Development and validation of HPLC-DAD method of determination piperidinium 2-( ( 5-( furan-2-yl )-4-phenyl-4 H-1 , 2 , 4-triazol-3-yl ) thio ) acetate in 1 % solution

Materials and methods. LC System was Agilent 1260 Infinity (degasser, binary pump, autosampler, thermostatted column compartment, diode array detector). Single quadrupole mass spectrometer Agilent 6120 with ionization in electrospray (ESI). Open LAB CDS Software. Column was Zorbax SB-C18; 30 mm × 4.6 mm; 1.8 μm. Injection volume was 2 μL. Isocratic mode. The mobile phase was water/acetonitrile (70:30) with 0.1 % methanoate acid. Standard samples were piperidinium 2-((5-(furan-2-yl)4-phenyl-4H-1,2,4-triazol-3-yl)thio)acetate, furan-2-carbohydrazide, 2-(furan-2-carbonyl)-N-phenylhydrazine-1-carbothioamide, 5-(furan-2-yl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione.

Determination of API in the manufacture and storage of solution for injection is an important task of modern pharmaceutical analysis.
Qualitative and quantitative methods of piperidinium 2-((5-(furan-2-yl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)acetate determination in the 1 % and 2.5 % solutions have been developed earlier.Іts ability to absorb light in the ultraviolet region of the spectrum was used for quantification of these compounds.Maximum absorption of aqueous solution was 280 nm.Distilled water was used as a solvent.Spectrophotometric method has low sensitivity and low selectivity [1].

The purpose
The purpose of this research is to develop new, highly sensitive and selective method for determination of these compounds in 1 % injection solution based on high performance liquid chromatography with diode-array detection.
Preparation of the mobile phase В. 1.00 mL methanoate acid was diluted to 1000.0 mL with acetonitrile.
Preparation of the test solution.10.00 mL of the solution sample to be examined was diluted to 100.0 mL.
Preparation of the solution for the chromatographic system suitability.5 mg of each impurity standard sample was dissolved in the solvent mixture (water/acetonitrile was 70:30) and was diluted to 100.0 mL with the solvent mixture.It was mixed (solution ІА).50 mg of standard sample of API was dissolved in the solvent mixture (water/acetonitrile was 70:30).1.00 mL of solution IA was added and it was diluted to 100.0 mL with the solvent mixture.It was mixed (solution ІВ).
ASSAY.Liquid chromatography conditions were described above.Reference solution was injected n times.RSD was calculated by API peak.Injection was stopped when RSD ≤ RSD max for content bias 5 % (requirements of Ph.Eur.

Вопросы фармации
The solution of standard sample and test solution were injected alternately set number of times (n).The results were used in following calculations average.The percentage content of piperidinium 2-((5-(furan-2-yl)-4-phenyl-4H-1,2,4triazol-3-yl)thio)acetate was calculated from the declared content of chemical reference substance.The retention time of API peak was about 2.7 minutes.
Preparation of the API model solutions for method validation. 1 % solution of API was prepared (isotonic by addition 0.55 g of sodium chloride to 100 mL).

Results and discussion
Substantiation method conditions Hydrazide, carbothioamide and thion are possible impurities which are intermediates in the API synthesis [4].

Optimization of the acetonitrile concentration in the mobile phase
Previously stationary and mobile phase have been selected.Chromatographic behavior of 1,2,4-triazoles deri vatives and intermediates in their synthesis was studied [5][6][7].
These patterns were used for graph construction of the capacity factor from the acetonitrile concentration dependence in the mobile phase for potential impurities and API on diode-array detector wavelength 254 nm (Fig. 1).
On the chart we can see that the maximum difference between the lines is about 30 % with the minimal capacity factor, i. e. the minimal retention time and minimal analysis time.Resolution (R s ) can be used to characterize the quality of separation.Experimental determination and calculation of the resolution (R s ) were conducted according to the Ph.Eur.2.2.46 and Ph.Ukr.2.2.29 using the OpenLAB CDS Software [8,9].R s ≥ 4.1 (between API peak and compound peak (3)) and R s ≥ 2.7 (between compound (4) and API peaks (1)).The separation between API peak (1) and carbothioamide (3), API peak (1) and thion (4) peaks are satisfactory, because conforming Ph.Ukr.2.2.29 (have to be ≥ 1) [8].
The selecting of analytical wavelength for quantification of the API was based on the UV spectrum study (Fig. 3).The absorption spectrum was measured in the cell of diode-array detector with substance elution by 30 % acetonitrile containing 0.1 % methanoate acid.
API peak purity was verified by mass spectrometric detector (Fig. 4).Peak purity report according to the mass spectrometric detector presented that: "The analysis found only one component, indicating a pure peak.Component 1: Peak at Scan 290.1.Top ions are 302, 303" The method uncertainty prediction.Calculation of uncertainty the solution standard sample (reference solution) preparation: -weighing standard sample of API: (0.1 mg/100 mg) × 100 = 0.1 %; -dilution of the solution in a volumetric flask 100.0 mL: 0.12 %.
Calculation of uncertainty of the test solution preparation: -measuring of the volume of 1 % test solution of API 10.00 mL: 0.5 %; -dilution of the solution in a volumetric flask 100.0 mL: 0.12 %.

Problems of pharmacy
Thus, the predicted value Δ SP did not meet Ph.Ukr.(0.537 % > 0.51 %).Therefore final analytical operation should be treated more stringent requirements.

Вопросы фармации
Validation characteristics of a method Validation of the method was performed according to the requirements of Ph.Ukr. in the standard method version by the standardized procedure [3,10,11].API content bias of 1 % injection solution is 5 %.
Linearity.Linear dependence graph in normalized coordinates, an equation of calibration graph and R 2 value is presented at the Fig. 5. Metrological characteristics of linear dependence for the method application range 80-120 % for nominal АPІ content is presented in the Table 1.Method is linear at entire range and meets Ph.Ukr.[3,10].
Precision and accuracy.Results of the precision and accuracy estimation of the API quantitative determination method are presented in the Table 2.The results show that the method meets the requirements of Ph.Ukr. to precision and accuracy [3,10].
Applying the developed method to quantify API in the 1 % injection solution 10.00 mL of test solution into 6 flasks was poured.It was diluted to 100.0 mL.
Reference solution was injected n times for determination of RSD.A number of the repeat injections ware determined according to the requirements for Ph.Eur.2.2.46 and Ph.Ukr.2.2.29 to RSD %.
Each solution measured by pipet obtained from 6 samples was injected.
Reference solution injection was done 5 times.Results are shown in Table 3.The resulting value did not exceed RSD requiments Ph.Ukr.2.2.29 to RSD % max with all values of n, starting with n = 2.So it was enough 2 times alternate injectons reference solution and test solution for each solution sample [3].
3. The results of the method validation show that it is specific and meet the requirements of linearity, precision and accuracy.
4. The results of API content determination in real samples of solutions for injection indicate that the method can be proposed for quality control of 1 % solutions for injection.

Fig. 5 .
Fig. 5. Linear dependence of the API peak area from concentration in the normalized coordinates.

Table 1 .
Metrological characteristics of linear dependence for the quantitative determination method of API in 1 % injection solution Y = bX + a

Table 2 .
The results of precision and accuracy estimation of the API quantitative determination method in the 1 % injection solution

Table 3 .
Results of chromatography system suitability test for RSD

Table 4 .
The results of the quantitative determination API in the 1 % injection solution