so the method developed was

simple and economical that can be adopted in regular Quality control test in Industries.

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