1

The National Ribat University

Faculty of Graduate Studies and Scientific Research

Simultaneous Determination of Loratadine and

Pseudoephedrine Sulphate in Tablets by Absorption

Correction UV Spectrophotometry

A Thesis Submitted in Partial Fulfillment of the Requirements for

Master Degree in Drug Quality Control

By: Abrar Ahmed Ismail Ahmed

Supervisor: Dr. Imad Osman Abu Reid

2017

2

DEDICATION

This work is dedicated to my loving parents, my sisters,

my brothers and to Isra Jamal.

I

Table of contents

Content Page No.

Table of contents I

Acknowledgement II

Abbreviations III

English Abstract IV

Arabic Abstract V

List of figures VI

List of tables VII

Chapter One

Introduction 1

Theoretical background 3

Objectives 4

Literature review 5

Chapter Two

Materials 8

Instrumental and Apparatus 8

Reagents and Solutions 8

Selection of λ max 9

Sample preparation 9

Chapter Three

Results 11

Selection of analysis wavelength 11

Linearity 12

Analysis of synthetic mixtures 15

Sample 18

Chapter Four

Conclusion 21

Reference 22

II

ACKNOWLEDGEMENTS

Foremost, I am highly grateful to AlmightyAllah for His blessing that

continue to flow into my life, and because of You, I made this through

against all odds.

With a great pleasure I would like to acknowledge at FirstMy supervisor,

Dr. Imad Abureid for the access and assistance in order to make my

fieldwork possible and especially for this confidence in me

I would also like to thank Dr. Ahmed Elsayed and Mohammed Al-Na'im

for their help.

III

Abbreviations

Molar absorptivity coefficient

A Absorbance

Am Absorbance of synthetic mixtures

B Path length

C Analyte concentration

FIA Flow Injection Analysis

HCl Hydrochloric acid

HPLC High Performance Liquid Chromatography

LOR Loratadine

PCR Principal Component Regression

PLS Partial least squares

PS Pseudoephedrine sulphate

RSD % Relative standard deviation %

SD Standard deviation

λ Wavelength

IV

ABSTRACT

UV Spectrophotometric absorbance correction method has been

developed for simultaneous estimation of Loratadine and

Pseudoephedrine sulphate in combined tablet dosage form.

The method was based on the absorbance correction equations for

analysis of both drugs using 0.1M HCl as solvent. Pseudoephedrine

sulphate has absorbance maxima at 256.6 nm and Loratadine has

absorbance maxima at 280 nm in 0.1M HCl it also shows absorbance at

256.6 nm. Since LOR is absorbing at 280 nm it was possible to detecting

its concentration directly from Beer - lambert law and also its

contribution the absorbance at 256.6 nm.

The proposed method was found to be accurate, the percent

concentration of the two actives in laboratory synthetic mixtures were

99.29% and 104.38% with relative standard deviation of 0.64% and

0.87% for PS and LOR respectively.

V

ملخـــص

نسبة اللوراتادين والسودوافيدرين في عينة الدواء هذه الطريقة لتساهم في تقدير تم تطوير التي تحتوي على المادتين معا.

بتحليل المادتين باستخدام حمض على معادلة تصحيح اإلمتصاص هذه الطريقة تعتمدالهيدروكلوريك كمادة مذيبة. اقصى امتصاصية للسودوافيدرين عند الطول الموجي

نانو متر 280نانومتر واقصى امتصاصية لللوراتادين على طول موجي مداه 256,6 وذلك لتحديد تركيز كل منهم تبعا لقانون بيرالمبار.

ن نسبة تركيز أمحاليل معده معمليا ووجد تم اختبار ثبتت الطريقة المقترحة دقتهاعندماأو 0.87و 0.64 وانحراف معياري نسبي %104.38و %99.29كل من المادتين

للسودوافيدرين واللوراتادين على التوالي.

VI

List of tables

Table No. Description Page No.

Table 1 Calibration data of pseudoephedrine sulfate at

256.6 nm

12

Table 2 Calibration data of loratadine at 256.6 nm 13

Table 3 Absorbance data of loratadine at 280 nm 14

Table 4 Absorbance data of synthetic mixtures at

(256.6 and 280 nm)

16

Table 5 Assay result of synthetic mixtures( Accuracy) 17

Table 6 Absorbance data of sample at (256.6 nm and

280 nm)

18

Table 7 Assay resultof commercial sample

19

VII

List of figures

Fig No. Description Page No

Figure 1 Chemical structure of Loratadine 5

Figure 2 Chemical structure of Pseudoephedrine 5

Figure 3 Overlain spectra of LOR and PS 11

Figure 4 Calibration graph of pseudoephedrine

sulphate at 256.6 nm

12

Figure 5 Calibration graph of loratadine at 256.6 nm

13

Figure 6 Calibration graph of loratadine at 280 nm

14

1

1.1 Introduction

Analysis of samples with numerous components presents a major

challenge in modern analysis[1]. Multicomponent analysis has become

one of the most appealing topics for analytical chemists in the last years,

in fields as clinical chemistry, drug analysis, pollution control,…. etc.[2].

Different analytical techniques can be applied for multicomponent

analysis including; spectrophotometry, chromatography, and

electrophoresis and UV Spectrophotometric absorbance correction

method simultaneous determinations of drugs. Because most analytes of

interest are accompanied in their dosage forms by other compounds

absorbing in the same spectral region, classical UV spectral

measurements could not be used for their determination [3]. The use of

traditional methods like extraction is quite difficult with accompanying

risks of analyte loss or contamination, expensive, and time consuming

[1]. UV spectrophotometric techniques are mainly used for

multicomponent analysis thus minimizing the cumbersome task of

separating interferons and allowing the determination of an increasing

number of analytes, consequently reducing analysis time and cost [4].

Multicomponent UV spectrophotometric methods are based on recording

and mathematically processing absorption spectra. They offer the

following advantages: [5] avoiding prior separation techniques e.g.

extraction, concentration of constituents, and cleanup steps that might be

required; spectral data are readily acquired with ease; the process is fast,

accurate, and simple; wide applicability to both organic and inorganic

systems; typical detection limits of 10-4 to 10-5 M and moderate to high

selectivity.

2

Combination drug products occupy a time-honored and important role in

therapeutics. When rationally formulated, fixed-combination drugs may

produce greater convenience, lower cost, and sometimes greater efficacy

and safety [6].

3

1.2 Theoretical background

When two analytes exist as a mixture, if the concentration of one of them

and its absorptivity at a wavelength free from interference by the other is

known, it is possible to calculate its contribution to the total absorbance

of the mixture at any other wavelength where both analytes absorb. The

concentration of the second analyte is then calculated from the corrected

absorbance (total absorbance at the wavelength of interference minus the

absorbance of the first analyte at this wavelength) [7].

4

1.3 Objectives

The objective of this research was:

1. To investigate the possibility of the application of absorbance

correction spectrophotometric method for the simultaneous

determination of loratadine and pseudoephedrine in combination.

2. To apply the method to estimate the concentrations of loratadine and

pseudoephedrine sulfate combined in tablet formulation.

5

1.4 Literature review

Loratadine (LOR) is ethyl 4-(8-chloro-5, 6-dihydro-11-H-benzo-[5, 6]

cyclohepta [1, 2-b] pyridine-11-ylidene)-1-piperidine-carboxylate

(Figure 1). It is a long-acting, non-sedative second generation H1

receptor blocker with no significant antimuscarinic activity. It is used for

the symptomatic relief of allergic conditions including rhinitis and

chronic urticaria [8].

Figure 1: Chemical structure of Loratadine

Pseudoephedrine sulphate (PS) is (1S, 2S)-2-(Methyl-amino)-1-

phenylpropan-1ol-sulfate [8] (Figure 2) is a direct and indirect

sympathomimetic. It is a stereoisomer of ephedrine and has a similar

action, but has been stated to have less pressor activity and fewer CNS

effects.

Figure 2: Chemical structure of Pseudoephedrine

Pseudoephedrine and its salts are given orally from symptomatic relief of

nasal congestion. It is commonly combined with other ingredients in

preparations intended for the relief of cough and cold symptoms [8].

6

Loratadine and pseudoephedrine sulphate are present together in dosage

form prescribed to relieve symptoms of allergic rhinitis [8].

Different analytical procedures have been reported for the simultaneous

determination of the two drugs in combinations:

A reversed phase liquid chromatographic and first derivative

spectrophotometric methods have been described for the determination

of LOR and PS. The HPLC method involved separation of the two

compound on µ-BondaPak C18 column. The calibration graphs are

linear in the range of 5-25 µg/ml for LOR and 240-720 µg/ml for PS; the

limits of detection are 0.16 mg/ml for LOR and 10 mg/ml for PS. The

mean percentage recoveries obtained for different synthetic mixture by

using this method are 97.6% with coefficient of variation 1.79 for LOR

and 101.6%with coefficient of variation 1.95 for PS [9].

Two UV spectrophotometric and one HPLC method have been

developed for simultaneous estimation of two-component drug mixture

of PS and LOR in combination tablet dosage form [10]. The first

developed method employs multi-wavelength spectroscopy using seven

mixed standards and 257.0 nm and 283.0 nm as two wavelengths for

estimation. The second method involves first derivative spectroscopy

using 308.6 nm and 263.0 nm as zero crossing point PS and LOR

respectively. The HPLC method was a reverse-phase chromatographic

method using Inertsil C18 column using Nimesulide was used as internal

stander. For HPLC method, linearity was observed in concentration

range of 0-200 mg/ml of loratadine and 100-2000 mg/ml of

pseudoephedrine hydrochloride.

7

LOR and PS were determined in pharmaceutical samples using non-

linear second-order data generated by a PH-gradient flow injection

analysis (FIA) system with diode-array detection. Determination of both

analytes was performed on the basis of differences between the acid-base

and spectral features of each drug species. Recoveries of 99.7%LOR and

95.6%PS were obtained [11].

Two chemometric method [12], principalcomponent regression (PCR)

andpartial least squares (PLS), were realized for the simultaneous

determination of PS and LOR in their combination. In the methods, the

concentration data matrix was prepared using the synthetic mixtures

containing these drugs in 0.1 M HCl. The absorbance datamatrix

corresponding to the concentration data matrix was obtained by the

measurements of absorbances at 18 wavelengths in the range 250 - 284

nm as Δ λ = 2 nm in principal component regression technique and at 18

wavelengths in the range 254 – 288 nm as Δ λ = 2 nm in partial least

squares technique in their zero order (original) spectra. Then, calibration

was obtained using the absorbance data matrix and concentration data

matrix for the prediction of concentration of pseudoephedrine sulphate

and loratadine in their binary mixture. The procedures do not require any

separation steps. Working range were found as 200.0 – 1200.0 µg/ml for

pseudoephedrine sulphate and 4.0 – 40.0 µg/ml for loratadine in both

methods.

Separation and quantification of LOR and PS was achieved utilizing a

combination of columns C18 (150 x 4.6 mm, 5 µm) followed by Tracer

Extrasil CN (15 x 4.6, 5 µm). The average recovery (n=5) was estimated

to be 99.8 % (RSD=1.21) and 99.5% (RSD=1.39) for LOR and PS,

respectively [13].

8

2.1 Material

Loratadine working standard was obtained as gift sample from

(Aabdalmonaem Pharmaceutical Industry-Khartoum-Sudan).

Pseudoephedrine sulphate working standard was obtained from

(SPIMC - Kingdom of Saudi Arabia).

Clarinase Tablets (Schering-Plough-Belgium) labeled to contain 5mg

Loratadine and 120 mg pseudoephedrine sulphate per tablet,

purchased from Saudi Arabia.

Concentrated Hydrochloric acid analytical grade (SDFCL -Mumbia,

India)

2.2 Instrument and apparatus

UV spectrophotometer UV 1800 (SHMADZU - Japan) double beam.

2.3 Reagents and solutions

2.3.1 Hydrochloric acid diluent (0.1 M)

8.5ml of concentrated hydrochloric acid were diluted to1000ml with

distilled water.

2.3.2 Loratadine standard stock solution

15 mg Loratadine working standard were accurately weighed, transferred

into 100 ml volumetric flask, dissolved and the volume was completed

with 0.1 M HCl (39*10-4 mole/L).

9

2.3.3 Pseudoephedrine standard stock solution

180 mg Pseudoephedrine sulphate working standard were accurately

weighed, transferred into 50ml volumetric flask, dissolved and the

volume was completed with 0.1M HCl (84*10-2 mole/L).

2.4 Selection of λ max

Five ml from each stock solution were transferred to separate 25 ml

volumetric flasks, each flask was then made to volume with 0.1 M HCl.

The resulted solution were scanned between 200-300 nm ranges. The

suitable wavelengths were determined.

2.5 Prepare of calibration curves

2.5.1 Loratadine Calibration curve

Aliquot volumes from the stock solution (1- 6 ml) were transferred into

six separate 25 ml volumetric flask, and diluted to volume with 0.1M

HCl (1.598*10-5 – 9.589*10-5 mole/L).

2.5.2 Pseudoephedrine sulphate Calibration curve

Aliquot volumes from the stock solution (1- 6 ml) were transferred into

six separate 25 ml volumetric flask, and diluted to volume with 0.1M

HCl (34*10-4 - 20*10-3 mole /L).

2.6 Laboratory synthetic mixtures

Six synthetic mixtures containg different concentration ratios of the two

drugs were prepared by quantity mixing different volumes from the

stock solution of analyte into the same 25 ml volumetric flasks, the final

volumes were made to mark with 0.1M HCl

10

2.7 Sample preparation

Ten tablets were accurately weighed and powdered; quantityof powdered

tablet equivalent to the average mass of one tablet (5 mg of LOR and

120mg PS) was weighed and transferred into 50 ml volumetric flask,

approximately 30 ml diluent were added and the mixture was sonicated

for15 minutes. Then mixture was diluted to volume with 0.1M HCl .The

resulting solution was filtered through a filter paper discarding the first

few ml of the filtrate ,this filtrated was again filtrated using 0.45 µ filter,

5ml from the clear solution were diluted to 25 ml with 0.1M HCl. The

absorbances of samples were measured at 256.6 and 280 nm.

11

3. Result and Discussion

3.1 Selection of analytical wavelengths

The spectraof PS and LOR (Fig 3) showed that at 280 nm LOR

absorbance was free from interference; while at the λ max of PS (256.6

nm), LOR was showing extensive interference. These two wavelengths

were selected for the application of the proposed method.

Figure 3: The overlain spectra of LOR (---) (conc. 7.99*10-5mole/L) and

PS ( ـــــــ) (conc. 0.00168 mole /L)

12

3.2 Linearity

3.2.1Linearity of pseudoephedrine sulfate at (256.6 nm)

The calibration curve of PS at 256.6 nm showed straight line relation

between the analyte concentration and absorbancewith a correlation

coefficient 0.9997.The calibration data is shown in Table 1 and the

calibration graph is displayed in Fig. 4

Table 1. Calibration data of pseudoephedrine sulfate at 256.6 nm

Figure 4 Calibration graph of pseudoephedrine sulphate at 256.6 nm

y = 396.17x + 0.0039

R² = 0.9997

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 0.0005 0.001 0.0015 0.002 0.0025

Ab

sorb

an

ce

concentration (mole/L)

Abso v conc

No. Conc.( Mole /L) Abs.

1 0.00034 0.136

2 0.00067 0.275

3 0.00101 0.403

4 0.00134 0.532

5 0.00168 0.665

6 0.00202 0.808

Slope 396.17

Intercept 0.0039

R2 0.9997

13

3.2.2 Linearity of loratadine at (256.6 nm)

The calibration curve of LOR at 256.6 nm showed straight line relation

between the analyte concentration and absorbance with a correlation

coefficient 0.9999. The calibration data is shown in Table 2 and the

calibration graph is displayed in Fig. 5

Table 2 Calibration data of loratadine at 256.6 nm

No. Conc. (Mole /L) Abs.

1 1.598E-05 0.146

2 3.197E-05 0.286

3 4.795E-05 0.425

4 6.393E-05 0.560

5 7.992E-05 0.701

6 9.589E-05 0.833

Slope 8607.2

Intercept 0.0103

R2 0.9999

Figure 5 Calibration graph of loratadine at 256.6 nm

y = 8607.2x + 0.0103

R² = 0.9999

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0.00E+00 2.00E-05 4.00E-05 6.00E-05 8.00E-05 1.00E-04 1.20E-04

Ab

sorb

an

ce

concentration (mole/L)

Abs. V Conc.

14

3.2.3 Linearity of loratadine at (280 nm)

The calibration data of LOR at 280 nm showed straight line relation

between the analyte concentration and absorbancewith a correlation

coefficient 1. The calibration data is shown in Table 3 andthe calibration

graph is displayed in Fig. 6

Table 3 Calibration data of loratadine at 280 nm

No. Conc. (Mole /L) Abs.

1 1.598E-05 0.156

2 3.197E-05 0.301

3 4.795E-05 0.448

4 6.393E-05 0.590

5 7.992E-05 0.738

6 9.589E-05 0.878

Slope 9050.5

Intercept 0.0122

R2 1

Figure 6 Calibration graph of loratadine at 280 nm

y = 9050.5x + 0.0122

R² = 1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0.00E+00 2.00E-05 4.00E-05 6.00E-05 8.00E-05 1.00E-04 1.20E-04

Ab

sorb

ance

concentration (mole /L)

Abs. V Conc.

15

The linearity data was used to calculate the molars absorptivity

coefficients of two analytes at the two selected wavelengths.

The molar absorptivity coefficients of LOR at 280 nm and 256.6 nm

were (9050.5 and 8607.2) respectively, while that of PS at 256.6 nm was

(396.17).

3.3 Analysis of the synthetic mixtures

Since the absorbance of LOR at 280 nm was shown to be free from

interfere by PS, its concentration in the mixture was calculated from

equation 1. Since

A= b C

and

CLOR = A/ b Equation 1

The concentration of PS was calculated from the absorbance of the

mixture at 256.6 nm using equation 2.

as

Am = PS b CPS+ LOR b CLOR

and

Am – ALOR = APS

then

CPS = ( Am – A LOR ) / PS b Equation 2

16

Table 4 Absorbance data of synthetic mixtures at (256.6 and 280 nm)

Mixture no. Abs.

256.6 nm 280 nm

1 1.362 0.743

2 0.826 0.453

3 0.696 0.455

4 0.685 0.30

5 0.832 0.597

6 0.70 0.598

The applied method showed very good accuracy for determination the

two analytes, as the actual concentration was very close to thetheoretical

concentration shown in Table 5.

The percentage content of LOR was 104.38% with RSD 0.87% and of

PS 99.29% with RSD 0.64%.

17

Table 5 Assay result of synthetic mixtures for accuracy

Mixtures no PS LOR

Theoretical (10-4) Actual (10-4) % content Theoretical(10-5) Actual(10-5) % content

1 16.80 16.54 98.45 7.94 8.21 103.40

2 10.08 9.98 99.00 4.76 5.01 105.25

3 6.72 6.65 98.95 4.76 5.03 105.67

4 10.08 10.09 100.09 3.18 3.32 104.40

5 6.72 6.67 99.26 6.35 6.59 103.78

6 3.36 3.36 100.00 6.35 6.59 103.78

Average 99.29% 104.38%

SD 0.64 0.91

RSD 0.64% 0.87%

18

3.4 Sample

for equation check the mixture

Table 6 Absorbance data of sample at (256.6 and 280 nm)

Sample no. Weight taken Theoretical actives weight Abs.

PS (mg) LOR (mg) 256.6 nm 280 nm

1 0.902 120.01 5.00 0.877 0.463

2 0.902 120.01 5.00 0.899 0.486

3 0.902 120.01 5.00 0.892 0.475

4 0.902 120.01 5.00 0.887 0.472

5 0.9021 120.03 5.00 0.88 0.465

6 0.9022 120.04 5.00 0.896 0.483

*Average weight / tablet = 0.9019g

19

The applied method showed very good accuracy for determination the two

analytes, as the actual concentration was very close to the theoretical

concentration for the synthetic mixtures as shown in Table 7,average content

of LOR was 100.25% with RSD 1.96% and of PS 98.64% with RSD 0.32%.

20

Table 7 Assay results of commercialsample

Sample No. PS (mg) LOR (mg)

Theoretical Actual % content Theoretical Actual % content

1 1.12 1.10 98.40 0.052 0.051 97.93

2 1.12 1.10 98.43 0.052 0.054 102.79

3 1.12 1.11 99.21 0.052 0.052 100.47

4 1.12 1.11 98.73 0.052 0.052 99.83

5 1.12 1.11 98.65 0.052 0.051 98.34

6 1.12 1.10 98.40 0.052 0.053 102.14

Average 98.64% 100.25%

SD 0.31 1.96

RSD 0.32% 1.96%

21

4.1 Conclusion

The Absorption correction is a straightforward procedure allowing the

accurate resolution of binary mixtures of compounds with overlapping

spectra.

The cost effectiveness and simplicity of the method render it as suitable

alternative to other expensive methods e.g. chromatographic methods

for the analysis of binary mixtures of compounds with overlapped

spectra in laboratories and countries where such sophisticated

equipment are not affordable.

The accuracy and simplicity of the method suggest its suitability in

cases where quick results are demanded e.g. as in-process analysis

procedure during blend analysis in industrial setups.

22

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23

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