Quantitative determination of the new substance 7-(4-fluorobenzyl)-3-thioxo-2,3-dihydro[1,2,4] triazolo[4,3-a]pyrazin-8(7H)-one as a  potential pharmaceutical agent

Krystyna Netosova1,

Oksana Zavada2,

Anna Materiienko3*,

Iryna Zhuravel4,

Serhii Baiurka1

1 Department of Drug and Analytical Toxicology, National University of Pharmacy, 53 Pushkinska Street, 61002, Kharkiv, Ukraine

2 Department of Medical and Bioorganic Chemistry, Kharkiv National Medical University, 4 Nauky Avenue, 61022, Kharkiv, Ukraine

2 Department of the Quality, Standartization and Certification of Medicines, National University of Pharmacy, 53 Pushkinska Street, 61002, Kharkiv, Ukraine

4 Department of Clinical Biochemistry, Forensic Toxicology and Pharmacy, Kharkiv Medical Academy of Post-graduate Education, 58 Amosova Street, 61176 Kharkiv, Ukraine

The article presents results of studies on the development of a quantification method for a  substance 7-(4-fluorobenzyl)-3-thioxo-2,3-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(7H)-one using the  method of non-aqueous potentiometric titration. Validation of the developed method has been carried out confirming the following characteristics: linearity, accuracy and precision that match the acceptance criteria for these measures.

Keywords: quantification, potentiometric titration,  [1,2,4]triazolo[4,3-А]pyrazin-8-ones, analytical validation


In the  last decade, the  number of new antimicrobial drugs, which are being introduced into the  world pharmaceutical market, has sharply decreased. This is due, first of all, to limited resources for conducting clinical trials of medicinal substances and the  rapid development of microorganism resistance to antimicrobial agents that certainly serves as a  deterrent to an uncontrolled arrival of new products onto the market.

Literature data indicate a  high antimicrobial activity of the compounds containing the nucleus of  [1,2,4]triazolo[4,3-a]pyrazine  [13]. The  synthetic scheme for synthesis of [1,2,3]triazolo[4,3-а] pyrazine derivatives based on substituted amides of oxalamic acids was developed. This scheme makes it possible to bring into the  structure, of the desired products, various substituents in positions 3 and 7 of heterocycle. The proposed approach allows the synthesis of large arrays of target products for the screening of pharmacological studies [4].

The microbiological screening of the  first synthesized derivatives of 3-thioxo-[1,2,4] triazolo[4,3-a]pyrazine, performed by us, made it possible to isolate substances with a  high antimicrobial and antifungal activity among compounds of this series [5]. According to the results of the microbiological screening for an in-depth study, the  substance 7-(4-fluorobenzyl)-3-thioxo-2,3-dihydro-[1,2,4]triazolo[4,3-а]pyrazin-8(7Н)-one has been selected (Fig.  1), the  antimicrobial activity of which was the  highest in relation to gram-negative bacteria (MICs 12.5 µg/ ml, MBCs 25.0 µg/ml) [5].

The aim of the  study is to develop a  method for quantitative determination of the  substance 7-(4-fluorobenzyl)-3-thioxo-2,3-dihydro-[1,2,4] triazolo[4,3-a]pyrazin-8(7H)-one with its subsequent validation for the  introduction of the  resulting substance into pharmaceutical practice. Article  [8] describes a  standardized procedure for validation of titrimetric methods, with its use for indicator titration, and the specifics of using this standardized procedure for the  most common method of titrimetric analysis  –  potentio-metric titration has not yet been experimentally described.


Fig. 1. The  chemical structure of 7-(4-fluorobenzyl)-3-thioxo-2,3-dihydro-[1,2,4]triazolo[4,3-а]pyrazin-8(7Н)-one


The test substance is a crystalline powder of white or almost white colour with a  grayish tinge and a  slight specific odour. The  solubility of the  obtained substance was established experimentally. The procedure for studying the solubility was performed according to the  requirements of SPhU. The substance of 7-(4-fluorobenzyl)-3-thioxo-2,3-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(7H)-one was found to be freely soluble in dimethylformamide, dimethylsulfoxide and triethylamine, slightly soluble in methanol and ethanol, and practically insoluble in water. An important step in the development of a drug is the development of accurate and reliable methods of its analysis. Due to the absence of a standard sample of the synthesized substance and on the  basis of its physicochemical properties, a direct method of determination was chosen for quantitative determination  –  potentiometric titration in a  non-aqueous medium. The  structure of the  7-(4-fluorobenzyl)-3-thioxo-2,3-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(7H)-one molecule contains nitrogen-containing heterocycles and exhibits basic properties. In the course of the work the approach of European Pharmacopoeia [7] was used, according to which the  greatest preference is given to direct methods of quantitative deter mination, which include potentiometric titration. Potentiometric titration is leading to determine the content of the active substance in pharmaceutical substances. Acidimetry in a non-aqueous medium allows the  weak bases and their salts to be determined with the necessary accuracy.

It was established experimentally that the most suitable solvent is a mixture of acetic acid and acetic anhydride, and the titrant is a pharmacopoeial titrated solution of 0.1 M perchloric acid.

Potentiometric titration was performed by using an automatic titrator 7025  M Titrino Metrohm (Switzerland) with a burette volume of 10 ml.

The amount of 7-(4-fluorobenzyl)-3-thioxo-2,3-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(7H)-one in the  substance in % was calculated by the formula

X= V0,02763K100100 m( 100W ) ,

where V is the volume of 0.1 M of perchloric acid solution, which was used for titration of the tested solution, ml; K is the correction factor to the molarity of titrant; 0.02763 is the number of grams of C12H9FN4OS, which corresponds to 1 ml of 0.1 M of perchloric acid solution; m is the mass of a sample weight; W is the  weight loss value on drying substance, %.

1 ml of 0.1 М of perchloric acid solution corresponds to 27.63 mg of C12H9FN4OS.


When using the  method of acid-base titration, the  uncertainty of the  result of the  final analytical operation is associated with taking a  sample of the  sample under investigation, determining the volume of the burette and determining the correction factor for the titrant solution [12]

Δ X,r = ( Δ m ) 2 + ( Δ Vt ) 2 + ( Δ C M ) 2 ,

where Δm is the sample uncertainty; ΔVt is the titration burette volume uncertainty; ΔCM is the uncertainty of the molar concentration of the titrated 0.1 M perchloric acid solution.

To prevent errors during titration, the weighed sample of the substance to be analysed should be taken from the  calculation so that 80  ±  10% of the volume of the burette is spent on titration [8].

As a result of experimental studies for the quantitative determination of 7-(4-fluorobenzyl)-3-thioxo-2,3-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-8(7H)-one the  following method was proposed: 0.250 g of the substance is dissolved in a mixture of 30 ml of acetic acid anhydrous P and 30 ml of acetic anhydride P, titrated with 0.1 M of perchloric acid solution potentiometrically till the  first jump in potential on the titration curve. The quantitative content of the main active substance in the substance should be from 99.0 to 101.0% in terms of dry substance. Characteristics of the  developed methods of acid-base titration are presented in Table 1.

Table 1. Characteristics of the developed acid-base titration method

Content tolerances, % Max Δas, % Max Δs, % Sample weight for analysis, m, g Max Δm, % Nominal titration volume, Vnom, ml ΔVt, % % of 10 ml burette volume ΔCM, %
99.0–101.0 1.0 0.32 0.25 0.080 8.95 0.20 89.5 0.063

The uncertainty of the result of the final analytical method for the  quantitative determination of 7-(4-fluorobenzyl)-3-thioxo-2,3-dihydro-[1,2,4] triazolo[4,3-a]pyrazin-8(7H)-one is 0.22%, which does not exceed the  maximum permissible uncertainty of the  analytical method (max Δas, %): 0.22 ≤ 1.00.

Earlier it was reported about the development of techniques for controlling concomitant impurities in the substance 7-(4-fluorobenzyl)-3-thioxo- 2,3-dihydro-[1,2,4]triazolo[4,3-а]pyrazin-8(7Н)- one  [6]. The  HPLC method was used to determine the  main impurities. Impurity А (1-(4-fluorobenzyl)-3-hydrazino-pyrazin-2(1H)- one) was a  semiproduct in the  synthesis of the  corresponding 3-thioxo-derivatives of  [1,2,4] triazolo[4,3-a]pyrazine. Impurity В (7-(4-fluorobenzyl)-[1,2,4]triazolo[4,3-a]pyrazine-3,8(2H, 7H)-dione) was an impurity of the decomposition of the  substance (oxidation under inappropriate storage conditions etc.) (Fig. 2). The total content of impurities in the  substance should not exceed 0.5%. Thus, the  accompanying impurities do not interfere with the  quantitative determination of the active substance in the substance by potentiometric titration.

On the basis of the previously obtained experimental data, the loss in mass during the drying of the substance was 0.2–0.4%. On the basis of experimental data, the weight loss during the drying of a substance is normalized at a level not exceeding 0.5%.


Fig. 2. The chemical structure of impurities A and B

For further use of the developed method, it was necessary to determine its validation parameters. Validation of the  developed method was carried out according to the  requirements of the  State Pharmacopoeia of Ukraine (SPhU) by studying its linearity, accuracy and precision.

The establishment of a linearity was performed by titration of nine different concentration samples in a  range of 80–120% of the  nominal sample of the test substance described in the method. Accordingly, a series of working solutions with concentrations of 80, 85, 90, 95, 100, 105 110, 115 and 120% of 250 mg was prepared. To study the reproducibility of the results in the process of studying the linearity, we took 3 samples for each point in a range of 80–120%. Using the  rule of concurrent validation, accuracy and precision were determined using the data obtained in the determination of linearity. To calculate the  normalized coordinates, we took the  nominal sample weight and the  nominal volume presented in Table  1. The results of studying the  linearity of the entire sample of 27 points and a comparison with the criteria [8] are presented in Table 2.

The results of the study of linearity allow us to conclude that the criteria |a| and |1–b| do not satisfy the requirement of statistical insignificance, but satisfy the requirement of practical acceptability of a linearity. The results of determining the precision and accuracy of the  developed methodology are presented in Table 3.

Table 2. Characteristics of the linearity Y = a + b∙X for the developed method

Parameter Value Statistical insignificance criterion Practical acceptability criteria Conclusion
a –0.92 |a| ≤ 0.131 not correspond
b 1.0111
|1–b| 0.0111 |1–b| ≤ 0.0013 not correspond
s0 0.255 ≤0.39 correspond
r 0.99982 ≥0.99959 correspond
r2 0.99965 ≥0.99917 correspond
δRL,80 0.039 ≤0.67 correspond
δRL,120 0.34 ≤0.67 correspond

Table 3. Accuracy and precision of the developed analytical method

X, % Y, % Z, %
80.08 80.22 100.18
80.24 80.45 100.26
79.92 79.78 99.82
85.12 85.47 100.42
85.24 85.81 100.67
84.88 84.47 99.52
89.84 89.50 99.62
90.08 89.94 99.85
90.32 90.28 99.95
94.88 95.20 100.33
94.68 94.53 99.84
95.12 95.42 100.31
100.24 100.45 100.21
100.00 100.11 100.11
99.96 99.89 99.93
104.72 104.80 100.08
105.12 105.25 100.12
105.04 105.03 99.99
109.92 110.17 100.23
110.08 110.73 100.59
110.24 110.84 100.54
114.96 115.31 100.30
114.68 114.97 100.25
114.92 115.53 100.53
120.26 120.45 100.24
119.92 120.56 100.53
120.20 120.45 100.21
x 100.17
SD 0.29
ΔR 0.19
δ = |–x – 100 0,17
Statistical insignificance, δ ≤ ΔR
0.17  0.19
Practical insignificance, δ ≤ 0.67
0.17 ≤ 0.67
Practical acceptability of precision (6 titrations), Δ R 1 3 Δ as
0.25  0.33

The developed method meets the requirements of accuracy and precision.

The obtained experimental data and the calculations based on them indicate that all the validation parameters of the developed method correspond to the necessary acceptance criteria.


1. Based on the  results of the  study, a  quantification technique for determination of the  basic substance in the  new pharmaceutical substance 7-(4-fluorobenzyl)-3-thioxo-2,3-dihydro-[1,2,4] triazolo[4,3-a]pyrazine-8(7H)-one was developed.

2. Validation of the  proposed technique was performed, which confirms matching the following characteristics: specificity, linearity, correctness, precision, convergence (repeatability) and intermediate (intralaboratory) precision matching the acceptance criteria for these measures.

Received 22 December 2020

Accepted 12 January 2021


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* Corresponding author. Email: anna.materienko@gmail.com

Krystyna Netosova, Oksana Zavada, Anna Materiienko, Iryna Zhuravel, Serhii Baiurka



Straipsnyje pateikiamas naujos medžiagos 7-(4-fluorobenzil)-3-tiokso-2,3-dihidro[1, 2, 4]triazolo[4,3-a]pirazin-8(7h)-ono kiekybinio nustatymo metodas naudojant nevandeninį potenciometrinį titravimą. Siūlomo metodo tinkamumas patvirtintas ištyrus pagrindines charakteristikas – tiesiškumą ir tikslumą.