INTRODUCTION:

The main remedies for epilepsy are anticonvulsants. One of the most common drugs used to treat epilepsy is carbamazepine. Evidence of its effectiveness in both symptomatic and cryptogenic epilepsy is that it has a positive effect on various forms of seizures: both simple and complex.

But along with this has a number of side effects that limit its use in the clinic, namely: headache, dizziness, drowsiness, depression of consciousness, inhibition of thinking, memory impairment, poor assimilation of new information, speech disorders, excitation, aggressive behavior, activation of psychosis, tinnitus, taste disturbances, involuntary movements, muscle weakness, decreased ability to work, peripheral neuritis, paresthesia, bradycardia, arrhythmia, worsening angina, congestive heart failure, thrombophlebitis, thromboembolism, jaundice, jaundice, electrolyte imbalance, hyponatremia, edema, leukopenia, thrombocytopenia, thyroid dysfunction. Today, one of the promising areas of medicine and pharmacy, which reduces the side effects of drugs, is the creation of combined drugs which include antioxidant in addition to the main active ingredient, which reduces side effects and in some cases leads to their leveling. One such drug is thiotriazoline - a broad-spectrum antioxidant¹. Most antiepileptic drugs are available in tablet form, so this dosage form was proposed to create a new fixed, combined drug whose active ingredients are carbamazepine and thiotriazoline². We have previously developed a method for standardizing the active substances of carbamazepine with thiotriazoline (1.5: 1) in the model mixture, tablet mass and tablets^3-7. In accordance with the requirements of the SPU, the next logical step was to carry out the validation for the developed methods on the following indicators: specificity, linearity, range of application, accuracy, correctness and robustness^8-12.

The aim of our work is to confirm the developed method of quantitative determination of active substances, namely carbamazepine and thiotriazoline in Carbatryl tablets.

materials aNd methods:

Certified substances of carbamazepine (manufacturer: Jubilant Organosys Ltd, India) and thiotriazoline (manufacturer: State Enterprise "Chemical Reagents Plant" of the Scientific and Technological Complex "Institute of Single Crystals" of the National Academy of Sciences of Ukraine) were used in the study. Chromatograph: LC-20 Prominence Shimadzu models in the following configuration: two LC-20AD pumps, SIL-20A autosampler, SPD-20AV detector, CTO-20A thermostat, CBM-20 ALITE system controller; Column: polymer column (Peek), size 100 mm x 4.6 mm, Chromolith SpeedROD RP-18e cat. No. 1.02129.0001 produced by Merk KGaA, Germany; analytical scales of the AUW 220D model, manufactured by Shimadzu, uncertainty of weighing results 0.033 mg^13-16.

Limits: the content of carbamazepine and thiotriazoline in one tablet of the drug should be from 142.5 mg to 157.5 mg and from 95 mg to 105 mg, respectively. Criteria for the suitability of the validation characteristics of the method were calculated for 5% tolerance of the content of active substances in the drug^17-22.

Preparation of the test solution and the comparison solution of carbamazepine and thiotriazoline was carried out according to the following method: in 10 volumetric flasks with a capacity of 100 ml we placed 30 mg of a mixture (carbamazepine with thiotriazoline) in the ratio (1.5:1). Then we added specified in table 1 amount of substances of thiotriazoline (series No. 410609) and carbamazepine (series No. 130223342) in flasks, 80 ml of methanol – to each flask, stirred for 20 minutes, the volume of the methanol mixture was adjusted to the mark and stirred for another 5 minutes. 2 ml of the resulting mixtures were centrifuged for 10 min at 8000 rpm. The upper transparent layer was used as model solutions.

Table 1. Acceptance criteria

Model sample No.	Thiotriazoline		Carbamazepine
Model sample No.	Weighed sample (mg)	Content, in% of nominal value	Sample weight (mg)	Content, in% of nominal value
0
1	22,51	79,91	33,84	80,09
2	23,90	84,85	35,95	85,08
3	25,33	89,92	38,07	90,10
4	26,75	94,96	40,35	95,50
5	28,19	100,07	42,23	99,94
6	29,55	104,90	44,44	105,17
7	30,97	109,94	46,49	110,03
8	32,40	115,02	48,62	115,07
9	33,73	119,74	50,76	120,13

Results:

Specificity. After preparing the solutions according to the method presented above, we analyze them using a chromatogram. Acceptance criteria: 1) On the chromatograms of the placebo solution of the drug (sample 0) there should be no peaks with the retention time coinciding with the retention time of carbamazepine with thiotriazoline on the chromatograms of the test solution; 2) The retention time of the carbamazepine and thiotriazoline peaks on the chromatograms of the test solution should coincide with the retention time of the carbamazepine and thiotriazoline peaks on the chromatograms of the carbamazepine and thiotriazoline comparison solution; 3) The peaks of carbamazepine and thiotriazoline on the chromatograms of the test solution should be well separated from the peaks of other possible impurities and the substances of carbamazepine and thiotriazoline^23-28.

The specificity of the method of determining the identification and quantification of carbamazepine and thiotriazoline is shown in Figure. 1.

The specificity of the method of quantitative and qualitative determination of carbamazepine and thiotriazoline is confirmed by the fact that:

- On the chromatogram of the placebo solution there are no peaks with retention time, coinciding with the retention time peaks of carbamazepine with thiotriazoline;

- The retention times of the carbamazepine and thiotriazoline peaks on the chromatograms of the test solution coincide with the retention times of the carbamazepine and thiotriazoline peaks on the chromatograms of the carbamazepine and thiotriazoline comparison solution;

- Complete separation of carbamazepine and thiotriazoline peaks is observed on the chromatograms of the test solution and the comparison solution of carbamazepine with thiotriazoline.

Based on all of the above, we can say that the method of identification and quantification of carbamazepine with thiotriazoline in Carbatryl by HPLC is specific^8-12.

Preparation of model test solutions.

The characteristics of correctness and precision were studied on model solutions of the drug with concentrations of thiotriazoline and carbamazepine, which correspond to 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% and 120% of their nominal content (Table 1).

The results of chromatographic model solutions are given in Table 2

Table 2. Results of chromatography of model solutions 1 - 9

Model solution No.	Thiotriazoline peak area	Carbamazepine peak area
1	274370	1686842
2	293235	1796039
3	308564	1897557
4	326235	2006039
5	343741	2109308
6	360785	2214510
7	377696	2321647
8	394932	2424862
9	412045	2531340

The linearity characteristic was studied in the range of carbamazepine and thiotriazoline concentrations from 80% to 120% relative to the nominal value.

The graph of linear dependence is presented in Figure. 2 and 3, and the results of calculations of the parameters of linear dependence in Table 3 and 4.

Figure 2. Linear dependence of the detected concentration of thiotriazoline on its introduced concentration in normalized coordinates

Figure 3. Linear dependence of the detected concentration of carbamazepine on its introduced concentration in normalized coordinates

Table 3. Metrological characteristics of the linear dependence of the detected concentration of thiotriazoline on its introduced concentration

Parameters	Value	Requirements 1	Requirements 2	Conclusion
b	1
S_b	0,00402
a	-0,00036	≤ \|0,99 \|	≤ \|2,56\|	Sustained by 1 criterion
S_a	0,52339
SD₀	0,20107
SD₀/b	0,20107	≤ \|0,84\|		Fulfilled
r	0,99991	> \|0,99988\|		Fulfilled

Table 4. Metrological characteristics of the linear dependence of the detected concentration of carbamazepine on its introduced concentration

Parameters	Value	Requirements 1	Requirements 2	Conclusion
b	0,99999
S_b	0,00402
a	0,0012	≤ \|0,77 \|	≤ \|2,56\|	Sustained by 1 criterion
S_a	0,40617
SD₀	0,15566
SD₀/b	0,7092	≤ \|0,84\|		Fulfilled
r	0,99994	> \|0,99988\|		Fulfilled

As can be seen from the presented data, the requirements for the parameters of linear dependence are met, i.e. the linearity of the method of quantitative determination of carbamazepine and thiotriazoline is confirmed in the concentration range from 80% to 120% of nominal value.

Correctness characterizes the degree of correspondence between the known content of the analyte in solution and its content in solution, determined by this method.

Convergence characterizes the precision of the method when carried out in the same conditions for a short period of time. At this stage, convergence is studied on 9 model mixtures, which cover the range of application of the method^{29, 30}.

The correctness and convergence of the method was checked by the method of "entered-found". The results of quantitative determination of carbamazepine and thiotriazoline in model solutions in the field of analytical concentrations and the results of calculations of metrological characteristics are presented in Table 5 and 6.

Table 5. The analysis results of model solutions containing from 80% to 120% of thiotriazoline relative to the nominal concentration, and their statistical processing

Solution No.	Introduced in% of nominal concentration (Xi, fact.,%)	Found in% of nominal concentration (Yi,%)	Found in% to entered Zi = 100 • (Yi / Xi)
1	79,91	79,769	99,824
2	84,85	85,268	100,493
3	89,92	89,737	99,796
4	94,96	94,888	99,924
5	100,07	99,990	99,920
6	104,90	104,958	100,055
7	109,94	109,888	99,953
8	115,02	114,912	99,906
9	119,74	119,900	100,134
Average, Zср, % =			100,06
Relative standard deviation, RSD_z, % =			0,212084
Relative confidence interval Δ_z % = t (95 %, 9 – 2) x RSD_z = 1,895 x 0,212 =			0,402
Critical value for the convergence results Δ_As, % =			1,6
Systematic error d % = \| Zср – 100 \| =			0,04
Criterion of insignificance of systematic error:
1) statistical insignificance: d < Δ_z : √9 = 0,402 : 3 = 0,134 % > 0,04 %			Fulfilled
If not fulfilled: 1), then d ≤ max d: 2) practical insignificance: δ% ≤ 0,32 ´ 1,6 = 0,51 % > 0,04 %			Fulfilled
General conclusion about the method			CORRECT

Table 6. Analysis results of model solutions containing from 80% to 120% of carbamazepine in relation to the nominal concentration, and their statistical processing

Solution No.	Introduced in% of nominal concentration (Xi, fact.,%)	Found in% of nominal concentration (Yi,%)	Found in% to entered Zi = 100 • (Yi / Xi)
1	80,09	80,071	99,976
2	85,08	85,248	100,197
3	90,10	90,061	99,957
4	95,50	95,204	99,690
5	99,94	100,100	100,160
6	105,17	105,088	99,922
7	110,03	110,168	100,125
8	115,07	115,061	99,992
9	120,13	120,109	99,983
Average, Zср, % =			100,002
Relative standard deviation, RSD_z, % =			0,152
Relative confidence interval Δ_z % = t (95 %, 9 – 2) x RSD_z = 1,895 x 0,152 =			0,289
Critical value for the convergence results Δ_As, % =			1,6
Systematic error d % = \| Zср – 100 \| =			0,002
Criterion of insignificance of systematic error:
1) statistical insignificance: d < Δ_z : √9 = 0,289 : 3 = 0,096 % > 0,02 %			Fulfilled
If not fulfilled 1), then d ≤ max d: 2) practical insignificance: δ% ≤ 0,32 ´ 1,6 = 0,51 % > 0,02 %			Fulfilled
General conclusion about the method			Correct

According to the data given in Table 5 and 6, it turns out that the method of quantitative determination of thiotriazoline and carbamazepine is characterized by sufficient accuracy and convergence (precision) in the entire range of concentrations (from 80% to 120%) and is correct.

As evidenced by the data in Table 5 and 6, in the range of concentrations of thiotriazoline and carbamazepine from 80% to 120% relative to the nominal concentration, the method of its quantification has no systematic error.

DISCUSSIONS:

Summing up the risk of all the above, we can say that the method is characterized by sufficient convergence, as the found value of the relative confidence interval of Δ_Ζ for thiotriazoline and carbamazepine does not exceed the critical value for convergence results (1.6%) (Tables 5, 6). Also, the method is characterized by sufficient accuracy, as it meets the criterion of insignificance of systematic error of the method. Systematic error of the method satisfies the requirements of statistical and practical insignificance. The high value of the correlation coefficient r = 0.99994 and 0.99991 satisfies the requirements of the acceptance criterion (r = 0.9998) and confirms the linearity of the relationship between taken and detected amount of thiotriazoline and carbamazepine in the range from 80% to 120% according to its nominal content in drugs. The requirements to the parameters of linear dependence (a, SD0 / b, r) of the method of determination of thiotriazoline and carbamazepine in the whole concentration range from 80% to 120% of the nominal value are met (Tables 3, 4)^8-12.

findings:

As a result of the carried out study, it was found that the developed method of standardization of active substances in Carbatryl tablets is valid for the following indicators: specificity, linearity, convergence, correctness and can be included in the Quality Control Methods project.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

THANKS TO:

The authors thank Zaporizhzhia State Medical University for its kind support during the studies.

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Received on 03.12.2021 Modified on 12.03.2022

Research J. Pharm. and Tech 2022; 15(11):5148-5153.

DOI: 10.52711/0974-360X.2022.00866