National Ribat University

Faculty of Graduate Studies and Scientific research

Multi-wavelength Spectrophotometric Determination

of Rifampicin and Isoniazid in Tablets

A Thesis Submitted in Partial Fulfillment of the Requirements for

Master Degree in Drug Quality Control

By: Samah Abdalla Nasr Ginawi

Supervisor: Dr. Imad Osman Abu Reid

2017

I

Dedication

I dedicated this research with all my love and appreciation to……

My parents: Allah rests their souls in heaven.

My brothers.

My little family.

II

Acknowledgements

I wish to express my gratitude for the assistance and guidance given to me by my

supervisor Dr. Imad. I am also grateful to Prof. Alrasheed (master cordinator) and all the

staff of the faculty of pharmacy at Al Ribat National University.

III

Contents

Dedication

Acknowledgements

Table of Contents

List of tables

List of figures

Abbreviations

Abstract (English)

Abstract (Arabic)

i

ii

iii

v

v

vi

vii

viii

Chapter 1: Introduction and Literature Review

1.1 Introduction

1

1.2 Theoretical background 2

1.3 Objectives 4

1.4 Literature Review 5

Chapter 2: Materials and Methods

2.1 Chemicals and Standards

10

2.2 Instruments

10

2.3 Samples and standard solutions preparation 10

2.3.1 Stock Standard solutions 10

2.3.2 Linearity standards 10

IV

2.3.3 Working standards 11

2.3.4 Laboratory synthetic mixtures 11

2.3.5 Sample preparation

11

2.4 General procedure

11

Chapter 3: Results and Discussion

3.1 Linearity over the selected wavelengths range 12

3.2 Determination of synthetic mixtures (accuracy) 13

3.3 Analysis of commercial sample 15

Chapter 4: Conclusion and References

4.1 Conclusion 17

4.3 References 18

V

List of Tables

Table 1. Rifampicin linearity data at selected wavelengths 13

Table 2. Isoniazid linearity data at selected wavelengths 13

Table 3. The absorbance data at the selected wavelengths 14

Table 4. The absorbance ratio data at the selected wavelengths 14

Table 5. The accuracy results of the synthetic mixtures 14

Table 6. Samples weight taken

15

Table 7. The absorbance data at the selected wavelengths (samples) 15

Table 8. The absorbance ratio data at the selected wavelengths

(samples)

16

Table 9. The assay results of samples 16

List of Figures

Figure 1. Chemical structure of Rifampicin 5

Figure 2. Chemical structure of Isoniazide 6

Figure 3. UV spectra of rifampicin (200 µg/ml) and isoniazide

(100µg/ml) in methanol:water.

12

VI

Abbreviations

UV Ultraviolet

M Molar concentration

MLRA Multi-Wavelength Linear Regression Analysis

RIF Rifampicin

INH Isoniazid

Nm Nanometer

RP-HPLC Reverse-Phase High Performance Liquid Chromatograph

PIPE Piperine

C18 Octadecylsilane

V/V Volume/Volume

min. Minute

ml Milliliter

ILS Inverse least squares

CLS Classical least squares

RPLC-PDA Reversed Phase Liquid Chromatography with Photo Diode Array

Detector

PEIC phenethyl isocyanate

FDC fixed-dose combination

HPLC High Performance Liquid Chromatograph

HPTLC High Performance Thin layer Chromatography

GCE Glassy carbon electrode

ECD Electrochemical detector

PLSR Partial least squares regression

µg Microgram

VII

Abstract

Rifampicin is a first line medication used as an anti-tubercular agent. It is active against

gram positive and gram negative bacteria.

Isoniazid is an anti-tubercular drug, which is mostly used in the treatment and

prevention of tuberculosis.

A simple, accurate and inexpensive method have used for the determination of

rifampicin and isoniazid in tablets. The method depend on utilizing the slope and the

intercept of the straight line obtained by plotting the ratio of the sample absorbances by

that of rifampicin standard of a known concentration against the absorbances ratio of

isoniazid standard and rifampicin standard.

The recovery of rifampicin (RIF) from the synthetic mixture was (100.28 - 106.35 %),

while the recovery of isoniazid (INH) was (101.64 -107.34 %).

The results obtained by applying the method to the analysis of the two analytes in

tablets were in good agreement with the label claim, 93.21% and 101.58% with relative

standard deviations of 0.118 % and 1.837 % for RIF and INH respectively.

VIII

لخالصةا

في عالج السل، وهو فعال ضد البكتريا الموجبه والسالبه لصبغه جرام. االول الدفاع خطريفامبسين هو

والعالج من السل. ايزونيازيد هو من اكثر االدوية استخداماً للوقايه

تم استخدام طريقه بسيطه، دقيقه و غير مكلفه لتحديد كمية الريفامبسين و االيزونيازيد في االقراص.

تعتمد الطريقه علي استخدام ميل وقاطع الخط المستقيم المتحصل عليه عن طريق تمثيل نسبة إمتصاص العينة

نسبة إمتصاص محلول االيزونيازيد المرجعي للريفامبسين لتركيز الريفامبسين المرجعي علي المحور السيني ضد

المرجعي علي المحور الصادي.

%100.72-%100.27ريفامبسين و 93.33%-%93.12النسبة المتحصل عليها بعد تحليل العينه هي

ايزونيازيد.

ه جداً مع ادعاء النتيجة المتحصل عليها بعد تطبيق هذه الطريقه لتحليل المركبين في االقراص تعتبر متوافق

%1.837-%0.118، وانحراف معياري نسبي %100.58و %93.21الشركة المنتجه لالقراص وهي

للريفامبسين وااليزونيازيد علي التوالي.

Chapter One

Introduction and Literature Review

1

1.1 Introduction:

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

When rationally formulated, fixed-combination drugs may produce greater convenience,

greater patient acceptability, multiple action, fewer side effects, lower cost, and sometimes

greater efficacy and safety (1).

The combination of drugs has therapeutic advantages; however, the combination of drugs

brings new challenges to the pharmaceutical industry with respect to stability studies of

combined drugs and their simultaneous analysis.

Different analytical techniques can be applied for multi-component analysis including;

spectrophotometry, chromatography and electrophoresis.

The use of traditional methods like extraction is quite difficult because extraction

techniques require large solvent consumption; with accompanying risks of analyte loss or

contamination, and possibility of incomplete separation, and above all the procedure may

be expensive and time consuming (2).

UV spectrophotometric techniques are mainly used for simultaneous multicomponent

analysis thus minimizing the cumbersome task of separating interferants and allowing the

determination of an increasing number of analytes, consequently reducing analysis time

and cost (3).

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 (4).

Multicomponent UV spectrophotometric methods are based on recording and

mathematically processing absorption spectra. They offer the following advantages:

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 10-5M and moderate to high selectivity (5).

2

1.2 Theoretical Consideration:

A number of methods have been developed to determine the composition of a binary

mixture spectrophotometrically. Most of these are directed at mixtures where one

component can be isolated from the other or they require a Beer’s law experiment to

measure the molar absorptivity of each of the substances in the mixture. However, Blanco,

et. al.( 6) described a method of resolving mixtures with overlapping spectra, called Multi-

Wavelength Linear Regression Analysis (MLRA) , without determining molar

absorptivities or complicated mathematics. Using Blanco’s method, the composition of a

binary mixture with overlapping spectra can be resolved with only three measurements, the

absorbance of a standard solution for each component, and the unknown mixture itself.

Assuming additivity, the absorbance of a mixture is the sum of the absorbances of its

components. If we have a mixture consisting of two components, 1 and 2, with an

unknown concentration of C 1 and C 2, then: Absorbance of the unknown mixture, A

mixture = A 1 + A 2 but applying Beer’s law: A1= Є1bC1 and A2 = Є2bC2

Substituting: A mixture = Є1bC1+ Є2bC2.

However, the absorbances of standard solutions of the same substances will follow the

same Beer’s law relationship and have the same molar absorbance, Є, and one centimeter

path length, b, as the unknown solutions under the same conditions.

Therefore, we can write:

A standard 1 = Є1bC standard 1 and A standard 2 = Є2bC standard 2

Rearranging these relationships:

Є1b = A standard1

C standard1 and Є2b =

A standard2

C standard2

Substituting,

A mixture = A standard1

C standard1 C1 + A standard 2 =

A standard2

C standard2 C2

Or

A mixture = C1

C standard1 A standard1 +

C2

C standard2 A standard2

3

Dividing by 𝐀 𝐬𝐭𝐚𝐧𝐝𝐚𝐫𝐝𝟏 and simplifying we obtain:

A mixture

C standard1 =

C1

C standard1+

C2

C standard2 𝑥

A standard2

A standard1

Therefore, a plot of

A mixture

C standard1 𝑣𝑒𝑟𝑠𝑢𝑠

A standard2

A standard1

Will give

a slope = C2

C standard2 and intercept =

C1

C standard1

That is, the concentration of the unknown component 2 (C2) in the mixture, equals the

slope times the concentration of the standard solution for component 2. Likewise, the

concentration of the unknown component 1 (C1) in the mixture equals the product of the

intercept times the concentration of the standard solution for component1.

Or simply

C1= intercept x C standard 1 and C2 = slope x C standard 2

4

1.3 Objectives:

The objectives of this research were:

To develop a new sectrophotometric method based on multi-wavelength linear

regression to overcome the problem of spectral overlap.

To apply this developed method for the estimation of RIF and INH combined tablets.

Rationale:

The individual spectra of rifampicin (RIF) and isoniazid (INH) shows considerable

overlapping over the wavelength range of 230 to 320 nm, accordingly application of the

classical spectrophotometric techniques for their determination in combined dosage form is

not possible.

5

1.4 Literature Review:

Rifampicin (RIF) is chemically, (7S, 9E, 11S, 12R, 13S, 14R, 15R, 16R, 17S, 18S, 19E,

21Z) -2, 15, 17, 27, 29 - pentahydroxy-11-methoxy-3, 7, 12, 14, 16, 18, 22-

heptamethyl-26-{(E) -[(4 methylpiperazin1yl) imino] methyl}-6,23-dioxo-8,30-dioxa-24

azatetracyclo [23.3.1.14,7.05, 28] triaconta 1 (28), 2, 4, 9, 19, 21, 25 (29), 26 –

octane – 13 - yl acetate. (Fig.1)

It is a semisynthetic derivative of Rifamycin B, obtained from Streptomyces mediterranei,

is an antibiotic used to treat a several types of bacterial infections.

Rifampicin is used for the treatment of tuberculosis in combination with other antibiotics,

such as pyrazinamide, isoniazid and ethambutol (7).

Figure 1: Chemical structure of Rifampicin

Isoniazid is a synthetic derivative of nicotinic acid also known

as isonicotinylhydrazide (INH); chemically it is Pyridine -4- carboxylic acid hydrazide.

(Fig.2)

It is pyridine carboxylic acid derivative and a synthetic analog of pyridoxine (8).

INH is a prodrug; mycobacterial catalase-peroxidase converts INH into an active

metabolite (9).

Is an antibiotic used as a first-line agent for the prevention and treatment tuberculosis. It is

widely used together with rifampicin, ethambutol and pyrazinamide among others, for the

chemotherapy of tuberculosis (10).

Figure 2: Chemical structure of isoniazid

6

The two drugs combination is not official in any pharmacopeia; hence no official analytical

method is available for their determination in combination.

Literature revealed that many methods are available for the determination of the two drugs

in combination or in combination with other drugs such as:

Simultaneous estimation of rifampicin and isoniazidin combined dosage form by UV

spectrophotometric method includes simultaneous equation method using 337.0 nm and

263.0 nm λ max of rifampicin and isoniazid respectively (11).

Spectrophotometric method was described for the determination of isoniazid and

rifampicin in their pure forms, pharmaceutical preparations and biological fluids. Method

used direct UV spectrophotometric measurement and the absorbencies at 264 and 474 nm

were used for isoniazid and rifampicin respectively (12).

Two methods were described for the determination of rifampicin and isoniazid in mixtures

by visible spectrophotometry and first derivative ultraviolet spectrophotometry. The

absorbance at 475 nm in buffer solution pH 7.4 was employed to determine rifampicin

after applying the three-point correction technique between 420 and 520 nm, while the

amplitude of the first-derivative spectrophotometric spectrum at 257 nm in HCl 0.012 M

was selected for the determination of isoniazid (13).

UV Spectrophotometric and RP- HPLC methods for simultaneous estimation of isonizid,

rifampicin and piperine in pharmaceutical dosage form were proposed. The

spectrophotometric method was based on absorption correction for the simultaneous

estimation of INH, RIF and PIPE in UV and Visible region using methanol and distilled

water as solvents. The wavelengths selected for the analysis were 262, 338 and 477 nm for

INH, PIPE and RIF respectively. In the RP – HPLC method successful separation of drugs

was achieved on LC18 100 A⁰ column (250 x 4.6 mm, 5 μ) using 0.01M Sodium

Dihydrogen Orthophosphate, pH 6.5 and acetonitrile (40:60, % v/v) as mobile phase with

flow rate of 0.9 mL/min. The wavelength of detection was 282nm (14).

Simultaneous determination of pyridoxine hydrochloride, isoniazid, pyrazinamide and

rifampicin in pharmaceutical formulation was performed on a 250 × 4.6 mm I.D.C18

column packed with 5 mm-in-size particles applying gradient elution with a mobile phase

composed of acetonitrile (A) and 15 mmol L-1 potassium dihydrogen phosphate buffer of

pH adjusted to 4.0 ± 0.1 with o-phosphoric acid (B). A: B ratio was 11:89 v/v for the initial

4.5 min, and then it was maintained at 50:50 v/v; the flow rate was 1 mL min-1. UV

detection was performed at 235 nm (15).

7

Two chemometric assisted UV spectrophotometric(inverse least square (ILS) and classical

least square (CLS)) and one RPLC-PDA (Reversed Phase Liquid Chromatography with

Photo Diode Array Detector) were found to be appropriate for determination of isonizid,

rifampicin and piperine in ternary mixture. The wavelength range 220-360nm with the

intervals of 10nm (Δλ=10nm) at 15 wavelength points. For the chemometric calibration, 20

ternary solutions were prepared as training set and 10 ternary solutions were prepared as

validation set. The chemometric methods do not require any separation step. The

chromatographic separation was achieved on a reversed-phase, Phenomenex Luna C18

column (250X4.6 mm, 5μ particle size). Gradient elution was carried out with a mobile

phase of 0.05M Disodium hydrogen phosphate buffer pH -7.0 (solution-A) and

Acetonitrile (solution-B).Chromatography was performed at ambient temperature using a

flow rate of 0.8 ml/min and a run time of 12 min. The flow rate was maintained at 0.8 ml

min−1, with PDA detection at 290nm for INH, RIF and PIP based on peak area (16).

HPLC/UV method for simultaneous quantification of four constituents in anti-tuberculosis

tablets by pre-column derivatization, using phenethyl isocyanate (PEIC) was described for

the simultaneous determination of the four anti-tuberculosis constituents: pyrazinamide,

isoniazid, rifampicin and ethambutol hydrochloride in anti-tuberculosis 4-FDC (fixed-dose

combination) tablets. The derivatives were efficiently separated using a mobile phase

gradient consisting of acetonitrile-phosphate buffer (8 mM, pH 6.8) at a flow rate of 1.0

ml/min. Quantification of constituents was carried out at wavelength 210 nm (17).

The High-performance liquid chromatographic method with gradient elution coupled to a

glassy carbon electrode (GCE)-based wall-jet/thin-layer electrochemical detector (ECD)

was described for the simultaneous analysis of isoniazid and rifampicin. The simultaneous

HPLC-ECD analysis of INH and RIF was performed using a reversed phase C18 column

(150 mm×4.6 mm, 5mm) using a gradient elution program at a flow rate of 1.0mL min-1,

UV detector wavelength was fixed at 268 nm and the ECD was placed behind the UV

detector, set at 0.9 V. The column was maintained at 40 ͦ C throughout the analysis and the

injection volume was 20μL (18).

Stability-indicating high-performance thin-layer chromatographic method has been

established and validated for analysis of isoniazid and rifampicin both as the bulk drugs

and in formulations. The compounds were separated on aluminum-backed silica gel 60

F254plates with n-hexane–2-propanol–acetone–am-monia–formic acid, 3:3.8:2.8:0.3:0.1

(v/v) as mobile phase was found to give compact spots for isoniazid and rifampicin (RF

8

values 0.59 ± 0.02 and 0.73 ± 0.04, respectively). Densitometric analysis of isoniazid and

rifampicin was performed at 254 nm (19).

A least-squares method in the matrix form was described for the simultaneous

determination of rifampicin and isoniazid in a mixture. The method allows the rapid

analysis of binary pharmaceutical formulations with minimum error. The concentration of

each component in the mixture has been determined spectrophotometrically by measuring

the absorbance of the mixture at 5-nm intervals from 230 to 290 nm. This method uses a

personal computer to solve the mathematical equations and to determine the drugs of

interest in each other’s presence in the dosage form with least error (20).

Partial least squares regression (PLSR) was used for the simultaneous quantification of

rifampicin (RIF) and isoniazid (INH) by visible spectrophotometry using a simple

derivatization reaction. In the presence of neocuproine, copper (II) is reduced by isoniazid

to a Cu (I)-neocuproine complex, which shows an absorption maximum at 455 nm. Under

these conditions, RIF shows an absorption maximum at 449 nm (21).

Simultaneous determination of rifampicin, isoniazid and pyrazinamide in combined

pharmaceutical dosage forms was achieved by first derivative spectroscopy. Rifampicin

was determined by measuring the signal at Zero crossing point for isonizid and

pyrazinamide (262.2 and 268.8 nm), isonizid was determined from the signal at the zero

crossing point for rifampicin and pyrazinamide (254.0 and 268.8 nm) and pyrazinamide is

determined from the signal at zero crossing point for isonizid and rifampicin (262.2 and

254.0 nm) respectively (22).

RP-HPLC method was developed for determination of rifampicin and isoniazid

simultaneously. The mobile phase consisted of methanol, acetonitrile and water in the ratio

60:20:20(v/v), in which satisfactory peak symmetry, resolved and free from tailing, at a

flow rate of 1 mL/min. The wavelength of detection was 254 nm and the column used was

Kromasil C18, (250 x 4.6 nm, 5 µm) (23).

HPTLC method for the simultaneous estimation of Rifampicin, Isoniazid and Pyridoxine

Hydrochloride in combined tablets dosage form was performed on aluminium plates

precoated with silica gel 60 G F254 as the stationary phase and the mobile phase used was a

mixture of Ethyl acetate: Methanol: Acetone: Acetic acid (5.5: 2.0: 2.0: 0.5, v/v).

Densitometric evaluation of the separated zones was performed using a UV detector at 254

nm in absorbance mode (24).

A spectrophotometric method was developed for the simultaneous determination of

isoniazid (INH) and rifampicin (RIF) in bulk and dosage forms. The method involved the

9

determination of INH in the presence of rifampicin using two wavelengths (238nm &

337nm). Beer’s law was obeyed in the concentration range 2.5-12.5µg/ml and 5-25µg/ml

with good linearity (0.9997 & 0.9999) and satisfactory limits of detection (0.70 &

0.26µg/ml) for INH and RIF respectively (25).

Chapter Two

Materials and Methods

10

2.1 Materials:

2.1 Chemicals and Standards:

Rifampicin and isoniazid were kindly donated by (GMC pharmaceutical industry-

Khartoum-Sudan), the purity of RIF was 99.2% and that of INH was 98.8% and used

without further purification.

(R150 + H 75) film coated tablets manufactured by (LUPIN LTD. Aurangabad,

Maharashtra, India) labeled to contain 150mg rifampicin and 75 mg isoniazid, was

obtained from local pharmacy.

Laboratory distilled water was used throughout the analysis.

Methanol analytical grade(Scharlau, Spain)

Methanol: Water (70:30) was used as diluent.

0.45µm nylon filter.

2.2 Instruments:

UV-Visible Single beam Spectrophotometer Model UV MINI 1240 (Shimadzu – Japan)

Electronic weighing balance (Shimadzu Corporation) was used for weighing the

standards and samples.

Ultrasonic bath

2.3 Samples and standard solutions preparation:

2.3.1 Stock Standards solutions:

Standard stock solutions containing Rifampicin (200 µg/ml) and Isoniazid (110 µg/ml)

were prepared separately, by accurately weighing about 20.0 mg of rifampicin and 11.0 mg

of isoniazid into 100 ml volumetric flask using methanol:water as a solvent.

All the stock solutions were scanned in range between 230-320 nm, to determine the

wavelength of maximum absorption for both the drugs. Rifampicin and isoniazid showed

absorbance maxima at 238 &263 nm respectively.

All the solutions were protected from light and were analyzed on the day of preparations.

2.3.2 Linearity standards:

Separate linearity standards of the two analytes were prepared by proper dilution of

suitable aliquots from their corresponding stock standard solutions with methanol: water to

give concentrations in the range of (4-20 µg/ml, rifampicin) and of (2-10 µg/ml, isoniazid).

11

2.3.3 Working standards:

Working standards were prepared by quantitative dilution with methanol: water of suitable

volumes from the stock standard solutions and used in different parts of the analytical

work.

2.3.4 Laboratory synthetic mixtures

Five synthetic mixtures containing different amounts of rifampicin and isoniazid were

prepared by proper dilution of aliquots from their corresponding stock standard solutions.

2.3.5 Sample preparation:

The twenty tablets were powdered using mortar and pestle. A quantity of the resulted

powder equivalent to about 150mg rifampicin and 75mg isoniazid was accurately weighed

and transferred into a 100 ml volumetric flask, 50 ml methanol: water were added and the

mixture was sonicated for 5 minutes then the volume was made to the mark with

methanol: water , the solution was filtered using 0.45 µm nylon filter. Five ml of the clear

filtrate were transferred into 50 ml volumetric flask and the volume was completed to the

mark with methanol: water, further 5 ml of this solution were transferred into 50 ml

volumetric flask and the volume was completed to the mark with methanol: water.

2.4 General procedure

The absorbences of the working standards, synthetic mixtures and samples were read at 10

nm intervals within the wavelength range of 230- 280 nm.

The concentration of rifampicin and isoniazid in the synthetic mixtures and the samples

were calculated according to the MLRA principle from the slope and intercept of the

straight line, using Microsoft Excel Spreadsheet.

Chapter Three

Results and Discussion

12

3. Results and Discusion:

The individual spectra of rifampicin and isoniazid (Fig.3), showed considerable

overlapping over the wavelength range of 230-320 nm, accordingly application of the

classical spectrophotometric techniques for their determination in combined dosage form is

not possible.

Figure 3: UV spectra of Rifampicin A (200 µg/ml) and Isoniazid B (100 µg/ml)

in Methanol:Water

Multi-wavelength linear regression analysis (6) is one of the approaches that can be used

when the overlapping between the spectra of the two analytes is very extensive. The

application of the techniques requires existence of a linear relation between the

concentration of the analytes and their absorbances over the wavelength range selected and

additivity of their absorbances at each wavelength.

3.1 Linearity over the selected wavelengths range

Both rifampicin (4-20 µg/ml) and isoniazid (2-10 µg/ml) showed good correlation between

the concentration and absorbance at each of the selected wavelength over the range of 230-

320 nm (r > 0.99) and very small intercept, the regression data of the two analytes is

presented in Tables 1 and 2. The presented data suggests possible of application of the

proposed method for the determination of rifampicin and isoniazid in combined in dosage

forms using MLRA (6).

13

Tables 1: Rifampicin linearity data at selected wavelengths

Tables 2: Isoniazid linearity data at selected wavelengths

µg/ml

Absorbance

230

nm

240

nm

250

nm

260

nm

270

nm

280

nm

290

nm

300

nm

310

nm

320

nm

2 0.0.06 0.061 0.063 0.072 0.065 0.043 0.017 0.005 0.008 0.004

4 0.104 0.113 0.127 0.144 0.132 0.085 0.034 0.014 0.012 0.005

6 0.173 0.174 0.188 0.212 0.195 0.128 0.058 0.025 0.018 0.01

8 0.218 0.226 0.25 0.282 0.26 0.171 0.07 0.037 0.023 0.013

10 0.257 0.279 0.306 0.344 0.318 0.209 0.09 0.048 0.026 0.015

Slope 0.025 1.0733 1.109 1.1199 0.930 0.659 0.4352 0.5936 0.430 0.6416

Intercept 0.012 -0.0037 -0.002 0.0016 -0.002 -0.001 -0.0016 -0.0061 0.006 -0.0018

R2 0.995 0.9974 0.999 0.9999 0.999 0.999 0.9962 0.9937 0.994 0.9929

3.2 Determination of synthetic mixtures (accuracy)

The method accuracy was tested by analyzing five laboratory prepared synthetic mixtures

containing different amounts of RIF and INH, the results obtained showed good

agreement between the actual and theoretical amounts of the two analytes.

RIF recovery from the synthetic mixture was (100.28 - 106.35 %), while the recovery of

INH was (101.64 -107.34 %).

The absorbance data at the selected wavelengths, the absorbance ratio data and the

summary of the accuracy results are shown in Tables 3, 4 and 5.

µg/ml

Absorbance

230

nm

240

nm

250

nm

260

nm

270

nm

280

nm

290

nm

300

nm

310

nm

320

nm

4.02 0.167 0.145 0.141 0.134 0.096 0.068 0.058 0.069 0.089 0.107

8.04 0.32 0.308 0.301 0.285 0.207 0.144 0.118 0.127 0.165 0.209

12.06 0.48 0.476 0.464 0.444 0.324 0.239 0.188 0.197 0.235 0.307

16.08 0.655 0.659 0.642 0.62 0.449 0.327 0.27 0.27 0.319 0.418

20.01 0.803 0.817 0.797 0.768 0.547 0.405 0.325 0.33 0.393 0.515

Slope 0.0402 1.0547 0.9752 0.9699 0.7137 0.7489 0.8007 0.9682 1.1446 1.3449

Intercept 0.0014 -0.031 0.000 -0.005 0.0033 -0.007 0.0024 0.0129 0.0129 -0.012

R2 0.9997 0.9999 0.9999 0.9999 0.9998 0.9997 0.9993 0.9997 0.9993 0.9999

14

Table 3: The absorbance data at the selected wavelengths

λ (nm) RIF INH m1 m2 m3 m4 m5

230 0.693 0.239 0.927 0.586 0.347 0.386 0.448

240 0.737 0.263 1.006 0.629 0.373 0.416 0.479

250 0.674 0.258 0.977 0.597 0.357 0.379 0.449

260 0.677 0.324 1.016 0.635 0.413 0.415 0.496

270 0.512 0.306 0.825 0.517 0.365 0.339 0.412

280 0.376 0.198 0.574 0.358 0.248 0.238 0.285

m = synthetic mixture

Table 4: The absorbance ratio data at the selected wavelengths

λ (nm) INH/RIF m1/RIF m2/RIF m3/RIF m4/RIF m5/RIF

230 0.3449 1.3449 0.8456 0.5007 0.5570 0.6465

240 0.3569 1.3650 0.8535 0.5061 0.5645 0.6499

250 0.3828 1.4000 0.8858 0.5297 0.5623 0.6662

260 0.4786 1.5007 0.9380 0.6100 0.6130 0.7326

270 0.5977 1.6113 1.0098 0.7129 0.6621 0.8047

280 0.5266 1.5266 0.9521 0.6596 0.6330 0.7580

Slope 1.0164 0.6162 0.8587 0.4253 0.6360

Intercept 1.0028 0.6381 0.2019 0.4081 0.4248

correlation coefficient 0.9927 0.9838 0.9986 0.9947 0.9993

Table 5: The accuracy results of the synthetic mixtures

Sample Rifampicin (µg/ml) %

content

Isoniazid (µg/ml) % content

Theoretical Found Theoretical Found

m1 20 20.06 100.28 11 11.1804 101.64

m2 12 12.76 106.35 6.6 6.778 102.7

m3 4 4.04 100.95 8.8 9.4457 107.34

m4 8 8.16 102.03 4.4 4.6783 106.33

m5 8 8.5 106.2 6.6 6.996 106

Rifampicin std (g %) 103.16 Isoniazid std (g %) 104.8

15

3.3 Analysis of commercial sample

The proposed method was applied to the analysis of three samples taken from commercial

tablets dosage. The weight of 10 tablets was 3.7144 g, the average weight of tablet was

found to be 0.37144 g.

Results obtained were in good agreement with the labeled amounts 93.21 % and 101.58 %

with relative standard deviations of 0.117692 % and 1.837463 % for RIF and INH

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

RIF and INH in tablets formulation. The tablets analysis data is presented in tables 6-9.

Table 6: Samples weight taken

Table 7: The absorbance data at the selected wavelengths (samples)

λ (nm) RIF INH S1 S2

S3

230 0.840 0.241 0.828 0.830 0.799

240 0.881 0.257 0.880 0.886 0.852

250 0.844 0.289 0.881 0.888 0.854

260 0.795 0.318 0.874 0.879 0.850

270 0.600 0.293 0.71 0.716 0.690

280 0.434 0.185 0.498 0.503 0.484

Sample wt taken

(gm)

mg active

RIF INH

S1 0.3725 150.428 75.214

S2 0.3723 150.347 75.174

S3 0.3721 150.267 75.133

Standard conc. µg/ml 150.3473 75.1736

S = sample

16

Table 8: The absorbance ratio data at the selected wavelengths (samples)

Table 9: The assay results of samples

RSD= Relative Standard Deviation (Std/M*100)

λ (nm) RIF/INH S1/RIF S2/RIF S3/RIF

230 0.2859 0.9822 0.9846 0.947805

240 0.2917 0.9989 1.0057 0.967083

250 0.3424 1.0438 1.0521 1.011848

260 0.4000 1.0994 1.1057 1.069182

270 0.4883 1.1833 1.1933 1.15

280 0.4263 1.1475 1.1590 1.115207

Slope 1.0076 1.0372 1.0027

Intercept 0.7006 0.6971 0.6674

correlation coefficient 0.9857 0.9814 0.9998

Sample Rifampicin ( µg/ml )

% content Isoniazid ( µg/ml )

% content Theoretical Found Theoretical Found

S1 0.00150428 0.0014 93.33 0.00075214 0.00075214 100.76

S2 0.00150347 0.0014 93.12 0.00075174 0.00075174 103.72

S3 0.00150267 0.0014 93.17 0.00075133 0.00075133 100.27

Average 93.21 Average 101.58

Standard deviation 0.11 Standard deviation 1.87

RSD% 0.117 RSD% 1.837

Chapter Four

Conclusion and References

17

4.1 Conclusion:

The MLRA is a straightforward procedure allowing the accurate resolution of binary

mixtures of compounds with overlapped 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 equipments are not affordable.

The accuracy and simplicity of the method suggest it suitability in cases where quick

results are demanded e.g. as in-process analysis procedure during blend analysis in

industrial setups.

18

4.2 References:

1. Crout JR, Fixed combination prescription drugs: FDA policy, J Clin Pharmacol, 1974;

14(5‐6): 249-254.

2. Bozdoǧan A, Acar AM, Kunt GK, Simultaneous determination of acetaminophen and

caffeine in tablet preparations by partial least-squares multivariate spectrophotometric

calibration), Talanta, 1992; 39(8): 977-979.

3. Saldanha TC,de Araújo MU,Neto BB,Chame HC. Simultaneous analysis of Co2+,

Cu2+, Mn2+, Ni2+and Zn2+ in the ultraviolet region Using 4 -(pyridil-2-azo) resorcinol

and

multivariate calibration. Anal Lett, 2000; 33(6): 1187-1202.

4. Korany MA, Wahbi AM, Mandour S, Elsayed MA. Determination of certain drugs in

multicomponent formulations by first derivative ultraviolet spectrophotometry). Anal Lett,

1985; 18: 21-34.

5. Skoog DA, Holler FJ, Crouch SR. Principles of Instrumental Analysis. 6th. ed., Canada;

Thomson Corporation: 2007.

6. Blanco.M, H. Iturriaga, S. Maspoch, and Tarin.P, MLRA , J Chemical Education178-

180)

7. Florey K. Analytical profiles of drug Substances, Vol.5, Academic Press, New York,

2005; 467 – 479.

8. Harvey RA, Champe PC. Pharmacology, 2nd ed., Lippincott Williams and Wilkins,

2000; pp. 331‐335.

9. Rang HP, Dale MM, Pitter JM, Flower RJJ. “RANG AND DALE’S Pharmacology”.

2007. 6th edn. Page 675.

10. Shinkich Shimizu et al. Ulmanns Encyclopedia of Industrial Chemistry, John

Wiley and sons, 2007.

11. Arifa Begum SK, Basava Raju D and Rama Rao N. Simultaneous estimation of

rifampicin and isoniazid in combined dosage form by a simple UV spectrophotometric

method. Scholars Research Library Der Pharmacia Lettre. 2013; 5 (3):419-426.

12. Barsoum N Barsoum, Manal S Kamel and Mohamed M A Diab.

Spectrophotometric Determination of Isoniazid and Rifampicin from Pharmaceutical

Preparations and Biological Fluids. Research Journal of Agriculture and Biological

Sciences. 2008; 4(5): 471-484.

19

13. Benetton S A, Kedor-Hackmann E.R.M. , Santoro M.I.R.M., Borges V.M.

Visible spectrophotometric and first-derivative UV spectrophotometric determination of

rifampicin and isoniazid in pharmaceutical preparations, Talanta .1998; 47 (3): 639 – 643.

14. Umag Shah, Ankith Jasani. UV Spectrophotometric and RP- HPLC methods for

simultaneous estimation of Isonizid, Rifampicin and Piperine in pharmaceutical dosage

form. International Journal of Pharmacy and Pharmaceutical Sciences. 2014; 6(10): 274-

280.

15. Dhal S K and Sharma R. Development and Validation of RP-HPLC Method for

Simultaneous Determination of Pyridoxine Hydrochloride, Isoniazid, Pyrazinamide and

Rifampicin in pharmaceutical Formulation. Chem. Anal. (Warsaw), 2009; 54:1487-1500.

16. Mansuri Shakeel S, Pathak Abhishek, Rajput Sadhana J. Development and

Validation of Chemometric Assisted UV Spectrophotometric and RPLC-PDA Methods for

the Simultaneous in Vitro Analysis of Isoniazid ,Rifampicin and Piperine in their

Pharmaceutical Formulation. Indo American Journal of Pharmaceutical Research, 2014;

4(1) 540-553.

17. Wang H, Cai C, Chu C, Liu J, Kong Y, Zhu M, ZhangT. A simple and rapid

HPLC/UV method for simultaneous quantification of four constituents in anti-tuberculosis

4-FDC tablets by pre- column derivatization. Asian Journal of Pharmaceutical Sciences

2012; 7(4): 303-309.

18. Hongling Yan, Yaping Zhou, Qingji Xie, Yi Zhang, Pei Zhang, Hualing Xiao,

Wen Wanga and Shuozhuo Yao. Simultaneous analysis of isoniazid and rifampicin by

high-performance liquid chromatography with gradient elution and wall-jet/thin-layer

electrochemical detection. Anal. Methods, 2014; (6):1530-1537.

19. Ali J., Ali N., Sultana Y., Baboota S., and Faiyaz S. Development and

Validation of Stability-Indicating HPTLC Method for Analysis of Anti-tubercular Drugs.

ACTA CHROMATOGRAPHICA, 2007; (18): 168-179.

20. Kumar K. Mahalanabis and Dipankar Basu. Application of the Least-squares

Method in the Matrix Form: Simultaneous Spectrophotometric Determination of

Rifampicin and lsoniazid in Binary Pharmaceutical Formulations. ANALYST. 1989;

114(10): 1311-1314.

21. Sandra Stets, Talita M. Tavares, Patricio G. Peralta-Zamora, Christiana A.

Pessoab and Noemi Nagata. Simultaneous Determination of Rifampicin and Isoniazid in

Urine and Pharmaceutical Formulations by Multivariate Visible Spectrophotometry. J.

Braz. Chem. Soc. 2013; 24(7): 1198-1205.

20

22. Rote A. R and Sharma A. K. Simultaneous Spectrophotometric Determination of

Rifampicin, Isoniazid and Pyrazinamide by First-Derivative UV Spectrophotometry in

Combined Pharmaceutical Dosage Forms. Indian journal of Pharmaceutical Sciences,

1997; 59(3):119-123.

23. Kumari M. Kusuma, Jyothi K. Kasthuri, Babu B. Hari, Satyanarayana P. V. V.

and Tchaleu B. Ngadjui. A Validated Liquid Chromatographic Method for the

Determination of Rifampicin and Isoniazid in Pharmaceutical Formulations. British

Journal of Pharmaceutical Research, 2015; 7(4): 299-307.

24. Puthusseri S hajahan and Mary Mathew. Validated HPTLC method for

simultaneous estimation of Rifampicin, Isonizid and Pyridoxine hyrochloride in

combined tablet dosage form. World Journal of Pharmaceutical Research, 2014; 3(10):523-

536.

25. Mohamed A.O. Mohamed, Shaza W. Shantier, Magdi A. Mohamed, Elrasheed

A. GadKariem, Esraa M.O. Ismail. Spectrophotometric Method for the Simultaneous

Determination of Isoniazid and Rifampicin in Bulk and Tablet Forms. Int. J. Pharm. Sci.

Rev. Res., 2015; 32(2): 154-156.