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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:
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.
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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,
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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
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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.
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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
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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
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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.
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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.
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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)
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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.
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Standard preparation for Fenofibrate:
Accurately Weighed and transferred 29 mg of Fenofibrate working Standards into 10 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 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:
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
dry volumetric flask, add 3/4 th of diluent , sonicated for 5 minutes and make up to the final
volume with diluent.
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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.
Accuracy:
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
dry volumetric flask, add 3/4 th of diluent , sonicated for 5 minutes and make up to the final
volume with diluent.
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 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
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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.
Test preparation at 100% Level:
Weighed 10 tablets and calculated the average weight of each tablet and crushed the tablets.
Then powder weight of 1500mg 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.
Test preparation at 100% Level:
Weighed 10 tablets and calculated the average weight of each tablet and crushed the tablets.
Then powder weight of 2250mg 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.
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.
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Preparation of Blank: Diluent is used as Blank.
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
dry volumetric flask, add 3/4 th of diluent , sonicated for 5 minutes and make up to the final
volume with diluent.
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.
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.
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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.
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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
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Fig : 1.4 Calibration curve of Ezetimibe
Fig : 1.5Calibration curve of Fenofibrate
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Fig : 1.6 Linearity 25% Chromatogram of Ezetimibe and Fenofibrate
Fig : 1.7 Linearity 50% Chromatogram Ezetimibe and Fenofibrate
Fig : 1.8 Linearity 75% Chromatogram Ezetimibe and Fenofibrate
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Fig : 1.9 Linearity 100% Chromatogram Ezetimibe and Fenofibrate
Fig : 2.0Linearity 125% Chromatogram Ezetimibe and Fenofibrate
Fig : 2.1Linearity 150% Chromatogram Ezetimibe and Fenofibrate
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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.
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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.
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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
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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.
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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
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2.7 Flow plus of Ezetimibe and Fenofibrate
2.8 Mobile phase minus of Ezetimibe and Fenofibrate
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2.9 Mobile phase Plus of Ezetimibe and Fenofibrate
2.9 Temperature Minus of Ezetimibe and Fenofibrate
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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
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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.
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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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