Використання тетраметилбензидину як індикатора у ензиматичному методі кількісного визначення етонію
The study considers the possibility of using 3,3',5,5'-tetramethylbenzidine (TMB) as an indicator in the enzymatic analysis for the quantitative determination of quaternary ammonium compounds on the example of ethonium. The feasibility of using TMB as an indicator in the kinetic photometri...
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| Published in: | Журнал органічної та фармацевтичної хімії |
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| Date: | 2024 |
| Volume: | 22 |
| Issue: | 4 |
| Pages: | 36-42 |
| ISSN: | 2518-1548 |
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| author | Kovalska, Olena V. Blazheyevskiy, Mykola Ye. Diadchenko, Vladyslav V. |
| author_facet | Kovalska, Olena V. Blazheyevskiy, Mykola Ye. Diadchenko, Vladyslav V. |
| author_institution_txt_mv | [
{
"author": "Olena V. Kovalska",
"institution": "National University of Pharmacy of the Ministry of Health of Ukraine",
"orcid": ""
},
{
"author": "Mykola Ye. Blazheyevskiy",
"institution": "National University of Pharmacy of the Ministry of Health of Ukraine",
"orcid": ""
},
{
"author": "Vladyslav V. Diadchenko",
"institution": "Military Institute of Armored Forces of the National Technical University “Kharkiv Polytechnic Institute”",
"orcid": ""
}
] |
| author_sort | Kovalska, Olena V. |
| baseUrl_str | https://ophcj.nuph.edu.ua/oai |
| collection | OJS |
| container_end_page | 42 |
| container_issue | 4 |
| container_start_page | 36 |
| container_title | Журнал органічної та фармацевтичної хімії |
| container_volume | 22 |
| datestamp_date | 2026-08-23T15:46:26Z |
| description | The study considers the possibility of using 3,3',5,5'-tetramethylbenzidine (TMB) as an indicator in the enzymatic analysis for the quantitative determination of quaternary ammonium compounds on the example of ethonium. The feasibility of using TMB as an indicator in the kinetic photometric method has been confirmed. Kinetic curves showing the relationship between the optical density and the ethonium concentration have been constructed. The reaction rates of acetylcholinesterase inhibition by ethonium have been estimated using the tangents of the angles of these curves with TMB as an indicator. The degree of enzyme inhibition has been calculated, and a linear relationship between the ethonium concentration and the degree of inhibition has been determined. This method was applied to determine the ethonium content in 0.1% ethonium gel. The relative standard deviation of the method does not exceed 2.5 %. The approach suggested offers a reliable and accurate method for the quantitative analysis of ethonium in dosage forms. |
| doi_str_mv | 10.24959/ophcj.24.321795 |
| first_indexed | 2025-07-23T04:43:38Z |
| format | Article |
| fulltext |
ISSN 2308-8303 (Print) / 2518-1548 (Online) 36
Original Research
http://ophcj.nuph.edu.ua
UDC 577.15:5615.2:547.2333.4
M. Ye. Blazheyevskiy1, O. V. Kovalska1, V. V. Diadchenko2
1 National University of Pharmacy of the Ministry of Health of Ukraine,
53 Hryhorii Skovoroda str., 61002 Kharkiv, Ukraine
2 Military Institute of Armored Forces of the National Technical University “Kharkiv Polytechnic Institute”,
2 Kyrpychova str., 61002 Kharkiv, Ukraine
The Use of Tetramethylbenzidine as an Indicator in the
Enzymatic Quantitative Determination of Ethonium
Abstract
The study considers the possibility of using 3,3’,5,5’-tetramethylbenzidine (TMB) as an indicator in the enzymatic analysis for
the quantitative determination of quaternary ammonium compounds on the example of ethonium. The feasibility of using
TMB as an indicator in the kinetic photometric method has been confirmed. Kinetic curves showing the relationship between
the optical density and the ethonium concentration have been constructed. The reaction rates of acetylcholinesterase inhibi-
tion by ethonium have been estimated using the tangents of the angles of these curves with TMB as an indicator. The degree
of enzyme inhibition has been calculated, and a linear relationship between the ethonium concentration and the degree
of inhibition has been determined. This method was applied to determine the ethonium content in 0.1 % ethonium gel.
The relative standard deviation of the method does not exceed 2.5 %. The approach suggested offers a reliable and accurate
method for the quantitative analysis of ethonium in dosage forms.
Keywords: ethonium; quaternary ammonium compounds; cholinesterase; acetylcholine
M. Є. Блажеєвський1, O. В. Ковальська1, В. В. Дядченко2
1 Національний фармацевтичний університет Міністерства охорони здоров’я України,
вул. Григорія Сковороди, 53, м. Харків, 61002, Україна
2 Національний технічний університет «Харківський політехнічний iнститут»,
вул. Кирпичова, 2, м. Харків, 61002, Україна
Використання тетраметилбензидину як індикатора в ензиматичному методі кількісного
визначення етонію
Анотація
У дослідженні розглянуто можливість використання 3,3’,5,5’-тетраметилбензидину (ТМБ) як індикатора в ензиматично-
му аналізі для кількісного визначення четвертинних амонієвих сполук на прикладі етонію. Підтверджено можливість
застосування ТМБ як індикатора в кінетичному фотометричному методі. Побудовано кінетичні криві, що демонстру-
ють залежність між оптичною густиною та концентрацією етонію. Швидкість реакції інгібування ацетилхолінестерази
етонієм було оцінено за тангенсами кутів цих кривих, із цим ТМБ використано як індикатор. Розраховано ступінь інгібу-
вання ферменту, а також визначено лінійну залежність між концентрацією етонію та ступенем інгібування. Цей метод за-
стосовували для визначення вмісту етонію в 0,1 % гелі етонію. Відносне стандартне відхилення методу не перевищує
2,5 %. Запропонований підхід забезпечує надійний та точний метод кількісного аналізу етонію в лікарських формах.
Ключові слова: етоній; холінестераза; ацетилхолін; четвертинні амонієві сполуки
Citation: Blazheyevskiy, M. Ye.; Kovalska, O. V.; Diadchenko, V. V. The Use of Tetramethylbenzidine as an Indicator in the Enzymatic
Quantitative Determination of Ethonium. Journal of Organic and Pharmaceutical Chemistry 2024, 22 (4), 36 – 42.
https://doi.org/10.24959/ophcj.24.321795
Received: 15 August 2024; Revised: 25 November 2024; Accepted: 11 December 2024
Copyright© 2024, M. Ye. Blazheyevskіy, O. V. Kovalska, V. V. Diadchenko. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0).
Funding: The authors received no specific funding for this work.
Conflict of interests: The authors have no conflict of interests to declare.
ISSN 2308-8303 (Print) / 2518-1548 (Online) 37
Journal of Organic and Pharmaceutical Chemistry 2024, 22 (4)
■ Introduction
3,3′,5,5′-Tetramethylbenzidine (TMB) is a wi-
dely used chromogen that is valued for its non-
carcinogenic nature and, upon oxidation, gives pro-
ducts with high absorption coefficients. This ma-
kes TMB a key component in enzyme-based as-
says, offering superior sensitivity and low de-
tection limits compared to many commercially
available chromogenic reagents. Notably, TMB
is the most widely used chromogenic substrate
in ELISA procedures that use horseradish per-
oxidase conjugates. The TMB substrate develops
a soluble blue reaction product that can be read
at 370 or 655 nm [1].
Additionally, many recent reports support and
justify the wide incorporation of TMB into ana-
lytical practice [2]. Thus, Sil et al. proposed a one-
step simple method for nanolevel detection of as-
corbic acid based on the inhibitory activity of ascor-
bic acid on horseradish peroxidase and hydrogen
peroxide supported by redox properties of TMB [3].
TMB was also used for the uric acid detection
with MnO2 nanosheets. In mildly acidic condi-
tions, MnO2 oxidizes TMB to its blue oxidation
product applied to human urine samples, achiev-
ing recovery rates of 93.1 – 102.4 % with a rela-
tive standard deviation below 3 % [4]. In 2022,
Zhang et al. developed a colorimetric detection
system for Cu2+ ions based on TMB and Ag(S2O3)2
3–
in an aqueous solution. Unlike Ag nanoparticles,
which could not oxidize TMB, Ag(S2O3)2
3– cata-
lyzed the reaction effectively, with optimal con-
ditions achieved at 800 μM for TMB, 400 μM for
Na2S2O3, for 40 min, and at 25 °C. This system,
with a detection range of 1 – 100 μM and a limit
of 100 nM, shows promise for monitoring the wa-
ter quality [5]. Important biogenic thiols, such
as glutathione (GSH), cysteine (CySH), and ho-
mocysteine (HcySH), were analyzed using TMB
as a chromogen in a simple spectrophotometric
method. Detection limits for GSH, CySH, and
HcySH were 1.04, 0.82, and 2.09 μM, respective-
ly, with successful application of the method to
human serum samples, achieving recovery rates
of 97 – 112 % [6]. Chandra and colleagues report-
ed the synthesis of fluorescent carbon quantum
dots (M-CQDs) through a simple hydrothermal
treatment of mustard seeds [7]. M-CQDs demon-
strated peroxidase-like activity, catalyzing the
oxidation of TMB in the presence of H2O2, thus
mimicking the natural horseradish peroxidase
activity. The process enabled the colorimetric
detection of H2O2 in the range of 0.02 – 0.20 mM
with a detection limit of 0.015 mM. Additional-
ly, reduction of oxidized TMB with ascorbic acid
allowed for a selective and sensitive detection of
ascorbic acid in the range of 10 – 70 μM, with a
detection limit of 3.26 μM. The method was suc-
cessfully applied to the ascorbic acid detection
in fresh fruits. Further advancements in TMB-
based systems include the application of oxidized
TMB nanobelts enhancing the colorimetric and
paper-based sensing of H2O2 [8], peroxidase-like
nanoenzymes, such as Fe3O4 nanoparticles [9],
CoFe2O4 nanoparticles [10], single-atom iron na-
nozyme [11], MOF-818 nanozyme containing tri-
nuclear copper centers [12], Ce2(WO4)3 nano-
sheets [13], Fe3O4@AuNPs [14], etc., all enhanc-
ing the H2O2 detection sensitivity through the for-
mation of oxidized TMB. The versatility of TMB
is reflected in its increasing popularity, with
2,889 publications between 2015 and 2024 in the
Scopus® database (www.scopus.com). Notably, the
number of publications surged by 1.5 times bet-
ween 2019 and 2024, demonstrates the growing
impact of TMB on analytical chemistry (Figure 1).
Previously, our laboratory developed a novel
biochemical kinetic-spectrophotometric method
for detecting cholinesterase inhibitors from the
group of quaternary ammonium compounds (QACs)
using the oxidation of p-phenetidine by hydro-
gen peroxide as the indicator reaction [15], in-
cluding a recent paper reporting the quantifica-
tion of Ethonium [16]. Considering all the advan-
tages that TMB can provide for the quantifica-
tion of this group of pharmaceuticals and its cen-
tral role in the development of sensitive and ef-
fective analytical methods, in this study we pre-
sent the results of using TMB as a chromogen
for the quantitative analysis of the QAC Etho-
nium in a 0.5 % gel formulation. The enzyme-
kinetic method based on the inhibition of cho-
linesterase activity and the use of oxidation of
TMB with hydrogen peroxide as an indicator re-
action was applied.
■ Materials and methods
Reagents and equipment
The following reagents were used in the
study:
• ETONIY® (Aethonium) powder (substance)
produced by OJSC Farmak, Kyiv, Ukraine.
C30H62Cl2N2O4; CAS: 21954-74-5; MW
585.736 g mol–1;
• 0.5 % Ethonium gel, 50 mL, batch No. 74 (Apr
2023) manufactured by APTEKA PAVLOVA
ISSN 2308-8303 (Print) / 2518-1548 (Online) 38
Журнал органічної та фармацевтичної хімії 2024, 22 (4)
Ltd (Odesa, Ukraine) with the composition
of ethonium (active pharmaceutical ingredi-
ent) 0.5 g; glycerol – 20.0 g; propylene glycol –
20.0 g; PEG 400 – 50.0 g; PEG 1500 – 10.0 g;
purified water – 10.0 ml. The content of the
active substance, according to the certificate,
is 4.4525 % (w/w);
• 3,3’,5,5’-Tetramethylbenzidine dihydrochlo-
ride (TMB), C16H20N2·2HCl; 98.5 % (Sigma-
Aldrich); MW 313.27 g mol–1;
• Disodium hydrogen phosphate dodecahydra-
te (Na2HPO4∙12H2O), puriss. p.a. (“ReaChem”,
Kharkiv, Ukraine);
• Stabilized hydrogen peroxide, 30 – 40 % solution,
puriss. p.a., (LLC Inter-Synthes, Boryslav, Uk-
raine) with the content of hydrogen peroxide de-
termined using permanganatometry according
to the State Pharmacopoeia of Ukraine [17];
• Acetylcholine chloride (Pharm Grade), 0.2 g
per amp/5 mL, manufactured by the State
Science Center of Virology and Biotechnolo-
gy “Vector”;
• A dry cholinesterase (EC 3.1.1.8) powder
from horse serum (SMU “Biomed”), 80 mg in
an ampoule (VI class, activity 28 AU mg–1).
The catalytic activity of 1 activity unit (AU)
is manifested in such an amount of this en-
zyme preparation that converts 1 μmol of the
substrate in 1 min under specified reaction
conditions.
• Ethanol 96 % v/v (USP, BP, Ph.Eur.) pure,
pharma grade.
• High-purity double distilled water was used
throughout the experiment.
The pH measurements were performed with
a combined glass electrode (SP20B) together with
an EAL-1М3.1 reference standard silver chloride
electrode.
The absorbance measurements were perfor-
med on an SF-26 spectrophotometer (λ = 420 nm,
l = 10 mm).
Preparation of solutions
0.2 M Phosphate buffer solution (pH 8.35)
Disodium hydrogen phosphate dodecahydra-
te (35.75 g) was dissolved in a 500 mL flask us-
ing double-distilled water. 0.1 M Solution of hyd-
rochloric acid (19 mL) was then added. The pH
of the final solution was controlled potentiomet-
rically.
0.02 M TMB solution
The substance of TMB (0.6265 g) was dissol-
ved in 40 % (v/v) ethanol solution in a 100 mL vo-
lumetric flask and diluted to the volume with the
same solvent. The mixture was heated to 45 °C
for complete dissolution and was stored in a tight-
ly closed dark glass bottle in a cool place.
10 % Hydrogen peroxide solution
The solution was prepared from a 30 – 40 % so-
lution of hydrogen peroxide by dilution with the re-
quired amount of double distilled water. The con-
tent of hydrogen peroxide in a 10 % working so-
lution was determined by permanganatometry.
Cholinesterase (ChE) solution
The accurately weighed content of an ampoule
containing the cholinesterase powder (80 mg)
was dissolved in double-distilled water (20.0 mL)
when heating gently on a water heater. The shelf
life of the solution was 1 day.
Acetylcholine chloride (ACh) solution
The solution with the initial concentration of
5.4·10–3 mol L–1 was prepared by dissolving the
ampoule content (0.2 g of acetylcholine) in 200 mL
of double-distilled water. For this purpose, the
ampoule was opened, and 4.0 mL of water was pi-
petted and added to the ampoule, and then shak-
en until acetylcholine was completely dissolved.
41 44
74 64
113 127
166
207
296 314 310
388
453
515
118
2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2025
P
a
p
e
rs
N
o
.
Year
24
Figure 1. The number of papers per year for the query “3,3’,5,5’-tetramethylbenzidine” (title, abstract, keywords) according to the
Scopus® database (2010-2025)
ISSN 2308-8303 (Print) / 2518-1548 (Online) 39
Journal of Organic and Pharmaceutical Chemistry 2024, 22 (4)
Then the solution was transferred into a 200 mL
volumetric flask and diluted to the volume with
double-distilled water.
0.1 % Ethonium solution
1.0000 g of the Ethonium substance was dis-
solved in a 1 L flask using double-distilled wa-
ter, the solution was heated to 40 – 45°C, 9.0 g of
sodium chloride was added to the solution, and
diluted to the volume.
Stock Solution of Ethonium (ET), 1×10–4 M
The accurately weighed powder of the Etho-
nium substance (0.058574 g) was dissolved in
500 mL of double-distilled water in a 1000 mL
volumetric flask. The solution was diluted to the
volume with the same solvent at +20 °C and
mixed thoroughly.
Ethonium Work Standard (WS) solution, 1·10–5 M
The accurately weighed powder of the etho-
nium substance (0.58574 g) was dissolved in 500
mL of ethanol solution in double-distilled water
(EtOH/H2O 30:70 v/v) in a 1 L volumetric flask
and diluted to the volume at +20 °C and mixed
thoroughly. Using a pipette, 10 mL of the result-
ing solution was taken and transferred to a 1 L
volumetric flask and diluted to the volume with
double-distilled water.
Ethonium WS Solution, 1×10–6 M
A 10 mL aliquot of Stock Solution of the drug
(1×10–4 mol L–1) was transferred into a 1 L volu-
metric flask and diluted to the volume with dou-
ble-distilled water at +20 °C.
The procedure for constructing the ki-
netic curves
Part 1 – working experiments «ACh + (ChE + ET)»
The buffer solution (2.00 mL, pH 8.35) was
added to each of the five 20 mL graduated test
tubes with a ground joint stopper. Then 0.50,
1.50, 3.00, 4.50, and 6.00 mL of the ethonium
1×10–6 M WS solution was added to the test tu-
bes, followed by 0.50 mL of the cholinesterase so-
lution. The content was thoroughly shaken, and
the test tubes were kept in a thermostat at +38 ºС
for 10 min. After that 1.0 mL of acetylcholine so-
lution and 5.90, 4.90, 1.90, 0.40 mL, and 5.4 mL of
double-distilled water were added to the five test
tubes, respectively. The content was mixed thor-
oughly and incubated again for 10 min at 38 ºС.
Then, a 10 % hydrogen peroxide solution (3.20 mL)
was added to each of the test tubes, and the latter
were incubated for 10 min at +38 ºС. After that
3.0 mL of 96 % ethanol and 0.50 mL of the TMB
solution were added, and the solution was shak-
en thoroughly and scanned photometrically on a
spectrophotometer at a wavelength of 420 nm in
a 1 cm cuvette over a 15-minute period. The phos-
phate buffer was used as a reference solution.
The relative rate of the reaction [[(ChE + ET) +
ACh] +H2O2 + TMB] (tgα (Inh), min–1) was deter-
mined as the slope of a linear section of the “op-
tical density (A) vs time (t, min)” kinetic curve.
Part 2 – control experiments #1 “ACh”
The buffer solution (2.00 mL, pH 8.35) was
added to a 20 mL graduated test tube, followed
by 6.9 mL of double-distilled water, 1.0 mL of
the acetylcholine solution, and 3.2 mL of the hy-
drogen peroxide solution. The solution was in-
cubated at 38 ºС for 10 min. After that 3.0 mL of
96 % ethanol and 0.5 mL of the TMB solution were
added to the test tube. The solution was shaken
thoroughly and scanned photometrically on a spec-
trophotometer at a wavelength of 420 nm in a 1 cm
cuvette over a 15-minute period. The phosphate
buffer was used as a reference solution. According
to the plotted “optical density (A) vs time (t, min)”
kinetic curve, the relative rate of the reaction
[(ACh + H2O2) + TMB] was determined as a slo-
pe of a linear section of the curve (tgα (ACh), min–1).
Part 3 – control experiments #2 “Ach+ChE”
2.0 mL of the buffer solution, 6.4 mL of double-
distilled water, 0.5 mL of the cholinesterase so-
lution, and 1.0 mL of the acetylcholine solution
were successively added to a 20 mL test tube with
a ground joint stopper and then thermostated at
38 ºС for 10 min. Further 3.2 mL of the hydro-
gen peroxide solution was added, and the mix-
ture was thoroughly shaken and thermostated
again at 38 ºС for 10 min. After that 3.0 mL of
96 % ethanol and 0.5 mL of the TMB solution were
added. Then the solution was scanned photomet-
rically on a spectrophotometer at a wavelength
of 420 nm in a 1 cm cuvette over a 15 min period.
The phosphate buffer was used as a reference
solution. According to the plotted “optical den-
sity (A) vs time (t, min)” kinetic curve, the rela-
tive rate of the reaction [(ChE + ACh) + H2O2 +
TMB] was determined as a slope of a linear sec-
tion of the curve (tgα (ACh + ChE), min–1).
The relative rates of the reactions (expressed
as tangents of the angles of slope) were used to
calculate the inhibition degree of the enzymatic
hydrolysis of ACh (U, %) in the presence of etho-
nium according to the following equation:
U(%) =
[tg�(Inh) – Ach + ChE)tg�� ]
× 100 %
[tg�� ) – Ach + ChE)tg�� ]Ach
where tgα (Inh) (min–1) is the relative reac-
tion rate of the TMB oxidation by peroxyacetic
ISSN 2308-8303 (Print) / 2518-1548 (Online) 40
Журнал органічної та фармацевтичної хімії 2024, 22 (4)
acid formed during the perhydrolysis of unre-
acted ACh in the working experiment at various
concentrations of the inhibitor (ET);
tgα (Ach) (min–1) is the relative reaction rate
of the TMB oxidation by peroxyacetic acid for-
med in the reaction of the ACh perhydrolysis in
the absence of the inhibitor and ChE (control ex-
periment #1);
tgα (Ach + ChE) (min–1) the relative reaction
rate of the TMB oxidation by peroxyacetic acid
formed in the reaction of the perhydrolysis of
unreacted ACh in the presence of ChE and in
the absence of the inhibitor (ET) (control experi-
ment #2).
The calculated values of U ( %) were used to
plot the “inhibition degree (U, %) vs ethonium
concentration (c, ng mL–1)” calibration graph
(Figure 3).
■ Results and discussion
Previously, a study was conducted to analyze
the parameters that may affect the effectiveness
of the approach proposed [18]. This allowed us
to determine the optimal working conditions
and concentrations of the reagents used in this
study.
Kinetic curves were plotted using experimen-
tal data showing the relationship between the op-
tical density and time, as presented in Figure 2.
The calibration graph (Figure 3) was con-
structed in the coordinates of the inhibition de-
gree (U, %) vs the concentration (c, ng mL–1).
From Figure 3, the linear dependence of the
inhibition degree on the concentration of the in-
hibitor was observed in the interval of 4 – 60 % (R
= 0.9) corresponding to the concentration of the
inhibitor 17 – 200 ng mL–1. The LOQ was defined
as the concentration corresponding to a 4 % de-
gree of inhibition, i.e., 17 ng mL–1.
The method for the quantitative deter-
mination of ethonium with TMB as an indi-
cator in a “Ethonium 0.5 % gel” formulation
0.1 g (accurate weight) of the ethonium gel
was dissolved in 1000 mL of double-distilled wa-
ter. Then 2.0 mL of the phosphate buffer solution,
5.0 mL of the gel solution, and 0.5 mL of the ChE
solution were successively added to a 20 mL test
tube. The mixture was thoroughly shaken and
incubated for 10 min at 38 ºС. After that 1.0 mL
of the ACh solution and 1.4 mL of double-distilled
water were added, and the content was carefully
mixed and incubated again for 10 min at 38 ºС.
Then, 3.2 mL of the hydrogen peroxide solution
was added, and the mixture was incubated again
at 38 ºС for 10 min. After that 3.0 mL of 96 % etha-
nol and 0.5 mL of the TMB solution were added
to the test tube. The optical density of the solu-
tion was measured at 420 nm in a 1 cm cuvette
for 15 min. According to the plot of the “optical
density vs time” dependence, the tangent of the
angle of slope for the linear section tgα (Inh) was
found in min–1. In parallel, two more experiments
were carried out. One of them involved acetyl-
choline and cholinesterase without the inhibitor,
another one was performed without the use of cho-
linesterase enzyme (control experiments #1 and #2
described in the previous section). As a result, the
0
0,1
0,2
0,3
0,4
0,5
0 10 20 30 40
A
b
s
o
rb
a
n
c
e
Time, min
Ach
Ach + ChE
1
2
3
4
5
Figure 2. Kinetic curves of the conjugated oxidation of TMB with hydrogen peroxide in the presence of mixtures ACh + ChE + Inh (1 – 5).
c (Ach) = 3.3×10–4 mol L–1; w(H2O2) =1.92 %; c (AChE) = 0.24 mg mL–1. w (ET): 17 ng mL–1 (1); 50 ng mL–1 (2); 100 ng mL–1 (3);
150 ng mL–1 (4); 200 ng mL–1 (5); c (ТМB) = 6.25×10–4 mol L–1; control experiments “Ach + ChE”, control experiments “ACh”
ISSN 2308-8303 (Print) / 2518-1548 (Online) 41
Journal of Organic and Pharmaceutical Chemistry 2024, 22 (4)
other two tangents tgα (Ach + ChE) and tgα (ACh),
respectively, were determined.
The content of ethonium in the gel formula-
tion, (w, %) was calculated by the formula:
w (%, w/w) =
0.00059574[tg tg� ��( ) – Ach + ChE)X ]×100 %
g× ][tg tg� ��(Ach) – Ach + ChE)
where 0.00059574 – is the mass of Ethonium
in a 10.00 mL aliquot of the Stock Solution of
the drug, g;
g – is the mass of the ethonium gel sample
taken for the analysis, g;
tgα (X) – is the relative reaction rate (the tan-
gent of the angle of slope of the kinetic curve), in
the working experiment with the sample solu-
tion of the drug studied (the Ach – ChE – Inh (Х)
+ (H2O2 – TMB) system), min–1;
tgα (Ach + ChE) – is the relative reaction rate
in the absence of the inhibitor (ET) (the tangent
of the angle of slope of the kinetic curve) in the
Ach – СhЕ + (H2O2 – TMB) system, min–1;
tgα (ACh) – is the relative reaction rate (the
tangent of the angle of slope of the kinetic curve)
in the working experiment without the use of the
inhibitor and cholinesterase, (the Ach – ChE –
Inh (WSS) + (H2O2 – TMB) system), min–1.
■ Conclusions
This paper proves the possibility of using
tetramethylbenzidine as a promising indicator
substance for the quantitative determination of
surface-active substances of the class of quater-
nary ammonium compounds.
The quantitative content of Ethonium as an
active ingredient in the 0.5 % gel formulation
and in the 0.1 % solution prepared ex tempore
was determined by the enzyme-kinetic method
using the effect of inhibiting the activity of the
enzyme cholinesterase. The inhibition was es-
timated by the residual acetylcholine using the
indicator reaction of the 3,3’,5,5’-tetramethyl-
benzidine oxidation with peracetic acid formed
in the perhydrolysis of acetylcholine. As a re-
sult, RSD did not exceed 2.5 % with accuracy
δ = –0.45….–2.00 % (δ < RSD). The LOQ value
was 17 ng mL–1.
y = 0.3092x – 2.9353
R = 0.9935²
0
10
20
30
40
50
60
70
0 50 100 150 200 250
D
e
g
re
e
o
f
in
g
ib
it
io
n
,
%
Etonium c
1
oncetration, ng mL
–
Figure 3. The dependence of the degree of inhibition on the concentration of Ethonium in the system (ACh + (ChE + Ethonium),
determined by the indicator reaction of the conjugated oxidation of TMB with hydrogen peroxide in the presence of residual
acetylcholine. c (ACh) = 3.3×10–4 mol L–1; w (H2O2) = 1.92 %; c (AChE) = 0.24 mg mL–1, c (TMB) = 6.25×10–4 mol L–1
Table. The results of the analysis of 0.5 % ethonium gel and 0.1 % solution according to the proposed procedure by the kinetic-
spectrophotometric enzyme method
The substance analyzed ET found (x ± Δx), %a RSD, % The quality
certificate data, % Accuracy, (δ, %)b
0.5 % Ethonium gel, 50 mL,
manufactured by APTEKA PAVLOVA Ltd
(Odesa, Ukraine)
0.443 ± 0.014 2.54 0.445 –0.45
0.100 % Ethonium solution, 1000 mL,
prepared ex tempore 0.098 ± 0.003 2.46 0.100 –2.00
Notes: a Mean of 5 measurements (P = 0.95); b δ = (x – μ) × 100 % × μ–1; μ is the actual content of ET according to the Certificate
ISSN 2308-8303 (Print) / 2518-1548 (Online) 42
Журнал органічної та фармацевтичної хімії 2024, 22 (4)
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Information about the authors:
Olena V. Kovalska (corresponding author), Ph.D. in Pharmacy, Associate Professor of Organization, Economy and Pharmacy Management
Department, Institute of Advanced Training of Pharmacy Specialists of the National University of Pharmacy of the Ministry of Health of
Ukraine; https://orcid.org/0000-0003-0113-7060; e-mail for correspondence: lena05021985@ukr.net; tel. +380970710498.
Mykola Ye. Blazheyevskіy, D.Sci. in Chemistry, Professor of the General Chemistry Department, National University of Pharmacy of the
Ministry of Health of Ukraine; https://orcid.org/0000-0002-8032-347X.
Vladyslav V. Diadchenko, Ph.D. in Chemistry, Associate Professor, Military Institute of Armored Forces of the National Technical
University “Kharkiv Polytechnic Institute”. https://orcid.org/0000-0002-5552-3031.
|
| id | oai:ojs.journals.uran.ua:article-321795 |
| institution | Journal of Organic and Pharmaceutical Chemistry |
| issn | 2518-1548 |
| keywords_txt_mv | |
| language | English |
| last_indexed | 2026-08-24T01:01:17Z |
| publishDate | 2024 |
| publisher | National University of Pharmacy |
| record_format | ojs |
| resource_txt_mv | ophcjnupheduua/f9/702276e7a553d3561f602e85c0a39af9.pdf |
| spelling | oai:ojs.journals.uran.ua:article-3217952026-08-23T15:46:26Z The Use of Tetramethylbenzidine as an Indicator in the Enzymatic Quantitative Determination of Ethonium Використання тетраметилбензидину як індикатора у ензиматичному методі кількісного визначення етонію Kovalska, Olena V. Blazheyevskiy, Mykola Ye. Diadchenko, Vladyslav V. ethonium quaternary ammonium compounds cholinesterase acetylcholine етоній холінестераза ацетилхолін четвертинні амонієві сполуки The study considers the possibility of using 3,3',5,5'-tetramethylbenzidine (TMB) as an indicator in the enzymatic analysis for the quantitative determination of quaternary ammonium compounds on the example of ethonium. The feasibility of using TMB as an indicator in the kinetic photometric method has been confirmed. Kinetic curves showing the relationship between the optical density and the ethonium concentration have been constructed. The reaction rates of acetylcholinesterase inhibition by ethonium have been estimated using the tangents of the angles of these curves with TMB as an indicator. The degree of enzyme inhibition has been calculated, and a linear relationship between the ethonium concentration and the degree of inhibition has been determined. This method was applied to determine the ethonium content in 0.1% ethonium gel. The relative standard deviation of the method does not exceed 2.5 %. The approach suggested offers a reliable and accurate method for the quantitative analysis of ethonium in dosage forms. У дослідженні розглянуто можливість використання 3,3',5,5'-тетраметилбензидину (ТМБ) як індикатора в ензиматичному аналізі для кількісного визначення четвертинних амонієвих сполук на прикладі етонію. Підтверджено можливість застосування ТМБ як індикатора в кінетичному фотометричному методі. Побудовано кінетичні криві, що демонструють залежність між оптичною густиною та концентрацією етонію. Швидкість реакції інгібування ацетилхолінестерази етонієм було оцінено за тангенсами кутів цих кривих, із цим ТМБ використано як індикатор. Розраховано ступінь інгібування ферменту, а також визначено лінійну залежність між концентрацією етонію та ступенем інгібування. Цей метод застосовували для визначення вмісту етонію в 0,1% гелі етонію. Відносне стандартне відхилення методу не перевищує 2,5%. Запропонований підхід забезпечує надійний та точний метод кількісного аналізу етонію у лікарських формах. National University of Pharmacy 2024-12-12 Article Article application/pdf https://ophcj.nuph.edu.ua/article/view/321795 10.24959/ophcj.24.321795 Journal of Organic and Pharmaceutical Chemistry; Vol. 22 No. 4 (2024); 36-42 Журнал органической и фармацевтической химии; Том 22 № 4 (2024); 36-42 Журнал органічної та фармацевтичної хімії; Том 22 № 4 (2024); 36-42 2518-1548 2308-8303 en https://ophcj.nuph.edu.ua/article/view/321795/313392 Copyright (c) 2025 Olena V. Kovalska, Mykola Ye. Blazheyevskiy, Vladyslav V. Diadchenko http://creativecommons.org/licenses/by/4.0 |
| spellingShingle | етоній холінестераза ацетилхолін четвертинні амонієві сполуки Kovalska, Olena V. Blazheyevskiy, Mykola Ye. Diadchenko, Vladyslav V. Використання тетраметилбензидину як індикатора у ензиматичному методі кількісного визначення етонію |
| title | Використання тетраметилбензидину як індикатора у ензиматичному методі кількісного визначення етонію |
| title_alt | The Use of Tetramethylbenzidine as an Indicator in the Enzymatic Quantitative Determination of Ethonium |
| title_full | Використання тетраметилбензидину як індикатора у ензиматичному методі кількісного визначення етонію |
| title_fullStr | Використання тетраметилбензидину як індикатора у ензиматичному методі кількісного визначення етонію |
| title_full_unstemmed | Використання тетраметилбензидину як індикатора у ензиматичному методі кількісного визначення етонію |
| title_short | Використання тетраметилбензидину як індикатора у ензиматичному методі кількісного визначення етонію |
| title_sort | використання тетраметилбензидину як індикатора у ензиматичному методі кількісного визначення етонію |
| topic | етоній холінестераза ацетилхолін четвертинні амонієві сполуки |
| topic_facet | ethonium quaternary ammonium compounds cholinesterase acetylcholine етоній холінестераза ацетилхолін четвертинні амонієві сполуки |
| url | https://ophcj.nuph.edu.ua/article/view/321795 |
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