Title of the Assignment:

“A review and comparison of RP-HPLC method with

other validated methods for the determination
of Fexofenadine with Pseudoephedrine in binary
pharmaceutical dosage forms”

Submitted by:
Student Id:
ARAVIND GURRAM 20046047
HASAN ALDEWACHI 20037207
MUHAMMD TARIQUL ISLAM 20040698
OZLEM KARAER 20027941

Academic Year: 2010/11

MSc Pharmaceutical Analysis

Module Tutor:
Dr. P H E Gardiner

Faculty of Health and Wellbeing

Department of Biosciences
Sheffield Hallam University
 TABLE OF CONTENTS
INTRODUCTION 1
EXPERIMENTAL 4
Materials and reagents 4
Instrumentation 4
Chromatographic conditions 5
Standard stock solutions and construction of calibration curves 5
Sample preparation 5
Optimization of the chromatographic condition 6
VALIDATION OF THE METHOD 6
System suitability 6
Specificity 7
Linearity 9
Limit of Detection (LOD) and Limit of Quantification (LOQ) 9
Precision 10
Accuracy 12
Application of the validated method in pharmaceutical dosage forms 12
CONCLUSION 12
REFERENCE 13

TABLE OF TABLES
Table 1 : System suitability results of the proposed method 7
Table 2: PSE and FEX calibration plots (mean of six injections) 9
Table 3: Summary of intra-day (repeatability) and inter-day Intermediate
precision) data 11
Table 4: Statistical analysis of assay results and recovery experiment in
commercial drug samples 11
TABLE OF FIGURES
Figure 1: Chemical Structure of the Fexofenadine 1
Figure 2: Chemical Structure of the Pseudoephedrine HCl 1
Figure 3: HPLC profile of Fexofenadine under stress conditions 8
INTRODUCTION
The analysis of multi-component mixtures without separation of the constituents
is rather a difficult task. Different methods have been used for the analysis of
binary mixtures as fexofenadine-pseudoephedrine (Allegra-D-) in tablets or
capsules.
Fexofenadine (FEX) (Figure 4) is a non-cardiotoxic and non-sedative terfenadine
metabolite, which acts as a selective second generation histamine H1 receptor
antagonist, relieving the uncomfortable manifestations of rhinitis. On the other
hand, Pseudoephedrine (PSE) is an adrenoreceptor agonist, useful for relieving
nasal congestion (Simpson and Jarvis, 2000). PSE (Figure 5) is a
sympathomimetic agent, structurally similar to ephedrine, used to relieve nasal
and sinus congestion and reduce air travel related otalgia in adults (Bharathi et
al., 2008)

Figure 4: Chemical Structure of the Fexofenadine

Figure 5: Chemical Structure of the Pseudoephedrine HCl

The pharmacological combination (1:2 w/w) between FEX and PSE is important
alternative to the less safe terfenadine-pseudoephedrine mixture; this
combination has been recently approved as a once-a-day formulation, and is
now widely used to relieve symptoms of allergic conditions, especially seasonal
allergic rhinitis (Mansfield, 2006).

A great interest has been paid in the last period to look for validated methods for
multi-component pharmaceutical dosage forms which contain binary, tertiary
mixtures to develop accurate, precise and reproducible methods with focusing
on the features of time saving ,simplicity and cost effectiveness as most
important factors for validation. In this essay, different methods of validating
pharmaceutical dosage forms containing FEX and PSE have been discussed to
reach to most suitable method for validating such a mixture. Many different
methods has been validated for formulations that contain both FEX and PSE and
these methods are the derivative spectrum ratio method which has been
developed by Mahgoub et al., (2002), the Ultra-Violet Partial Least Square (UV-
PLS) method which is developed by Maggio et al., (2007) and the RP-HPLC
method which is developed by Karakuş et al., (2008).

A derivative spectrophotometry method is based on the use of first derivative of

the ratio spectrum which is obtained by dividing the absorption spectrum of the
mixture by that of one of the components and it has found great application in
pharmaceutical, clinical, biochemical analysis Karpinska (2004), although of this
great importance of this method in the analysis of multi-component samples it
need high resolution for the quantification of the analytes and is not simultaneous
as it should be carried out separately which is one of the most important
limitation to derivative ratio spectrum method that is attributed to the fact that
absorbance ability of FEX obeys Beer’s law in the pharmacological dosage form,
at lower concentrations where PSE is poorly absorbing so cannot be quantified
with acceptable accuracy and precision. On the contrary, more concentrated
samples allow proper quantification of PSE (Maggio et al., 2007). In addition to
that it has many disadvantages:
- If the spectra are strongly overlapped , the derivative technique may not
be able to cope with the level of interference (Mahgoub et al., 2002)
- Low reproducibility attributed to dependence of derivative
spectrophotometry on instrumental parameters (Karpi´nska, 2004).
- Non robust parameter of the parameters selected for the elaborated
method (Karpi´nska, 2004).

Maggio et al., (2007) has conducted alternative method which rely on chemo-
metric evaluation of spectral data of the analytes in a selected UV region in their
pharmaceutical forms for determination of FEX and PSE in their combined tablet
formulations, and the method represents an "improvement over the first
derivative of spectral ratio (DSR) technique and allows high sample throughput
with minimum reagent consumption and waste generation". Also it achieves
simultaneous determination of FEX and PSE in the commercial tablet but even
though this method does not produce results that are equivalent statistically to
those obtained by HPLC and the reason for that non equivalence is the non
modelled compounds in the tablet that may act as interferents (Maggio et al.,
2007).

Another validated method for this binary mixture is the reverse phase HPLC
method which depends on chromatographic separation of FEX and PSE using
cetrizine (CET) as internal standard. This proposed method has many of the
important features that are required in methods to be validated, like
- Time saving (short analysis time < 5 minutes) ,
- Appropriate limit of detection (LOD) 0.75 Mg/ml for PSE and 0.27 Mg/ml
for FEX.
- Precision of the method is less than 1% in all instances.
- No interference from any components of pharmaceutical dosage forms or
degradation products was observed.
 - Provide linear response within the concentration range 10-80 mg/ml for
PSE and 5–40 Mg/ml for FEX (Karakuş et al., 2008).

Using CET (for PSE-FEX binary mixtures) as internal standard for the
quantitative determination, reduce the possible analytical errors due to the
sample dilution and injection procedures and improved the sensitivity of the
method (Karakuş et al., 2008).
As a result the proposed method can be considered a specific, accurate, precise
in capsule or tablets. There are also other validated methods for determination of
FEX in pharmaceutical dosage form but these methods are fit only for FEX like
capillary electrophoresis methods for the determination of FEX in capsules
(Breier et al., 2005) or tablets (Mikus et al., 2005) as well as anodic voltammetry
method for determination of FEX (Golcu et al., 2005) in addition FEX can be
quantified by ion complex reaction (Amin et al., 2010) also The combined FEX-
PSE formulation has been studied in plasma samples by HPLC with electro-
spray ionization and tandem mass spectrometry (Bharathi et al., 2008). But this
essay is focusing on this combination in pharmaceutical forms only so that the
latter method will not be discussed here.

EXPERIMENTAL
Materials and reagents
Pseudoephedrine hydrochloride (PSE) and Fexofenadine hydrochloride (FEX) is
the key component of this experiment. Gradient grade of methanol and
acetonitrile were used. Analytical grade of triethylamine (TEA) and
orthophosphoric acid (85%) also used. Mobile phase and other solutions were
prepared by distilled water. The experiment also includes the following
pharmaceutical dosage forms: 60 mg of FEX and 120 mg of PSE present in
Allegra-D® (12h extended release tablets).
Instrumentation
Instrument equipped with a quaternary solvent delivery system and a photodiode
array detector was contained in the liquid chromatographic system. A 20 µl
sample loop of Rheodyne syringe loading sample injector was used for the
injection of analytes. At ambient temperature, on a reversed phase Zorbax
Eclipse XDB-C8 column (150mm x 4.6 mm; 5µm particle size) separation was
performed. Zorbax C8 analytical guard column (12.5mm x 4.6 mm) packed with
the same sorbent was used. The isocratic modes were employed in all the
experiment

Chromatographic conditions
Various proportions of methanol, acetonitrile and triethylamine (TEA) solution
(0.5%) were tried as a mobile phase. Orthophosphoric acid is used for the
adjustment of the desired pH (3.0-5.0) of TEA solution (0.5%), before mixing with
organic solutions. TEA solution (0.5%, pH 4.5)-methanol-acetonitrile (50:20:30,
v/v/v) were found to be optimum mobile phase which was prepared by filtering
through a 0.45 µm membrane and degassed by ultrasonification. A flow rate of
1.5 ml/min was used for the solvent release. A wavelength of 218 nm was
chosen for the detection of analytes.

Standard stock solutions and construction of calibration curves

Standard calibration solutions were considered at seven different levels which
were 25%, 50%, 75%, 100%, 125%, 150% and 200% of the test concentration. A
constant concentration of 20µg/ml of CET (internal standard) was used in
preparing the binary mixture of FEX (5-40 µg/ml) and PSE (10-80 µg/ml) in the
mobile phase. For each concentration six replicate injections were made. The
peak area ratios of the drug to that of internal standard, against the drug
concentration were used to plot for construction of the calibration curves of FEX-
PSE binary mixtures.
Sample preparations
The finely divided powder of accurately weighed, determined mean weight of the
twenty capsules or tablets was taken. To a 50ml volumetric flask an amount of
one capsule or tablet content was transferred, 40ml of methanol was added and
soniacated for 30min, with methanol it is diluted to 50ml and a solution of 10ml
from this is centrifuged for 15min at 3000rpm.
A 1 ml aliquot from supernatant was then decanted to another 10 ml volumetric
flask. Fixed concentrations of FEX 5 µg/ml and PSE 20 µg/ml were obtained by
adding the appropriate amounts of internal standard.

Optimization of the chromatographic conditions

Three columns (Zorbax C8 5 µm, 150 mm x 4.6 mm; Kromasil C18 5 µm, 250
mm x 4.6 mm; Symmetry C18 5 µm, 150 mm x 4.6 mm), two organic solvents
(acetonitrile and methanol) and five different pH values (3.0-5.0) with and without
ion pairing agent (hexane sulphonate) were tested during the optimization of the
separation method. Hexane sulphonate was tried to overcome the weak retention
of PSE but very late elution or no peaks for FEX was observed. As a result of pH
screening, the optimum mobile phase was chosen as TEA solution (0.5%, pH
4.5)-methanol-acetonitrile (50:20:30, v/v/v). The flow rate of 1.5 ml/min and a
wavelength of 218 nm were found as optimum mobile phase flow rate and
detection wavelength.

VALIDATION OF THE METHOD:

According to Ermer (2005), "Method validation is the process of confirmation that
an analytical procedure is fit for is intended use i.e. for addressing analytical
problem. It is also the process of fixing the performance parameters and
limitations of a method and determination of effects which might alter these
parameters and to what extend". The method which has been selected is broadly
validated in respect of specificity, linearity, limit of detection and limit of
quantification, accuracy, precision, and system suitability. The recent validated
method which is developed by Karakuş et al (2008) has been used in the
analysis of pharmaceutical dosage forms including FEX and PSE where CET is
used as an internal standard.

System suitability:
The purpose of the system suitability test is to ensure that the performance of the
analytical system is suitable for the purpose of use in terms of instrument,
reagents, columns, analysts. The USP chromatography general chapter states:
"System suitability tests are an integral part of gas and liquid chromatographic
methods. They are applied to confirm that the chromatographic system is
extremely reproducible and the peaks of interest are resolved very well. The tests
are conducted depending on the idea that the equipment, electronics, analytical
operations and samples to be analyzed comprise an integral system that can be
assessed as such." In that method validation the system suitability test was
performed by repeating injections from fresh standard solutions six times and
analyzing FEX for its peak area, theoretical plates and tailing factors. The system
suitability criteria are the following:
• Relative standard deviation of peak areas
• Retention times, tR (<1%)
• Peak resolution, R (>2) between FEX and PSE
• Number of theoretical plate, N (≥2000) for FEX and PSE
• USP tailing factors, T (<1.5)

Table 5: System suitability results of the proposed method (Karakuş, S., et al 2008,
p298)
Compound N R T Relative Standard Deviation

tR peak area
PSE 2434 - 1.13 0.24 0.70
FEX 4896 12.53 1.21 0.13 0.21
Required N≥2000 R>2 T<1.5 R.S.D<1%
limits
According to result shown in , the developed method executes the criterions
mentioned above within the acceptable limits.

Specificity:
Ermer (2005) stated that, the specificity is the capability of method to estimate
accurately the analyte in the constituents such as impurities, degradants,
reagents, and enantiomers. In this method validation the specificity of the method
has been confirms in three ways. These are:
• Recovery studies with standard addition have verifies that the
interferences from ingredients in the formulation have no effect on the
proposed method.
• Photodiode array detection of the peaks of interest obtained from the
recovery experiment at seven different points supported the idea that the
peaks are uniform. This is the evidence of no co-eluting peaks.
• Accelerated Degradation studies by using acid, base, heat, UV (254nm)
and direct day light are carried out to grant evidence for the specificity of
the proposed method. 3.4% degradation of PSE was observed under
acidic stressed conditions (0.5N HCl at 80°C) while there is no
degradation under basic (0.5N NaOH at 80°C). More than 10%
degradation occurred when FEX was exposed to acidic or basic stressed
condition. Moreover, there is no significant thermal or photo degradation
observed for both PSE and FEX well resolved degradant peaks (Figure )
are another evidence of specificity of the method.
Figure 3: HPLC profile of Fexofenadine under stress conditions, (A) pure FEX
sample; (B) FEX sample in acidic cnditio;n ; (C) FEX sample in basic condition.
(Karakuş, S., et al 2008, p299)

Linearity:
The linearity of analytical method is the ability to elicit measurement results such
as absorbance that are directly proportional to the concentration of analyte in the
sample within the given range. The linearity data can be procured by preparing
the stock solution of the analyte and making serial dilutions to get necessary
sample with desired concentration. In this method validation the linearity data
were attained by analyzing seven different concentrations of FEX and PSE within
the range 5.0-40.0 µg/ml and 10.0-80.0 µg/ml respectively while retaining the
concentration of internal standard (CET) at a value of 20µg/ml. The injections for
each concentration were triplicated. The linearity of the calibration curves was
observed on two different days for repeatability and intermediate precision. A
linear curve was obtained by plotting ratio of the peak area of FEX or PSE to
peak area of internal standard vs. concentration within the given ranges
mentioned above. A linear simple regression was practiced by using least square
method see .

Table 6: PSE and FEX calibration plots (mean of six injections) (Karakuş, S., et al
2008, p300)
PSE FEX
Linearity range (µg/ml) 10-80 5-40
slope 0.0293 0.0776
Intercept -0.0086 -0.0195
Correlation coefficient ® 0.9998 0.9999
R.S.D. % of slope 0.41 0.39
R.S.D. % of intercept 0.74 0.96
Limit of detection (µg/ml) 0.75 0.27
Limit of quantification (µg/ml) 2.26 0.83

Limit of Detection (LOD) and Limit of Quantification (LOQ):

Limit of detection can be defined as the lowest concentration of analyte in a
sample which can be detected but not necessarily measured as an accurate
amount under the stipulated measurement condition and quantification of
analytical method is the smallest concentration of analyte in a sample which can
be quantified with an acceptable precision, accuracy and linearity under the
stipulated condition. These limits can be obtained in three ways,
• Visual evaluation

• Signal-to-noise ration

• The use of standard deviation of the response and the slope of calibration
curve

In this method validation study, the third approach was applied according
following equations:
The values of limit of detection and limit of quantification of the proposed method
are given in .

Precision:
According to Ermer (2005), "The precision of an analytical procedure can be
defined as the closeness of agreement between a series of independent
measurements results obtained from several sampling from the identical
homogenous sample under the stipulated condition". And he also stated that,
precision is the degree of the scatter of repeated test result and based on the
dispersion of random errors. Therefore it is not an indication of closeness of
those test result of true value. In this method validation the precision of proposed
method was evaluated in terms of repeatability (intra-day) and intermediate
precision (inter-day). The analysis of FEX and PSE samples were replicated five
times at low, medium and high concentrations. To assess the intermediate
precision the experiment was carried out for two weeks. According to Table 7, for
the repeatability precision the %R.S.D. values of measurement is between 0.27%
and 0.71%. For intermediate precision the %R.S.D. of measurement result does
not exit 0.80%. These are the indication of good precision of the proposed
method.
Table 7: Summary of intra-day (repeatability) and inter-day (Intermediate
precision) data (Karakuş, S., et al 2008, p300)
Compound Theoretical Intra-day measured Inter-day measured
concentration concentration (µg/ml)a concentration (µg/ml)b
(µg/ml)
Mean R.S.D % Mean R.S.D %
PSE 12 11.91 0.27 11.89 0.76
40 39.73 0.60 39.69 0.49
72 71.91 0.71 72.88 0.35
FEX 6 6.06 0.54 5.99 0.77
20 20.00 0.50 19.99 0.80
36 35.61 0.69 35.56 0.41
a
Mean values was obtained from five different sample standard for each concentration
b
Inter-day precision was calculated from five different runs over 2 week period.
Table 8: Statistical analysis of assay results and recovery experiment in
commercial drug samples (Karakuş, S., et al 2008, p300)
Allegra-D® 12h extended release
tablets
PSE FEX
Analysis of pharmaceutical dosage forms
Label claim (mg) 120 60
Mean of the amount found (mg) a 119.22 58.87
confidence limit b ±1.07 ±0.22
recovery % 99.35 98.11
R.S.D. % 0.86 0.35
Recovery analysis using standard addition
method
Added (mg) 30 15
Mean of the amount found (mg) a 30.29 14.85
confidence limit b ±0.80 ±0.08
recovery % 100.98 98.97
R.S.D. % 0.55 0.54
a b
Mean values were obtained from six replicates Calculated value for 95% confidence level
Accuracy:
According to Chitlange (2007), Accuracy shows how close a measurement result
is to the accepted value so it covers the influence both precision (random errors)
and bias (common systematic error). Accuracy can be validated by recovery
studies in which known amounts of an analyte are spiked into a solution including
excipients, degradants, and reagents. The acquired amount of the analyte can be
then compared with the actual spike amount. This can be estimated as
percentage recovery. In this method validation known amounts of FEX and PSE
on to known concentration of commercial tablets (Allegra-D®). For PSE 100.98%
recovery with a R.S.D. value of 0.55% was found in Allegra-D® tablets whereas
98.97% recovery for FEX with R.S.D. value of 0.54% (see Table 8). These high
percentages are indication of accuracy of the proposed method. So for
quantitative and routine analysis of the PSE and FEX this method can be applied
in commercial drug sample.

Application of the validated method in pharmaceutical dosage forms:

The propose method was used in determination of FEX and PSE in extended
release tablet Allegra-D®. It can be seen from the Table 8 that there is only slight
difference between the claimed amounts (mg) and mean of the amount found
(mg) with acceptable R.S.D. values given by the pharmacopoeias.

CONCLUSION:

When the developed HPLC method is compared with mentioned method in this
study it has advantages of simplicity, reliability, repeatability, sensitivity, short
analysis time and cheap reagents. The use of cetrizine as internal standard
lowers the possible analytical errors which are brought by the sample dilution and
injection steps. As a result the proposed method can be used to carry out
quantitative analysis of binary mixture of PSE and FEX.

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BHARATHI, V. D., et al. (2008). LC–MS–MS assay for simultaneous

quantification of fexofenadine and pseudoephedrine in human plasma.
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