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Reddy Memorial College of Pharmacy
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.
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
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, solubility’s, optical activities or refractive indices.
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
A.M.Reddy Memorial College of Pharmacy
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY3.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.Reddy Memorial College of Pharmacy Page 4 .• 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. a separation is achieved. The stationary phase can also take two forms solid and liquid. 4 A. together with Liquid Solid Chromatography (LSC) and Liquid Liquid Chromatography (LLC).M. Those components held preferentially in the stationary phase are retained longer in the system than those that are distributed in the mobile phase. a stationary phase and a 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. which provides two subgroups of GC and LC namely Gas-Solid Chromatography (GSC) and Gas-Liquid Chromatography (GLC). 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).
and quantify compounds. 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. Fast and effective development of rugged analytical HPLC methods is more efficiently undertaken with a thorough understanding of HPLC principles.Reddy Memorial College of Pharmacy . a pump that moves the mobile phase(s) through the column. 6. precision. TYPES OF HPLC Based on modes of chromatography: • Normal phase chromatography Page 5 A.In the modern pharmaceutical industry. HPLC utilizes a column that holds chromatographic packing material (stationary phase). accuracy. and a detector that shows the retention times of the molecules. ease of automation and eliminates tedious extraction and isolation procedures. development and production. HPLC is a major analytical tool applied at all stages of drug discovery. the molecules being analyzed. specificity. 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. 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. theory and instrumentation. Most of the drugs in multi component dosage forms are analyzed by HPLC method because of several advantages like rapidity. and the solvent(s) used. Some of the advantages of HPLC are. Retention time varies depending on the interactions between the stationary phase.M.
M.Reddy Memorial College of Pharmacy Page 6 .• 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. REVERSED PHASE CHROMATOGRAPHY A. 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. o Polar compounds are retained for longer times and take more time to elute because of their higher affinity with the stationary phase. non-polar compounds travel faster and are eluted first because of the lower affinity between the non-polar compounds and the stationary phase. o In this technique. o 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.
food and biomedical sciences. phosphate. pharmaceutical. there is no interaction between the sample compounds and the column packing material. •Ionic dyes. butyl silane (C4) etc. octyl silane (C8). o Instead. Separation occurs according to their molecular mass o Molecules larger than the pore opening do not diffuse into the particles. The technique is well suited for: • The separation of inorganic and organic anions and cations in aqueous solution. etc. SIZE EXCLUSION CHROMATOGRAPHY (SEC) o In SEC.Reddy Memorial College of Pharmacy Page 7 .). o The different columns used are octa decyl silane (ODS or C18). 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. while molecules smaller than the pore opening enter the particle and are separated. the column packing contains ionic groups (e. o In this mode. formate. tetra alkyl ammonium) and the mobile phase is an aqueous buffer (e. 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. molecules diffuse into pores of a porous medium. o Polar compound gets eluted first and non-polar compounds are retained for longer time. (in the order of increasing polarity of the stationary phase). As most of the drugs and pharmaceuticals are polar in nature. they are not retained for longer times and hence elute faster.M. ION EXCHANGE CHROMATOGRAPHY: o In ion exchange. and proteins can be separated by ion exchange technique. sulfonic.g.g.o It is the most popular mode for analytical and preparative separations of compounds of interest in chemical. A. biological. amino acids.
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) A.M.Large molecules elute first. Smaller molecules elute later. 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.Reddy Memorial College of Pharmacy Page 8 . The SEC technique is used by 10-15% of chromatographers.
Solvent reservoir A modern HPLC apparatus is equipped with one or more glass or stainless steel reservoirs. heptane. to which is added a small amount of a more polar solvent. Solvents with large dipole A. The reservoir is often equipped with an online degasser which removes the dissolved gasses usually oxygen and nitrogen. with the most polar solute being the first to elute. typically water. distillation system. The mobile phases used in normal-phase chromatography are based on non-polar hydrocarbons. 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. to which a less polar solvent such as acetonitrile or methanol is added. such as hexane. which interfere by forming bubbles. The mobile phases used in reversed-phase chromatography are based on a polar solvent. such as 2-propanol.M. system devices for heating.Reddy Memorial College of Pharmacy Page 9 . • In a reverse-phase separation the order of elution is reversed. Solvent selectivity is controlled by the nature of the added solvent. Degasser may consist of vacuum pumping system. or octane.
and pulse-free flow of mobile phase. which must be damped as its presence is manifested as base line noise on the chromatogram. Solvents that are good proton donors. such as alcohols. such as methylene chloride and 1. such as chloroform. Pump System: The purpose of the pump or solvent delivery system is to ensure the delivery of a precise. Two check valves control the flow of solvent. Reciprocating pumps have a disadvantage of producing pulsed flow. M-cresol and water interact preferentially with basic solutes such as amines and sulfoxides. amines.2-dichloroethane interact preferentially with solutes that have large dipole moments. constant. ethers.moments.Reddy Memorial College of Pharmacy Page 10 .M. reproducible. and amines. tend to interact best with hydroxylated molecules such as acids and phenols. Advantages of this pump include their small internal volume. and sulfoxides. nitriles. 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. high output A. and solvents that are good proton acceptors. such as nitrocompounds.
In addition. pressure output. the mobile phase is contained in a collapsible container housed in a vessel that can be pressurized by a compressor gas.Reddy Memorial College of Pharmacy Page 11 . In rheodyne 7125 valve. On turning to ‘inject’. Displacement pumps also produce a flow that tends to be independent of viscosity and backpressure. Pumps of this kind are inexpensive and pulse free. filling the sample loop which is a small piece of stainless steel tube connected between ports. After replacing the fitting the system is again pressurized. They suffer from limited capacity. c) Pneumatic pumps In pneumatic pumps. 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. dependence of flow rate on solvent viscosity and column backpressure. commonly 10-50 µL are used. the output is pulse free. sample from a microlitre syringe is loaded into the needle port. In addition. ready adaptability to gradient elution. Sample injection system The earliest and simple means of sample introduction was syringe injection through a self-sealing electrometric septum. 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.pressure. 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. With these devices sample is first transferred at atmospheric pressure from a syringe into a sample loop. they are not amenable to gradient elution and are limited to pressures less than about 2000 psi. A variety of loop volumes are available. Any excess goes to waste from another port. Columns: A. Commercial chromatographs use valves for sample injection. Disadvantages include limited solvent capacity (250 ml) and considerable inconvenience when solvents must be changed. and independent of column backpressure and viscosity of solvent.M. the loop contents are flushed on to the column.
has been limited by the difficulty of preparing columns with internal diameters less than 10 mm. a length of 7. Second is clogging of particulate material injected with the sample to the analytical column.000–60. an analytical column responsible for the separation and a guard column.000 theoretical plates/m. The development of open tubular columns. First is binding of solutes irreversibly to the stationary phase degrade the column’s performance by decreasing the available stationary phase. Micro columns use less solvent and because the sample is diluted to a lesser extent produce larger signals at the detector.6 mm.5 mm and a cost one-tenth of that for the corresponding analytical column are typical. however. The guard column is placed before the analytical column to protect it from contamination.HPLC typically includes two columns. Typical column efficiencies are 40. Because they are intended to be sacrificial. To minimize these problems. Open tubular micro columns also have been developed with internal diameters of 1–50 mm and lengths of approximately 1 m. These columns are packed with 3–10 mm porous silica particles that may have an irregular or spherical shape. These columns which contain no packing material may be capable of obtaining column efficiencies of up to 1 million theoretical plates. Guard columns usually contain the same particulate packing material and stationary phase as the analytical column.M.1 mm and 4. a guard column is placed before the analytical column. These columns are made from fused silica capillaries with internal diameters of 44–200 mm and lengths of up to several meters. and lengths ranging from approximately 30 mm to 300 mm. Micro columns packed with 3–5 mm particles have been prepared with column efficiencies of up to 250. The most commonly used columns for HPLC are constructed from stainless steel with internal diameters between 2. Guard columns: Two problems tend to shorten the lifetime of an analytical column. A. but are significantly shorter and less expensive. guard columns are replaced regularly.000 theoretical plates. Analytical columns: It is the most important part of HPLC technique which decides the efficiency of separation.Reddy Memorial College of Pharmacy Page 12 .
COLUMNS USED IN HPLC (Fig: 2) Stationary Phases: In Liquid–Liquid Chromatography the stationary phase is a liquid film coated on a packing material consisting of 3–10 mm porous silica particles. which is the more commonly encountered form of HPLC. The properties of a stationary phase are determined by the nature of the organosilane’s alkyl group.M. the stationary phase is non-polar and the mobile phase is polar. The combination of a polar stationary phase and a non-polar mobile phase is called normal phase chromatography. The stationary phase may be partially soluble in the mobile phase. If R is a polar functional group then the stationary phase will be polar. To prevent unwanted interactions between the solutes and any unreacted – SiOH groups the silica frequently is “capped” by reacting it with Si (CH3)3Cl. To prevent this loss of stationary phase it is covalently bound to the silica particles. causing it to “bleed” from the column over time.Reddy Memorial College of Pharmacy Page 13 . the mobile phase is a non-polar or moderately polar solvent. where R is an alkyl or substituted alkyl group. Since the stationary phase is polar. such columns are designated as end-capped. In reverse phase chromatography. The most common A. Bonded stationary phases are attached by reacting the silica particles with an organochlorosilane of the general form Si (CH3)2RCl.
polar Page 14 A. Peptides proteins Reverse-phase C8 Octyl C6H5 CN Phenyl Polar aromatic fatty acids. PAH. alkaloids Reverse-phase material C18 Octadecyl Fatty acids. Vitamins Reverse-phase and ion pair.Reddy Memorial College of Pharmacy .M.non-polar stationary phases use an organochlorosilane for which the R group is an n-octyl (C8) or n-octadecyl (C18) hydrocarbon chain. Cyano Normal and Reverse-phase. 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.
Solute property detectors respond to some property of solutes such as UV absorbing. NH2 Amino weak ion exchange Carbohydrates. Aromatic compounds Normal. fluorescence or diffusion current which are not possessed by the mobile phase. Cation exchange. OH SA Diol Sulphonic acid Detectors The function of the detector in HPLC is to monitor the mobile phase emerging from the column. organic acids.Reddy Memorial College of Pharmacy . Reverse. separation of cations. proteins. Bulk property detectors respond to mobile phase bulk property such as refractive index.compounds NO2 Nitro Normal and Reverse-phase. dielectric constant or density. Most common HPLC detectors • • • • • UV-Visible absorbance detector (UV-VIS) Photo-diode array detector (PDA) Fluorescence detector Electrochemical detector (ECD) Refractive Index detector (RI) Page 15 A. chlorinated pesticides Normal.M. 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. PAH. Reverse phase peptides.
) Page 16 A. Parameters such as plate count.• • • 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. excipients.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. generally k1>2.M.Reddy Memorial College of Pharmacy . degradation product. columns. 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. internal standard. analysts) is suitable for the intended application. etc. resolution and reproducibility are determined and compared against the specifications set for the method. tailing factors. System suitability is the checking of a system to ensure the system performance before or during the analysis of unknown compounds.
tR = retention volume at the apex of the peak (solute) and t0 = void volume of the system. Capacity factor can be determined by using the formula.Reddy Memorial College of Pharmacy Page 17 . Capacity factor (k1): Capacity factor is the ratio of the reduced retention volume to the dead volume.M. Capacity factor is a measure of how well the sample molecule is retained by a column during an isocratic separation. A. The ideal value of k1 ranges from 2-10. capacity factor (k') changes by 10 percent for a temperature change of 5º C.Tailing Factor (T) Theoretical plates (N) T of ≤=2 In general should be > 2000 1. 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 (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 (Fig: 3) Where.
Reddy Memorial College of Pharmacy Page 18 . a =V2 – V1 / V1 – V0 = k1(2) / k1 (1) Where. The ideal value of ’a’ is 2. Higher values are associated with excessively brood peaks and unacceptably long run times. In general if the selectivity of two components is equal to 1. This parameter is independent of the column efficiency it only depends on the nature of the components. a) is a measure of relative retention of two components in a mixture. and the ratio of its adjusted retention times. V0 is the void volume of the column. Selectivity (a): The selectivity (or separation factor. V1 and V2 are the retention volumes of the first and the second peak respectively. eluent composition and adsorbent surface chemistry.M. Selectivity represents the separation power of particular adsorbent to the mixture of these particular components. then there is no way to separate them by improving the column efficiency.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). eluent type. It can be calculated by using formula. Selectivity is the ratio of the capacity factors of both peaks. Lower values may give inadequate resolution. A. Capacity factor (k1) values are sensitive to: • • • • • • Solvent strength Composition Purity Temperature Column chemistry Sample 2.
Resolution between two peaks (Fig: 5). For baseline separation. The resolution Rs of two neighboring peaks is defined as the ratio of the distance between two peak maxima. It is calculated by using the formula. Higher the number of theoretical plates more efficient is the column. Resolution can be improved by increasing column length.5.M. Columns with N ranging from A.Selectivity (Fig: 4) 3. 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. It is the difference between the retention times of two solutes divided by their average peak width. changing the eluent or stationary phase. increasing temperature. Column Efficiency (N): Efficiency N of a column is measured by the number of theoretical plates per meter. the ideal value of Rs is 1.Reddy Memorial College of Pharmacy Page 19 . It is a measure of band spreading of a peak. Where. Resolution (Rs): Resolution is the parameter describing the separation power of the complete chromatographic system relative to the particular components of the mixture. 4. decreasing particle size.
1 should be achievable.9 to 1. 5.M. Efficiency is calculated by using the formula.000 plates / meter are ideal for a good system. and molecular weight of the analyte. flow-rate of mobile phase. divided by the corresponding front half width a gives the asymmetry factor (Fig: 7) Asymmetric Factor For a well packed column. (Fig:6) Where. column temperature. an asymmetry factor of 0.Reddy Memorial College of Pharmacy Page 20 . particle size in column.5. A. viscosity of mobile phase. tR is the retention time and W is the peak width.000 to 100. Peak asymmetry factor (Af): Peak asymmetry factor (Af). Parameters which can affect N include Peak position. can be used as a criterion of column performance. The peak half width b of a peak at 10 % of the peak height.
M. energy and cost of virtually all-instrumental methods. In addition.no :8)Asymmetric Factor METHOD DEVELOPEMENT FOR RP-HPLC11-13 During the development of method. mobile or stationary phase composition. computer driven automated method development. Computer driven The manual approach involves varying one experimental variable at a time. This univariate approach to system optimization is slow. Selection of mobile phase A. Mode of separation 2. temperature. efficiency is optimized while experimental input is minimized. peak shape. they are capable of significantly reducing the time. the initial sets of conditions that have evolved from the Literature survey are improved or maximized in terms of resolution. time consuming and potentially expensive.Reddy Memorial College of Pharmacy Page 21 . and recording changes in response . asymmetry. and pH. however it may provide a much better understanding of the principles. elution time. detection wavelength. while holding all others constant. Manual 2. Computer driven automated approaches can be applied to many applications. theory involved and interactions of the variables.(Fig. plate counts. The various parameters that need to be optimized during method development 1. limit of quantitation and overall ability to quantify the specific analyte of interest. capacity. Selection of stationary phase 3. detection limit. Optimization of a method can follow either of two general approaches: 1.The variables might include flow rates. In the second approach.
As the particle size decreases the surface area available for coating increases. Generally longer columns provide better separation due to higher theoretical plate numbers.e. base deactivated silane (C18) BDS phenyl. Selection of detector Selection of mode of separation In reverse phase mode. the mobile phase is comparatively more polar than the stationary phase. The particle size and the nature of the column packing 3. amino etc. The nature of the analyte is the primary factor in the selection of the mode of separation.Reddy Memorial College of Pharmacy Page 22 appropriate choice of separation column includes three different . Reversed phase mode of chromatography facilitates a wide range of columns like dimethyl silane (C2). Selection of separation system 2. 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. nitro. octadecylsilane (C18). butylsilane (C4). the most preferred mode is reverse phase. Selection of stationary phase / column Selection of the column is the first and the most important step in method development.4. The approaches 1. A second factor is the nature of the matrix. reproducibility A. cyanopropyl (CN). For the separation of polar or moderately polar compounds. The physical parameters of the column i. Columns with 5-µm particle size give the best compromise of efficiency.M. octylsilane (C8).
Columns that provide symmetrical peaks are always preferred while peaks with poor asymmetry can result in. For a given stationary phase.M.Reddy Memorial College of Pharmacy Page 23 . The following are the parameters. 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.6 mm Peak shape is equally important in method development. the nature and the composition of which has to be judiciously selected in order to get appropriate and required solute retention. the solute retention is governed by the solute distribution factor. 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. 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 A.and reliability. The selectivity will be particularly altered if the buffer pH is close to the pKa of the analytes. In liquid chromatography. solute – mobile phase and the mobile phase – stationary phase. the column selected had a particle size of 5 µm and a internal diameter of 4. In this case. the retention of the given solute depends directly upon the mobile phase. which reflects the different interactions of the solute – stationary phase.
silica may dissolve. A. while pH values above 8. Characteristics that are to be fulfilled by a detector to be used in HPLC determination are. if necessary. The strength of the buffer can be increased.Buffer and its strength play an important role in deciding the peak symmetries and separations. commonly employed buffers are Phosphate buffers prepared by using salts like KH2PO4.Na2HPO4.Reddy Memorial College of Pharmacy Page 24 .0. Most widely used solvents in reverse phase chromatography are Methanol and Acetonitrile. Acetic acid buffers prepared using CH3COOH. oxidation. fluorescence.NaH2PO4.0. 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. Experiments were conducted using buffers having different pH to obtain the required separations. K2HPO4. This is due to the fact that the siloxane linkage area cleaved below pH 2. 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. reduction etc. The solvent strength is a measure of its ability to pull analytes from the column. Mobile phase composition Most chromatographic separations can be achieved by choosing the optimum mobile phase composition. to achieve the required separations.M.etc Phosphoric acid buffers prepared by using H3PO4. It is generally controlled by the concentration of the solvent with the highest strength.0 as most columns does not withstand to the pH which are outside this range. Selection of detector The detector was chosen depending upon some characteristic property of the analyte like UV absorbance. Some of the most. The retention times also depend on the molar strengths of the buffer – Molar strength is increasingly proportional to retention times. conductance. etc. Acetate buffers – Ammonium acetate.0 to 8. It is important to maintain the pH of the mobile phase in the range of 2. Sodium acetate.
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.M. Higher wavelengths give greater selectivity. For the greatest sensitivity λmax should be used. A. High sensitivity. An understanding of the UV light absorptive properties of the organic impurities and the active pharmaceutical ingredient is very helpful. UV wavelengths below 200 nm should be avoided because detector noise increases in this region.Reddy Memorial College of Pharmacy Page 25 .
Choose detector and Detector settings 4. Need for special HPLC Procedure.(Fig 9) Strategy for Method Development 1.Reddy Memorial College of Pharmacy . sample. etc 3. Choose LC method. estimate best Separation separaons 5. Qualitative method Page 26 calibration purified material A. Recover 7b. Introduction on sample Define separation goals 2. Preliminary run.Quantitative 7c. Optimize separation condition 6. Check for problems or requirements for special procedure 7a. pretreatment.M.
M.8.Reddy Memorial College of Pharmacy Page 27 . Validate method for release into routine laboratory A.
3.M. 1-diyl]bis(phosphonic acid) A.0 DRUG PROFILE ZOLEDRONICACID [1-hydroxy-2-(1H-imidazol-1-yl) ethane-1.Reddy Memorial College of Pharmacy Page 28 .
2. at a temperature not exceeding 250C IV. Metabolism: it was not metabolized by liver 4.Reddy Memorial College of Pharmacy Page 29 .P. Distribution: zoledronic acid has a large volume of distribution of around 3lit/Kg.2007 and official method is HPLC 1. Pharmacokinetics: RESULTS White to Half white powder 4. Storage: Store in air tight containers. Strength available: Zometa -4mg/vial Zolodonat -4mg/vial III. Elimination: it was excreted by kidney in an unchanged form II. Plasma protein binding is about 65%.5-7 sparingly Soluble freely soluble C18 H24 N2 O7P2 348.TESTS i) Description ii) pH Solubility: a) Soluble in water b) Soluble in NaOH v) Molecular formula vi) Molecular weight vii) Official Status I. Distributes freely between plasma and RBC. 3.M. Absorption: zoledronic acid is rapidly absorbed after oral dose.9 It is official in B. Adverse effects: Pruritis A. with peak plasma concentration occurring after about 2hrs.
Uses and Administration It was used in the treatment of bone cancer and it was administered through I.Reddy Memorial College of Pharmacy Page 30 . Nausea & vomiting Hypocalcaemia Rise in body temperature Dementia Anxiety V.V. A.M. route.
4.8 mL/min and the detection was done at 210 nm. 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. The method produced linear responses in the concentration range of 0.812 min.0 LITERATURE REVIEW Mastanamma S K et al.6 mm x 5 m length).. A. (2012)27. The retention time of the drug was 3.M.01M phosphate buffer (pH-3) (60:40 v/v) as the mobile phase at a flow rate of 0.Reddy Memorial College of Pharmacy Page 31 .using a mixture of methanol and 0. Chromatography was carried out on an ODS C18column (250 x 4. The method was found to be reproducible for analysis of the drug in parentral preparations.25 to 60 g/mL of zoledronic acid.
ZHENG Guo-gang et al.200 to 1. 3 with 50% phosphoric acid RESULTS: A. (2011)29. The separation was achieved on a 5μ C18 column (250 X 4. selectivity and precision to assay Zoledronic acid in commercial pharmaceutical injection dosage form. 0. 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.L.02mmol/mL tetrabutyl ammonium hydroxide and 0. The retention time of imidazol-1-ylacetic acid and zoledronic acid were 7. The flow rate was maintained at 1. developed To establish an HPLC method for the determination of zoledronic acid for injection.6 mm) using mobile phase consist of buffer (4. Raghu et al. Complete separation was achieved for the parent compound Zoledronic acid. A UV-Vis detector set at 215 nm was used to monitor the eluate.. 02mmol/mL ammonium dihydrogen phosphate adjusted pH 2. The drug product was subjected to oxidation.2 and 10.0 g of tetra butyl ammonium hydrogen sulphate (TBAHS) in 1000 mL of water) and methanol in the ratio of 900:100 v/v. developed a novel.Reddy Memorial College of Pharmacy Page 32 . photo-stability. The detection of the constituents was done at 215 nm using UV detector. 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. The ion-pair buffer solution was a mixture of 0. METHOD:The analysis was achieved by using Diamonsil C18 column with acetonitrile. the impurities and excipients in an overall analytical run time of approximately 20 minutes.. (2005)30. and heat to apply the stress conditions. (2012)28.999 indicates linearity of the method within the limits. The 100% aqueous mobile phase consisted of only diluted formic acid without any ion-pair substance.2 min respectively. Maheswara Reddy et al.M.5 g of Di-potassium hydrogen phosphate anhydrous and 2.0 mL min -1.200 mg mL-1(25% to 150% of Zoledronic acid concentration). The newly developed method has the requisite accuracy.. The developed method can be applicable for regular qualitative analysis. The method was found to be linear over the concentrations range from 0.ion-pair buffer solution(25 : 75 ) as mobile phase and UV-detector at wavelength 210nm.7 mL min-1 and by using a new generation Allsep® anion exchange column. Recovery studies were satisfactory and the correlation coefficient. The proposed chromatographic conditions employed an isocratic elution of mobile phase at constant eluent flow rate of 0. hydrolysis.
pH =2. WANG Ling-ling et al. For human blood plasma. 4% ). CONCLUSIONS: This method is simple. The method was successfully applied for the analysis of spiked urine and blood plasma samples as well as samples from two osteoporosis patients. METHODS Ion-pair RP-HPLC coupled with evaporative light-scattering detection was performed on a BDS C8 column A.75×10(-7) mol/L in the MRM mode.Reddy Memorial College of Pharmacy Page 33 . It consists of a derivatisation of the bisphosphonate with trimethylsilyl diazomethane under multiple methylester formation. sensitive. The average recovery rate was 100. Results:The calibrated linear curve of zoledronic acid was within 5 . For calibration purposes.125μg·mL-1. accurate and suitable for the determination of zoledronic acid for injection.M.5 decades starting at the limit of quantification.0 mL·min-1 and the detection at 218 nm. in contrast to the non-derivatisedanalyte. The average recovery was 100.5×10(-7) mol/L were determined.5μm) .2x10(-7) mol/L.6 mm. The related substances of zoledronic acid were completely separated from zoledronic acid. The linear dynamic range comprised 3. 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.2008)31. an eluate composed of 0. limit of quantification (LOQ) is 3.2007)33. (2005 . a LOD of 1×10(-7) mol/L and a LOQ of 2. 0).50%.03% tetrabutyl-ammonium hydroxide solution (pH =2. an eluate consisted of acetonitrile-tetrahydrofuran-0. easily be separated by reversed phase liquid chromatography due to its reduced polarity. For human urine. 55 adjusted with phosphoric acid) for zoledronic acid assay. 0% (RSD = 0. a flow rate of 1. CONCLUSION: This reliable RP-HPLC method can be used in quality control of zoledronic acid. 55 adjusted with phosphoric acid)-acetonitrile (100:7) for the related substances. 05 mol·L-1 ammonium dihydrogen phosphate (4: 1: 100.. developed To establish a HPLC method for assay of zoledronic acid for injection and its related substances. a C18 ODS column (250 mm×4. a deuterated internal standard has been synthesized in a three-step synthesis starting with d(4)-imidazole.3% with RSD of 0. developed To establish a HPLC method for the determination of zoledronic acid and its preparations... the limit of detection (LOD) is 1.The linear range was from 50 to 240 μg/mL (r = 1. Katrin Veldboer et al. Methods:The reversedphase HPLC condition was as follows. Detection is performed by electrospray tandem mass spectrometry. The formed derivative can. (2011)32. JIANG Ye et al. (2006 .
The assay shows a LLOQ 0.69% (n = 9). 8 or 16 mg of zoledronic acid. CONCLUSION The method is rapid. plasma. for zoledronic acid injection. The drug was found to be stable in other stress conditions attempted.(4.4% with RSD of 0. The developed HPLC method was validated with respect to response function. oxidation. 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.For zoledronic acid for injection. Francois Legay et al. accuracy 97%).9999). A radioimmunoassay has been developed to determine zoledronic acid concentration in human serum. In 23 patients receiving 4. The drug substance was subjected to stress conditions of hydrolysis. and urine. The degradation of zoledronic acid was observed under oxidative stress at higher temperature.Reddy Memorial College of Pharmacy Page 34 . highly potent bisphosphonate drug under clinical evaluation. the average recovery was 100. Zoledronic acid disposition in plasma and the recovery of only 40-50% of the dose in A. adjusted to pH 7.6 mm× 150 mm..2% with RSD of 0. (2002)35. It can be also used to test the stability samples of zoledronic acid. The mobile phase consisted of methanolbuffer solution (5 mmol·L-1 ammonium acetate and 10 mmol·L-1 amylamine. 5 ng/ml in urine (21%.0 with acetic acid) (3:97). Rao BM et al. developed Zoledronic acid is a new. 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.5μm) at room temperature. 98%). followed by a prolonged gradual decline to concentrations near the LLOQ.4 μg·mL-1(r = 0. (2005)34. at a flow rate of 1.04 -891. precision. specificity and robustness. accuracy. RESULTS: The calibration curve was linear in the range of 99. an ion-pairing agent and methanol(95:5) as mobile phase. drug concentrations in plasma were dose proportional and showed a multiphasic profile.0 mL·min-1. the average recovery was 100. 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.. 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.M.76% (n = 9).4 ng/ml in serum or plasma (interassay%CV=17%. photolysis and thermal degradation. simple and accurate for the determination of zoledronic acid in material and in its preparations.
Sarath chandiran et al.. linearity and range. sensitive and selective method is described for the determination of itraconazoleand its metabolite in human plasma.urine are consistent with the rapid and extensive uptake by and slow release from bone in parallel with renal clearance.8 (51:45:4 v/v) at flow rate of 1. developed a simple. (2009)37. Miconazole as internal standard by using HTLCMS/MS. The high recovery and low relative standard deviation confirm the suitability of the method for the routine determination of ketoconazole in bulk drug. and Limit of quantitation (LOQ). 4.05-0. The accuracy of the method is 99. Calibration plots were linear over the concentration range 1-50μg/ml. F. precision. and C) in capsule formulations. 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. 5μm) using mobile phase water : acetonitrile : buffer ph 6.. Isocratic elution was employed using a mixture of methanol and water (40:60. The precision of this method reflected by relative standard deviation of replicates is 0. Al-Rimawi et al.3%. developed A simple and stability-indicating liquid chromatographic method was developed and validated for the analysis of Fluconazole and its related compound (A.M. Liquid chromatography with a UV detector at a wavelength of 260 nm using a reversed-phase C18 column was employed in this study. This new method was validated in accordance with USP requirements for new methods for assay determination. Quantification was achieved with photodiode array detection at 238 nm over the concentration range of 1-50 μg/ml. Rakesh Kumar Jat et al. developed A simple. accurate rapid and precise RPHPLC method has been developed and validated for determination of ketoconazole in bulk drug. rapid. The retention times were 2. linearity and range.15 mg/ml of Fluconazole. (2011)38.0 ml/min at ambient temperature. The current method demonstrates good linearity over the range of 0. B. and reproducible.6 mm. (2008)36. precision. reliable.713 min. The RP-HPLC separation was achieved on Promosil C-18. The method was validated statistically and applied successfully for the determination of ketoconazole.5mm. (250 mm. selectivity.Reddy Memorial College of Pharmacy Page 35 . which include accuracy.61%. for ketoconazole. Low LOQ of the related compounds using this method enables the detection and quantitation of these impurities at low concentration. 50μm) HTLC column by injecting 15μL sample and chromatographic separation isperformed with C18 Reverse phase A. Validation studies revealed that method is specific. The method consists of a online coupling of extraction with cyclone P (50mm x0.. v/v). specificity. typically shown by bisphosphonates.
The total run time for sample analysis was 1. The method was fully validated in terms of specificity sensitivity. Ketoconazole could immediately be determined at 250 nm after injection of diluted shampoo. the other excipients of the shampoo did not interfere in the assays for both substances. 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. (2002)39. developed Ketoconazole is an antifungal agent. Different selectivity towards ketoconazole and formaldehyde was observed when applying other C columns.column using 90:10 Acetonitrile: 10mM Ammonium Formate Buffer (pH 6. 45/55 (v/v). 5 μ) reversed-phase column. 5 mm) C column 8 and a mobile phase containing acetonitrile–phosphate buffer 0. Method validation was performed on both assays. Chiranjeevi Bodepudi et al.6 mm. Vander Heyden et al.0. which is the active ingredient in a shampoo primarily used for the treatment of seborrhatic dermatitis (anti-dandruff shampoo).6 mm.5ml of human plasma per assay. The shampoo also contains imidazolidinyl urea as a formaldehyde releasing preservative. The method produced linear responses in the concentration range of 10 to 50μg/ml for both Fluconazole and Tinidazole. The Tailing factors of Fluconazole and Tinidazole were A.4dinitrophenylhydrazine solution. This fact.5 min and the lower limit of quantification was 1ng/mL for both drug and its metabolite.Reddy Memorial College of Pharmacy Page 36 . The injection volume was 20μl. Stability assessment was also included. however. Formaldehyde was measured at 345 nm after derivatisation with a 2..5 for Fluconazole and 3. The validated method was applied in bioavailability and bioequivalence study Y. did not affect the assays of both 8substances. The analysis was carried out on a Kromasil stainless steel C18 (250 x 4. precision..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.. accuracy and stability over a concentration range of 1 to 500g/ml for both Drug and its metabolite using 0. pH 4. 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.M.025 M.The retention time of the drug was 2. The finally selected isocratic system consisted of an Interchrom Nucleosil (25034. At the selected conditions. The aim of this study was to develop a HPLC system that allows the determination of both ketoconazole and formaldehyde. (2005)40.1 for Tinidazole.
The method can be successfully applied for estimation of sirolimus from in-vitro elution studies of sirolimus eluting stents. Linear calibration curves were obtain for each compound across a range of 50. V.00 ng/ml.02M Phosphate buffer at PH 5. (2009)43.. The retention times of Letrozole and fluconazole the internal standard were 4.3% for 1. 3. The PDA detection was done at 278nm with analytical run time less than 6 min. simultaneous estimation of ketoconazole and sirolimus in therapeutic drug monitoring and other pharmacokinetic studies. The assay exhibited good linear relationship.1% ratios run isocratically through a C18 (250mm × 4. Extraction was performed using dichloromethane under nitrogen atmosphere and the separation of sirolimus and ketoconazole was accomplished by reverse phase chromatography. A Anil Kumar et al. The average mean recovery was found to be 98. The method was found to be applicable for determination of the drug in tablets. developed Sirolimus and Ketoconazole are used in organ transplantation regimen and potential metabolic interactions of these drugs were reported when administered concomitantly. It uses less biological material and applicable. 10μg/ml concentrations. Greater than 85% recoveries were obtain for Letrozole.5ng/ml and the limit of Quantification was 37. developed A rapid high-performance liquid chromatography method has been developed for bioanalytical method development and validation of letrozole in human plasma.depentent diseases. The intra and interday relative standard deviation (%RSD) were <5%. 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.6mm. The stationary phase A. a potent aromatase inhibitor for treatment of oestrogen . the limit of detection was 12. letrozole (CGS 20 267).47 min respectively.Reddy Memorial College of Pharmacy Page 37 .5 and 25% acetonitrile as the mobile phase.2009)41..29 and 7. (2008 . 5.84% and 1.5ng/ml for Letrozole. water and glacial acetic acid at 90:10:0. LLOQ was 10ng/ml with 0. Letrozole was found with symmetrical peak shapes on a analytical column Phenomenex Luna C18 column using 75% 0. developed A simple. (2012)42. Parul Parmar et al. 5μm) reverse phase analytical column.found to be 1 and 1.3 respectively.M..1-10μg/ml. 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.55-120.28% of accuracy and precision over the concentration range of 0. The mobile phase consists of a combination of methanol.Sekar et al. precise.
6mm i. the total time of analysis was less than 6min. Extracts were further cleaned-up and concentrated via solid phase extraction. At a flow rate of 0. The samples were deprived of proteins and lipids by treating with acetonitrile and consequent extraction in n-hexane.d.used was precoated silica gel 60F 254 . The limit of detection and the limit of quantification for clotrimazole were found to be 50 ng/spot and 200 ng/spot.were finally overcome using a 3. (2011)45. Limits of detection and quantification for both analytes were equal to 0.01 and 0.M.Reddy Memorial College of Pharmacy Page 38 . imidazole has pK(a) 6. 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 optimal mobile phase for separation of clotrimazole. 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. Farzaneh Ahmad Khan Beigi et al. Hájková R et al. The proposed method can be successfully used to determine the drug content of bulk drug and marketed formulation of tablet. The analytes were determined quantitatively using a validated high performance liquid chromatography method. The calibration curve was found to be linear between 200 to 1000 ng/spot for clotrimazole.. Agilent Technologies). The method was validated in terms of linearity.5. The mobile phase used was a mixture of cyclohexane : toluene : methanol : triethyleamine (8:2:0. precision and specificity. v/v) with pH* conditioned by phosphoric acid to 3.9 compared to relatively more acidic (2-chlorophenyl)diphenylmethanol . The method was applied for routine analysis (batch analysis and stability tests) in commercial spray solution. The detection of spot was carried out at 262 nm. degradation products imidazole and (2-chlorophenyl)diphenylmethanol and ibuprofen as internal standard consists of a mixture of acetonitrile and water (65:35.7. (2007)44.5mum Zorbax((R)) SB-Phenyl column (75mmx4. The complications with different acido-basic properties of the analysed compounds in HPLC separation .1-1. respectively..2 v/v/v/v).while clotrimazole has pK(a) 4. accuracy. Important parameters influencing the extraction efficiency were A. The method was linear in the range of 0. respectively.5:0..5mlmin(-1) and detection at 210nm.0 µg mL -1 for both analytes.1 µg mL-1.
in pure forms and in pharmaceutical formulations.78% using SPE. The differences in the retention times (tR) of the three azoles permit their use as internal standard for each other. A. 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.6 mm.1.M. developed High-performance liquid chromatographic technique has been developed for the determination or some azolcsantifungals namely. 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.)] using a mobile phase containing acetonitrilc+25 mM trishydroxy methyl amino methane in phosphate butter (pH 7)= 55:45 (v/v). CZ was well separated from its acid degradation products and quantified by densitometric scanning at 260 nm. The TLCfractionation was performed on a precoated silica gel F254 plates using a solvent system consisting of chloroform+acetone+ammonia (25%) (7:1:0.Reddy Memorial College of Pharmacy Page 39 . the proposed method was applied to the analysis of milk samples. 25 cm x 4. clotrimazole (CZ). Satisfactory recoveries obtained in the range of 95. viz (2-chlorophenyl)-diphenyl methanol and imidazole.. with UV-detection at 260 nm. i.investigated and then optimized. In addition. ketoconazole (KZ) and fluconazole (FZ). by volumes).9-101. E M Abdel-Moety et al.d. The analyzed drugs were separated on a reversed-phase column [Bondapak C18 (10 microm. (2002)46.
pH selection iii. Flow rate selection b) Validation and optimization of parameters i.Reddy Memorial College of Pharmacy Page 40 .0 PLAN OF WORK It includes a) Method development i. System specificity A. Column selection iv.5. Selection of wavelength ii. System suitability ii.M.
Precision a. ruggedness A.iii. Accuracy vii. Range vi.Reddy Memorial College of Pharmacy Page 41 . Method repeatability b.M. Method reproducibility iv. Robustness viii. Linearity v.
Triethylamine ii.M.0 MATERIALS AND METHODS a) Equipments i.Reddy Memorial College of Pharmacy Page 42 . Sonicator b) Reagents and standards i. HPLC ( waters 2695 ) ii. UV double beam spectrophotometer ( Schimadzu ) iii. PH meter iv. Orthophosphoric acid A.6.
iii. Sometimes “gradient elution” also preferable. Zoledronic acid working standard c) Introduction to method i. Solvent delivery system selection: Separation using with one solvent called “isocratic elution” always preferable. Buffer & its strength b. Water for injection iv. iii. % of carbon load e. a.mobile phases are mixed at predetermined ratio A. Length & diameter b. ii. Solubility: It includes solubility of drug substance in different solvents and different PH conditions. Particle size & shape d. Wavelength selection: Spectral profile is useful in understanding absorption characteristics which helps in selection of detector and wavelength for analysis. Low pressure gradient elution :. Mobile phase selection and its optimization: Its selection is done always in combination with selection of column. Column selection :A column has been selected for analytic purpose by considering following parameters a. Following parameters should be taken into consideration while doing selecting and optimizing the mobile phase a. Pore volume & surface area v. Buffer/mobile phase pH c. Packing material c.M.Reddy Memorial College of Pharmacy Page 43 . Mobile phase composition iv.
Reddy Memorial College of Pharmacy Page 44 . viii.5ml/min vii. Diluents Selection: It will be based upon a) Extraction efficiency b) Peak symmetry c) Resolution of impurities from API peak A.M. High pressure gradient elution :.b. Temperature selection: Generally Ambient Column Temperature Was Preferable To Optimize Chromatographic Conditions.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.
M.A.Reddy Memorial College of Pharmacy Page 45 .
2 with ortho phosphoric acid & filter through 0.Take 2ml of this solution into 25 ml volumetric flask & make up the volume with mobile phase & filter iii. A.Reddy Memorial College of Pharmacy Page 46 . Take 5ml of this solution into another 25ml volumetric flask & make up to volume with mobile phase. iv.M.22µ membrane filter & degas ii.0 METHOD DEVELOPMENT a) Method description i. Preparation of placebo-Weigh accurately about 1.7.12g of sodium citrate in 10ml volumetric flask & add 5ml mobile phase & sonicate to dissolve & dilute with same mobile phase .10g of mannitol& 0. 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.Mix 2 ml tri methyl amine in 1000ml water for injection & adjust pH to 3. Preparation of mobile phase /Buffer. Take 5 ml from this & again make up with mobile phase in another 25ml volumetric flask. Preparation of Standard – Weigh 22mg of zoledronic acid marking Standard into 25ml volumetric flask & make up with mobile phase.
1.M.a.2) A.b) Optimization of method development parameters i.Reddy Memorial College of Pharmacy Page 47 .1) TRAIL -2 CHROMATOGRAM (7.1. Selection of wavelength TRAIL-1 CHROMATOGRAM (7.a.
a.Reddy Memorial College of Pharmacy Page 48 .1.TRAIL -3 CHROMATOGRAM (7.M.1.a.3) TRAIL -4 CHROMATOGRAM (7.4) A.
2 3.T Tailing factor 1.1.30 2.2.387 3. So it was selected as optimum wavelength for this specified drug. ii. Selection Of pH Buffer pH 3.a.0 CHROMATOGRAMS AT 3.Reddy Memorial College of Pharmacy Page 49 .30 1.395 Table no: 7.M.1) A.387 3.Result: After reviewing above chromatograms maximum absorption occurs at 215 nm by using PDA detector.6 Retention time (mins) 3.4 3.2 pH TRAIL -1 CHROMATOGRAM (7.
a.2) A.TRAIL -2 CHROMATOGRAM (7.M.Reddy Memorial College of Pharmacy Page 50 .2.
2.M.a.4) A.3) TRAIL -4 CHROMATOGRAM (7.2.TRAIL -3 CHROMATOGRAM (7.a.Reddy Memorial College of Pharmacy Page 51 .
CHROMATOGRAMS AT PH 3.M.4 A.Reddy Memorial College of Pharmacy Page 52 .
M.2) TRAIL -3 CHROMATOGRAM (7.Reddy Memorial College of Pharmacy Page 53 .a.a.3.3.1) TRAIL -2 CHROMATOGRAM (7.a.3.3) A.TRAIL -1 CHROMATOGRAM (7.
4) CHROMATOGRAMS AT 3.a.M.4.3.6 PH : TRAIL -1 CHROMATOGRAM (7.TRAIL -4 CHROMATOGRAM (7.a.Reddy Memorial College of Pharmacy Page 54 .1) A.
2) TRAIL -3 CHROMATOGRAM (7.4.4.M.Reddy Memorial College of Pharmacy Page 55 .a.a.3) A.TRAIL -2 CHROMATOGRAM (7.
TRAIL -4 CHROMATOGRAM (7.4.a.M.4) A.Reddy Memorial College of Pharmacy Page 56 .
2.5. iii.14 4.Result: From the above results peak shape was found to be good at PH at 3.Reddy Memorial College of Pharmacy Page 57 .2 and 3.4 respectively.2 will be regarded as suitable pH.5 Area 2224305 1333103 Table no: 7.M.1) A. but considering column stability PH at 3.3 2.0 Tailing factor 1.a.T Chromatograms using Hypersil BDS C18 TRAIL -1 CHROMATOGRAM (7. Column selection Type of column Hypersil BDS C18 Unicem US C18 Retention time ( mins) 3.
2) TRAIL -3 CHROMATOGRAM (7.TRAIL -2 CHROMATOGRAM (7.5.3) A.M.a.a.Reddy Memorial College of Pharmacy Page 58 .5.
M.a.4) Chromatograms using Unicem US C18 :TRAIL -1 CHROMATOGRAM (7.TRAIL -4 CHROMATOGRAM (7.5.a.6.1) A.Reddy Memorial College of Pharmacy Page 59 .
6.3) A.TRAIL -2 CHROMATOGRAM (7.2) TRAIL -3 CHROMATOGRAM (7.a.M.6.Reddy Memorial College of Pharmacy Page 60 .a.
0 1.8 Table no: 7.6.a. iv.TRAIL -4 CHROMATOGRAM (7. area and tailing factor has been satisfactorily found with Hypersil BDS C18 column.4 2.4) Result: After reviewing results there has been better separation has been observed between API and other peaks.Reddy Memorial College of Pharmacy Page 61 . Flow rate selection Flow rate ( mL/ min) 1.2 Retention time (mins) 3. Hence it will be selected. Retention time.M.T Area 2245388 1865507 A.3.
CHROMATOGRAMS AT 1.M.a.Reddy Memorial College of Pharmacy Page 62 .a.2) A.7.0 (mL/ min) FLOW RATE TRAIL -1 CHROMATOGRAM (7.7.1) TRAIL -2 CHROMATOGRAM (7.
a.Reddy Memorial College of Pharmacy Page 63 .M.4) A.7.7.TRAIL -3 CHROMATOGRAM (7.a.3) TRAIL -4 CHROMATOGRAM (7.
8.2) A.a.a.CHROMATOGRAMS AT 1.1)CHROMATOGRAM TRAIL -2 CHROMATOGRAM (7.Reddy Memorial College of Pharmacy Page 64 .M.8.2 (ML/MIN) FLOW RATE :TRAIL -1 (7.
M.4) A.8.TRAIL -3 CHROMATOGRAM (7.3) TRAIL -4 CHROMATOGRAM (7.a.a.8.Reddy Memorial College of Pharmacy Page 65 .
2ml/min.2min 10.M. Hence the flow rate of 1.4. λmax: 215nm A. CHROMATOGRAPHIC CONDITIONS 1.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 can be selected for this method validation. Solvent composition Water for injection and Ortho phosphoric acid 2. the composition of mobile phase is decided as 95:5 (v/v). 5.Reddy Memorial College of Pharmacy Page 66 Retention time 3.no 1.347min 4. buffer (95:5%v/v) Methanol and phosphate buffer (60:40%v/v) Tetrabutyl ammonium and methanol (900:100v/v) Table no: 7. Column: HypersilBDS C18 ( 250x 4. 3.4min .T After reviewing the above data.6mm) 2. Mobile phase composition S.
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 A. Injection volume : 20µl 5.3.2mL/min 4. Run time :10 min 8.Reddy Memorial College of Pharmacy Page 67 .0 METHOD VALIDATION13. accuracy and preciseness of its intended purpose. International Conference on Harmonization defines validation as ‘Establishing documented evidence.M. 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. Column temperature: 35°c 6. Flow rate : 1. 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”.
System suitability peak for zoledronic acid A. 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. recorded and trended throughout the course of the validation. these may include tailing factors. During validation where applicable system suitability parameters are calculated.1a.1.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. retention times. and theoretical plates. resolution. capacity factors.Reddy Memorial College of Pharmacy Page 68 .M. and precision of standard peak areas and comparison to a confirmation standard. A system suitability evaluation usually contains its own set of parameters. For Chromatographic assays.
Reddy Memorial College of Pharmacy Page 69 .2b.1.system suitability peaks for zoledronic acid dilutions A.M.8.
1T.18 5962 Result: system suitability parameter meets acceptance criteria 8. degradation products A. Result 0.Reddy Memorial College of Pharmacy Page 70 .1.2 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.41 1.M.Parameter % RSD Tailing factor Platecount Acceptance criteria NMT 2 NMT 2 NLT 2000 Table no: 8.
To determine specificity during the validation of blanks. Prepare triplicate sample preparations with appropriate levels of excipients as placebo sample and inject into HPLC.Reddy Memorial College of Pharmacy Page 71 .and excipients.M. System specificity peaks for zoledronicacid using blank A. Acceptance criteria: It should not show any interference from blank and placebo at retention times of zoledronic acid peak 8. sample matrix (placebo) and known related impurities are analyzed to determine whether interferences occur.1a. Procedure: • • Prepare and inject water as blank in triplicates to the system.2.
System specificity peaks for zoledronic acid using placebo 8.8. System specificity peaks for zoledronicacid using sample dilutions A. System specificity peaks for zoledronic acid using Standard dilutions 8.Reddy Memorial College of Pharmacy Page 72 .4d.M.18.104.22.168c.2b.
1T Page 73 A.Reddy Memorial College of Pharmacy .M.2.Result: Table no: 8.
182 6.433 3.S.431 3. As parameters the standard deviation.429 3.184 6.432 % 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.433 3. Intermediate precision includes the influence of additional random effects A. Repeatability expresses the analytical variability under the same operating conditions over a short interval of time (intra-day).M.182 3. 8. Hence the method was specific and selective for estimation of zoledronic acid in 4mg per vial injection dosage form.” Precision may be considered at three levels • Repeatability • Intermediate precision and • Reproducibility Precision should be obtained preferably using authentic samples.Reddy Memorial College of Pharmacy Page 74 .430 3. At least nine determinations covering the specified range or six determinations at 100 % test concentration should be performed. the relative standard deviation (coefficient of variation) and the confidence interval should be calculated for each level of precision.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 6.3 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.
analysts or equipment etc.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.Reddy Memorial College of Pharmacy Page 75 . but can be taken into account for standardization of analytical procedures.M. Reproducibility i.1a. Method Repeatability Peaks For Zoledronic Acid A.3.within laboratories. the precision between laboratories (collaborative or inter laboratory studies) is not required for submission.e. according to the intended use of the procedure for example different days. 8.
no T-1 T-2 A.09 Page 76 .09 4.Reddy Memorial College of Pharmacy Results (assay) 4.RESULT: S.M.
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%. 8.08 4.3.08 4.2b.08 4. 8.3.005 0.112 It was found that % RSD of 6 samples were within limit & hence the method is precise.08 4. Method Reproducibility Peaks For Zoledronic Acid Working Standard A.T 4.Reddy Memorial College of Pharmacy Page 77 .1.M.08 0.T-3 T-4 T-5 T-6 Average Standard deviation % RSD 8.
Reddy Memorial College of Pharmacy Page 78 .A.M.
Reddy Memorial College of Pharmacy Page 79 .3c.M.8.3. Method Reproducibility Peaks For Zoledronic Acid Dilutions A.
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
A.M.Reddy Memorial College of Pharmacy
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
A.M.Reddy Memorial College of Pharmacy Page 81
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
A.M.Reddy Memorial College of Pharmacy
CHROMATOGRAMS FOR LINEARITY A.Reddy Memorial College of Pharmacy Page 83 .M.
M.A.Reddy Memorial College of Pharmacy Page 84 .
Reddy Memorial College of Pharmacy Page 85 .M. Linearity & Range For Zoledronic Acid A.1a.5.8.
100% & 120% of nominal concentration (test conc 0.Result: It was found that correlation coefficient.6 ACCURACY The accuracy of a measurement is defined as the closeness of the measured value to the true value. Typically accuracy is represented and determined by recovery studies but there are three ways to determine accuracy. 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%. slope.accuracy was then calculated from test results as % of analyte recovered by assay. 8. y – intercept were within limits from range 80% .Reddy Memorial College of Pharmacy Page 86 . Hence test method was found linear and range for estimation of zoledronic acid in injection.M. A. 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.16 mg/ml). • Comparison to a reference standard • Recovery of the analyte spiked into blank matrix or • Standard addition of the analyte.120% of target concentration.
Reddy Memorial College of Pharmacy Page 87 .8.1a.6.M. Accuracy Peaks for Zoledronicacid A.
415 3.2 Accuracy – 120% .6.420 3.417 3.2 Accuracy – 100% .Reddy Memorial College of Pharmacy .418 3.2.427 3.1 Accuracy – 80% .423 3.D Accuracy – 80% .416 3.418 3.T Sample no.1 Accuracy – 120% .3 Accuracy – 100% .2 Accuracy – 80% .6.3 8.M.414 Area 1665880 1652011 1653849 2102494 2106121 2107040 2539464 2532733 2527766 8. Spiked level Weight of zoledronic Weight of zoledronic Sample area Amount recovered % of Recovery Page 88 A. 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.3 Accuracy – 120% .1.Acceptance criteria: Accuracy (Recovery) for average of triplicate in each concentration level should be within 98 – 102%.1 Accuracy – 100% .T Retention time (min) 3.
55 11.66% 100.51 100.1 2 3 1 2 3 1 2 3 80% 100% 120% acid added 11.73 100. flow rate etc.73% 100.43 17.7 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.42 for 100% & 100.34 100.51% 100.43 acid found 11.53 17.55% 99.56% 101. mobile phase composition. Conditions such as pH .43 17. 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.53 99.53% It was found that accuracy (recovery) for average of triplicates from each concentration levels were within 98 – 102% (98. 8.47 100. This is an important parameter with respect to the transferability of the method following validation. 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.34% 100. and instrument settings) and provides an indication of its reliability during normal usage.00 100.34 100. temperature.66 100.53 14.M.62 11.38 98.53 1665880 1652011 1653849 2102494 2106121 2107040 2539464 2532733 2527766 99.56 101. pH.87 for 80%.00% 100. It is performed by varying diff. A. 100. It is part of the formal methods validation process.53 14.51 100.62 14..38% 98.62 11.56 101. temperature.73 100.Reddy Memorial College of Pharmacy Page 89 .g.00 100.75 for 120% levels respectively) and meets acceptance criteria.45 11.55 99.
M. 1 2 3 Tailing factor Average % RSD Area (3.3 pH) 831885 1872787 2237816 1.1a Robustness at normal flowrate using placebo A.9 ml/min) 889648 2056379 2439575 1.24 1795201 0.04 Area (3.1 pH) 810274 1852889 2210100 1.7.23 1647496 0.19 1614700 0.1 ml/min) 727543 1680448 1997801 1.22 1468597 0.24 1604820 0.23 1624421 1. Robustness at different pH: 8.T Injection no.7.Reddy Memorial College of Pharmacy Page 90 .99 8.85 Area (3.2 ml/min) 801782 1813142 2229178 1.7.82 Area (1.1. 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 flowrate: 8.2 pH) 805518 1814170 2194771 1.T Injection no.2. 1 2 3 Tailing factor Average % RSD Area (1.64 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.63 Area (0.1.
8.2b Robustness at normal flowrate of zoledronicacid working Standard A.1.Reddy Memorial College of Pharmacy Page 91 .7.M.
A.Reddy Memorial College of Pharmacy Page 92 .M.
8.M.1.Reddy Memorial College of Pharmacy Page 93 .7.3c Robustness peaks for zoledronic acid in normal flowrates using Standard dilutions A.
4d Robustness Peaks For Zoledronic Acid In Normal Flowrate Using Sample Dilutions A.8.7.1.M.Reddy Memorial College of Pharmacy Page 94 .
A.Reddy Memorial College of Pharmacy Page 95 .M.
7.2.1ml using placebo A.M.Reddy Memorial College of Pharmacy Page 96 .1a Robustness at flowrate 1.8.
2b Robustness at flow rate 1.M.22.214.171.124ml using zoledronic acid 8.8.1ml using Standard dilutions A.3c Robustness at flow rate 1.2.Reddy Memorial College of Pharmacy Page 97 .
126.96.36.199d Robustness at flow rate 1.1ml using sample dilutions
A.M.Reddy Memorial College of Pharmacy Page 98
A.M.Reddy Memorial College of Pharmacy
188.8.131.52a Robustness at flow rate 0.9ml using placebo
A.M.Reddy Memorial College of Pharmacy
8.M.Reddy Memorial College of Pharmacy Page 101 .3.7.9ml using zoledronic acid A.2b Robustness at flow rate 0.
7.4d Robustness at flow rate 0.Reddy Memorial College of Pharmacy Page 102 .M.3.9ml using sample dilutions A.9ml using Standard dilutions 8.3c Robustness at flow rate 0.7.3.8.
M.Reddy Memorial College of Pharmacy Page 103 .A.
1a Robustness at Normal pH Using Placebo A.8.4.Reddy Memorial College of Pharmacy Page 104 .M.7.
8.7.M.7.2b Robustness at normal pH using zoledronicacid 8.4.Reddy Memorial College of Pharmacy Page 105 .4.3c Robustness at normal pH using Standard dilutions A.
Reddy Memorial College of Pharmacy Page 106 .4.8.4d Robustness at normal pH using sample dilutions A.7.M.
A.M.Reddy Memorial College of Pharmacy Page 107 .
1a Robustness at pH 3.M.5.7.Reddy Memorial College of Pharmacy Page 108 .8.1 using placebo A.
184.108.40.206 using zoledronic acid 8.2b Robustness at pH 3.M.Reddy Memorial College of Pharmacy Page 109 .7.1 using Standard dilutions A.7.3c Robustness at pH 3.
8.1 using sample dilutions A.7.Reddy Memorial College of Pharmacy Page 110 .4d Robustness at pH 3.M.5.
Reddy Memorial College of Pharmacy Page 111 .A.M.
3 using placebo A.Reddy Memorial College of Pharmacy Page 112 .M.1a Robustness at pH 220.127.116.11.
8.7.2b Robustness at pH 3.6.Reddy Memorial College of Pharmacy Page 113 .3 using zoledronic acid 8.3c Robustness at pH 3.3 using Standard dilutions A.6.M.7.
6.M.8.Reddy Memorial College of Pharmacy Page 114 .7.4d Robustness at pH 3.3 using sample dilutions A.
A.M.Reddy Memorial College of Pharmacy Page 115 .
7.Reddy Memorial College of Pharmacy Page 116 .M.7.1a Robustness by column change using placebo A.8.
7.M.3c Robustness by column change using Standard dilutions A.7.2b Robustness by column change using zoledronic acid 8.Reddy Memorial College of Pharmacy Page 117 .8.7.7.
M.4d Robustness by column change using sample dilutions A.7.Reddy Memorial College of Pharmacy Page 118 .7.8.
A.M.Reddy Memorial College of Pharmacy
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.
A.M.Reddy Memorial College of Pharmacy
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
A.M.Reddy Memorial College of Pharmacy
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. A.M.Reddy Memorial College of Pharmacy Page 122 .9.
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. The study of ruggedness made by conducting by different alliance systems and by two analysts the results was shown in table.From the specificity studies it was conformed that there will be no interference of excipients has been observed.Reddy Memorial College of Pharmacy Page 123 . % of recovery in accuracy studies has been obtained in range of 97-103%. A.% RSD obtained in case of system precision was 0..e. Validation of proposed method was verified by accuracy studies. Validation of proposed method was verified by system precision & method precision studies. 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.M. pH and temperature indicated that the analytical method remains unaffected. on verified by changing parameters such as flow rate..
Reddy Memorial College of Pharmacy Page 124 .0 SUMMARY AND CONCLUSION: Development of new analytical method for the determination of drug in pharmaceutical dosage forms is more important in pharmacokinetic.10. toxicological and biological studies.M. The pharmaceutical industry is under increased security from the government and the public interested groups to A. Today pharmaceutical analysis entails much more than the analysis of active pharmaceutical ingredients or the formulated product.
setting specifications and method validation. specificity. purity and quality of the product. precision and accuracy are in estimation of drugs. content and purity of the products. Simplicity. efficacious product that fulfill unmet medical needs. As a result. efficacy. A. sensitivity.Reddy Memorial College of Pharmacy Page 125 . The commonly used tests of pharmaceutical analysis generally entail compendia testing method development. low cost. safety. The pharmaceutical analyst plays a major rule in assuring identity. The need for pharmaceutical analysis is driven largely by regulatory requirements. Therefore of achieving the selectivity. 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. Analytical testing is one of the more interesting ways for scientist to take part in quality process by providing actual data on the identity. New methods are now being developed with a great deal of consideration to worldwide harmonization. speed. the quality assurance and quality control departments play major role in bringing out a safe and effective drug or dosage form. In industry. new products can be assured to have comparable quality and can be brought to international markets faster.contain costs and at consistently deliver to market safe.M. Pharmaceutical analysis occupies a vital role in statuary certification of the drugs and their formulation either by the industry or the regulatory authorities.
Goal Publishing House.23rded.11.Publications. 2. H.pp 1. Introduction to Analytic al Chemistry” .0 BIBLIOGRAPHY 1.Reddy Memorial College of Pharmacy Page 126 .Stenlake.“Instrumental methods of chemical analysis.S. C. Meerut .B.B. J.1-4 A.M. pp.Beckett. New Delhi. 1997. B.K Sharm . “Practical Pharmaceutical Chemistry”. 2004. Part two. 4thed.
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Vol. kumar SS. Chinese journal of drugs 2006-08. Veerender M. 879 (22): Page no: 2073-80. Journal of Pharmaceutical and Biomedical Analysis [2005.janardhanreddy. Srinivasu MK.M. “Assay of zoledronic acid for injection and its related substances by HPLC”. Rani ChP. Arne Streitbürger. A. in human serum. 2012.“simple RP-HPLC pharmaceutical method for related substances of zoledronic acid in products. ZHENG Guo-gang. Raman Venkataramanan.HU Ying-he. Sonia Gauron. Rao BM. “Determination of zoledronic acid for injection by HPLC”. Therapeutic Drug Monitoring (impact factor: 2.k. . Sripal Reddy Mada. Chinese pharmaceutical journal 2005-07. volume 34 (6) 2011.30 (4):897-911.raveendrareddy. 2002 Nov 7. 2005. GAO Song.Reddy Memorial College of Pharmacy Page 129 . J Chromatogr B AnalytTechnol Biomed Life Sci. KatrinVeldboer. J Pharm Biomed Anal. 33. Marilyn Torch. 36. “Determination of zoledronic acid in human urine and blood plasma using liquid chromatography/electrospray mass spectrometry”. Kumar PR. Christina Ravera. Uwe Karst. JendrikHardes. L. nandansrinivasan. Chandrasekhar KB. 476-489(14) 30. GhislaineGosset. 30(3):314-9. TorstenVielhaber. 2011. Zhang Xiao-qing. WANG Ling-ling . pp. Shimin Zhang. 06/2008. ZONG Guo-jun. Francois Legay. XuZhi-ru. 29.49).p. 34.28. Helmut Ahrens. a new potent heterocyclic bisphosphonate. “Determination of zoledronic acid and its preparations by RP-HPLC”. Journal of pharmaceutical practice.“A new analytical method for estimation of zoledronic acid in commercial pharmaceutical injections by Ion-Exchange (iec) High Performance Liquid Chromatography”. plasma and urine”. Jiang Ye. “Development and validation of a High-Performance Liquid Chromatographic assay for the determination of Fluconazole in human whole blood using solid phase extraction”. 35. Raghu. 31. 39(3-4):781-790]. “Development and validation of a highly sensitive RIA for zoledronic acid.Arabian Journal of Chemistry. FabienneDeckert. 32. HansjörgWiegand.Maheswarareddy. Horst Schran. Rakesh K Goyal. Ulrike Pfaar. FANG Ying-zhi. Journal of liquid chromatography & related technologies. “A validated stability indicating ion-pair RP-LC method for zoledronic acid”.
71(4):451-4. 3 (3). 1. 3(2): 316-328. “Rapid and sensitive HPLC method for the determination of sirolimus with ketoconazole as internal standard and its further applications”. Mahmood Payehghadr. Nguyen Minh Nguyeta. Massarta. Plaizier-Vercammen. S.c J. M.Venkatesh. P. 39. P Srinivas. R. “Bioanalytical method development and validation of letrozole by RP-HPLC method”. 73(3):483-489].R. Farzaneh Ahmad Khan Beigi. 2009 Jul. Detaevernierb. N. and C) in Capsule Formulations by HPLC with UV Detection”. E.37. ChiranjeeviBodepudi*.M. Jordan Journal of Chemistry Vol.Shanmugasundaram. Sklenárová H. 43. Hájková R. 4 (4). K Spandana. A Anil Kumar.Udhayakumar. 45. 2008-2009. 38. V. Y. M. J VidyaSagar. Sarathchandiran.*. pp 13091317. “Novel Reverse Phase HPLC Method development and validation of Fluconazole and Tinidazole in a combined tablet dosage form”. N. 4(1): 70-73. 40. “Development and validation of HPTLC method for the estimation of clotrimazole in bulk drug and tablet formulation”.Sekar. Talanta [2007. IJPRD. Parul Parmar.Reddy Memorial College of Pharmacy Page 130 .Jayaseelan. Journal of Chromatography A.VijeyAanandhi. P. 2011. Mohammad Imani. K.Perumal. “Development and Validation of Analytical Method for Fluconazole and Fluconazole Related Compounds (A. KalyanObula Reddy M. 44. Jayaveera and Raghunadha Reddy. Svecová P. A. “Development and validation of HPLC method for determination of clotrimazole and its two degradation products in spray formulation”. Matysová L. Indian J Pharm Sci. Vol. Al-Rimawi. S. 41. Swati Bantu. Rama. International Journal of Pharmaceutical Sciences and Drug Research 2012. B.P. “Simultaneous determination of ketoconazole and formaldehyde in a shampoo: liquid chromatography method development and validation”. International Journal of ChemTech Research. 42.L. I. Vol. 958 (2002) 191–201. Solich P. pp. “Development and Validation of High-Throughput Liquid Chromatography –Tandem Mass Spectrometric Method for Quantification of Itraconazole and its Metabolite in Human Plasma”. Heday at Hosseini. “SPE-HPLC method for determination of ketoconazole and A. 2009. D. Scholars Research Library Der Pharmacia Lettre. N. F. Vander Heydena .Subash. 357-365. Ankita Mehta. Vol.
9 São Paulo Sept. E M Abdel-Moety. A. Braz.clotrimazole residues in cow's milk”. F I Khattab. Soc. Chem.Reddy Memorial College of Pharmacy Page 131 . 2011. A M Abou Al-Alamein. J. vol. IJPS 2002. 57(11):931-8. K M Kelani. “Chromatographic determination of clotrimazole. 46.M.22 no. ketoconazole and fluconazole in pharmaceutical formulations”.
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