INTRODUCTION:

Palonosetron^1-3 is an antiemetic medication works by blocking the action of a chemical messenger (serotonin) in the brain which cause nausea and vomiting during anti-cancer treatment (chemotherapy) or after surgery. (Figure 1).

Figure 1: Structure of Palonosetron HCl

Definitions of this prescription in different structures are used in adults for the expectation of squeamishness and disgorging identified with conventionally emetogenic infection chemotherapy. Composing outline revealed that lone few HPLC strategies^4-11 are represented the affirmation of palonosetron HCl in measurements structures. The above logical strategies^7-11 that were accounted for in the writing for the assurance of palonosetron HCl in plans realized high base commotion, long following, long maintenance times and low affectability and this made the essayist to develop another dependability showing RP-HPLC technique in details as per ICH rules¹².

MATERIALS AND METHODS:

Chemicals and Solvents:

Standard powder form (99.9% pure) of palonosetron HCl were obtained as gifted sample from Ajanta Pharma Ltd, and its market formulation [Palozac capsules containing Palonosetron HCl 500µg] was purchased from local pharmacy respectively. Ultra-pure water was obtained from a Millipore system (Bedford, MA, USA). Acetonitrile (HPLC Grade), methane sulfonic acid (AR Grade), Potassium dihydrogen phosphate (HPLC Grade), HCl (HPLC Grade), ortho phosphoric acid (HPLC Grade) and Tri ethylamine (HPLC Grade) were purchased from Qualigens Ltd., Mumbai.

Instrumentation:

The present HPLC analysis was carried out on Agilent 1200 Series HPLC System consisting of a G1379B Vacuum Degasser, a G1312A Binary Pump, a G1329A Autosampler, a G1330B ALS Thermostat, G1316A TCC Thermostatted Column Compartment, a G1315D DAD Detector and Agilent C18 column with dimensions 250x4.6mm,5µm respectively. High precision analytical balance was used for weighing purpose. The resultant data of each chromatographic run was recorded and processed with chemstation software.

Mobile phase preparation:

Prepared a filtered and degassed mixture of acetonitrile and buffer (pH-2.60±0.05) in the proportion of 50:50 (%v/v) in current assay respectively.

Buffer (pH - 2.60±0.05):

Weigh and dissolve 3.48gm of Potassium dihydrogen phosphate in a 1000ml water added add 1ml of triethylamine and 1.0ml methane sulfonic acid and mix well and finally make up the volume with water, Adjust the pH adjusted to 2.60±0.05 with dil. ortho phosphoric acid solution. Filter the solution through 0.45 membrane filter or equivalent and degas.

Diluent preparation:

In the present study acetonitrile and water in the ratio of 10:90 (%v/v) is used as diluent.

Preparation of standard solutions:

Precisely, weigh and move 10mg of palonosetron HCl powder (99% pure) into a 100ml clean dry volumetric flask, include 70ml of diluent, sonicated for 5 minutes and makeup to the final volume with similar diluent (Standard stock solution). Appropriate aliquots from the standard stock solution were pipetted into a progression of 10ml volumetric flasks independently and diluted sufficient with the diluent to acquire inside the concentration of range 10 - 30µg/ml for palonosetron HCl separately.

Preparation of sample solution:

5 capsules of Palozac capsules containing Palonosetron HCl 500µg obtained from the nearby drug store were utilized. Their shells were expelled, and the fine powder was gathered and weighed. Test solution (Stock) was set up by moving equivalently weighed 10mg of the fine powder of the formulation into a 100ml clean and a dry volumetric flask containing 70ml of diluent and sonicated totally and the volume made sufficient with the same dissolvable (diluent). From the above solution(stock) reasonable aliquots were pipetted and moved into diverse dry 10ml volumetric flasks and diluted to the mark with a similar diluent [10-30µg/ml for palonosetron HCl]. 20μL volumes of these sample arrangements were infused multiple times into the HPLC system and the peak areas were recorded.

RESULTS AND DISCUSSION:

i. Method Development:

In working up the present RP-HPLC strategy for palonosetron HCl, a deliberate examination of the effect of various factors was grasped by moving one parameter without a moment's delay and keeping each and every other condition enduring that consolidates picking the proper wavelength and decision of stationary and versatile stages.

For this of solutions of palonosetron HCl containing 100% level fixation were readied utilizing the diluent and the above-arranged arrangement was filtered on UV spectrophotometer between 200-400 nm utilizing diluent as clear and the particular λmax was recorded and the λmax was seen as 237nm for palonosetron HCl as this wavelength gave greatest UV-reaction. In this examination, distinctive developmental starters were performed with C18 sections of different sorts, made by different makers. Finally, the fantastic division and peak shapes for palonosetron HCl were gotten with the use of the switch stage segment [Agilent C18 column (250mmx4.6 mm I.D; particle size 5µm)] and this section was picked as stationary stage in the present look at. In order to get a sharp peak, low following element and benchmark separation of the palonosetron HCl, different tests were finished by changing the course of action of various solvents. In this understanding mix of acetonitrile and Buffer (pH-2.60±0.05) in different blends were attempted as portable stages using the picked switched stage C18 column. From these examination mix of acetonitrile and buffer (pH-2.60±0.05) in the extent of 50:50%v/v was shown as sensible portable stage as for different mixes since, the chromatographic peaks got with this versatile stage were especially described and settled, for all intents and purposes free from following. Stream paces of the versatile stage were changed from 0.5 – 1.5ml/min separately for ideal partition of the palonosetron HCl. Tentatively it was discovered that 1.0ml/min stream rate was perfect for the effective elution of the above analyte.

ii. Chromatographic Conditions:

Structure the above formative investigations the separation of palonosetron HCl in detailing was strikingly accomplished on utilizing Agilent C18 column (250mm x 4.6mm I.D; molecule size 5µm) with mobile phase comprising of acetonitrile and buffer (pH-2.60 ± 0.05) in the proportion of 50:50% v/v at a flow rate was 1.0ml/min with UV detection of 237nm at encompassing temperature. The retention time for palonosetron HCl was observed to be 3.873min separately.

iii. Forced Degradation Studies:

The forced degradation or stress studies were performed at the fixation 20μg/ml of palonosetron HCl (API) within the sight of excipients to demonstrate the soundness showing property of the present proposed RP-HPLC method.

Intentional degradation was attempted to stress condition of acid hydrolysis (0.1N HCl at 70°C for 24hrs), base hydrolysis (0.1N NaOH at 70°C for 24hrs), and thermal (at 80°C for 72hrs to evaluate the ability of the proposed method to separate palonosetron HCl from their degradation products. All stressed samples were analyzed by developed RP-HPLC method. Photodiode array (PDA) detector was employed to check and ensure the homogeneity and purity of the palonosetron HCl peak in all forced degradation sample solutions. For all the stability studies, the formation of degradable product was confirmed by comparing to chromatogram of the standard solution of palonosetron HCl kept under normal conditions.

Acid and base-induced degradation:

This acidic and basic degradation was performed in the dark in order to exclude the possible degradative effect of light. 10ml of sample stock solution of palonosetron HCl was taken in a round bottom flask. 10mL of 0.1N HCl and 10mL of 0.1N NaOH were added separately to the stock solution. The reaction was carried out for 70^oC for 24hrs. Later the sample was cooled at room temperature and the response was halted, weakened with the mobile phase and blended well. This arrangement was infused into the HPLC system.

Thermal stress:

This study was performed by keeping powdered drug content at around 80˚C for 72 hours. After this it was allowed to come at room temperature. This powdered drug was used for prepare stock solution preparation as per procedure described in page and the same was used in this study.

Method Validation:

The created RP-HPLC method is furthermore endorsed according to ICH guidelines¹² for the trial of palonosetron HCl using the accompanying parameters.

a. Specificity:

Specificity of the proposed RP-HPLC method was tried against standard and test mixes within the sight of blank and placebo under streamlined test conditions. The chromatograms of blank and placebo blend uncovered no extra co-eluting peaks with the particular peaks of palonosetron HCl in the standard and test solutions, inferring that the proposed RP-HPLC method is explicit.

b. System Suitability:

System reasonableness parameters like number of theoretical plates, HETP and peak tailing were resolved for palonosetron HCl by utilizing the above characterized chromatographic conditions and the qualities were accounted in Table 1.

Table 1: Results of system suitability studies

Parameter	Palonosetron HCl
Retention time	3.873
Theoretical plates	8279
Tailing factor	1.19
% RSD	0.053

c. Linearity and Detector Response:

The linearity of the proposed RP-HPLC method was assessed by purging 20µl of five different concentrations of working standard arrangements of palonosetron HCl into the recommended chromatographic system under the chromatographic conditions portrayed previously. The chromatograms acquired at every concentration were recorded and their peak areas were resolved. A calibration plot of palonosetron HCl was plotted with the peak area ratios acquired versus the theoretical concentrations individually [Figure 2]. The linearity of the calibration chart was approved by the high estimations of correlation coefficient 0.9993 with slope and intercept estimations of 2372642 and -10742781 for palonosetron HCl respectively. The LOD and LOQ of palonosetron HCl were observed to be 0.012µg/ml and 0.040µg/ml and are accounted respectively.

Figure 2. Linearity plot of palonosetron HCl

d. Precision:

The precision investigations of the proposed RP-HPLC method were found out by infusing six duplicate infusions (Intra-day precision ponders) of working standard and sample solutions of one fixed fixation (125% focus level). The Intra-day precision studies results demonstrated the %RSD estimations of 0.62 for palonosetron HCl individually uncovering the high precision of the proposed RP-HPLC method.

e. Accuracy:

The accuracy of the present RP-HPLC method was performed at three levels, in which sample stock solutions were spiked with standard medication arrangement containing 50, 100 and 150% of the labeled amount of palonosetron HCl and in tablets as well. Three duplicate samples of every fixation level were arranged and analyzed under endorsed test conditions. The % recovery at every focus level (n = 3), was resolved and detailed in Table 2. The % recovery was acquired for palonosetron HCl was gone from 98 - 102% individually uncovering the proposed RP-HPLC method is right.

Table 2: Results of accuracy studies

S. No.	Concentration(µg/ml)	Area	Recovery
1	10	12324986	100.41
2		12229205	101.42
3		12311614	100.37
4	20	37393903	99.69
5		37026647	99.86
6		36955066	100.72
7	30	57721401	101.26
8		57525783	100.14
9		57912129	101.60
*Average			100.61

*Average of three determinations

f. Robustness Studies:

The power of the proposed method was checked by making purposeful changes to certain parameters, for example, the mobile phase flow rate, pH of the solution and detection wavelength. The elements chose in the present examination were the adjustment in the pH of the buffer in mobile phase by ±0.2units, change in stream rate by ±0.2ml/min and the adjustment in detection wavelength by ±2nm separately. As the peak areas of palonosetron HCl were not clearly influenced by moment varieties in the chromatographic conditions it is finished up, that the created RP-HPLC method was observed to be powerful with the system reasonableness parameters inside the breaking points and appeared in Table 3 individually.

Table 3: Results of Robustness studies

Flow Variation(±0.2ml/min)
S. No.	Flow rate	Area	Result (%)
1	0.80 ml/min	56881186	99.02
2	1.20 ml/min	38149245	99.22
pH Variation (±0.2 units)
S. No.	pH	Area	Result (%)
1	2.4	48489022	100.79
2	2.8	44980128	99.39
Wavelength Variation((±2nm)
S. No.	Wavelength	Area	Result (%)
1	235nm	57190931	99.13
2	239nm	35278255	99.21

g. Ruggedness:

The toughness of the proposed RP-HPLC method was assessed by a different analyst and different instrument in a similar lab. The % RSD for peak areas of palonosetron HCl were determined and the exploratory outcomes uncovered that the %RSD of 0.54 was inside the points of confinement demonstrating that the created RP-HPLC method was observed to be tough.

h. Forced degradation studies:

Palonosetron HCl test was dropped to 84.77%, 90.90% and 94.08% in acid hydrolysis, alkali hydrolysis, and thermal degradation. In all the above-portrayed stress conditions showed that all peaks are all around isolated from palonosetron HCl peak and additionally discovered that, that there is no obstruction from degradants, encouraging blunder free measurement of palonosetron HCl. The chromatograms acquired in the wake of oppressing test answer for acid hydrolysis, alkali hydrolysis, and thermal degradation are introduced in Figures 3.a-c and the outcomes got are condensed in Table 4.

Figure 3.a

Figure 3.b

Figure 3. a and b: Chromatogram representing acid stress (a) and alkali stress (b)

Figure 3.c: Chromatogram representing thermal stress

Table 4: Results of Forced degradation studies

S. No.	Condition	Result (%)	% Degradation
1	Acid	84.77	15.23
2	Base	90.90	9.10
3.	Thermal	94.08	5.92

Analysis of Market Formulation:

Analysis of marketed oral tablets [Palozac capsules containing Palonosetron HCl 500mg] was carried out using the above said optimized mobile phase and HPLC conditions. The % content of palonosetron HCl in the prescribed capsules were calculated and were found to be 99.99±0.41 respectively. Calculated sstudent’s t-test (0.46) for accuracy and F-test (2.25) for precision revealed no significant difference between the developed RP-HPLC assay method and the literature method^[11]at the 95% confidence level with respect to accuracy and precision. The results are given in Table 5 respectively.

CONCLUSION:

Taking everything into account, a straightforward isocratic RP-HPLC method has been created and subsequently approved for the assurance of palonosetron HCl in formulations. The separation was carried on C18 Agilent column (250mm x 4.6mm I.D; molecule size 5µm) with mobile phase comprising acetonitrile and buffer (pH - 2.60±0.05) in the proportion of 50:50 (%v/v) at a stream rate of 1.0ml/min with UV detection of 237nm in encompassing temperature individually. Basing on the approval results (within the ICH standards), the effortlessness in the separation methodology, low volume of infusion and shorter run times it is inferred that the proposed RP-HPLC test method was discovered appropriate for the normal examination of palonosetron HCl in other conventional formulations.

ACKNOWLEDGEMENTS:

The creators are grateful to the Department of Chemistry, Bharath Institute of Higher Education and Research, Selaiyur, Chennai, Tamilnadu, India for giving specialized help during this examination.

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Received on 25.01.2020 Modified on 26.03.2020

Research J. Pharm. and Tech 2021; 14(3):1305-1309.

DOI: 10.5958/0974-360X.2021.00231.6