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 20046047HASAN ALDEWACHI 20037207MUHAMMD TARIQUL ISLAM 20040698OZLEM KARAER 20027941

Academic Year: 2010/11MSc Pharmaceutical Analysis

Module Tutor:Dr. P H E Gardiner

Faculty of Health and WellbeingDepartment 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

limits

N≥2000 R>2 T<1.5 R.S.D<1%

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

concentration

(µg/ml)

Intra-day measured

concentration (µg/ml)a

Inter-day measured

concentration (µg/ml)b

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 0.41a Mean values was obtained from five different sample standard for each concentrationb 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 0.54a Mean values were obtained from six replicates b 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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