Al Ribat National University

Faculty of Pharmacy

Spectrophotometric determination of Chlorzoxazone and Paracetamol

in Tablets by H-point Standards Addition Using Both Standards Addition

and Absorbance Difference

By

Mohammed Abdeen Mohammed Salih

B. Pharm (U of K)2013

A Thesis Submitted in Partial Fulfillment of The requirements for Master Degree

in Drug Quality Control

Supervisor

Dr. Imad Osman Abu Reid

2016

DEDICATION

This thesis is dedicated to my lovely mother and father

who taught me that difficult tasks can be accomplished one

step at a time.

I dedicate this thesis with special thanks to my lovely

family member and friends, who have always helped me,

encouraged me and believed in me.

Contents

Table of Contents i

Acknowledgements v

Abbreviations vi

Abstract in English viii

Abstract in Arabic ix

List of tables x

List of figures xi

Table of Contents

Chapter one: Introduction And Literature Review

1.1 Introduction: 1

1.2 literature review: 2

1.2.1 Paracetamol 2

1.2.1.1 UV/vis spectrophotometric methods 2

1.2.1.2 Flow-injection spectrophotometric methods: 3

1.2.1.3 Multivariate spectrophotometric methods: 3

1.2.1.4 Derivative spectrophotometric methods: 3

1.2.1.5 Infrared spectrophotometric methods 3

1.2.1.6 Spectrofluorimetric methods: 4

1.2.1.7 Chemiluminescence methods: 4

1.2.1.8 Electroanalytical methods: 4

1.2.1.9 Chromatographic methods: 5

1.2.1.10 Capillary electrophoretic methods: 5

1.2.2 Chlorzoxazone: 5

1.2.3 Analysis of CLX and PCT in combination with other drugs 6

1.2.4 Analysis of chlorzoxazone and paracetamol combination in dosage forms: 6

1.3 Analysis of multicomponent formulation: 7

1.3.1 Chromatographic separation methods: 8

1.3.1.1 Gas Chromatography: 8

1.3.1.2 High Performance Liquid Chromatography: 8

1.3.1.3 Capillary Electrophoresis: 8

1.3.2 Ultraviolet-Visible (UV-VIS) Spectrophotometry: 8

1.3.2.1 Simultaneous Equation method: 8

1.3.2.2 The Absorption Ratio Method (Isoabsorptive Point Method): 9

1.3.2.3 Derivative Spectroscopic Method: 9

1.3.2.4 Multicomponent Mode Method: 9

1.3.2.5 Area Under Curve Method: 9

1.4 H-point standard additions method(HPSAM) Theoretical Consideration: 9

1.4.1 Basic principle 9

1.4.2 HPSAM by addition of both analytes: 12

1.4.3 HPSAM using absorbance increment: 15

1.5 Objectives: 17

Chapter 2: Materials and Methods

2.1 Instrumentation 18

2.2 Materials 18

2.3 Reagents and standards 18

2.3.1 Sodium hydroxide 0.1M 18

2.3.2 Standards stock solutions 18

2.3.3 Working standard mixture 18

2.3.4 Laboratory synthetic mixtures 18

2.4 Samples preparation 18

2.4.1 Tablets 18

2.4.2 Capsules 19

2.5 Procedures: 19

2.5.1 Optimum wavelength selection 19

2.5.3 Linearity at the selected wavelengths 19

2.5.4 General procedure 19

2.5.5 Accuracy: 20

2.5.6 Precision: 20

2.5.6.1 Repeatability: 20

2.5.6.2 Intermediate precision: 20

Chapter 3: Results and discussion

3.1 Optimum wavelengths selection 21

3.2 Suitability for HPSAM application 25

3.3 Analysis of laboratory synthetic mixtures: 26

3.4 Precision 28

3.4.1 Repeatability 28

3.4.2 Intermediate precision: 30

3.5 Analysis of Capsules 32

Chapter 4: Conclusion and references

4.1 Conclusion 33

4.2 References 34

Acknowledgements

This research project would not have been done without ALLAH and the support of many

people.

I would like to thanks my supervisor Dr. Imad Osman Abu Reid for his patience, support

during this program and guidance to accomplish this research.

Special thanks to Prof. Elrasheed A.Gadkariem for being helpful, supportive and for his

valuable advices in anything concerned with M.Sc programme.

I will not forget every teacher, doctor and professor who has taught me a letter in my life,

without them I could not have reached this level.

My gratitude also goes to the staff of Azal laboratories for their help and attention.

List of Abbreviations

Abbreviation

Description

PCT

Paracetamol

CLX Chlorzoxazone

NIR

Near Infrared

HPSAM

H-Point Standard Additions Method

CLS

Classical Least- Squares

ILS

Inverse Least-Squares

PLS Partial Least-Squares

PCR

Principal Components Regression

MCR Mean Centering Of Ratio

PCCA Pure Component Contribution Algorithm

CL Chemiluminescence

FI

Flow Injection

HPLC High Performance Liquid Chromatography

RP-HPLC Reversed-Phase High Performance Liquid

Chromatographic.

TLC Thin-Layer Chromatography

GLC Gas Liquid Chromatography

LC Liquid Chromatography

CE

Capillary Electrophoretic

MEKC

Micellar Electro Kinetic Chromatography

FDNB

1-Fluoro-2,4-Dinitrobenzene

MOSA

Method Of Standard Addition

ZCP

Zero Crossing Points

TYB Total Youden Blank

DCQC 2,6-Dichloroquinone Chlorimide

MBTH 3-Methyl-2-Benzothiazolinone Hydrazone

Abstract

Simple, specific, accurate and precise spectrophotometric method was developed for the

simultaneous determination of Paracetamol (PCT) and Chlorzoxazone (CLX) in their tablet

dosage forms. The proposed H-Point Standard Addition Method (HPSAM) using both

Standards addition and absorbance difference; involves addition of the both analyte to the

binary mixture solution. The absorbance of each solution was measured at the selected

wavelength pair (244 and 270.6nm) for CLX, (255 and 277.4nm) for PCT. Concentrations

of PCT and CLX in the samples were obtained from intersection of straight line (obtained

by plotting ΔA value against the added concentration) with the negative side of the

abscissa. The calibration curves were linear over the concentration range of 2-12and 4-14

µg/mL for PCT and CLX, respectively. This method was tested by analyzing synthetic

mixtures of the above drugs and was applied to commercial pharmaceutical preparation of

the subjected drugs, the assay values were 103.00 % and 102.29% for CLX and PCT with

relative standard deviation 1.68% and 2.29 % respectively. There were no significant

difference (at P = 0.05 , n = 6 ) between the analysis results obtained on two different days

, the calculated t-value was less than the t- tabulated. This confirms the precision of the

proposed method. Moreover, the proposed method was also applied for the determination

of CLX and PCT in capsules formulation, the results obtained were in good agreement with

the labeled amounts 100.08% and 100.08% with relative standard deviations of 1.60% and

1.37% for CLX and PCT respectively.

المستخلص

تم تطوير طريقة قياس طيفيه بسيطة ,متخصصه , ذات مصداقية ودقيقة للتحليل المتزامن للباراسيتامول

المقترحة ـ كال من طريقة H اٳلضافة القياسية للنقطةتشمل طريقة والكلورزوكسازون الموجودان في شكل أقراص.

ٳضافة المحاليل القياسية وطريقة فرق االمتصاصيةـ وتشمل ايضا اضافة المادتين المراد تحليلهما الى الخليط الثنائي

244الذي يحتوي على المادتين. تم قياس امتصاص كل محلول تم تحضيره في االطوال الموجية التي تم اختيارها )

نانومتر( للبراسيتامول. 277.4و 255نانومتر( للكلورزوكسازون,) 270.6و

تم حساب تركيز الباراسيتامول والكلورزوكسازون من تقاطع الخط المستقيم ) المتحصل عليه من خالل مد قيمة فارق

عايرة خطيين على مدى االمتصاصية ضد التركيز المضاف( مع الجانب السالب من المحور السيني. وقد كان منحنيا الم

مايكروجرام/مل للباراسيتامول والكلورزوكسازون على التوالي. تم اختبار هذه الطريقة من 14-4و 12 - 2تركيز

خالل تحليل مخاليط اصطناعية من العقارين المذكورين أعاله وتم تطبيقها على مستحضر صيدالني تجاري يحتوي

% للكلورزوكسازون والباراسيتامول 102.29% و 103.00يمة النقاوة هي على الدوائين المعروضين. وكانت ق

( n= 6 و p = 0. 95 % على الترتيب. لم يكن هناك فارق معنوي ) عند2.29و %1.68بانحراف معياري نسبي

كد دقة هذه وهذا ما يؤ .المحسوبة كانت أقل من المجدولة t بين التائج المتحصل عليها خالل يومين مختلفين, حيث قيمة

عالوة على ذلك فقد تم أيضا تطبيق الطريقة المقترحة في حساب الباراسيتامول والكلورزوكسازون .الطريقة المقترحة

ل عليها في توافق جيد مع القيمة المنصوص عليها بنسبة 100.08الموجودان في شكل كابسوالت. كانت النتائج المتحص

.% للكلورزوكسازون والباراسيتامول على التوالي1.37% و 1.06ن نسبيين % بانحرافين معياريي100.04% و

List of Tables

Table 1. Linearity data at 244 nm and 270.6 nm for Paracetamol: 22

Table 2. Linearity data at 255 nm and 277.4 nm for Chlorzoxazone: 23

Table 3. Linearity results of PCT and CLX at the selected

wavelength

24

Table 4. Linearity data at 255 nm and 277.4 nm for Paracetamol 24

Table 5. Linearity data at 244 nm and 270.6 nm for paracetamol: 25

Table 6. Linearity results of PCT and CLX at the selected

wavelength

25

Table 7. Equations of crossed line for each synthetic mixture 26

Table 8. Analysis result of the synthetic mixtures using ΔA values 27

Table 9. Summary of synthetic mixture analysis results 27

Table 10. Paracetamol -added concentration in µg/ml and ∆A of each

test

28

Table 11. Chlorzoxazone; Added concentration in µg/ml and ∆A of

each test

29

Table 12. Results of the repeatability analysis using ΔA method 30

Table 13. Results of the intermediate precision analysis using ΔA

method

31

Table 14. Statistical analysis of the precision data 31

Table 15. Results of capsules analysis using ΔA method 32

Table 16. Summary of capsules analysis of results 32

List of Figures

Figure 1. Chemical structure of Paracetamol 1

Figure2. Chemical structure of Chlorzoxazone 2

Figure3. Spectrum of analytes X and Y and sample S, and location

of one pair of wavelengths λ1, and λ2, and one pair of

wavelengths λ3 and λ4

11

Figure 4. Plot of the H-point standard additions method. C added is

the added analyte concentration

12

Figure 5. HPSAM lines obtained when the double standard addition

is employed

13

Figure 6. The plot of HPSAM 15

Figure 7. The plot of the HPSAM with ∆A values. 16

Figure 8. Individual spectra of Chlorzoxazone 10 µg/ml and

Paracetamol 10 µg/ml.

21

Figure 9. Calibration curve of Paracetamol at λ 244 nm and

270.6 nm

22

Figure 10. Calibration curve of Chlorzoxazone at λ 255 nm and

277.4 nm

23

Figure 11. Calibration curve of Paracetamol at λ 255 nm and 277.4

nm

24

Figure 12. Calibration curve of Chlorzoxazone at λ 244 nm and

270.6nm

25

Figure 13. Linear regression plot of added concentration

(µg/ml)against ∆A for Paracetamol

29

Figure 14. Linear regression plot of added concentration (µg/ml)

against ∆A for Chlorzoxazone

29

1.1 Introduction:

Different dosage forms containing drug combination are available in the market. Due to

their greater patient acceptability, increased potency, multiple action, fewer side effects

and quicker relief, they have acquired a lot of importance nowadays (1).

Spectrophotometric techniques are mainly used for multicomponent analysis thus

minimizing the cumbersome task of separating interferents and allowing the determination

of an increasing number of analytes, consequently reducing analysis time and cost(2).

Multicomponent UV spectrophotometric methods are based on recording and

mathematically processing absorption spectra. They offer the following advantages (3):

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. The

spectrophotometric quantitative analysis of mixtures often involves resolution of two

components with partially overlapped spectra. The greater the extent of overlapping the

more difficult the resolution is rendered.

Paracetamol (PCT) is an acylated aromatic amide, which was firstly introduced into

medicine as an antipyretic/analgesic by Von Mering in 1893 and has been in use as an

analgesic for home medication for over 30 years and is accepted as a very effective

treatment for the relief of pain and fever in adults and children. It is the most used medicine

after acetylsalicylic acid in many countries as an alternative to aspirin and phenacetin.

Paracetamol is also known as acetaminophen (N-acetyl-p-aminophenol, 4-

acetamidophenol) Fig(1); it is a major ingredient in numerous cold and flu medications and

many prescription analgesics(4).

Figure 1: Chemical structure of Paracetamol

Chlorzoxazone (CLX),Fig(2) belongs to the class of organic compounds known as

benzoxazolones. These are organic compounds containing a benzene fused to an oxazole

ring (a five-member aliphatic ring with three carbon atoms, one oxygen atom, and one

nitrogen atom) bearing a ketone group, its IUPAC name is 5-chloro-2,3-dihydro-1,3-

benzoxazol-2-one(5)

Figure 2: Chemical structure of Chlorzoxazone

Chlorzoxazone and paracetamol combination is indicated as an adjunct to other measures,

such as rest and physical therapy, for relief of pain and muscle spasm associated with acute,

painful musculoskeletal conditions(6)

1.2 literature review:

1.2.1 Paracetamol

The extensive review on the analytical methods used for the analysis of paracetamolalone

and in combination with other drugs and in biological fluid(4);revealed that variety of

techniques and methods were used, these include:

1.2.1.1 UV/vis spectrophotometric methods:

The spontaneous oxidation of alkaline mixtures of p-aminophenol and phenol with

molecular oxygen to form indophenol has been made the basis of a colorimetric procedure

for the determination of paracetamol via its hydrolysis product, p-aminophenol(7).

When there are no significant spectral interferences, the determination of paracetamol in

pharmaceutical products can be carried out by direct UV absorption spectrophotometry,

such as in the PCT tablets monograph in the British Pharmacopoeia (8). However, when

formulated with other UV absorbing substances such as excipients or active substances,

where spectral overlapping is possible, separation techniques such as high performance

liquid chromatography (HPLC) are usually necessary, as prescribed, for instance, in several

of the acetaminophen articles in the USP (9).

Direct UV/Vis spectrophotometry is by far the instrumental technique of choice in

industrial laboratories, owing mainly to its simplicity, often demanding low cost

equipment. The majority published spectrophotometric methods for the determination of

paracetamol are based on indophenol dye, and Schiff’s base formation, nitrosation and

subsequent chelation, oxidation, oxidative coupling with some special reagent, UV

absorption, pH-induced spectral changes(4).

1.2.1.2 Flow-injection spectrophotometric methods:

Various flow injection analysis along with suitable detection technique have also been

reported for the determination of paracetamol. A simple flow-through UV optisensing

device was developed for the determination of paracetamol based on its transient retention

and concentration on a suitable active solid support (Sephadex QAE A-25 anion-exchange

resin) packed in the flow cell and the continuous monitoring of its native absorbance on

the solid phase at 264 nm(10-12).

1.2.1.3 Multivariate spectrophotometric methods:

In recent years, multivariate calibration techniques have been widely applied to UV/Vis

spectral data, classical least-squares (CLS), inverse least-squares (ILS) and methods such

as principal components regression (PCR) and partial least-squares (PLS) are increasingly

being used in conjunction with flow injection techniques(4).

A fast analytical procedure was proposed for the simultaneous determination of

paracetamol in a mixture with caffeine and acetylsalicylic acid by means the PLS, the

method involves the use of eight standard mixtures of the three compounds assayed(13).

Also, paracetamol, chlorpheniramine and pseudoephedrine were determined by CLS and

PLS(14).

ILS method in matrix form which is K-matrix representation of Beer’s law was presented

for simultaneous determination of ibuprofen and paracetamol without prior separation from

each other(15).

1.2.1.4 Derivative spectrophotometric methods:

Derivative method is used mainly for simultaneous determination of two or more

compounds in the same mixtures without preliminary separation; there is a lot of published

paper about the use of derivative methods to the determination of paracetamol in mixtures

with other compounds.

First derivative spectrophotometry and absorbance ratio method in the zero-order spectra

were used for determination of tablets containing paracetamol and analgine; The relative

standard deviation of the derivative method was found to be 0.35% for analgine and 0.31%

for PCT and 0.49% and 1.26% for analgine and PCT respectively for the absorbance ratio

method(16).

1.2.1.5 Infrared spectrophotometric methods:

A near infrared (NIR) spectroscopic method for the simultaneous determination of five

active principles present in a drug for alleviating influenza symptoms (paracetamol,

ascorbic acid, dextromethorphan hydrobromide, caffeine and chlorpheniramine maleate)

was developed(17).

A procedure for the direct FTIR spectrometric determination of PCT is described [159].

The method was based on the solubilization of paracetamol in a 10% (v/v) ethanol in

dichloromethane solution and direct absorbance measurement at 1515 cm−1, using the

baseline established at 1900 cm−1 for measurement correction. The procedure can be

carried out in both the stopped-flow and flow injection modes.(18)

1.2.1.6 Spectrofluorimetric methods:

Direct spectrofluorimetric determinations of PCT require a previous and adequate

derivatisation step. a stopped-flow method with fluorescence detection for the

determination of PCT based on its oxidation with hexacyanoferrate(III), was developed; a

kinetic study of the reaction is developed measuring the initial rate of change of the

fluorescence intensity of the oxidised product formed at 241 and 426 nm excitation and

emission wavelengths, respectively.(19)

1.2.1.7Chemiluminescence methods:

Due to its inherent high sensitivity and low detection limit, chemiluminescence (CL) has

been applied to the analysis of many biomedical important analytes. The CL analysis can

be measured from the CL induced by the reaction of analyte with CL reagents or the

inhibition of CL that resulted from the reaction of analyte with CL reagents or oxidants

prior to the CL reaction. Most of the reported procedures use the well-known luminol,

peroxyoxalate or lucigenin CL reaction systems.(4)

A rapid and precise continuous-flow method was described for the determination of PCT

based on the chemiluminescence produced by its reaction with Ce(IV) in acidic

solution(20)

1.2.1.8 Electroanalytical methods:

A flow injection (FI) kinetic potentiometric method for the determination of phenolic

(acetaminophen and isoxsuprine) and hydrazino (isoniazid) drugs was described; The

method was based on the reaction of l-fluoro-2,4-dinitrobenzene (FDNB) with the analytes

in a weakly alkaline medium, which proceeds through the liberation of fluoride from the

reagent. The slow reactions with phenols are catalysed by micelles of

cetyltrimethylammonium bromide. The reaction rate was monitored with a fluoride-

selective electrode in a wall-jet configuration and is used to construct a calibration graph,

using the fixed-time approach. The response time and the long-term stability of the

electrode were found to be adequate for such kinetic determinations(21)

1.2.1.9 Chromatographic methods:

HPLC method was developed Using a C18 stationary phase with methanol–water (1:2, v/v)

mixture as mobile phase at the flow rate of 1.78 ml/min with detection at 193.3 nm;

sulphamethoxazole is used as an internal standard(22)

Another method is; stability-indicating GLC-procedure for the determination of

acetaminophen and aspirin in suppositories was described. Analysis was performed using

a flame ionization detector and a column of 2% OV-225 on Chromosorb W. Nitrogen was

used as the carrier(23)

A thin-layer chromatography–UV scanning densitometric technique is used for the

simultaneous determination of paracetamol and chlorzoxazone(24)

1.2.1.10 Capillary electrophoretic methods:

Capillary electrophoretic (CE) offers possible advantages over LC in terms of separation

time, solvent pollution and analysis expense. It has been reported as a powerful tool for a

wide range of analysis, including many applications to the determination of drugs, such as

the main component determinations, drug-related impurities estimations, chiral

separations, etc.(4). A micellar electro kinetic chromatography (MEKC) method was

established for determination of paracetamol and chlorpheniramine maleate in cold tablets.

Separation of both drugs, as well as other seven cold remedy ingredients, was achieved in

25.5 min, the effective capillary length, the separating voltage and the temperature was

optimized, detection was by a diode array detector at 214 nm.(25)

1.2.2 Chlorzoxazone:

The USP monograph for CLX tablet described HPLC method, using water, acetonitrile and

glacial acetic acid in ratio (70:30:1) as mobile phase ,phenacetin in acetonitrile as internal

standard, detector at 280 nm and 4mm x 30cm column containing packing L1. The flow

rate is about 1.5 ml/min(26)

Several methods are available for the determination of CLX alone and in combination with

many other drugs specially non-steroidal anti-inflammatory drugs, these include:

Electrochemical method based on cyclic and square wave voltammetric techniques has

been used for the determination of CLX(27)

Three different methods were described for colorimetric determination of chlorzoxazone

in pure form and in formulations using based on the oxidative coupling reaction of the

hydrolysis product of chlorzoxazone with 3-methyl-2-benzothiazolinone hydrazone

(MBTH) in the presence of Fe (III), N, N-dimethyl-p-phenylenediamine (DMPD) in the

presence of periodate (IO4) and 2,6-dichloroquinone chlorimide (DCQC) (28)

1.2.3 Analysis of CLX and PCT in combination with other drugs

Reversed-phase HPLC has been used for the simultaneous estimation of acetaminophen,

ibuprofen and chlorzoxazone in formulations. The method was carried out on a Kromasil®

C8 column using a mixture of 0.2% triethylamine:acetonitrile (adjusted to pH 3.2 using

dilute orthophosphoric acid), the detection was carried out at 215 nm using ketoprofen as

internal standard(29)

Chlorzoxazone, paracetamol and diclofenac sodium combination determination was

carried out using isocratic reverse phase high performance liquid chromatographic. The

chromatographic separation was performed on an inertsil C18 column (250 mm × 4.6 mm

i.d 5 µm particle size) using a mobile phase consisted of a mixture of phosphate buffer

(0.02 M KH2PO4, pH adjusted to 3.7 using orthophosphoric acid), acetonitrile and

methanol in the ratio of (25: 25: 50) at a flow rate of 1.0 mL/min. The wavelength was set

at 220 nm(30).

Chlorzoxazone, paracetamol and aceclofenac combination was determined using a

reversed-phase liquid chromatographic method, this method used a Zorbax SB C18, 250 ×

4.6 mm, 5 μm analytical column and a mobile phase made of acetonitrile and buffer (40:60,

v/v), buffer containing 50mM ortho-phosphoric acid; pH of the buffer is adjusted to 6 with

10% w/v sodium hydroxide solution. The instrumental settings are at a flow rate of 1

mL/min; the column temperature is 25°C, and detector wavelength is 270 nm.(31)

Three spectrophotometric methods were described for the same combination, the three

methods were; novel pure component contribution algorithm (PCCA) along with mean

centering of ratio spectra (MCR) and the factor based partial least squares (PLS)

algorithms.(32).

1.2.4 Analysis of chlorzoxazone and paracetamol combination in dosage forms:

Different methods have been described for the simultaneous determination of paracetamol

and chlorzoxazone in mixtures.

Two spectrophotometric methods have been developed for the simultaneous determination

of chlorzoxazone and acetaminophen in their combined dosage forms, the first, an

absorbance ratio technique using the Isoabsorptive point as one of the wavelengths,

together with “Q curve” analysis. The second was a difference spectrophotometric method

based on measurement of absorbance of an alkaline solution relative to that of an acidic

solution of identical concentration of the sample at two different wavelengths(33).

Derivative spectrophotometry was also used for the simultaneous determination of

paracetamol-chlorzoxazone, binary mixture, second derivative spectrophotometry permits

simple, rapid, and direct determination of these binary mixtures without previous

separations(34)

Orthogonal functions-ratio spectrophotometry has been developed and applied to the

determination of paracetamol and chlorzoxazone in authentic mixtures and capsules(35)

Thin layer chromatography densitometric method was also reported for simultaneous

determination of PCT with CLX in multicomponent mixtures. The mobile phase was ethyl

acetate: methanol: ammonia 25% (85:15:5 v/v) (24).

Simultaneous estimation of PCT and CLX in tablet dosage form was carried out using X

Terra® C18 column (150 mm ×4.6 mm id, 5 μm particle size) as stationary phase,

Acetonitrile: Methanol: HPLC grade water [20: 10: 70 v/v/v] as a mobilephase with flow

rate of 0.7 ml/min. Quantification was achieved with Photo Diode Array detector at 270

nm(36)

Gas liquid chromatography method was described for the estimation of PCT and CLX in

combined dosage forms using 10% OV-17 column and flame ionisation detector;

phenacetin is used as internal standard(37).

New chemometric approaches were introduced into the high performance liquid

chromatographic (HPLC) determination of chlorzoxazone and paracetamol in the tablets

and spiked human plasma. These chemometric approaches contain the application of

classical least squares (CLS), principle component regression (PCR), and partial least

squares (PLS) calibrations to the multi-wavelength HPLC peak area obtained by plotting

the chromatograms at the five wavelengths. The multi-chromatograms were obtained by

using the photodiode array detector at 225 (A), 240 (B), 255 (C), 270 (D), and 285 (E) nm.

The algorithms of CLS, PCR, and PLS were applied to the multi-chromatogram data to

construct the HPLC-CLS, HPLC-PCR, and HPLC-PLS calibrations. A mixture of

acetonitrile and 0.1 M ammonium carbonate (60:40, v /v) on a Waters Symmetry w C18

Column 5 mm 4.6 250 mm at a flow rate of 0.8 mL /min was used as a mobile phase to

separate and determine chlorzoxazone and paracetamol in samples. Hydrochlorothiazide

was used as an internal standard in this chromatographic separation(38).

1.3 Analysis of multi-component formulation:

The determination of mixtures has always been an interesting question for analysts; so

many selective analytical techniques have been put forward.

1.3.1 Chromatographic separation methods:

Chromatography separates complex mixtures with great precision. There are quite a

number of chromatographic techniques that have been developed to analyze complex

mixtures; these include:

1.3.1.1 Gas Chromatography:

In gas chromatography, the components of a vaporized sample are separated as

aconsequence of being partitioned between a gaseous mobile phase and a liquid or solid

stationary phase held in a column. There are two types of gas chromatography; gas liquid

chromatography (GLC) and gas solid chromatography (GSC). With GLC, the stationary

phase is a nonvolatile liquid bonded to the inside of the column or to a fine solid support,

whereas GSC is based on a solid stationary phase in which retention of analytes occurs

because of physical adsorption(39)

1.3.1.2 High Performance Liquid Chromatography:

High performance liquid chromatography(HPLC) is the term used to describe liquid

chromatography in which the liquid mobile phase is mechanically pumped through a

column that contains the stationary phase. HPLC is a powerful tool in analytical chemistry. It

has been used extensively in chemical analysis(40, 41)

1.3.1.3 Capillary Electrophoresis:

The electrophoretic separation technique is based on the principle that under the influence

of an applied potential field different species in solution will migrate at different velocities

from one another.(42, 43)

1.3.2 Ultraviolet-Visible (UV-VIS) Spectrophotometry:

Spectrophotometric multi-component analysis involves recording and mathematically

processing of absorption spectra for samples that consist of several components

contributing to the overall spectrum in proportion to their individual absorptivities and

concentrations(44).

The UV-Visible spectroscopic methods for estimation of drugs combinations are:

1.3.2.1 Simultaneous Equation method:

This method is used for determination of drug combination that contain two drugs each of

which absorbs at the λmax of the other by the technique of simultaneous equations (verodt's

method), beer lambert's law should be obeyed for application of this technique.(45)

1.3.2.2 The Absorption Ratio Method (Isoabsorptive Point Method):

The absorbance ratio method is a modification of the simultaneous equations procedure. It

depends on the property that, for a substance, which obeys Beer’s law at all wavelength,

the ratio of absorbances at any two wavelengths is a constant value independent of

concentration or path length. In the quantitative assay of two components in admixture by

the absorbance ratio method, absorbances are measured at two wavelengths, one being the

λ-max of one of the components (λ2) and other being a wavelength of equal absorptivity

of two components (λ1), i.e. an iso-absorptive point(45)

1.3.2.3 Derivative Spectroscopic Method:

Derivative spectroscopy involves the conversion of a normal spectra to its first, second or

higher derivative spectra. The normal spectrum is known as fundamental, zero order or D0

spectra(45).

For the quantitative estimation of binary mixtures by the derivative spectroscopy, first of

all we have to find out the Zero Crossing Points (ZCP) for both the components (A and B).

Now select ZCP for A and B so that at that particular ZCP other component shows

remarkable absorbance. Now prepare calibration curve of A at the ZCP of B and of B at

the ZCP of A. Find out the unknown concentration using calibration curves

1.3.2.4 Multi-component Mode Method:

This method requires two wavelengths. One wavelength is selected such that one drug

shows maximum absorbance while the other drug shows considerable absorbance. The

second wavelength is selected such that other drug shows maximum absorbance while the

first one shows considerable absorbance.

Choosing wavelengths at which the differences in molar absorptivities are large, leads to

attain greater accuracy in this analysis.(46)

1.3.2.5 Area Under Curve Method:

The total area under the curve of a mixture at a particular wavelength range is equal to the

sum of area under curve of the individual components at same wavelength range. By

applying “Cramers Rule” and “Matrix Method”, the concentration of component of a

mixture can be determined(46)

1.4 H-point standard additions method(HPSAM)Theoretical Consideration:

1.4.1 Basic principle

HPSAM is a chemometrics methods often used in analytical chemistry. A modification of

the standard additions method, presented by Foster et al(47), was proposed in 1988 by

Reigand Falco (48) in order to obtain an unbiased analyte concentration when both analyte

and interferants are present in a sample. It also permits the determination of an interferant

known to be present. HPSAM is based on the principle of dual-wavelength

spectrophotometry and the standard addition method (48, 49) . HPSAM can determine the

two components simultaneously with extensive or even coincident overlapping

spectra(50)and/or Total Youden Blank (TYB). The TYB represents the constant error of

the method, extrapolated to zero sample level, being independent of the size of sample

taken and not attributable to the analyte(51, 52)

In that method, additions of pure analyte X were made to the sample. Absorbance

increments were used when only the analyte concentration was required(53). This variant

allows the elimination of the blank bias error due to the use of absorbent blanks(54, 55)

The following principles have to be followed for selection of appropriate wavelengths for

applying HPSAM:

At these selected wavelengths the analyte signals must be linear with the concentrations

and the interference signal must remain equal, in the case where the analyte concentrations

are changed, the analytical signal obtained from the mixture containing the analyte and the

interfering should be equal to the sum of the individual signals of the two components. In

addition, the difference in the slopes of the two straight lines measured at two selected

wavelengths must be as large as possible while the difference in the slopes of the two

straight lines measured at the other pair of wavelengths must be as small as

possible(preferred to be equal zero) in order to get good accuracy and sensitivity.(56)

Let us suppose a binary mixture S of the compounds X and Y. Their spectra are shown in

Fig. 3.Absorbance of the sample at each wavelength will be the addition of the individual

absorbances of the species X and Y at this wavelength:

Where ɛx j and .ɛy,j are the molar absortivity coefficients for the species X and Y at λj;

𝑪𝑿,𝒋𝟎 and 𝑪𝑿,𝒋

𝟎 are the concentrations of the species X and Y in the sample; 𝑨𝑿,𝒋𝟎 and 𝑨𝒀,𝒋

𝟎

are the individual absorbance of the species X and Y in the sample.

Previously HPSAM basis develops a procedure to quantify an X analyte in presence of a

Y direct interferant and/or TYB, by the construction of two X analyte standard addition

plots, with Mx,1, and Mx,2 slopes, at two previously selected wavelengths (λ1, λ2), which

intersect at the H-point, with (-CH, AH)coordinates Fig(4). H-Point depends on the analyte

concentration 𝑪𝑿,𝒋𝟎 by means of this expression(48, 49):

If λ1 andλ2, are selected in such a way that the Y interferent absorbance values are equals

(Fig. 3):

then the abscissa of the H-point will be the X analyte concentration in the sample, 𝑪𝑿𝟎 :

Figure 3: Spectrum of analytes X and Y and sample S, and location of one pair of

wavelengths λ1, and λ2, and one pair of wavelengths λ3 andλ4

The interferent determination is made from the AH value (previously corrected the TYB

value, if it is present) and a calibration graph of the interferent.

Figure 4: Plot of the H-point standard additions method. C added is the added analyte

concentration

1.4.2 HPSAM by addition of both analytes:

Basic principle of this method is to analyze each component of the mixture in presence of

the other one as interferent, using analytical signals obtained at two wavelengths where the

absorbance is the same for the interferent and different for the analyte

The required data to apply the method are the absorbance of the sample and the absorbance

of the sample spiked with known amounts of X and Y species at previously selected

wavelengths. Let us suppose that h, and h, are selected according to Eq. 3 (Fig. 3). The

relation of the added concentrations between species X and Y, 𝑪𝒀𝒊 /𝑪𝑿

𝒊 , is the same in all the

solutions prepared to apply the method (because the additions are made from astandard

mixture of both). Then, the equations of the lines that describe the absorbance of the

successive standard additions by application of the HPSAM versus the added concentration

of analyte X at the two previously selected wavelengths will be (Fig. 5):

For λ1 :

For λ2:

Where:

A1 and A2 : the measured absorbance at the two previously selected wavelengths.

Mx,1 , My,1 , Mx,2 and My,2 : are the slopes due to the addition of species X and Y in the

lines obtained at λ1, and λ2.

𝑪𝒀𝒊 and 𝑪𝑿

𝒊 ,: are the concentration of species X and Y added in the i solution.

n: is the number of additions.

When i = 0 corresponds with the solution where only exists sample (there is no standard

added).

Fig. 5. HPSAM lines obtained when the double standard addition is employed

If we represent the analytical signal, absorbance at the two previously selected wavelengths

λ1, and λ2, versus the concentration added of species X, we will obtain two lines, with

intercepts 𝑨𝑿,𝟏𝟎 +𝑨𝒀,𝟏

𝟎 and 𝑨𝑿,𝟐𝟎 +𝑨𝒀,𝟐

𝟎 and slopes

and

The slopes obtained are the ones obtained if the addition was made of analyte X alone, with

the addition of a constant, which depends on the relative concentration of each species in

the standard and the absorbance of the species Y at the selected wavelength. Both lines

intersect at the H point, with coordinates (- CH(X), AH(Y)) where Cn(x) is the unbiased analyte

X concentration. If we operate in Eqs.(5)and(6), and taking into account that Ay,1 = Ay,2and

then My 1 = My,2(Eq. 3):

From where:

Where:

𝑪𝑯(𝑿)= 𝑪𝑿𝟎 : is the unbiased analyte concentration in the sample, because Eq. 8 depends

only of variables related with the analyte, and it is equivalent to:

The incorrigible error due to the presence of one interferent, in spite of its concentration is

not constant, has been transformed into a constant systematic error, which is easily

valuable, as the HPSAM basis predicts. In effect, if we substitute Eq. 9 in Eqs. 5 or 6, we

can prove that AH(Y)is equivalent to:

In the same way as for species X we obtain for the species Y the next expressions, if two

wavelengths λ3, and λ4, are selected in such a way that the species X presents the same

absorbance:

Analogousto expressions (8) and (10), we obtain that:

1.4.3 HPSAM using absorbance increment:

Application of the HPSAM under the condition that the absorbance values of the interferent

at the two wavelengths are the same (Ay,1 = Ay,2), and by subtracting Eqs. 5 from 6, and

taking into account that Ay,1 = Ay,2and then My 1 = My,2 yields:

According to this equation, the absorbance increment depends exclusively on the analyte

concentration, so the plot of ΔA1.2.VS. 𝑪𝑿𝒊 willbe a straight line of ordinate 𝑨𝑿,𝟏

𝟎 -𝑨𝑿,𝟐𝟎 and

slope Mx,1-Mx,2(Fig. 6).The analyte concentration canthus be calculated from ΔA values

by applying the HPSAM to the intercept of the straight line, at the H-point of which

Figure 6: The plot of HPSAM

The ΔA value obtained after each addition will be exclusively related to the analyte

concentration as the interferent absorbance will be the same at the two wavelengths, so its

contribution to ΔA will be zero even if the intensity of its analytical signal changes on

successive analyte additions because of interaction with the analyte. Therefore, the

analytical signals will be free from constant systematic errors (and also from proportional

errors thanks to the features of the MOSA)

Consequently, when only the analyte concentration must be calculated or only the sample

matrix spectrum is known, a single calibration plot of ΔA against the added analyte

concentration allows one to calculate the unknown concentrations free from any bias error

from the intercept of the line in the same way as with the MOSA. but with no systematic

errors.

Figure 7 illustrates the two possibilities of using the HPSAM plots to calculate the analyte

concentration.

Figure 7: The plot of the HPSAM with ∆A values.

On the other hand, routine work usually involves determining the analyte in a given sample

by using the single-calibration method, which requires the sample signal (As), the analytical

signal (Ar) and the concentration of standard used (Cr) to be known. Thus, the analyte

concentration can be calculated from spectroscopic data by using the equation:

The variables required to obtain unbiased results by applying the MOSA are As and the

analytical signal (A1) yielded by a solution containing the sample plus a certain known

added concentration of pure analyte (Ca). The equation to be applied in this case is:

Applying the HPSAM to ∆A values requires them to be known for the original sample and

the solution including the added pure analyte at the two selected wavelengths, as well as

the analyte concentration added. The equation to be used will thus be:

1.5 Objectives:

The combination of paracetamol and chlorzoxazone is not official in any pharmacopoeia;

hence no official method is available for their simultaneous estimation in their combined

synthetic mixture or dosage forms.

The spectra of PCT and CLX (Fig.8), shows significant overlapping over the range of 220-

320 nm, however this overlapping is possible to manipulate using the conditions required

for the application of HPSAM.

The objective of this search was to:

1- Develop and validate HPSAM method based on standard addition of both analytes

utilizing the absorbance difference (∆A) method.

2- To apply the developed method for the simultaneous determination of the two actives in

tablets and capsules

2.1 Instrumentation

• UV-Vis absorption spectra were measured on computer controlled Schimadzu

UV-1800 spectrophotometer with the use of 1.0 cm quartz cells.

• Sensitive balance (Sartorius - Germany)

• Ultrasonic (Life-care equipment - India)

• Centrifuge (Sartorius - Germany)

2.2 Materials

• Sodium hydroxide pellets (Scharlau- Spain)

• Methanol analytical grade (Scharlau - Spain)

• Laboratory produced distilled water was used throughout this work

• Paracetamol working standard was a gift from (Azal Pharmaceutical Company -

Sudan) and Chlorzoxazone working standard was a gift from (Blue Nile

Pharmaceutical Company - Sudan).

• Nilogesic Tablets labeled to contain 250 mg chlorzoxazone and 300 mg

paracetamol per tablet(Blue Nile Pharmaceutical Company - Sudan)

• RelaxoneCapsuleslabeled to contain250 mg chlorzoxazone and 300 mg

paracetamol per capsule ( Jamjoom Pharma-KSA).

2.3 Reagents and standards

2.3.1 Sodium hydroxide 0.1M

Was prepared by dissolving 4 gm of sodium hydroxide pellets in 1000 ml volumetric flask

using purified water.

2.3.2 Standards stock solutions

Standard stock solutions of paracetamol (300 µg/ml) and Chlorzoxazone (300 µg/ml) were

prepared separately by dissolving 30 mg each in 100 ml methanol in a volumetric flask.

2.3.3 Working standard mixture

Working standard solution containing both components was prepared by transferring 5 ml

from each stock solution into a 100 ml volumetric flask and making the volume to the mark

with 0.1 N NaOH.

2.3.4 Laboratory synthetic mixtures

Synthetic samples containing different concentration ratios of PCT and CLX were prepared

by proper dilution of aliquots from the standards stock solutions.

2.4 Samples preparation

2.4.1 Tablets

The homogenized powder from twenty tablets with an average weight equivalent to 300

mg paracetamol and 250 mg Chlorzoxazone was transferred into a 100 ml volumetric flask,

about 70 ml methanol were added, the content of the flask was mechanically shaken for 20

minutes and then sonicated for another 10 minutes(avoid heat generated by the sonicator).

The contents of the flasks were allowed to cool and volume was completed with methanol.

A suitable portion of this mixture was centrifuged at 1500 rpm for 10 minutes, 2 ml of the

clear supernatant solution were transferred into 100 ml volumetric flask and the volume

was completed to the mark with 0.1 N sodium hydroxide.

2.4.2 Capsules

The content of twenty capsules was accurately weighed and mixed well. A quantity of the

resulted powder equivalent to about 250 mg chlorzoxazone was accurately weighed and

transferred into a 100 ml volumetric flask, 70 ml methanol were added and the mixture was

mechanically shaken for 20 minutes then the volume was made to the mark with methanol.

A suitable portion of this mixture was centrifuged at1500 rpm for 10 minutes, 2 ml of the

clear supernatant solution were transferred into 100 ml volumetric flask and the volume

was completed to the mark with 0.1 N sodium hydroxide.

2.5 Procedures:

2.5.1 Optimum wavelength selection

Two separate solutions each containing 150 µg/ml of PCT and CLX were prepared by

diluting 5 ml from their respective standard stock solutions to 100 ml with 0.1 N NaOH.

The UV spectra of the two solutions were recorded over the range 220-320 nm. From the

obtained spectra the pairs of wavelengths satisfying the requirement of Eq.8 for PCT and

Eq. 12 for CLX were selected from the spectra.

2.5.3 Linearity at the selected wavelengths

A calibration series was prepared for PCT and CLX by transferring different volumes from

their respective standard working solutions (100 µg/mL) into two separate series of 50-mL

volumetric flasks, then the volume was completed using 0.1 N NaOH. The prepared

solutions were scanned in the range of 220–320 nm and the absorbance values at the

selected two pairs of wavelength (244, 270.6 nm) and (255, 277.4 nm) was plotted against

their corresponding concentration. Slope and other regression line parameter were

calculated.

2.5.4 General procedure

Two ml of the sample solution/synthetic mixture were transferred into each of six 25 ml

volumetric flasks; each of the six flasks was spiked with a different volume of working

standard mixture (2, 3, 4, 5, 6 ml) except one. The volumes of the flasks were made to mark

with 0.1 N NaOH. The absorbance of each solution was measured at the selected

wavelength pair. Concentrations of PCT and CLX in the samples were obtained from

intersection of straight line (obtained by plotting ΔA value against the added concentration)

with the negative side of the abscissa.

All graphs and calculations were performed using Microsoft Excel spread sheet.

2.5.5 Accuracy:

Six synthetic mixtures containing different ratios of PCT and CLX were prepared by

mixing different volumes from their stock solutions.

2.5.6 Precision:

2.5.6.1Repeatability:

Six homogeneous tests samples from the tablet formulation were determined using the

proposed method following the general procedure described earlier.

2.5.6.2 Intermediate precision:

The determination was repeated on another six samples on different day using freshly

prepared reagent and standards.

3.1 Optimum wavelengths selection

Figure 8: Individual spectra of Chlorzoxazone 10 µg/ml mg/ml and Paracetamol10

µg/ml.

Paracetamol showed equal absorbance at the wavelengths 244nm and 270.6nmaccordingly

it has been selected for the determination of CLX while chlorzoxazone gave equal

absorbanceat255nm and 277.4nm, hence was chosen for the determination of PCT.

The suitability of each selected wavelengths was confirmed by comparing the calibration

graphs (Fig. 10 and 11) produced for each analyte at the two wavelengths where it gave

equal absorbance (i.e. 244 nm and 270.6nm for paracetamol,255nm and 277.4nm for

chlozoxazone).

The obtained calibration plots were linear, approximately superimposed on each other

and their slope ratios were close to one Table 3.These findings confirmed the suitability

of the selected pairs for the two analytes according to HPSM as the requirement of Eq. 8

and 12 above were satisfied(56).

Table 1: Linearity data at 244 nm and 270.6 nm for Paracetamol:

Paracetamol

Cnoc(mg/ml) Absorbance

ΔA 244 nm 270.6 nm

0.002 0.120 0.120 0.000

0.004 0.232 0.233 -0.001

0.006 0.356 0.356 0.000

0.008 0.468 0.471 -0.003

0.010 0.581 0.586 -0.005

0.012 0.700 0.706 -0.006

Figure 9: Calibration curve of Paracetamol at λ 244nm and 270.6nm

Table ( 2 ) Linearity data at 255 nm and 277.4 nm for Chlorzoxazone:

Chlorzoxazone

Cnoc(mg/ml) Absorbance

ΔA 255 nm 277.4 nm

0.004 0.100 0.097 0.003

0.006 0.153 0.150 0.003

0.008 0.205 0.199 0.006

0.010 0.256 0.250 0.006

0.012 0.306 0.297 0.009

0.014 0.354 0.343 0.011

y = 57.986x + 0.0036R² = 0.9998

y = 58.629x + 0.0016R² = 0.9999

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.005 0.01 0.015

ab

sora

nce

conc (mg/ml)

Paracetamol at λ 270.6nm and 244nm

270.6nm

244nm

Figure 10: Calibration curve of Chlorzoxazone at λ 255 nm and 277.4 nm

Table 3: Linearity results of PCT and CLX at the selected wavelength

Parameter Paracetamol Chlorzoxazone

Wavelength (nm) 244 nm 270.6 nm 255 nm 277.4 nm

Concentration range (µg/ml) (2 to 12) µg/ml (4 to 14) µg/ml

Slope (b) 57.986 58.6286 25.429 24.6

Slope ratio 0.99 1.03

Intercept (a) 0.0036 0.0016 0.0001 0.0013

Coefficient of determination (R²) 0.9998 0.9999 0.9997 0.9994

y = 25.429x + 0.0001R² = 0.9997

y = 24.6x + 0.0013R² = 0.9994

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 0.005 0.01 0.015

Ab

sorb

ance

Conc(mg/ml)

Chlorzoxazone at λ 255nm and 277.4nm

255 nm

277.4 nm

Table 4: Linearity data at 255 nm and 277.4 nm for Paracetamol:

Paracetamol

Cnoc(mg/ml) Absorbance

ΔA 255 nm 277.4 nm

0.002 0.144 0.095 0.049

0.004 0.282 0.185 0.097

0.006 0.430 0.284 0.146

0.008 0.568 0.374 0.194

0.010 0.705 0.466 0.239

0.012 0.852 0.564 0.288

Figure 11: Calibration curve of paracetamol at λ 255nm and 277.4nm

Table5: Linearity data at 244 nm and 270.6 nm for Chlorzoxazone:

Chlorzoxazone

Cnoc(mg/ml) Absorbance

ΔA 244 nm 270.6 nm

0.004 0.219 0.055 0.164

0.006 0.338 0.084 0.254

0.008 0.449 0.112 0.337

0.010 0.563 0.140 0.423

y = 70.671x + 0.0021R² = 0.9999

y = 46.829x + 0.0002R² = 0.9998

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 0.002 0.004 0.006 0.008 0.01 0.012 0.014

Ab

sorb

an

ce

Conc(mg/ml)

Paracetamol at λ 255nm and 277.4nm

255 nm

277.4 nm

0.012 0.675 0.165 0.510

0.014 0.778 0.191 0.587

Figure 12: Calibration curve of chlorzoxazone at λ 244nm and 270.6nm

Table 6: Linearity results of PCT and CLX at the selected wavelength

Parameter Paracetamol at Chlorzoxazone at

Wavelength (nm) 255 nm 277.4 nm 244 nm 270.6 nm

Concentration range (µg/ml) (2 to 12) µg/ml (4 to 14) µg/ml

Slope (b) 70.67 46.828 56 13.58

Slope ratio 1.5 4.12

Intercept (a) 0.00213 0.0002 -0.00033 0.0013

Coefficient of determination (R²) 0.9999 0.9998 0.9996 0.9992

3.2 Suitability of HPSAM application

The calibration graphs obtained for each analyte at the two wavelengths were found to be

suitable for its analysis as they produced straight lines with large enough slope difference

(Table 6), accordingly the requirement for application of HPSAM is satisfied (56).

3.3 Analysis of laboratory synthetic mixtures:

To check the accuracy of the method six synthetic samples containing different ratios of

PCT and CLX were prepared from their stock solutions and analysed following the

general procedure.

Table 7: Equations of crossed line for each synthetic mixture:

y = 56x - 0.0003R² = 0.9996

y = 13.586x + 0.0022R² = 0.99930

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 0.005 0.01 0.015

Ab

sorb

ance

Conc(mg/ml)

Chlorzoxazone at lamda 244nm and 270.6nm

244 nm

270.6 nm

Table 8: Analysis result of the synthetic mixtures using ΔA values

Table 9: Summary of synthetic mixture analysis results

Test Chlorzoxazone Paracetamol

1 101.15 101.26

2 102.08 98.33

3 99.38 98.13

4 103.60 97.50

5 102.33 99.67

6 98.40 99.80

Averg 101.16 99.12

STDV 1.95 1.38

RSD 1.92 1.40

The results showed good agreement between the expected concentration and the actual

ones with low relative standard deviation, indicating that the method is accurate enough to

be applied for the analysis of the two analytes in dosage forms.

3.4 Precision

3.4.1 Repeatability

The concentration of each active was graphically obtained from the intersection of the

straight line (obtained by plotting ∆A value against the added concentration) with negative

side of the x-axis as shown representative graphs Fig 12 and 13 for PCT and CLX

determination, respectively. The concentration of each analyte was also obtained by

solving its corresponding linear regression equation ∆A is equal to zero or simply it is equal

to intercept/slope of the regression line.

The method showed good repeatability for determination of both CLX and PCT. The assay

values were 104.29% and 100.94% for CLX and PCT with relative standard deviation

1.68% and 2.38% respectively. These results are summarized in Table 12.

Table 10: Paracetamol -added concentration in µg/ml and ∆A of each test:

Added

Conc(µg/ml)

Paracetamol

∆A (255-277.4 nm)

Test-1 Test-2 Test-3 Test-4 Test-5 Test-6

0.0 0.119 0.122 0.121 0.117 0.121 0.119

1.2 0.150 0.153 0.151 0.148 0.151 0.148

1.8 0.163 0.168 0.166 0.162 0.164 0.163

2.4 0.178 0.182 0.182 0.178 0.180 0.178

3.0 0.194 0.196 0.196 0.192 0.194 0.191

3.6 0.208 0.211 0.211 0.207 0.207 0.207

Figure 13: Linear regression plot of added concentration (µg/ml)against ∆A for

Paracetamol

0

0.05

0.1

0.15

0.2

0.25

-6.0 -4.0 -2.0 0.0 2.0 4.0 6.0

∆A

Added concentration in µg/ml

Test-1

Test-2

Test-3

Test-4

Test-5

Test-6

Table 11: Chlorzoxazone; Added concentration in µg/ml and ∆A of each test:

Added

conc(µg/ml)

Chlorzoxazone

∆A (244-270.6 nm)

Test-1 Test-2 Test-3 Test-4 Test-5 Test-6

0.0 0.170 0.168 0.167 0.172 0.170 0.171

1.2 0.219 0.217 0.215 0.223 0.221 0.219

1.8 0.241 0.243 0.240 0.248 0.243 0.245

2.4 0.265 0.266 0.265 0.272 0.268 0.269

3.0 0.290 0.290 0.289 0.296 0.293 0.290

3.6 0.314 0.313 0.313 0.319 0.314 0.315

Figure 14: Linear regression plot of added concentration (µg/ml)against ∆A for

chlorzoxazone

Table 12: Results of the repeatability analysis using ΔA method

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

-6.0 -4.0 -2.0 0.0 2.0 4.0

∆A

Added concentration in µg/ml

Test-1

Test-2

Test-3

Test-4

Test-5

Test-6

3.4.2 Intermediate precision:

The method showed good intermediate precision analysis results for the determination of

both CLX and PCT. The assay values were 103.00 % and 102.29% for CLX and PCT

with relative standard deviation 1.68% and 2.29 % respectively. These results are

summarized in Table 13.

There were no significant difference (at P = 0.05 , n = 6 ) between the analysis results

obtained on two different days , the calculated t-value was less than the t- tabulated as

shown in Table 14. This confirms the precision of the proposed method(57).

Table 13: Results of the intermediate precision analysis using ΔA method

Table 14:Statistical analysis of the precision data

% of labeled amount

Chlorzoxazone Paracetamol

sample day1 day2 day1 day2

1 106.50 105.00 100.00 102.90

2 103.25 104.50 102.08 97.92

3 102.75 103.75 100.42 104.17

4 102.50 102.75 97.06 104.17

5 104.50 101.00 104.17 101.67

6 106.25 101.00 101.88 102.92

Average 104.29 103.00 102.29 100.94

STDEV 1.76 1.72 2.34 2.40

RSD% 1.68 1.67 2.29 2.38

Calculated t 1.29 0.99

Tabulated t 2.23 2.23

P = 0.05 n = 6

3.5 Capsules

The proposed method was also applied for the determination of CLX and PCT in capsules

formulation.

The results obtained were in good agreement with the labeled amounts100.08% and

100.04% with relative standard deviations of 1.60% and 1.37% for CLX and PCT

respectively. This supports the suitability the proposed method for the determination of

CLX and PCT in capsules formulation as well.

Table 15: Results of capsules analysis using ΔA method

Table 16: Summary of capsules analysis of results

Test Chlorzoxazone Paracetamol

1 99.75 102.29

2 101.00 100.42

3 98.25 99.38

4 98.50 98.13

5 100.50 99.79

6 102.50 100.21

Average 100.08 100.04

STDEV 1.60 1.37

RSD% 1.60 1.37

4.1 Conclusion

It was observed that PCT and CLX in their mixture have the overlapping absorption spectra

in the spectral region of 220 and 320 nm. Therefore, the simultaneous spectrophotometric

determination of PCT and CLX substances in their synthetic and commercial tablets and

capsules was performed by using this method. The proposed method does not require prior

separation step. From the previous discussion, it could be concluded that the proposed

method is accurate, precise and, unlike the other spectrophotometric methods applied on

this mixture, can cancel the matrix effect during the analysis of PCT and CLX in their

available dosage form. The method is suitable and valid for application in laboratories

lacking liquid chromatographic instruments.

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