pharmaceutical analysis

A.M.Reddy Memorial College of Pharmacy

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.


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


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


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


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.


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.


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)


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.


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.


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.


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)


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.


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.


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,


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,


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


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,


Resolution between two peaks (Fig: 5).

Where, tR (1) and tR (2) are the retention times of components 1 and 2 and

W1 and W2 are peak width of components 1 and 2.

4. Column Efficiency (N): Efficiency N of a column is measured by the number of

theoretical plates per meter. It is a measure of band spreading of a peak. Higher the

number of theoretical plates more efficient is the column. Columns with N ranging from

5,000 to 100,000 plates / meter are ideal for a good system. Parameters which can affect

N include Peak position, particle size in column, flow-rate of mobile phase, column

temperature, viscosity of mobile phase, and molecular weight of the analyte.

Efficiency is calculated by using the formula.

(Fig:6)

Where, tR is the retention time and

W is the peak width.


5. Peak asymmetry factor (Af): Peak asymmetry factor (Af), can be used as a criterion of

column performance. The peak half width b of a peak at 10 % of the peak height, divided

by the corresponding front half width a gives the asymmetry factor

(Fig: 7) Asymmetric Factor

For a well packed column, an asymmetry factor of 0.9 to 1.1 should be achievable.

(Fig.no :8)Asymmetric Factor

METHOD DEVELOPEMENT FOR RP-HPLC11-13

During the development of method, the initial sets of conditions that have evolved

from the Literature survey are improved or maximized in terms of resolution, peak shape,

plate counts, asymmetry, capacity, elution time, detection limit, limit of quantitation and

overall ability to quantify the specific analyte of interest.

Optimization of a method can follow either of two general approaches:

1. Manual

2. Computer driven


The manual approach involves varying one experimental variable at a time, while

holding all others constant, and recording changes in response .The variables might

include flow rates, mobile or stationary phase composition, temperature, detection

wavelength, and pH. This univariate approach to system optimization is slow, time

consuming and potentially expensive, however it may provide a much better

understanding of the principles, theory involved and interactions of the variables.

In the second approach, computer driven automated method development,

efficiency is optimized while experimental input is minimized. Computer driven

automated approaches can be applied to many applications. In addition, they are capable

of significantly reducing the time, energy and cost of virtually all-instrumental methods.

The various parameters that need to be optimized during method development

1. Mode of separation

2. Selection of stationary phase

3. Selection of mobile phase

4. Selection of detector

Selection of mode of separation

In reverse phase mode, the mobile phase is comparatively more polar than the

stationary phase. For the separation of polar or moderately polar compounds, the most

preferred mode is reverse phase. The nature of the analyte is the primary factor in the

selection of the mode of separation. A second factor is the nature of the matrix.

Selection of stationary phase / column

Selection of the column is the first and the most important step in method

development. The 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


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


stationary phase, the retention of the given solute depends directly upon the mobile phase,

the nature and the composition of which has to be judiciously selected in order to get

appropriate and required solute retention. The mobile phase has to be adapted in terms of

elution strength (solute retention) and solvent selectivity (solute separation) Solvent

polarity is the key word in chromatographic separations since a polar mobile phase will

give rise to low solute retention in normal phase and high solute retention in reverse

phase LC. The selectivity will be particularly altered if the buffer pH is close to the pKa of

the analytes.

The following are the parameters, which shall be taken into consideration while

selecting and optimizing the mobile phase.

Buffer

pH of the buffer

Mobile phase composition

Buffer and it strength

Buffer and its strength play an important role in deciding the peak symmetries and

separations. Some of the most, commonly employed buffers are

Phosphate buffers prepared by using salts like KH2PO4,

K2HPO4,NaH2PO4,Na2HPO4,etc

Phosphoric acid buffers prepared by using H3PO4.

Acetate buffers – Ammonium acetate, Sodium acetate, etc.

Acetic acid buffers prepared using CH3COOH.

The retention times also depend on the molar strengths of the buffer – Molar

strength is increasingly proportional to retention times. The strength of the buffer can be

increased, if necessary, to achieve the required separations. The solvent strength is a

measure of its ability to pull analytes from the column. It is generally controlled by the

concentration of the solvent with the highest strength.

pH of the buffer


pH plays an important role in achieving the chromatographic separations as it

controls the elution properties by controlling the ionization characteristics. Experiments

were conducted using buffers having different pH to obtain the required separations.

It is important to maintain the pH of the mobile phase in the range of 2.0 to 8.0 as

most columns does not withstand to the pH which are outside this range. This is due to

the fact that the siloxane linkage area cleaved below pH 2.0, while pH values above 8.0,

silica may dissolve.

Mobile phase composition

Most chromatographic separations can be achieved by choosing the optimum

mobile phase composition. This is due to the fact that fairly large amount of selectivity

can be achieved by choosing the qualitative and quantitative composition of aqueous and

organic portions. Most widely used solvents in reverse phase chromatography are

Methanol and Acetonitrile.

Selection of detector

The detector was chosen depending upon some characteristic property of the

analyte like UV absorbance, fluorescence, conductance, oxidation, reduction etc.

Characteristics that are to be fulfilled by a detector to be used in HPLC determination are,

High sensitivity, facilitating trace analysis

Negligible baseline noise to facilitate lower detection

Large linear dynamic range

Low dead volume

Non destructive to sample

Inexpensive to purchase and operate

All pharmaceutical ingredients do not absorb UV light equally, so that selection of

detection wavelength is important. An understanding of the UV light absorptive

properties of the organic impurities and the active pharmaceutical ingredient is very

helpful.

For the greatest sensitivity λmax should be used. UV wavelengths below 200 nm

should be avoided because detector noise increases in this region. Higher wavelengths

give greater selectivity.


(Fig 9) Strategy for Method Development


1. Introduction on sample

Define separation goals


2. Need for special HPLC

Procedure, sample, pretreatment, etc

3. Choose detector and

Detector settings

4. Choose LC method;

Preliminary run; estimate best

Separation separaons

5. Optimize separation condition

6. Check for problems or requirements

for special procedure

7a. Recover

purified material

7b.Quantitative

calibration

7c. Qualitative

method

8. Validate method for release into

routine laboratory

3.0 DRUG PROFILE

ZOLEDRONICACID


[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


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.


4.0 LITERATURE REVIEW


Mastanamma S K et al., (2012)27, Developed a rapid and reproducible reverse phase

high performance liquid chromatographic method has been developed for the estimation

of zoledronic acid in its pure form as well as in pharmaceutical dosage forms.

Chromatography was carried out on an ODS C18column (250 x 4.6 mm x 5 m

length),using a mixture of methanol and 0.01M phosphate buffer (pH-3) (60:40 v/v) as

the mobile phase at a flow rate of 0.8 mL/min and the detection was done at 210 nm. The

retention time of the drug was 3.812 min. The method produced linear responses in the

concentration range of 0.25 to 60 g/mL of zoledronic acid. The method was found to be

reproducible for analysis of the drug in parentral preparations.

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


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


http://veldboer.k.lib.bioinfo.pl/auth:Veldboer,K

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


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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


http://lib.bioinfo.pl/auth:Legay,F


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


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


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.


http://lib.bioinfo.pl/auth:Abdel-Moety,EM

5.0 PLAN OF WORK


It includes

a) Method development

i. Selection of wavelength

ii. pH selection

iii. Column selection

iv. Flow rate selection

b) Validation and optimization of parameters

i. System suitability

ii. System specificity

iii. Precision

a. Method repeatability

b. Method reproducibility

iv. Linearity

v. Range

vi. Accuracy

vii. Robustness

viii. ruggedness


6.0 MATERIALS AND METHODS


a) Equipments

i. HPLC ( waters 2695 )

ii. UV double beam spectrophotometer ( Schimadzu )

iii. PH meter

iv. Sonicator

b) Reagents and standards

i. Triethylamine

ii. Orthophosphoric acid

iii. Water for injection

iv. Zoledronic acid working standard

c) Introduction to method

i. Solubility: It includes solubility of drug substance in different solvents and

different PH conditions.

ii. Wavelength selection: Spectral profile is useful in understanding absorption

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

iii. Mobile phase selection and its optimization: Its selection is done always in

combination with selection of column. Following parameters should be taken into

consideration while doing selecting and optimizing the mobile phase

a. Buffer & its strength

b. Buffer/mobile phase pH

c. Mobile phase composition

iv. Column selection :A column has been selected for analytic purpose by

considering following parameters

a. Length & diameter


b. Packing material

c. Particle size & shape

d. % of carbon load

e. Pore volume & surface area

v. Solvent delivery system selection: Separation using with one solvent called

“isocratic elution” always preferable. Sometimes “gradient elution” also

preferable.

a. Low pressure gradient elution :- mobile phases are mixed at

predetermined ratio

b. High pressure gradient elution :- mobile phases are pumped at

different flow rate

vi. Flow rate selection:

It will be selected upon

a) Retention time

b) Column back pressure

c) Separation of impurities

d) Peak symmetry

Note: It should be not more than 2.5ml/min

vii. Temperature selection:

Generally Ambient Column Temperature Was Preferable To Optimize

Chromatographic Conditions.

viii. Diluents Selection:

It will be based upon

a) Extraction efficiency

b) Peak symmetry

c) Resolution of impurities from API peak


7.0 METHOD DEVELOPMENT


a) Method description

i. Preparation of mobile phase /Buffer- Mix 2 ml tri methyl amine in 1000ml water

for injection & adjust pH to 3.2 with ortho phosphoric acid & filter through 0.22µ

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.


b) Optimization of method development parameters

i. Selection of wavelength

TRAIL-1 CHROMATOGRAM (7.a.1.1)

TRAIL -2 CHROMATOGRAM (7.a.1.2)


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



CHROMATOGRAMS AT PH 3.4




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



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




Chromatograms using Unicem US C18 :TRAIL -1 CHROMATOGRAM (7.a.6.1)


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



CHROMATOGRAMS AT 1.2 (ML/MIN) FLOW RATE :TRAIL -1

(7.a.8.1)CHROMATOGRAM



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


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


8.1.1a. System suitability peak for zoledronic acid


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


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


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


Result: Table no: 8.2.1T


S.no Sample name Retention time

(min)

% of interference

1 Blank – 1 Nil Nil



1 Placebo – 1 6.182 Nil



1 Standard – 1 3.431 Nil



1 Sample – 1 3.433 Nil



It was found that there was no interference observed from placebo and diluents

and meets acceptance criteria.

Hence the method was specific and selective for estimation of zoledronic acid in

4mg per vial injection dosage form.

8.3 PRECISION

“The precision of an analytical procedure expresses the closeness of agreement

(degree of scatter) between a series of measurements obtained from multiple sampling of

the same homogeneous sample under the prescribed conditions.”

Precision may be considered at three levels

Repeatability

Intermediate precision and

Reproducibility

Precision should be obtained preferably using authentic samples. As parameters

the standard deviation, the relative standard deviation (coefficient of variation) and the

confidence interval should be calculated for each level of precision.


Repeatability expresses the analytical variability under the same operating

conditions over a short interval of time (intra-day). At least nine determinations covering

the specified range or six determinations at 100 % test concentration should be

performed. Intermediate precision includes the influence of additional random effects

within laboratories, according to the intended use of the procedure for example different

days, analysts or equipment etc.

Reproducibility i.e. the precision between laboratories (collaborative or inter

laboratory studies) is not required for submission, but can be taken into account for

standardization of analytical procedures.

8.3.1 METHOD REPEATABILITY

Repeatability expresses precision under same operating conditions over a short

interval of time by conducted the study as per test procedure in same laboratory by same

analyst & by using same equipment

Procedure:-

i) Repeatability has been assessed using minimum of 6 determinations at 100 % of test

concentration

ii) Precision of test method has been conducted by assay of 6 replicate sample

preparations of zoledronic acid injections & calculate % RSD for 6 samples

Acceptance Criteria:

RSD of 6 sample preparations should be NMT 2%


8.3.1a. Method Repeatability Peaks For Zoledronic Acid

RESULT:

S.no Results (assay)


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


8.3.3c. Method Reproducibility Peaks For Zoledronic Acid Dilutions


Results :


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


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:


“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


CHROMATOGRAMS FOR LINEARITY


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.


8.6.1a. Accuracy Peaks for Zoledronicacid


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









8.6.1.T


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.


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


% RSD 0.85 1.04 0.99

8.7.1.1a Robustness at normal flowrate using placebo


8.7.1.2b Robustness at normal flowrate of zoledronicacid working Standard


8.7.1.3c Robustness peaks for zoledronic acid in normal flowrates using Standard

dilutions


8.7.1.4d Robustness Peaks For Zoledronic Acid In Normal Flowrate Using Sample

Dilutions


8.7.2.1a Robustness at flowrate 1.1ml using placebo


8.7.2.2b Robustness at flow rate 1.1ml using zoledronic acid


8.7.2.3c Robustness at flow rate 1.1ml using Standard dilutions


8.7.2.4d Robustness at flow rate 1.1ml using sample dilutions


8.7.3.1a Robustness at flow rate 0.9ml using placebo


8.7.3.2b Robustness at flow rate 0.9ml using zoledronic acid


8.7.3.3c Robustness at flow rate 0.9ml using Standard dilutions


8.7.3.4d Robustness at flow rate 0.9ml using sample dilutions


8.7.4.1a Robustness at Normal pH Using Placebo


8.7.4.2b Robustness at normal pH using zoledronicacid


8.7.4.3c Robustness at normal pH using Standard dilutions


8.7.4.4d Robustness at normal pH using sample dilutions


8.7.5.1a Robustness at pH 3.1 using placebo


8.7.5.2b Robustness at pH 3.1 using zoledronic acid


8.7.5.3c Robustness at pH 3.1 using Standard dilutions


8.7.5.4d Robustness at pH 3.1 using sample dilutions


8.7.6.1a Robustness at pH 3.3 using placebo


8.7.6.2b Robustness at pH 3.3 using zoledronic acid


8.7.6.3c Robustness at pH 3.3 using Standard dilutions


8.7.6.4d Robustness at pH 3.3 using sample dilutions


8.7.7.1a Robustness by column change using placebo


8.7.7.2b Robustness by column change using zoledronic acid


8.7.7.3c Robustness by column change using Standard dilutions


8.7.7.4d Robustness by column change using sample dilutions


8.0 RUGGEDNESS:

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


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


Plate count NLT 2000 5919


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.


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.


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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