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 the enzyme 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 HMGCoA 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)-, -dihydroxy-5-(1-

methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H-pyrrole-1heptanoic acid, calcium salt (2:1) trihydrate. The empirical formula of atorvastatin calcium is (C33H34FN2O5)2Ca3H2O 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: calcium carbonate, 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 breastfeeding.

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 enzyme catalyzing 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-,,6-trihydroxy-2

methyl-8-(2-methyl-1-

oxobutoxy)-, monosodium salt,[1S[1(S*,S*), 2,6,8(R*),8a]].

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 a partition 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 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. 1.2.2.3 Pravastatin is used to treat high cholesterol. Pravastatin is also used to lower the risk of stroke, heart attack, or other heart complications 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 C24H25FNO4Na, 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 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. 1.3.3 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. 1.3.4.2 Before using this medication, tell your doctor or pharmacist your medical history, especially of: liver disease, kidney disease, alcohol use. 1.3.4.3 Before having surgery, tell your doctor or dentist about all the products you use (including prescription drugs, nonprescription drugs, and herbal products). 1.3.4.4 Limit alcoholic beverages. Daily use of alcohol may increase your risk for liver problems, especially when combined with fluvastatin. Ask your doctor or pharmacist for more information. 1.3.4.5 Older adults may be more sensitive to the side effects of this drug, especially muscle problems. 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, urinating more 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. 1.4.4.2 Before using this medication, tell your doctor or pharmacist your medical history, especially of: liver disease, kidney disease, alcohol use. 1.4.4.3 Before having surgery, tell your doctor or dentist about all the products you use (including prescription drugs, nonprescription drugs, and herbal products). 1.4.4.4 Limit alcoholic beverages. Daily use of alcohol may increase your risk for liver problems, especially when combined with rosuvastatin. Ask your doctor or pharmacist for more information. 1.4.4.5 Older adults may be more sensitive to the side effects of this drug, especially muscle problems. 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 breastfeeding.

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 all types 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 3.1.2 The HPLC-grade methanol and orthophosphoric acid High-purity water was prepared using Milli-QPlus waterpurification system 3.1.3 Reference Substances. The PS (99.10%), FVS (99.90%), ATC (99.70%), and RC (99.90%) 3.2 Samples Sample A B C D E F G H I 3.3 Placebos placebos A B C D E F G Composition lactose, micro crystalline cellulose, povidone, croscamellose sodium, and magnesium stearate previously mentioned excipients mixing green lacquer LB-451 magnesium stearate, sodium bicarbonate, talc, microcrystalline cellulose, modified maize starch, and calcium carbonate calcium carbonate, Micro crystalline cellulose, lactose monohydrate, croscarmellose sodium, polysorbate 80,hyprolose,magnesium stearate,YS-1-7040 color white opadry, and simethicone emulsion USP lactose monohydrate, micro crystalline cellulose, calcium phosphate tribasic, crospovidone, magnesium stearate, hypromellose, triacetin, titanium dioxide, and ferric oxide. Type of sample 10.0mg PS tablet 20.0mg PS table 40.0mg PS tablet 20.0mg FVS capsule 40.0mg FVS capsule 10.0mg ATC tablet 20.0mg ATC tablet 10.0mg RC tablet 20.0mg RC tablet

H 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 Instrument Injection system Oven Detector Column Mobile phase pH of mobile phase flowrate PARAMETER HPLC equipped with solvent delivery system Auto injector fitted with 20-l loop Column thermostat ultraviolet visible(UV-VIS) photodiode array at 238nm LiChrospher C18 column (125.4mm,5mm) methanol:water (60:40), (v/v), for PS and RC and 70:30, (v/v), for FVS and ATC adjusted to 3.0 with orthophosphoric acid 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 (n3) 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 Whatmanns 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 Whatmanns 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 241oC and 261oC. 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 3.5.9 Stress testing 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) 10.13) (Snyder, Kirkland, and Glajch 1997). The data provide conclusive evidence of linearity between concentration and instrumental response. Linearity Plot of Fluvastatin Sodium 1600000
1400000

Linearity Plot of Pravastatin Sodium 1200000 Peak Response

1000000 800000 600000 400000 200000 0 0 10 20 30 y = 49126x + 31589 R2 = 0.9994

1000000 800000 600000 400000 200000 0 0.0 10.0 20.0 30.0

Peak Response

1200000

y = 57811x 140816 R2 = 0.9995

Concentration (g/mL)

Concentration (g/mL)

Linearity Plot of Atorvastatins Calcium

1200000.0 1000000.0 peak response 800000.0 600000.0 400000.0 200000.0 0.0 0.0 5.0 10.0 15.0 20.0 25.0 concentration mg/l y = 49792x - 5172.4 R = 1

Linearity Plot of Rosuvastatin Calcium 1400000.0

1200000.0 1000000.0 800000.0 600000.0 400000.0 200000.0 0.0 0.0 5.0 10.0 15.0 20.0 25.0

peak response

y = 52828x 2597.3 R2 = 0.9990 Concentration (g/mL)

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).

4.7 Robustness The robustness of an analytical procedure is a measurement of its capacity to remain unaffected by small and deliberate variations in method parameters and provides an indication of its reliability during normal usage (International Conference on Harmonization 2005). Upon deliberate changes in the analytical parameters, no significant changes were observed in the instrumental responses, and the RSD values were less than 1% in all cases (Table 5). Thus, the proposed methods can be considered reliable and robust.

4.8 Stress Testing Forced degradation is an important part of drug product development. The stress testing is defined as a stability testing performed in more drastic conditions than those used for accelerated stability tests (Klick et al. 2005). No interferences were observed in the chromatograms after neutral hydrolysis (Figs. 4a, 4e, 5a, and 5e). Modification in the chromatograms can be observed

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.

REFERENCES 1. Villa J; Pratley RE (June 2010). "Ezetimibe/simvastatin or atorvastatin for the treatment of hypercholesterolemia in patients with the metabolic syndrome: the VYMET study". Curr. Diab. Rep. 10 (3): 1735. doi:10.1007/s11892-010-0107-5. PMID 20425579 2. McCormack T; Harvey P, Gaunt R, Allgar V, Chipperfield R, Robinson P (July 2010). "Incremental cholesterol reduction with ezetimibe/simvastatin, atorvastatin and rosuvastatin in UK General Practice (IN-PRACTICE): randomised controlled trial of achievement of Joint British Societies (JBS-2) cholesterol targets". Int. J. Clin. Pract. 3. Hermann M; Bogsrud MP, Molden E, Asberg A, Mohebi BU, Ose L, Retterstl K (June 2006). "Exposure of atorvastatin is unchanged but lactone and acid metabolites are increased several-fold in patients with atorvastatin-induced myopathy". Clin. Pharmacol. Ther. 79 (6): 5329. doi:10.1016/j.clpt.2006.02.014. PMID 16765141. 4. Williams D; Feely J (2002). "Pharmacokinetic-pharmacodynamic drug interactions with HMG-CoA reductase inhibitors". Clin Pharmacokinet 41 (5): 34370. doi:10.2165/00003088-200241050-00003. PMID 12036392. 5. Keller DM (2010-06-27). "PLANET I and II: Atorvastatin beats rosuvastatin for protecting kidneys in diabetic and nondiabetic patients". theheart.org. Retrieved 2011-03-31.