Assignment Clinical Norliza Baharom

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MASTER OF SCIENCE

ANALYTICAL CHEMISTRY AND INSTRUMENTAL ANALYSIS

SCGS 6323

CLINICAL AND PHARMACEUTICAL

“Development and Validation of Stability Indicating HPLC Methods for Quantitative

Determination of Pravastatin, Fluvastatin, Atorvastatin, and Rosuvastatin in

Pharmaceuticals”

LECTURER:

PROF DR CHUAH CHENG HOCK

PREPARED BY

NORLIZA BINTI BAHAROM

SUBMITTED

24 APRIL 2012



Development and Validation of Stability Indicating HPLC Methods for Quantitative

Determination of Pravastatin, Fluvastatin, Atorvastatin, and Rosuvastatin in

Pharmaceuticals

INTRODUCTION

1.0 STATIN

Statins (or HMG-CoA reductase inhibitors) are a class of drugs that reduce cholesterol

in individuals who have dyslipidemia (abnormal fats in the blood) and thus are at risk for

cardiovascular disease. Dyslipidemia may involve an elevation of total cholesterol, a

reduction of low density lipoprotein (LDL) cholesterol and/or triglycerides, or a reduction

of high density lipoprotein (HDL) cholesterol in blood. Statins work by blocking theenzyme in the liver that is responsible for making cholesterol. This enzyme is called

hydroxy-methylglutaryl-coenzyme A reductase (HMG-CoA reductase).

Cholesterol is described as a soft wax-like fatty substance that is found in the blood

stream and in cells. It is important to note that cholesterol is a naturally existing substance

in all individuals from birth and its presence is actually necessary for promoting an overall

healthy body. About 75% of cholesterol is produced by the liver and other cells in the

body, and 25% comes from food.

Cholesterol can have a negative impact on health, when there is too much bad LDL

cholesterol circulating in the blood system. Contributing factors to high LDL levels may

be unhealthy foods, genetics, lack of physical activity, and smoking. Triglycerides are a

form of body fat that increases due to being overweight or obese, and physical inactivity,

amongst others factors. High triglycerides levels may contribute towards heart disease and

diabetes. HDL cholesterol is known as good cholesterol as it protects the heart against

heart attacks: it is important to have an HDL level greater than 40 mg/dL.

As previously mentioned, cholesterol contributes to cardiovascular disease as well as

neurological and peripheral vascular disease. The way this occurs is by atherosclerosis, a

condition, where over a course in time, cholesterol builds up in arteries and forms

hardened plaques. If plaques rupture, blood clots may form on the plaque and block the

arteries. The clots also may dislodge and circulate within the body, block distant arteries,



and ultimately reduce the flow of blood and oxygen through the arteries and to organs.

Clots situated in the coronary arteries may give rise to angina or a heart attack. Clots in

the carotid artery (the artery that supplies blood to the brain) may result in a stroke, and

clots affecting the lower extremities such as the legs may result in peripheral arterial

disease.

Statins are among the most commonly prescribed drugs in medicine. The statins (or

HMG-CoA reductase inhibitors) are a class of drugs that lower cholesterol levels in

people. They lower cholesterol by inhibiting the enzyme HMG-CoA reductase, which is

the rate-limiting enzyme of the mevalonate pathway of cholesterol synthesis. Inhibition of

this enzyme in the liver results in decreased cholesterol synthesis as well as increased

synthesis of LDL receptors, resulting in an increased clearance of low-density lipoprotein

(LDL) from the bloodstream. The first results can be seen after one week of use and the

effect is maximal after four to six weeks. Statins act by competitively inhibiting HMG-

CoA reductase, the first committed enzyme of the HMG-CoA reductase pathway. Because

statins are similar to HMG-CoA on a molecular level they take the place of HMG-CoA in

the enzyme and reduce the rate by which it is able to produce mevalonate, the next

molecule in the cascade that eventually produces cholesterol, as well as a number of other

compounds. This ultimately reduces cholesterol via several mechanisms. The types of

statin drugs are, Lipitor (atorvastatin), Lescol (fluvastatin), Mevacor (lovastatin), Livalo

(pitavastatin), Pravachol (pravastatin), Zocor (simvastatin), Crestor (rosuvastatin)

1.1 Atorvastatin (Lipitor)

1.1.1 Chemistry of atorvastatin

Lipitor is a synthetic lipid-lowering agent. Atorvastatin is an inhibitor

of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase.

This enzyme catalyzes the conversion of HMG-CoA to mevalonate, an

early and rate-limiting step in cholesterol biosynthesis. Atorvastatin

calcium is [R-(R*, R*)]-2-(4-fluorophenyl)-β, δ-dihydroxy-5-(1-

methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1Hpyrrole-1-

heptanoic acid, calcium salt (2:1) trihydrate. The empirical formula of

atorvastatin calcium is (C33H34FN2O5)2Ca•3H2O and its molecular

weight is 1209.42. Its structural formula is:



Figure 1: the chemical structure of Atorvastatin

Atorvastatin calcium is a white to off-white crystalline powder

that is insoluble in aqueous solutions of pH 4 and below. Atorvastatin

calcium is very slightly soluble in distilled water, pH 7.4 phosphate

buffer, and acetonitrile; slightly soluble in ethanol; and freely soluble in

methanol. Lipitor tablets for oral administration contain 10, 20, 40, or

80 mg atorvastatin and the following inactive ingredients: calciumcarbonate, USP; candelilla wax, FCC; croscarmellose sodium, NF;

hydroxypropyl cellulose, NF; lactose monohydrate, NF; magnesium

stearate, NF; microcrystalline cellulose, NF; Opadry White YS-1-7040

(hypromellose, polyethylene glycol, talc, titanium dioxide); polysorbate

80, NF; simethicone emulsion.

Figure 2 : Pack and tablet of Atorvastatin (Lipitor) 40mg in

pharmaceutical industry.



1.1.2 uses of Atorvastatin

1.1.2.1 Atorvastatin is a cholesterol-lowering medication that blocks

the production of cholesterol (a type of fat) in the body.

1.1.2.2 Atorvastatin reduces low-density lipoprotein (LDL) cholesterol

and total cholesterol in the blood. Lowering your cholesterol can

help prevent heart disease and hardening of the arteries,

conditions that can lead to heart attack, stroke, and vascular

disease.

1.1.2.3 Atorvastatin is used to treat high cholesterol. Atorvastatin is also

used to lower the risk of stroke, heart attack, or other heart

complications in people with coronary heart disease or type 2

diabetes.

1.1.3 The side effects of the Atorvastatin

1.1.3.1 Remember that your doctor has prescribed this medication

because he or she has judged that the benefit to you is greater

than the risk of side effects. Many people using this medication

do not have serious side effects.

1.1.3.2 This drug may infrequently cause muscle problems (which can

rarely lead to a very serious condition called rhabdomyolysis).

Tell your doctor immediately if you develop any of these

symptoms: muscle pain/tenderness/weakness (especially with

fever or unusual tiredness), change in the amount of urine.

1.1.3.3 This medication may rarely cause liver problems. If you notice

any of the following rare but serious side effects, tell your

doctor immediately: yellowing eyes/skin, dark urine, severe

stomach/abdominal pain, persistent nausea/vomiting.

1.1.3.4 A very serious allergic reaction to this drug is rare. However,

seek immediate medical attention if you notice any symptoms of

a serious allergic reaction, including: rash, itching/swelling

(especially of the face/tongue/throat), severe dizziness, trouble

breathing.



1.1.4 The precautions when taking the pravastatin

1.1.4.1 If you have any other allergies. This product may contain

inactive ingredients, which can cause allergic reactions or other

problems. Talk to your pharmacist for more details.

1.1.4.2 Before using this medication, tell your doctor or pharmacist

your medical history, especially of: liver disease, kidney

disease, alcohol use.

1.1.4.3 Before having surgery, tell your doctor or dentist about all the

products you use (including prescription drugs, nonprescription

drugs, and herbal products).

1.1.4.4 Limit alcoholic beverages. Daily use of alcohol may increase

your risk for liver problems, especially when combined with

atorvastatin. Ask your doctor or pharmacist for more

information.

1.1.4.5 Older adults may be more sensitive to the side effects of this

drug, especially muscle problems.

1.1.4.6 This medication must not be used during pregnancy.

Atorvastatin may harm an unborn baby. Therefore, it is

important to prevent pregnancy while taking this medication.

Consult your doctor for more details and to discuss using at least

2 reliable forms of birth control (such as condoms, birth control

pills) while taking this medication. If you become pregnant or

think you may be pregnant, tell your doctor immediately.

1.1.4.7 It is unknown if this medication passes into breast milk. Because

of the possible risk to the infant, breast-feeding while using this

drug is not recommended. Consult your doctor before breast-

feeding.

1.2 Pravastatin (Pravachol)

1.2.1 Chemistry of Pravastatin

Pravachol (pravastatin sodium) is one of a class of lipid-lowering

compounds, the statins, which reduce cholesterol biosynthesis. These

agents are competitive inhibitors of HMG-CoA reductase, the enzymecatalyzing the early rate-limiting step in cholesterol biosynthesis,



conversion of HMG-CoA to mevalonate. Pravastatin sodium is

designated chemically as 1-Naphthalene-heptanoic acid,

1,2,6,7,8,8ahexahydro-β,δ,6-trihydroxy-2 methyl-8-(2-methyl-1-

oxobutoxy)-, monosodium salt,[1S[1α(βS*,δS*), 2α,6α,8β(R*),8aα]].

Figure 3; the chemical structure for Pravastatin

Pravastatin sodium is an odorless, white to off-white, fine or

crystalline powder. It is a relatively polar hydrophilic compound with apartition coefficient (octanol/water) of 0.59 at a pH of 7.0. It is soluble

in methanol and water ( > 300 mg/mL), slightly soluble in isopropanol,

and practically insoluble in acetone, acetonitrile, chloroform, and ether.

Pravachol is available for oral administration as 10 mg, 20 mg,

40 mg, and 80 mg tablets. Inactive ingredients include: croscarmellose

sodium, lactose, magnesium oxide, magnesium stearate,

microcrystalline cellulose, and povidone. The 10 mg tablet also

contains Red Ferric Oxide, the 20 mg and 80 mg tablets also contain

Yellow Ferric Oxide, and the 40 mg tablet also contains Green Lake

Blend (mixture of D&C Yellow No. 10-Aluminum Lake and FD&C

Blue No. 1-Aluminum Lake).



Figure 4: 10 mg of Pravachol

1.2.2 Uses of Pravastatin

1.2.2.1 Pravastatin is a cholesterol-lowering medication that blocks the

production of cholesterol (a type of fat) in the body.

1.2.2.2 Pravastatin reduces low-density lipoprotein (LDL) cholesterol




disease.

1.2.2.3 Pravastatin is used to treat high cholesterol. Pravastatin is also

used to lower the risk of stroke, heart attack, or other heartcomplications in people with coronary heart disease.

1.2.3 The side effects of the pravastatin

1.2.3.1 The effect when allergies with the pravachol, hives, difficulty

breathing, swelling of your face, lips, tongue or throat.

1.2.3.2 the serious side effects are, chest pain (muscle pain, tenderness,

or weakness with fever or flu symptoms and dark colored urine),

nausea, stomach pain, low fever, loss of appetite, dark urine,

clay-colored stools, jaundice (yellowing of the skin or eyes).

1.2.3.3 Less serious side effects are, mild stomach pain, constipation,

diarrhea, heartburn, gas, bloating, upset stomach, tired feeling,

headache, dizziness, stuffy nose, cold or flu symptoms, skin rash

or general pain.

1.2.4 the precautions when taking the pravastatin

1.2.4.1 This medication can cause birth defects in an unborn baby. Do

not use if you are pregnant. Use an effective form of birth



control, and tell your doctor if you become pregnant during

treatment.

1.2.4.2 Do not take pravastatin if you have liver disease, or if you are

breast-feeding.

1.2.4.3 Before taking pravastatin, tell your doctor if you have diabetes,

underactive thyroid, kidney disease, a muscle disorder, or if you

drink 3 or more alcoholic beverages per day.

1.2.4.4 Avoid eating foods that are high in fat or cholesterol. Pravastatin

will not be as effective in lowering your cholesterol if you do

not follow a cholesterol-lowering diet plan.

1.2.4.5 Avoid drinking alcohol while taking pravastatin. Alcohol can

raise triglyceride levels, and may also damage your liver while

you are taking pravastatin.

1.2.4.6 There may be other drugs that can interact with pravastatin. Tell

your doctor about all the prescription and over-the-counter

medications you use. This includes vitamins, minerals, herbal

products, and drugs prescribed by other doctors.

1.3 Fluvastatin (Lescol)

1.3.1 chemistry of Fluvastatin

Lescol is a water-soluble cholesterol lowering agent which acts through

the inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA)

reductase. Fluvastatin sodium is [R*,S*-(E)]-(±)-7-[3-(4-fluorophenyl)-

1-(1-methylethyl)-1H-indol-2-yl]-3,5-dihydroxy-6-heptenoic acid,

monosodium salt. The empirical formula of fluvastatin sodium is

C24H25FNO4•Na, its molecular weight is 433.46. This molecular

entity is the first entirely synthetic HMG-CoA reductase inhibitor, and

is in part structurally distinct from the fungal derivatives of this

therapeutic class.



Figure 5: The Chemical structure of Fluvastatin

Fluvastatin sodium is a white to pale yellow, hygroscopic

powder soluble in water, ethanol and methanol. LESCOL is supplied as

capsules containing fluvastatin sodium, equivalent to 20 mg or 40 mg of

fluvastatin, for oral administration. LESCOL XL is supplied as

extended-release tablets containing fluvastatin sodium, equivalent to 80

mg of fluvastatin, for oral administration.

Figure 6: 40mg Lescol

1.3.2 Uses of Fluvastatin

1.3.2.1 Fluvastatin is a cholesterol-lowering medication that blocks the

production of cholesterol (a type of fat) in the body.



1.3.2.2 Fluvastatin reduces low-density lipoprotein (LDL) cholesterol




disease.

1.3.3 the Side Effects of Fluvastatin

1.3.3.1 The signs of an allergic reaction: hives; difficulty breathing;

swelling of your face, lips, tongue, or throat.

1.3.3.2 these serious side effects (muscle pain, tenderness, or weakness

with fever or flu symptoms and dark colored urine)

1.3.3.3 Less serious side effects may include (mild stomach pain, gas,

bloating, stomach upset, heartburn), nausea, constipation or

diarrhea.

1.3.4 The precautions when taking the Fluvastatin

1.3.4.1 Before taking fluvastatin, tell your doctor or pharmacist if you

are allergic to it. This product may contain inactive ingredients,

which can cause allergic reactions or other problems.









fluvastatin. Ask your doctor or pharmacist for more information.



1.3.4.6 This medication must not be used during pregnancy. Fluvastatin

may harm an unborn baby. Therefore, it is important to prevent

pregnancy while taking this medication.

1.3.4.7 This medication passes into breast milk and may have

undesirable effects on a nursing infant. Breast-feeding while



using this drug is not recommended. Consult your doctor before

breast-feeding.

1.4 Rosuvastatin (Crestor)

1.4.1 Chemistry of Rosuvastatin

Crestor (rosuvastatin calcium) is a synthetic lipidlowering agent for oral

administration. The chemical name for rosuvastatin calcium is bis[(E)-

7-[4-(4-fluorophenyl)-6-isopropyl-2-[methyl (methylsulfonyl) amino]

pyrimidin-5-yl](3R,5S)-3,5-dihydroxyhept-6-enoic acid] calcium salt.

The empirical formula for rosuvastatin calcium is (C22H27FN306S)2Ca

and the molecular weight is 1001.14. Rosuvastatin calcium is a white

amorphous powder that is sparingly soluble in water and methanol, and

slightly soluble in ethanol. Rosuvastatin calcium is a hydrophilic

compound with a partition coefficient (octanol/water) of 0.13 at pH of

7.0.

Figure 7: The Chemical structure of Rosuvastatin

Each tablet contains: microcrystalline cellulose NF, lactose

monohydrate NF, tribasic calcium phosphate NF, crospovidone NF,

magnesium stearate NF, hypromellose NF, triacetin NF, titanium

dioxide USP, yellow ferric oxide, and red ferric oxide NF.



Figure 8: 20 mg of Crestor

1.4.2 Uses of rosuvastatin

1.4.2.1 Rosuvastatin is a cholesterol-lowering medication that blocks

the production of cholesterol (a type of fat) in the body. It works

by reducing levels of "bad" cholesterol (low-density lipoprotein,

or LDL) and triglycerides in the blood, while increasing levels

of "good" cholesterol (high-density lipoprotein, or HDL).

1.4.2.2 Rosuvastatin is used to treat high cholesterol in adults and

children who are at least 10 years old. Lowering your

cholesterol can help prevent heart disease and hardening of the

arteries, conditions that can lead to heart attack, stroke, and

vascular disease.

1.4.3 The side effects of rosuvastatin.

1.4.3.1 The signs of an allergic reaction: hives; difficulty breathing;

swelling of your face, lips, tongue or throat.

1.4.3.2 the serious side effects (muscle pain, tenderness or weakness

with fever or flu symptoms and dark colored urine, urinatingmore or less than usual), nausea, stomach pain, low fever, loss

of appetite, dark urine, clay-colored stools, jaundice (yellowing

of the skin or eyes), chest pain or swelling in your hands or feet.

1.4.3.3 Less serious side effects are weakness, dizziness, mild nausea,

constipation, diarrhea, sore throat, runny or stuffy nose, memory

loss, headache or pain or burning when you urinate.



1.4.4 The precautions when taking the Rosuvastatin

1.4.4.1 Before taking rosuvastatin, if you have any other allergies. This

product may contain inactive ingredients, which can cause

allergic reactions or other problems.









rosuvastatin. Ask your doctor or pharmacist for more

information.



1.4.4.6 This medication must not be used during pregnancy.

Rosuvastatin may harm an unborn baby. Therefore, it is

important to prevent pregnancy while taking this medication.

Consult your doctor for more details and to discuss using at least

2 reliable forms of birth control (such as condoms, birth control

pills) while taking this medication. If you become pregnant or

think you may be pregnant, tell your doctor immediately.

1.4.4.7 It is unknown if this medication passes into breast milk. Because

of the possible risk to the infant, breast-feeding while using this

drug is not recommended. Consult your doctor before breast-

feeding.



2.0 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

2.1 Introduction of HPLC

High Performance Liquid Chromatography (HPLC) is one mode of

chromatography, one of the most used analytical techniques. Chromatographic

process can be defined as separation technique involving mass-transfer between

stationary and mobile phase. HPLC utilises a liquid mobile phase to separate the

components of a mixture. The stationary phase can be a liquid or a solid phase.

These components are first dissolved in a solvent, and then forced to flow through

a chromatographic column under a high pressure. In the column, the mixture

separates into its components. The amount of resolution is important, and is

dependent upon the extent of interaction between the solute components and the

stationary phase. The stationary phase is defined as the immobile packing material

in the column. The interaction of the solute with mobile and stationary phases can

be manipulated through different choices of both solvents and stationary phases.

As a result, HPLC acquires a high degree of versatility not found in other

chromatographic systems and it has the ability to easily separate a wide variety of

chemical mixtures. (fig.1)

Initially, pressure was selected as the principal criterion of modern liquid

chromatography and thus the name was "high pressure liquid chromatography" or

HPLC. This was, however, an unfortunate term because it seems to indicate that

the improved performance is primarily due to the high pressure. This is, however,

not true. In fact, high performance is the result of many factors: very small



particles of narrow distribution range and uniform pore size and distribution, high

pressure column slurry packing techniques accurate low volume sample injectors,

sensitive low volume detectors and, of course, good pumping systems. Naturally,

pressure is needed to permit a given flow rate of the mobile phase.

2.2 Theory of HPLC

HPLC is a dynamic adsorption process. Analyte molecules, while moving

through the porous packing beads, tend to interact with the surface adsorption

sites. Depending on the HPLC mode, the different types of the adsorption forces

may be included in the retention process: Hydrophobic (non-specific) interactions

are the main ones in reversed-phase (RP) separations. Dipole-dipole (polar)

interactions are dominant in normal phase (NP) (mode. Ionic interactions are

responsible for the retention in ion-exchange chromatography. All these

interactions are competitive. Analyte molecules are competing with the eluent

molecules for the adsorption sites. So, the stronger analyte molecules interact with

the surface. The weaker the eluent interaction, the longer the analyte will be

retained on the surface. SEC (size-exclusion chromatography) is another case. It is

the separation of the mixture by the molecular size of its components. The basic

principle of SEC separation is that the bigger the molecule, the less possibility

there is for it to penetrate into the adsorbent pore space. So, the bigger the

molecule the less it will be retained.

There are many ways to classify liquid column chromatography. If this

classification is based on the nature of the stationary phase and the separation

process, three modes can be specified.

a. Adsorption chromatography: the stationary phase is an adsorbent (like silica

gel or any other silica based packing) and the separation is based on repeated

adsorption-desorption steps.

b. Ion-exchange chromatography: the stationary bed has an ionically charged

surface of opposite charge to the sample ions. This technique is used almost

exclusively with ionic or ionizable samples. The stronger the charge on the

sample, the stronger it will be attracted to the ionic surface and thus, the longer



it will take to elute. The mobile phase is an aqueous buffer, where both pH and

ionic strength are used to control elution time.

c. Size exclusion chromatography: the column is filled with material having

precisely controlled pore sizes, and the sample is simply screened or filtered

according to its solvated molecular size. Larger molecules are rapidly washed

through the column; smaller molecules penetrate inside the porous of the

packing particles and elute later. This technique is also called gel filtration or

gel permeation chromatography. Concerning the first type, two modes are

defined depending on the relative polarity of the two phases: normal and

reversed-phase chromatography. In normal phase chromatography, the

stationary bed is strongly polar in nature (e.g. silica gel), and the mobile phase

is nonpolar (suchas n-hexane). Polar samples are thus retained on the polar

surface of the column packing for longer than less polar materials.

Reversed-phase chromatography is the inverse of this. The stationary bed is

(nonpolar) in nature, while the mobile phase is a polar liquid, such as mixtures of

water and methanol or acetonitrile. Here the more nonpolar the material is, the

longer it will be retained. Reverse phase chromatography is used for almost 90%

of all chromatographic applications. Eluent polarity plays the major role in alltypes of HPLC. There are two elution types: isocratic and gradient. In the first

type, constant eluent composition is pumped through the column during the whole

analysis. In the second type, eluent composition (and strength) is steadily changed

during the run.

2.3 Intrumentation of HPLC

HPLC instrumentation includes a pump, injector, column, detector and data

system. The heart of the system is the column where separation occurs. Since the

stationary phase is composed of micrometre size porous particles, a high pressure

pump is required to move the mobile phase through the column. The

chromatographic process begins by injecting the solute onto the top of the column.

Separation of components occurs as the analytes and mobile phase are pumped

through the column. Eventually, each component elutes from the column as a

narrow band (or peak) on the recorder. Detection of the eluting components is

important, and this can be either selective or universal, depending upon the



detector used. The response of the detector to each component is displayed on a

chart recorder or computer screen and is known as a chromatogram. To collect,

store and analyse the chromatographic data, computer, integrator, and other data

processing equipment are frequently used.



3.0 Method validation of pharmaceutical product

Proper validation of analytical methods is important for pharmaceutical analysis when

ensurance of the continuing efficacy and safety of each batch manufactured relies solely on

the determination of quality. The ability to control this quality is dependent upon the ability

of the analytical methods, as applied under well-defined conditions and at an established level

of sensitivity, to give a reliable demonstration of all deviation from target criteria. Method

development and validation can be simultaneous, but they are two different processes, both

downstream of method selection. The methods were validated according to the United States

Pharmacopeia 31st edition (2008) and ICH Guidance for Industry (International Conference

on Harmonization 2005). The objective of validation of an analytical procedure is to

demonstrate that it is suitable for its intended purpose. A tabular summation of the

characteristics applicable to identification, control of impurities and assay procedures is

included. Other analytical procedures may be considered in future additions to this document.

The objective of the analytical procedure should be clearly understood since this will govern

the validation characteristics which need to be evaluated. Typical validation characteristics

which should be considered are listed below:

a. Specificity

Specificity is the ability to assess unequivocally the analyte in the presence of

components which may be expected to be present. Typically these might include

impurities, degradants, matrix, etc.

b. Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement between

the value which is accepted either as a conventional true value or an accepted

reference value and the value found. This is sometimes termed trueness.c. Precision

The precision of an analytical procedure expresses the closeness of agreement (degree

of scatter) between a series of measurements obtained from multiple sampling of the

same homogeneous sample under the prescribed conditions. Precision may be

considered at three levels: repeatability, intermediate precision and reproducibility.

Precision should be investigated using homogeneous, authentic samples. However, it

is not possible to obtain a homogeneous sample it may be investigated using

artificially prepared samples or a sample solution. The precision of an analytical



procedure is repeatability expresses the precision under the same operating conditions

over a short interval of time. Repeatability is also termed intra-assay precision.

d. Detection Limit

The detection limit of an individual analytical procedure is the lowest amount of

analyte in a sample which can be detected but not necessarily quantitated as an exact

value.

e. Quantitation Limit

The quantitation limit of an individual analytical procedure is the lowest amount of

analyte in a sample which can be quantitatively determined with suitable precision and

accuracy. The quantitation limit is a parameter of quantitative assays for low levels of

compounds in sample matrices, and is used particularly for the determination of

impurities and/or degradation products.

f. Linearity

The linearity of an analytical procedure is its ability (within a given range) to obtain

test results which are directly proportional to the concentration (amount) of analyte in

the sample.

g. Range

The range of an analytical procedure is the interval between the upper and lower

concentration (amounts) of analyte in the sample (including these concentrations) for

which it has been demonstrated that the analytical procedure has a suitable level of

precision, accuracy and linearity.

h. Robustness

The robustness of an analytical procedure is a measure of its capacity to remain

unaffected by small, but deliberate variations in method parameters and provides an

indication of its reliability during normal usage.



- signifies that this characteristic is not normally evaluated, + signifies that this

characteristic is normally evaluated, (1) in cases where reproducibility (see glossary) has

been performed, intermediate precision is not needed, (2) lack of specificity of one

analytical procedure could be compensated by other supporting analytical procedure(s) (3)may be needed in some cases



3.0 EXPERIMENTAL

3.1 Reagents

3.1.1 The HPLC-grade methanol and orthophosphoric acid

3.1.2 High-purity water was prepared using Milli-QPlus water-

purification system

3.1.3 Reference Substances. The PS (99.10%), FVS (99.90%), ATC

(99.70%), and RC (99.90%)

3.2 Samples

Sample Type of sample

A 10.0mg PS tablet

B 20.0mg PS table

C 40.0mg PS tablet

D 20.0mg FVS capsule

E 40.0mg FVS capsule

F 10.0mg ATC tablet

G 20.0mg ATC tablet

H 10.0mg RC tablet

I 20.0mg RC tablet

3.3 Placebos

placebos Composition

A lactose, micro crystalline cellulose, povidone,

croscamellose sodium, and magnesium stearate B

C previously mentioned excipients mixing green

lacquer LB-451

D magnesium stearate, sodium bicarbonate, talc,

microcrystalline cellulose, modified maizestarch, and calcium carbonate

E

F calcium carbonate, Micro crystalline cellulose,

lactose monohydrate, croscarmellose sodium,

polysorbate 80,hyprolose,magnesium

stearate,YS-1-7040 color white opadry, and

simethicone emulsion USP

G

H lactose monohydrate, micro crystalline

cellulose, calcium phosphate tribasic,

crospovidone, magnesium stearate,

hypromellose, triacetin, titanium dioxide, and

ferric oxide.

I



3.4 Instrumentation

3.4.1 The HPLC methods were performed using an equipment consisting

of a solvent-delivery system, an auto injector fitted with 20-mL

loop, an online degasification system, a column thermostat oven,

and an ultraviolet visible(UV-VIS) photodiode array detector.

ITEM PARAMETER

Instrument HPLC equipped with solvent delivery system

Injection system Auto injector fitted with 20-µl loop

Oven Column thermostat

Detector ultraviolet visible(UV-VIS) photodiode array at

238nmColumn LiChrospher C18 column (125.4mm,5mm)

Mobile phase methanol:water (60:40), (v/v), for PS and RC and

70:30, (v/v), for FVS and ATC

pH of mobile

phase

adjusted to 3.0 with orthophosphoric acid

flowrate 1.0mL/min

3.5 Procedure

3.5.1 System Suitability

Standard solutions containing 12.0mg/mL (PS), 20.0mg/mL (FVS),

and 14.0mg/mL (ATC and RC) were prepared by dilution in the

respective mobile phases. System suitability was determined from

six replicate injections of each standard solution.

3.5.2 Specificity

The selectivity of the methods was assessed by comparing the

chromatograms obtained with placebo solutions using equivalent

concentrations of 10.0, 20.0, and 40.0mg/mL of PS, 20.0 and

40.0mg/mL of FVS, and 10.0 and 20.0mg/mL of ATC and RC.

Injections were made in triplicate.

3.5.3 Linearity and range

3.5.3.1 Reference standard stock solutions were prepared separately,

by weighing exactly 10.0mg of PS and FVS and 12.5mg of



ATC and RC, in duplicate, and transferring them to 25-mL

volumetric flasks.

3.5.3.2 Approximately 23mL of respective mobile phase were

added, and the mixtures were sonicated for 10min.

3.5.3.3 The volumes were then completed with the respective

mobile phases. Aliquots of 0.1, 0.2, 0.3, 0.4, and 0.5mL for

PS and 0.3, 0.4, 0.5, 0.6, and 0.7mL for FVS were

transferred separately to 10-mL volumetric flasks.

3.5.3.4 Aliquots of 0.3, 0.5, 0.7, 0.9, and 1.1mL for ATC and RC

were transferred separately to 25-mL volumetric flasks.

Volumes were completed with respective mobile phases.

3.5.3.5 Final concentrations were 4.0, 8.0, 12.0, 16.0, and

20.0mg/mL of PS; 12.0, 16.0, 20.0, 24.0, and 28.0mg/mL of

FVS; and 6.0, 10.0, 14.0, 18.0, and 22.0mg/mL for ATC and

RC. The calibration plots were constructed by plotting mean

response (n¼3) vs. respective concentrations.

3.5.4 Precision

3.5.4.1 Twenty tablets and capsules of each pharmaceutical dosage

form were weighed separately, powered, and homogenized.

Amounts of powder equivalent to 6.0mg (PS), 10.0mg

(FVS), and7.0mg (ATC and RC) were separately and

exactly weighed and transferred to 50-mL volumetric flasks.

3.5.4.2 Approximately 35mL of the respective mobile phases were

added, and the mixtures were sonicated for 10min. The

volume of each volumetric flask was completed with

respective mobile phase, and the final solutions were filtered

through Whatmann’s filter paper no. 1.

3.5.4.3 The solutions were refrigerated at 4oC. Ten-1.0mL aliquots

of each statin solutions (PS, FVS, ATC, and RC) were

transferred separately to 10mL volumetric flasks. The

volumes were completed with respective mobile phases. The

final concentrations were approximately 12.0mg/mL (PS),

20.0mg/mL (FVS) and 14.0mg/mL (ATC and RC).



Solutions were filtered through 0.45-mm filters (Millex HV,

Millipore, Milford, USA)

3.5.4.4 Before injection into the system. Standard solutions

containing 12.0mg=mL (PS), 20.0mg=mL (FVS), and

14.0mg=mL (ATC and RC) were prepared as described.

3.5.4.5 Triplicate determinations were made with standard and

sample solutions of each flask, each day, on three

consecutive days, and mean peak areas were determined.

3.5.5 Accuracy

3.5.5.1 10.0mg were separately and exactly weighed and transferred

separately to 25-mL volumetric flasks. Standard and sample

solutions were prepared separately as described to obtain

solutions containing 400.0mg=mL of each statin. Three

aliquots of 0.1, 0.2, and 0.3mL (PS); 0.3, 0.4, and 0.5mL

(FVS); and 0.15, 0.25, and 0.35mL (ATC and RC) of each

standard solution, and three 0.1-mL aliquots of each sample

were transferred to 10-mL volumetric flasks. Solutions were

filtered through Whatmann’s filter paper no. 1.

3.5.5.2 Final concentrations were 8.0, 12.0, and16.0mg=mL (PS);

16.0, 20.0, and 24.0mg=mL (FVS); and 10.0, 14.0,and

18.0mg=mL (ATC and RC). All solutions were filtered

through0.45-mm filters, (Millex HV, Millipore, Milford,

USA) before injection into the system.

3.5.6 Robustness Test

3.5.6.1 The mobile phases were constituted of methanol:water:

61.2:38.8 (v:v) for PS, 58.8:41.2 (v:v) for RC, 71.4:28.6

(v:v) for FVS, and 68.6:31.4 (v;v) for ATC, with pH

adjusted to 3.0 with orthophosphoric acid at a flow rate of

0.9 and 1.1mL=min.

3.5.6.2 The injection volumes were fixed at 15 and 25mL; UV

detection was made at 234 and 243nm.

3.5.6.3 All analyses were made at room temperature, and column

temperature was set at 24±1o

C and 26±1o

C. The mobile



phases were prepared fresh and vacuum-filtered through a

0.45-mm filter, Millex HV.

3.5.6.4 The standard solutions containing 12.0mg/mL of PS,

20.0mg/mL of FVS, and 14.0mg/mL of ATC and RC were

injected in triplicate, and system suitability parameters such

as capacity factor, selectivity, resolution, and peak tailing

factor were evaluated.

3.5.7 Stability Test

Standard and sample solutions were prepared separately as

described to obtain solutions containing 12.0mg/mL (PS),

20.0mg/mL (FVS), and 14.0mg/mL (ATC and RC). These solutions

were stored in the refrigerator at 4oC, and triplicate measurements

were made on three days.

3.5.8 Stress testing

3.5.9 Neutral hydrolysis was made in water, chemical oxidations were

done with 3% H2O2, acid hydrolysis with 1.0mol/L HCl, and

alkaline hydrolysis was performed with 1.0mol=L NaOH. The

solutions were heated at 80oC for 2h.

3.5.10 Adequate dilutions were made from each solution after stressing

time. Solutions from acid and alkaline hydrolysis were neutralized

before analysis.

3.5.11 Volumes were completed with respective mobile phases. The final

concentrations of solutions were 12.0mg/mL (PS), 20.0mg/mL

(FVS), and 14.0mg/mL (ATC and RC).



4.0 RESULT AND DISCUSION

4.1 system suitability

System suitability tests are an integral part of liquid chromatographic method.

They are used to verify that the resolution and reproducibility of the

chromatographic system are adequate for the analysis to be done (United States

Pharmacopeia 2008). Standard solutions were injected, and the relative

standard deviations (RSD) for each parameter were determined. In all cases,

RSD values were less than 2%, which proves the reliability of the methods for

proposed applications (Table).

4.2 Specificity

Placebo formulations containing all ingredients except PS, FVS, ATC,

and RC were prepared for these assays. The placebo solutions were treated

using the same procedure used for the samples. It was observed that

independent of concentration (10.0, 20.0, and 40.0mg/ml equivalent of PS,

20.0 and 40.0mg/mL of FVS, 10.0 and 20.0mg/mLof ATC and RC), the

excipients do not interfere in the analysis (Figs. 2 and 3).





4.3 Linearity and Range

Calibration plots for proposed methods were evaluated and checked by

analyzing standard solutions at five concentration levels, ranging from 4.0 to

20.0mg/mL (PS), 6.0 to 22.0mg/mL (FVS), and 6.0 to 22.0mg/mL (ATC and

RC), respectively. The correlation coefficients (r) were >0.999. Therefore,

statins presented good linearity. The calibration curves were established by

least square linear regression (Snyder, Kirkland, and Glajch 1997). The F test

was applied, and values of 957.40 (PS), 425.81 (FVS), 8810.88 (ATC), and

721.28 (RC) were obtained for the proposed methods (F (0.05) ¼ 10.13)

(Snyder, Kirkland, and Glajch 1997). The data provide conclusive evidence of

linearity between concentration and instrumental response.

0

200000

400000

600000

800000

1000000

1200000

0 10 20 30

Linearity Plot of Pravastatin

Sodium

P e a k

R e s p o n s e

Concentration (µg/mL)

y = 49126x + 31589

R2 = 0.9994

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

0.0 10.0 20.0 30.0

Linearity Plot of Fluvastatin

Sodium

Concentration (µg/mL)

P e a k R e s p o n s e

y = 57811x – 140816R2 = 0.9995

y = 49792x - 5172.4

R² = 1

0.0

200000.0

400000.0

600000.0

800000.0

1000000.0

1200000.0

0.0 5.0 10.0 15.0 20.0 25.0

p e a k

r e s p o n s e

concentration mg/l

Linearity Plot of Atorvastatins

Calcium

0.0

200000.0

400000.0

600000.0

800000.0

1000000.0

1200000.0

1400000.0

0.0 5.0 10.0 15.0 20.0 25.0

Linearity Plot of Rosuvastatin

Calcium

p e a k

r e s p o n s e

Concentration(µg/mL)

y = 52828x – 2597.3

R2 = 0.9990



4.4 Detection Limit (DL) and Quantitation Limit (QL)

The DL and QL were determined based on the standard deviation among

response and slope of the curve (International Conferenceon Harmonization

2005). The theoretical values for QL were confirmed experimentally. The DL

and QL were 1.22 and 3.08mg/mL (PS), 2.02 and 6.12mg/mL (FVS), 0.44 and

1.34mg/mL (ATC), and 1.55 and 4.70mg/mL (RC), respectively. These low

values indicated good sensitivity of the methods.

4.5 Precision

Figures 2 and 3 show the chromatograms of samples A, D, F, and H. The

precision was determined by repeatability (intraday) and expressed as RSD.

Interday data was evaluated by one-way analysis of variance (ANOVA)

(Bolton 1990). To compare the variability among responses on three

consecutive days, ANOVA analysis was used. The low intraday and interday

RSD values prove the repeatability and intermediate precision of the proposed

methods, respectively.

4.6 Accuracy

According to ICH guidelines, the standard solution addition should be done in

a range from 80 to 120% of the nominal concentration (International



Conference on Harmonization 2005).The accuracy of the methods was

evaluated at three concentration levels. Triplicate determinations were made at

each concentration level. The accuracy is expressed as percentage of standard

recovered from sample matrix (International Conference on Harmonization

2005).The mean recoveries of investigated statins were found to be in the

range of 97.80% and 102.75%, indicating good accuracy for chromatographic

methods (Table 4).





after chemical oxidation tests (Figs. 4b,4f, 5b, and 5f). After acid and alkaline

hydrolysis, chromatograms show many additional peaks, due to the

degradation products (Figs.4c, 4g, 4d, 4h, 5c, 5g, 5d, and 5h). The proposed

methods were appropriate for quantitative determination of statins in the

presence of its degradation products, because all these products could be

separated, as can be observed in the chromatograms. For this reason, they can

be used as stability-indicating methods (Table 6).

4.9 Stability of Solutions

The normal time of analysis in a quality-control laboratory is around 6h, so it is

essential to evaluate the stability of standard and sample solutions to obtain

reliable results. Standard and sample solutions were refrigerated at 4oC for

three consecutive days (precision), and the obtained results were compared

with freshly prepared solutions. No differences were observed in the

instrumental responses under the described conditions.

CONCLUSION

In conclusion, two sensitive and selective stability-indicating methods have been

developed and validated for the analysis of PS, FVS, ATC, and RC. Based on peak purity

results, obtained after forcibly degraded samples using the described methods, it can be

concluded that the absence of coeluting peak along with the main peak of statins indicates

that the developed methods are specific for quantitative determination of statins in the

presence of its degradation products. The proposed methods presented excellent accuracy,

sensitivity, and reproducibility. Even though no attempt was made to identify the

degradation products, the proposed methods can be used as stability-indicating methods

and can be used for routine quality-control analyses of PS, FVS, ATC, and RC in

pharmaceuticals.



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PMID 20425579

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3. Hermann M; Bogsrud MP, Molden E, Asberg A, Mohebi BU, Ose L, Retterstøl K

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metabolites are increased several-fold in patients with atorvastatin-induced

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PMID 16765141.

4. Williams D; Feely J (2002). "Pharmacokinetic-pharmacodynamic drug interactions

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