IAMSPH 3 (1) (2022) Pages:.

95-101

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International Archives of Medical Sciences and Public Health

Published: Pena Cendekia Insani

Determination of Isoniazid and Pyridoxine Hydrochloride Levels in

Tablets with Ultraviolet Spectrophotometry by Successive Ratio Derivative
1 2
Rida Evalina Tarigan Yustifin Sarumaha
1,2
Department of Pharmaceutical Chemistry, Institut Kesehatan Helvetia Medan

Article Info ABSTRACT

Article history: Isoniazid and pyridoxine hydrochloride are a combination of anti-
Received : 12 October 2021 tuberculosis drugs that are often used as standard therapy. The
Revised : 19 Nopember 2021 advantage of this drug combination is to obtain treatment
Accepted : 09 December 2021 effectiveness and prevent the emergence of resistance. Examination of
the quality of medicinal preparations, determination of the content
of efficacious substances is an important part so that a reliable
Keywords: analytical method is needed as well as tools and operational costs
Isoniazid, Pyridoxine that are relatively cheaper, easy to implement, but can provide
Hydrochloride, Ultraviolet results with good accuracy and precision. This study aims to
Spectrophotometry, Successive determine the levels of isoniazid and pyridoxine hydrochloride in
Ratio Derivative tablet preparations using successive ratio derivative ultraviolet
spectrophotometry. isoniazid (10 g/mL) was then transformed into
derivative 1 at 4. The results showed that the levels of isoniazid
101.4313% and pyridoxine hydrochloride 104.7125%. Based on the
results of the research conducted, it can be concluded that the levels
of isoniazid and pyridoxine hydrochloride meet the requirements
for levels according to the Indonesian Pharmacopoeia Edition VI.
This is an open access article under the CC BY-SAlicense.

Corresponding Author:
Rida Evalina Tarigan,
Institut Kesehatan Helvetia Medan
Email: [email protected]

1. INTRODUCTION
In general, Anti Tuberculosis Drugs (OAT) used are isoniazid, rifampin, ethambutol,
pyrazinamide and streptomycin. This group of drugs is called the primary drug used as
standard therapy. To obtain the effectiveness of treatment and prevent the emergence of
resistance, it is necessary to avoid the use of monotherapy, by combining several types of
drugs in sufficient quantities and in appropriate doses according to the treatment category.
However, without quality assurance, the bioavailability of the drug will be reduced, the risk of
toxicity or under- dose and over-dose can facilitate the development of drug resistance (1).

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  E-ISSN: 2798-5156 P-ISSN: 2798-

Determination of levels of efficacious substances in medicinal preparations is an important

part in agencies that carry out drug levels determination such as the Food and Drug
Supervisory Agency (BPOM) and the drug industry so that reliable analytical methods are
needed as well as tools and operational costs that are relatively cheaper and easier to
implement but can be implemented. gives good results and precision (2). The assay of the
single form of isoniazid and pyridoxine hydrochloride tablets can be determined by the high
performance liquid chromatography (HPLC) method (3). Isoniazid can be determined by
ultraviolet spectrophotometry in aqueous solution at a wavelength of 266 nm and in 0.1N HCl
at a wavelength of 265 nm. Pyridoxine hydrochloride can be determined by ultraviolet
spectrophotometry in a solution of pH 7 at a wavelength of 254 nm and in 0.1N HCl at a
wavelength of 291 nm (4).
Research that has been carried out by Solanki, et al (2015) in the development of two new
spectrophotometric methods in determining the ternary mixture of formulation tablets used as
antihypertensive therapy Olmesartan Medoxomil (OLM), Amlodipine Besilate (AMLO), and
Hydrochlorthiazide (HCTZ) with methanol solvent using two the same method, namely the
double divisor ratio spectra method (DDRSM) and successive ratio derivative (SRD),
provides accurate, sensitive, simple, precise and reliable results. This method does not require
a long preparation time, a large number of solvents, and can determine the compound drug
mixture without prior separation (5).
Research conducted by Youssef S. H, et al (2018) in the analysis of paracetamol,
pseudoephedrine and cetirizine in capsule preparations using spectrophotometric techniques,
with double distilled water solvent using the SRD method, which gives accurate, selective and
sensitive results. This method does not require a long preparation time, a large number of
solvents, and can determine overlapping spectra at the same time without prior separation (6).

2. RESEARCH METHODE
This research was conducted at the USU Faculty of Pharmacy Research Laboratory in
October-November 2021. The tools used in this research are complete ultraviolet-visible
spectrophotometry (Shimadzu 1800) with a personal computer (PC) equipped with UV probe
2.42 software, MS Excel, sonicator (Branson), analytical balance (Sartorius), measuring flask
(Pyrex). , measuring cup (Pyrex), beaker glass (Pyrex), maat pipette (kimax), mortar and
pestle. The materials used in this study were tablets containing isoniazid 400 mg and
pyridoxine hydrochloride 10 mg Pehadoxin Forte® (PT. Pharpros), isoniazid (BPFI),
pyridoxine hydrochloride (BPFI), 0.1 N HCl and aquadest. The samples of this study were
tablets containing isoniazid 400 mg and pyridoxine hydrochloride 10 mg Pehadoxin Forte®
(PT. Pharpros) which were obtained from a pharmacy in Medan city. The data obtained is in
the form of absorbance spectrum measurement results which are calculated with the help of
UV Probe and Ms Excel software.

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 International Archives of Medical Sciences and Public 
3. RESULT AND ANALYSIS

Absorption Spectrum
0,5165

0,4000

0,2000

0,0000
200,00 250,00 300,00 350,00 400,00
nm.
0,8794

0,8000

0,6000

0,4000

0,2000

0,0000
200,00 250,00 300,00 350,00 400,00
nm.

0,8794

0,8000

0,6000

0,4000

0,2000

0,0000
200,00 250,00 300,00 350,00 400,00
nm.

Image 1. Absorption spectrum (a) Isoniazid (b) Pyridoxine Hydrochloride, Maximum

absorption spectrum (c) Isoniazid (d) Pyridoxine Hydrochloride(e) overlapping spectrum
Isoniazid 10µg/mL Pyridoxine Hydrochloride 10 g/mL and standard mixture

Derivative Ratio Spectrum

  E-ISSN: 2798-5156 P-ISSN: 2798-

226,0103

0,0000

-500,0000

-1000,0000

-1120,8019
200,00 250,00 300,00 350,00 400,00
nm.

2713,5306

2000,0000

1000,0000

0,0000

-439,8367
200,00 250,00 300,00 350,00 400,00
nm.

Image 2. Spectrum ratio derivatives (a) isoniazid (b) pyridoxine hydrochloride

This method begins with the selection of a divisor and analysis wavelength. The
concentration of the divider used in this study is isoniazid 10 g/mL and pyridoxine
hydrochloride 10 g/mL. After dividing all the spectra by their respective spectra, the ratio
spectrum of isoniazid and pyridoxine hydrochloride is obtained. , then the spectrum is derived
so that the derivative ratio spectrum is obtained to determine the analysis of isoniazid and
pyridoxine hydrochloride (8).

Determination of Wavelength Analysis

1,3499

1,0000

0,0000

-1,0000

-1,2573
200,00 250,00 300,00 350,00 400,00
nm.

Figure3. Overlapping spectrum ratios of isoniazid and pyridoxine hydrochloride derivatives

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International Archives of Medical Sciences and Public 

In determining the wavelength of isoniazid, the zero-order spectrum of isoniazid is divided

by the spectrum of pyridoxine hydrochloride (10 g/mL) to record the first ratio spectrum and
then converted to derivative 1 at 4, then the stored spectrum is further divided by pyridoxine
hydrochloride (10 g/mL ) to record the second ratio spectrum and the result is transformed into
derivative 1 at 4. Then the maximum or minimum wavelength is selected which gives the best
correlation value, in which case the wavelength is 266.0 nm with a correlation coefficient value of
0.9966 given in the spectrum of derivative 1 at 4. In determining the wavelength of pyridoxine
hydrochloride, the zero-order spectrum of pyridoxine hydrochloride is divided by the spectrum
of isoniazid (10 g/mL) to record the first ratio spectrum and then converted to derivative 1 at
4, then the stored spectrum is divided again with isoniazid (10 g/mL) to record the second ratio
spectra ua and the result is transformed into derivative 1 at 4. Then the maximum or minimum
wavelength that gives the best correlation value is selected, in which case the wavelength is 279.0
nm with a correlation coefficient value of 0.9990 given to the derivative 1 spectrum at 4 (8)(11).

Method Validation
Accuracy, Precision (RSD), Linearity, Limit of Detection (LOD) and Limit of Quantity (LOQ)
Table 1. Accuracy, Precision (RSD), Linearity, Limit of Detection (LOD) and Limit of
Quantity (LOQ)

No. Parameter Metode successive ratio derivative

Isoniazid Piridoksin Hidroklorida
1 Akurasi (%) 98,7909 100,0205
2. Presisi (RSD)(%) 0,3485 0,1013
3. Linearitas (r) 0,9966 0,9990
4. LOD (μg/mL) 1,0747 1,3687
5. LOQ (μg/mL) 3,5833 4,5626
Table 1 shows that the successive ratio derivative method meets the validation
requirements for the parameters of accuracy, precision, linearity as well as the limit of
detection (LOD) and limit of quantification (LOQ). very good between concentration and
absorbance. This indicates that as the concentration increases, the absorbance value also
increases. The accuracy value obtained shows that this method meets the requirements for
method validation (accuracy value requirements are 98%-102%)(7)(9)( 12). Precision shows
that the method gives results that are close to each other even though it has been tested in
several replications. The precision parameter is reflected from the resulting RSD value, and
from the results obtained the successive ratio derivative method meets the validation
requirements (RSD <2 %) (7). The detection limit is the lowest analyte concentration in the
sample that can still be detected, while the quantitation limit is defined as the lowest analyte
concentration in the sample that can still meet the careful and thorough criteria (8)(10)(13).

Determination of the concentration of a mixture of isoniazid and pyridoxine hydrochloride in

tablet preparations
Table 2. Levels of isoniazid and pyridoxine hydrochloride
Persyaratan
Successive Ratio
Klaim Farmakope Edisi
Komponen Derivative
Dilabel (mg) VI
2020
% Mg % mg
Isoniazid 101,4313 405 400 90-110 360-440
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Piridoksin 104,7125 10 10 95-115 9,5-11,5

Hidroklorida
Table 2 shows that the tablet preparations meet the requirements where the content of the
substance is in the range of 90-110% for isoniazid and 95-115% for pyridoxine hydrochloride
according to the Indonesian Pharmacopoeia edition VI (2020), this shows that the successive
ratio derivative method can determine the levels in tablet preparations. with overlapping
spectra without prior separation (5)(11).

4. CONCLUSION
Based on the research conducted, it can be concluded that the determination of the levels of
isoniazid and pyridoxine hydrochloride in tablet preparations using the ultraviolet
spectrophotometric method using successive ratio derivatives meets the requirements of the
Indonesian Pharmacopoeia Edition VI..

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International Archives of Medical Sciences and Public 

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