analysis means identification, characterization and determination of drugs
in mixtures such as
pharmaceutical formulations and biological
fluids. There is large
number of cardiovascular and anti-diabetic drugs are introduced into the
market at an alarming rate. These drugs may be either new
entity in the market or partial structural modifications of the
existing drugs. Newer analytical methods are developed for these drugs in their
combined dosage form because of the following
The drug combinations may not be
official in any official compendia
Literature survey reveal no analytical
methods are reported
Analytical methods is
hard in drug combinations due to the
interference from excipient.
the other hand, the existing procedure may,
Needed expensive instruments, the
reagents, solvents for analysis
time consuming or tedious
extraction or separation steps
rapid, accurate, reliable and sensitive
newly developed analytical method finds their importance in various fields like
Government and private Research institutions
Quality control department in industries
Approved drug testing laboratories
Bio-pharmaceutics and Bio-equivalence studies
Clinical pharmacokinetic studies
Estimation of Drugs in combined
Administration of two
or more drugs at a time becomes crucial for several therapeutic considerations.
The combined dosage form have gained lot of importance now days due to greater patient acceptability, increased
potency, multiple action, fewer side
and quicker relief. The quantitative determining of combined dosage forms(CDF)
are complex in nature as one
component does not interfere with the estimation of the other. There is a need of
analysis of such formulations without prior separation. For the
estimation of multi-component formulation, the instrumental techniques commonly
employed, are spectrophotometry, GLC, HPTLC, HPLC etc. these methods
are based upon the measurement of
specific and nonspecific physical
properties of the substances. Of all the above techniques HPLC
is most widely considered quantitative
analysis of drugs in CDF.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
all the chromatographic techniques HPLC8 is one of the most widely used
analytical techniques providing superior qualitative and quantitative results, reproducibility and higher detection
sensitivity and high reliability of analytical
data. In high performance liquid chromatography
(HPLC) the liquid mobile phase is passed through the stationary phase under
pressure. Chromatographic separation in HPLC
is the result of specific
interactions between sample molecules with both stationary and mobile phases.
HPLC offers a great variety of stationary phases, which allows
a greater variety of these selective interactions and more possibilities for separation. A
simple HPLC system consisting of a solvent reservoir to hold the mobile phase,
a pumping system to pressurize the mobile phase, and injector to introduce a
small volume of the sample mixture under high pressure, a column containing
stationary phase, a detector to detect the presence of components as they exit the column, and a recorder to record the detector signal.
used for analysing
with high polarity or ionic samples
with high molecular weight
unstable and decomposable substances
In HPLC there are various
modes of separations.
normal phase mode
stationary phase polar & mobilephase nonpolar, for this nonpolar compounds
eluted first reason lowaffinityof nonpolar compounds & polar stationaryphase. take more time to
separate . The stationary phases used are SiO2, Al2O3,
-NH2, -CN, -Diol, -NO2.The mobile phases used are
heptanes, hexane, cyclohexane, chloroform, dichloromethane.
Reversed phase mode in analytical field of
chemicalbiological pharmaceuticalbiomedical sciences, stationary phase non
polargroups attached silicagel & mobile
phase polarsolvent. Coloumns are ODS viz:
C8 & C4 C18 The mobile phases used are water, acetonitrile, methanol and buffers.
Ion exchange chromatography, the
stationary phase contains cation exchanger (SO -, COO-)
and anion exchanger (NR +,NHR +), used separating of
inorganic ions, organic acids, organic bases, proteins and nucleic acids.
Ion pair chromatography
For acidic substances tetraalkyl
ammonium salts and for basic substances alkyl sulfonates are used as ion pair
10 involves the covalent attachment of immobilized biochemical to solid
support highly specific biochemical interactions for separation. When sample is passed through the
column only solutes that selectively bind to complementary ligand is retained while other sample components elute
without retaining in column. The
stationary phase contains specific groups of molecules
called ligands, This technique was used
to seperate biological samples like proteinsenzymes
&antibodies in High mixtures.
separation in molecular mass. LSM first and the SSM elute later, it
later defined into GPC and GFC.
Instrumentation in HPLC 11, 12
phase reservoir system with pumps
Mobile phase reservoir system with pumps
HPLC pump is very important component
of the system. Pumps are required to
deliver a constant flow of mobile
phase at pressure ranging from 14.6 to 8000 psi. Pumps capable of pressures up to 8000 psi provide a wide range of mobile phase flowrate typically from 0.01- 10 ml / min.
viscosity backpressure nondepending with
35 -400 ?l ,outputpressure 10,000 psi
&constantflowrate & pulsed flow.
pump:. less than 2000 psi.
There are two
type of elution process, i.e. isocratic and gradient,
In this system, the things are kept
constant throughout the run. In the case of pumping of mobile phase, the mobile
phase composition is kept constant throughout the run. The nominal flow rate
accuracy required is ±1% of the set flow.
some change purposely incorporated during the particular sample run to achieve a better or/and faster
separation. In case of pumping mobile phase, the composition of
mobile phase is continuously varied during the particular run.
The gradient accuracy of ±1% of
the step gradient composition is typical.
Columns in HPLC are fabricated withstand high pressure. The internal diameter of analytical column are 4-5 mm and of length
10-30 cm. The shorter column with 3-6 cm in length
is also available. The particle sizes in column packing are generally 3 or 5 µm. For analysis of single component
shorter columns are sufficient but for
separation two or more components
longer column are preferable.
There are three
main types of column packing in HPLC.
Totally Porous silica particles
The ideal detector should respond to a particular property of the
substance being separated, it should be sensitive and give a linear response
range over a wide concentration range.
basically two types
Property Detectors &Solute Property Detectors:.
used in HPLC analysis of pharmaceutical substances are
Photometric detectors: These
normally operate in ultraviolet region of spectrum and most widely used. These
detectors consist of light source, dispersing element, flow cell,
photomultiplier cell or diode. These are of five types
wavelength detectors Multi wavelength detectors Variable wavelength detector
Programmable detector Diode array detectors
detector it is more sensitive when compared photometric detectors.
Refractive index detectors these detectors
respond to change in bulk property of refractive index of solution of the
component in mobile phase. Its sensitivity is less when compared to bulk
Electrochemical detectors are detector based on
electrochemical principles involving amperometry, voltammetry, and
are several detectors available in the market. However UV Visible detector,
photo diode array detector, fluorescence detector, conductometric and
coulometric detector are more commonly used. The new ELSD detector is proving
to be important detector, while the MS detector is outstanding.
Fig.1.1. Instrumentation of HPLC
analysis in HPLC 8
quantitative analysis in HPLC can performed three methods
The external Standard is the same
substance as that being analysed in the sample.
This method is most commonly used for analysis. The external Standard must be pure and its composition must be
known through prior analysis. By injecting Standard
solution in different concentration peak
response is plotted vs.
concentration. Unknown samples are analysed in similar manner and their
concentration determined from the calibration
The effect of minor variables in separation parameters, peak size and
injection errors. In this method of
analysis, a fixed concentration of known
compound addconcentration separate peaks.
have following properties
have completely resolved peaks without interference from other compound present
have similar chemical properties as the compound of interest.
mimic the analyte in any sample preparation
be stable, compatible with column packing, mobile phase and chemically inert
towards components of sample.
be easily available and with reasonably high
calibration is produced by analyzing various concentrations of pure drug with a
fixed concentration of internal Standard. From the chromatogram response factor
Similarly the analyte is to added to the fixed
concentration of internal Standard and response factor is calculated
RF = Ax/AISTD
internal Standard. From the interpolation of calibration curve the
concentration of analyte calculated.
of internal Standard addition method is not always advantageous as precision is
less when compared to external Standard method.
these method different concentrations of.
The response from Standard addition
to extrapolated to zero addition concentration. The negative intercept on the
X-axis will be the concentration of analyte. The important aspect in this method is detector response should be adequate
to produce S/N ratio >10 otherwise results are not precise.
Method Development and design of
separation method 13
Plate number (N): It
is an important property of the column. It reflects
its quality of separation and its
ability to produce sharp, narrow peaks and good
resolution of peaks. Column with N ranging from
5,000 to 1,00,000 plates/meter are ideal for a good system. The factors
influencing plate number are
Ø Larger columns
viscosity of mobile phase
Resolution factor (RS)
the quality of separation of adjacent bands. It is defined as distance centers of band peaks divided by average band width.
Rt2- Rt1/ 0.5(W1 + W2)
&Rtension2 retention time and W1 & W2
which influences the resolution factor are
Capacityfactor Selectivity factorColumn efficiency
efficiency: It represents
the number of theoretical plates per meter (N) and HETP. HETP height equivalent
to theoretical plates is a section in a column in which
mobile phase and stationary phase are
equilibrium. Lower the HETP value more efficiency is the column
HETP = L/N
length of column
= no. of theoretical plates
Its value depends upon particle size, flow rate,
viscosity of mobile phase and quality of packing
Peak Assymetry (As): It
is a practical measurement of peak shape. Peak asymmetry is measured at 10% of
full peak height. Good columns produce peaks with As values of 0.95-1.1.
VALIDATION OF ANALYTICAL METHOD 14-17
is the assessment of the defined test method. The result of any successful
validation is a comprehensive set of data
support the suitability of test method for its intended use.
parameters are:Specificity Precision LinearityAccuracyLimit of detection
Limit of quantitationRuggednessRobustness Stability solutions
inject sample as well
as other related compounds decomposition/ degradation products, intermediates,
solvent, chemicals used in analysis.
All compounds should separate from main
to demonstrate the lack of interference
stressed placebo to
show that degradation products from excipients will not interfere with
degradation products of drug substance.
sample subjected to
degradation studies to achieve 10-20 % of degradation of analyte in conditions of 0.1 N HCl, 0.1 N NaOH, 3% H2O2 ,50 °C , UV radiation. All the degradation products if any should be
separated from analyte.
Repeatability of method can be determined by multiple
replicate preparations of sample.
These can be done either by multiple
sample preparations (n=6) in same experiment or by preparing three replicates
at three different concentrations. % RSD should be less than 2 %.
RSD should be less than 2 %.
the precision between laboratories.
of analytical procedure is its ability
to obtain test results that is directly proportional to the concentration of
analyte in the sample. The following results
should be reported slope,
correlation coefficient, y intercept and residual sum of squares.
is to verify that the method performance is not affected by typical changes in normal
experiments. The method parameters that is investigated for robustness are
column (lot, age, brand)
phase composition (pH ± 0.05 unit, percent organic ± 2 % )
(detection wavelength ± 2 nm , column temperature ± 5 °C, flow rate ± 0.2 ml/min)
operators in same laboratory
equipment in same laboratory
source of reagents and solutions
source of column
Ø Different laboratory
solutions used for the analysis is required to be
stable for a period more than 24 hr to
produce precise and reliable results.
Advantages of analytical method validation
of analytical results and assurance of quality
capability of the method can be confirmed by analysts using the
about importance of protocols for
for improvement in quality of work
opportunity for training to QC staff
in scientific communication on
STUDIES AND STABILITY INDICATING ANALYTICAL METHOD 18
pharmaceutical molecules chemical stability is a matter of great worth as it make impact on safety and efficacy of the drug product. To study the FDA and
ICH laid guidelines for the requirement of analytical
stability testing data. Knowledge about stability of molecule beneficial
in choosing proper formulation and packaging requirements as well as storage
conditions maintenance and shelf
life, which is essential in filling documentation for regulatory.
Forced degradation studies the drug products and drug
substances undergo degradation at conditions more intense than accelerated conditions
and thus produce degradation products
which can be used to assess the
stability of the molecule. According to the
ICH guideline the intention in stress
testing is further helps investigation of the
intrinsic stability of the molecule and elucidating
possible degradation pathways, and for validation of the stability indicating
method used. For filling the new drug moiety in registration dossier it is
mandatory to perform stability studies. The duration for stability studies in long term studies is 12
and for accelerated stability studies it is 6
months. The intermediate studies are performed for 6 months can be at mild
conditions when compare to accelerated studies. So it will take long
time for separation, identification and quantification of degradation products.
When compared to stability studies in
forced degradation studies the degradation
products are generating in shorter time. The samples generated from forced
degradation studies was further useful for the development of the stability
indicating methods which can be applied
for the analysis of samples generated from accelerated and long term stability studies.
degradationstudies are carried out achieve following purposes:
investigate degradation pathways of drug substances and drug products.
differentiate degradation products of drug products from those of non-drug product in a formulation.
structural elucidation of degradation products.
assess the intrinsic stability of a
drug substance in formulation.
reveal the drug degradation mechanisms in pure and formulations
examine the stability indicating nature of a
understand the chemical properties of drug molecules.
produce more stable formulations.
predict stability-related problems
stability indicating method (SIM) is
an analytical procedure which is used to estimate the decrease in the concentration of the active pharmaceutical ingredient
(API) in drug product when it exposed to various degradation conditions. As per FDA guidance document a
stability-indicating method is a validated quantitative analytical procedure
that can be used for the study in variation in the stability of the drug substances and drug products with time. A
stability-indicating method accurately measures the changes in active
ingredients concentration without interference from other generated degradation
products, related impurities and formulation excipients. The suitable platform
in pre-formulation studies, stability studies and proper storage conditions for
drug substance and drug product.
is one of the two most common mechanisms of drug
degradation over a wide range of pH. Hydrolysis is a chemical
process that involves degradation of a chemical compound by reaction with
water. Hydrolytic reactions are mainly acid or base catalyzed. For hydrolytic
study acidic, neutral and basic conditions should be employed which susceptibly
attacks at ionizable functional groups present in the molecule. The type and concentrations
of acid or base used for hydrolysis is
based on the stability of the drug
substance. Generally HCl or H2SO4 in the concentration range of 0.1–1 M are
acid hydrolysis and NaOH or
KOH in the concentration range of 0.1–1M
for base hydrolysis. If the
compounds have low solubility in water, then inert water miscible co- solvents
can be used. Hydrolytic stress studies is normally performed at
room temperature and if there is
no sign of degradation then elevated temperature (50–70 °C) are recommended. The duration of stress testing should not exceed more than one week.
reactions are one of the two most common mechanisms of drug degradation.
Hydrogen peroxide is most widely used reagent for oxidation of drug
substances in forced degradation
studies but other oxidizing agents such as metal ions, oxygen, and radical
initiators (e.g., azobisisobutyronitrile, AIBN) can also be used. Selection of
an oxidizing agent, its concentration, and conditions depends on the drug
substance. It is recommended to
expose sample with 0.3–3 % hydrogen peroxide at neutral pH and room temperature
in dark for seven days or 5- 20 % degradation could potentially produce
relevant degradation products.
degradation is a degradation that results from
exposure of drug substance or
drug product to UV or visible light in the wavelength range of 300-800 nm.
Exposure to radiation at wavelengths < 300 nm is not needed because pharmaceutical won't expose to such light during its life cycle. The drug substance or drug product should be exposed to a minimum of 1.2 million lux-hrs and 200 Wh/m2 light. The maximum allowable