DEVELOPMENT AND VALIDATION OF A METHOD FOR SIMULTANEOUS QUANTITATIVE DETERMINATION OF ALBENDAZOLE AND PRAZIQUANTEL IN COATED TABLETS “AP-HELMIN”

research is to develop methods for the qualitative and quantitative analysis of albendazole and praziquantel in coated tablets “AP-helmin”. Following the recommendations of the European Pharmacopoeia, the identification of albendazole, as well


Introduction
Parasitic diseases are one of the most common among the population.Recently, there has been a clear trend towards an increase in the incidence of invasive etiology, in particular, helminthiasis.According to the latest data from the World Health Organization, helminthiasis affects about 4.5 billion people.In Europe, one in three people is affected.85-95 % of the adult population of the United States have pathogens of parasitic infections (according to Dr. R. Andersen).
At the Department of Pharmaceutical Technology of Drugs of the National University of Pharmacy there was created the medicine in the form of coated tablets "AP-helmin", the API of which are albendazole and praziquantel in a ratio 1:4.This medicine is intended for the treatment of helminthiasis of the digestive system in adults.The effect of the therapeutic dose on the body was confirmed in models of ascariasis in piglets, spontaneous toxocariasis and dipilidiosis in dogs and spontaneous paraspidoderosis and hymenolepidosis in white rats.The absolute efficacy of coated tablets "AP-helmin" in all studied cases of intestinal helminthiasis was established, which indicates their high level of specific pharmacological activity.Indicators of haematological studies in piglets free from intestinal helminths before and 24 and 72 hours after drug administration were within the physiological norm.Clinical examination of experimental animals confirmed the low degree of toxicity of the drug [1,2].The novelty of the developed drug is protected by a patent of Ukraine for invention No. 124898, "Anthelmintic medicine based on albendazole and praziquantel" (December 8, 2021) and utility model patent of Ukraine No. 142220", Anthelmintic medicine based on albendazole and praziquantel" (May 25, 2020) [3].
Developing qualitative and quantitative analysis methods in the composition of medicine is a mandatory point of quality control.Thus, the purpose of this research is to develop methods for the qualitative and quantitative analysis of albendazole and praziquantel in coated tablets "AP-helmin".
Following the recommendations of the European Pharmacopoeia, the identification of albendazole, as well as praziquantel, should be performed by infrared absorption spectrophotometry or gas-liquid chromatography [4].The resulting spectrum should be identical to the standard sample of the substance albendazole or the standard sample of the substance praziquantel, respectively.
Validation is performed to confirm the method of quantitative determination of albendazole and praziquantel by liquid chromatography's compliance with the criteria of acceptability in tablets "AP-Helmin." Validation was performed according to the requirements of the State Pharmacopoeia of Ukraine (SPhU) [5].
The research was performed in the State Research Laboratory for Quality Control of Medicines (National University of Pharmacy, Kharkiv, Ukraine) in the period June 2021-February 2022.

Planning (methodology) of research
In order to meet the set aim of the research, the following tasks of the study were identified (Fig. 1).
The equipment used for validation has been fully certified (Table 1).

Materials and methods
Reagents.As the reagent of the research, water for chromatography and acetonitrile for chromatography (p.UN1648, valid until 08.2022) were used.
Method of quantitative determination of albendazole and praziquantel.Quantitative determination of albendazole and praziquantel was conducted according to SPU, method 2.2.29.
Test solution.Place 8.0 mg of powdered tablet mass in a volumetric flask (50.0 ml), add 40 ml of methanol R, sonicate for 10 min.Then, bring the volume of the solution with the same solvent to the mark, mix and filter through the paper filter "blue tape".Place 5.0 ml of the obtained solution in a volumetric flask (100.0 ml) and bring to the mark with methanol R.
Reference solution а.Place 250.0 mg of PSS albendazole in a volumetric flask (50.0 ml), dissolve in 30 ml of methanol R, sonicate for 10 minutes Then bring the volume of the solution with the same solvent to the mark, mix and filter through paper filter "blue tape".
Reference solution b.Place 400.0 mg of PSS praziquantel is placed in a volumetric flask (50.0 ml), dissolve in 30 ml of methanol R, and sonicate for 10 minutes.Then bring the volume of the solution with the same solvent to the mark, mix and filter through paper filter "blue tape".
Reference solution c. Place 2.0 ml of reference solution a and 5.0 ml of reference solution b in a volumetric flask (100.0 ml), bring to the mark with methanol R and mix.
The solutions are used freshly prepared.Before chromatography, the solutions are filtered through a membrane filter with a pore size of not more than 0.45 μm.
Chromatography is performed on a liquid chromatograph with a spectrophotometric detector under the following conditions: -column size 150×4.6mm, filled with octadecylsilyl silica gel for chromatography R (for example, Symmetry C18, size 150х4,6 mm, by "Waters") with pre-column, 5 μm, for which the conditions of the suitability of the chromatographic system; -mobile phase flow rate:1.5 ml/min; -column temperature: 45 °С; -wavelength detection at 225 nm; -injection volume: 20 μl.
The chromatographic system is considered suitable using the following conditions for the reference solution: -peak symmetry coefficient of albendazole and praziquantel ranges from 0.8 to 1.5; -separation coefficient of the peaks of albendazole and praziquantel is not less than 1.5.
Formula 1 calculates the content of albendazole (X 1 ) or praziquantel (X 2 ) in one tablet in mg, based on the tablet's average weight: where S 1 -average area of the peaks of albendazole (praziquantel), calculated from the chromatograms of the test solution; S 0 -average area of the peaks of albendazole (praziquantel), calculated from the chromatograms of the reference solution; m -weight of the tablet portion taken to prepare the test solution, mg; m 0 -weight of the PSS albendazole (PSS praziquantel) portion taken to prepare the reference solution, mg; Р -content of the main substance in PSS albendazole (PSS praziquantel) taken to prepare a reference solution, %.

Forecast of uncertainty of analysis results.
The maximum allowable total relative uncertainty of the method of analysis of finished drugs Δ AS % is associated with the tolerance of the content of the analyzed substance in accordance with the specification (B).
In the case of coated tablets "AP-helmin", the quantitative content of albendazole and praziquantel should be within ±5 % (according to the requirements of the quality control methods (QCM) project for the drug-coated tablets "AP-helmin", therefore max Δ AS is ≤1.6 %.
The forecast of the uncertainty of sample preparation was calculated based on the general requirements of the SPU for laboratory equipment (Tables 2, 3).
Determination of the uncertainty of the final analytical operation Δ FAO is carried out for the test solution and the comparison solution.When calculating confidence intervals, one-sided Student's coefficient for a probability of 95 % and the corresponding number of degrees of freedom are used.Confidence intervals for the comparison solution and the test solution are calculated for the average of 5 results (the maximum number of measurements according to the MQQ of the medicine, methods of SPhU 5.3 N.2, 2.4 and 2.6 were used).
( ) ( ) According to the requirements of the suitability of the chromatographic system of the quantitative determination method, the relative standard deviation of five parallel determinations should be no more than 1.0 %.When n=5, t(95 %, n-1)=2.1318: The total uncertainty of the final analytical operation: Complete uncertainty of the method of analysis of albendazole Δ AS %: The complete uncertainty of the praziquantel analysis methodology Δ AS %: Thus, the calculated total uncertainty of the analysis method Δ AS % is less than maxΔ AS (1.56 %<maxΔ AS =1.6 % and 1.48 %<maxΔ AS =1.6 %), which meets the requirements for this parameter.
Therefore, the uncertainty of sample preparation and analysis as a whole should ensure sufficient measurement accuracy.

Result
Validation studies.Specificity.The specificity of the method was confirmed by comparing the chromatograms of the reference solution, test solution, blank solution, and placebo solution.
The retention time of the albendazole and praziquantel peaks on the chromatograms of the test solution corresponds to the retention time of the albendazole and praziquantel peaks on the chromatograms of the reference solution (approximately 6.0 min and 7.4 min, respectively).
The blank chromatogram revealed no peaks whose retention time coincided with the retention time of the albendazole and praziquantel peaks.There are no peaks on the chromatograms of placebo solutions that would coincide with the peaks of albendazole and praziquantel.Chromatograms of the blank solution, reference solutions, placebo solution and test solution are shown in Fig. 2-7.Linearity.
The quantification method should be linear within the range of application, which should overlap the possible values of the concentrations of the active substance.SPU sets the range of application of quantitative methods as 80-120 %.
To confirm the method's linearity, 9 model solutions were prepared, the concentration of which varies evenly within the range of application (step -5 %).
Calculations and criteria are given for normalized values of the standard solution (Tables 4, 5) by the formulas (3), (4): (3) In Fig. 8, a graph of the linear dependence of the analytical signal on the actual concentration of the solu-tion, constructed in normalized coordinates, based on the data in the Table 4 is presented.In Fig. 9, a graph of the linear dependence of the analytical signal on the actual concentration of the solution, constructed in normalized coordinates based on the data in Tab. 5 is presented.
Eligibility criteria are given in Tables 6, 7.

Correctness.
To determine the correctness within the range of use of the analytical method, 9 test solutions were pre-pared, observing all stages of the analytical method.The concentration of albendazole and praziquantel in solutions ranged from 80 % to 120 %.The determination of the correctness parameters and eligibility criteria are presented in Tables 8, 9.If not carried out, the criterion of practical insignificance: δ≤0.512 (0.03≤0.512)

General conclusion about the method Correct
Fulfilment of the criteria of correctness for the determination of albendazole and praziquantel in coated tablets "AP-helmin" by liquid chromatography are given in Table 10.

Precision.
Precision was investigated on the tested solutions prepared for the definition of criterion "Correctness".The precision criteria of a quantitative determination of albendazole and praziquantel of standard solution are fulfilled (Table 11).

Intra-laboratory precision.
Results of the study of 6 trials of one sample by two analysts on different days during one working week using different measuring vessels are used.Determination of intra-laboratory precision parameters and calculation of its criteria are presented in Tables 12, 13.Fulfilment of the criteria of intra-laboratory precision for determining albendazole and praziquantel in coated tablets "AP-helmin" by liquid chromatography are given in Table 14.

Robustness.
Study of the stability of the test solution and reference solution was performed after 24 hours.The results are presented in Tables 15, 16.Thus, the differences between the obtained values of the peak areas should not exceed the criterion of insignificance compared to the maximum allowable uncer-tainty of the analysis results (Δ AS, insig ), i.e. 0.512 %.According to the determination results, the test solution was stable for 24 hours.Therefore, the findings found that the chromatographic system was compliant with the requirements of the test to verify the suitability of the chromatographic system for albendazole and praziquantel quantitative determination in coated tablets "AP-helmin" (Table 17).

Discussion
The obtained results confirm that the method of quantitative determination of albendazole and praziquantel in coated tablets "AP-helmin" is specific.
The findings confirm that the method of quantitative determination of albendazole and praziquantel in coated tablets "AP-helmin" in the concentration range from 80 % to 120 % is linear; it satisfies the criteria of acceptability of the validation indicator "Correctness".The method is characterized by sufficient convergence, as the value of the relative confidence interval of the value is 0.69 and 0.52 %, which is less than the critical value for the convergence of results (1.6 %) and satisfies the criteria of acceptability of the validation indicator "Precision".The chosen method of quantitative determination of albendazole and praziquantel in coated tablets "AP-helmin" meets the eligibility criteria of the test "Intra-laboratory precision" as well.
The obtained results coincide with similar studies by other authors.In particular, spectrophotometric methods for determining pure albendazole and in the composition of solid and liquid dosage forms are sensitive, fast, economically available and subject to validation [6][7][8][9][10][11][12].In the simultaneous determination of albendazole and other anthelmintic agents, for example, ivermectin, other researchers often turn to LC, GLC, and HPLC [13][14][15][16][17].However, no validation data is provided.The number of publications on the study on the determination of praziquantel is lower compared to the number of publications on albendazole.The gas-liquid chromatography method is considered the most effective for this substance [18,19].Materials on the simultaneous determination of albendazole and praziquantel are extremely scarce.Available publications on the use of spectrophotometric methods [20].There are few reliable studies on the validation of these methods because the combination of these substances in the proposed ratio (1:4) is new, which confirms the relevance and timeliness of the described validation studies.
Practical relevance.The practical relevance of the results lies in validating the method for the joint qualitative determination of albendazole and praziquantel by the method of liquid chromatography in the coated tablets "AP-helmin".
Research limitations.The presented validation results of the method for joint quantitative determination of albendazole and praziquantel are limited to the dosage form of coated tablets, the technology of their preparation, and the ratio of the APIs.
Prospects for further research.Prospects of further research include 2 main directions: 1 -investigation of the validating parameters of joint quantitative determination of albendazole and praziquantel in other dosage forms as they are developed; 2 -investigation of the validating parameters of quantitative determination of albendazole in the dosage form of chewable pastilles under the conditional name "Albenpast".

Conclusions
According to the findings it can be concluded that the method of liquid chromatography is considered validated and can be used for quantitative analysis of albendazole and praziquantel in the coated tablets "APhelmin" in accordance with the project of QCM for this drug.All calculated parameters meet the required validation criteria.

Fig. 1 .
Fig. 1.General methodology of the research

Fig. 9 .
Fig. 9. Graph of linear dependence (Y i =b*X i +a) for standard praziquantel solution

Table 1
Equipment used in the research

Table 2
Calculation of uncertainty of sample preparation for analysis of albendazole (Δ sр )

Table 3
Calculation of uncertainty of sample preparation for analysis of praziquintel (Δ sр ) Fig. 2. Chromatogram of the blank solution

Table 4
Calculation of linearity parameters of the method of quantitative determination of the standard solution of albendazole according to the project of QCM

Table 5
Calculation of linearity parameters of the method of quantitative determination of the standard solution of praziquantel according to the project of QCM

Table 6
Data of verification of linearity of the method of quantitative determination of albendazole

Table 8
Calculation of the correctness parameters of the standard solution of albendazole

Table 9
Calculation of the correctness parameters of the standard solution of praziquantel

Table 14
Results of the assessment of intra-laboratory precision

Table 16
Determination of the stability of albendazole and praziquantel in the test solution

Table 17
Compliance with the requirements of the test to verify the suitability of the chromatographic system for mutual albendazole and praziquantel quantitative determination