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Please cite this article in press Sharmila Dusia et al., Method Development and Validation of Donepezil
Hydrochloride by RP-HPLC, Indo Am. J. P. Sci, 2018; 05(05).
INTRODUCTION:
Analytical chemistry is used to determine the about the presence or absence of certain components.
qualitative and quantitative composition of material A quantitative analysis provides numerical
under study. Both these aspects are necessary to information as to the relative amount of one or more
understand the sample material. Analytical chemistry of this component. For analyzing the drug samples in
is divided into two branches quantitative and bulk, pharmaceutical formulations and biological
qualitative. A qualitative analysis gives us the fluids, different analytical methods are routinely
information about the nature of sample by knowing being used.
The plate theory: the sample molecules. This is suitable for the
According to Martin and Synge, a chromatographic separation of charged molecules only. Changing the
system consists of discrete layers of theoretical pH and salt concentration can modulate the retention.
plates. At each of these, equilibration of the solute
between the mobile and stationary phases occurs. The
movement of solute is considered as a series of Ion Pair Chromatography:
stepwise transfers from plate to plate. This technique is also referred to as Reversed Phase
The rate theory: Ion Pair Chromatography or Soap Chromatography.
This theory considers the dynamics of the solute It may be used for the separation of ionic compounds
particles as it passes through the void space between and this method can also substitute for Ion Exchange
the stationary phase particles in the system as well its Chromatography. Strong acidic and basic compounds
kinetic as it is transferred to and from the stationary may be separated by reversed phase mode by forming
phase. ion pairs (coulumbic association species formed
between two ions of opposite electric charge) with
TYPES OF CHROMATOGRAPHY suitable counter ions.
Normal Phase Chromatography:
In Normal Phase mode the stationary phase is polar Affinity Chromatography:
and the mobile phase is non polar in nature. In this This technique uses highly specific biochemical
technique, non polar compounds travel faster and are interactions for separation. The stationary phase
eluted first. This is because of the lower affinity contains specific groups of molecules which can
between the non polar compounds and the stationary absorb the sample if certain steric and charge related
phase. Polar compounds are retained for longer times conditions are satisfied. This technique can be used to
because of their higher affinity with the stationary isolate proteins, enzymes as well as antibodies from
phase. These compounds, therefore take more times complex mixtures.
to elute. Normal phase mode of separation is
therefore, not generally used for pharmaceutical Size Exclusion Chromatography:
applications because most of the drug molecules are It separates molecules according to their molecular
polar in nature and hence take longer time to elute. mass. Largest molecules are eluted first and the
smallest molecules last. This method is generally
Reversed Phase Chromatography: used when a mixture contains compounds with a
It is the most popular mode for analytical and molecular mass difference of at least 10%. This mode
preparative separations of compound of interest in can be further subdivided into gel permeation
chemical, biological, pharmaceutical, food and chromatography (with organic solvents) and gel
biomedical sciences. In this mode, the stationary filtration chromatography (with aqueous solvents).
phase is non polar hydrophobic packing with octyl or
octa decyl functional group bonded to silica gel and METHOD DEVELOPMENT (2-4):
the mobile phase is polar solvent. The polar Analytical method development and validation play
compound gets eluted first in this mode and non polar important roles in the drug discovery and
compounds are retained for longer time. As most of manufacture of pharmaceuticals. These methods used
the drugs and pharmaceuticals are polar in nature, to ensure the identity, purity, potency, & performance
they are not retained for longer times and hence elute of drug products. There are many factors to consider
faster. The different columns used are octa decyl when developing methods. The initially collect the
silane (ODS) or C18, C8, C4, (in the order of information about the analyte’s physiochemical
increasing polarity of the stationary phase). An properties (pKa, log P, solubility) and determining
aqueous mobile phase allows the use of secondary which mode of detection would be suitable for
solute chemical equilibrium (such as ionization analysis. The majority of the analytical development
control, ion suppression, ion pairing and effort goes into validating a stability indicating HPLC
complexation) to control retention and selectivity. method. The goal of the HPLC-method is to separate
and quantify the main active drug, any reaction
Ion Exchange Chromatography: impurities, all available synthetic intermediates and
The stationary phase contains ionic groups like any degradants.
NR3+, SO3-which interact with the ionic groups of
Selection of diluents is based on the solubility of Keq = [H3O+] [A-] / [H2O] [HA]
analyte. The analyte must be soluble in the diluents Now in dilute solutions of acid, [H2O] stays roughly
and must not react with any of the diluents constant. Therefore define a new equilibrium
components. The diluent should match to the starting constant the acidity constant Ka.
eluent composition of the assay to ensure that no
peak distortion will occur, especially for early eluting Ka = [H3O+] [A-] / [HA]
components. pH and pKa plays an important role in This is also in logarithmic form are follows: pKa =
HPLC method development. The pH value is defined −log10 Ka.
as the negative of the logarithm to base 10 of the It turns that the pKa of an acid is the pH at which it is
concentration of the hydrogen ion, exactly half dissociated. This can be shown by
pH = - log10 [H3O+] rearranging the expression for Ka:
The acidity or basicity of a substance is defined most
typically by the pH value. Selecting a proper pH for pH = pKa – log([AH]/[A-])
velocities of compounds moving through the column The drug substance being analyzed should be stable
are constant. Analyte eluent and analyte-stationary in solution (diluent). During initial method
phase interactions are also constant throughout the development, preparations of the solutions in amber
whole run. This makes isocratic separations more flasks should be performed until it is determined that
predictable, although the separation power (the the active component is stable at room temperature
number of compounds which could be resolved) is and does not degrade under normal laboratory
not very high. The peak capacity is low; and the conditions. The sample solution should be filtered;
longer the component is retained on the column, the the use of a 0.22 or 0.45 µm pore size filter is
wider is the resultant peak. generally recommended for removal of particulates.
Ref: http://infolinkads.com/Number-of-theoretical-plates.html.
ACCURACY: REPEATABILITY:
Accuracy is the nearness of a measured value to the Repeatability is the variation experienced by a single
true or accepted value. Accuracy indicates the analyst on a single instrument. It does not distinguish
deviation between the mean value found and the true between variations from the instrument or system
value. It is determined by applying the method to alone and from the sample preparation process.
samples to which known amounts of analyte have During validation, repeatability is performed by
been added. These should be analyzed against analyzing multiple replicates of an assay composite
standard and blank solutions to ensure that no sample by using the analytical method. The recovery
interference exists. The accuracy is then calculated value is calculated. Intermediate precision is the
from the test results as a percentage of the analyte variation within a laboratory such as different days,
recovered by the assay. It may often be expressed as with different instruments, and by different analysts.
the recovery by the assay of known, added amounts The precision is then expressed as the r elative
of analyte. standard deviation.
QUANTITATION LIMIT:
The limit of Quantitation (LOQ) or Quantitation limit
of an individual analytical procedure is the lowest
amount of analyte in a sample that can be
quantitatively determined with suitable precision and
accuracy. For analytic al procedures such as HPLC
that exhibit baseline noise, the LOQ is generally
estimated from a determination of S/N ratio (10:1)
and is usually confirmed by injecting standards which
give this S/N ratio and have an acceptable percent
relative standard deviation as well. fig.7 Donepezil Tablets
Mechanism of action:
SPECIFICITY:
Specificity is the ability to assess unequivocally the Donepezil is a piperidine derivative that is a
analyte in the presence of components that may be centerally active, reversible inhibitor of
expected to be present such as impurities, acetylcholinesterase. This drug is structurally
degradation products, and excipients. Specificity unrelated to other anticholinesterase agents.
measures only the desired component without Donepezil's proposed mechanism of action involves
interference from other species that might be present; the reversible inhibition of cholinesterases (eg.
separation is not necessarily required. acetylcholinesterase), which prevents the hydrolysis
of acetycholine, and leads to an increased
RANGE: concentration of acetylcholine at cholinergic
Range is defined as the interval between the upper synapses. Evidence suggests that the
and lower concentrations of analyte in the sample for anticholinesterase activity of donepezil is relatively
which it has been demonstrated that the analytical specific for acetylcholinesterase in the brain.
results which show that donepezil and/or its plasma at concentrations equal to about 20% of
metabolites do not inhibit the metabolism of donepezil. Over a period of 10 days, approximately
warfarin, theophylline, digoxin, or cimetidine in 57% of the total radioactivity was recovered in urine
humans. Concurrent administration of cimetidine or and faeces, with about 17% of the donepezil dose
digoxin in pharmacokinetic studies does not affect recovered in the urine as unchanged, while 28%
the metabolism of donepezil. remained unrecovered. Donepezil plasma
Excretion : Donepezil is excreted in the urine intact. concentrations decline with a half-life of
Plasma radioactivity expressed as a percent of the approximately 70 hours. There is no evidence to
administered dose, following administration of C14 - suggest enterohepatic recirculation of donepezil
labeled donepezil, was present primarily as intact and/or any of its metabolites. Sex, smoking history
donepezil (53%), 6-O-desmethyl donepezil (11%), and race have no clinically significant influence on
which has been reported to inhibit AChE to the same plasma concentrations of donepezil.
extent as donepezil in vitro and was found in the
DRUG INTERACTIONS
The risk or severity of adverse effects can be
16-Bromoepiandrosterone increased when 16-Bromoepiandrosterone is
combined with Donepezil.
The risk or severity of adverse effects can be
increased when 19-norandrostenedione is
19-norandrostenedione combined with Donepezil.
Chemicals used:
Table.6 List of chemicals used
NAMES CHEMICALS USED
Methanol Merck Speciality Pvt .Ltd Mumbai
Water Merck Speciality Pvt .Ltd Mumbai
Acetontrile Merck Speciality Pvt .Ltd Mumbai
TRAIL: 1
TRAIL:3
OPTIMIZED CHROMATOGRAM:
Discussion: Symmetric peak with asymmetry factor not more than 2 and theoretical plates was obtained.
Reproducible peak with excellent peak characteristics was obtained with rapid retention time of 4.40 minutes.
METHOD VALIDATION:
SYSTEM SUITABILITY:
Inference: The obtained experimental values in system suitability trials (n=6) were found to be within the limits
proposed by ICH guidelines.
SPECIFICITY:
Concentration(µg/ml) Area
10 7256776
20 12882676
30 18923041
40 19439750
50 31396449
Correlation coefficient 0.999
Slope(m) 548364.2
Intercept(c) 1528812
ACCURACY:
Inference: The results represent the high percent recovery values indicating that the proposed method is accurate.
PRECISION:
Interday Precision:
Interday Precision:
Inference: The % RSD for Intraday precision and interday precision for Donepezil were found to be 0.09 and 0.44
which indicates the method is precise.
Limit of Detection (LOD) and Limit of Quantification (LOQ)
The LOD value was found at 1.8 μg/ml concentration where the signal to noise ratio was 3:1 and the LOQ value was
found at 2.8 μg/ml where the signal to noise ratio was 10:1.
ROBUSTNESS:
Flow rate variation:
Fig.43 Chromatogram for Flow rate Fig.44 Chromatogram for Flow rate
variation (0.8ml/min) variation (0.4ml/min)
Fig.45 Chromatogram for mobile phase Fig.46 Chromatogram for mobile phase
composition variation (70:30) composition variation (50:50)
Inference: All the experimental values for robustness obtained fall into the acceptance criteria.
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