INTRODUCTION:

Parasites are the organisms which require other organisms to survive. Some parasite grows, reproduce or invade organs systems that make their hosts sick resulting in a parasitic infection. The drugs which are used for worm infection are known as antihelmintic drugs¹.Praziquantel, pyrental pamoate and febantel are the antihelmintic drugs which are being used in combination for the study.

Praziquantal is (RS)-2-(Cyclohexylcarbonyl)-1, 2, 3, 6, 7, 11b-hexahydro-4H-pyrazino [2, 1-A] isoquinolin- 4-onean generally being used for treatment against diseases caused due to parasitic infections². Praziquantel works by causing severe spasms and paralysis of the worms' muscles. This paralysis is accompanied and probably caused by a rapid Ca 2+influx inside the schistosome. These morphological alterations are accompanied by an increased exposure of schistosome antigens at the parasite surface. The worms are then either completely destroyed in the intestine or passed in the stool. Glutathione S-transferase (GST), an essential detoxification enzyme in parasitic helminths, drug target against schistosomiasis. Various analytical methods have been reported for the estimation of Praziquantel as alone as well as in combination with other drugs. It include HPLC method for Praziquantel^3,4 and with Pyrantel pamoate⁵, Febantel⁶, Oxantel,pamoate⁷, Ivermection ^8,9

Pyrental pamoate is designated chemically as 4-[(3-Carboxy-2-hydroxynaphthalen-1-yl)methyl]-3-hydroxynaphthalene-2-carboxylic acid; 1-methyl-2-[(E)-2-thiophen-2-ylethenyl]-5,6-dihydro-4H-pyrimidine which is being used to treat worm infections¹⁰ .It acts as a depolarizing neuromuscular blocking agent. Various analytical methods have been reported for the estimation of Pyrental pamoate as alone as well as in combination with other drugs for HPLC method with Mebendazole¹¹ and Fenbendazole¹².

Febantel is chemically designated as methyl N-[N'-[2-[(2-methoxyacetyl) amino]-4-phenylsulfanylphenyl]-N methoxycarbonyl carbamimidoyl] carbamate which is being used to treat parasitic infection¹³. Its mechanism of action is by blocking of microtubules perturbs the uptake of glucose, which eventually empties the glycogen reserves¹⁴. This blocks the whole energy management mechanism of the worms that are paralyzed and die or are expelled.

Several methods have been reported for individual as well as in combination for HPLC, UV spectroscopy but no individual method was reported for the estimation of these three drugs in combination by RP-HPLC method.

Therefore, main objectives of the study were to develop simple, accurate and sensitive RP-HPLC method for estimation of praziquantel, pyrental pamoate and febantel in tablet dosage form. Validation of the developed method was done according to ICH guidelines.

MATERIALS AND METHODS:

Instruments and Apparatus:

HPLC analysis was carried out by using chromatographic system (Thermo Separations, equipped with PDA detector, C18, (250*4.6) mm; 5µm column and Peak ABC 21 CFR complies as Software. Analytical balance (Labman), volumetric flasks, pipettes of borosilicate glass was used in the study.

Reagents and chemicals:

The commercial fixed dose combination product and bulk powder were procured from local market. HPLC grade water, acetonitrile, methanol, AR grade KH₂PO₄and TEA were procured from Finar chemical ltd and EMSURE ltd.

Chromatographic Condition:

1. Column: Zorbax C18 (250*4.6) mm;5µm

2. Mobile Phase: Buffer (pH 3.0): ACN (60:40v/v)

3. Flow Rate: 1.0 ml/min

4. Detection Wavelength: 240 nm

5. Run time: 10 min

6. Injection volume: 20.0 μl

Preparation of 0.05M phosphate buffer:

6.8gm of KH₂PO₄was weighed accurately and transferred to 1000 ml volumetric flask. 500 ml HPLC water was added and shaken to dissolve and diluted up to the mark with HPLC grade water. pH was adjusted at 3.0 with triethylamine.

Preparation of Mobile phase:

Accurately measured 600 ml of prepared 0.05M phosphate buffer was taken and mixed with 400 ml of Acetonitrile to make ratio of 60:40% v/v. The solution was degassed by sonication.

Fig. 1. Structure of Pyrental Pamoate

Fig. 2. Structure of Praziquantel

Fig. 3. Structure of Febantel

Preparation of Stock Solution:

12.5 mg of Praziquantel, 36 mg of Pyrantel Pamoate, 37.5 mg of Febantel standard is weighed accurately and transferred into 50 ml volumetric flask and dissolved with few ml of mobile phase, then volume was made up to the mark with mobile phase and mixed thoroughly.

Preparation of Calibration curve:

Calibration curves were plotted over a concentration range of 6.25-18.75µg/ml,18-54 µg/ml and 18.75-56.25 µg/ml of praziqunatel, Pyrental pamoate and Febantel respectively.

Preparation of Sample Solution:

A 193 mg of sample was weighed accurately and transferred into 50 ml volumetric flask and dissolved with few ml of mobile phase, then volume was made up to the mark with mobile phase and mixed thoroughly and further from sample solution (C) 5ml of solution was diluted to 25ml and was make up to the mark with mobile phase.

System Suitability Solutions:

System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. System suitability test parameters to be established for a particular procedure depend on the type of procedure being validated. System suitability tests are an integral part of liquid chromatography. These tests include tests for number of theoretical plates, Resolution and tailing factor. The data for system suitability parameter are shown in the table.1.

Table:1

Parameter	Praziquantel	Pyrental Pamoate	Febental
Retention time (min)	2.587	5.970	8.401
Theoretical Plate	7071	19262	21203
Tailing Factor	0.82	1.02	1.00
Resolution	22.58	11.88

Method Validation^15-17

1. Linearity:

The linearity means obtained test results from the applied analytical process is relative to selected concentration range of analyte in the taken sample.

2. Precision (Intraday and Interday):

The precision of the method was demonstrated by interday and intraday variation studies. Intraday precision was determined by analyzing the samples three times in the same day and interday precision by analyzing the drugs three successive days.

3. Repeatability:

Six repeated injections of standard solutions were made and the response factor of drug peaks and percentage RSD were calculated.

4. LOD and LOQ:

ICH guidelines provide equation of measurement of LOD and LOQ. The equation is:

LOD = 3.3 X σ/S

LOQ = 10 X σ/S

Where, σ = std. deviation of response,

S= Slope of calibration curve.

5. Accuracy (% Recovery):

Standard solution of praziquantel, pyrental pamoate and febantel were repeatedly injected (n=3) for the measurement of the precision for the instrument. Precision was calculated for peak area, retention time and tailing factor. %RSD or coefficient of variance should not more than 2%.

6. Robustness:

By measurement of robustness it is derived that method is unaffected by the change in parameters like: organic solvent, pH or flow rate.

Analysis of Formulation:

Take tablet powder equivalent to 12.5mg of Praziquantel, 36mg of Pyrental pamoate and 37.5mg of febantel was transferred to 100ml volumetric flask, and made up volume up to the mark with mobile phase. The solution was filtered through Whatman filter paper no. 42 and discarded the first few drops of filtrate. 1ml of solution was diluted to 10ml with mobile phase. The Solution was injected 20µl. The areas of resulting peak were measured at 240nm.

RESULTS AND DISCUSSION:

In present study several mobile phase compositions were tried. A satisfactory separation and good peak symmetry was found in a mixture of Buffer: ACN (60:40% v/v) at pH 3.0 at flow rate of 1ml/min. The optimum wavelength for detection was set at 240nm at which much better detector response for three drugs were obtained.

Linearity:

The linearity of the response for Praziquantel, pyrental pamoate and febantel were determined by preparing and injecting standard solution of Praziquantel, pyrental pamoate and febantel. The calibration curve of Praziquantel, pyrental pamoate and febantel shown in Figure 4 and 5 respectively indicate that the response is linear over the concentration range. Correlation coefficient (r) was found 0.999 for Praziquantel 0.999 for Pyrental Pamoate and 0.999 for Febantel.

Fig 4. Calibration curve of Praziquantel

Fig.5. Calibration curve of pyrental pamoate and febantel

Precision (Intraday and Interday):

The RSD values of intraday (0.875-1.443, 0.608-0.818 and 0.671-1.182%) and interday (0.342-0.855, 0.571-0.985 and 0.548- 0.921%) variations for praziquantel, pyrental pamoate and febantel respectively at 240 nm.

Repeatability:

The RSD values for praziquantel, pyrental pamoate and febantel were found to be 0.182, 0.506 and 0.374%, respectively at 240 nm and Low value of relative standard deviation indicates that the proposed method was repeatable.

LOD and LOQ:

Limit of detection and limit of quantification for praziquantel, pyrental pamoate and febantel were found out which are shown below in the table-2.

Table-2: LOD and LOQ data

Drug	LOD	LOQ
Praziquantel	0.22 µg/ml	0.69 µg/ml
Pyrental Pamoate	0.40 µg/ml	1.22 µg/ml
Febantel	0.04 µg/ml	0.15 µg/ml

Accuracy (% Recovery):

The recovery experiment was performed by the standard addition method. The low value of standard deviation indicates that the proposed method was accurate. Results of recovery studies were shown in Table 3.

Table-3: Recovery data for Praziquantel, pyrental pamoate and febantel

Drug	Amount Taken (µg/ml)	Amount Added (µg/ml)	Amount recovered (µg/ml)	% recovery ± S.d.(n= 3)
Praziquantal	43	6.25	6.21	99.36±0.423
	43	12.5	12.47	99.76±0.211
	43	18.75	18.56	99.02±0.670
Pyrental Pamoate	43	18	17.89	99.30±0.705
	43	36	35.92	99.77±0.490
	43	54	53.86	99.73±0.145
Febantel	43	18.75	18.71	99.80±0.376
	43	37.5	37.35	99.61±0.241
	43	56.25	56.16	99.83±0.205

Robustness:

The method was found to be robust. The %RSD was found below 2% for each of three parameter altered deliberately. The data is shown in Table 4.

Table-4: Robustness Study

Parameters		Area
Parameters		Praziquantel	Pyrental Pamoate	Febantel
pH (± 0.2)	2.8	1715552	3278398	7712259
	3.0	1729586	3258589	7722985
	3.2	1742852	3298687	7814987
	Mean± SD	1729330±13651.8	3278558±20049.5	7750077±56458.95
	%RSD	0.789	0.611	0.728
Flow Rate (±0.02 mL/Min)	0.98mL/Min	1702289	3169158	7702982
	1.0 mL/Min	1725281	3269985	7722358
	1.02 mL/Min	1732487	3247865	7873145
	Mean± SD	1720019±15671.692	3229003±52994	7766162±93155.42
	%RSD	0.916	1.641	1.199
Mobile Phase Composition Buffer:ACN (±2 mL)	58:42	1713245	3208970	7701058
	60:40	1722587	3214472	7714981
	62:38	1748424	3280545	7871571
	Mean ± SD	1728085.3±18222.63	3234662±39830.7	7762537±94682.77
	%RSD	1.054	1.231	1.219

Application to Pharmaceutical Dosage Form:

Applicability of the proposed method was tested by analyzing the tablet formulation. The results are shown in Table 5.

Table-5: Analysis of Tablet formulation

Tablet

Worm Trap

Label Claim

Praziquantel

(50 mg)

Pyrental Pamoate (144mg)

Febantel

(150mg)

Assay (% label of labelclaim*) Mean ± S.D

96.82±0.676

97.66±0.521

96.92±0.749

CONCLUSION:

The developed method was validated for various parameters like accuracy, recovery, linearity as per ICH guidelines. The result obtained were within the acceptance criteria for the respective parameters. The proposed method was applied for the estimation of praziquantel, pyrental pamoate and febantel in marketed formulations. The assay results conformed to the label claim of the formulation.

ACKNOWLEDGEMENT:

The authors are also thankful to Saraswati Institute of Pharmaceutical Sciences for providing necessary equipment, facility and chemicals to complete research work and sincere thanks to my highly respected and esteemed, Principal, Dr Shrenik Shah, Director and HOD of PG Department Dr Ankit B Chaudhary. I would like to express thanks to my parents without their encouragement love and blessings I would not have reached this level manuscript.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 20.05.2021 Modified on 27.07.2021

Research J. Pharm. and Tech. 2022; 15(8):3535-3539.

DOI: 10.52711/0974-360X.2022.00593