Pharmaceutical Analysis

A.M.
Reddy Memorial College of Pharmacy
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1.0
INTRODUCTION
Pharmaceutical Analysis plays a vital role in the Quality Assurance and Quality Control of bulk drugs and formulations. It is a specialized branch of analytical chemistry which involves separating, identifying and determining the relative amounts of components in a sample matrix1. It is mainly involved in the qualitative identification or detection of compound sand the quantitative measurement of the substances present in bulk and pharmaceutical preparations2. Qualitative analysis reveals the chemical identity of the sample. Quantitative analysis establishes the relative amount of analytes in numerical terms. A separation step is usually a necessary part of both qualitative and quantitative analysis1. Methods of detecting analytes2: 1.
2. 3.
Physical means Mass, color, refractive index, thermal By electromagnetic radiation Absorption, emission, scattering By an electric charge Electro chemistry, mass spectrometry Separation of analytes by precipitation, extraction or
conductivity
ClassicalMethods2: 1. distillation.
2. Qualitative analysis by reaction of analytes with reagents that yielded products
that could be recognized by their colours, boiling or melting points, solubilitys, optical activities or refractive indices.
3.
Quantitative
analysis
by
gravimetric
or
by
titrimetric
techniques. Instrumental methods: Measurement of physical properties of analytes such as conductivity, electrode potential, light absorption or emission, mass to charge ratio and fluorescence, began to be used for quantitative analysis of variety of inorganic and biochemical analytes. Highly efficient chromatographic and electrophoresis techniques began to replace distillation, extraction and precipitation for the separation of components of complex mixtures prior to their qualitative or quantitative determination. These newer methods for separating and determining chemical species are known collectively as instrumental methods of analysis.
A.M.Reddy Memorial College of Pharmacy Page 2
Most of the instrumental methods fit into one of the three following categories viz., spectroscopy, electrochemistry and chromatography. Advantages of instrumental methods:
Small samples can be used. High sensitivity is obtained. Measurements obtained are reliable. Determination is very fast Even complex samples can be handled easily
Limitations of instrumental methods2: An initial or continuous calibration is required as sensitivity and accuracy depends on the instrument or the wet chemical method

Cost of equipment is large Concentration range is limited Specialized training is needed Sizable space is required
Principle types of chemical instrumentation5 A) Spectrometric techniques

Ultraviolet and visible spectro photometry Fluorescence and phosphorescence spectro photometry. Atomic spectrometry (emission and absorption) Infrared spectro photometry Raman spectroscopy X-Ray spectroscopy Nuclear Magnetic Resonance spectroscopy Electron spin resonance spectroscopy
B) Electrochemical techniques Potentiometry Voltametric techniques Stripping techniques. Amperometric techniques Electro gravimetry
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A.M.Reddy Memorial College of Pharmacy
Conductance techniques
C) Chromatographic techniques
Gas Chromatography High Performance Liquid Chromatography Thin Layer Chromatography
D) Miscellaneous techniques

Thermal analysis Mass spectrometry Kinetic techniques
E) Hyphenated techniques3

GC-MS(Gas Chromatography Mass Spectrometry) ICP-MS(Inductive Coupled Plasma- Mass Spectrometry) GC-IR(Gas Chromatography Infrared Spectroscopy) LC-MS(Liquid Chromatography Mass Spectrometry) CHROMATOGRAPHY
Chromatography by classical definition is a separation process where resolution is achieved by the distribution of the components of a mixture between two phases, a stationary phase and a mobile phase. Those components held preferentially in the stationary phase are retained longer in the system than those that are distributed in the mobile phase. As a consequence solutes are eluted from the system in the order of their increasing distribution coefficients with respect to the stationary phase, a separation is achieved. The mobile phase can be a gas or a liquid which gives rise to the two basic forms of chromatography namely Gas Chromatography (GC) and Liquid Chromatography (LC). The stationary phase can also take two forms solid and liquid, which provides two subgroups of GC and LC namely Gas-Solid Chromatography (GSC) and Gas-Liquid Chromatography (GLC), together with Liquid Solid Chromatography (LSC) and Liquid Liquid Chromatography (LLC). HIGH PERFORMANCE LIQUID CHROMATOGRAPHY3, 4
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In the modern pharmaceutical industry, HPLC is a major analytical tool applied at all stages of drug discovery, development and production. Fast and effective development of rugged analytical HPLC methods is more efficiently undertaken with a thorough understanding of HPLC principles, theory and instrumentation. High-Performance Liquid Chromatography (or) High Pressure Liquid
Chromatography (HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC utilizes a column that holds chromatographic packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules. Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent(s) used. Most of the drugs in multi component dosage forms are analyzed by HPLC method because of several advantages like rapidity, specificity, accuracy, precision, ease of automation and eliminates tedious extraction and isolation procedures. Some of the advantages of HPLC are, Speed (analysis can be accomplished in 20 minutes or less) Greater sensitivity (various detectors can be employed) Improved resolution (wide variety of stationary phases) Reusable columns (expensive columns but can be used for many samples) Ideal for the substances of low volatility Easy sample recovery, handling and maintenance Instrumentation lends itself to automation and quantitation (less time and less labour) Precise and reproducible
Suitable for preparative liquid chromatography on a much larger scale5, 6.
TYPES OF HPLC Based on modes of chromatography: Normal phase chromatography

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Reversed phase chromatography
Based on principle of separation: Adsorption chromatography Ion exchange chromatography Size exclusion chromatography Affinity chromatography Chiral phase chromatography
Based on elution technique: Isocratic separation Gradient separation
Based on the scale of operation2, 3: Analytical HPLC Preparative HPLC
NORMAL PHASE CHROMATOGRAPHY

o
In normal phase mode the nature of stationary phase is polar and the mobile phase is non-polar.
In this technique, non-polar compounds travel faster and are eluted first because of the lower affinity between the non-polar compounds and the stationary phase.
Polar compounds are retained for longer times and take more time to elute because of their higher affinity with the stationary phase.
Normal phase mode of separation is not generally used for pharmaceutical applications because most of the drug molecules are polar in nature and hence take longer time to elute.
REVERSED PHASE CHROMATOGRAPHY
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o It is the most popular mode for analytical and preparative separations of compounds of interest in chemical, biological, pharmaceutical, food and biomedical sciences.
o
In this mode, the stationary phase is non-polar hydrophobic packing with octyl or octa decyl functional group bonded to silica gel and the mobile phase is a polar solvent.
Polar compound gets eluted first and non-polar compounds are retained for longer time. As most of the drugs and pharmaceuticals are polar in nature, they are not retained for longer times and hence elute faster.
The different columns used are octa decyl silane (ODS or C18), octyl silane (C8), butyl silane (C4) etc. (in the order of increasing polarity of the stationary phase).
ION EXCHANGE CHROMATOGRAPHY: o In ion exchange, the column packing contains ionic groups (e.g. sulfonic, tetra alkyl ammonium) and the mobile phase is an aqueous buffer (e.g. phosphate, formate, etc.). o Reversible exchange of ions takes place between the similarly charged ions and that of ion exchangers o Ion exchange is used by about 20% of the liquid chromatographers. The technique is well suited for: The separation of inorganic and organic anions and cations in aqueous solution. Ionic dyes, amino acids, and proteins can be separated by ion exchange technique. SIZE EXCLUSION CHROMATOGRAPHY (SEC) o In SEC, there is no interaction between the sample compounds and the column packing material. o Instead, molecules diffuse into pores of a porous medium. Separation occurs according to their molecular mass o Molecules larger than the pore opening do not diffuse into the particles, while molecules smaller than the pore opening enter the particle and are separated.
Large molecules elute first. Smaller molecules elute later. The SEC technique is used by 10-15% of chromatographers, mainly for polymer characterization and for protein characterization. o This mode can be further subdivided into a) Gel permeation chromatography (with organic solvents) b) Gel filtration chromatography (with aqueous solvents)
2.0 INSTRUMENTATION7-10:
The essential parts of the High Performance Liquid Chromatography are: Solvent reservoir Mobile phase Pump system Sample injection system Column Detector
SCHEMATIC DIAGRAM OF HPLC6 (Fig: 1)
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Solvent reservoir A modern HPLC apparatus is equipped with one or more glass or stainless steel reservoirs. The reservoir is often equipped with an online degasser which removes the dissolved gasses usually oxygen and nitrogen, which interfere by forming bubbles. Degasser may consist of vacuum pumping system, distillation system, system devices for heating, and a solvent stirrer Mobile phase The elution order of solutes in HPLC is governed by polarity In a normal-phase separation the least polar solute spends proportionally less time in the polar stationary phase and is the first solute to elute from the column. The mobile phases used in normal-phase chromatography are based on non-polar hydrocarbons, such as hexane, heptane, or octane, to which is added a small amount of a more polar solvent, such as 2-propanol. Solvent selectivity is controlled by the nature of the added solvent.
In a reverse-phase separation the order of elution is reversed, with the most polar solute being the first to elute. The mobile phases used in reversed-phase chromatography are based on a polar solvent, typically water, to which a less polar solvent such as acetonitrile or methanol is added. Solvents with large dipole
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moments,
such
as
methylene
chloride
and
1,2-dichloroethane
interact
preferentially with solutes that have large dipole moments, such as nitrocompounds, nitriles, amines, and sulfoxides. Solvents that are good proton donors, such as chloroform, M-cresol and water interact preferentially with basic solutes such as amines and sulfoxides, and solvents that are good proton acceptors, such as alcohols, ethers, and amines, tend to interact best with hydroxylated molecules such as acids and phenols. Pump System: The purpose of the pump or solvent delivery system is to ensure the delivery of a precise, reproducible, constant, and pulse-free flow of mobile phase.
Classification of pumps: HPLC pump can be classified in to the following groups according to the manner in which they operate:
Constant flow rate pump (or) constant displacement pump
i) Reciprocating piston pump ii) Syringe drive pump
Constant pressure pump
i) Simple gas displacement pump ii) Pneumatic amplifier pump a) Reciprocating pump Reciprocating pumps usually consist of a small chamber in which the solvent is pumped by the back and forth motion of a motor driven piston. Two check valves control the flow of solvent. Reciprocating pumps have a disadvantage of producing pulsed flow, which must be damped as its presence is manifested as base line noise on the chromatogram. Advantages of this pump include their small internal volume, high output
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pressure, ready adaptability to gradient elution, and independent of column backpressure and viscosity of solvent. b) Displacement pump Displacement pumps usually consist of large syringe like chambers equipped with a plunger that is activated by a screw driven mechanism powered by stepping motor. Displacement pumps also produce a flow that tends to be independent of viscosity and backpressure. In addition, the output is pulse free. Disadvantages include limited solvent capacity (250 ml) and considerable inconvenience when solvents must be changed. c) Pneumatic pumps In pneumatic pumps, the mobile phase is contained in a collapsible container housed in a vessel that can be pressurized by a compressor gas. Pumps of this kind are inexpensive and pulse free. They suffer from limited capacity, pressure output, dependence of flow rate on solvent viscosity and column backpressure. In addition, they are not amenable to gradient elution and are limited to pressures less than about 2000 psi. Sample injection system The earliest and simple means of sample introduction was syringe injection through a self-sealing electrometric septum. In stop flow injections the flow of solvent is stopped momentarily and fitting at column head is removed and the sample is injected directly into the head of column packing. After replacing the fitting the system is again pressurized. Commercial chromatographs use valves for sample injection. With these devices sample is first transferred at atmospheric pressure from a syringe into a sample loop. Turning the valve from load to inject position connects the sample loop into the highpressure mobile phase stream where by the contents of the sample loop are transferred on to the column. In rheodyne 7125 valve, sample from a microlitre syringe is loaded into the needle port, filling the sample loop which is a small piece of stainless steel tube connected between ports. Any excess goes to waste from another port. On turning to inject, the loop contents are flushed on to the column. A variety of loop volumes are available, commonly 10-50 L are used. Columns:
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HPLC typically includes two columns, an analytical column responsible for the separation and a guard column. The guard column is placed before the analytical column to protect it from contamination. Guard columns: Two problems tend to shorten the lifetime of an analytical column. First is binding of solutes irreversibly to the stationary phase degrade the columns performance by decreasing the available stationary phase. Second is clogging of particulate material injected with the sample to the analytical column. To minimize these problems, a guard column is placed before the analytical column. Guard columns usually contain the same particulate packing material and stationary phase as the analytical column, but are significantly shorter and less expensive; a length of 7.5 mm and a cost one-tenth of that for the corresponding analytical column are typical. Because they are intended to be sacrificial, guard columns are replaced regularly.
Analytical columns: It is the most important part of HPLC technique which decides the efficiency of separation. The most commonly used columns for HPLC are constructed from stainless steel with internal diameters between 2.1 mm and 4.6 mm, and lengths ranging from approximately 30 mm to 300 mm. These columns are packed with 310 mm porous silica particles that may have an irregular or spherical shape. Typical column efficiencies are 40,00060,000 theoretical plates/m. Micro columns use less solvent and because the sample is diluted to a lesser extent produce larger signals at the detector. These columns are made from fused silica capillaries with internal diameters of 44200 mm and lengths of up to several meters. Micro columns packed with 35 mm particles have been prepared with column efficiencies of up to 250,000 theoretical plates. Open tubular micro columns also have been developed with internal diameters of 150 mm and lengths of approximately 1 m. These columns which contain no packing material may be capable of obtaining column efficiencies of up to 1 million theoretical plates. The development of open tubular columns, however, has been limited by the difficulty of preparing columns with internal diameters less than 10 mm.
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COLUMNS USED IN HPLC (Fig: 2)
Stationary Phases: In LiquidLiquid Chromatography the stationary phase is a liquid film coated on a packing material consisting of 310 mm porous silica particles. The stationary phase may be partially soluble in the mobile phase, causing it to bleed from the column over time. To prevent this loss of stationary phase it is covalently bound to the silica particles. Bonded stationary phases are attached by reacting the silica particles with an organochlorosilane of the general form Si (CH3)2RCl, where R is an alkyl or substituted alkyl group. To prevent unwanted interactions between the solutes and any unreacted SiOH groups the silica frequently is capped by reacting it with Si (CH3)3Cl; such columns are designated as end-capped. The properties of a stationary phase are determined by the nature of the organosilanes alkyl group. If R is a polar functional group then the stationary phase will be polar. Since the stationary phase is polar, the mobile phase is a non-polar or moderately polar solvent. The combination of a polar stationary phase and a non-polar mobile phase is called normal phase chromatography. In reverse phase chromatography, which is the more commonly encountered form of HPLC, the stationary phase is non-polar and the mobile phase is polar. The most common
non-polar stationary phases use an organochlorosilane for which the R group is an n-octyl (C8) or n-octadecyl (C18) hydrocarbon chain. Most reverse phase separations are carried out using a buffered aqueous solution as a polar mobile phase.
BONDED STATIONARY PHASES FOR HPLC Table: 1 STATIONARY PHASE Silica FUNCTIONAL GROUP Si-OH
APPLICATIONS Normal phase material Pesticides, alkaloids Reverse-phase material
C18
Octadecyl Fatty acids, PAH, Vitamins Reverse-phase and ion pair, Peptides proteins Reverse-phase
C8
Octyl
C6H5 CN
Phenyl Polar aromatic fatty acids. Cyano Normal and Reverse-phase, polar
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compounds NO2 Nitro Normal and Reverse-phase, PAH, Aromatic compounds Normal, Reverse, NH2 Amino weak ion exchange Carbohydrates, organic acids, chlorinated pesticides Normal, Reverse phase peptides, proteins. Cation exchange, separation of cations.
OH SA
Diol Sulphonic acid
Detectors The function of the detector in HPLC is to monitor the mobile phase emerging from the column. The output of the detector is an electrical signal that is proportional to some property of the mobile phase and/or the solutes8 LC detectors are basically of two types. Bulk property detectors respond to mobile phase bulk property such as
refractive index, dielectric constant or density. Solute property detectors respond to some property of solutes such as UV
absorbing, fluorescence or diffusion current which are not possessed by the mobile phase. Most common HPLC detectors UV-Visible absorbance detector (UV-VIS) Photo-diode array detector (PDA) Fluorescence detector Electrochemical detector (ECD) Refractive Index detector (RI)
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Mass detectors (MS) Conductometric detector Chiral detector (Polarimetric & circular dichrosim) Evaporative light scattering detector (ELSD) Radiochemical detector
SYSTEM SUITABILITY PARAMETERS5: The purpose of the system suitability test is to ensure that the complete testing system (including instrument, reagents, columns, analysts) is suitable for the intended application. System suitability is the checking of a system to ensure the system performance before or during the analysis of unknown compounds. Parameters such as plate count, tailing factors, resolution and reproducibility are determined and compared against the specifications set for the method. The parameters that are affected by the changes in chromatographic conditions are,
Capacity factor (k1) Selectivity (a) Resolution Column efficiency (N) and Peak asymmetry / tailing factor (Af)
Parameters Capacity factor (k1) Repeatability Relative retention Resolution (Rs)
Recommendations The peak should be well-resolved from other peaks and the void volume, generally k1>2.0 RSD = 1% for N = 5 is desirable Not essential as long as the resolution is started Rs of >2 between the peak of interest and the closest eluting potential interferent (impurity, excipients, degradation product, internal standard, etc.)
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Tailing Factor (T) Theoretical plates (N)
T of =2 In general should be > 2000
1. Capacity factor (k1): Capacity factor is the ratio of the reduced retention volume to the dead volume. Capacity factor k1 is defined as the ratio of the number of molecules of solute in the stationary phase to the number of molecules of the same in the mobile phase. Capacity factor is a measure of how well the sample molecule is retained by a column during an isocratic separation. The ideal value of k1 ranges from 2-10. Capacity factor can be determined by using the formula,
Capacity factor (Fig: 3) Where, tR = retention volume at the apex of the peak (solute) and t0 = void volume of the system.
Capacity factor (k1) changes are typically due to: Variations in mobile phase composition Changes in column surface chemistry (due to aging) Changes in operating temperature In most chromatography modes, capacity factor (k') changes by 10 percent for a temperature change of 5 C.
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Adjusting capacity factor (k1): Good isocratic methods usually have a capacity factor (k1) in the range of 2 to 10 (typically between 2 and 5). Lower values may give inadequate resolution. Higher values are associated with excessively brood peaks and unacceptably long run times. Capacity factor (k1) values are sensitive to: Solvent strength Composition Purity Temperature Column chemistry Sample
2. Selectivity (a): The selectivity (or separation factor, a) is a measure of relative retention of two components in a mixture. Selectivity is the ratio of the capacity factors of both peaks, and the ratio of its adjusted retention times. Selectivity represents the separation power of particular adsorbent to the mixture of these particular components. This parameter is independent of the column efficiency it only depends on the nature of the components, eluent type, eluent composition and adsorbent surface chemistry. In general if the selectivity of two components is equal to 1, then there is no way to separate them by improving the column efficiency. The ideal value of a is 2. It can be calculated by using formula, a =V2 V1 / V1 V0 = k1(2) / k1 (1) Where, V0 is the void volume of the column, V1 and V2 are the retention volumes of the first and the second peak respectively.
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Selectivity (Fig: 4) 3. Resolution (Rs): Resolution is the parameter describing the separation power of the complete chromatographic system relative to the particular components of the mixture. Resolution can be improved by increasing column length, decreasing particle size, increasing temperature, changing the eluent or stationary phase. The resolution Rs of two neighboring peaks is defined as the ratio of the distance between two peak maxima. It is the difference between the retention times of two solutes divided by their average peak width. For baseline separation, the ideal value of Rs is 1.5. It is calculated by using the formula,
Resolution between two peaks (Fig: 5). Where, tR (1) and tR (2) are the retention times of components 1 and 2 and W1 and W2 are peak width of components 1 and 2. 4. Column Efficiency (N): Efficiency N of a column is measured by the number of theoretical plates per meter. It is a measure of band spreading of a peak. Higher the number of theoretical plates more efficient is the column. Columns with N ranging from
5,000 to 100,000 plates / meter are ideal for a good system. Parameters which can affect N include Peak position, particle size in column, flow-rate of mobile phase, column temperature, viscosity of mobile phase, and molecular weight of the analyte. Efficiency is calculated by using the formula.
(Fig:6) Where, tR is the retention time and W is the peak width. 5. Peak asymmetry factor (Af): Peak asymmetry factor (Af), can be used as a criterion of column performance. The peak half width b of a peak at 10 % of the peak height, divided by the corresponding front half width a gives the asymmetry factor
(Fig: 7) Asymmetric Factor For a well packed column, an asymmetry factor of 0.9 to 1.1 should be achievable.
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(Fig.no :8)Asymmetric Factor METHOD DEVELOPEMENT FOR RP-HPLC11-13 During the development of method, the initial sets of conditions that have evolved from the Literature survey are improved or maximized in terms of resolution, peak shape, plate counts, asymmetry, capacity, elution time, detection limit, limit of quantitation and overall ability to quantify the specific analyte of interest. Optimization of a method can follow either of two general approaches: 1. Manual 2. Computer driven The manual approach involves varying one experimental variable at a time, while holding all others constant, and recording changes in response .The variables might include flow rates, mobile or stationary phase composition, temperature, detection wavelength, and pH. This univariate approach to system optimization is slow, time consuming and potentially expensive, however it may provide a much better understanding of the principles, theory involved and interactions of the variables. In the second approach, computer driven automated method development, efficiency is optimized while experimental input is minimized. Computer driven automated approaches can be applied to many applications. In addition, they are capable of significantly reducing the time, energy and cost of virtually all-instrumental methods. The various parameters that need to be optimized during method development 1. Mode of separation 2. Selection of stationary phase 3. Selection of mobile phase
4. Selection of detector Selection of mode of separation In reverse phase mode, the mobile phase is comparatively more polar than the stationary phase. For the separation of polar or moderately polar compounds, the most preferred mode is reverse phase. The nature of the analyte is the primary factor in the selection of the mode of separation. A second factor is the nature of the matrix. Selection of stationary phase / column Selection of the column is the first and the most important step in method development. The approaches 1. Selection of separation system 2. The particle size and the nature of the column packing 3. The physical parameters of the column i.e. the length and the diameter Some of the important parameters considered while selecting chromatographic columns are a) Length and diameter of the column b) Packing material c) Shape of the particles d) Size of the particles e) % of carbon loading f) Pore volume g) Surface area h) End capping The column is selected depending on the nature of the solute and the information about the analyte. Reversed phase mode of chromatography facilitates a wide range of columns like dimethyl silane (C2), butylsilane (C4), octylsilane (C8), octadecylsilane (C18), base deactivated silane (C18) BDS phenyl, cyanopropyl (CN), nitro, amino etc. Generally longer columns provide better separation due to higher theoretical plate numbers. As the particle size decreases the surface area available for coating increases. Columns with 5-m particle size give the best compromise of efficiency, reproducibility
appropriate choice of separation column includes three different
and reliability. In this case, the column selected had a particle size of 5 m and a internal diameter of 4.6 mm Peak shape is equally important in method development. Columns that provide symmetrical peaks are always preferred while peaks with poor asymmetry can result in, Inaccurate plate number and resolution measurement Imprecise quantitation Degraded and undetected minor bands in the peak tail Poor retention reproducibility Selection of mobile phase The primary objective in selection and optimization of mobile phase is to achieve optimum separation of all the individual impurities and degradants from each other and from analyte peak. In liquid chromatography, the solute retention is governed by the solute distribution factor, which reflects the different interactions of the solute stationary phase, solute mobile phase and the mobile phase stationary phase. For a given stationary phase, the retention of the given solute depends directly upon the mobile phase, the nature and the composition of which has to be judiciously selected in order to get appropriate and required solute retention. The mobile phase has to be adapted in terms of elution strength (solute retention) and solvent selectivity (solute separation) Solvent polarity is the key word in chromatographic separations since a polar mobile phase will give rise to low solute retention in normal phase and high solute retention in reverse phase LC. The selectivity will be particularly altered if the buffer pH is close to the pKa of the analytes. The following are the parameters, which shall be taken into consideration while selecting and optimizing the mobile phase.
Buffer pH of the buffer Mobile phase composition
Buffer and it strength

Buffer and its strength play an important role in deciding the peak symmetries and separations. Some of the most, commonly employed buffers are
Phosphate
buffers
prepared
by
using
salts
like
KH2PO4,
K2HPO4,NaH2PO4,Na2HPO4,etc
Phosphoric acid buffers prepared by using H3PO4.
Acetate buffers Ammonium acetate, Sodium acetate, etc.

Acetic acid buffers prepared using CH3COOH.
The retention times also depend on the molar strengths of the buffer Molar strength is increasingly proportional to retention times. The strength of the buffer can be increased, if necessary, to achieve the required separations. The solvent strength is a measure of its ability to pull analytes from the column. It is generally controlled by the concentration of the solvent with the highest strength. pH of the buffer pH plays an important role in achieving the chromatographic separations as it controls the elution properties by controlling the ionization characteristics. Experiments were conducted using buffers having different pH to obtain the required separations. It is important to maintain the pH of the mobile phase in the range of 2.0 to 8.0 as most columns does not withstand to the pH which are outside this range. This is due to the fact that the siloxane linkage area cleaved below pH 2.0, while pH values above 8.0, silica may dissolve. Mobile phase composition Most chromatographic separations can be achieved by choosing the optimum mobile phase composition. This is due to the fact that fairly large amount of selectivity can be achieved by choosing the qualitative and quantitative composition of aqueous and organic portions. Most widely used solvents in reverse phase chromatography are Methanol and Acetonitrile. Selection of detector The detector was chosen depending upon some characteristic property of the analyte like UV absorbance, fluorescence, conductance, oxidation, reduction etc. Characteristics that are to be fulfilled by a detector to be used in HPLC determination are,
High sensitivity, facilitating trace analysis Negligible baseline noise to facilitate lower detection Large linear dynamic range Low dead volume Non destructive to sample Inexpensive to purchase and operate All pharmaceutical ingredients do not absorb UV light equally, so that selection of detection wavelength is important. An understanding of the UV light absorptive properties of the organic impurities and the active pharmaceutical ingredient is very helpful. For the greatest sensitivity max should be used. UV wavelengths below 200 nm should be avoided because detector noise increases in this region. Higher wavelengths give greater selectivity.
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(Fig 9) Strategy for Method Development 1. Introduction on sample Define separation goals 2. Need for special HPLC
Procedure, sample, pretreatment, etc 3. Choose detector and Detector settings
4. Choose LC method; Preliminary run; estimate best Separation separaons
5. Optimize separation condition 6. Check for problems or requirements for special procedure
7a. Recover
7b.Quantitative
7c. Qualitative method

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calibration purified material A.M.Reddy Memorial College of Pharmacy
8. Validate method for release into routine laboratory
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3.0 DRUG PROFILE ZOLEDRONICACID
[1-hydroxy-2-(1H-imidazol-1-yl) ethane-1, 1-diyl]bis(phosphonic acid)
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TESTS i) Description ii) pH Solubility: a) Soluble in water b) Soluble in NaOH v) Molecular formula vi) Molecular weight vii) Official Status I. Pharmacokinetics:
RESULTS White to Half white powder 4.5-7
sparingly Soluble freely soluble C18 H24 N2 O7P2 348.9 It is official in B.P.2007 and official method is HPLC
1. Absorption: zoledronic acid is rapidly absorbed after oral dose, with peak plasma
concentration occurring after about 2hrs.

2. Distribution: zoledronic acid has a large volume of distribution of around 3lit/Kg,
Distributes freely between plasma and RBC. Plasma protein binding is about 65%.
3. Metabolism: it was not metabolized by liver 4. Elimination: it was excreted by kidney in an unchanged form
II. Strength available: Zometa -4mg/vial Zolodonat -4mg/vial III. Storage: Store in air tight containers, at a temperature not exceeding 250C IV. Adverse effects: Pruritis
Nausea & vomiting Hypocalcaemia Rise in body temperature Dementia Anxiety V. Uses and Administration It was used in the treatment of bone cancer and it was administered through I.V. route.
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4.0 LITERATURE REVIEW Mastanamma S K et al., (2012)27, Developed a rapid and reproducible reverse phase high performance liquid chromatographic method has been developed for the estimation of zoledronic acid in its pure form as well as in pharmaceutical dosage forms. Chromatography was carried out on an ODS C18column (250 x 4.6 mm x 5 m length),using a mixture of methanol and 0.01M phosphate buffer (pH-3) (60:40 v/v) as the mobile phase at a flow rate of 0.8 mL/min and the detection was done at 210 nm. The retention time of the drug was 3.812 min. The method produced linear responses in the concentration range of 0.25 to 60 g/mL of zoledronic acid. The method was found to be reproducible for analysis of the drug in parentral preparations.
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L. Maheswara Reddy et al., (2012)28, developed a novel, selective and sensitive reverse phase-high performance liquid chromatography (RP-HPLC) method has been developed for the validated estimation of imidazol-1-yl-acetic acid in zoledronic acid formulations. The separation was achieved on a 5 C18 column (250 X 4.6 mm) using mobile phase consist of buffer (4.5 g of Di-potassium hydrogen phosphate anhydrous and 2.0 g of tetra butyl ammonium hydrogen sulphate (TBAHS) in 1000 mL of water) and methanol in the ratio of 900:100 v/v. The flow rate was maintained at 1.0 mL min -1. The detection of the constituents was done at 215 nm using UV detector. The retention time of imidazol-1-ylacetic acid and zoledronic acid were 7.2 and 10.2 min respectively. Recovery studies were satisfactory and the correlation coefficient, 0.999 indicates linearity of the method within the limits. The developed method can be applicable for regular qualitative analysis. Raghu et al., (2011)29, developed A new ion exchange high performance liquid chromatographic (IEC) method has been developed and validated for quantitative determination of Zoledronic acid in pharmaceutical injection dosage form. Complete separation was achieved for the parent compound Zoledronic acid, the impurities and excipients in an overall analytical run time of approximately 20 minutes. The proposed chromatographic conditions employed an isocratic elution of mobile phase at constant eluent flow rate of 0.7 mL min-1 and by using a new generation Allsep anion exchange column. A UV-Vis detector set at 215 nm was used to monitor the eluate. The 100% aqueous mobile phase consisted of only diluted formic acid without any ion-pair substance. The drug product was subjected to oxidation, hydrolysis, photo-stability, and heat to apply the stress conditions. The method was found to be linear over the concentrations range from 0.200 to 1.200 mg mL-1(25% to 150% of Zoledronic acid concentration). The newly developed method has the requisite accuracy, selectivity and precision to assay Zoledronic acid in commercial pharmaceutical injection dosage form. ZHENG Guo-gang et al., (2005)30, developed To establish an HPLC method for the determination of zoledronic acid for injection. METHOD:The analysis was achieved by using Diamonsil C18 column with acetonitrile- ion-pair buffer solution(25 : 75 ) as mobile phase and UV-detector at wavelength 210nm. The ion-pair buffer solution was a mixture of 0.02mmol/mL tetrabutyl ammonium hydroxide and 0. 02mmol/mL ammonium dihydrogen phosphate adjusted pH 2. 3 with 50% phosphoric acid RESULTS:
The linear range was from 50 to 240 g/mL (r = 1. 0). The average recovery was 100. 0% (RSD = 0. 4% ). CONCLUSIONS: This method is simple, sensitive, accurate and suitable for the determination of zoledronic acid for injection. WANG Ling-ling et al., (2006 - 2008)31, developed To establish a HPLC method for assay of zoledronic acid for injection and its related substances. Methods:The reversedphase HPLC condition was as follows; a C18 ODS column (250 mm4.6 mm,5m) , an eluate consisted of acetonitrile-tetrahydrofuran-0. 05 molL-1 ammonium dihydrogen phosphate (4: 1: 100, pH =2. 55 adjusted with phosphoric acid) for zoledronic acid assay, an eluate composed of 0.03% tetrabutyl-ammonium hydroxide solution (pH =2. 55 adjusted with phosphoric acid)-acetonitrile (100:7) for the related substances, a flow rate of 1.0 mLmin-1 and the detection at 218 nm. Results:The calibrated linear curve of zoledronic acid was within 5 - 125gmL-1. The average recovery rate was 100.3% with RSD of 0.50%. The related substances of zoledronic acid were completely separated from zoledronic acid. CONCLUSION: This reliable RP-HPLC method can be used in quality control of zoledronic acid. Katrin Veldboer et al., (2011)32, developed A new method for the analysis of 1-hydroxy2-imidazol-1-yl-phosphonoethyl phosphoric acid (zoledronic acid) in urine and blood samples has been developed. It consists of a derivatisation of the bisphosphonate with trimethylsilyl diazomethane under multiple methylester formation. The formed derivative can, in contrast to the non-derivatisedanalyte, easily be separated by reversed phase liquid chromatography due to its reduced polarity. Detection is performed by electrospray tandem mass spectrometry. For calibration purposes, a deuterated internal standard has been synthesized in a three-step synthesis starting with d(4)-imidazole. For human urine, the limit of detection (LOD) is 1.2x10(-7) mol/L, limit of quantification (LOQ) is 3.7510(-7) mol/L in the MRM mode. For human blood plasma, a LOD of 110(-7) mol/L and a LOQ of 2.510(-7) mol/L were determined. The linear dynamic range comprised 3.5 decades starting at the limit of quantification. The method was successfully applied for the analysis of spiked urine and blood plasma samples as well as samples from two osteoporosis patients. JIANG Ye et al., (2005 - 2007)33, developed To establish a HPLC method for the determination of zoledronic acid and its preparations. METHODS Ion-pair RP-HPLC coupled with evaporative light-scattering detection was performed on a BDS C8 column
(4.6 mm 150 mm,5m) at room temperature. The mobile phase consisted of methanolbuffer solution (5 mmolL-1 ammonium acetate and 10 mmolL-1 amylamine, adjusted to pH 7.0 with acetic acid) (3:97), at a flow rate of 1.0 mLmin-1. RESULTS: The calibration curve was linear in the range of 99.04 -891.4 gmL-1(r = 0.9999).For zoledronic acid for injection, the average recovery was 100.4% with RSD of 0.76% (n = 9); for zoledronic acid injection, the average recovery was 100.2% with RSD of 0.69% (n = 9). CONCLUSION The method is rapid, simple and accurate for the determination of zoledronic acid in material and in its preparations. Rao BM et al., (2005)34, developed the present paper describes the development of a stability indicating high performance liquid chromatographic (HPLC) assay method for zoledronic acid in the presence of its impurities and degradation products generated from forced decomposition studies. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The degradation of zoledronic acid was observed under oxidative stress at higher temperature. The drug was found to be stable in other stress conditions attempted. Successful separation of the drug from the degradation products formed under stress conditions was achieved on a C18 column using a mixture of phosphate buffer that contains 7 mM tetra butyl ammonium hydrogen sulphate, an ion-pairing agent and methanol(95:5) as mobile phase. The developed HPLC method was validated with respect to response function, precision, accuracy, specificity and robustness. The developed HPLC method to determine the related substances and assay determination of zoledronic acid can be used to evaluate the quality of regular production samples. It can be also used to test the stability samples of zoledronic acid. Francois Legay et al., (2002)35, developed Zoledronic acid is a new, highly potent bisphosphonate drug under clinical evaluation. A radioimmunoassay has been developed to determine zoledronic acid concentration in human serum, plasma, and urine. The assay utilizes rabbit polyclonal antisera against a zoledronic acid-BSA conjugate and a [125I] zoledronic acid derivative as tracer in a competitive format adapted to microtiter plates. The assay shows a LLOQ 0.4 ng/ml in serum or plasma (interassay%CV=17%, accuracy 97%), 5 ng/ml in urine (21%, 98%). In 23 patients receiving 4, 8 or 16 mg of zoledronic acid, drug concentrations in plasma were dose proportional and showed a multiphasic profile, followed by a prolonged gradual decline to concentrations near the LLOQ. Zoledronic acid disposition in plasma and the recovery of only 40-50% of the dose in
urine are consistent with the rapid and extensive uptake by and slow release from bone in parallel with renal clearance, typically shown by bisphosphonates. Rakesh Kumar Jat et al., (2008)36, developed a simple, accurate rapid and precise RPHPLC method has been developed and validated for determination of ketoconazole in bulk drug. The RP-HPLC separation was achieved on Promosil C-18, (250 mm, 4.6 mm, 5m) using mobile phase water : acetonitrile : buffer ph 6.8 (51:45:4 v/v) at flow rate of 1.0 ml/min at ambient temperature. The retention times were 2.713 min. for ketoconazole. Calibration plots were linear over the concentration range 1-50g/ml. Quantification was achieved with photodiode array detection at 238 nm over the concentration range of 1-50 g/ml. The method was validated statistically and applied successfully for the determination of ketoconazole. Validation studies revealed that method is specific, rapid, reliable, and reproducible. The high recovery and low relative standard deviation confirm the suitability of the method for the routine determination of ketoconazole in bulk drug. F. Al-Rimawi et al., (2009)37, developed A simple and stability-indicating liquid chromatographic method was developed and validated for the analysis of Fluconazole and its related compound (A, B, and C) in capsule formulations. Liquid chromatography with a UV detector at a wavelength of 260 nm using a reversed-phase C18 column was employed in this study. Isocratic elution was employed using a mixture of methanol and water (40:60, v/v). This new method was validated in accordance with USP requirements for new methods for assay determination, which include accuracy, precision, specificity, linearity and range. The current method demonstrates good linearity over the range of 0.05-0.15 mg/ml of Fluconazole. The accuracy of the method is 99.3%. The precision of this method reflected by relative standard deviation of replicates is 0.61%. Validation of the same method for Fluconazole related compounds analysis was also performed according to USP requirements for quantitative determination of impurities which include accuracy, precision, linearity and range, selectivity, and Limit of quantitation (LOQ). Low LOQ of the related compounds using this method enables the detection and quantitation of these impurities at low concentration. Sarath chandiran et al., (2011)38, developed A simple, sensitive and selective method is described for the determination of itraconazoleand its metabolite in human plasma, Miconazole as internal standard by using HTLCMS/MS. The method consists of a online coupling of extraction with cyclone P (50mm x0.5mm, 50m) HTLC column by injecting 15L sample and chromatographic separation isperformed with C18 Reverse phase
column using 90:10 Acetonitrile: 10mM Ammonium Formate Buffer (pH 6.8) as gradient mobile phase followed by quantification with Tandem Mass spectrometry (MS/MS) in selective reaction monitoring mode using Electro sprayionization mode (ESI) as an interface. The method was fully validated in terms of specificity sensitivity, precision, accuracy and stability over a concentration range of 1 to 500g/ml for both Drug and its metabolite using 0.5ml of human plasma per assay. Stability assessment was also included. The total run time for sample analysis was 1.5 min and the lower limit of quantification was 1ng/mL for both drug and its metabolite. The validated method was applied in bioavailability and bioequivalence study Y. Vander Heyden et al., (2002)39, developed Ketoconazole is an antifungal agent, which is the active ingredient in a shampoo primarily used for the treatment of seborrhatic dermatitis (anti-dandruff shampoo). The shampoo also contains imidazolidinyl urea as a formaldehyde releasing preservative. The aim of this study was to develop a HPLC system that allows the determination of both ketoconazole and formaldehyde. The finally selected isocratic system consisted of an Interchrom Nucleosil (25034.6 mm, 5 mm) C column 8 and a mobile phase containing acetonitrilephosphate buffer 0.025 M, pH 4.0, 45/55 (v/v). Ketoconazole could immediately be determined at 250 nm after injection of diluted shampoo. Formaldehyde was measured at 345 nm after derivatisation with a 2,4dinitrophenylhydrazine solution. At the selected conditions, the other excipients of the shampoo did not interfere in the assays for both substances. Method validation was performed on both assays. Different selectivity towards ketoconazole and formaldehyde was observed when applying other C columns. This fact, however, did not affect the assays of both 8substances. Chiranjeevi Bodepudi et al., (2005)40, developed A precise and feasible highperformance liquid chromatographic (HPLC) method for the analysis of the Fluconazole and Tinidazole in a combined tablet dosage form has been developed. The analysis was carried out on a Kromasil stainless steel C18 (250 x 4.6 mm, 5 ) reversed-phase column, using a mixture of Acetonitrile: Water (55:45%v/v) as the mobile phase using a low pressure gradient mode with flow rate at 1ml/min. The injection volume was 20l..The retention time of the drug was 2.5 for Fluconazole and 3.1 for Tinidazole. The method produced linear responses in the concentration range of 10 to 50g/ml for both Fluconazole and Tinidazole. The Tailing factors of Fluconazole and Tinidazole were
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found to be 1 and 1.3 respectively. The method was found to be applicable for determination of the drug in tablets. V.Sekar et al., (2008 - 2009)41, developed A rapid high-performance liquid chromatography method has been developed for bioanalytical method development and validation of letrozole in human plasma. letrozole (CGS 20 267), a potent aromatase inhibitor for treatment of oestrogen - depentent diseases. Letrozole was found with symmetrical peak shapes on a analytical column Phenomenex Luna C18 column using 75% 0.02M Phosphate buffer at PH 5.5 and 25% acetonitrile as the mobile phase. The retention times of Letrozole and fluconazole the internal standard were 4.29 and 7.47 min respectively. Linear calibration curves were obtain for each compound across a range of 50.55-120.00 ng/ml. the limit of detection was 12.5ng/ml and the limit of Quantification was 37.5ng/ml for Letrozole. Greater than 85% recoveries were obtain for Letrozole. The intra and interday relative standard deviation (%RSD) were <5%. It uses less biological material and applicable. A Anil Kumar et al., (2012)42, developed Sirolimus and Ketoconazole are used in organ transplantation regimen and potential metabolic interactions of these drugs were reported when administered concomitantly. An analytical method based on high-performance liquid chromatography (HPLC) with photo diode array (PDA) detection was developed for quantification of sirolimus using ketoconazole as internal standard. Extraction was performed using dichloromethane under nitrogen atmosphere and the separation of sirolimus and ketoconazole was accomplished by reverse phase chromatography. The mobile phase consists of a combination of methanol, water and glacial acetic acid at 90:10:0.1% ratios run isocratically through a C18 (250mm 4.6mm, 5m) reverse phase analytical column. The PDA detection was done at 278nm with analytical run time less than 6 min. The average mean recovery was found to be 98.3% for 1, 3, 5, 10g/ml concentrations. The assay exhibited good linear relationship. LLOQ was 10ng/ml with 0.84% and 1.28% of accuracy and precision over the concentration range of 0.1-10g/ml. The method can be successfully applied for estimation of sirolimus from in-vitro elution studies of sirolimus eluting stents, simultaneous estimation of ketoconazole and sirolimus in therapeutic drug monitoring and other pharmacokinetic studies. Parul Parmar et al., (2009)43, developed A simple, precise, accurate and rapid high performance thin layer chromatographic method has been developed and validated for the determination of clotrimazole in bulk drug and tablet dosage form. The stationary phase
used was precoated silica gel 60F 254 . The mobile phase used was a mixture of cyclohexane : toluene : methanol : triethyleamine (8:2:0.5:0.2 v/v/v/v). The detection of spot was carried out at 262 nm. The method was validated in terms of linearity, accuracy, precision and specificity. The calibration curve was found to be linear between 200 to 1000 ng/spot for clotrimazole. The limit of detection and the limit of quantification for clotrimazole were found to be 50 ng/spot and 200 ng/spot, respectively. The proposed method can be successfully used to determine the drug content of bulk drug and marketed formulation of tablet. Hjkov R et al., (2007)44, developed A novel simple isocratic HPLC method with UV detection for the determination of three compounds in spray solution (active component clotrimazole and two degradation products imidazole and (2chlorophenyl)diphenylmethanol) using ibuprofen as an internal standard was developed and validated. The complications with different acido-basic properties of the analysed compounds in HPLC separation - while clotrimazole has pK(a) 4.7, imidazole has pK(a) 6.9 compared to relatively more acidic (2-chlorophenyl)diphenylmethanol - were finally overcome using a 3.5mum Zorbax((R)) SB-Phenyl column (75mmx4.6mm i.d., Agilent Technologies). The optimal mobile phase for separation of clotrimazole, degradation products imidazole and (2-chlorophenyl)diphenylmethanol and ibuprofen as internal standard consists of a mixture of acetonitrile and water (65:35, v/v) with pH* conditioned by phosphoric acid to 3.5. At a flow rate of 0.5mlmin(-1) and detection at 210nm, the total time of analysis was less than 6min. The method was applied for routine analysis (batch analysis and stability tests) in commercial spray solution. Farzaneh Ahmad Khan Beigi et al., (2011)45, developed A simple confirmatory method for HPLC-UV determination of ketoconazole and clotrimazole residues in cow's milk after solid phase extraction (SPE) is reported in this article. The samples were deprived of proteins and lipids by treating with acetonitrile and consequent extraction in n-hexane. Extracts were further cleaned-up and concentrated via solid phase extraction. The analytes were determined quantitatively using a validated high performance liquid chromatography method. The method was linear in the range of 0.1-1.0 g mL -1 for both analytes. Limits of detection and quantification for both analytes were equal to 0.01 and 0.1 g mL-1, respectively. Important parameters influencing the extraction efficiency were
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investigated and then optimized; the proposed method was applied to the analysis of milk samples. Satisfactory recoveries obtained in the range of 95.9-101.78% using SPE. E M Abdel-Moety et al., (2002)46, developed High-performance liquid chromatographic technique has been developed for the determination or some azolcsantifungals namely, clotrimazole (CZ), ketoconazole (KZ) and fluconazole (FZ), in pure forms and in pharmaceutical formulations. The proposed HPLC-method can be successfully applied as a stability indicating method for the determination of CZ in presence of its acid degradation products; viz (2-chlorophenyl)-diphenyl methanol and imidazole. The analyzed drugs were separated on a reversed-phase column [Bondapak C18 (10 microm, 25 cm x 4.6 mm, i.d.)] using a mobile phase containing acetonitrilc+25 mM trishydroxy methyl amino methane in phosphate butter (pH 7)= 55:45 (v/v), with UV-detection at 260 nm. The differences in the retention times (tR) of the three azoles permit their use as internal standard for each other. In addition, a coupled TLC-densitometric method has been also applied as a stability indicating method to separate and quantify CZ alone or in presence of byproducts impurities and/or its acid degradation products. The TLCfractionation was performed on a precoated silica gel F254 plates using a solvent system consisting of chloroform+acetone+ammonia (25%) (7:1:0.1, by volumes), CZ was well separated from its acid degradation products and quantified by densitometric scanning at 260 nm.
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5.0 PLAN OF WORK It includes a) Method development i. Selection of wavelength

ii. pH selection
iii. Column selection iv. Flow rate selection b) Validation and optimization of parameters i. System suitability ii. System specificity
iii. Precision a. Method repeatability b. Method reproducibility iv. Linearity v. Range vi. Accuracy vii. Robustness
viii. ruggedness
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6.0 MATERIALS AND METHODS

a) Equipments
i. HPLC ( waters 2695 ) ii. UV double beam spectrophotometer ( Schimadzu )

iii. PH meter
iv. Sonicator b) Reagents and standards i. Triethylamine ii. Orthophosphoric acid

iii. Water for injection iv. Zoledronic acid working standard c) Introduction to method
i. Solubility: It includes solubility of drug substance in different solvents and
different PH conditions.
ii. Wavelength selection: Spectral profile is useful in understanding absorption
characteristics which helps in selection of detector and wavelength for analysis.

iii. Mobile phase selection and its optimization: Its selection is done always in
combination with selection of column. Following parameters should be taken into consideration while doing selecting and optimizing the mobile phase a. Buffer & its strength
b. Buffer/mobile phase pH
c. Mobile phase composition

iv. Column selection :A column has been selected for analytic purpose by
considering following parameters a. Length & diameter
b. Packing material c. Particle size & shape d. % of carbon load e. Pore volume & surface area
v. Solvent delivery system selection: Separation using with one solvent called
isocratic elution always preferable. Sometimes gradient elution also preferable. a. Low pressure gradient elution :- mobile phases are mixed at predetermined ratio
b. High pressure gradient elution :- mobile phases are pumped at different flow rate vi. Flow rate selection: It will be selected upon a) Retention time b) Column back pressure c) Separation of impurities d) Peak symmetry Note: It should be not more than 2.5ml/min vii. Temperature selection: Generally Ambient Column Temperature Was Preferable To Optimize
Chromatographic Conditions.
viii. Diluents Selection:
It will be based upon a) Extraction efficiency b) Peak symmetry c) Resolution of impurities from API peak
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7.0 METHOD DEVELOPMENT a) Method description

i.
Preparation of mobile phase /Buffer- Mix 2 ml tri methyl amine in 1000ml water for injection & adjust pH to 3.2 with ortho phosphoric acid & filter through 0.22 membrane filter & degas
ii. Preparation of placebo-Weigh accurately about 1.10g of mannitol& 0.12g of
sodium citrate in 10ml volumetric flask & add 5ml mobile phase & sonicate to dissolve & dilute with same mobile phase .Take 2ml of this solution into 25 ml volumetric flask & make up the volume with mobile phase & filter
iii. Preparation of Standard Weigh 22mg of zoledronic acid marking Standard into
25ml volumetric flask & make up with mobile phase. Take 5 ml from this & again make up with mobile phase in another 25ml volumetric flask.
iv. Preparation of sample Transfer contents of 5 vials into 25 ml volumetric flask by
rinsing vials 2-3 times with mobile phase & sonicated & make up again. Take 5ml of this solution into another 25ml volumetric flask & make up to volume with mobile phase.
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b) Optimization of method development parameters i. Selection of wavelength TRAIL-1 CHROMATOGRAM (7.a.1.1)
TRAIL -2 CHROMATOGRAM (7.a.1.2)
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Result: After reviewing above chromatograms maximum absorption occurs at
215 nm
by using PDA detector. So it was selected as optimum wavelength for this specified drug.
ii. Selection Of pH
Buffer pH 3.2 3.4 3.6
Retention time (mins) 3.387 3.387 3.395 Table no: 7.1.T
Tailing factor 1.30 1.30 2.0
CHROMATOGRAMS AT 3.2 pH
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CHROMATOGRAMS AT PH 3.4

CHROMATOGRAMS AT 3.6 PH : TRAIL -1 CHROMATOGRAM (7.a.4.1)


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Result: From the above results peak shape was found to be good at PH at 3.2 and 3.4 respectively, but considering column stability PH at 3.2 will be regarded as suitable pH. iii. Column selection
Type of column Hypersil BDS C18 Unicem US C18
Retention time ( mins) 3.14 4.0
Tailing factor 1.3 2.5
Area 2224305 1333103
Table no: 7.2.T Chromatograms using Hypersil BDS C18 TRAIL -1 CHROMATOGRAM (7.a.5.1)
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Chromatograms using Unicem US C18 :TRAIL -1 CHROMATOGRAM (7.a.6.1)
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Result: After reviewing results there has been better separation has been observed between API and other peaks. Retention time, area and tailing factor has been satisfactorily found with Hypersil BDS C18 column. Hence it will be selected. iv. Flow rate selection
Flow rate ( mL/ min) 1.0 1.2
Retention time (mins) 3.4 2.8 Table no: 7.3.T
Area 2245388 1865507
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CHROMATOGRAMS AT 1.0 (mL/ min) FLOW RATE TRAIL -1 CHROMATOGRAM (7.a.7.1)
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CHROMATOGRAMS AT 1.2 (ML/MIN) FLOW RATE :TRAIL -1 (7.a.8.1)CHROMATOGRAM

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Result: For the selection of flow rate parameters such as retention time and area were found to be good at flow rate of 1.2ml/min. Hence the flow rate of 1.2ml/min can be selected for this method validation. 5. Mobile phase composition S.no 1. Solvent composition Water for injection and Ortho phosphoric acid 2. 3. buffer (95:5%v/v) Methanol and phosphate buffer (60:40%v/v) Tetrabutyl ammonium and methanol (900:100v/v) Table no: 7.4.T After reviewing the above data, the composition of mobile phase is decided as 95:5 (v/v). CHROMATOGRAPHIC CONDITIONS
1. Column: HypersilBDS C18 ( 250x 4.6mm) 2. max: 215nm
Retention time 3.347min
4.2min 10.4min
3. Flow rate : 1.2mL/min 4. Injection volume : 20l 5. Column temperature: 35c 6. Run time :10 min
8.0 METHOD VALIDATION13, 14 Method validation is an integral part of the method development, it is the process by which a method is tested by the developer or user for reliability, accuracy and preciseness of its intended purpose. International Conference on Harmonization defines validation as Establishing
documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics. According to ICH, typical analytical performance characteristics that should be considered in the validation of different types of methods are: System suitability Specificity Precision Linearity Range Accuracy Robustness Ruggedness
8.1 SYSTEM SUITABILITY
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System suitability is the evaluation of the components of an analytical system to show that the performance of a system meets the standards required by a method. A system suitability evaluation usually contains its own set of parameters. For Chromatographic assays, these may include tailing factors, resolution, and precision of standard peak areas and comparison to a confirmation standard, capacity factors, retention times, and theoretical plates. During validation where applicable system suitability parameters are calculated, recorded and trended throughout the course of the validation. PROCEDURE: injected Standard preparations (6 replicate injections) into
chromatograph & recorded the system suitability parameters as per test procedure Acceptance criteria: i) % RSD for 6 replicates injection of peak response of zoledronic acid from Standard preparation should NMT 2 ii) Tailing factor for zoledronic acid peak should NMT 2 iii) Theoretical plate count of peak should be more than 2000
8.1.1a. System suitability peak for zoledronic acid
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8.1.2b.system suitability peaks for zoledronic acid dilutions

Parameter % RSD Tailing factor Platecount
Acceptance criteria NMT 2 NMT 2 NLT 2000 Table no: 8.1.1T.
Result 0.41 1.18 5962
Result: system suitability parameter meets acceptance criteria 8.2 SPECIFICITY Specificity is the ability to assess unequivocally the analyte in the presence of components that may be expected to be present such as impurities, degradation products
and excipients. To determine specificity during the validation of blanks, sample matrix (placebo) and known related impurities are analyzed to determine whether interferences occur. Procedure: Prepare and inject water as blank in triplicates to the system. Prepare triplicate sample preparations with appropriate levels of excipients as placebo sample and inject into HPLC. Acceptance criteria: It should not show any interference from blank and placebo at retention times of zoledronic acid peak
8.2.1a. System specificity peaks for zoledronicacid using blank
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8.2.2b. System specificity peaks for zoledronic acid using placebo
8.2.3c. System specificity peaks for zoledronic acid using Standard dilutions
8.2.4d. System specificity peaks for zoledronicacid using sample dilutions
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Result:
Table no: 8.2.1T

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S.no 1 2 3 1 2 3 1 2 3 1 2 3
Sample name Blank 1 Blank 2 Blank 3 Placebo 1 Placebo 2 Placebo 3 Standard 1 Standard 2 Standard 3 Sample 1 Sample 2 Sample 3
Retention time (min) Nil Nil Nil 6.182 6.184 6.182 3.431 3.429 3.430 3.433 3.433 3.432
% of interference Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil
It was found that there was no interference observed from placebo and diluents and meets acceptance criteria. Hence the method was specific and selective for estimation of zoledronic acid in 4mg per vial injection dosage form. 8.3 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 obtained preferably using authentic samples. As parameters the standard deviation, the relative standard deviation (coefficient of variation) and the confidence interval should be calculated for each level of precision. Repeatability expresses the analytical variability under the same operating conditions over a short interval of time (intra-day). At least nine determinations covering the specified range or six determinations at 100 % test concentration should be performed. Intermediate precision includes the influence of additional random effects
within laboratories, according to the intended use of the procedure for example different days, analysts or equipment etc. Reproducibility i.e. the precision between laboratories (collaborative or inter laboratory studies) is not required for submission, but can be taken into account for standardization of analytical procedures. 8.3.1 METHOD REPEATABILITY Repeatability expresses precision under same operating conditions over a short interval of time by conducted the study as per test procedure in same laboratory by same analyst & by using same equipment
Procedure:i) Repeatability has been assessed using minimum of 6 determinations at 100 % of test concentration ii) Precision of test method has been conducted by assay of 6 replicate sample preparations of zoledronic acid injections & calculate % RSD for 6 samples Acceptance Criteria: RSD of 6 sample preparations should be NMT 2%
8.3.1a. Method Repeatability Peaks For Zoledronic Acid

RESULT: S.no T-1 T-2

Results (assay) 4.09 4.09

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T-3 T-4 T-5 T-6 Average Standard deviation % RSD 8.3.1.T
4.08 4.08 4.08 4.08 4.08 0.005 0.112
It was found that % RSD of 6 samples were within limit & hence the method is precise.
8.3.2 METHOD REPRODUCIBILITY Procedure:Reproducibility of Precision of test method has been conducted 6 determinations of same batch of samples tested in method repeatability by different analyst with same HPLC & column etc Acceptance criteria:% RSD of average assay results obtained by analytical group/another laboratory analyst should be NMT 3%.
8.3.2b. Method Reproducibility Peaks For Zoledronic Acid Working Standard
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8.3.3c. Method Reproducibility Peaks For Zoledronic Acid Dilutions
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Results : S.No T-1 T-2 T-3 T-4 T-5 T-6 Average Standard deviation % RSD 8.3.2.T It was found that RSD of 6 sample preparations of by analyst were less than 2% &RSD between assay results obtained by both analysts found less than 3% & hence test method was found to be precise. Results 4.14 4.14 4.13 4.12 4.12 4.13 4.13 0.008 0.193
8.4 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. It may be demonstrated directly on the analyte or on spiked samples using at least five concentrations over the whole working range. Besides a visual evaluation of the analyte signal as a function of the concentration, appropriate statistical calculations are recommended, such as a linear regression. The parameters slope and intercept, residual sum of squares and the coefficient of correlation should be reported. A graphical presentation of the data and the residuals is recommended. Procedure:A series of solutions of zoledronicacid Standards in concentration ranges from 80%-120% level were prepared & plot a graph to concentration vs area & determined the correlation coefficient
Result:8.4.1g
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8.4.1.T Linearity level (%) 80 90 100 110 120 Obtained concentration (mg/ml) 11.62 13.07 14.53 15.98 17.43 8.4.2.T Area 1628999 1843361 2055112 2265904 2468984
Slope Intercept Correlation coefficient Regression
144740 -5.0042 1.000 1.000
8.5 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. Range is normally expressed in the same units as test results (e.g. percent, parts per million) obtained by the analytical method. The working range of an analytical procedure is usually derived from the results of the other validation characteristics. It must include at least the expected or required range of analytical results, the latter being directly linked to the acceptance limits of the specification or the target test concentration. Result: Linearity level (%) 80 100 120 8.5.1.T Obtained concentration (mg/ml) 11.62 14.53 17.43 8.5.2.T Area 1628717 2053312 2466400
Correlation coefficient Y intercept Slope
1.000 -44731 144167
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CHROMATOGRAMS FOR LINEARITY
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8.5.1a. Linearity & Range For Zoledronic Acid

Result: It was found that correlation coefficient, slope, y intercept were within limits from range 80% - 120% of target concentration. Hence test method was found linear and range for estimation of zoledronic acid in injection.
8.6 ACCURACY The accuracy of a measurement is defined as the closeness of the measured value to the true value. In a method with high accuracy a sample (whose true value is known) is analyzed and the measured value is identical to the true value. Typically accuracy is represented and determined by recovery studies but there are three ways to determine accuracy, Comparison to a reference standard Recovery of the analyte spiked into blank matrix or Standard addition of the analyte. Procedure: It was determined by applying method in triplicate samples of mixtures of placebo to which known amount of working Standard was added at different levels of about 80%, 100% & 120% of nominal concentration (test conc 0.16 mg/ml).accuracy was then calculated from test results as % of analyte recovered by assay.
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8.6.1a. Accuracy Peaks for Zoledronicacid
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Acceptance criteria: Accuracy (Recovery) for average of triplicate in each concentration level should be within 98 102%. Result: Zoledronic acid accuracy studies: Sample name Zoledronic acid Zoledronic acid Zoledronic acid Zoledronic acid Zoledronic acid Zoledronic acid Zoledronic acid Zoledronic acid Zoledronic acid Sample I.D Accuracy 80% - 1 Accuracy 80% - 2 Accuracy 80% - 3 Accuracy 100% - 1 Accuracy 100% - 2 Accuracy 100% - 3 Accuracy 120% - 1 Accuracy 120% - 2 Accuracy 120% - 3 8.6.1.T Retention time (min) 3.427 3.423 3.420 3.418 3.418 3.417 3.416 3.415 3.414 Area 1665880 1652011 1653849 2102494 2106121 2107040 2539464 2532733 2527766
8.6.2.T Sample no. Spiked level Weight of zoledronic Weight of zoledronic Sample area Amount recovered % of Recovery
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1 2 3 1 2 3 1 2 3
80% 100% 120%
acid added 11.62 11.62 11.62 14.53 14.53 14.53 17.43 17.43 17.43
acid found 11.55 11.45 11.47 100.34 100.51 100.56 101.00 100.73 100.53
1665880 1652011 1653849 2102494 2106121 2107040 2539464 2532733 2527766
99.38 98.55 99.66 100.34 100.51 100.56 101.00 100.73 100.53
99.38% 98.55% 99.66% 100.34% 100.51% 100.56% 101.00% 100.73% 100.53%
It was found that accuracy (recovery) for average of triplicates from each concentration levels were within 98 102% (98.87 for 80%, 100.42 for 100% & 100.75 for 120% levels respectively) and meets acceptance criteria.
8.7 ROBUSTNESS : Robustness is defined as the measure of the ability of an analytical method to remain unaffected by small but deliberate variations in method parameters (e.g. pH, mobile phase composition, temperature, and instrument settings) and provides an indication of its reliability during normal usage. This is an important parameter with respect to the transferability of the method following validation. Determining robustness is a systematic process of varying a parameter and measuring the effect on the method by monitoring system suitability or the analysis of samples. It is part of the formal methods validation process.
It is performed by varying diff. Conditions such as pH , temperature, flow rate etc., a) Influence of variations on flow rate A study to establish influence/effect of variations of flow rate study was conducted as per test method & evaluated analytical method robustness by determined the system suitability parameters for following typical variations from set procedures.
Robustness flowrate: 8.7.1.T Injection no. 1 2 3 Tailing factor Average % RSD Area (1.2 ml/min) 801782 1813142 2229178 1.19 1614700 0.82 Area (1.1 ml/min) 727543 1680448 1997801 1.22 1468597 0.63 Area (0.9 ml/min) 889648 2056379 2439575 1.24 1795201 0.64
b) Influence of variations of pH in mobile phase
It is a study to establish the influence /effect of variations of p H in mobile phase. Study was conducted as per test method & evaluated analytical method robustness by determined the system suitability parameters for the following typical variations from set procedures. Robustness at different pH: 8.7.2.T Injection no. 1 2 3 Tailing factor Average % RSD Area (3.2 pH) 805518 1814170 2194771 1.24 1604820 0.85 Area (3.1 pH) 810274 1852889 2210100 1.23 1624421 1.04 Area (3.3 pH) 831885 1872787 2237816 1.23 1647496 0.99
8.7.1.1a Robustness at normal flowrate using placebo
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8.7.1.2b Robustness at normal flowrate of zoledronicacid working Standard
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8.7.1.3c Robustness peaks for zoledronic acid in normal flowrates using Standard dilutions
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8.7.1.4d Robustness Peaks For Zoledronic Acid In Normal Flowrate Using Sample Dilutions
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8.7.2.1a Robustness at flowrate 1.1ml using placebo
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8.7.2.2b Robustness at flow rate 1.1ml using zoledronic acid
8.7.2.3c Robustness at flow rate 1.1ml using Standard dilutions
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8.7.2.4d Robustness at flow rate 1.1ml using sample dilutions

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8.7.3.1a Robustness at flow rate 0.9ml using placebo
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8.7.3.2b Robustness at flow rate 0.9ml using zoledronic acid
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8.7.3.3c Robustness at flow rate 0.9ml using Standard dilutions
8.7.3.4d Robustness at flow rate 0.9ml using sample dilutions

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8.7.4.1a Robustness at Normal pH Using Placebo
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8.7.4.2b Robustness at normal pH using zoledronicacid
8.7.4.3c Robustness at normal pH using Standard dilutions
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8.7.4.4d Robustness at normal pH using sample dilutions
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8.7.5.1a Robustness at pH 3.1 using placebo
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8.7.5.2b Robustness at pH 3.1 using zoledronic acid
8.7.5.3c Robustness at pH 3.1 using Standard dilutions
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8.7.5.4d Robustness at pH 3.1 using sample dilutions

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8.7.6.1a Robustness at pH 3.3 using placebo
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8.7.6.2b Robustness at pH 3.3 using zoledronic acid
8.7.6.3c Robustness at pH 3.3 using Standard dilutions
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8.7.6.4d Robustness at pH 3.3 using sample dilutions

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8.7.7.1a Robustness by column change using placebo
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8.7.7.2b Robustness by column change using zoledronic acid
8.7.7.3c Robustness by column change using Standard dilutions

8.7.7.4d Robustness by column change using sample dilutions

8.0 RUGGEDNESS:
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The ruggedness of an analytical method is the degree of reproducibility of test results obtained by analysis of same samples by different analysts. It is a measure of reproducibility of test results under normal operational conditions from analyst to analyst The ruggedness of analytical method was determined by analysis of aliquots from homogenous lots by different analyst by different equipment & different days. This have been compared to precision of assay by different analysts. Acceptance criteria:RSD for 6 sample preparations should be NMT 2.0% System Suitability Parameters of Analyst-1: 8.8.1.T Name of parameter % RSD for replicate injections of peak response of zoledronic acid from Standard preparation Tailing factor Plate count NMT 2 NLT 2000 1.27 5830 Acceptance criteria NMT 2 Result 0.80
System Suitability Parameters of Analyst-2: 8.8.2.T Name of parameter % RSD for replicate injections of peak response of zoledronic acid from Standard preparation Tailing factor Plate count NMT 2 NLT 2000 1.24 5919 Acceptance criteria NMT 2 Result 0.62
Result:The % RSD of 6 sample preparations of zoledronic acid injection 4mg/vial is 0.16.
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The % RSD of between average assay results obtained by both analyst was found to be 1.17. It was found that RSD of 6 sample preparations of both analyst on different days were less than 2% & RSD between average assay results obtained by both analyst found less than 2%.hence test method was precise & rugged for zoledronic acid & it meets acceptance criteria
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9.0 RESULTS AND DISCUSSION The goal of the study is to validate HPLC method for the assay of zoledronic acid injection in parenterals by using most commonly employed c18 column using UV detector at appropriate wavelength.
From the specificity studies it was conformed that there will be no interference of excipients has been observed. Validation of proposed method was verified by accuracy studies. % of recovery in accuracy studies has been obtained in range of 97-103%. Validation of proposed method was verified by system precision & method precision studies.% RSD obtained in case of system precision was 0..112% & in method precision % of zoledronic acid was found to be less than 2 % which meets acceptance criteria & results has been shown in tabulated form in above. From the linearity studies specified concentration has been determined & it shows its linearity in range of 80 -120 % concentration levels The results obtained from robustness studies i.e., on verified by changing parameters such as flow rate, pH and temperature indicated that the analytical method remains unaffected. The study of ruggedness made by conducting by different alliance systems and by two analysts the results was shown in table.
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10.0 SUMMARY AND CONCLUSION: Development of new analytical method for the determination of drug in pharmaceutical dosage forms is more important in pharmacokinetic, toxicological and biological studies. Today pharmaceutical analysis entails much more than the analysis of active pharmaceutical ingredients or the formulated product. The pharmaceutical industry is under increased security from the government and the public interested groups to
contain costs and at consistently deliver to market safe, efficacious product that fulfill unmet medical needs. The pharmaceutical analyst plays a major rule in assuring identity, safety, efficacy, purity and quality of the product. The need for pharmaceutical analysis is driven largely by regulatory requirements. The commonly used tests of pharmaceutical analysis generally entail compendia testing method development, setting specifications and method validation. Analytical testing is one of the more interesting ways for scientist to take part in quality process by providing actual data on the identity, content and purity of the products. New methods are now being developed with a great deal of consideration to worldwide harmonization. As a result, new products can be assured to have comparable quality and can be brought to international markets faster. Pharmaceutical analysis occupies a vital role in statuary certification of the drugs and their formulation either by the industry or the regulatory authorities. In industry, the quality assurance and quality control departments play major role in bringing out a safe and effective drug or dosage form. The current good manufacturing practice (CGMP) and the Food Drug Administration (FDA) guideline insist for adoption of sound methods of analysis with greater sensitivity and reproducibility. Therefore of achieving the selectivity, speed, low cost. Simplicity, sensitivity, specificity, precision and accuracy are in estimation of drugs.
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11.0 BIBLIOGRAPHY
1. H.Beckett, J.B.Stenlake, Practical Pharmaceutical Chemistry, 4thed, Part two,
C.B.S.Publications, New Delhi, 1997,pp 1.

2. B.K Sharm ,Instrumental methods of chemical analysis, Introduction to Analytic
al Chemistry ,23rded, Goal Publishing House, Meerut , 2004, pp.1-4
Page 126
3. H.H. Willard, L.L. Merritt, J.A. Dean, F.A. Settle, Instrumental Methods of
Analysis, 7thed, CBS publishers and Distributors, New Delhi, 1986, pp.518-521, 580-610.
4. John Adamovies, Chromatographic Analysis of Pharmaceuticals, Marcel
Dekker Inc. New York, 2nded, 2000, pp.74, 5-15.

5. Tips on Liquid chromatography, Waters, www.waters.com , referred date: 15-5-
2011. 6. J. Swarbrick , JC. Boylan, Encyclopedia of pharmaceutical technology. Vol. I. New York, Marcel Dekker Inc, 1998, pp. 217-224. 7. S. Lindsay, High Performance Liquid Chromatography, 1st ed. Londo, John wiley and sons Publication, 1991, pp. 30-45.
8. David Harvey Modern Analytical Chemistry, 1st ed. United States of America,
Harcourt Brace & Company 1997, pp.578-80

9. Yuri Kazakevich, Rosario Lobrutto HPLC for pharmaceutical sciences.United
States of America, A John Wiley and Sons, Inc, Publication, 2007, pp.3 10. R. Phyllis Brown, Advances in Chromatography, 2002, pp. 1,26-7,30-1
11. PD. Sethi, High-performance liquid chromatography, 1sted, 2001, pp. 101-103.
12. F. Settle, Handbook of instrumental techniques for analytical chemistry. Singapore: Pearson education Ltd, 199, pp. 148. 13. LR. Snyder, JJ. Kirkland, JL. Glajch, Practical HPLC method development, 2nded. Wiley International publication, 1997, pp. 1-50.
14. David M Bliesner, Validating Chromatographic Methods - A Practical Guide,
New Jersy, John Wiley and Sons Inc 2006, pp. 1, 9-12. 15. 16. 2012 17. 18. www.medicinenet.com/zoledronic acid referred date:12-02-2012 www.drugbank.com/zoledronic acid referred date :12-02-2012 www.rxlist.com/zoledronic acid/html referred date : 23-01-2012 Indian pharmacopiea vol-2 2010 edition page -625 referred date:23-01-
19. D. Anantha Kumar, J.V.L.N. SeshagiriRao and G.SrinivasaRao, Simultaneous
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20. B. Jayakar, M. Kumar, C. Saravanan and M. V. Kumudhavalli,
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development and validation of RP-HPLC method for simultaneous determination of Lamivudine and Zidovudine,Journal of Chemical and Pharmaceutical Research, 2010, 2(1): 478-481.
21.Ch. Balasekarreddy, BahlulZ.Awen, Ch.Baburao*, N.Sreekanth and P.Ramalingam,
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22. D. Anantha Kumar, M.V. Naveen Babu, J.V.L.N. SeshagiriRao* and V.
JayathirthaRao, Simultaneous determination of Lamivudine,Zidovudine and Nevirapine in tablet dosageforms by RP-HPLC method, RasayanJ.Chem, 2010, 3(1): 94-99.
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and Abacavir in the mixture by International Journal of Pharmaceutical Sciences

and Research, 2010, 1(7):82-86. 24. T. Sudha*, V. R. Ravikumarand P. V. Hemalatha, Validated HPTLC method for
simultaneous determination of Lamivudine and AbacavirSulphate in tablet dosage form International Journal of Pharmaceutical Sciences and Research, 2010 , 1 (11): 107-111.
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27. S. K. Mastanamma*, G. Suresh, D. SinduPriya and J. V. L. N. SeshagiriRao,
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29. Raghu, nandansrinivasan,A new analytical method for estimation of zoledronic
acid in commercial pharmaceutical injections by Ion-Exchange (iec) High Performance Liquid Chromatography, Journal of liquid chromatography & related technologies, , volume 34 (6) 2011, pp. 476-489(14)
30. ZHENG Guo-gang, FANG Ying-zhi, Determination of zoledronic acid for
injection by HPLC, Journal of pharmaceutical practice, 2005;

31. WANG Ling-ling , GAO Song, ZONG Guo-jun,HU Ying-he, Assay of
zoledronic acid for injection and its related substances by HPLC, Chinese journal of drugs 2006-08;
32. KatrinVeldboer, TorstenVielhaber, Helmut
Ahrens, JendrikHardes, Arne
Streitbrger, Uwe Karst, Determination of zoledronic acid in human urine and blood plasma using liquid chromatography/electrospray mass spectrometry, J Chromatogr B AnalytTechnol Biomed Life Sci. 2011; Vol. 879 (22): Page no: 2073-80.
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34. Rao BM, Srinivasu MK, Rani ChP, kumar SS, Kumar PR, Chandrasekhar
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A.M.

Reddy Memorial College of Pharmacy

Principle types of chemical instrumentation5 A) Spectrometric techniques

A.M.Reddy Memorial College of Pharmacy

Gas Chromatography High Performance Liquid Chromatography Thin Layer Chromatography

Thermal analysis Mass spectrometry Kinetic techniques

A.M.Reddy Memorial College of Pharmacy

TYPES OF HPLC Based on modes of chromatography: Normal phase chromatography

A.M.Reddy Memorial College of Pharmacy

Reversed phase chromatography

Based on elution technique: Isocratic separation Gradient separation

Based on the scale of operation2, 3: Analytical HPLC Preparative HPLC

NORMAL PHASE CHROMATOGRAPHY

REVERSED PHASE CHROMATOGRAPHY

A.M.Reddy Memorial College of Pharmacy

SCHEMATIC DIAGRAM OF HPLC6 (Fig: 1)

A.M.Reddy Memorial College of Pharmacy

A.M.Reddy Memorial College of Pharmacy

Constant flow rate pump (or) constant displacement pump

i) Reciprocating piston pump ii) Syringe drive pump

Constant pressure pump

A.M.Reddy Memorial College of Pharmacy

A.M.Reddy Memorial College of Pharmacy

A.M.Reddy Memorial College of Pharmacy

COLUMNS USED IN HPLC (Fig: 2)

APPLICATIONS Normal phase material Pesticides, alkaloids Reverse-phase material

A.M.Reddy Memorial College of Pharmacy

Diol Sulphonic acid

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Parameters Capacity factor (k1) Repeatability Relative retention Resolution (Rs)

A.M.Reddy Memorial College of Pharmacy

Tailing Factor (T) Theoretical plates (N)

T of =2 In general should be > 2000

A.M.Reddy Memorial College of Pharmacy

A.M.Reddy Memorial College of Pharmacy

A.M.Reddy Memorial College of Pharmacy

appropriate choice of separation column includes three different

Buffer pH of the buffer Mobile phase composition

Buffer and it strength

Acetate buffers Ammonium acetate, Sodium acetate, etc.

A.M.Reddy Memorial College of Pharmacy

Procedure, sample, pretreatment, etc 3. Choose detector and Detector settings

4. Choose LC method; Preliminary run; estimate best Separation separaons

7c. Qualitative method

calibration purified material A.M.Reddy Memorial College of Pharmacy

8. Validate method for release into routine laboratory

A.M.Reddy Memorial College of Pharmacy

3.0 DRUG PROFILE ZOLEDRONICACID

[1-hydroxy-2-(1H-imidazol-1-yl) ethane-1, 1-diyl]bis(phosphonic acid)

A.M.Reddy Memorial College of Pharmacy

RESULTS White to Half white powder 4.5-7

concentration occurring after about 2hrs.

A.M.Reddy Memorial College of Pharmacy

A.M.Reddy Memorial College of Pharmacy

A.M.Reddy Memorial College of Pharmacy

A.M.Reddy Memorial College of Pharmacy

A.M.Reddy Memorial College of Pharmacy

5.0 PLAN OF WORK It includes a) Method development i. Selection of wavelength

A.M.Reddy Memorial College of Pharmacy

6.0 MATERIALS AND METHODS

i. HPLC ( waters 2695 ) ii. UV double beam spectrophotometer ( Schimadzu )

iv. Sonicator b) Reagents and standards i. Triethylamine ii. Orthophosphoric acid

characteristics which helps in selection of detector and wavelength for analysis.