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Analytical Chemistry
Analytical Chemistry
ANALYTICAL CHEMISTRY:
“Analytical Chemistry deals with methods for determining the chemical composition of
samples of matter. A qualitative method yields information about the identity of atomic
or molecular species or the functional groups in the sample: a quantitative method in
contrast, provides numerical information as to the relative amount of one or more of
these components”.
Quantitative chemical analysis is an important tool to assure that the raw material
used and the intermediate products meet the required specifications. Drug analysis is
the base for the determination of the product.
Every year number of drugs is introduced into the market, also quality is
important in every product or service but it is vital in medicines as it involves life15.
The number of drugs introduced into the market is increasing every year. These
drugs may be either new entities or partial structural modification of the existing one 29.
Very often there is a time delay from the date of introduction of a drug into the
market to the date of its inclusion in pharmacopoeias.
Varieties of analytical methods are used for the analysis of drugs in bulk,
formulations and biological samples. In pharmaceutical industry,
spectrophotometric and chromatographic methods have gained the significance
in recent years.
Importance of Analytical Methods
1. Official pharmacopoeia may not reveal an analytical procedure for the drugs or
its combination.
1.1. Spectrophotometry
When a molecule absorbs ultraviolet radiation of frequency v sec -1, the electron
in the molecules undergoes transition from a lower to a higher energy level or molecular
orbital, the energy difference is given by
E= hʋ Equation 1
Where,
E = Energy of the photon
ʋ = Frequency of the monochromatic radiation
h = Plank’s constant.
Absorption Law
As per the Beer’s law discussed above, there is a direct proportionality between
the absorbance and concentration. A plot of absorbance versus concentration is
expected to be a straight line passing through origin. However, this is not always true;
there are certain limitations.
The law does not hold for all species under every condition. Many a times
instead of a straight line, a curvature in the plot may be observed as shown in figure
1.2.
The upward curvature, curve (a), is known as positive deviation and the
downward curvature, curve(c), as negative deviation.
Figure 2: Beer-Lambert’s Law Plot; the Curvatures Show Deviations from the
Law
In a single beam instrument all of the light passes through the sample cell. It
contains Monochromators and phototube as detectors and design to measure %
transmittance or absorbance.
This was the earliest design, but is still in common use in both teaching and
industrial labs.
• Deuterium lamp
• Hydrogen lamp
In a double-beam instrument, the light is split into two beams before it reaches
the sample. One beam is used as the reference; the other beam passes through the
sample.
Double beam instruments became quite popular in the early days of spectrophotometry
due to the instability of light sources, detectors, and the associated electronics 37.
1. Transmittance (T)
It is the ratio of intensity of transmitted light to that of incident light.
T= It / lo Equation 3
2. Absorbance (A)
It is the negative logarithm of transmittance to the base 10.
A = -log10T
= -log10Io/It Equation 4
Applications
1. Solutions of transition metal ions can be colored (i.e. absorb visible light)
because electrons within the metal atoms can be excited from one electronic
state to another. The color of metal ion solutions is strongly affected by the
presence of other species, such as certain anions or ligands. For instance, the
color of a dilute solution of copper sulphate is a very light blue; adding ammonia
intensifies the color and changes the wavelength of maximum absorption (λmax).
1.2. Chromatography
1. Adsorption Chromatography:
Separation is mainly due to the interaction between solute and surface on the
adsorbent. In this, stationary phase is solid and mobile phase is liquid.
e.g.: TLC, HPTLC, HPLC, PC
Next, the components of this sample mixture are separated on the column, a
process monitored with a flow-through detector as the isolated components emerge
from the column.
The HPLC is classified into two modes depending on the relative polarity of the
two phases viz. normal and reverse- phase chromatography.
There are two elution types in HPLC they are isocratic and gradient. In
isocratic elution composition of solvent is pumped through the column during complete
analysis. In gradient system eluent composition and strength is steadily changed during
the run7.
Quantification
With an internal standard method, compound of known purity that does not
cause interference in the analysis is added to the sample mixture. Quantification is
based on the response ratio of compound of interest to the internal standard vs. the
response ratio of a similar preparation of the reference standard (HPLC or GC). This
technique is rarely used for TLC methods.
Where,
W x = Width of the peak determined at either 5% or 10% above baseline
f = Distance between peak maximum and peak front at W x
t o = Elution time of void volume or non-retained components
t r= Retention time of the analyte, R
tw = Peak width measured at baseline of the extrapolated straight sides to
baseline.
K '= (tR-to) / to.
T = Wx / 2f Equation 8
N = 16 (tR/w)2 = L / H Equation 9
N is fairly constant for each peak on a chromatogram with a fixed set of operating
conditions. H or HETP, the height equivalent of a theoretical plate, measures the
column efficiency per unit length (L) of the column.
Dead Volume means any empty space or unoccupied volume, the presence of
which can lead to disastrous losses in efficiency. There will be dead volume in the
column itself, which will be the space that is not occupied by the stationary phase.
The other sources of dead volume are the injection unit, the tubing and fittings at
each end of the column, and the detector cell.
Retention time is the difference in time between the point of injection and
appearance of peak maxima. Retention time is the time required for 50% of a
component to be eluted from a column. Retention time is measured in minutes of
seconds. Retention time is also proportional to the distance moved on a chart paper,
which can be measured in cm or mm.
Retention volume is the mobile required to elute 50% of the component from the
column. It is the product of retention time and flow rate6.
Parameter Recommendation
Applications of HPLC
1) PreparativeHPLC:
2) Analytical HPLC:
Here the focus is to obtain information about the sample compound, which
includes relative comparison, quantification and resolution of a compound.
For this purpose a clean peak of known sample assay has to be observed from the
chromatogram. Selection of column mobile phase and flow rate matter to certain level in
this process by comparing with reference compound does identification and it can be
assured by combining two or more detection methods18.
4) Purification:
5) Chemical Separation:
This can be accomplished using HPLC by utilizing the fact that, certain
compounds have different migration rates given at a particular column and mobile
phase. The extent or degree of separation is mostly determined by the choice of
stationary phase and mobile phase.
5) Quantification:
Methods are developed for new products when no official methods are available.
Alternate methods for existing products are developed to reduce the cost and time for
better precision and ruggedness. Trial runs are conducted, method is optimized and
validated. When alternate method proposed is intended to replace the existing
procedure comparative laboratory data including merit / demerits are made available 21.
c) When multiple components are to be analyzed in the sample matrix, the number
of components is noted, data is assembled and the availability of standards for
each one is determined.
d) Only those methods (Spectroscopic, MS, GC, HPLC etc.,) that are compatible
with sample stability are considered.
2. Method Requirements
The goals or requirements of the analytical method that need to be developed
are considered and the analytical figures of merit are defined. The required detection
limits, selectivity, linearity, range, accuracy and precision are defined.
4. Choosing a Method
a) Using the information in the literatures and prints, methodology is adapted. The
methods are modified wherever necessary. Sometimes it is necessary to acquire
additional instrumentation to reproduce, modify, improve or validate existing
methods for in-house analytes and samples.
b) If there are no prior methods for the analyte in the literature, from analogy, the
compounds that are similar in structure and chemical properties are investigated
and are worked out. There is usually one compound for which analytical method
already exist that is similar to the analyte of interest.
Data thus generated become part of the methods validation package submitted
to Center for Drug Evaluation and Research (CDER). Simply, method validation is the
process of proving that an analytical method is acceptable for its intended purpose.
The process of validation and method design also should be clearly in the
development cycle before important data are generated. Validation should be on going
in the form of re-validation with method changes.
Validation Parameters
1) Accuracy,
5) Selectivity / Specificity,
6) Robustness / Ruggedness,
(1) Accuracy:
Dosage form assays commonly provide accuracy within 3-5% of the true value.
The ICH documents recommend that accuracy should be assessed using a minimum of
(2) Precision:
The linearity of an analytical method is its ability to elicit test results that are
directly (or by a well-defined mathematical transformation) proportional to the analyte
concentration in samples within a given range. Linearity usually expressed in terms of
the variance around the slope of regression line calculated according to an established
mathematical relationship from test results obtained by the analysis of samples with
varying concentrations of analyte.
The linear range of detectability that obeyed Beer’s law is dependent on the
compound analyzed and the detector used. The working sample concentration and
samples tested for accuracy should be in the linear range. The claim that the method is
linear is to be justified with additional mention of zero intercept by processing data by
linear least square regression. Data is processed by linear least square regression
declaring the regression co-efficient and b of the linear equation y = mx + c together
with the correlation coefficient of determination r2. For the method to be linear the r2
value should be close to 1.
The range of an analytical method is the interval between the upper and lower
levels of the analyte (including these levels) that have been demonstrated to be
determined with precision, accuracy and linearity using the method as written.
Limit of Detection: - The limit of detection is the parameter of limit tests. It is the lowest
level of analyte that can be detected, but not necessarily determined in a quantitative
fashion, using a specific method under the required experimental conditions. The limit
test thus merely substantiates that the analyte concentration is above or below a certain
level.
𝟑.𝟑𝝈
𝑳𝑶𝑫 = Equation 11
𝑺
𝟏𝟎𝝈
𝑳𝑶𝑸 = Equation 12
𝑺
Hence one basic difference in the selectivity and specificity is that, while the
former is restricted to qualitative detection of the components of a sample, the latter
means quantitative measurement of one or more analyte.
Stability of the sample, standard and reagents is required for a reasonable time
to generate reproducible and reliable results. For example, 24 h stability is desired for
solutions and reagents that need to be prepared for each analysis.
System suitability test provide the added assurance that on a specific occasion
the method is giving, accurate and precise results. System suitability test are run every
time a method is used either before or during analysis. The results of each system
suitability test are compared with defined acceptance criteria and if they pass, the
method is deemed satisfactory on that occasion.
Range 80 –120 %
1. Linear Regression:
Once linear relationship has been shown to have a high probability by the value of
the correlation coefficient ‘r’ then the best straight line through the data points has to be
estimated. This can be often done by visual inspection of graph but in many cases it is
far more sensible to evaluate the best straight line by linear regression20.
Y = mx + c Equation 13
To obtain regression line ‘y ‘ on ‘x ‘ the slope ‘m’ of the line intercept ‘c’ on the y
axis are given by the following formula.
𝐍 ∑ 𝐱𝐲 − (∑ 𝐱) (∑ 𝐲)
𝐦= Equation 14
𝐍 ∑ 𝐱 𝟐 − (∑ 𝐱)𝟐
(∑ 𝐲) (∑ 𝐱 𝟐 ) – (∑ 𝐱) (∑ 𝐱𝐲)
𝐜 = Equation 15
𝐍 ∑ 𝐱 𝟐 − (∑ 𝐱)𝟐
2.Correlation Coefficient:
𝐧 ∑ 𝐱𝟏 𝐲𝟏 − ∑ 𝐱𝟏 𝐲𝟏
𝐫= 𝟏 ⁄
Equation 16
{[𝐧 ∑ 𝐱𝟏 𝟐 − (∑ 𝐱𝟏 𝟐 ][𝐧 ∑ 𝐲𝟏 𝟐 − (∑ 𝐲𝟏 𝟐 ]} 𝟐
The fit must be quite poor before or become smaller than about 0.98 and is really
very poor when less than 0.9.
̅)𝟐 /𝒏 − 𝟏
S=∑(𝒙 − 𝒙 Equation 17
Where,
S = Standard deviation
̅
𝒙 = Mean or arithmetic average.
n = Number of observations
𝑺𝑫
% 𝑹𝑺𝑫 = ̅
∗ 𝟏𝟎𝟎 Equation 18
𝒙
Where,
SD = Standard deviation
The standard error of mean can be defined as the value obtained by the division
of standard deviation by square root of number of observations. It is mathematically
expressed as,
𝑺𝑫
𝑺. 𝑬 = Equation 19
√𝒏
Where,
SD = Standard deviation.
n = number of observations.
From the stages of drug development to marketing and post marketing, analytical
techniques play a great role in understanding the physical and chemical stability of the
drug, impact on the selection and design of the dosage form, assessing the stability of
the drug molecules, quantitation of the impurities and identification of those impurities
which are above the established threshold essential to evaluate the toxicity profiles of
these impurities to distinguish these from that of the API, when applicable and
assessing the content of drug in the marketed products.
Newer analytical methods are developed for new chemical entities and for
existing drugs either alone or in combinations for the following reasons:
Quest for new drug discovery is an ongoing R&D work. The assurance of quality
is achieved through analysis of the drug product. Now-a-days the pharmaceutical
dosage forms are widely presented with multipleactive componentsi.e.in combined
dosage forms. This has opened new task for analyst to develop simultaneous estimation
of different drugs in such combined dosage forms.
Nebivolol and Indapamide both are official in Indian Pharmacopoeia 2010 respectively.
1. Since only few methods have been reported in the literature for the quantitative
estimation of Nebivolol and Indapamide, there is scope for investigation of new
analytical methods for Nebivolol and Indapamide in bulk and pharmaceutical
dosage forms and many of the previously published methods are not directly
applicable for this issue and need more investigation for method development
and validation.
2. Consequently focus of the present study was aimed to develop the following
analytical methods for Nebivolol and Indapamide with high sensitivity, accuracy
and precision.
The validated analytical method shall then be applied for the individual and
simultaneous estimation of Nebivolol and Indapamide in dosage forms.
Among the several analytical techniques like HPLC, GC, NMR, MS,
Spectrophotometry, IR available for the assay of drugs, the HPLC is simple, sensitive,
selective, rapid, and economical method.
3.Dosage containing drugs Atenolol and Indapamide are used to treat the symptoms of
Hypertension, a condition technically known as increase in Blood pressure. Several
chromatographic methods have been reported for simultaneous estimation of these
drugs and individual drug. In the present study a simple, sensitive, accurate and
effective Reverse Phase High-Performance liquid chromatographic (RP- HPLC) method
was developed for the determination of Atenolol and Indapamide in combined dosage
form. The analysis was resolved by using different chromatographic conditions by
altering mobile phase and flow rate mechanism and HPLC system consisting of Inertsil
ODS (C18, 150*4.6mm), 5nm column at a wavelength of 242 nm. The retention time for
the drugs was found for Atenolol 2.2 min and for Indapamide 4.3 min respectively. As in
simultaneous estimation of these drugs it was found that a confined release can be
formulated. The effect of different polymers on release of the sustained release tablets
of the Atenolol and Indapamide was evaluated. The IR spectrum study revealed that
there is no disturbance in the principal peaks of pure drugs. This confirms the integrity
4. This research is concerned with the development of simple, specific, accurate and
reproducible isocratic reversed phase high performance liquid chromatographic (RP-
HPLC) method which is subsequently validated using ICH recommendations for the
simultaneous estimation of Perindopril Erbumine (PE) and Indapamide (ID) in combined
tablet dosage form. The determination was carried for a runtime of 20min at 40˚C on
Inertsil ODS- 3V column having 250mm x 4.6mm i.d. with 5μm particle size and
potassium dihydrogen phosphate buffer adjusted to pH 3.0 using ortho phosphoric acid
and acetonitrile (60:40 v/v) was used as eluent at a constant flow rate of 1.0ml/min with
UV detection wavelength of 215nm. The retention time of PE and ID was about 11.9
and 4.9min with correlation coefficient of 0.9992 and 0.9990 respectively. The linearity
was established at 8-24µg/ml for PE and 2.5-7.5µg/ml for ID and the mean recovery for
both drugs were found to be 100.3% at a load volume of 50µl.(Damera Vamshi
et.al.2013)
5 .An isocratic reversed-phase liquid chromatograpic assay method was developed for
the quantitative determination of amlodipine besylate (AML) and indapamide (IND) in
combined dosage form. A Brownlee C-18, 5 μm column with a mobile phase containing
0.02 M potassium dihydrogen phosphate–methanol (30+70, v/v) total pH-adjusted to 3
using o-phosphoric acid was used. The flow rate was 1.0 mL min −1 and effluents were
monitored at 242 nm. The retention times of amlodipine besylate and Indapamide were
5.9 min and 3.6 min, respectively. The proposed method was validated with respect to
linearity, accuracy, precision, and robustness. The method was successfully applied to
the estimation of amlodipine besylate and Indapamide in combined tablet dosage
forms.(DA shah et.al.2007)
4. A simple, sensitive, precise and specific Reverse Phase High Performance Liquid
Chromatographic method was developed and validated for the determination of
Nebivolol and Indapamide in bulk and tablet dosage form. It was found that the
excipient in the tablet dosage form does not interfere in the quantification of active drug
by proposed method. The HPLC separation was carried out by reverse phase
chromatography by Inertsil ODS C-18 (150 x 4.6 mm) column with a mobile phase
composed of Buffer: Acetonitrile (60:40) in isocratic mode at a flow rate of 1ml/min. The
detection was monitored at 282 nm. The calibration curve for Nebivolol and Indapamide
was linear from 25-225 µg/ml and 7.5-67.5 µg/ml respectively. The inter-day and intra-
day precision was found to be within limits. The proposed method has adequate
sensitivity, reproducibility and specificity for the determination of Nebivolol and
Indapamide.(Barot PH et.al.2013)
5. Nebivolol HCl and Indapamide tablet is newly approved combination by CDSCO for
the treatment of treatment of hypertension in patients whose blood pressure is not
adequately controlled by monotherapy on 12 May 2010. Stability indicating HPLC
method was developed using C18 (250 mm x 4.6 mm i.d., 5? m particle size) column
6. A simple, rapid, accurate and reliable HPTLC method has been established for
simultaneous estimation of Nebivolol and Indapamide in pharmaceutical dosage form.
Method was developed on silica gel 60 F254 TLC plates using ethyl acetate: methanol:
dil. ammonia, (8.5: 0.8: 1.0 v/v/v), as mobile phase. This system resolved compact
bands for Nebivolol (Rf ~0.43) and Indapamide (Rf~0.64). The developed HPTLC
method was validated in accordance with ICH guideline in terms of linearity and range,
accuracy, precision, specificity, sensitivity and robustness. The validated linearity
ranges found were 500–4000 mg /spot (r2= 0.9994) and 300–1050 mg/spot (r2=
0.9989) for Nebivolol and Indapamide, respectively. The method was found sensitive,
precise, accurate and specific and be used for quantitative estimation of both the
analytes in commercial pharmaceutical dosage form. ( P.U.Patel et.al.2011 )
7. A simple, sensitive, precise and specific Reverse Phase High Performance Liquid
Chromatographic method was developed and validated for the determination of
Nebivolol and Indapamide in bulk and tablet dosage form. It was found that the
excipient in the tablet dosage form does not interfere in the quantification of active drug
by proposed method. The HPLC separation was carried out by reverse phase
chromatography by Inertsil ODS C-18 (150 x 4.6 mm) column with a mobile phase
8. High efficiency and less run time are the basic requirements of high-speed
chromatographic separations. To fulfill these requirements, a new separation technique,
ultra-performance liquid chromatography (UPLC), has shown promising developments.
A rapid, specific, sensitive, and precise reverse-phase UPLC method is developed for
the determination of Amlodipine (AMLO) and Indapamide (INDP) in tablet dosage form.
The chromatographic separation was achieved on Acquity UPLC BEH C18 column (2.1
mm× 100 mm, 1.7 μm) using mobile phase of Buffer (1% Glacial acetic acid) :
Acetonitrile (58:42, v/v), at a flow rate of 0.25 mL/min at an ambient temperature. UV
detection was carried out at 240 nm with injection volume of 2 µl. The retention time for
Amlodipine (AMLO) and Indapamide (INDP) was found 1.56 min and 2.58 min
respectively. Linearity was observed in range of 10-50 µg/ml and 3-15 µg/ml for AMLO
and INDP respectively. The method is validated according to the ICH guidelines and is
applied successfully for the determination of both the rugs in tablet formulation as well
as bulk.(N.Khandelwal et.al.2011)
9. A reverse phase high performance liquid chromatographic method was developed for
the simultaneous estimation of Nebivolol HCl (NEBI) and Indapamide (INDA) in tablet
formulation. The separation was achieved by C18 column (250×4.6 mm, 5 μm Particle
Size) and the mobile phase consisting of 0.074M potassium dihydrogen phosphate (pH
adjusted to 3.0±0.1 using orthophosphoric acid), acetonitrile and triethaylamine in the
proportion of 40:60:0.1 (v/v/v). Detection was carried out at 282 nm. Retention time of
Nebivolol HCl and Indapamide was found to be 3.403 and 7.907 min, respectively.
Linearity for Nebivolol HCl was found in the range of 50-150 μg/ml and Indapamide was
in the range of 15-45 μg/ml. The percentage recoveries obtained for Nebivolol HCl and
4.1. Materials
Drug Sample:
Chemicals Required:
Distilled water
For HPLC
Marketed Formulations:
Nebivolol (NEBICIP., CIPLA)
Nebivolol……………………………......5mg
Indapamide………………………………… 1.5mg
4.2 Instruments
Figure:4.3.1Structure of Nebivolol
Nebimac (Macleods)
Nebinex (Glenmark)
9. Physical Properties
9.1. Colour : white
9.2. State/Form : solid
9.3. Solubility : Soluble in methanol and water
Slightly soluble in acetone.
9.4. Melting point : 159.2-160.7 °C
9.5. pKa :13.52
9.6. Other characteristics :
Stability : Stable at normal temperature and pressure
11. Pharmacokinetics:
13. Toxicity:
The most common signs and symptoms associated with Nebivolol over dosage are
bradycardia and hypertension. Other important advise events reported with Nebivolol
over dosage include cardiac failure, dizziness, hypoglycemia, fatigue and vomiting.
Other adverse events associated with β-Blockers over dose include bronchospasm and
heart block. The largest known investigation of Nebivolol worldwide involved a patient
who ingested upto 500 mg of Nebivolol along with several 100 mg tablets of acetyl
salicylic acid in a suicide attempt.
This section contains uses of this drug that are not listed in the approved
professional labeling for the drug but that may be prescribed by your health
care professional. Use this drug for a condition that is list.
15. Storage: Store between 680 and 770F, protect from light.
2. Category : Hypertension
3.Chemical Structure :
Diurix,
Inditor,
Natridac.
8. Physical Properties
10.Pharmacokinetics
11. Absorption: Cmax is approximately 115-260 ng/ml. The Tmax is 2h.
12. Distribution: 71% to 79% is protein bound
13. Metabolism: Extensively metabolized.
14. Elimination: The t1/2is 26h.More than 70% is excreted in urine, and 23% is
excreted in GI tract, Probably including the bilary route.
11.Side Effects:
Diarrhea
Trouble in sleeping
Tired feeling
headache
Dizziness
Loss of appetite
Stomach upset.
12. Toxicity:
Side effects include electrolyte imbalance (potassium or salt depletion due to too much
fluid loss), nausea, stomach disorders, vomiting, and weakness.
This medication is used to treat high blood pressure. Indapamide is also used to
reduce extra salt and fluid in the body (edema) caused by a
certain heart problem (congestive heart failure). Lowering high blood
pressure helps prevent strokes, heart attacks, and kidney problems. Decreasing
extra salt and fluid in the body helps to decrease swelling and breathing
problems from congestive heart failure and increases your ability to exercise.
Indapamide is a "water pill" (diuretic) that increases the amount of urine you
make. Getting rid of extra water and salt may help to relax the blood vessels so
that blood can flow more easily. These effects help to lower blood pressure and
decrease the amount of work the heart must do to pump blood.
14. Storage:
Store at 25°C (77°F), excursions permitted to 15-30°C (59-86°F) Protect from moisture.
A. Method Development
1. Solvent Selection:
In order to select suitable solvent for determination of Nebivolol, various solvents
were selected for the solubility studies and it was found that Nebivolol was freely soluble
in Methanol and sparingly soluble in Acetone and Water. In the present investigation
methanol was used as an initial solvent and further dilutions was made with water.so
methanol and water were selected as a solvents.
3. Determination of λ max:
1 ml of stock solution-II was transferred to 10ml volumetric flask and the volume
was adjusted to the mark with water to obtain the solution of concentration containing
10µg/ml. The solution was scanned in the UV region of 200-400nm, which shows
maximum absorbance at 281nm and the absorbance curve was given in figure 7.
1. Accuracy:
Accuracy is the closeness of the test results obtained by the method to the true
value. To assess the accuracy of the proposed method, recovery experiment was
performed at three different levels i.e. 50%, 100% and 150%. To the pre-anlysed
sample solution a known amount of standard drug solution was added at three different
levels and absorbances were recorded. The recovery values were summarized in table
6
2. Precision:
The precision of an analytical method is the degree of agreement among
individual test results when the method is applied repeatedly to multiple samplings of
homogenous samples. It provides an indication of random error results and was
expressed as coefficient of variation (CV).
Intra and Inter-day Precision:
The precision of the proposed method was ascertained by actual determination
of six replicates of fixed concentration (50µg/ml) of the drug within the Beer’s range and
variation of results within the same day (intra-day), variation of results between day
(inter-day) in different environmental conditions (Room temperature, Refrigerating
conditions) were analyzed and was shown in table 7 and table 7.1.
3. Linearity:
This is the method ability to obtain results which are either directly, or after
mathematical transformation proportional to the concentration of the analyte within a
given range.
The linearity of the method was demonstrated over the concentration range of 10
- 50 µg/ ml of the target concentration. Aliquots of 1, 2, 3, 4 and 5 µg/ ml are prepared
from Stock solution-II; Calibration curve was plotted and presented in figure 8.
5. Ruggedness:
Ruggedness is the degree of reproducibility of results obtained by the analysis of
the same sample under a variety of normal test conditions i.e. different analysts,
laboratories, instruments, reagents, assay temperatures etc.
The solution of 15µg/ml was prepared and analyzed with change in the analytical
conditions like different instruments (LABINDIA 3000+ and Elico SL 210) and different
analysts (Analyst-1 and Analyst-2). The results were given in table 8.
A. Method Development
1. Solvent Selection:
In order to select suitable solvent for determination of Indapamide, various
solvents were selected for the solubility studies and it was found that Indapamide was
freely soluble in Methanol, acetone and acetonitrile. In the present investigation
Methanol was selected as a solvent.
3. Determination of λ max:
1 ml of stock solution-II was transferred to 10 ml volumetric flask and the volume
was adjusted to the mark with methanol to obtain the solution of concentration
containing 10µg/ml. The solution was scanned in the UV region of 200-400nm, which
shows maximum absorbance at 240nm and the absorbance curve was given in figure 9
1. Accuracy:
Accuracy is the closeness of the test results obtained by the method to the true
value. To assess the accuracy of the proposed method, recovery experiment was
performed at three different levels i.e. 80%, 100% and 120%. To the preanlysed sample
solution a known amount of standard drug solution was added at three different levels
and absorbances were recorded. The recovery values were summarized in table 12.
5. Ruggedness:
Ruggedness is the degree of reproducibility of results obtained by the analysis of
the same sample under a variety of normal test conditions i.e. different analysts,
laboratories, instruments, reagents, assay temperatures etc.
1. Solvent Used
Methanol
Nebivolol: Appropriate aliquots ranging from 10-50µg/ml was pipetted out in to a series
of 10mL volumetric flasks. The volume was made up to the mark with the solvent.
Absorbance of the above solutions was measured at 281nm and a calibration curve of
absorbance against concentration was plotted.
1. Accuracy:
Accuracy is the closeness of the test results obtained by the method to the true
value. To study the accuracy, 20 tablets were weighed and powdered and analysis of
the same was carried out. Recovery studies were carried out by addition of known
amount of Standard Nebivolol and Indapamide to the sample at three different
concentration levels i.e. 80%, 100% and 120% (Standard addition method). The results
were given in table 18.
2. Precision:
The precision of an analytical method is the degree of agreement among
individual test results when the method is applied repeatedly to multiple samplings of
homogenous samples. It provides an indication of random error results and was
expressed as relative standard deviation (coefficient of variation)
The linearity of an analytical method is its ability to elicit test results that are
directly proportional to the concentration of analyte in the sample with in a given range.
Appropriate aliquots ranging from 10-50µg/ml and 2-10µg/ml were pipetted out
separately from the ‘standard stock solutions in to a series of 10ml volumetric flasks.
The volume was made up to the mark with methanol to obtain a concentration range.
Absorbance of the above solutions was measured at 281nm and 240nm respectively.
4. Ruggedness:
It was found that methanol and water gives satisfactory results as compared to
other mobile phases. This mobile phase system was tried with different proportions and
using different flow rates. Finally, the optimal composition of the mobile phase was
obtained in the ratio of Phosphate buffer pH (3.0): Acetonitrile (40:60) with a flow rate of
0.8ml/min.
Peak areas were recorded for all the peaks and a standard calibration curve of
area against concentration was plotted as concentration of the drug Vs peak area
(figure 25 and 26). The results were shown in table 25. Both the drugs follow the
Beer‘s Lambert‘s lawin the concentration range of 10-50μg/ml of Nebivolol and 2-
10μg/ml of Indapamide.
C. Method Validation
The method was validated according to ICH Q2 B guidelines for validation of
analytical procedures in order to determine system suitability, linearity, sensitivity,
precision, accuracy and robustness for the analytes.
1. System Suitability:
The system suitability parameters were evaluated from standard chromatograms
by calculating the % RSD from six replicate injections for Nebivolol and Indapamide
retention times and peak areas.
System suitability was carried out by injecting 100% concentration (sample
having 50µg/ml of Nebivolol and 10µg/ml of Indapamide) into the HPLC system. This
was repeated for six times under similar condition. The tailing factor (T) and no. of
theoretical plates (N) obtained were shown in figure 28and the results were given in
table 28 and 29.
6. Precision:
The precision of an analytical method was studied by performing intraday and
inter day precision.
Intraday Precision
Variation of results within the same day was analyzed. Intraday precision was
determined by analyzing a set of six combined standard solutions of Nebivolol (50µg/ml)
and Indapamide (10 µg/ml) in linearity range as 100% concentration at three different
8. Robustness:
The evaluation of robustness should be considered during the development
phase and depends upon the type of procedure under study. It should show the
reliability of analysis with respect to deliberate variations in method parameters like
different column temperate, different analytical wavelength, different flow rate. The
solution containing 50μg/ml of Nebivolol and 10 μg/ml of Indapamide was injected into
sample injector of HPLC three times under different parameters like deliberate
variations in flow rate (±0.2ml/min) and detection wavelength (± 2 nm).
0.6
0.5
0.3
0.2
0.1
0
0 100 200 300 400
Wavelength(nm)
Concentration
S. Absorbance
of
No. at 281nm
Nebivolol(µg/ml) Mean S.D %RSD
1 0 0 0 0 0
*n=No.of determinants
0.6
0.5
R² = 0.9994
0.3
0.2
0.1
0
0 10 20 30 40 50 60
Concentration ( ug/ml)
Amount
Amount Found Amount Found
S. No Taken
(µg/ml) (%w/w)
(µg/ml)
1 50 49.98 99.86
Amount Total
Added (µg/ml) Amount %
S. No Level of
Recovered Recovery
Recovery Test Std
(µg/ml) (w/w)
99.0
1 50% 15 5 20.2
99.5
2 100% 15 25 39.8
99.8
3 150% 15 45 59.9
Absorbance
S. Conc. Interday
No (µg/ml) Intraday Precision (Day 1) Precision
0hrs 2hrs 4hrs 6hrs 8hrs Day 2
1 50 0.533 0.584 0.554 0.551 0.559 0.573
2 50 0.539 0.581 0.567 0.561 0.554 0.573
3 50 0.531 0.567 0.552 0.567 0.567 0.575
4 50 0.536 0.589 0.561 0.554 0.555 0.578
5 50 0.532 0.572 0.534 0.562 0.560 0.560
6 50 0.540 0.573 0.563 0.560 0.551 0.563
Mean 0.535 0.577 0.557 0.560 0.557 0.568
S.D 0.004708 0.0081 0.006765 0.004320 0.005609 0.007554
%RSD 0.87 1.40 1.21 0.77 1.0 1.32
Conc.
Instrument1 Instrument 2
(µg/ml) Analyst 1 Analyst 2
0.538 0.533 0.536 0.531
1
0.9
0.8
0.7
0.6
Absorbance
ƛmax:240nm
0.5
0.4
0.3
0.2
0.1
0
0 50 100 150 200 250 300 350
Wavelength(nm)
Concentration
S. Absorbance
of Indapamide Mean S.D %RSD
No. at 240nm
(µg/ml)
1 0 0 0 0 0
0.6
0.5
y = 0.0628x + 0.008
Absorbance
0.4 R² = 0.9993
0.3
0.2
0.1
0
0 2 4 6 8 10 12
Concentration (ug/ml)
Amount
Amount Found Amount Found
S. No Taken
(µg/ml) (%w/w)
(µg/ml)
1 10 9.8 98
Amount Total
Added (µg/ml) Amount %
S. No Level of
Recovered Recovery
Recovery Test Std
(µg/ml) (w/w)
Conc.
S.No Instrument1 Instrument 2
(µg/ml) Analyst 1 Analyst 2
1 15 0.647 0.649 0.635 0.649
2 15 0.639 0.642 0.640 0.645
3 15 0.642 0.645 0.643 0.642
4 15 0.645 0.648 0.645 0.640
5 15 0.643 0.652 0.642 0.643
6 15 0.642 0.649 0.641 0.648
Mean 0.643 0.647 0.641 0.644
SD 0.00275 0.00350 0.00340 0.00350
%RSD 0.42 0.54 0.53 0.54
ƛmax=226 nm
Concentration
Absorbance
S. No. of Nebivolol Mean S.D %RSD
at 281nm
(µg/ml)
1 0 0 0 0 0
Concentration
S. Absorbance
of Indapamide Mean S.D %RSD
No. at 240nm
1 0 0 0 0 0
*n=No.of determinants
0.6
0.5
R² = 0.9998
0.3
0.2
0.1
0
0 10 20 30 40 50 60
-0.1
Concentration (ug/ml)
0.6
0.5
Absorbance
0.4
y = 0.0551x + 0.0079
0.3 R² = 0.9993
0.2
0.1
0
0 2 4 6 8 10 12
Concentration (ug/ml)
*Y=mX+C where X is the concentration of drug in µg/ml and Y is the absorbance at the respective λmax
Amount of Total
Amount of
Standard Amount % Recovery
Recovery Test Added
Drug Added Recovered (w/w)
Level (µg/ml)
(µg/ml) (µg/ml)
Conc
S.No Instrument1 Instrument 2
(µg/ml) Analyst 1 Analyst 2
1 50 0.556 0.542 0.552 0.542
Conc
S.No Instrument1 Instrument 2
(µg/ml) Analyst 1 Analyst 2
1 10 0.554 0.546 0.556 0.545
ƛmax= 226 nm
Flow Retention
Mobile phase
Trials Rate Drug Time Area
Composition
ml/min (min)
Mode of
Isocratic
Operation
Column
Ambient
temperature
Detection
226nm
wavelength
Injection
20 µl
volume
Detector UV-Detector
Linearity:
6 µg/ml of Indpamide)
18000000
Nebivolol
16000000
14000000
12000000 y = 405859x + 60888
R² = 0.9999
peak area
10000000
8000000
6000000
4000000
2000000
0
0 10 20 30 40 50 60
concentration(µg/ml)
120000000
60000000
40000000
20000000
0
0 2 4 6 8 10 12
Concentration( ug/ml)
System Suitability
Figure 28: RP-HPLC Chromatogram to Show System Suitability Parameters
Nebivolol Indapamide
S. No Conc. Conc.
Rt (min) Peak Area Rt (min) Peak Area
(µg/ml) (µg/ml)
Nebivolol(5mg) Indapamide(1.5mg)
S. No Peak Areas
1 10754216 10785421
2 10758452 10569854
3 10698545 10698542
4 10756321 10365412
5 10895622 10845962
6 10595623 10569852
SD 107500 104488
Rt Peak Area
1 2.957 11725891
2 2.921 11742783
4 2.945 11734562
5 2.929 11736555
6 2.935 11765832
SD 0.01262 1378.8
1 2.980 15516782
2 2.974 15465845
3 2.986 15626585
Analyst-2
4 2.973 15658552
5 2.979 15498555
6 2.976 15698526
SD 0.00477 15929.0
Rt Peak Area
1 4.337 91071911
2 4.329 91075623
4 4.319 91048562
5 4.320 91056562
6 4.317 91086541
SD 0.00864 136277
1 4.340 91061810
2 4.332 91056212
3 4.324 91043252
Analyst-2
4 4.328 91056852
5 4.337 91045562
6 4.319 91065321
SD 0.00792 137719
The objectives of the proposed work was to develop some new and sensitive
analytical methods for the determination of Nebivolol and to validate the methods
according to USP and ICH guidelines and applying the same for its estimation in
pharmaceutical formulations.
The optical characteristics such as absorption maxima, Beer’s law limits, molar
absorptivity, slope (b), intercept (C), correlation coefficient (r2) obtained from different
concentrations, percent relative standard deviation, LOD and LOQ values were given in
table 4 with satisfactory results. The quantitative estimation was carried out on
formulation. The quantitative results obtained were subjected to statistical analysis to
find out standard deviation and standard error values. The percentage purity of the
marketed formulation was found to be 99.86%w/w. The results were given in table 5.
The accuracy of the methods was confirmed by the recovery studies, by adding
known amount of the pure drug to the formulation and the analytical data were given in
table 6.
The % RSD values were less than 2 indicating the precision of the methodology
and low standard error values indicates the accuracy of the method. The statistical
data’s were given in tables 7 and 7.1.
Results obtained for the proposed methods confirm the suitability of these methods for
pharmaceutical dosage forms. The other active ingredients and excipients usually
present in the pharmaceutical dosage forms did not interfere in the estimation, when
commercial dosage forms were analyzed by these methods.
The objectives of the proposed work was to develop some new and sensitive
analytical methods for the determination of Indapamide and to validate the methods
according to USP and ICH guidelines and applying the same for its estimation in
pharmaceutical formulations.
The optical characteristics such as absorption maxima, Beer’s law limits, molar
absorptivity, slope (b), intercept (C), correlation coefficient (r2) obtained from different
concentrations, percent relative standard deviation, LOD and LOQ values were shown
in table 10 with satisfactory results.
The accuracy of the methods was confirmed by the recovery studies, by adding
known amount of the pure drug to the formulation and the analytical data are presented
in table 12.
The % RSD values are less than 2 indicating the precision of the methodology.
The statistical data’s were given in tables 13 and 13.1. Results obtained for the
proposed methods confirm the suitability of these methods for pharmaceutical dosage
forms. The other active ingredients and excipients usually present in the pharmaceutical
dosage forms did not interfere in the estimation, when commercial dosage forms were
analyzed by these methods.
The percentage recovery was found to be between 99.0 –99.8%w/w, shows that
the method was free from the interference of excipients used in the formulation.
The objective of the proposed work was to develop UV simultaneous method for
the determination of Nebivolol and Indapamide, and to validate the methods according
to ICH guidelines and applying the same for its estimation in marketed formulations.
This is a very simple method and requires knowledge of molar absorptivity of the
components which should be determined very accurately. It only requires measurement
of absorbance at 281 nm and 240 nm which were selected as the maximum
absorbance, the absorption curve was shown in figure 11 and few simple calculations,
which can be done manually. The percent label claim in given tablet formulation was
found to be 98.9% w/w for Nebivolol and 101% w/w for Indapamide, which were found
to be satisfactory and the results were given in table 17. %RSD were found to be below
2.
The method was validated as per ICH norms. Accurate results were obtained by
utilizing the proposed methods for the quantitation of Nebivolol and Indapamide and a
good agreement with the results obtained by the reported methods was found. For UV
spectrophotometric method, linearity was obtained in the concentration range of 10-
50µg/ml for Nebivolol and 2-10µg/ml for Indapamide and the results were given in
tables 15,15.1 and 16 with calibration curves shown in figure 12 and 13.
High % recovery greater than 98% (99-98.3% w/w for Nebivolol and 99.1-99.8%
w/w for Indapamide) showed that the method is free from the interference of excipients
used in the formulation. The results were given in table 18. The value of standard
deviation and % RSD were found to be < 2%, indicated the high precision of the
method. The results were given in tables 19, 19.1, 20 and 20.1.
Detection limit and quantitation limit were determined from the standard deviation
of y-intercepts of six calibration curves and average slope of the same. LOD and LOQ
of Nebivolol were found to be 0.25µg/ml and 1.52µg/ml at 281nm. LOD and LOQ of
The objective of this study was to develop a rapid and sensitive RP-HPLC
method for the analysis of Nebivolol and Indapamide in bulk drug and pharmaceutical
dosage form by using the most commonly employed C-18 column with UV-detection.
Initially, various mobile phase compositions were tried to elute the drug. Mobile
phase ratio and flow rate were selected based on peak parameters (height, capacity,
theoretical plates, tailing or symmetry factor), run time and resolution. The system with
phosphate buffer (PH3.0): Acetonitile (40: 60 v/v) and 1ml / min flow rate was selected.
The optimum wavelength selected was 226 nm from the overlain spectra
obtained which was shown in figure 14 at which better detector response for the drug
was obtained. The retention time for Nebivolol and Indapamide was found to be 2.9 min
and 4.2 min respectively which were shown in figure 18 and19. The linearity was
observed in concentration range of 10-50 µg/ ml for Nebivolol and 2-10 µg/ ml for
Indapamide.Calibration curves of the respective drugs were shown in figure 25, 26and
results were given in tables 25 and 26.
Analytical testing is one of the more interesting ways for scientists to take part in
quality process by providing actual data on the identity, content and purity of the drug
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.
In the present work, an attempt was made to provide a newer, simple, accurate
and low cost spectrophotometric and HPLC methods for the effective quantitative
determination of Nebivolol and Indapamide as an active pharmaceutical ingredients as
well as in pharmaceutical preparations in their single and combined dosage forms,
without the interferences of other constituent in combined formulations. Hence it is
planned to develop both HPLC and Spectrophotometric methods. The results were
summarized as follows.
Results
Parameter
Nebivolol Indapamide
λ max(nm) 281 240
Beer’s Law Range (µg/ml) 10-50 2-10
Regression Equation Y=0.0111x+0.065 Y=0.0628x+0.0008
Correlation Coefficient (r2) 0.9994 0.9994
Molar Extinction
114.8 256.6
Coefficient (lit.mol-1cm-1)
Sandelle’s Sensitivity (µg.
0.0154 0.0130
cm-2/0.001 abs units)
% Recovery (w/w) 99.0-99.8 99.5-99.8
LOD (µg/ml) 0.25 0.43
LOQ (µg/ml) 1.52 0.72
Assay (% Purity) w/w 99.86 101.3
At At
Precision (%RSD) At room At room
Refrigerating Refrigerating
temperature temperature
condition condition
Ruggedness (%RSD)
Analyst 1 0.35 0.53
Analyst 2 0.77 0.54
Instrument 1 0.40 0.42
Instrument 2 0.45 0.54
Results
Parameter
Nebivolol Indapamide
λ max(nm) 281 240
Beer’s Law Range (µg/ml) 10-50 2-10
Regression Equation Y=0.0117x+0.0031 Y=0.051x+0.0079
Correlation Coefficient (r2) 0.9998 0.9997
Molar Extinction Coefficient
560 673.9
(lit.mol-1cm-1)
Sandell’s Sensitivity (µg.
0.01222 0.01920
cm-2/0.001 abs units)
% Recovery (w/w) 99.-98.3 99.1-99.8
LOD (µg/ml) 0.41 0.26
LOQ (µg/ml) 1.25 2.32
Assay (% Purity) w/w 98.9 101.3
At At
Precision (%RSD) At room At room
Refrigerating Refrigerating
temperature temperature
condition condition
Ruggedness (%RSD)
Analyst 1 0.54 0.49
Analyst 2 0.46 0.25
Instrument 1 0.48 0.46
Instrument 2 0.60 0.44
Results
Parameter
Nebivolol Indapamide
Detection Wavelength(nm) 226
Rt (min) 2.9 4.2
Beer’s Law Range (µg/ml) 10-50 10-50
Regression Equation Y=405059x+60888 Y=1E+07X+278611
Correlation Coefficient (r2) 0.9999 0.9992
% Recovery (w/w) 99.8-100.0 98.6-99.4
LOD (µg/ml) 0.158 0.342
LOQ (µg/ml) 0.92 0.72
Assay (% purity) w/w 99.6 99.3
Precision (%RSD)
Intraday Precision 0.58 0.77
Robustness (%RSD)
Flow Rate 0.8ml/min 0.58 0.70
Flow Rate 1.2ml/min 0.604 1.41
Detection Wavelength at
0.60 0.18
228nm
Detection Wavelength at
0.32 0.75
224nm
Ruggedness (%RSD)
Analyst 1 0.10 0.61
Analyst 2 0.12 0.22
Development of methods to achieve the final goal of ensuring the quantity of drug
substances and drug products is not a trivial undertaking. The capabilities of the
methods developed were complementary to each other. Hence they can be regarded as
simple, specific and sensitive methods for the estimation of Nebivolol and Indapamide in
single and combined pharmaceutical dosage forms. The developed UV
Spectrophotometric methods and RP-HPLC methods were validated according to ICH
guidelines and were found to be applicable for the routine analysis of Nebivolol and
Indapamide in their single and combined dosage forms.
The proposed UV methods were simple, sensitive and reliable with good
precision and accuracy. This method was specific while estimating the commercial
formulations without interference of excipients and other additives. Hence, this method
can be used for the estimation of Nebivolol and Indapamide in bulk samples and their
pharmaceutical formulations individually and in combination by simultaneous estimation
method.
The developed and validated RP-HPLC method was found to be economical due to the
use of water as a solvent in mobile phase. The low solvent consumption (1mL/min),
along with short analytical run time of 12.0 minutes lead to an environmental friendly
chromatographic procedure that allows the analysis of a large number of samples in a
short period of time. This method has been found to be better than previously reported
methods, due to its wider range of linearity, use of readily available mobile phase, lack
of extraction procedures. Hence above method can be used in quality control for routine
analysis of finished products of Nebivolol and Indapamide simultaneously without any
interference.
The RP-HPLC method developed and validated allows a simple and fast quantitative
determination of Nebivolol and Indapamide from their formulations. All the validation
parameters were found to be within the limits according to ICH guidelines. The
proposed method was found to be specific for the drugs of interest irrespective of the
excipients present and the method was found to be simple, accurate, precise, rugged,
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