Simultaneous Spectrophotometric Estimation of Rifampicin, Isoniazid and Pyrazinamide in their Pharmaceutical Dosage Form

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Asian J. Research Chem. 13(2): March-April 2020
1
ISSN 0974-4169(Print) www.ajrconline.org
0974-4150(Online)
RESEARCHARTICLE
Simultaneous Spectrophotometric Estimation of Rifampicin, Isoniazid and
Pyrazinamide in their Pharmaceutical Dosage Form
Sourav Khawas, Sampurna Parui, Suddhasattya Dey, Dr. Sudip Kr. Mondal, Saptarshy Sarkar*
Dr. B. C. Roy College of Pharmacy and Allied Health Sciences, Durgapur-713206, West Bengal, India
*Corresponding Author E-mail: saptarshysarkar@gmail.com
ABSTRACT:
A simple, accurate, precise, reproducible and economical UV spectrophotometric method was developed and
validated for the estimation of Isoniazid (INH), Rifampicin (RIF) and Pyrazinamide (PYZ) in their bulk and
pharmaceutical dosage form. First order derivative spectrophotometric method was used. Wavelength for Isoniazid
is 721nm (zero crossing point of Rifampicin and Pyrazinamide), for Rifampicin is 322nm (zero crossing point of
Isoniazid and Pyrazinamide) and for Pyrazinamide it is 256nm (zero crossing point of Isoniazid and Rifampicin).
Beer Lambert’s law was obeyed within the concentration range of 0.75-3.75µg/ml for Isoniazid, 1.5-7.5µg/ml for
Rifampicin and 4-12µg/ ml for Pyrazinamide. The proposed method was been validated statistically as per the ICH
guidelines for linearity, accuracy, precision, specificity, LOD and LOQ. The method developed and validated
successfully for the quantitative analysis of Isoniazid, Rifampicin and Pyrazinamide in bulk and dosage form.
KEYWORDS: Isoniazid, Rifampicin, Pyrazinamide, validation, UV Spectroscopy.
1. INTRODUCTION:
Formulations containing Rifampicin, Isoniazid and
Pyrazinamide are widely available in the market. To
perform an assay of these formulations various methods
have been developed, however after going through
various research articles it has been observed that a
method for simultaneous estimation of the combination of
these three drugs by UV-VIS spectrophotometry has not
been developed yet.
Rifampicin1 chemically known as
(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-
methylpiperazin-1-yl)iminomethyl]-6,23-dioxo-8,30-
dioxa-24-azatetracyclo[23.3.1.14,7.05,28]triaconta-
1(29),2,4,9,19,21,25,27-octaen-13-yl acetate having
molecular formula C43H58N4O12 and molecular weight of
822.9 g/mol.
Received on 20.12.2018 Modified on 21.02.2019
Accepted on 04.04.2019 ©AJRC All right reserved
Asian J. Research Chem. 2020; 13(2):
DOI:
Fig 1: Structure of Rifampicin
Rifampicin or rifampin is a semisynthetic derivative of
Rifamycin B obtained from Streptomyces mediterranei. It
is bactericidal to M. tuberculosis and commonly used in
combination with other agents as therapy of tuberculosis.
M. lapreae is highly sensitive but some are moderately
susceptible. Rifampicin is an antibiotic that inhibits DNA-
dependent RNA polymerase which is encoded by rpo B
gene and blocks its polymerizing function. Specifically, it
interacts with bacterial RNA polymerase but does not
inhibit the mammalian enzyme. It is bactericidal and has
a very broad spectrum of activity against most gram-
positive and gram-negative organisms and specifically
Mycobacterium tuberculosis.

Asian J. Research Chem. 13(2): March-April 2020
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Isoniazid2 chemically known pyridine-4-carbohydrazide
having chemical formula of C6H7N3O with a molecular
weight of 137.139g/mol.
Fig 2: Structure of Isoniazid
Isoniazid is the most reliable and most commonly used
medication for tuberculosis. Isoniazid is mycobactericidal
in nature. Isoniazid is a carbohydrazide obtained by
formal condensation between pyridine-4-carboxylic acid
and hydrazine.It derives from an isonicotinic acid.
Isoniazid blocks the synthesis of mycolic acids, major
components of the mycobacterial cell wall. Isoniazid is a
pro drug which gets converted to a reactive metabolite by
the help of enzyme catalase peroxidase (encoded by katG
gene). This free radical inhibits Enoyl ACP reductase
enzyme which is responsible for synthesis of mycolic acid
thus a cell wall deficient mycobacterium is formed,
another minor mechanism by which it inhibits enzyme
DHFRase. So, Isoniazid is a mycobactericidal drug.
Pyrazinamide3 is chemically known as pyrazine-2-
carboxamide having molecular formula C5H5N3O and
molecular weight of 123.113g/mol.
Fig 3: Structure of Pyrazinamide
Pyrazinamide is a first line antituberculotic medication,
but is used only in combination with other antituberculotic
medications such as isoniazid or rifampicin. It is weakly
mycobactericidal but gets converted to a strong one in
acidic medium. It is more active against intracellular
bacilli. Pyrazine carboxamide is a monocarboxylic acid
amide resulting from the formal condensation of the
carboxy group of pyrazinoic acid (pyrazine-2-carboxylic
acid) with ammonia It has a role as an antitubercular agent
and a prodrug. Pyrazinamide kills or stops the growth of
certain bacteria that cause tuberculosis (TB). It is used
with other drugs to treat tuberculosis. It is a highly
specific agent and is active only against Mycobacterium
tuberculosis. The drug is active only at a slightly acidic
pH. Pyrazinamide gets activated to Pyrazinoic acid in the
bacilli where it interferes with fatty acid synthase FAS I.
This interferes with the bacterium ability to synthesize
new fatty acids, required for growth and replication. It has
a good sterilizing activity. Pyrazinamide is particularly
active against slowly multiplying intracellular bacilli
(unaffected by other drugs) by an unknown mechanism of
action. Its bactericidal action is dependent upon the
presence of bacterial pyrazinamidase, which removes the
amide group to produce active pyrazinoic acid.
Pyrazinamide is an important component of multidrug
therapy for tuberculosis.
2. MATERIAL AND METHODS:
2.1. Chemicals and Reagents:
Isoniazid, Rifampicin and Pyrazinamide were used.
Marketed pharmaceutical dose Rifater tablet was used
manufactured by Sanofi India Ltd. containing Isoniazid
50mg, Rifampicin 120mg, Pyrazinamide 300mg.
Methanol of analytical grade were used as solvent
2.2. Instrumentation:
The proposed work was carried out on a Shimadzu UV-
visible spectrophotometer (model UV-1700 series),
which possesses a double beam double detector
configuration with a1 cm quartz matched cell. All
weighing was done on electronic balance.
2.3. Solubility:
The drugs Isoniazid, Rifampicin and Pyrazinamide are
soluble in methanol. Thus, methanol was chosen as the
solvent for developing the medium.
2.4. Introduction to UV Method Development:
Spectrophotometry is largely favoured particularly by
little scale businesses as the expense of the gear is less and
the support issues are insignificant. UV
spectrophotometer principle follows the Beer-Lambert
Law. This law states that whenever a beam of
monochromatic light is passed through a solution with an
absorbing substance, the decreasing rate of the radiation
intensity along with the thickness of the absorbing solution
is actually proportional to the concentration of the solution
and the incident radiation.
2.5. Preparation of Stock Solution:
Standard stock solution of Isoniazid, Rifampicin and
Pyrazinamide was prepared by dissolving 10mg of
Isoniazid in 10ml of methanol to produce a concentration
of 1000µg/ml. 1ml of this stock solution was taken and
then diluted up to 10ml by using methanol to produce a
concentration of 100µg/ml which is the standard stock
solution. Again, 1ml of this stock solution was taken and
then diluted up to 10ml by using methanol to produce a
concentration of 10µg/ml which is the standard stock
solution.
2.6. Preparation of Working Standard Solution:
2.7. From the above stock solution of concentration 10
µg/ml of Isoniazid, 0.75ml was diluted to 10ml with
methanol to prepare a concentration of 0.75µg/ml.
Similarly, 1.5ml, 2.25ml, 3ml & 3.75ml were diluted to

Asian J. Research Chem. 13(2): March-April 2020
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10ml with methanol to prepare concentrations of
1.5µg/ml, 2.25µg/ml, 3µg/ml & 3.75µg/ml respectively.
From the above stock solution of Rifampicin of
concentration 10µg/ml,1.5ml was diluted to 10ml with
methanol to prepare a concentration of 1.5µg/ml.
Similarly, 3ml, 4.5ml, 6ml & 7.5ml were diluted to 10ml
with methanol to prepare concentrations of 3µg/ml,
4.5µg/ml, 6µg/ml & 7.5µg/ml respectively.
From the above stock solution of concentration 10µg/ml
of Pyrazinamide 4ml was diluted to 10ml with methanol
to prepare a concentration of 4µg/ml. Similarly,8ml was
diluted to 10ml with methanol to prepare concentrations
of 8 µg/ml. Then from the above stock solution of
concentration 100µg/ml of Pyrazinamide 1.2ml was
diluted to 10ml with methanol to prepare a concentration
of 12µg/ml. Similarly, 1.6ml & 2ml, were diluted to 10ml
with methanol to prepare concentrations of 16µg/ml &
20µg/ml.
2.8. Determination of zero crossing point:
All the prepared solution of the drugs of different
concentrations were scanned in UV-VIS
Spectrophotometer in the range 800-200nm using
methanol as a blank. After scanning of the samples, the
zero-crossing point for the respective drugs were
determined from the graphs obtained after the scanning by
the UV-VIS Spectrophotometer Zero crossing point is the
point of the graph where the sample shows zero
absorbance. We check for points where two drug samples
show zero absorbance but the third drug sample shows
absorbance at this point. The zero-order graph of
Isoniazid was transformed to first order derivative graph
and the point selected 721nm. For Rifampicin the zero-
order graph was transformed to first order derivative
graph and the point selected was 322nm. For
Pyrazinamide the zero-order graph was transformed to
first order derivative graph and the point selected was 256
nm.
Overlay spectra of the drugs:
Overlay of first order derivative graph of Isoniazid,
Rifampicin and Pyrazinamide
2.9. Preparation of Calibration Curve of Isoniazid:
The calibration curve was prepared by taking the above
solutions of concentration ranging from 0.75-3.75µg/ml.
Then, the calibration curve was plotted by taking
concentration on x-axis and absorbance from the first
order derivative graph obtained after UV-VIS
spectrophotometric scanning of the solutions on the y-
axis. The curve showed linearity in the concentration
range of 0.75-3.75µg/ml. The correlation coefficient (r²)
was found to be 0.9983
2.10. Preparation of Calibration Curve of Rifampicin:
The calibration curve was prepared by taking the above
solutions of concentration ranging from 1.5-7.5µg/ml.
Then, the calibration curve was plotted by taking
concentration on x-axis and absorbance from the first
order derivative graph obtained after UV-VIS
spectrophotometric scanning of the solutions on the y-
axis. The curve showed linearity in the concentration
range of 1.5-7.5µg/ml. The correlation coefficient (r²) was
found to be 0.9967
2.11. Preparation of Calibration Curve of
Pyrazinamide:
The calibration curve was prepared by taking the above
solutions of concentration ranging from 4-20µg/ml. Then,
the calibration curve was plotted by taking concentration
on x-axis and absorbance from the second order derivative
graph obtained after UV-VIS spectrophotometric
scanning of the solutions on the y- axis. The curve showed
linearity in the concentration range of 4-20µg/ml. The
correlation coefficient (r²) was found to be 0.9927.
2.12. Method Validation4:
Validation is a process of establishing documented
evidence, which provides a high degree of assurance that
a specific activity will consistently produce a desired
result or product meeting its predetermined specifications
and quality characteristics. The validation for UV method
development was performed using parameters like
Linearity, Accuracy, Precision, Robustness, Ruggedness,
and Limit of detection (LOD), Limit of quantification
(LOQ) (Table no. 1).
2.12.1. Linearity:
Various aliquots were prepared form the stock solution of
Isoniazid ranging from 0.75-3.75µg/ml, Rifampicin
ranging from 1.5-7.5µg/ml and Pyrazinamide 4-20µg/ml.
The samples were scanned in UV-VIS Spectrophotometer
using methanol as blank. It was found that the selected
drug shows linearity between 0.75-3.75µg/ml in case of
Isoniazid, Rifampicin between 1.5-7.5µg/ml and
Pyrazinamide between 4 - 20µg/ml. (Table 8)
2.12.2. Accuracy:
The accuracy of the method was determined by preparing
solutions of different concentrations that is 80%, 100%
and 120% in which the amount of marketed formulation
was kept constant and the amount of pure drug was varied
respectively. The solutions were prepared in triplicates
and the accuracy was indicated by % recovery. (Table 2)

Asian J. Research Chem. 13(2): March-April 2020
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Fig 4A: Overlay spectra of Isoniazid, Rifampicin and Pyrazinamide
Fig 4B: Calibration curve of Isoniazid
Fig 4C: Calibration curve of Rifampicin
Fig 4D: Calibration curve of Pyrazinamide
Table No.1: Linearity Table of Isoniazid, Rifampicin, Pyrazinamide in Working Standard:
Concentration (µg/ml) (INZ)
Abs (INZ)
Conc. (µg/ml) (RIF)
Abs (RIF)
Conc. (µg/ml) (PYZ)
Abs (PYZ)
0.75
0.0112
1.5
0.0811
4
0.1062
1.5
0.0291
3
0.1176
8
0.2313
2.25
0.0465
4.5
0.1561
12
0.3018
3
0.0687
6
0.2054
16
0.39
3.75
0.0872
7.5
0.2488
20
0.4824
Table no.2: Accuracy Readings of Isoniazid, Rifampicin and Pyrazinamide
OBSERVATION/RESULT
No. of
preparation
s
Conc. (µg/ml)
% Recovery
Statistical Result
Formulation
Pure Drug
Mean
SD
%RSD
Drug
IN
Z
RIF
PY
Z
IN
H
RI
F
P
Y
Z
INH
RI
F
PY
Z
IN
H
RI
F
PY
Z
IN
H
RIF
PY
Z
IN
H
RIF
PY
Z
S1: 80%
3.7
5
7.5
20
3
6
1
6
102.3
10
0.8
100
.4
S2: 80%
3.7
5
7.5
20
3
6
1
6
101.0
8
10
0.6
100
.02
10
1.
1
10
0.9
7
100
.2 9
1.1
5
0.4
7
0.2
0
1.1
3
0.4
6
0 2
0
S3: 80%
3.7
5
7.5
20
3
6
1
6
100.0
1
10
0.5
100
.4
S4: 100%
3.7
5
7.5
20
3.7
5
7.5
2
0
99.7
99.
9
99.
8
S5: 100%
3.7
5
7.5
20
3.7
5
7.5
2
0
99.9
10
0.2
100
.1
99.
9 7
10
0.0
6
99.
93
0.3
0
0.1
5
0.1
5
0.3
0
0.1
5
0.1
5
S6: 100%
3.7
5
7.5
20
3.7
5
7.5
2
0
100.3
10
0.1
99.
9
S7: 120%
3.7
5
7.5
20
4.5
9
2
4
100.2
10
0.2
100
.1
S8: 120%
3.7
5
7.5
20
4.5
9
2
4
100.3
99.
7
100
.3
10
0.
2
99.
93
100
.1 7
0.4
3
0.3
2
0.1
1
0.4
3
0.3
2
0.1
1
S9: 120%
3.7
5
7.5
20
4.5
9
2
4
99.5
99.
8
100
.1

Asian J. Research Chem. 13(2): March-April 2020
5
2.12.3. Precision:
Precision of the technique was exhibited by intraday and
interday variety ponders. In intraday variety contemplate,
6 distinct arrangements of same fixation that is 2.25µg/ml
were set up if there should arise an occurrence of INH, 6
unique arrangements of RIF of focus 4.5µg/ml were
readied, 6 unique arrangements of PYZ of 12 µg/ml and
broke down multiple times in multi day i.e. morning,
evening and evening and the absorbances were noted. The
outcome was demonstrated by % RSD (Table 3). In the
interday variety think about, arrangements of same
fixations were arranged and examined multiple times for
three continuous days and the absorbances were noted.
The outcome was shown by % RSD (Table 3).
2.12.4. Robustness:
Two different analysts performed the scanning of the
samples at different conditions of temperature and the %
recovery followed by % RSD was obtained. (Table 4)
2.12.5. Ruggedness:
Two different analysts performed the scanning of the
samples and after obtaining the % recovery, %RSD was
calculated. (Table 4)
Table no. 3: Intraday Precision and Inter day precision
Conc. (µg/ml)
Recovered Conc. (µg/ml)
1
Recovered Conc. (µg/ml)
2
Recovered Conc. (µg/ml)
3
Avg %RSD
INH
RIF
PY
Z
INH
RIF
PYZ
INH
RIF
PYZ
INH
RIF
PYZ
INH
RIF
PYZ
2.25
4.5
12
2.21
4.41
11.96
2.23
4.42
11.98
2.21
4.45
12.01
2.25
4.5
12
2.21
4.42
11.96
2.23
4.42
12.01
2.23
4.43
11.99
2.25
4.5
12
2.21
4.41
12.01
2.22
4.43
12.01
2.20
4.45
11.99
2.25
4.5
12
2.22
4.42
11.97
2.23
4.42
11.96
2.22
4.5
12.03
2.25
4.5
12
2.20
4.42
11.99
2.21
4.42
11.96
2.23
4.5
11.97
2.25
4.5
12
2.22
4.42
11.99
2.23
4.42
11.97
2.23
4.43
12.05
%RSD
0.34%
0.20%
0.16%
0.40%
0.10%
0.19%
0.57%
0.72%
0.25%
0.43%
0.34%
0.2%
Inter day precision
Conc. (µg/ml)
Day 1
Day 2
Day 3
%RSD
INH
RIF
PYZ
INH
RIF
PYZ
INH
RIF
PYZ
INH
RIF
PYZ
INH
RIF
PYZ
2.25
4.5
12
2.25
4.45
11.96
2.29
4.6
12.0
5
2.24
4.41
12.1
0.74
%
0.79
%
0.38
%
Table 4: Results Showing Robustness & Ruggedness of Method
Room Temp.
Temp. 18°
Conc. (µg/ml)
Recovered Conc. (µg/ml)
Statistical Analysis
(%RSD)
Recovered Conc. (µg/ml)
Statistical Analysis
(%RSD)
INZ
RIF
PY
Z
INZ
RIF
PYZ
INZ
RIF
PY
Z
INZ
RIF
PYZ
INZ
RIF
PY
Z
2.25
4.5
12
2.29
4.42
11.99
1.23%
0.32%
0.19
2.24
4.45
11.96
0.74%
0.79%
0.38
%
2.25
4.5
12
2.21
4.45
12.03
2.29
4.6
12.05
2.25
4.5
12
2.22
4.41
11.09
6
2.25
4.41
12.1
2.12.6. Limit of Detection (LOD):
LOD was determined by signal is to noise ration which is
termed as S/N is 10/3. Therefore, according to the formula
3.3 LOD = LOQ. LOD for Isoniazid was found to be
0.006µg/ml, for Rifampicin 0.047µg/ml and for
Pyrazinamide, 0.0301µg/ml. (Table 5)
2.12.7. Limit of Quantification (LOQ):
LOQ was determined by taking the least concentration
where the machine shows sensitivity i.e. three times to
that of the base line and noted as the LOQ. LOQ was
found to 0.0198µg/ml for Isoniazid 0.15µg/ml for
Rifampicin and Pyrazinamide 0.099µg/ml was recorded.
These are minimum concentration in which these three
drugs can be quantitated. (Table 5)
2.13. Assay of INH, RIF and PYZ tablets
(RIFATER®):
A quantity of powder equivalent to 3.75mg of INH, 7.5mg
of RIF and 20mg of PYZ was taken in a 100ml volumetric
flask and it was dissolved and diluted up to the mark with
methanol. The resultant solution was ultrasonicated for 15
minutes. The solution was then filtered using Whatmann
filter paper No.40. From the filtrate, appropriate dilutions
were made in ethanol to obtain the desired concentration.
This solution was then analysed in UV and the result was
indicated by % recovery given in table 5.

Asian J. Research Chem. 13(2): March-April 2020
6
Fig 5: first order spectra of marketed formulation
3. RESULTS AND DISCUSSION:
The developed method was found to be precise as the %
RSD values for intra-day and inter-day were found to be
less than 2%. Good recoveries (100.4% to 102.3%) of the
drug were obtained at each added concentration,
indicating that the method was accurate. The LOD and
LOQ were found to be in sub-microgram level indicating
the sensitivity of the method. The method was also found
to be robust and rugged as indicated by the % RSD values
which are less than 2%. The results of Assay show that
the amount of drug was in good agreement with the label
claim of the formulation as indicated by % recovery
(101.6%, 100.8% & 101.2%). Summary of validation
parameters of proposed spectrophotometric method is
shown in table 5.
Table No.5: Summary of validation
PARAMETER
RESULT
ISONIAZID
RIFAMPICIN
PYRAZINAMIDE
Linearity indicated by correlation coefficient
0.9983
0.9967
0.9927
Precision indicated by %RSD
0.94%
1.33%
0.551%
Accuracy indicated by % recovery
102.3
100.8
100.4
Limit of Detection
0.006 µg/ml
0.047 µg/ml
0.0301µg/ml
Limit of Quantification
0.0198 µg/ml
0.15µg/ml
0.099µg/ml
Range(µg/mL)
0.75-3.75
1.5-7.5
4-20
Linear regression equation
y=0.0255x-0.0089
y=0.0282x+ 0.0348
y=0.0228x+ 0.029
Robustness indicated by %RSD
0.74%
0.79%
0.38%
Assay indicated by % recovery for tablet
101.6
100.8
101.2
From the above study we can conclude that the proposed
method was accurate, precise, simple, sensitive, robust
and cost effective and can be applied successfully for the
estimation of Isoniazid, Rifampicin and Pyrazinamide in
bulk and marketed formulation. Till date not a suitable
method has been developed for the simultaneous
estimation of these three drugs.
4. ACKNOWLEDGEMENT:
The authors are grateful to the management of Dr. B. C.
Roy College of Pharmacy and A.H.S. for providing
solvents, apparatus, instrument and suitable facilities that
was required to carry out the project work. We would also
like to extend our gratitude to the management of The
Mission Hospital, Durgapur, for providing the required
pharmaceutical products for our project.
5. REFERENCES:
1. https://www.drugbank.ca/drugs/DB00951 accessed on 16/9/2019.
2. https://www.drugbank.ca/drugs/DB01045 accessed on 16/9/2019.
3. https://www.drugbank.ca/drugs/DB00339 accessed on 16/9/2019.
4. ICH, Q2 (R1) validation of analytical procedures: text and
methodology, International conference on harmonization;
Nov.1996. Tripathi KD. Essentials of Medical Pharmacology.7th
ed. New Delhi: Jaypee Brothers; 2013: 765-769.