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
117
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, 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,14 R,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 01.12.2019 Modified on 18.01.2020
Accepted on 10.02.2020 ©AJRC All right reserved
Asian J. Research Chem. 2020; 13(2):117-122.
DOI: 10.5958/0974-4150.2020.00024.3
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
118
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.

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Similarly, 1.5ml, 2.25ml, 3ml and3.75ml were diluted to
10ml with methanol to prepare concentrations of
1.5µg/ml,2.25µg/ml, 3µg/ml and 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 of1.5µg/ml.
Similarly, 3ml, 4.5ml, 6ml and 7.5ml were diluted to
10ml with methanol to prepare concentrations of 3µg/ml,
4.5µg/ml, 6µg/ml and 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 and 2ml, were diluted to
10ml with methanol to prepare concentrations of
16µg/ml and 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
120
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 preparations
Conc. (µg/ml)
% Recovery
Formulation
Pure Drug
Drug
INZ
RIF
PYZ
INH
RIF
PYZ
INH
RIF
PYZ
S1: 80%
3.75
7.5
20
3
6
16
102.3
100.8
100.4
S2: 80%
3.75
7.5
20
3
6
16
101.08
100.6
100.02
S3: 80%
3.75
7.5
20
3
6
16
100.01
100.5
100.4
S4: 100%
3.75
7.5
20
3.75
7.5
20
99.7
99.9
99.8
S5: 100%
3.75
7.5
20
3.75
7.5
20
99.9
100.2
100.1
S6: 100%
3.75
7.5
20
3.75
7.5
20
100.3
100.1
99.9
S7: 120%
3.75
7.5
20
4.5
9
24
100.2
100.2
100.1
S8: 120%
3.75
7.5
20
4.5
9
24
100.3
99.7
100.3
S9: 120%
3.75
7.5
20
4.5
9
24
99.5
99.8
100.1
Table no.2: Cont……
OBSERVATION/RESULT
No. of preparations
Statistical Result
Mean
SD
%RSD
Drug
INH
RIF
PYZ
INH
RIF
PYZ
INH
RIF
PYZ
S1: 80%
S2: 80%
101. 1
100.9 7
100.2 9
1.1 5
0.4 7
0.2 0
1.1 3
0.4 6
0 2 0
S3: 80%
S4: 100%
S5: 100%
99.9 7
100.0 6
99.93
0.3 0
0.1 5
0.1 5
0.3 0
0.1 5
0.1 5
S6: 100%
S7: 120%
S8: 120%
100. 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%

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121
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)
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 3.3LOD= LOQ.LOD for Isoniazid was found to
be 0.006µg/ml, for Rifampicin0.047µg/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.
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
PYZ
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
12.05
2.24
4.41
12.1
0.74
%
0.79
%
0.38
%
Table 4: Results Showing Robustness and 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
PYZ
INZ
RIF
PYZ
INZ
RIF
PY
Z
INZ
RIF
PYZ
INZ
RIF
PYZ
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
11.096
2.25
4.41
12.1

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122
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
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% and 101.2%). Summary of
validation parameters of proposed spectrophotometric
method is shown in table 5.
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.