The development and validation of HPLC-DMD method for intermediate products impurities determination of morpholinium 2-((4-(2-methoxyphenyl)-5-(pyridine-4-yl)-4 H -1,2,4-triazole-3-yl)thio)acetate in bulk drug

Purpose. A development and validation of new sensitive, high efficient and selective HPLC determination method of intermediates technological contaminations in bulk drug of morpholin-4-ium 2-((4-(2-methoxyphenyl)-5-(pyridine-4-yl)-4 H- 1,2,4-triazole-3-yl) thio)acetate (active pharmaceutical ingredient – API). Materials and methods. LC System was Agilent 1260 Infinity (degasser, binary pump, autosampler, column thermostat, diode array detector) Open LAB CDS Software. Column was Zorbax SB-C18; 30 mm × 4.6 mm; 1.8 µm. Injection volume was 5 µL. Isocratic mode. The mobile phase was water/acetonitrile (84:16) with 0.1 % methanoic acid. Standard samples were morpholinium 2-((4-(2-methoxyphenyl)-5-(pyridin-4-yl)-4 H- 1,2,4-triazol-3-yl)thio)acetate, pyridine-4-carbohydrazide, 2-isonicotinoyl- N- (2-me- thoxyphenyl)hydrazine-1-carbothioamide, 4-(2-methoxyphenyl)-5-(pyridin-4-yl)-2,4-dihydro-3 H- 1,2,4-triazole-3-thione. Results. A new criterion for choosing chromatographic separation condition was proposed. It is absolute value of retention factors differences (|Δ k |). Six different curves which show dependence of absolute value of retention factors differences (|Δ k |) for each compound from the acetonitrile in mobile phase was built at registration of the signal on diode-array detector. A chromatographic separation optimal condition of impurities and API in drug bulk was found with satisfied resolution. UV spectra of API and impurities were determined. Method of the quantitative determination of the impurities was elaborated. Total sample preparation uncertainty was predicted. Method was validated according to European and Ukrainian Pharmacopeia. It was applied for real bulk drug samples. Conclusions. Chromatography separation of impurities and API was done. A method was complied with linearity criteria, specificity, precision and accuracy. The results of impurity determination in bulk drug indicated, that method can be used for the quality control of bulk drug.


Introduction
Heterocyclic systems which are based on 1,2,4-triazole are interesting for modern pharmaceutical chemistry [1]. They have antioxidant, hepatoprotective and other activities, in addition some of them are already registered and are used in the practice. The morpholinium 2-((4-(2-methoxyphenyl)-5-(pyridine-4-yl)-4H-1,2,4-triazole-3-yl)thio)acetate is active pharmaceutical ingredient (API) of new drug. It is under registration and introduction to industry. That`s why the development of determination methods for its impurities, which effect on its pharmacological properties is an important task of modern pharmaceutical science, it has interest and practical significance.
Nowadays we know quantitative determination method of studied API and impurities in bulk drug with high performance liquid chromatography (HPLC). Method based on 5 µm sorbent. It has low efficiency and selectivity. It considers only single impurity, has low sensitivity and time-consumable. Spectrophotometric method of determination it API in 1 and 2.5 % water solutions doesn`t allow to determine the impurities [2].
The most effective method of impurity determination in pharmaceutical preparations and bulk drugs is HPLC with 1.8 µm size of sorbent particles.
Preparation of the mobile phase A. 1.00 mL of methanoic acid was added to volumetric flask with capacity 1000.0 mL, dissolved in 100.0 mL of highly purified water. The volume of solution was brought to mark by using the same solvent and mixed. Preparation of the mobile phase B. 1.00 mL of methanoic acid was added to volumetric flask with capacity 1000.0 mL, dissolved in 100.0 mL of acetonitrile, the volume of solution was brought to mark by using the same solvent and mixed.
Preparation of soluition for chromatography system suitability test. A weight of morpholinium 2-((4-(2-methoxyphenyl)-5-pyridine-4-yl)-4H-1,2,4-triazole-3-yl)thio)acetate standard sample (250.0 mg) was weighed accurately and transferred to volumetric flask with capacity 100.0 mL, dissolved in the highly-purified water and acetonitrile (84:16). 1.00 mL of the solution IA was added to it flask. The volume of solution was brought to mark by using the same solvent and carefully mixed.
Preparation of the test solution. An aliquot of 250.0 mg of the bulk drug sample was added to volumetric flask with capacity 100.0 mL then, dissolved in the 50.0 mL compound of highly-purified water and acetonitrile (84:16), brought the volume of solution to mark by the same solvent and carefully mixed.
Solutions of 2,3,4 standard samples are chromatographed, RSD is counted for each sample area, chromatography is stopped ,when received values of RSD do not exceed the value RSD max, which are calculated according to Ph. Eur. 2.2.46 and Ph. Ukr. 2.2.29. (The Supplement 1) for content limit of the compound (2) B = 16 %, content limits of the compound (3) and compound (4) is B = 5 % [3,4].
The solution of standard samples 2, 3, 4 and investigated solution are alternately chromatographed with (n) times, average values are used in further calculations.
When it is chromatographed at mentioned conditions, the retention time of pick API should be about 4.6 min, compound 2 (about 0.7 min) compound 3 (about 3.9 min) compound 4 (about 5.6 min).

Results and Discussion
Interpretation of the method conditions. The specific impurities of compound (2,3,4) can get into bulk drug of API in process of synthesis [1]. These impurities are identified by chromatography with mass spectrometric detection. The picks of appropriate impurities are detected at appropriate SIM with m/z 138, 303, 285, they are also complied in retention time on appropriate standards ( Fig. 1-3).
Chromatography determination conditions of these compounds should ensure them separation from API.
Optimization of concentration acetonitrile in the mobile phase.
Earlier we have discussed and suggested stationary phase and mobile phase, made an investigation of chromatographic behavior of a series of derivatives of 1,2,4-triazole and intermediate products of synthesis [5][6][7].
Based on received facts, we have built the graph of dependence of retention (capacity) factor k from concentration of acetonitrile in mobile phase for potential impurities and API at registration of the signal on diode-array detector at wavelength 254 nm (Fig. 4).
On this graph we can see, that maximum difference between curves is about 16-18 %. Index, which shows the quality of separation is the resolution (R s ). Experimental determination and calculation of the resolution (R s ) are conducted according to Ph. Eur. and Ph. Ukr. with using Open LAB CDS Software [3,4,8]. The separation between picks of API (1) and carbotiamide (3), API (1) and thione (4) is most critical at this research. A dependence of resolution for most problematic separations (compounds 3-1 and 1-4) from concentration of acetonitrile in the eluent was studied. According to Ph. Ukr. the results are considered sufficient if the resolution is more than 1.0. All resolution values were over 1.0, but resolutions were maximal and standard deviations of both resolutions were minimal at the 16 % (Table 1). Therefore, the optimal content of acetonitrile is 16 %. Alternatively, we proposed new criteria for choosing of separation condition. It is retention factors differences (|Δk|). Number of combinations for separation of two compounds for total number of four compounds are equal:

Вопросы фармации
Six different curves which show dependence of absolute value of retention factors differences (|Δk|) for each compound from the acetonitrile in mobile phase was built at registration of the signal on diode-array detector (wavelength 254 nm).
Study of UV spectra of API and impurities needs to choose the analytic wavelength, which can be used for determination of appropriate compounds ( Fig. 6-8).
The hydazide (0.694 min), carbothioamide (3.859 min), thione (5.566 min) and some unidentified impurities were found on the chromatogram of model solution of the bulk drug with impurities addition at 272 nm (API concentration is 0.5 g/L, injection volume is 5 µL) (Fig. 9). The retention time of API was 4.919 min.
Validation of impurities determination method in bulk drug. Prediction of uncertainty of method.
Uncertainty calculation of preparation of the solution of   (2) was selected 16 %, and at validation criteria calculation of the compound (3) and (4) was selected most stringent requirements (5 %), because results were complied with its requirements.

Linearity.
Metrological characteristics of linear dependence for using method range (25-125 %) from the nominal content of appropriate impurity are shown at tables 2-4. Linearity was complied with requirements of Ph. Ukr. Selectivity.
Pick of API was fully separated with picks of compound 2, 3, 4 R ≥ 2.96 (between picks of API and compound 3) and R ≥ 2.65 (between picks of the compound 4 and API).
Precision and accuracy.

The use of method for quantitative determination of API in bulk drug
In the investigated series of bulk drug the impurity of carbothioamide (compound 3) was not detected. That is why we have conducted determination of compounds (2) and (4).The solution was chromatographed 5 times. Results are shown at table 8, 9. Received value of RSD did not exceed the calculated one, accordingly to Ph. Ukr. requirements to RSD % max for the maximal uncertainty 16 % (compound 2) and 5 % (compound 4) at all values n, beginning from n = 2. Therefore, it is enough for alternate chromatography of the comparing and test solutions for each investigated sample of bulk drug [8]. The humidity of the bulk drug is identified on a loss of weight during the drying, it was 0.1 %.