ArticlePDF Available

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

Authors:
  • IQ City Institute of Pharmaceutical Sciences
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
2
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
3
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
4
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
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.
... The regression coefficient (R 2 ) was 0.9996, and the relative standard deviation (RSD) for all points was below 5%. PZA calibration standards were prepared in methanol (MeOH) at concentrations ranging from 4.0 to 20.0 µg.mL −1 [96]. The R 2 was 0.9992, and the RSD for all points was less than 3%. ...
Article
Full-text available
Background/Objectives: This study aims to develop a sustainable and environmentally friendly drug delivery system by synthesizing a novel drug–drug eutectic mixture (DDEM) of acetylsalicylic acid (ASA) and pyrazinamide (PZA) using a green and efficient mechanochemical approach. Methods: The DDEM was characterized using various techniques, including differential scanning calorimetry (DSC), thermogravimetry and differential thermal analysis (TG-DTA), powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FT-IR), and Raman spectroscopy. Binary phase diagrams and Tammann’s triangle analysis determined the eutectic point. Density functional theory (DFT) calculations were performed on the starting compounds. The new system was evaluated for aqueous solubility, dissolution, and hygroscopicity. Results: A V-shaped binary phase diagram indicated the formation of a DDEM with a 2:1 molar ratio of ASA to PZA. A positive mixing enthalpy suggested a quasi-eutectic structure. The solubility of ASA and PZA increased by 61.5% and 85.8%, respectively, in the DDEM compared to the pure drugs. Conclusions: These findings highlight the potential of DDEMs to enhance drug properties and delivery. The synergistic interaction between ASA and PZA in the eutectic mixture may further improve therapeutic efficacy, warranting further investigation.
... To analyze and control the concentration of PZA in biological body samples, many analytical approaches have been reported. Almost many of the reported approaches are based on chromatography [13][14][15][16] and UV-Vis spectroscopy [17,18], whereas other methods like capillary electrophoresis [19][20][21] and electrochemical sensors [22][23][24] have also been reported for PZA determination. Simioni et al. [24] reported the fabrication of an electrochemical sensor for PZA detection using the nanodiamond dispersion for modification of the GCE surface, while the Rachel Fanelwa group [25] modified the GCE with graphene oxide and AgNPs nanocomposite for PZA detection. ...
Article
Full-text available
Graphite electrode (GE) is an alternative, commercially available, and ready-to-use electrode for a wide range of electroanalytical applications. Electrochemical activation of GE is an efficient step in the preparation of high-performance electrochemical (bio-)sensors. In the present study and the continuation of our research project in the lab about the effect of activation of GE surface on the alteration of electrode structure (formation of different functional groups) and subsequent influence on the sensitive determination of various analytes, a simple and low-cost electrochemical sensor based on the graphite electrode extracted from the battery is developed to measure pyrazinamide (PZA), an antibiotic that is mostly used in treating tuberculosis. Two activation strategies including potentiostatic and potentiodynamic were tested and according to the results, utilizing the potentiodynamic strategy represents good performance in the sensitive detection of PZA. Morphological characterization of activated GE was done using scanning electron microscopy (SEM). A comparison of the effective surface area of the activated and bare GE revealed that the activation process increased the effective surface area of the electrode by 1.6 times. The electroanalytical response of PZA at the activated GE surface was studied utilizing cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques. The impact of various parameters on the reduction current response of PZA was investigated and it was found that the developed electrochemical sensor can successfully determine PZA within the concentration ranges of 1.31–29.81 µM under the optimized conditions and the limit of detection (LOD) was calculated to be 0.89 µM as well. Analysis of real samples such as pharmaceutical formulations and human serum demonstrated excellent recoveries, revealing the promising capability of the proposed sensor for PZA determination.
... Besides these tedious and time-consuming sample treatments, chiral analysis by an indirect method in the enantiomer selective assays of (±)-ABZSO was also performed, in which the fractions of different enantiomers (±) were collected. The enantiomers were again chromatographed through a chiral stationary phase after drying by evaporation, [26,29] except for studies assayed on Chiral Pak AD® column in normal elution mode and other developed methods [36][37][38][39][40]. Therefore to derogate between different steps throughout sample treatment and at the same time to study the major metabolites of albendazole in biological factors, high performance liquid chromatography (HPLC) method was developed and validated for albendazole (ABZ), albendazole sulphoxide (ABZSO) and albendazole sulfone (ABZSO2) in rat plasma. ...
Article
Full-text available
To develop a quick, simple and reproducible dual column high performance liquid chromatography (HPLC) method to determine the albendazole and its metabolites in rat plasma. Albendazole (ABZ), albendazole sulfoxide (ABZSO) and albendazole sulfone (ABZSO2) were analyzed in rat plasma by high performance liquid chromatography using UV-detector. Preparation of plasma samples was carried out by protein precipitation using 8.25% perchloric acid. This method involves two different mobile phases with two different columns and different wavelengths. Estimation of Albendazole was done using Enable C18 column (250 mm × 4.6 mm, 5μm: SpinCo Biotech Pvt. Ltd.), mobile phase acetonitrile: water in the ratio 60: 40, wavelength 225nm and Praziquantel as an internal standard (IS). The retention time for Albendazole and Praziquantel was 3.7 and 6.4 minutes respectively. But the estimation of Albendazole Sulfoxide, Albendazole Sulfone were done by using Phenomenex C18 Luna column (250 mm × 4.6 mm, 5μm: USA), mobile phase acetonitrile: methanol: phosphate buffer (20mM) in the ratio 20: 25: 55. The pH was adjusted to 6.9 using 0.1N NaOH solution, wavelength 290nm and oxfendazole an internal standard (IS). The retention time for Albendazole Sulfoxide, Albendazole Sulfone, and Oxfendazole was 5.5, 7.0 and 8.2 minutes respectively. Both the methods were validated over the range from 0.005-5µg/mL for Albendazole, 0.05-80µg/mL for Albendazole Sulfoxide and Albendazole Sulfone. Both the method showed % RSD and % DEV lower than 15% for all the analytes. The limit of quantitation was 0.005µg/mL for Albendazole whereas 0.05µg/mL for Albendazole Sulfoxide and Albendazole Sulfone. Metabolites of albendazole were analyzed in rat plasma samples using a single dose of Albendazole 50mg/kg was determined application of this method was also used to found the pharmacokinetic studies.
... Besides these tedious and time-consuming sample treatments, chiral analysis by an indirect method in the enantiomer selective assays of (±)-ABZSO was also performed, in which the fractions of different enantiomers (±) were collected. The enantiomers were again chromatographed through a chiral stationary phase after drying by evaporation, [26,29] except for studies assayed on Chiral Pak AD® column in normal elution mode and other developed methods [36][37][38][39][40]. Therefore to derogate between different steps throughout sample treatment and at the same time to study the major metabolites of albendazole in biological factors, high performance liquid chromatography (HPLC) method was developed and validated for albendazole (ABZ), albendazole sulphoxide (ABZSO) and albendazole sulfone (ABZSO2) in rat plasma. ...
... Besides these tedious and time-consuming sample treatments, chiral analysis by an indirect method in the enantiomer selective assays of (±)-ABZSO was also performed, in which the fractions of different enantiomers (±) were collected. The enantiomers were again chromatographed through a chiral stationary phase after drying by evaporation, [26,29] except for studies assayed on Chiral Pak AD® column in normal elution mode and other developed methods [36][37][38][39][40]. Therefore to derogate between different steps throughout sample treatment and at the same time to study the major metabolites of albendazole in biological factors, high performance liquid chromatography (HPLC) method was developed and validated for albendazole (ABZ), albendazole sulphoxide (ABZSO) and albendazole sulfone (ABZSO2) in rat plasma. ...
Article
Full-text available
To develop a quick, simple and reproducible dual column high performance liquid chromatography (HPLC) method to determine the albendazole and its metabolites in rat plasma. Albendazole (ABZ), albendazole sulfoxide (ABZSO) and albendazole sulfone (ABZSO2) were analyzed in rat plasma by high performance liquid chromatography using UV-detector. Preparation of plasma samples was carried out by protein precipitation using 8.25% perchloric acid. This method involves two different mobile phases with two different columns and different wavelengths. Estimation of Albendazole was done using Enable C18 column (250 mm × 4.6 mm, 5μm: SpinCo Biotech Pvt. Ltd.), mobile phase acetonitrile: water in the ratio 60: 40, wavelength 225nm and Praziquantel as an internal standard (IS). The retention time for Albendazole and Praziquantel was 3.7 and 6.4 minutes respectively. But the estimation of Albendazole Sulfoxide, Albendazole Sulfone were done by using Phenomenex C18 Luna column (250 mm × 4.6 mm, 5μm: USA), mobile phase acetonitrile: methanol: phosphate buffer (20mM) in the ratio 20: 25: 55. The pH was adjusted to 6.9 using 0.1N NaOH solution, wavelength 290nm and oxfendazole an internal standard (IS). The retention time for Albendazole Sulfoxide, Albendazole Sulfone, and Oxfendazole was 5.5, 7.0 and 8.2 minutes respectively. Both the methods were validated over the range from 0.005-5µg/mL for Albendazole, 0.05-80µg/mL for Albendazole Sulfoxide and Albendazole Sulfone. Both the method showed % RSD and % DEV lower than 15% for all the analytes. The limit of quantitation was 0.005µg/mL for Albendazole whereas 0.05µg/mL for Albendazole Sulfoxide and Albendazole Sulfone. Metabolites of albendazole were analyzed in rat plasma samples using a single dose of Albendazole 50mg/kg was determined application of this method was also used to found the pharmacokinetic studies
... One of the major components of leprosy MDT is rifampicin 5 , which in certain case in several countries, M. leprae resistance toward this drug issue has been reported albeit only a few 1 . Rifampicin has been standardized and formulized for leprosy, thus could be combined with another treatments [6][7][8][9] . Rifampicin also utilized in tuberculosis and hepatic damage patients as well, so this is considered semi-broad spectrum antibiotic 10,11 . ...
Article
Full-text available
Leprosy persists to be a health problem in Indonesia, especially in the provinces of North Maluku, West Papua and Papua. Early diagnosis and complete treatment with multidrug therapy (MDT) remain the key strategy for reducing the disease burden. One of the major components of MDT is rifampicin which in certain cases in several countries, M. leprae resistance to this drug issue has been reported albeit only a few. This research aimed to detect and analyze polymorphism in M. leprae rpoB gene that was isolated from leprosy patients in three provinces: North Maluku Province, West Papua Province and Papua Province, Indonesia. The identification of mutations in the M. leprae rpoB gene was carried out by aligning the results of DNA sequencing with the reference strain. The 3D structure of rpoB was derived using the Swiss Model. The T450A, S456L, and H451Y variants of RNA Polymerase B subunits were constructed using FoldX based on the wild-type structure. The structures were repaired, and protein stability was evaluated using foldX under the Yasara viewer. The QC of the rpoB M. leprae homology models was conducted with Ramachandran Plot modeling using PROCHECK. The difference in binding affinity between native protein and T450A, S456L, and H45I variants were analyzed using molecular docking. rpoB gene of M. leprae contains a mutation found in nucleotide of 1348 bp. The mutation triggered the conversion of the amino acid Threonine to Alanine in the amino acid to 450 rpoB subunit B. The structure of 3D RNA Polymerase Subunit B was constructed using rpoB Mycobacterium tuberculosis with PDB code 5UH5 as template. According to Ramachandran Plot, the percentage of residues in the most favored regions are 91.9%, and there was no significant number of residues in the disallowed regions. The results of molecular docking showed that the T450A variant had the same binding affinity with the native protein which was-8.9 kcal. Binding affinity on the S456L and H451Y variants increased by-7.3 kcal and-8.2 kcal, respectively. According to Molecular Docking analysis, T450A variant did not affect the energy binding between RNA polymerase and rifampicin.
Article
Efficient and cost-effective UV spectrophotometric techniques have been devised using methanol and phosphate buffer pH 7.4 to quantify Rifampicin concentrations in the Self Nanoemulsifying Drug Delivery System (SNEDDS), as shown by the drug loading efficiency. Rifampicin has a peak wavelength of 474nm with a concentration of 10-50µg/mL. The relationship between the concentration and the absorbance is linear, as shown by a high correlation value (r) of 0.9998. The average percent recovery of Rifampicin falls between the range of 99.138% to 101.349%. The approach devised is suitable for analyzing Rifampicin in nanoparticle formulations.
Article
Tuberculosis may be considered as a grave and persistent threat to global health.One of the major challenges in the treatment of tuberculosis is drug resistance, or sometimes, multiple drug resistance.The combination of Pyridoxine HCl, Isoniazid, Trimethoprim, and Sulfamethoxazole has recently been approved by a regulatory agency as a potent treatment for multiple drug-resistant tuberculosis.Hence a successful attempt was made to develop and validate a novel reverse phase high performance liquid chromatographic method for simultaneous determination of Pyridoxine HCl, Isoniazid, Trimethoprim, and Sulfamethoxazole.A mixture of acetonitrile and phosphate buffer in a ratio of 10:90 was employed as the mobile phase for the first 4 minutes in gradient programming, followed by adjusting the ratio of acetonitrile and phosphate Buffer to 25:75 until 8 minutes while maintaining the pH of the mobile phase at 4 throughout the measurement.With this developed method, the retention times of Pyridoxine HCl, Isoniazid, Trimethoprim, and Sulfamethoxazole were found to be 3.19± 0.0574, 3.65±0.0374, 4.88±0.0167, and 7.96±0.0989, respectively. The range of values was found to be 5µg/ml to 30µg/ml, 10µg/ml to 30µg/ml, 12µg/ml to 36µg/ml, and 10µg/ml to 50µg/ml for Pyridoxine HCl, Isoniazid, Trimethoprim, and Sulfamethoxazole, respectively. All the system suitability parameters and validation parameters results were within the acceptance criteria, in accordance with ICH guidelines for analytical method validation.
Article
There are several limitations in using AFB and GeneXpert to evaluate the treatment of TB patients, one of which is influenced by sputum quality. Therefore, an alternative method is needed to help evaluate the treatment of TB patients. This study aimed to analyze the correlation of the Procalcitonin test with AFB and GeneXpert for evaluating the treatment of TB patients and the performance of Procalcitonin as a marker of TB patient treatment progress. A prospective cohort study was conducted from May to September 2022 at the West Nusa Tenggara General Hospital, Indonesia. Sputum and blood samples were collected from 36 patients who were confirmed positive for TB by GeneXpert MTB/RIF examination, then examined for procalcitonin and AFB before being given treatment and after the intensive phase of treatment. Procalcitonin tested with VIDAS Biomerieux and VIDAS BRAHMS PCT kit. Procalcitonin did not correlate with AFB (p=0.064, r= 0.327) and GeneXpert before treatment (p=0.169, r=0.245), but correlated with AFB (p=0.013, r=0.427) and GeneXpert MTB/RIF (p=0.020, r=0.405) after the intensive phase of treatment. Procalcitonin test with a cut-off value of 0.07 detected negative AFB cases after treatment with a sensitivity of 28.6 and a specificity of 96.2%. The procalcitonin cut-off value of 0.07 also detected negative Xpert MTB/RIF after treatment with a sensitivity of 16.7% and a specificity of 100%. The performance of Procalcitonin for detecting negative smear and negative Xpert MTB/RIF after the intensive phase of treatment is classified as having high specificity, but its sensitivity is still low. Future studies are needed to evaluate the performance of Procalcitonin compared to bacterial cultures.
Article
Nitrosamine impurities have been detected in various pharmaceutical products in recent days. Various sartans, ranitidine, nizatidine and metformin have been recalled from the markets due to the high limit of nitrosamine impurities. When assessing the danger of human cancer, pharmaceutical products ability to regulate potentially mutagenic and carcinogenic contaminants is crucial. The risk of their mutagenic and carcinogenic potential has increased with the recent finding of nitrosamine impurities in various commercially available medications. Nitrosamine is the substance deemed to be a potential human carcinogen by the International Agency for Research on Cancer (IARC). Impurities in nitrosamines have been shown to be mutagenic and carcinogenic; even very low exposure levels to these impurities can cause cancer. These impurities may be created by a reagent, catalyst, solvent, or raw materials employed in the manufacturing process and end up in drug substances or drug products. Angiotensin II receptor blocker (ARB) medications with nitrosamine impurities have caused widespread health problems. Risk management of nitrosamine impurity is necessary to control the level of this impurity in drug substance, drug product and APIs. By using risk management tools like fishbone diagram which is used to identify and organize the possible sources of nitrosamines in medicines. For detection of nitrosamine impurity different countries develops their own analytical methods.
ResearchGate has not been able to resolve any references for this publication.