Method for Trifuzol-Neo assay determination by GC-MS

A.A. Yaroshenko, V.V. Parchenko, O.A. Bihdan*, O.I. Panasenko,

Yu V. Karpenko, E.O. Karpun

Zaporizhzhya State Medical University.

*Corresponding Author E-mail: abigdana@gmail.com

ABSTRACT:

The derivatives of 1,2,4-triazole are of the great scientific interest in the fields of pharmaceutics, agricultural chemistry, and construction The production of Trifuzol-Neo has already been launched, and it is now commercially available. Compounds based on 1,2,4-triazole may also be useful in veterinary practice due to their antiviral activity. Trifuzol-Neo, or piperidinium ([5-(2-furanyl)-4-phenyl-4Н-1,2,4-triazol-3-yl] thio) acetate (Figure 1), has already proved to be an effective immunostimulator for poultry.Since that, the quality control of raw materials and the ready product is required during the production stage. Currently, there is only one method for determination of the assay of Trifuzol-Neo, which involves HPLC-DMD (Center of Medical Research Information and Patent and Licensing Practice of Ukraine. The main disadvantages of HPLC-DMD are the use of relatively high amounts of costly solvents (acetonitrile in this case), considerable baseline instability, and time-consuming system stabilization. Gas-chromatography is able to avoid these constrictions since it involves gas as a mobile phase, which is a more cost-effective and reproducible alternative. The newly developed GC/MS method for Trifuzol-Neo assay determination consists of the following steps: weighing 20.0mg of Trifuzol-Neo powder, dillution in 20mL methanol, injection of 0.5uL of the obtained solution to GC column (at least three times). The same operations are done with a Trifuzol-Neo standard.

KEYWORDS: Trifuzol-Neo, 1,2,4-triazol, antimicrobial activity, gas-chromatography, anticorrosive properties.

INTRODUCTION:

Among the large number of synthetic organic compounds that can be promising in drug production, 1,2,4-triazole derivatives, as biologically active substances, deserve special attention¹. The properties of the derivatives of the represented heterocyclic system are very diverse, and therefore inspire different scientists to search². Scientists have proven the presence of antimicrobial, anti-inflammatory and analgesic properties in new thiosemicarbazides with a fragment of 1,2,4-triazole³. Moderate antimicrobial activity against S. aureus, S. epidermidis, E. coli, and P. Aeruginosa was detected in new nitroderivatives⁴.

The active substance of the known effective anticancer drug "Letrazole" is a derivative of 1,2,4-triazole. Surfaces were created to visualize excited state properties such as highest occupied molecular orbital's, lowest unoccupied molecular orbital's and electrostatic potential (ESP) mapped density. The most feasible position for letrozole to act as a highly potent and totally selective aromatase inhibitor was found to be-116.271466 au (−72961.512600kcal/mol)⁵. The further introduction of the most promising compounds into practice or in-depth study of known substances requires a comprehensive approach. An important fragment of such scientific research is analytical scientific tests of potential substances. Chromatographic studies are used in various areas of human activity, helping to analyze samples efficiently and quickly. A simple, precise and accurate RP-HPLC method coupled with PDA detector has been developed for the estimation of erythrosine (E127) in food stuffs and tablet dosage forms determination was preformed on phenomenex C18 Gemini column (150X4.6mm), 5μ particle size⁶. Using gas chromatography, the authors analyzed the active components present in the ethanol extract of Delonix elata (L.) Gamble (Family: Caesalpiniaceae) and Clerodendrum phlomidis L, (Family: Verbenaceae)⁷. Also, the chromatographic method of analysis has proved effective in the study of the active pharmaceutical ingredient (AFI) of the drug «Rivaroxaban», an anticoagulant used for the prevention of venous thromboembolism⁸. Five products of hydrolytic decomposition of the active pharmaceutical ingredient «Prasugrel», an effective means of preventing blood clots, have been identified by gas chromatography with mass detection⁹. Ethylenediaminetetraacetic acid (EDTA) is widely used for the different purposes in pharmaceutical (bulk drug and formulations) industry. The authors propose a new effective and rapid method of identification of this component by liquid chromatography by the ability to form "chelates"¹⁰.

The derivatives of 1,2,4-triazole are of the great scientific interest in the fields of pharmaceutics, agricultural chemistry, and construction. In terms of pharmaceutics, 1,2,4-triazoles, especially their thione and thiol derivatives, exhibit a wide range of biological activities, including antimicrobial, antifungal, anti-inflammatory, anti-cancer, and diuretic¹¹. According to previous publication [12], compounds (4-((5-nitrofuran-2-yl) methyleneamino)-1-propyl-4H-1,2,4-triazolium bromide) and (N-((5-nitrofuran-2-yl)methylene)-4H-4-amino-1,2,4-triazolidium chloride) possess antimicrobial activity/ Research contains -((4-amino-5-R-4H-1,2,4-triazol-3-yl)thio)acetohydrazides as potential diuretic drugs [13]. New article demonstrated that (2-(5-(adamantan-1-yl)-4H-1,2,4-triazol-3-ylthio) acetates are promising antihypoxic medicines [14]. Other researchers study anticorrosive properties of 1,2,4-triazoles, which is particularly valuable in construction. New plan of synthesis pay special attention to 4-amino-3,5-bis(disubstituted)-1,2,4-triazole derivatives, which inhibited corrosion in a sufficient extent in their study [15]. However, compounds based on 1,2,4-triazole may also be useful in veterinary practice due to their antiviral activity. Trifuzol-Neo, or piperidinium ([5-(2-furanyl)-4-phenyl-4Н-1,2,4-triazol-3-yl]thio)acetate (Figure 1),  has already proved to be an effective immunostimulator for poultry¹⁶.

Figure 1: The structure of piperidinium ([5-(2-furanyl)-4-phenyl-4Н-1,2,4-triazol-3-yl]thio)acetate.

The production of Trifuzol-Neo has already been launched, and it is now commercially available. Since that, the quality control of raw materials and the ready product is required during the production stage. Currently, there is only one method for determination of the assay of Trifuzol-Neo, which involves HPLC-DMD. The main disadvantages of HPLC-DMD are the use of relatively high amounts of costly solvents (acetonitrile in this case), considerable baseline instability, and time-consuming system stabilization. Gas-chromatography is able to avoid these constrictions since it involves gas as a mobile phase, which is a more cost-effective and reproducible alternative.

The newly developed GC/MS method for Trifuzol-Neo assay determination consists of the following steps: weighing 20.0mg of Trifuzol-Neo powder, dilution in 20mL methanol, injection of 0.5uL of the obtained solution to GC column (at least three times). The same operations are done with a Trifuzol-Neo standard.

The combination of gas chromatography and mass-spectrometry brings more accuracy in both identification and quantitation of the compounds of interest, and Trifuzol-Neo in particular. However, mass-spectrometry detection is not required, and, thus, this method may be adjusted for the application with flame-ionization detector, which is more common in laboratory practice. This paper presents the first GC/MS method for determination of Trifuzol-Neo assay.

PURPOSE OF THE RESEARCH:

Develop a method for quantitative determination (assay) of piperidinium ([5-(2-furanyl)-4-phenyl-4Н-1,2,4-triazol-3-yl]thio)acetate (the API of Trifuzol-Neo) by GC/MS and validate the developed procedure.

MATERIALS AND METHODS:

Objects of research, solvents, and equipment.

The object of the research was the substance of Trifuzol-Neo. The sample substances for the study were obtained from German-Ukrainian Research-And-Production Firm “Brovapharma” (Brovary, Ukraine). In this study, methanol was used as a solvent due to a good solubility of the substance in it and suitable GC behavior, such as acceptable vapor volume at high injection temperatures.

Analytical equipment: Agilent 7890B (USA) GC system coupled with Agilent 5977B (USA) single quadruple mass-spectrometry detector. The injector is replaced with Gerstel CIS 4 cooled injection system with C506 controller (Germany). Components of the substance were separated on J&W Agilent DB-5ms (USA) GC column.

Method for determination of assay of Trifuzol-Neo

The following procedure was developed to determine the assay of Trifuzol-Neo.

Determination is performed using gas chromatography system coupled to MS-detector.

Test solution. Take 20.0mg of the substance to be examined, add 20.0mL of methanol R and dissolve.

Reference solution. Take 20.0mg of the trifuzol-neo CRS, add 20.0mL of methanol R and dissolve.

Carrier gas: helium for chromatography R.

Split ratio: 1:50

Flow rate: 2mL/min.

Column:

· size: l = 30m, Ø = 0.25mm;

· stationary phase: [phenyl][methyl]polysiloxane R (0.25µm).

Detector: MS.

Temperatures:

	Time (min)	Temperature (°C)
Column	0 - 4	60 → 260
	4 - 5	260
	5 - 9	260 → 290
Injection port		310
GC/MS Interface		290
Ionizer		230
Quadruple		150

Injection: 0.5µL, at least three times.

Elution order: piperidinium cation – around 1.87 min, ([5-(2-furanyl)-4-phenyl-4Н-1,2,4-triazol-3-yl]thio)acetate (acidic residue) – around 7.09 min.

Calculate trifuzol-neo assay in per cent m/m using the following expression:

S_x × m_st P × 100

--------------------

S_st × m_st × (100 – W)

Sx=	average area of the peak due to the acidic residue of the substance on chromatograms obtained with test solution;
Sst =	average area of the peak due to the acidic residue of the substance on chromatograms obtained with reference solution;
mx =	weighed quantity of the substance of trifuzol-neo, g;
mst=	weighed quantity of the trifuzol-neo CRS, g;
P =	percentage of the active ingredient in the trifuzol-neo CRS, %;
W =	percentage of water in the substance, %.

The assay of trifuzol-neo must be in range of 97–103%.

Method validation procedure

The following validation procedure was developed according to the recommendations of the State Pharmacopoeia of Ukraine; it was used to determine specificity, linearity, accuracy, and precision.

According to the State Pharmacopoeia of Ukraine, linearity, precision, and accuracy tests should be held simultaneously. To conduct the study, ten solutions of the trifuzol-neo CRS were prepared: nine model solutions with concentrations in the usable range between 80% and 120% (with 5% step), and one standard solution for reference.

Specificity:

Specificity is required to distinguish the substance of interest from other substances that may be present in the sample. Specificity is evaluated taking into account the peaks on the chromatogram and their resolution if the peak of the substance interferes with other peaks on the chromatogram.

Linearity:

Linearity is determined to approve the linear dependence between the sample concentration (C) and the corresponding signal (A). The following expressions reflect the linear dependence:

A_i                                    C_i

---× 100 = b × -----  × 100 + a

A_st                       C_st

Y_i = b ×X_i + a

Ai=	average area of the peak due to the acidic residue of the substance on chromatograms obtained with model solution;
Ast =	average area of the peak due to the acidic residue of the substance on chromatograms obtained with standard solution;
Ci=	concentration of model solution, %;
Cst =	concentration of standard solution, %;
Yi =	relation of the  to  multiplied by 100, %;
Xi =	relation of the  to  multiplied by 100, %;
b =	slope of the linear dependence for the obtained regression line;
a=	intercept of the linear dependence for the obtained regression line.

The intercept and slope are obtained through the following expressions:

To find the intercept, standard deviations of intercept and slope should be found first:

Residual variance  is calculated using the relation between experimental values and the values calculated using the determined intercept a and slope b:

Residual variance due to horizontal axis should not exceed the maximum uncertainty of the analysis, which is 3.0% in this case:

Test for statistical significance sets the limit for a (there is no limit for b, however):

Standard deviation of concentrations, required for finding the general index of correlation, is obtained through the expression:

The general index of correlation can be found using the following expression:

Limits for the general index of correlation are determined by the following expression:

Accuracy and Precision:

Accuracy shows if the method yields accurate results, while precision demonstrates its ability being reproducible. To assess accuracy, test for statistical significance is used; if the former fails, test for practical significance is used:

In precision assessment, one-sided confidence interval should not exceed the maximum uncertainty of the analysis :

RESULTS:

Three chromatograms were obtained for each of the 10 sample solutions. Fig. 2 demonstrates a typical chromatogram of piperidinium ([5-(2-furanyl)-4-phenyl-4Н-1,2,4-triazol-3-yl]thio)acetate obtained by GC/MS. The very first peak is piperidinium, the very last peak is the acidic residue of the substance. Their mass spectra are demonstrated in Figures 3 and 4.

Figure 2: Typical GC/MS chromatogram of piperidinium ([5-(2-furanyl)-4-phenyl-4Н-1,2,4-triazol-3-yl]thio)acetate.

Figure 3: Mass spectrum of the piperidinium.

Figure 4: Mass spectrum of the acidic residue.

Table 1: Accuracy and precision results.

№ mod. sol.	Quantity, g	Injected (rel. to ST), Xi, %	Avg. peak area	Found (rel. to ST), Yi, %
ST	0.0200	---	6513303	---	---
1	0.0159	79.50	5141027	78.93	99.28
2	0.0168	84.00	5432869	83.41	99.30
3	0.0180	90.00	5846622	89.76	99.74
4	0.0191	95.50	6204940	95.27	99.75
5	0.0199	99.50	6450806	99.04	99.54
6	0.0210	105.00	6904742	106.01	100.96
7	0.0221	110.50	7288766	111.91	101.27
8	0.0231	115.50	7614375	116.90	101.22
9	0.0243	121.50	7925937	121.69	100.16
Avg.,Z̅, %					100.14
Relative standard deviation, sz, %					0.81
δ% ≤ ΔZ / 3 =  0.50					0.14
ΔZ ≤ max ΔAs = 3.00 %					1.50

Mass quantities, injected and found concentrations, and peak areas along with results for accuracy and precision tests are designated in Table 1, while Table 2 contains the calculated parameters of linearity.

Table 2: Linearity results.

Parameter	Value	Acceptance criterion	Conclusion
a	-4.082	|a| ≤ 4.800	conforms
sa	0.920	---	---
b	1.043	---	---
sb	0.009	---	---
s0^2	0.368	---	---
Sy	14.293	---	---
Sx,0	0.353	≤ 1.583	conforms
Rc	0.9997	≥ 0.9938	conforms

DISCUSSION:

It was concluded from the experiments that the compound of piperidinium ([5-(2-furanyl)-4-phenyl-4Н-1,2,4-triazol-3-yl]thio)acetate has two peaks on the chromatogram which was confirmed by the peaks’ mass spectra. All calculations were performed due to the peak of acidic residue (mass of 243.0) on all chromatograms.

Results of accuracy, precision (Table 1), and linearity (Table 2) demonstrate that the method conforms to all requirements of the validation procedure. Namely, the value of one-sided confidence interval Δ_Z does not exceed the value of the maximum uncertainty of the analysis Δ_As. Moreover, the method passes the test for statistical significance, and δ% does not exceed the limit value. These evidences indicate that the obtained average value Z̅ is valid and represents reliable results which conform to the requirements for accuracy and precision of the method. Considering that the absolute value of the slope a and residual variance due to horizontal axis S_x,0 do not exceed the limits and the general index of correlation  is acceptable, it may be concluded that the method reliably yields linear results in range of 80-120% of the substance. Therefore, accuracy, precision, and linearity meet the requirements of the State Pharmacopoeia of Ukraine, which prompts that the newly developed method for Trifuzol-Neo assay determination is valid.

CONCLUSION:

A new GC/MS method of determination of Trifuzol-Neo assay has been developed and validated according to the State Pharmacopoeia of Ukraine. The method allows conducting qualitative and quantitative determination of the API of Trifuzol-Neo using the combination separation capabilities of gas chromatography and identification by mass spectrometry. As compared to the HPLC-DMD method, which is currently the only method for Trifuzol-Neo assay determination, GC/MS technique involves way smaller amounts of solvents, less time for sample preparation, and provides more reliable and reproducible results, which is very important in terms of quantitative analysis. The validation procedure confirmed that the method yields acceptable accuracy, precision, and linearity. Therefore, a new method may be used as a reliable alternative for the HPLC-DMD method of Trifuzol-Neo assay determination.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 01.04.2020           Modified on 11.10.2020

Accepted on 03.02.2021         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(9):4523-4528.

DOI: 10.52711/0974-360X.2021.00787