INTRODUCTION:

Eribulin mesylate is assigned chemically as (2R,3R,3aS,7R,8aS,9S,10aR,11S,12R,13aR, 13bS, 15S,18S,21S,24S,26R, 28R,29aS)-2-[(2S)-3-Amino-2-hydroxypropyl]-3-methoxy-26-methyl-20,27-dimethylidenehexacosahydro-11, 15:18,21:24,28-triepoxy-7,9-ethano-12,15-methano-9H,15H-furo[3,2-i] furo [2',3':5,6] pyrano [4,3- b][1,4]dioxacyclopentacosin-5(4H)-one monomethanesulfonate (salt). It is a white powder and freely soluble in water, methanol, ethanol, 1-octanol, benzyl alcohol, dimethyl sulfoxide, N-methylpyrrolidone, dichloromethane and ethylacetate, soluble in acetone, sparingly soluble in acetonitrile, practically insoluble in tert-butyl methyl ether, n-heptane and n-pentane. Eribulin mesylate was freely soluble at pH 3-7, soluble at pH 9 and slightly soluble at pH 11.

Eribulin is a simplified synthetic analogue of halichondrin B, a natural polyether macrolide produced in marine sponges ^{1,
2, 3}. Eribulin suppresses microtubule growth without affecting tubulin depolymerisation resulting in sequestration of microtubules into non functional aggregates. This leads to an irreversible mitotic block and thereby cell cycle arrest in the G2-M phase and apoptosis ^{4, 5, 6}. Probably due to its unique mechanism of action, different to the one of vinca alkaloids or paclitaxel, Eribulin has shown an antitumor activity in paclitaxel-resistant ovarian cancer cell lines⁷.

Eribulin mesylate used for monotherapy treatment of locally advanced or metastatic breast cancer with tumour progression after treatment with at least two chemotherapy regimens containing an anthracycline and a taxane⁸. Molecular formula is C₄₀H₅₉NO₁₁ and Molecular weight 826.090 g/mol. The chemical structure of Eribulin mesylate shown in Figure1.

Figure 1. Chemical structure of Eribulin Mesylate

The literature survey reveals that only one HPLC method was reported till date ⁹. Only a few methods reported to date for the estimation of Eribulin mesylate in biological fluid were carried out by LC-MS/MS ^{10, 11}.

Hence we tried to develop stability indicating HPLC method for Eribulin mesylate. The present work describes a simple, stability indicating HPLC method for the determination of Eribulin mesylate in Eribulin mesylate in parenteral dosage form according to ICH guidelines ^{12, 13}.

MATERIALS AND METHODS:

Chemicals and Reagents:

All chemicals and reagents used were analytical grade. Potassium dihydrogen orthophosphate, potassium hydroxide pellets, Acetonitrile, Ethanol, Hydrochloric acid, Sodium hydroxide pellets, Hydrogen peroxide (30%) and HPLC grade water, reagents and chemicals were procured from merck chemicals. Mumbai, India. Ultrapure water, purified via Mill-Q water purification system (Millipore, Bedford, France) was used throughout the work.

Instrumentation:

Separation and determination of the analytes were performed using Waters HPLC model: 2690 & 2695 quaternary solvent systems with PDA detector, Bandelin ultrasonic bath, pH Meter (Thermo Orion Model) and Analytical Balance (Metller Toledo Model) were used in the present study.

Preparation of potassium hydroxide solution:

Accurately weighed and transferred 5.6328 g of potassium hydroxide pellets into a 100 mL of milli-Q water sonicate to dissolve and mixed well.

Preparation of pH 6.5 phosphate buffer solution:

Accurately weighed and transferred 2.7241 g of potassium dihydrogen orthophosphate into a 1000 mL of milli-Q water and mixed well pH was adjusted to 6.5 with potassium hydroxide solution. Filtered the solution with 0.45 µm membrane filter and sonicate to degas.

Preparation of mobile phase:

Prepared a mixture of 700 mL of pH 6.5 potassium dihydrogen orthophosphate buffer solution and 300 mL of acetonitrile in the ratio of 70:30 (%volume/volume) mixed well and sonicate to degas.

Preparation of diluent:

Prepared a mixture of 950 mL of water and 50 mL of ethanol in the ratio of 95:5 (% volume/volume) mixed well and sonicate to degas.

Preparation of standard solution:

Accurately weighed and transferred 25.16 mg of Eribulin mesylate working standard into a 25 mL volumetric flask sonicated to dissolved the contents and made upto the volume with diluent. Further diluted this solution 5 mL in to 50 mL volumetric flask and made up the volume with diluent and mixed well.

Preparation of pooled placebo solution (1.0 mg / 2 mL)

Mixed the contents of 5 vials.

Preparation of placebo solution:

Transferred 5 mL of pooled placebo solution into a 50 mL volumetric flask, made the volume up to the mark with diluent and mixed well.

Preparation of pooled sample solution (1.0 mg / 2 mL)

Mixed the contents of 5 vials.

Preparation of sample solution:

Transferred 5 mL of pooled sample solution into a 50 mL volumetric flask, made the volume up to the mark with diluent and mixed well.

METHOD DEVELOPMENT:

UV-spectroscopic analysis of Eribulin mesylate drug substance was showed that maximum UV absorbance (λmax) at 200 nm respectively. To develop a suitable and robust HPLC method for the determination of Eribulin mesylate, different mobile phase pH was employed to achieve the good peak shape. Quite a few analytical columns (Agilent Zorbax SB-C18, 150 x 4.6 mm, 5μm; Inertsil ODS-3V, 150 x 4.6 mm, 5μm; Hypersil BDS C18, column (150 mm x 4.6 mm; 5 μ particle size, Waters X-Bridge Shield RP18 (150 x 4.6 mm, 3.5µ) and movable segment arrangements (pH 6.5 phosphate buffer and acetonitrile in dissimilar proportions and run velocities) were examined for developing a novel high performance liquid chromatographic method for the determination of Eribulin in bulk and injection dosage form. The analytical column, mobile phase composition, flow rate and associated chromatographic circumstances were determined taking into account the values of system suitability (peak area response, number of theoretical plates and peak tailing). Considering those parameters for the separation and analysis of Eribulin, it was concluded that optimum HPLC conditions consisting of a mobile phase containing pH 6.5 phosphate buffer and acetonitrile (70:30 v/v) with a flow rate of 0.8 mL/min, column temperature of 40°C, injection volume of 10 μL and detection wavelength 200 nm. These chromatographic conditions were selected for validation studies.

Optimised chromatographic conditions:

Chromatographic analysis was performed on Waters X-Bridge Shield-RP18 (150 x 4.6 mm, 3.5µ) and the mobile phase consists of pH 6.5 phosphate buffer and acetonitrile in the ratio of (70:30 v/v). The column temperature was maintained at 40°C and sample cooler temperature was maintained at 5°C, injection volume 10µL, flow rate 0.8mL/min and wavelength 200 nm, run time 25 minutes.

RESULTS AND DISCUSSION:

The developed RP-HPLC method extensively validated for assay of Eribulin mesylate in Eribulin mesylate parenteral formulation using the following parameters.

Specificity^{14, 15} and System suitability

Blank and Placebo interference:

A study to establish the interference of blank and placebo were conducted. Diluent and placebo was injected into the chromatograph in the defined above chromatographic conditions and the blank and placebo chromatograms were recorded.

Force Degradation studies:

A study was conducted to demonstrate the effective separation of degradants/impurities from Eribulin. Separate portions of sample and placebo solutions were exposed to the following stress conditions to induce degradation. Stressed and unstressed samples were injected into the HPLC system with photo diode array (PDA) detector. Degradation study results were presented in Table 2.

System precision^{16, 17}

System precision was demonstrated by preparing standard solution as per the optimised method and chromatographed the same into HPLC system in six replicate injections of standard solution. The peak areas of analyte were recorded for these standard injections. The system precision was evaluated by computing the % relative standard deviation for the peak area of these standard injections. The observations are tabulated below in Table 3.

Table3. System precision results

S. No.	No.of injections	Area response
1	Inj-1	1210762
2	Inj-2	1216225
3	Inj-3	1225458
4	Inj-4	1211096
5	Inj-5	1226204
6	Inj-6	1216342
	Average	1217681
	SD	6168.08
	%RSD	0.51

The relative standard deviation of six replicate standard solution results found to be within the specification limit (≤ 2.0%) i.e.0.51%.

Method precision:

Method precision was demonstrated by prepare six samples of Eribulin mesylate injection (1 mg/2.0 mL as per optimised method and injected in to the chromatographic system. The precision of the method was evaluated by calculate the individual % assay, mean % assay and % relative standard deviation for each set of samples. The results of the precision study are tabulated below in Table 4.

Table4. Method precision results

S. No.	No. of Preparations	% Assay
1	Preparation 1	101.8
2	Preparation 2	100.9
3	Preparation 3	101.5
4	Preparation 4	102.0
5	Preparation 5	100.5
6	Preparation 6	101.0
Average		101.3
SD		0.5273
%RSD		0.52

Individual assay and overall assay within the specification limit (between from 90% to 110%). The relative standard deviation of six sample solution results found to be within the specification limit (≤ 2.0%) i.e.0.52%.

Linearity of detector response^18-23

The linearity of an analytical method is its ability to obtain test results which has a definite mathematical relation to the concentration of analyte. The linearity of response for Eribulin was determined in the range of 50% to 150 % (50.36-151.08 µg/ml for Eribulin). The calibration curve of analytical method was assessed by plotting concentration versus peak area and represented graphically.

Table5. Linearity studies for Eribulin

S. No	Linearity Level	Concentration (ppm)	Area response
1	Linearity at 50%	50.36	601991
2	Linearity at 75%	75.54	894062
3	Linearity at 100%	100.72	1191768
4	Linearity at 125%	125.9	1480657
5	Linearity at 150%	151.08	1785258
Correlation coefficient (r²)			0.9999
Intercept			9495.6000
Slope			11.728.0739
100% Y-intercept			0.80

Figure 4. Calibration curve for Eribulin mesylate

The Correlation coefficient (r²) was found to be 0.9999. Therefore the HPLC method was found to be linear standard curve were calculated and given in Figure 4 to demonstrate the linearity of the proposed method. From the data obtained which is given in Table 5 the method was found to be linear within the proposed range.

Accuracy ²⁴

The accuracy of the test method was demonstrated by preparing recovery samples of Eribulin mesylate at 50% to 150% of the target concentration level. The recovery samples were prepared in triplicate preparations on Eribulin mesylate API spiked to placebo, analyzed as per the proposed method for each concentration level. The above samples were chromatographed and the percentage recovery of each sample was calculated for the amount added.

Table 6. Recovery studies for Eribulin mesylate by proposed method

% Level	(µg) Recovered	(µg) Added	% Recovery	% Mean Recovery
Accuracy at 50 %-1	25.11	25.18	99.7	99.7
Accuracy at 50 %-2	25.19	25.25	99.8
Accuracy at 50 %-3	25.22	25.34	99.5
Accuracy at 100 %-1	100.59	100.72	99.9	99.7
Accuracy at 100 %-2	100.25	100.51	99.7
Accuracy at 100 %-3	100.21	100.64	99.6
Accuracy at 150 %-1	150.55	150.64	99.9	100.0
Accuracy at 150 %-2	150.38	150.28	100.1
Accuracy at 150 %-3	150.27	150.39	99.9

Evaluated the precision of the recovery at each level by computing the relative standard deviation of three preparations for 50% and 150% level recovery samples results. The data obtained which given in Table 6, the method was found to be accurate.

Solution stability of analytical solutions²⁵

Solution stability of standard and sample solutions was established at various conditions such as bench top at room temperature and in refrigerator 2-8°C. The stability of standard and sample solutions was determined by comparison of initial prepared standard and sample solutions with freshly prepared standard solutions. The data obtained which given in Table 7 to Table 9.

Table7. Results for solution stability of standard

Time Interval	Similarity factor
Time Interval	Room temperature (25-28°C)	Refrigerator (2-8°C)
Initial	NA	NA
12hrs	1.01	1.00
24hrs	1.04	1.01

Table 8. Results for solution stability of sample at Room temperature (25-28°C)

Time Interval	%Assay	%Assay difference
Initial	101.8	NA
12hrs	101.1	0.8
24hrs	100.5	1.3

Table 9. Results for solution stability of sample in Refrigerator (2-8°C)

Time Interval	%Assay	% Assay difference
Initial	101.8	NA
12hrs	101.3	0.5
24hrs	101.1	0.7

Solution stability of standard and sample solutions at RT and Refrigerator different time intervals studied, from the above results, it is concluded that standard solution is stable upto 12 hours at bench top and 24 hours in refrigerator condition (2-8°C). Sample solutions are stable up to 24 hours in both the conditions (bench top (RT) and refrigerator condition (2-8°C).

Robustness studies:

To validate the method robustness the chromatographic performance at changed conditions was evaluated compared to nominal conditions of the method. Standard solution was injected at each of the following changed conditions. The data obtained which given in Table 10.

Table 10. Robustness studies Results

Parameter		Theoretical plates	Tailing factor	% RSD
Flow variation ± 10%	0.7 mL	8543	1.1	0.61
Flow variation ± 10%	0.9 mL	10184	1.0	0.45
Temperature variation ± 5^oC	45^°C	9942	1.1	0.39
Temperature variation ± 5^oC	35°C	8891	1.2	0.55
pH variation ± 0.2	6.7	9145	1.0	0.33
pH variation ± 0.2	6.3	8347	1.2	0.43
Mobile phase Variation ± 10%	73:27 v/v	7962	1.3	0.78
Mobile phase Variation ± 10%	67:33 v/v	11478	1.0	0.24

Method is robust for changes like flow rate, column oven temperature, pH variation and organic phase variation of mobile phase.

CONCLUSION:

The developed method was validated for various parameters as per ICH guidelines like specificity (forced degradation)^26-28, system suitability, precision, linearity, accuracy, solution stability and robustness ²⁹. The results obtained were within the acceptance criteria. So, it can be concluded that the developed method is simple, precise, cost-effective, eco-friendly, safe and can be successfully employed for the routine analysis of Eribulin mesylate in bulk and pharmaceutical dosage forms.

CONFLICT OF INTERESTS:

The authors claim that there is no conflict of interest.

ACKNOWLEDGMENT:

The authors are grateful to the Department of Chemistry, University College of Sciences, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur. Andhra Pradesh, India, for providing facilities to carry out this research work.

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Received on 22.03.2025 Revised on 12.07.2025

Accepted on 20.09.2025 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2336-2342.

DOI: 10.52711/0974-360X.2026.00335

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

S. No	Name	Retention Time (min)	Theoretical plates	Tailing factor
1	Blank	Not Detected	Not Applicable	Not Applicable
2	Placebo solution	Not Detected	Not Applicable	Not Applicable
3	Standard solution	10.44	9274	1.0
4	Sample solution	10.52	NA	NA

S. No.	Degradation condition	(%) Assay	Purity Angle	Purity Threshold	Peak Purity
1	Control sample	101.3	0.251	0.425	Pass
2	Acid degradation (0.5N HCl/60°C /2 hrs)	92.8	0.138	0.387	Pass
3	Base Degradation (0.5N NaOH/BT/24hrs)	99.5	0.221	0.527	Pass
4	Peroxide Degradation/ (3% H2O2/BT/24hrs)	100.3	0.478	0.621	Pass
5	Thermal Degradation/60°C/24hrs	95.3	0.229	0.542	Pass
6	Humidity Degradation/95%RH/24hrs	100.8	0.108	0.327	Pass
7	Photolytic Degradation 1.2 million Lux hours/ 200 watt hours/m2 for 7 days	97.5	0.207	0.576	Pass