pharmaceutical analysis
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A.M.Reddy Memorial College of Pharmacy Page 1
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. Physical means – Mass, color, refractive index, thermal conductivity
2. By electromagnetic radiation – Absorption, emission, scattering
3. By an electric charge – Electro chemistry, mass spectrometry
ClassicalMethods2:
1. Separation of analytes by precipitation, extraction or 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.
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.
Most of the instrumental methods fit into one of the three following categories viz.,
spectroscopy, electrochemistry and chromatography.
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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
Conductance techniques
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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
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
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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
Reversed phase chromatography
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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.
o 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.
o Polar compounds are retained for longer times and take more time to elute
because of their higher affinity with the stationary phase.
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.
REVERSED PHASE CHROMATOGRAPHY
o It is the most popular mode for analytical and preparative separations of
compounds of interest in chemical, biological, pharmaceutical, food and
biomedical sciences.
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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.
o 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.
o 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.
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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 moments, such as methylene chloride
and 1,2-dichloroethane interact preferentially with solutes that have large dipole
moments, such as nitro- compounds, 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.
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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
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.
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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 high-
pressure 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 µL are used.
Columns:
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 column’s 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.
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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 3–10 mm porous silica
particles that may have an irregular or spherical shape. Typical column efficiencies are
40,000–60,000 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 44–200 mm and lengths
of up to several meters. Micro columns packed with 3–5 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 1–50 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.
COLUMNS USED IN HPLC (Fig: 2)
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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. 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 organosilane’s 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.
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BONDED STATIONARY PHASES FOR HPLC
Table: 1
STATIONARY
PHASE
FUNCTIONAL
GROUPAPPLICATIONS
Silica Si-OHNormal phase material
Pesticides, alkaloids
C18 OctadecylReverse-phase material
Fatty acids, PAH, Vitamins
C8 OctylReverse-phase and ion pair, Peptides
proteins
C6H5 PhenylReverse-phase
Polar aromatic fatty acids.
CN CyanoNormal and Reverse-phase, polar
compounds
NO2 NitroNormal and Reverse-phase, PAH,
Aromatic compounds
NH2 Amino
Normal, Reverse,
weak ion exchange Carbohydrates,
organic acids, chlorinated pesticides
OH DiolNormal, Reverse phase peptides,
proteins.
SA Sulphonic acid Cation exchange, separation of cations.
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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)
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,
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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 RecommendationsCapacity factor (k1) The peak should be well-resolved from other peaks and the void
volume, generally k1>2.0Repeatability RSD ≤= 1% for N ≥= 5 is desirable
Relative retention Not essential as long as the resolution is startedResolution (Rs) Rs of >2 between the peak of interest and the closest eluting
potential interferent (impurity, excipients, degradation product, internal standard, etc.)
Tailing Factor (T) T of ≤=2Theoretical plates (N) 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,
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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 5º C.
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
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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.
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,
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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.
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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.
(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
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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 appropriate choice of separation column includes three different
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
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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-µm particle size give the best compromise of efficiency, reproducibility
and reliability. In this case, the column selected had a particle size of 5 µm 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
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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
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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 λmax 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
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1. Introduction on sample
Define separation goals
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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
purified material
7b.Quantitative
calibration
7c. Qualitative
method
8. Validate method for release into
routine laboratory
3.0 DRUG PROFILE
ZOLEDRONICACID
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[1-hydroxy-2-(1H-imidazol-1-yl) ethane-1, 1-diyl]bis(phosphonic acid)
TESTS RESULTS
i) Description White to Half white powder
ii) pH 4.5-7
Solubility:
a) Soluble in water sparingly Soluble
b) Soluble in NaOH freely soluble
v) Molecular formula C18 H24 N2 O7P2
vi) Molecular weight 348.9
vii) Official Status It is official in B.P.2007 and official method is HPLC
I. Pharmacokinetics:
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
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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
A.M.Reddy Memorial College of Pharmacy Page 30
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.
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 5μ 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-yl-
acetic 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
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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 μg/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 reversed-
phase HPLC condition was as follows; a C18 ODS column (250 mm×4.6 mm,5μm) , an
eluate consisted of acetonitrile-tetrahydrofuran-0. 05 mol·L-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 mL·min-1 and the detection at 218 nm. Results:The calibrated linear curve of
zoledronic acid was within 5 - 125μg·mL-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-hydroxy-
2-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
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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
3.75×10(-7) mol/L in the MRM mode. For human blood plasma, a LOD of 1×10(-7)
mol/L and a LOQ of 2.5×10(-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,5μm) at room temperature. The mobile phase consisted of methanol-
buffer solution (5 mmol·L-1 ammonium acetate and 10 mmol·L-1 amylamine, adjusted to
pH 7.0 with acetic acid) (3:97), at a flow rate of 1.0 mL·min-1. RESULTS: The
calibration curve was linear in the range of 99.04 -891.4 μg·mL-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
A.M.Reddy Memorial College of Pharmacy Page 33
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 RP-
HPLC 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,
5μm) 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 1-50μg/ml. Quantification was
achieved with photodiode array detection at 238 nm over the concentration range of 1-50
μg/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
A.M.Reddy Memorial College of Pharmacy Page 34
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, 50μm) HTLC column by injecting
15μL 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 acetonitrile–phosphate 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 2,4-
dinitrophenylhydrazine 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
A.M.Reddy Memorial College of Pharmacy Page 35
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 high-
performance 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 μ) 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 20μl..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 50μg/ml for both
Fluconazole and Tinidazole. The Tailing factors of Fluconazole and Tinidazole were
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
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90:10:0.1% ratios run isocratically through a C18 (250mm × 4.6mm, 5μm) 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, 10μg/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 0.1-10μg/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.
Hájková 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 (2-
chlorophenyl)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
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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 µg mL -1 for both
analytes. Limits of detection and quantification for both analytes were equal to 0.01 and
0.1 µg mL-1, respectively. Important parameters influencing the extraction efficiency were
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 TLC-
fractionation 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
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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
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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
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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
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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 0.22µ
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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TRAIL -3 CHROMATOGRAM (7.a.1.3)
TRAIL -4 CHROMATOGRAM (7.a.1.4)
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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 Retention time (mins) Tailing factor
3.2 3.387 1.30
3.4 3.387 1.30
3.6 3.395 2.0
Table no: 7.1.T
CHROMATOGRAMS AT 3.2 pH
TRAIL -1 CHROMATOGRAM (7.a.2.1)
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TRAIL -2 CHROMATOGRAM (7.a.2.2)
TRAIL -3 CHROMATOGRAM (7.a.2.3)
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TRAIL -4 CHROMATOGRAM (7.a.2.4)
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CHROMATOGRAMS AT PH 3.4
TRAIL -1 CHROMATOGRAM (7.a.3.1)
TRAIL -2 CHROMATOGRAM (7.a.3.2)
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TRAIL -3 CHROMATOGRAM (7.a.3.3)
TRAIL -4 CHROMATOGRAM (7.a.3.4)
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CHROMATOGRAMS AT 3.6 PH : TRAIL -1 CHROMATOGRAM (7.a.4.1)
TRAIL -2 CHROMATOGRAM (7.a.4.2)
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TRAIL -3 CHROMATOGRAM (7.a.4.3)
TRAIL -4 CHROMATOGRAM (7.a.4.4)
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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 Retention time
( mins)
Tailing factor Area
Hypersil BDS C18 3.14 1.3 2224305
Unicem US
C18
4.0 2.5 1333103
Table no: 7.2.T
Chromatograms using Hypersil BDS C18
TRAIL -1 CHROMATOGRAM (7.a.5.1)
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TRAIL -2 CHROMATOGRAM (7.a.5.2)
TRAIL -3 CHROMATOGRAM (7.a.5.3)
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TRAIL -4 CHROMATOGRAM (7.a.5.4)
Chromatograms using Unicem US C18 :TRAIL -1 CHROMATOGRAM (7.a.6.1)
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TRAIL -2 CHROMATOGRAM (7.a.6.2)
TRAIL -3 CHROMATOGRAM (7.a.6.3)
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TRAIL -4 CHROMATOGRAM (7.a.6.4)
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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) Retention time (mins) Area
1.0 3.4 2245388
1.2 2.8 1865507
Table no: 7.3.T
CHROMATOGRAMS AT 1.0 (mL/ min) FLOW RATE
TRAIL -1 CHROMATOGRAM (7.a.7.1)
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TRAIL -2 CHROMATOGRAM (7.a.7.2)
TRAIL -3 CHROMATOGRAM (7.a.7.3)
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TRAIL -4 CHROMATOGRAM (7.a.7.4)
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CHROMATOGRAMS AT 1.2 (ML/MIN) FLOW RATE :TRAIL -1
(7.a.8.1)CHROMATOGRAM
TRAIL -2 CHROMATOGRAM (7.a.8.2)
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TRAIL -3 CHROMATOGRAM (7.a.8.3)
TRAIL -4 CHROMATOGRAM (7.a.8.4)
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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 Solvent composition Retention time
1. Water for injection and
Ortho phosphoric acid
buffer (95:5%v/v)
3.347min
2. Methanol and phosphate
buffer (60:40%v/v)
4.2min
3. Tetrabutyl ammonium and
methanol (900:100v/v)
10.4min
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. λmax: 215nm
3. Flow rate : 1.2mL/min
4. Injection volume : 20µl
5. Column temperature: 35°c
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
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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
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
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8.1.1a. System suitability peak for zoledronic acid
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8.1.2b.system suitability peaks for zoledronic acid dilutions
Parameter Acceptance criteria Result
% RSD NMT 2 0.41
Tailing factor NMT 2 1.18
Platecount NLT 2000 5962
Table no: 8.1.1T.
Result: system suitability parameter meets acceptance criteria
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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 Sample name Retention time
(min)
% of interference
1 Blank – 1 Nil Nil
2 Blank – 2 Nil Nil
3 Blank – 3 Nil Nil
1 Placebo – 1 6.182 Nil
2 Placebo – 2 6.184 Nil
3 Placebo – 3 6.182 Nil
1 Standard – 1 3.431 Nil
2 Standard – 2 3.429 Nil
3 Standard – 3 3.430 Nil
1 Sample – 1 3.433 Nil
2 Sample – 2 3.433 Nil
3 Sample – 3 3.432 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.
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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%
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8.3.1a. Method Repeatability Peaks For Zoledronic Acid
RESULT:
S.no Results (assay)
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T-1 4.09
T-2 4.09
T-3 4.08
T-4 4.08
T-5 4.08
T-6 4.08
Average 4.08
Standard deviation 0.005
% RSD 0.112
8.3.1.T
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 :
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S.No Results
T-1 4.14
T-2 4.14
T-3 4.13
T-4 4.12
T-5 4.12
T-6 4.13
Average 4.13
Standard deviation 0.008
% RSD 0.193
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.
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 (%) Obtained concentration
(mg/ml)
Area
80 11.62 1628999
90 13.07 1843361
100 14.53 2055112
110 15.98 2265904
120 17.43 2468984
8.4.2.T
Slope 144740
Intercept -5.0042
Correlation coefficient 1.000
Regression 1.000
8.5 RANGE:
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“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: 8.5.1.T
Linearity level (%) Obtained concentration
(mg/ml)
Area
80 11.62 1628717
100 14.53 2053312
120 17.43 2466400
8.5.2.T
Correlation coefficient 1.000
Y – intercept -44731
Slope 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 Sample I.D Retention time
(min)
Area
Zoledronic acid Accuracy – 80% - 1 3.427 1665880
Zoledronic acid Accuracy – 80% - 2 3.423 1652011
Zoledronic acid Accuracy – 80% - 3 3.420 1653849
Zoledronic acid Accuracy – 100% - 1 3.418 2102494
Zoledronic acid Accuracy – 100% - 2 3.418 2106121
Zoledronic acid Accuracy – 100% - 3 3.417 2107040
Zoledronic acid Accuracy – 120% - 1 3.416 2539464
Zoledronic acid Accuracy – 120% - 2 3.415 2532733
Zoledronic acid Accuracy – 120% - 3 3.414 2527766
8.6.1.T
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8.6.2.T
Sample
no.
Spiked
level
Weight of
zoledronic
acid added
Weight of
zoledronic
acid found
Sample
area
Amount
recovered
% of
Recovery
1 80% 11.62 11.55 1665880 99.38 99.38%
2 11.62 11.45 1652011 98.55 98.55%
3 11.62 11.47 1653849 99.66 99.66%
1 100% 14.53 100.34 2102494 100.34 100.34%
2 14.53 100.51 2106121 100.51 100.51%
3 14.53 100.56 2107040 100.56 100.56%
1 120% 17.43 101.00 2539464 101.00 101.00%
2 17.43 100.73 2532733 100.73 100.73%
3 17.43 100.53 2527766 100.53 100.53%
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.
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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. Area (1.2 ml/min) Area (1.1 ml/min) Area (0.9 ml/min)
1 801782 727543 889648
2 1813142 1680448 2056379
3 2229178 1997801 2439575
Tailing factor 1.19 1.22 1.24
Average 1614700 1468597 1795201
% RSD 0.82 0.63 0.64
b) Influence of variations of p H in mobile phase
It is a study to establish the influence /effect of variations of pH 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. Area (3.2 pH) Area (3.1 pH) Area (3.3 pH)
1 805518 810274 831885
2 1814170 1852889 1872787
3 2194771 2210100 2237816
Tailing factor 1.24 1.23 1.23
Average 1604820 1624421 1647496
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% RSD 0.85 1.04 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
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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
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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
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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
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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
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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
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8.7.7.3c Robustness by column change using Standard dilutions
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8.7.7.4d Robustness by column change using sample dilutions
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8.0 RUGGEDNESS:
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 Acceptance criteria Result
% RSD for replicate
injections of peak response
of zoledronic acid from
Standard preparation
NMT 2 0.80
Tailing factor NMT 2 1.27
Plate count NLT 2000 5830
System Suitability Parameters of Analyst-2: 8.8.2.T
Name of parameter Acceptance criteria Result
% RSD for replicate
injections of peak response
of zoledronic acid from
Standard preparation
NMT 2 0.62
Tailing factor NMT 2 1.24
Plate count NLT 2000 5919
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Result:-
The % RSD of 6 sample preparations of zoledronic acid injection 4mg/vial is 0.16.
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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