Development of an Optimal Method for the Quantitative
Determination of 1-(ß phenylethyl)-4-amino-1,2,4-triazolium Bromide in a
solution for Injection
Kucherenko Liudmyla, Derevianko Natalia, Borsuk
Serhii, Khromylova Olga,
Bihdan Oleksii, Dmytro Skoryna
Department of Pharmaceutical, Organic and Bioorganic
Chemistry,
Zaporizhzhia State Medical and Pharmaceutical
University, The City of Zaporizhzhia, Ukraine.
*Corresponding Author E-mail:
borsuksergejjj@gmail.com
ABSTRACT:
Cardiovascular diseases are currently the leading
cause of death and disability worldwide. Each year, their prevalence and
severity continue to grow. As a result, developing new treatments and
preventive measures for cardiovascular conditions has become a critical focus
in medicine and pharmacy today. Today, drugs in the form of injections are used
in most cases for the treatment of patients in hospital conditions. Therefore,
an injectable dosage form was created for the new original compound of
1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide, which exhibits
antihypertensive, antiischemic, and antioxidant properties. The purpose of our
work is to develop an optimal method for the quantitative determination of
1-(ß-phenylethyl)-4-amino-1,2,4-triazolium bromide in an injectable dosage form
by the method of high-performance liquid chromatography (HPLC). During the
research, we used a laboratory sample of a solution for injections with a concentration
of 5 mg/ml, the active ingredient is 1-(ß-phenylethyl)-4-amino-1,2,4-triazolium
bromide. A standardized substance of 1-(ß-phenylethyl)-4-amino-1,2,4-triazolium
bromide with a content of 99.95% was used as a working standard sample. The
research was carried out on the basis of the chemical and physical control
laboratory of the Technical Control Department, SE "Chemical Reagent
Plant" (city of Kharkiv). The main stage of the work was the development
of the method of standardization of the active substance by the HPLC method. As
a result of the conducted research, an eluent and a column were selected, with
the help of which, under certain conditions, it is possible to carry out
simultaneous identification and quantitative determination. According to the
developed methods, we analyzed all the series prepared in laboratory
conditions. During the work, the content of
1-(ß-phenylethyl)-4-amino-1,2,4-triazolium bromide in the injection solution
was determined, which is in the range from 4.83 to 5.25 mg in 1 ml. According
to the demands of the State Pharmacopoeia of Ukraine, the content of
1-(ß-phenylethyl)-4-amino-1,2,4-triazolium bromide in the injection solution
should be from 4.5 mg to 5.5 mg in 1 ml. As can be seen from the above data,
the obtained results meet all the demands of the State Pharmacopoeia of
Ukraine. Thus, in the course of the work, a method was developed for the
quantitative determination of 1-(ß-phenylethyl)-4-amino-1,2,4-triazolium
bromide in an injectable dosage form using the HPLC method. The developed
method is planned to be used for continuous quality control of the developed
dosage form.
KEYWORDS: Liquid chromatography, Injection solution, Quantitative
determination, 1-(ß-phenylethyl)-4-amino-1,2,4-triazolium bromide, Arterial hypertension,
Ischemia.
INTRODUCTION:
Strategies for effectively treating arterial
hypertension (AH) have evolved alongside our understanding of its underlying
mechanisms. Initially, emphasis was placed on reducing blood pressure (BP),
which laid the foundation for establishing "target blood pressure"
goals. In today’s pharmacology and pharmacy fields, the focus extends beyond
merely lowering BP to exploring the organ-protective effects of
antihypertensive drugs1.
This includes evaluating both current medications and
newly developed compounds in Ukraine that could compete with imported options.
Structural and functional changes in the myocardium and vascular remodeling in
AH patients are known to increase cardiovascular risk. Consequently, developing
antihypertensive drugs with cardioprotective, antioxidant, and NO-mimetic
properties in various formulations presents a promising avenue for advancement1-2.
Thus, the above prompted the creation of a
fundamentally new potential domestic antianginal and antihypertensive drug. The leading scientists of Ukraine jointly
with the employees of the Department of Pharmaceutical Chemistry of ZDMFU under
the leadership of Professor Ivan Antonovych Mazur obtained a new original
compound - 1-β-phenylethyl-4-amino-1,2,4-triazolium bromide, which
exhibits antihypertensive, anti-ischemic and antioxidant properties2.
Pharmacological activity and toxicity studies have
already been carried out for the new potential original medicinal product,
which includes 1-(ß-phenylethyl)-4-amino-1,2,4-triazolium bromide. The research
results showed that 1-(ß-phenylethyl)-4-amino-1,2,4-triazolium bromide belongs
to the IV class of toxicity (low toxicity compounds). It was established that
1-(ß-phenylethyl)-4-amino-1,2,4-triazolium bromide not only exhibits
antihypertensive activity in terms of strength, significantly superior to that
of metoprolol, but also significant cardioprotective activity. Also, the
medicinal substance has an NO-mimetic effect, which is absent in metoprolol,
which significantly enhances its protective effect on the target organ, the
heart, in case of arterial hypertension3-5.
The success of these pharmacological studies led to
the creation of an injectable form containing the active compound
1-(ß-phenylethyl)-4-amino-1,2,4-triazolium bromide. In subsequent stages,
developing quality control methods for this new pharmaceutical became
essential. For accurate measurement of the active ingredient in the drug form
(a 5mg/ml solution for injection in 2ml ampoules), the HPLC method was proposed
due to its numerous advantages over other modern analytical techniques6.
The purpose of
our research is to develop a method for the quantitative determination of
1-(ß-phenylethyl)-4-amino-1,2,4-triazolium bromide in an injectable dosage form
by HPLC.
materials aNd methods:
In this study, we utilized six series of a 5mg/ml
injection solution containing the active compound
1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide, prepared under
laboratory conditions. For the working standard, we employed a certified sample
of 1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide with a purity of
99.95%, provided by the State Enterprise "Chemical Reagent Plant" of
the Scientific and Technological Complex "Institute for Single
Crystals" of the NAS of Ukraine. Currently, the HPLC method is commonly
used for identifying and quantifying active ingredients in finished dosage
forms, making it our method of choice for this analysis7-9.
Results:
The first stage of the development of the methodology
was the study of the chromatographic behavior of the active substance
1-(ß-phenylethyl)-4-amino-1,2,4-triazolium bromide; as a result, an eluent and
a column were selected, with the help of which, under certain conditions, it is
possible to carry out simultaneous identification and quantitative
determination10-13.
Thus, for the quantitative determination of
1-(ß-phenylethyl)-4-amino-1,2,4-triazolium bromide in the injectable dosage
form, it was proposed to use the following conditions:
· Steel column 4.6mm × 250mm ZorbaxSB-C18
with a particle size of 5μm, or similar;
· Composition of the mobile phase is
programmed according to the data in Table 1;
· Speed of the mobile phase is 0.8ml/min;
· Column temperature – 30○С;
· Wavelength of registration – 210nm.
Table 1. Composition of the mobile phase
|
Time (min.)
|
Content of
mobile phase A
|
Content of
mobile phase B
|
|
0 – 5
|
100
|
0
|
|
5 – 15
|
100 – 0
|
0 – 100
|
|
15 – 25
|
0
|
100
|
|
25 – 30
|
0 – 100
|
100 – 0
|
|
30 – 35
|
100
|
0
|
To prepare mobile phase A, transfer 490.0ml of
phosphate buffer solution at pH 2.5 into a 500.0ml volumetric flask and top up
to the mark with acetonitrile. Filter the resulting solution through a 0.45
μm membrane filter.
For mobile phase B, add
300.0ml of phosphate buffer solution at pH 2.5 to a 500.0ml volumetric flask
and fill to the mark with acetonitrile. Then, filter this solution through a
membrane filter with a 0.45μm pore size.
Evaluating chromatographic system suitability.
A chromatographic system is
deemed suitable if it meets the following criteria:
·
The
chromatographic system should have an efficiency of at least 3,000 theoretical
plates, based on the peak of 1-(β-phenylethyl)-4-amino-1,2,4-triazolium
bromide in the chromatograms of comparison solution 2.
·
The
main peak’s symmetry coefficient, derived from the
1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide peak in the
chromatograms of comparison solution 2, should not exceed 2.0.
· The relative standard
deviation for the peak of 1-(β-phenylethyl)-4-amino-1,2,4-triazolium
bromide from the chromatograms of comparison solution 2 should be 2.0% or
lower.
Preparation of a pH 2.5 Phosphate Buffer
Solution.
To prepare this buffer, dissolve 2.04g of sodium
dihydrogen phosphate and 1.0g of sodium octyl sulfonate in 1000.0 ml of water.
Adjust the solution to a pH of 2.5±0.1 by adding phosphoric acid with stirring.
Test solution.
Transfer 2ml of the test solution into a 100 ml
volumetric flask, then dilute to the mark with water and mix thoroughly.
Comparison solution (standard solution). Accurately weigh about 10.0mg of a standard sample of
1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide and place it in a 100
ml volumetric flask. Dissolve in 70ml of water and then dilute to the mark with
the same solvent. This solution should be freshly prepared14-18. Chromatography
Procedure. Inject 50μl each of the test solution and comparison
solution 2 into a liquid chromatograph equipped with a UV detector, running
enough chromatograms under the specified conditions to achieve adequate
results. Example chromatograms for both solutions are displayed in Figures 1-2.
Figure 1. Chromatogram of the studied working solution
(1-(ß-phenylethyl)-4-amino-1,2,4-triazolium bromide injection solution, series
1)
Figure 2. Chromatogram of the working standard solution
(comparison solution)
The content of
1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide (X) in one milliliter,
in mg, is calculated by the formula:
where:
S – the average value of the peak areas of
1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide calculated from the
chromatograms of the tested solution;
S0 – the average value of the peak areas of
1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide calculated from the
chromatograms of the comparison solution 2;
m0 – weight of a standard sample of
1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide, in mg;
mх– weight of the sample of the tested solution, ml (2 ml)
P – content of the main substance in the standard
sample of 1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide, in percent.
The content of 1-(β-phenylethyl)-4-amino-1,2,4-triazolium
(X) bromide in one milliliter should be: at the time of release - from
4.75mg to 5.25mg; during the shelf life - from 4.5mg to 5.5mg.
The results of the conducted studies of the
quantitative content of the active substance in the injectable dosage form are
shown in Table 2.
Table 2. Results of quantitative determination of
1-(ß-phenylethyl)-4-amino-1,2,4-triazolium bromide in injection solution (6
series) by the method of HPLC
|
Sl.No.
|
1-(ß-phenylethyl)-4-amino-1,2,4-triazolium
bromide
|
|
Peak area
|
Average peak area
|
Found, mg
|
Statistical
indicators
|
|
1
|
13354548
|
13395926
|
5,07
|
 =5,025
Sx =
0,15
t (0,95)=2,02
∆х
=0,31
∆=0,124
 ±∆ =
5,025 ± 0,124
e=2,49%
|
|
13359687
|
|
13417143
|
|
13470708
|
|
13377544
|
|
2
|
12908450
|
12946753
|
4,90
|
|
12979945
|
|
12893120
|
|
13032514
|
|
12919736
|
|
3
|
12719210
|
12761800
|
4,83
|
|
12683413
|
|
12770399
|
|
12815867
|
|
12820111
|
|
4
|
13213488
|
13184551
|
4,99
|
|
13176946
|
|
13203648
|
|
13217892
|
|
13110781
|
|
5
|
13868413
|
13871522
|
5,25
|
|
13946510
|
|
13768728
|
|
13826663
|
|
13947296
|
|
6
|
13583232
|
13501614
|
5,11
|
|
13571183
|
|
13485882
|
|
13510766
|
|
13357007
|
|
СР
|
13240292
|
13144918
|
|
|
13066182
|
|
13240995
|
|
13234275
|
|
12942846
|
DISCUSSIONS:
According to the above-mentioned method, we analyzed
all series (6 series) of injection solutions prepared in laboratory conditions.
During the work, the content of 1-(ß-phenylethyl)-4-amino-1,2,4-triazolium
bromide in the injection solution was determined. As can be seen from the data
in Table 1, as a result of research and calculations, the content of the active
substance was found, which is in the range from 4.83 to 5.25mg in 1 ml.
According to the requirements of the State Pharmacopoeia of Ukraine19-21,
the content of 1-(ß phenylethyl)-4-amino-1,2,4-triazolium bromide in the injection
solution should be from 4.5mg to 5.5mg in 1 ml. As can be seen from the above
data, the obtained results meet all the requirements of the State Pharmacopoeia
of Ukraine.
findings:
During this work, a method was established for the
quantitative analysis of 1-(ß-phenylethyl)-4-amino-1,2,4-triazolium bromide in
an injectable formulation using HPLC. This method is intended for use in the
stepwise quality control of the injectable form containing the active
ingredient, 1-(ß-phenylethyl)-4-amino-1,2,4-triazolium bromide. Once validated,
the quantitative analysis procedure for this compound will be proposed to the
manufacturing facility for inclusion in the quality control protocols for the
newly developed pharmaceutical product.
CONFLICT OF INTEREST:
The
authors declare that they have no conflicts of interest related to this study.
ACKNOWLEDGEMENT:
The authors thank Zaporizhzhia State Medical and
Pharmaceutical University (Zaporizhzhia, Ukraine) for its kind support during
research. Also, thanks for the
cooperation to Lyudmila Dudnik, head of the physical and chemical control
laboratory of the technical control department of the State Enterprise
"Chemical Reagents Plant", Kharkiv, Ukraine.
references:
1.
Metabolitotropic
mechanisms of the cardioprotective action of new antianginal and
antihypertensive drug "Hypertril" in experimental myocardial ischemia.
Bielenichev І.F., Kucherenko L.І, Nahorna О.О. Odesa
Medical Journal. 2014; 6 (146): 22-26.
2.
Therapy of post-COVID-19
syndrome: improving the efficiency and safety of basic metabolic drug treatment
with tiazotic acid (thiotriazoline). Igor Belenichev; Lyudmyla Kucherenko;
Sergii Pavlov. Pharmacia 69(2). – Sofia, Bulgaria, 2022. – P. 509-516.
3.
Use of Thiocetam in the
complex therapy of combat contusion of the brain. І. F. Bielenichev, K.
Yu. Nerianov, V. І. Chernii, D. А. Sereda, L. I. Kucherenko, N. V.
Derevianko, K. І. Kandybei // Current Issues of Pharmaceutical and
Medical Science and Practice. - 2023. - Т. 16, No.2 (42). – P. 170-174. -
DOI: 10.14739/2409-2932.2023.2.281460.
4.
Development of a method
for quantitative determination of the content of 4-amino-4H-1,2,4-triazole
impurity in the active pharmaceutical ingredient
1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide. H. V. Heorhievskii,
О. А. Zinchenko, L.I. Kucherenko, L. І. Shapovalova . Farmakom.
2013; 1: 32–37.
5.
Development of a
quantitative determination method for the step-by-step control of the
production of Hypertril tablets / L.I. Kucherenko, N.V. Parniuk, Z.B. Moriak //
Pharmaceutical journal. – 2015. – No. 2. – P. 60-63.
6.
Regarding the
Standardization of Antihypertensive tablets / l.i. kucherenko, n.v. parniuk,
z.b. moriak // Current Issues of Pharmaceutical and Medical Science and
Practice. – 2015. – No. 3. – P. 25-29.
7.
Regarding Conducting the
"Dissolution" Test for Hypertril Tablets Using the HPLC Method /
Kucherenko L.I., Moriak Z.B., Cherkovska L.H. // Current Issues of
Pharmaceutical and Medical Science and Practice. - 2019. – Т.12, No.1
(29). - P.42-46.
8.
Validation of the
quantitation methods of 1-(β-phenylethyl)-4-amino-1,2,4-triazole bromide
substance by spectrophotometric method // Kucherenko L.I., Khromylova O.V.,
Parniuk N.V. // Asian Journal of Pharma-ceutical and Clinical Re-search. - Vol
11, No. 2. – 2018. - P.231-234.
9.
Kucherenko L. Bidnenko
O. Khromylova O. Validation of the spectrophotometric method for the
determination of quantitative composition of S-2,6-diagenoxic acid of
3-methyl-1,2,4-triazolyl-5-thioacetate. Asian J Pharm Clin Res. 2018; 11 (9):
91-94. doi.org/10.22159/ajpcr.2018.v11i9.22684
10.
Murugan S, Chandra
Sekhar R. Method Development and Validation of Fingolimod in Bulk and Tablet
Dosage Form by RP-HPLC. Research J. Pharm. and Tech. 2017; 10(8): 2573-2576.
doi: 10.5958/0974-360X.2017.00456.5
11.
Tejaswi Gilakamsetti,
Poojitha Nalluri, Ramya Kuber Banoth. Development and Validation of a Novel
RP-HPLC Method for Estimation of Bexagliflozin in Pure and Pharmaceutical
Formulation. Research Journal of Pharmacy and Technology. 2024; 17(10): 4758-4.
doi: 10.52711/0974-360X.2024.00733
12.
Leena Sawaikar, Pankaj
Kapupara. Development and Validation of a Stability indicating RP-HPLC Method
for the Estimation of Chlorthalidone and Cilnidipine in Combined Pharmaceutical
Dosage Form. Research J. Pharm. and Tech. 2020; 13(5): 2376-2380. doi: 10.5958/0974-360X.2020.00427.8
13.
Priyanka M. Pandya,
Pankaj P. Kapupara, Ketan V. Shah. Development and Validation of an Analytical
Method for Simultaneous Estimation of Clotrimazole and its Impurity by RP-HPLC.
Research J. Pharm. and Tech. 2018; 11(3): 894-898. doi: 10.5958/0974-360X.2018.00165.8
14.
Shiv Kumar Gupta, Neetu
Sachan, Phool Chandra, Arun Kumar Sharma. A Simple and Sophisticated RP-HPLC
Approach for Quantifying of Irinotecan in API and in Pharmaceutical Dosage
Form. Research Journal of Pharmacy and Technology. 2024; 17(10):4722-6. doi: 10.52711/0974-360X.2024.00728
15.
Bhanu Biswas, Manish
Kumar, Jai Bharti Sharma, Vipin Saini, Shailendra Bhatt. Method Development and
Validation for Estimation of Teneligliptin in Tablet Dosage Form by RP-HPLC.
Research J. Pharm. and Tech. 2020; 13(4): 1774-1778. doi: 10.5958/0974-360X.2020.00320.0
16.
Baokar Shrikrishna,
Ranpise Nisharani. Analytical Method Development and Validation for
Simultaneous Estimation of Montelukast and Ebastine by HPLC. Research J. Pharm.
and Tech. 2015; 8(1): Jan. 01-05. doi: 10.5958/0974-360X.2015.00001.3
17.
P. Ravi Sankar, K.
Saisneha Latha, A. Bhavani Sailu, SK. Taheera, B. Madhuri. Development and
Validation of RP-HPLC Method for the Determination of Pazopanib Hydrochloride
(A Tyrosine Kinase Inhibitor) in Pharmaceutical Dosage Form. Research J. Pharm.
and Tech 2021; 14(3): 1549-1554. doi: 10.5958/0974-360X.2021.00273.0
18.
Palani Shanmugasundaram,
Kamarapu S. K. RP-HPLC Method for the Simultaneous Estimation and Validation of
Amlodipine Besylate and Atenolol in Bulk and Tablet Dosage Form in Biorelevant
Dissolution Medium (Fassif). Research J. Pharm. and Tech 2017; 10(10):
3379-3385. doi: 10.5958/0974-360X.2017.00601.1
19.
State Enterprise
«Ukrainian Scientific Pharmacopoeial Center for Drug Quality». State
Pharmacopoeia of Ukraine: in 3 volumes. 2nd edit. 2015; 1 – 1128 P.
20.
State Enterprise
«Ukrainian Scientific Pharmacopoeial Center for Drug Quality». State
Pharmacopoeia of Ukraine: in 3 volumes. 2nd edit. 2015; 2. – 724 P.
21.
State Enterprise
«Ukrainian Scientific Pharmacopoeial Center for Drug Quality». State
Pharmacopoeia of Ukraine: in 3 volumes. 2nd edit. 2015; 3. – 732 P.
|
Available online from July 05, 2025
|
|
This work is
licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0
International License. Creative Commons License.
|
|