INTRODUCTION:

Despite antiepileptic drugs (AEDs) have the relatively wide range¹, only 65-70% of the patients can control epileptic attacks². The problem of refractory epilepsy is also remains acute³. The search for new AEDs, which could meet the criteria of high efficiency and a favorable safety, is a pressing issue.

Considering the above mentioned problem, a promising anticonvulsant, 1-(4-methoxyphenyl)-5-[2-[4-(4-methoxyphenyl)piperazin-1-yl]-2-oxo-ethyl]pyrazolo[3,4-d]pyrimidin-4-one, having the code name "Epimidin" (Figure 1) was synthesized⁴ and patented⁵.

Figure 1: Structure of Epimidine – perspective anticonvulsant

High anticonvulsant activity of Epimidine has been proven by using chemotoxic models – pentylenetetrazole (PTZ), caffeine, picrotoxin seizures, as well as in chronic epileptogenesis conditions – PTZ-kindling. The substance has demonstrated moderate activity in the model of MES-induced seizures and strychnine convulsions. Wide range of effective anticonvulsant doses (50-250mg/kg intragastrically), mild sedative and anxiolytic effect without muscle relaxant properties, positive cognitive effect, pronounced anti-inflammatory and analgesic properties, the absence of addictive potential, and toxicity class V according to Hodge and Sterner classification⁶ substantiate the necessity of the further study of Epimidine as a promising API.

An integral part of a new API pharmaceutical development is identification of impurities that may adversely affect pharmaco-technological parameters, pharmaco-toxicological profile, and cause the API or dosage forms side effects⁷. International Conference on Harmonization guidelines Q3A⁸ contains recommendations concerning the content, identification and qualification of impurities in new APIs obtained by chemical synthesis. According to the ICH, impurities associated with API are classified into the following categories: organic impurities – starting materials, process-related impurities, intermediates and degradation products; inorganic – salts, catalysts heavy metals; other materials (filter aids and charcoal) and residual solvents⁹. Organic impurities may occur during the manufacture and/or storage of substance and require clear regulation and control of the content^10,11. Analytical control of technological impurities requires a detailed analysis of all synthesis stages, as well as basic and secondary chemical processes. Impurity fate mapping draft is often necessary to predict, profile and regulate impurities even for every synthesis intermediate^12,13.

The literature data concerning the presence and determination methods of concomitant impurities in structurally similar substances were analyzed. For example, the presence of concomitant impurities of both different tautomeric forms of the initial 2,4-diamino-6-hydroxypyrimidine, and tautomers of the final synthesis product were found in Pemetrexed substance [4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrole [2,3-d] pyrimidin-5-yl) ethyl] benzoyl] -glutamic acid^14,15. In Pazopanib hydrochloride - 5-[[4-[(2,3-dimethylindazol-6-yl)-methylamino]pyrimidin-2-yl]amino]-2-methylbenzene-sulfonamide substance the presence of both intermediates and genotoxic impurities of synthesis by-products was specific¹⁶. To confirm the adequacy of the proposed methods for concomitant impurities determination in Zidovudine, the mentioned compounds were synthesized¹⁷.

Stability assessment¹⁸ is also important ICH recommendation during the process of APIs development. Determination of changes in API quality and estimation of degradation impurities under the influence of various environmental conditions, such as temperature, pH, humidity, light, etc., allows assessing the internal stability of the substance, determining the API processing, optimal storage conditions and recommending shelf life. Methods and approaches for determination of API impurities and degradation products include high performance liquid chromatography (HPLC), liquid chromatography–tandem mass spectrometry (LC/MS/MS)¹⁹, gas/liquid chromatography, thin-layer chromatography, as well as niche methods, e.g.: ultra high performance liquid chromatography (UHPLC), capillary electrophoresis, Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS), inductively coupled plasma-mass spectrometry (ICP-MS), gas chromatography (GC), etc²⁰. HPLC with UV and MS detection is priority method for detection, identifications, isolation, separation, quantitation, which is shown in determination of impurities of pyrimidine-containing substances both for new APIs^21-23, and for dosage forms^24,25, as well as in biological fluids²⁶.

HPLC is traditionally used for analysis of pyrimidine-containing Ambrisentan²⁷, Fluorouracil²⁸, anelated pyrimidine derivatives – Quinazoline^29,30, condensed – pyrazolopyrimidine – Zaleplon³¹, piperazine substituted derivatives^32-34. The data analysis concerning representative HPLC methods has shown that most of them are designed for water- and/or alcohol-soluble substances, while analytical methods for sparingly soluble substances having pyrazolopyrimidine structure are almost absent. Therefore, the aim of the given research was development and validation of the method for determination of related impurities of the synthesis of antiepileptic API – 1-(4-methoxyphenyl)-5-[2-[4-(4-methoxyphenyl)piperazin-1-yl]-2-oxo-ethyl]pyrazolo[3,4-d]pyrimidin-4-one (Epimidine) using HPLC method, and its stability testing under stress conditions.

MATERIAL AND METHODS:

Materials and instrumentation:

Liquid chromatography separation was performed using a Shimadzu Nexera X2 LC-30AD HPLC system (Shimadzu, Japan) composed of a quaternary pump, an on-line degasser, a column temperature controller, the SIL-30AC autosampler (Shimadzu, Japan); the CTO-20AC thermostat (Shimadzu, Japan) as well as the SPD-M20A diode array detector (DAD).

Another instruments such as Ultrasonic Cleaner Set for ultra-sonication using (Wise Clean WUC-A06H, Witeg Labortechnik GmbH, Germany), Libra UniBloc AUW120D (Shimadzu Analytical Scale, Japan). Volumetric glassware complies with Class A requirements of the SPhU³⁵ were used in the investigation.

¹H NMR spectra were recorded on a Varian Mercury-400 (Varian Inc., Palo Alto, CA, USA) spectrometer (400 MHz) in hexadeuterodimethyl sulfoxide (DMSO-d6) using tetramethylsilane (TMS) as an internal standard (chemical shifts are in ppm). The elemental analysis was performed on a Euro Vector EA-3000 (Eurovector SPA, Redavalle, Italy) microanalyzer. Elemental analyses were within ± 0.4% of the theoretical values.

Synthesis of related substances:

Ethyl 5-amino-1-(4-methoxyphenyl)-1H-pyrazole-4-carboxylate (related substance А, Scheme)

1.75g (0.01mole) of 4-methoxyphenylhydrazine hydrochloride and 1.53ml triethylamine (0.011mole) were added to solution of 1.57ml (0.01mole) of (2E)-ethyl 2-cyano-3-ethoxyacrylate (1mole) in 5ml of isopropanol. The mixture was hit during 2 hours at a temperature of 60°С. The reaction mixture was cooled to room temperature, isopropanol was vacuum-evaporated, and the residue was diluted in 20ml of water. The formed precipitate was filtered, washed with water and dried. The substance was crystallized from isopropyl alcohol.

Yield: 98%, melting point (mp) 180-2°C; ¹H NMR (400 MHz, DMSO-d₆, δ (ppm)): 7.68 (1H, s, CH-3), 7.42 (2H, d J=8.2, H-3',5'), 7.09 (2H, d, J=8.2, H-2',6'), 4.22 (2Н, q, ОCH₂), 3.85 (3H, s, OCH₃), 1.28 (3H, t, СН₃). Found, m/z: 262.18 [M+H]⁺. The Anal. Calcd. was for C₁₃H₁₅N₃O₃: C, 59.81; H 5.81; N, 16.1; we found: C, 59.78; H 5.80; N, 16.2. UV–vis (СН₃ОН) max 238 nm.

1-(4-Methoxyphenyl)-5H-pyrazolo[3,4-d]pyrimidin-4-one (related substance В)

2.61g (0.01mole) of ethyl 5-amino-1- (4-methoxyphenyl) -1H-pyrazole-4-carboxylate was hit in 2.0ml (0.03mole) formamide at a temperature of 120°С within 24 hours. The reaction mixture was cooled to room temperature. The formed precipitate was filtered and crystallized from isopropyl alcohol.

Yield: 89%, melting point (mp) 188-90°C; ¹H NMR (400 MHz, DMSO-d₆, δ (ppm)): 12.52 (1H, br. s, NH-3), 8.32 (1H, s, CH-3), 8.12 (1H, s, CH-5), 7.89 (2H, d J=8, H-3',5'), 7.12 (2H, d, J=8, H-2',6'), 3.82 (3H, s, OCH₃). ¹³C NMR (126 MHz, DMSO-d₆): δ 158.1, 157.3, 145.7, 141.9 (2), 132.0, 114.9 (2), 112.6, 111.6, 108.0, 55.8. Found, m/z: 243.08 [M+H]⁺. The Anal. Calcd. was for C₁₂H₁₀N₄O₂: C, 59.50; H 4.16; N, 23.16; we found: C, 59.30; H 4.14; N, 23.16. UV–vis (СН₃ОН) max 236 nm.

2-Chloro-1-[4-(4-methoxyphenyl)piperazin-1-yl]ethanone (related substance С)

9.6ml (0.12mole) of chloroacetyl chloride was added dropwise to the solution of 19.2 (0.1mole) of 1- (4-methoxyphenyl) piperazine in 25ml dioxane using an ice bath. After reaction is completed, the solution is diluted with fourfold volume of water and filtered. The additional cleaning is not required. MF: C₁₃H₁₇ClN₂O₂, MW:268.74. Melting point (mp) 93-95°C³⁶.

HPLC, conditions and reagents:

HPLC grade acetonitrile (Sigma-Aldrich GmbH, Switzerland), was used in the analysis. HPLC grade water was obtained from a water purifying system (Millipore, Bedford, MA, USA). Other chemicals and solvents were of analytical grade. All the solvents used for mobile phase were filtered through membrane (0.22 m pore size) and degassed before use. Epimidine (Fig. 1) was synthesized with a purity of 99.5%, which was established by non-aqueous titration.

Chromatographic separation was performed on an ACE C18 column 250mm x 4.6mm, 5µm particle size (YMC) with a pre-column. The mobile phase A – 2.0g/L sodium phosphate dibasic solution with 5.0ml triethylamine, adjusted to pH 7.0 with dilute phosphoric acid R; the mobile phase B – methanol R.

The separation was carried out by a multi-layered gradient elution mode using the following gradient program:

0-15 min 65→25% А, 35→75% В; 15-17min 25% А, 75% В; 17-18 min 25→65% А, 75→35% В; 18-25 min 65% А, 35% В. The flow rate was maintained at 1ml/min with UV detection at 240nm. The sample injection volume was 10µl and the column temperature was maintained at 45^◦C.

All solutions were prepared immediately before use according to the procedures described below.

Test solution. 20.0mg of substance Epimidin (accurately weighted) was placed into a 20.0ml volumetric flask, 8 ml of dimethylsulfoxide R were added, ultrasonicated for 15 minutes, made up to the mark with the methanol R and mixed.

Reference solution a. 5.0mg of impurity A (accurately weighted) was placed into a 20.0ml volumetric flask, 8 ml of dimethylsulfoxide R were added, ultrasonicated for 15 minutes, made up to the mark with the methanol R and mixed.

Reference solution b. 5.0mg of impurity B (accurately weighted) was placed into a 20.0ml volumetric flask, 8 ml of dimethylsulfoxide R were added, ultrasonicated for 15 minutes, made up to the mark with the methanol R and mixed.

Reference solution c. 5.0mg of impurity C (accurately weighted) was placed into a 20.0ml volumetric flask, 8 ml of dimethylsulfoxide R were added, ultrasonicated for 15 minutes, made up to the mark with the methanol R and mixed.

Reference solution d. 1.0 of test solution was placed into a 100.0ml volumetric flask, made up to the mark with the methanol R and mixed.

Reference solution e. 5.0ml of reference solution d were placed into a 50.0ml volumetric flask, made up to the mark with the methanol R and mixed thoroughly.

System suitability solution. 1.0ml of reference solution a, 1.0ml of reference solution b, 1.0ml of reference solution c and 1.0ml of test solution were placed into a 25.0ml volumetric flask, made up to the mark with the methanol R and mixed.

LC/MS, conditions and reagents:

The LC/MS studies for impurities were carried out with Acquity H-class UPLC system (Waters, Milford, USA) equipped with Acquity UPLC BEH C18 column (2.1×50 mm, 1.7μm) (Waters, Milford, USA).

Gradient elution was performed with 0.1% formic acid water solution (solvent A) and acetonitrile (solvent B) with the flow rate set to 0.5ml/min. Linear gradient profile was applied with following proportions of solvent B: initial – 5%, 7 min – 50%, 7.5 min -100%, 9 min -100%, 9.10 min – 5% followed by 5 min re-equilibrium at initial.

Xevo TQD triple quadrupole mass spectrometer detector (Waters Millford, USA) was used to obtain MS/MS data. Positive electrospray ionization was applied with the following settings: capillary voltage – 1.5 kV, source temperature –150°C, desolvation temperature –350°C, desolvation gas flow – 650 l/h, cone gas flow – 25 l/h. Collision energy and cone voltage was optimized for each compound separetely. Collision energy varied in range from 6 eV to 20 eV and cone voltage was selected from 8 V to 38 V.

Degradation study:

Degradation of Epimidine was examined in acidic, basic, oxidative and temperature conditions. Epimidine samples (20mg) were treated in parallel with hydrochloric acid (1.0 M, 2ml), sodium hydroxide (1.0 M, 2ml), 3% hydrogen peroxide (2ml) and 100ºC temperature for 2 h. The acid and base treated samples were neutralized with the same volume of base and acid, respectively. The obtained solutions were diluted to the testing concentration with methanol and analyzed by HPLC and LC/MS.

RESULTS AND DISCUSSION:

At the first stage of research, the method of Epimidine substance synthesis was analyzed, and potential impurities at all stages of synthesis were determined. Ethyl 5-amino-1-(4-methoxyphenyl)-1H-pyrazole-4-carboxylate (А) – the first main synthesis intermediate, 1-(4-methoxyphenyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4-one (В) – the second one, and 2-chloro-1-[4-(4-methoxyphenyl)piperazin-1-yl]ethanone (C) – alkylating agent adding at the last stage of synthesis (Fig. 2) were detected as the most probable impurities in the final API. The initial 4-methoxyphenylhydrazine hydrochloride and ethyletoxymethylencyanocetate, used for carboxylate (A) synthesis, did not determined due to 98% yield and the following multi-stage purification of each of the intermediates and the final product make their presence impossible.

Table 2: Linearity, LOD and LOQ parameters of the developed HPLC assay method

Component	Concentration (μg/ml)	Equation	Correlation coefficient r²	RSD, %	LOD (ng/ml)	LOQ (ng/ ml)
Epimidin	33.87-0.27	y = 49888.3x+9176.48	0.999951	1.18	56	169
Impurity A	12.5-0.098	y = 121457x+31511.00	0.999943	1.09	30	90
Impurity B	33.87-0.27	y = 57018.4x+9835.36	0.999945	1.20	32	98
Impurity C	0.42-26.7	y=25497.3x+3753.20	0.9999828	0.89	30	90

Table 3: Accuracy parameters and precision study results of developed HPLC method

Compound	Concentration (µg/ml)	Intra Day (n=3)		Inter Day (n=6)
Compound	Concentration (µg/ml)	RSD (%)	Accuracy (%)	RSD (%)	Accuracy (%)
Epimidin	33.87	0.21	99.70	0.76	100.07
	4.23	0.83	101.18	0.90	100.57
	1.06	0.67	99.06	0.85	99.87
Impurity A	12.50	0.23	99.68	0.26	100.32
	1.56	1.35	101.92	1.12	101.23
	0.78	0.91	98.72	1.02	99.82
Impurity B	33.87	0.17	100.24	0.39	100.02
	4.23	1.16	101.65	0.95	100.98
	1.06	1.35	98.11	0.85	99.64
Impurity C	0.84	0.97	99.97	0.99	100.47
	3.36	1.02	99.66	1.05	100.25
	26.70	0.83	100.23	0.87	100.10

Accuracy and Precision:

The RSD% were found to be less than 1.5% for intra-day and inter-day precision for all analyzed components. The recovery was 100±2% for all samples. The results summarized in Table 3. All the data indicate that the method is highly accurate and precise for the determination of Epimidine related impurities.

Robustness:

The robustness of the method was examined by small variations of critical parameters, such as column temperature (±5°С), pH (±0.5) and flow rate of mobile phase (±10%). Table 4 shows changes in the impurities separation degree.

Table 4: The impurities separation degree after chromatography parameters changing

Flow rate, ml/min	t, ^oC	pH	B→C	C→A	A→ Epimidine
1.0	45	6.5	7.07	4.70	8.65
1.0	45	7.5	8.39	4.75	8.42
1.0	40	7.0	9.35	6.32	12.06
1.0	50	7.0	11.02	6.05	11.59
0.9	45	7.0	10.15	5.95	11.07
1.1	45	7.0	11.51	6.82	12.66

The results indicate that the method was robust with respect to the key figures of merit.

All the parameters meet the necessary eligibility criteria. The method is validated and can be used for determination of related impurities in Epimidine substance.

Stability of test solution:

To study Epimidine solution stability, a sample was studied for the individual and total impurities at every 4 hours to 24 hours against a freshly prepared spiked sample. It was found that the there is no changes in the impurity level of the test sample against a freshly prepared solution. The solution was stable up to 24 hours under the proposed experiment conditions.

LC/MS analysis:

To raise the selectivity of the study, LS/MS/MS mode was used for identification of the related impurities and the main substance. Using this mode, ions of pre-set mass accumulated and mass spectra were recorded. The LS/MS/MS results of the substances have shown peaks of m/z 475.1, 243.0, 262.0 and 269.1 (M+H)+, correspond to protonated molecular ions of Epimidine and B, A and C impurities, respectively. The molecular weight of the substances was determined as 474, 242, 261, 268 Da.

Epimidine degradation study:

The drug substance was thoroughly examined for stability assessments under various stress conditions such as high temperature, as well as the influence strong acid and base and oxidizing agents. The obtained solutions were analyzed by HPLC/MS. The results of the analyzed solutions for accelerated degradation under stressful conditions are shown in Table 5.

Table 5: Results of HPLC analysis of related impurities in the substance

Impurities	RT min, (relative RT)	Amount, %
Impurities	RT min, (relative RT)	No degradation	0.1 М NaOH	0.1 М HCl	3 % H₂O₂	t^oC
Imp 1	7.69 (0.54)	0.03	-	-	0.03	0.03
Imp B	8.98 (0.63)	0.03	-	-	0.04	0.15
Imp С	10.96 (0.77)	0.10	-	-	-	-
Imp 2	12.55 (0.88)	0.01	-	-	-	-
Imp 3	15.65 (1.11)	0.09	-	-	-	0.45
Imp 4	17.27 (1.21)	0.07	-	-	-	0.15
Imp 5	3.86 (0.27)	-	11.74	0.51	0.38	-
Imp 6	4.55 (0.32)	-	-	0.04	0.25	-
Imp 7	6.64 (0.47)	-	10.15	0.37	0.21	-
Imp 8	9.75 (0.68)	-	-	-	0.06	-
Imp 9	10.33 (0.72)	-	35.86	24.21	1.92	-
Imp 10	12.44 (0.87)	-	2.49	3.80	0.29	0.04
The amount of impurities		0.33	60.24	28.93	3.18	0.72

After peroxidation, unidentified impurities 5-10 are formed, similar to alkaline and acid hydrolysis. The additional impurity 8 with retention time 9.75 (0.68) appeared, and the amount of impurity 1 with retention time 7.69 (0.54) was still 0.03% (Fig. 6). Besides, higher concentration of the impurity B was determined – 0.04%, comparing to the solution without degradation (0.03%), which may point to a partial cleavage of the alkyl moiety during oxidation of Epimidine substance.

Temperature degradation leads to formation of the smallest and insignificant total amount of impurities – 0.72% (Fig. 6). The amount of impurity 1 remains unchanged, but impurity B concentration slightly increases from 0.03 to 0.15%. An increase in concentration is also observed for 3 and 4 impurities – from 0.09 to 0.45 and from 0.07 to 0.15%, respectively. Also, an impurity 10 appears which is typical for all four solutions after degradation. As it is shown on stress conditions chromatograms, compared with chromatogram of the substance test solution, Epimidine is not resistant to peroxidation, alkaline and acid decomposition, but it is still resistant to temperature degradation.

CONCLUSIONS:

A new HPLC method has been developed and validated for the determination of related substances for new perspective anticolvunsant agent Epimidine. The developed method is specific, accurate, precise and linear across the analytical range according to ICH recommendations. The described method can be used for identification of poorly water- and alcohol-soluble pyrazolopyrimidine derivatives during new APIs development. Synthesis and degradation impurities profile under the stress conditions was studied. The obtained results will allow establishing the substance shelf life in future. It was found that Epimidine substance is not resistant to peroxidation, alkaline and acid decomposition, which should be considered during the dosage form development. Epimidine is well-resistant to temperature degradation, which allows storage the substance at room temperature.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Vossler, D. G., Weingarten, M., Gidal, B. E. American epilepsy society treatments committee. Summary of antiepileptic drugs available in the United States of America: working toward a world without epilepsy. Epilepsy Currents. 2018; 18.4_suppl: 1-26.

2. Vogt, V.L., Äikiä, M., Del Barrio, A., Boon, P., Borbély, C., Bran, E., Braun, K., Carette, E., Clark, M., Cross, J.H., Dimova, P., Fabo, D., Foroglou, N., Francione, S., Gersamia, A., Gil-Nagel, A., Guekht, A. Current standards of neuropsychological assessment in epilepsy surgery centers across Europe. Epilepsia. 2017; 58(3): 343-355.

3. Janmohamed, M., Brodie, M.J., Kwan, P. Pharmacoresistance - Epidemiology, mechanisms, and impact on epilepsy treatment. Neuropharmacology. 2020; 168: 107790

4. Severina, A. I., Georgiyants, V. А., Shtrygol, S. Yu., Kavraiskyi, D. P. Synthesis of N-substituted derivatives of 1-(4-methoxyphenyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one as potential anticonvulsants. Scripta scientifica pharmaceutica. 2016; 3(2): 36-40.

5. The invention patent №116226 A61K 31/505 (2006.01), A61P 21/00, C07D 231/54 (2006.01) C07D 471/04 (2006.01). 5-R-1-Aryl-1,5-dihydro-4H-pyrazolo [3,4-d] pyrimidin-4-ones, having anticonvulsant activity. Severina H. I., Kavraiskyi D. P., Shtrygol S. Yu., Georgiyants V. А. National University of Pharmacy. Declared 06.07.2015 № a 2015 06662. Published 26.02.2018. Bulletin 4.

6. Hodge, H. C., Sterner, J. H. Tabulation of toxicity classes. American Industrial Hygiene Association Quarterly. 1949; 10(4): 93-96.

7. Jain, D., Basniwal, P. K. Forced degradation and impurity profiling: recent trends in analytical perspectives. Journal of Pharmaceutical and Biomedical Analysis. 2013; 86: 11-35.

8. ICH Harmonised Tripartite Guideline. Impurities in new drug substances Q3A (R2). In: Proceedings of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland. 2006.

9. Roy, J. Pharmaceutical impurities—a mini-review. AAPs PharmSciTech, 2002; 3.2: 1-8. (https ://doi.org/10.1208/pt030 206—99943)

10. Venkannaa, G., Madhusudhan, G., Mukkanti, K., Sampath Kumar, Y. Identification synthesis of process-related impurities (substances) ethyl-4-(1-hydroxy-1-methylethyl)-2-propyl-imidazole-5-carboxylate [key intermediate of Olmesartan medoxomil (Antihypertensive drug)]. Asian Journal of Research in Chemistry. 2015; 8(5): 307-317.

11. Yamgar, R., Sawant, S. Synthesis and characterization of novel impurities in Paroxetine Hydrochloride hemihydrate and adopting QbD principles to built in quality in process of final drug substance. Asian Journal of Research in Chemistry. 2012; 5(3): 329-335.

12. Li, Y., Liu, D. Q., Yang, S., Sudini, R., McGuire, M. A., Bhanushali, D. S., Kord, A. S. Analytical control of process impurities in Pazopanib hydrochloride by impurity fate mapping. Journal of Pharmaceutical and Biomedical Analysis. 2010; 52(4): 493-507.

13. Pirrone, G. F., Mathew, R. M., Makarov, A. A., Bernardoni, F., Klapars, A., Hartman, R., Limanto J., Regalado, E. L. Supercritical fluid chromatography-photodiode array detection-electrospray ionization mass spectrometry as a framework for impurity fate mapping in the development and manufacture of drug substances. Journal of Chromatography B. 2018; 1080: 42-49.

14. Kjell, D. P., Hallberg, D. W., Kalbfleisch, J. M., McCurry, C. K., Semo, M. J., Sheldon, E. M., Spitler T. J., Wang, M. Determination of the source of the N-methyl impurity in the synthesis of pemetrexed disodium heptahydrate. Organic Process Research & Development. 2005; 9(6): 738-742.

15. Hongliu Wu. Pemetrexed intermediate and preparation method thereof. China Unexamined APPLIC, open to Public inspection CN20071024889. 06 Jul 2007.

16. Liu, D. Q., Chen, T. K., McGuire, M. A., Kord, A. S. Analytical control of genotoxic impurities in the pazopanib hydrochloride manufacturing process. Journal of Pharmaceutical and Biomedical Analysis. 2009; 50(2): 144-150.

17. Vadgaonkar, A. R., Kasture, V. S., Gosavi, S. A., Pawar, S. S., Jadhav, G. P. (2014). Synthesis, isolation, characterisation and quantification of process related impurity of zidovudine. Rasayan Journal Chemistry. 2014; 7(2): 181-189.

18. Guidance for Industry: Q1A(R2) Stability Testing of New Drug Substances and Products, United States Department of Health and Human Services, Food and Drug Administration, November 2003.

19. Gupta, A., Rawat, S., Gandhi, M., Yadav, J.S. Method development and acid degradation study of doxofylline by RP-HPLC and LC-MS/MS. Asian Journal of Pharmaceutical Analysis. 2011; 1(1): 10-13.

20. Holm, R., Elder, D. P. Analytical advances in pharmaceutical impurity profiling. European Journal of Pharmaceutical Sciences, 2016; 87: 118-135.

21. Liu, M., Wang, J., Liu, P. HPLC method development, validation, and impurity characterization of a potent antitumor nucleoside, T-dCyd (NSC 764276). Journal of Pharmaceutical and Biomedical Analysis, 2016; 131: 429-435.

22. Swathi, P., Rajeswar Dutt, K., V Rao, K. N., Alagar Raja, M. RP-HPLC Method development and validation for estimation of Sofosbuvir in pure and tablet dosage form. Asian Journal of Pharmacy and Technology. 2017; 7 (3): 153-156.

23. Prakash, S. S., Kalyan, K., Raju, S.A., Danki, L.S.. Development and validation of spectrophotometric methods for the estimation of Gemcitabine in bulk drug and its pharmaceutical formulations. Research Journal of Pharmacy and Technology. 2011; 4(6): 951-954.

24. Annapurna, M. M., Baswani R., Pradhan D.P. New Stability Indicating RP-UFLC Method for the Determination of Flucytosine – An Anti Fungal Agent. Acta Scientific Pharmaceutical Sciences, 2019; 3(5): 84-90.

25. Satyanarayana, L., Naidu, S.V., Narasimha Rao, M., Reddy Suma Latha. The estimation of Nilotinib in capsule dosage form by RP-HPLC. Asian Journal of Pharmaceutical Analysis. 2011; 1(4): 100-102.

26. Markelj, J., Zupančič, T., Pihlar, B. Optimization of high performance liquid chromatography method for simultaneous determination of some purine and pyrimidine bases. Acta Chimica Slovenica. 2015; 63(1): 8-17.

27. Narayana, M. B. V., Chandrasekhar, K. B., Rao, B. M. (2014). A validated specific stability-indicating RP-HPLC assay method for ambrisentan and its related substances. Journal of Chromatographic science, 2014; 52(8): 818-825.

28. Pasha, S. I., Ibrahim, M., Balram, V. M. Stability Indicating Analytical Method Development, Validation, Method Transfer And Impurity Profile (Related Substances) of 2,4-Dihydroxy-5-Fluoropyrimidine by Liquid Chromatography. International Journal of Pharmaceutical Research and Applications, 2016; 1(1): 53-59

29. Sharma, M., Upadhyay, N. K., Mahindroo, N. Stability-indicating HPLC Method for Determination of 7, 8, 9, 10-tetrahydroazepino [2, 1b] quinazolin-12 (6H)-one, a Potential Anticancer Agent. Indian Journal of Pharmaceutical Sciences, 2017; 78(6): 769-774.

30. Kumar, P., Dhande, P., Mazlee, M., Yaman, S. M., Chandran, N., Makhtar, M., Shekhawat, D., Lanke, S., Kumar, R., Mhetre, S. A controlled, efficient and robust process for the synthesis of an epidermal growth factor receptor inhibitor: Afatinib Dimaleate. Chemical Reports, 2019; 1(1): 3-12.

31. Metwally, F. H., Abdelkawy, M., Abdelwahab, N. S. Application of spectrophotometric, densitometric, and HPLC techniques as stability indicating methods for determination of Zaleplon in pharmaceutical preparations. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2007; 68(5): 1220-1230.

32. Suganthi, A., Ravi, T.K. Stability indicating RP-HPLC method for the determination of fenoverine in bulk and formulation. Research Journal of Pharmacy and Technology, 2011; 4(2): 237-241.

33. Raveendra Babu, G., Srinivasa Rao, J., Suresh Kumar, K., Jayachandra Reddy, P. Stability indicating Liquid Chromatographic Method for Aripiprazole. Asian Journal of Pharmaceutical Analysis. 2011; 1(1): 03-07.

34. Kiran Kumar, V., Appala Raju, N., Namratha Rani, Seshagiri Rao, J.V.L.N., Satyanarayana, T. The estimation of Irinotecan HCl in parenterals by RP-HPLC. Asian Journal of Research in Chemistry. 2009; 2(1): 54-56.

35. State Pharmacopoeia of Ukraine: in 3 volumes. Kharkiv: State enterprise “Ukrainian scientific pharmacopoeial center of medicines quality”, 2-nd ed., Vol. 1., 2014

36. PubChem Identifier: CID 2497546. URL: https://pubchem.ncbi.nlm.nih.gov/compound/ 2497546.

37. ICH harmonized tripartite guideline Q2 (R1). Validation of Analytical Procedures: Text and Methodology Q2 (R1). In: Proceedings of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland, 2005.

38. ICH Harmonised Tripartite Guideline. Impurities in new drug products Q3B (R2). In: International Conference On Harmonisation Of Technical Requirements For Registration Of Pharmaceuticals For Human Use, Geneva, Switzerland. 2006.

Received on 19.05.2020 Modified on 14.12.2020

Research J. Pharm. and Tech. 2021; 14(6):3223-3231.

DOI: 10.52711/0974-360X.2021.00561

Parameter	Requirements	Results
Resolution between the peaks due to impurity B and A	≥15.0	17.78
Resolution between the peaks due to impurity A and Epimidine	≥10.0	11.56
The tailing factor for Epimidine (Reference solution D )	≤1.5	1.1
The tailing factor for impurity A	≤1.5	1.07
The tailing factor for impurity B	≤1.5	1.15
The tailing factor for impurity C	≤1.5	1.03


Figure 5: Chromatogram of Epimidin solution after alkaline and acid hydrolysis


Figure 6: Chromatogram of Epimidin solution after peroxidation and temperature degradation