International Journal of Pharmacognosy and Chemistry

Development and Validation of New Analytical Method for The Simultaneous Estimation of Netupitant And Palonosetron In Pharmaceutical Dosage Form

Potluri Surendra*¹, P.Sreenivasa Prasanna^{2 ,}K. Thejomoorthy³

¹ Department of Pharmaceutical analysis, M.L.College of Pharmacy, S. Konda-523101.

² Principal, M.L.College of Pharmacy, S.Konda-523101.

³ Head, Department of Pharmaceutical analysis, M.L.College of Pharmacy, S. Konda-523101.

Abstract

A simple, Accurate, precise method was developed for the simultaneous estimation of the Netupitant and Palonosetron in Pharmaceutical dosage form. The chromatogram was run through Std Discovery C18250 x 4.6 mm, 5m. Mobile phase containing Buffer 0.1% OPA (2.2ph): Acetonitrile taken in the ratio 55:45 was pumped through the column at a flow rate of 1 ml/min. The buffer used in this method was 0.1% OPA. The temperature was maintained at 30°C. The optimized wavelength selected was 220 nm. The retention time of Netupitant and Palonosetron was found to be 2.308min and 3.093min. %RSD of the Netupitant and Palonosetron were and found to be 0.9 and 0.6 respectively. %Recovery was obtained as 99.51% and 99.29% for Netupitant and Palonosetron respectively. LOD, LOQ values obtained from regression equations of Netupitant and Palonosetron were 1.84, 0.01, and 5.59, 0.03 respectively. Regression equation of Netupitant  is y = 7232.8x + 3439.3., and y = 28857x + 97.732 of Palonosetron. Retention times were decreased and run time was decreased, so the method developed was simple and economical that can be adopted in regular Quality control tests in Industries.

Keywords: Netupitant, Palonosetron, RP-HPLC

Article History

Received on: 015-03-2021

Revised on: 02-05-2021

Accepted on: 10-05-2021

DOI: https://doi.org/10.46796/ijpc.vi.158

*Corresponding Author

Potluri Surendra

Department of Pharmaceutical analysis,

M.L.College of Pharmacy, S. Konda

Email: mlcollegeofpharmacy@gmail.com

This article is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Copyright © 2021 Author(s) retain the copyright of this article. 

Introduction

Netupitant  (NTP)  is  a  novel  antiemetic  [1,2]  drug  in  the  combination  of  NTP/ palonosetron  (PLS).  It  is  used  to  the  prevention  of  acute  and  delayed  chemotherapy-induced  nausea  and  vomiting  [3],  including  highly  emetogenic  [4]  chemotherapy  [5]  such  as  with  cisplatin  [6].  5-hydroxytryptamine  (5-HT3)  receptors  [7]  are  located  on  the  nerve  terminals  [8]  of  the  vagus  [9]  in  the  periphery  and  centrally  in  the  chemoreceptor  [10]  trigger  zone  of  the  area  postrema  [11].  It  is  thought that chemotherapeutic agents produce nausea and vomiting by releasing serotonin [12] then activate 5-HT3 receptors located on vagal afferents [13] to initiate the vomiting reflex. Netupitant is chemically called as 2-[3,5-bis (trifluoromethyl) phenyl]-N,2- dimethyl- N- [4-(2-methylphenyl) -6- (4-methylpiperazin-1-yl) pyridin-3-yl] propenamide. It Delayed emesis (vomiting) has been largely associated with the activation of tachykinin family neurokinin 1 (NK1) receptors (broadly distributed in the central and peripheral nervous systems) by substance P. As shown in in vitro and in vivo studies, netupitant inhibits substance P mediated responses. The structure is shown in figure 01.

Figure 01: Chemical structure of Netupitant

Palonosetron chemically called as (5S)-3-[(3S)-1-azabicyclo[2.2.2]octan-3-yl]-3-azatricyclo [7.3.1.05,¹³] trideca- (12), 9(13), 10-trien-2-one.it is a selective serotonin 5-HT₃ receptor antagonist. The antiemetic activity of the drug is brought about through the inhibition of 5-HT₃ receptors present both centrally (medullary chemoreceptor zone) and peripherally (GI tract). This inhibition of 5-HT₃ receptors in turn inhibits the visceral afferent stimulation of the vomiting center, likely indirectly at the level of the area postrema, as well as through direct inhibition of serotonin activity within the area postrema and the chemoreceptor trigger zone. The chemical structure shown in figure 02

Figure 02: Chemical structure of Palonosetron

The   review   of   literature   revealed   that   several   analytical   methods  have  been  reported  for  NTP  and  PLS  in  spectrophotometry,  high-performance  liquid  chromatography  (HPLC),  high-performance  thin-layer  chromatograph y  [14-22]  individually,  and  in  the  combination.  To date, there are  no  reports  for  stability-indicating  simultaneous estimation and forced degradation study of NTP and PLS.

Materials and methods

Materials

Netupitant and Palonosetron pure drugs (API), Combination Netupitant and Palonosetron  capsules (Flumed N), Distilled water, Acetonitrile, Phosphate buffer, , Methanol, Potassium dehydrogenate  ortho phosphate buffer,  Ortho-phosphoric acid. All the above chemicals andsolvents are from Rankem

Instruments

Electronics Balance-Denver, p^H meter -BVK enterprises, India, Ultrasonicator-BVK enterprises, WATERS HPLC 2695 SYSTEM equipped with quaternary pumps,Photo Diode Array detector and Auto sampler integrated with Empower 2 Software , UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2 mm and 10mm and matched quartz cells integrated with UV win 6 Software was used for measuring absorbances of Netupitant and Palonosetron  solutions.

Methods

Diluent

Based up on the solubility of the drugs, diluent was selected, Acetonitrile and Water taken in the ratio of 50:50

Preparation of Standard stock solutions

Accurately weighed 150 mg of Netupitant ,  0.25mg of Palonosetron  and transferred to individual 50 ml volumetric flasks separately. 3/4 th of diluents was added to both of these flasks and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution 1and 2. (3000µg/ml of Netupitant  and 5µg/ml of Palonosetron )

Preparation of Standard working solutions (100% solution)

1ml from each stock solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (300µg/mlNetupitant  of and 0.5µg/ml of Palonosetron )

Preparation of Sample stock solutions

5 capsules were weighed and the average weight of each capsule was calculated,then the weight equivalent to 1 capsule was transferred into a 100ml volumetric flask, 5ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (3000µg/ml of Netupitant  and 5µg/ml of Palonosetron )

Preparation of Sample working solutions (100% solution)

1ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent.(300µg/ml of Netupitant  and 0.5µg/ml of Palonosetron )

Preparation of buffer

0.1% OPABuffer:1ml of Conc Ortho Phosphoric acid was diluted to 1000mlwith water.

Method Validation [23,24, 26, 27,28,29]

System suitability parameters

The system suitability parameters were determined by preparing standard solutions of Netupitant  (300ppm) and Palonosetron  (0.5ppm) and the solutions were injected six times and the parameters like peak tailing, resolution and USP plate count were determined.

The % RSD for the area of six standard injections results should not be more than 2%.

Specificity

Checking of the interference in the optimized method.We should not find interfering peaks in blank and placebo at retention times of these drugs in this method. So this method was said to be specific.

Precision

Preparation of Standard stock solutions 

Accurately weighed 150 mg of Netupitant ,  0.25mg of Palonosetron  and transferred to individual 50 ml volumetric flasks separately. 3/4 th of diluents was added to both of these flasks and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution 1and 2. (3000µg/ml of Netupitant  and 5µg/ml of Palonosetron )

Preparation of Standard working solutions (100% solution)

1ml from each stock solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (300µg/mlNetupitant  of and 0.5µg/ml of Palonosetron )

Preparation of Sample stock solutions

5 capsules were weighed and the average weight of  each capsule was calculated,then the weight equivalent to 1 capsule was transferred into a 100 ml volumetric flask, 5ml of diluents was  added and sonicated for 25 min, further the volume was  made up with diluent and filtered by HPLC filters(3000µg/ml of Netupitant  and 0.5µg/ml of Palonosetron )

Preparation of Sample working solutions (100% solution)

1ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent.(300µg/ml of Netupitant  and 0.5µg/ml of Palonosetron )

Linearity

Preparation of Standard stock solutions

Accurately weighed 150 mg of Netupitant ,  0.25mg of Palonosetron  and transferred to individual 50 ml volumetric flasks separately. 3/4 th of diluents was added to both of these flasks and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution 1and 2. (3000µg/ml of Netupitant  and 5µg/ml of Palonosetron )

25% Standard solution

0.25ml each from two standard stock solutions was pipetted out and made up to 10ml. (75µg/ml of Netupitant  and 0.125 µg/ml of Palonosetron )

50% Standard solution

0.5ml each from two standard stock solutions was pipetted out and made up to 10ml. (150µg/ml of Netupitant  and 0.25µg/ml of Palonosetron )

75% Standard solution

0.75ml each from two standard stock solutions was pipetted out and made up to 10ml. (225µg/ml of Netupitant  and 0.375µg/ml of Palonosetron )

100% Standard solution: 1.0ml each from two standard stock solutions was pipetted out and made up to 10ml. (300µg/ml of Netupitant  and 0.5µg/ml of Palonosetron )

125% Standard solution

1.25ml each from two standard stock solutions was pipetted out and made up to 10ml. (375µg/ml of Netupitant  and 0.625g/ml of Palonosetron )

150% Standard solution

1.5ml each from two standard stock solutions was pipettede out and made up to 10ml (450µg/ml of Netupitant  and 0.75g/ml of Palonosetron )

Accuracy

Preparation of Standard stock solutions

Accurately weighed 150 mg of Netupitant ,  0.25mg of Palonosetron  and transferred to individual 50 ml volumetric flasks separately. 3/4 th of diluents was added to both of these flasks and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution 1and 2. (3000µg/ml of Netupitant  and 5µg/ml of Palonosetron )

Preparation of 50% Spiked Solution

0.5ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.

Preparation of 100% Spiked Solution

1.0ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.

Preparation of 150% Spiked Solution

1.5ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.

Acceptance Criteria

The % Recovery for each level should be between 98.0 to 102

Robustness

Small deliberatechanges in method like Flow rate, mobile phase ratio, and temperature are made but there were no recognized change in the result and are within range as per ICH Guide lines.

Robustness conditions like Flow minus (0.9ml/min), Flow plus (1.1ml/min), mobile phase minus, mobile phase plus, temperature minus (25°C) and temperature plus(35°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit.

LOD sample Preparation

0.25ml each from two standard stock solutions was pipetted out and transferred to two separate 10ml volumetric flasks and made up with diluents. From the above solutions 0.1ml each of Netupitant, Palonosetron , solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluents

LOQ sample Preparation: 0.25ml each from two standard stock solutions was pipetted out and transferred to two separate 10ml volumetric flask and made up with diluent. From the above solutions 0.3ml each of Netupitant, Palonosetron , and solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluent.

Degradation studies [25,30,31]

Oxidation

To 1 ml of stock solution of Netupitant and Palonosetron , 1 ml of 20% hydrogen peroxide (H2O2) was added separately. The solutions were kept for 30 min at 60⁰c. For HPLC study, the resultant solution was diluted to obtain 300µg/ml& 0.5µg/ml solution and 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Acid Degradation Studies

To 1 ml of stock ssolution Netupitant and Palonosetron , 1ml of 2N Hydrochloricacidwasadded and refluxed for 30 mins at 60⁰c.The resultant solution was diluted to obtain 300 µg/ml & 0.5µg/ml solution and 10 µl solutions were injected into  the system and the chromatograms were recorded to assess the stability of sample.

Alkali Degradation Studies

To 1 ml of stock solution Netupitant and Palonosetron , 1 ml of 2N sodium hydroxidewasadded and refluxed for 30mins at 60⁰c. Theresultantsolutionwas diluted to obtain 300µg/ml& 0.5µg/ml solution and 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Dry Heat Degradation Studies

Thestandarddrug solution was placed inovenat 105°C for1h tos tudy dry heat degradation.ForHPLCstudy,the resultant solution was diluted to 300µg/ml& 0.5µg/ml solution and10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Photo Stability studies

The photochemical stability of the drug was also studied by exposing the 3000µg/ml Netupitant  & 5µg/ml Palonosetron  solution to UV Light by keeping the beaker in UV Chamber for 1days or 200 Watt hours/m² in photo stability chamber^. For HPLC study, the resultant solution was diluted to obtain 300µg/ml& 0.5µg/ml solutions and 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Neutral Degradation Studies

Stress testing under neutral conditions was studied by refluxing the drugin water for 1 hrs at a temperature of 60º. For HPLC study, the resultant solution was diluted to 300 µg/ml& 0.5µg/ml solution and 10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Results And Discussion

Optimized method : Chromatographic conditions:

Mobile phase : 55% 0.1% OPA buffer: 450% Acetonitrile

Flow rate : 1ml/min

Column :  Discovery C18 (4.6 x 250mm, 5µm)

Detector wave length : 240nm

Column temperature :  30°C

Injection volume :  10mL

Run time :   6min

Diluent :  Water and Acetonitrile in the ratio 50:50

Results : Both peaks have good resolution, tailing factor,

theoretical plate count and resolution.

Fig 03 Optimized Chromatogram

Observation

Netupitant and Palonosetron were eluted at 2.325 min and 3.027 min respectively with good resolution. Plate count and tailing factor was very satisfactory, so this method was optimized and to be validated.

System suitability

All the system suitability parameters were within the range and satisfactory as per ICH guidelines

Table: 01 Systemsuitability parameters forNetupitant and Palonosetron

Discussion

According to ICH guidelines plate count should be more than 2000, tailing factor should be less than 2 and resolution must be more than 2. All the system suitable parameters were passed and were within the limits.

Validation

Specificity

Figure No. 04 Chromatogram of blank

Figure No. 05 Chromatogram of placebo

Fig: 06 Typical chromotogram

Discussion

Retention times of Netupitant and Palonosetron  were 2.308 min and 3.093 min respectively. We did not found and interfering peaks in blank and placebo at retention times of these drugs in this method. So this method was said to be specific.

Linearity 

Table 02: Linearity table forNetupitant  and Palonosetron

 Fig: 07 Calibrationcurve of Netupitant 

Fig: 08 Calibration curve of Palonosetron

Discussion

Six linear concentrations of Netupitant  (150-450µg/ml) and Palonosetron  (0.125-0.75µg/ml) were injected in a duplicate manner. Average areas were mentioned above and linearity equations obtained for Netupitant  was y = 7232.8x + 3439.3and of Palonosetron  was y = 28857x + 97.732Correlation coefficient obtained was 0.999 for the two drugs.

Precision

System Precision

Table 03: System precision table of Netupitant  and Palonosetron

Discussion

From a single volumetric flask of working standard solution six injections were given and the obtained areas were mentioned above. Average area, standard deviation and % RSD were calculated for two drugs.% RSDobtained as 0.9%and 0.6% respectively for Netupitant and Palonosetron  .As the limit of Precision was less than “2” the system precision was passed in this method.

Repeatability

Table: 04 Repeatability table of Netupitant  and Palonosetron

Discussion

Multiple sampling from a sample stock solution was done and six working sample solutions of same concentrations were prepared, each injection from each working sample solution was given and obtained areas were mentioned in the above table. Average area, standard deviation and % RSD were calculated for two drugs and obtained as 0.2% and 0.3% respectively for Netupitant  and Palonosetron . As the limit of Precision was less than “2” the system precision was passed in this method

Intermediate precision (Day_Day Precision)

Table: 05 Intermediate precision table of Netupitant  and Palonosetron

Discussion

Multiple sampling from a sample stock solution was done and six working sample solutions of same concentrations were prepared, each injection from each working sample solution was given on the next day of the sample preparation and obtained areas were mentioned in the above table. Average area, standard deviation and % RSD were calculated for two drugs and obtained as 0.9% and 0.2% respectively for Netupitant  and Palonosetron . As the limit of Precision was less than “2” the system precision was passed in this method.

Accuracy

Table: 06 Accuracy table of Netupitant

Table: 07 Accuracy table of Palonosetron

Discussion

Three levels of Accuracy samples were prepared by standard addition method. Triplicate injections were given for each level of accuracy and mean %Recovery was obtained as 99.51% and 99.29% for Netupitant and Palonosetron  respectively.

Sensitivity

Table: 08 Sensitivity table of Netupitant  and Palonosetron

Robustness

Table : 09 Robustness data for Netupitant  and Palonosetron .

Discussion

Robustness conditions like Flow minus (0.9ml/min), Flow plus (1.1ml/min), mobile phase minus (50B:50A), mobile phase plus (60B:40A), temperature minus (25°C) and temperature plus(35°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit.

Assay

Akynzeo, bearing the label claim Netupitant  300mg, Palonosetron  0.5mg.Assay wasperformed with the above formulation. Average % Assay for Netupitant and Palonosetron  obtained was 99.34% and 99.58% respectively

Table: 10 Assay Data of Netupitant

Table: 11 Assay Data of Palonosetron

Fig 09 Chromatogram of working standard solution

Fig: 10 Chromatogram of working sample solution

Degradation data

Table 12 Robustness data for Netupitant  and Palonosetron

Conclusion

A simple, Accurate, precise method was developed for the simultaneous estimation of the Netupitant and Palonosetron in pharmaceutical dosage form. Retention time of Netupitant and Palonosetron were found to be 2.308min and 3.093min. %RSD of the Netupitant and Palonosetron  were and found to be 0.9 and 0.6 respectively. %Recovery was obtained as 99.51% and 99.29% for Netupitant and Palonosetron  respectively. LOD, LOQ values obtained from regression equations of Netupitant and Palonosetron  were 1.84, 0.01 and 5.59, 0.03 respectively. Regression equation of Netupitant  is y = 7232.8x + 3439.3., and y = 28857x + 97.732 of Palonosetron. Retention times were decreased and run time was decreased, so the method developed was simple and economical that can be adopted in regular Quality control test in Industries.

Author Contribution

All authors are Contributed Equally.

Funding

No Funding

Conflict of Intrest

Authors are Declered no Conflict of Intrest.

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