INTRODUCTION:

Diroximelfumarate is a new drug from the fumarate class formulated to treat various relapsing forms of Multiple sclerosis . Diroximelfumarate is indicated for the treatment of relapsing forms of multiple sclerosis (MS) in adults; specifically active secondary progressive disease and clinically isolated syndrome, as well as relapsing-remitting MS. Diroximelfumarate relieves the neurological symptoms of relapsing MS with less gastrointestinal effects than its bioequivalent counterpart, dimethyl fumarate. It is important to note that diroximel fumarate can cause angioedema, anaphylaxis, hepatotoxicity, flushing, lymphopenia, and Progressive Multifocal Leukoencephalopathy (PML).

Fig 1. Structure of Diroximel Fumarate

Literature survey revealed that only a few analytical methods were reported for the estimation Diroximel Fumarate. Bio analytical methods (LC-MS/MS) have been reported for the quantification of Diroximel Fumarate in biological fluids.⁹ There is no RP-HPLC method for the analysis of Diroximel Fumarate.

MATERIALS AND METHODS:

Materials:

Diroximel Fumarate pure drug was obtained as gift sample from Biophore pharma, Hyderabad. Diroximel Fumarate Capsules (VUMERITY) were purchased from local market. HPLC grade acetonitrile, and distilled water were purchased from Merck, Mumbai. Analytical grade phosphate buffer, ortho-phosphoric acid were obtained from Rankem laboratories, Hyderabad.

Instrument:

The liquid chromatographic system was performed on waters HPLC 2965 system equipped with auto injector and PDA Detector. Empower software was used for data acquisition, processing and reporting.

Chromatographic conditions:

The method development was achieved on Waters C18 column (250 x 4.6mm, 5µm). Mobile phase was freshly prepared by mixing water (85% OPA) and acetonitrile in the ratio of 70:30 v/v. The flow rate was maintained at 1.0mL/min. Detector wavelength was monitored at 215 nm, and the injection volume was 20μL and run time was kept 5 min.

Preparation of standard stock solution:

Accurately weighed 10mg of Diroximel Fumarate was dissolved in 10ml mobile phase (1000µg/ml of Diroximel Fumarate standard stock solution). From the above standard stock solution 0.125ml was transferred into a 10ml volumetric flask and made up to the mark with mobile phase. (125µg/ml of Diroximel Fumarate).

Preparation of Sample stock solutions:

Average weighed amount equivalent to each tablet was calculated. Weight equivalent to 18.49mg was transferred into a 100ml volumetric flask and made up to the mark with mobile phase (125µg/ml of Diroximel Fumarate). The solution was sonicated for 25 min and filtered by HPLC filters. Further pipette out 0.125ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with mobile phase. (125µg/ml of Diroximel Fumarate).

Method validation:

System suitability parameters:

System suitability was evaluated in terms peak tailing and USP plate count by injecting 6 replicates of 10 μg/mL Diroximel Fumarate standard drug concentration. The calculated % RSD for the area of six standard injections results should not be more than 2%.

Specificity:

The specificity of developed method was evaluated by injecting blank sample (mobile phase) to demonstrate the absence of interference with elution of Diroximel Fumarate standard solution. (125μg/mL).

Linearity:

Six different concentrations ranging from 62.5-187.5 μg/mL of Diroximel Fumarate standard stock solution were prepared and injected. Calibration curve was constructed by plotting mean response factor against the respective concentration. The method was evaluated by determination of the correlation coefficient and intercept value.

Accuracy:

Recovery assessment was obtained by using standard addition technique which was performed by adding known quantities of pure standards at three different levels in 50%, 100% and 150% to the pre analysed sample formulation. From the amount of drug found and amount of drug recovered, percentage recovery was calculated.

Precision:

Diroximel Fumarate standard concentrations (125 μg/mL) were analyzed for 6 times in consecutive days (inter day precision) and 6 times during the same day (intra-day precision). The precision of proposed method was obtained by calculating the relative standard deviation (RSD) values for intra-day and inter-day analysis with acceptance criteria of ≤ 2% RSD.

Robustness:

Robustness of the method was evaluated by small but deliberate changes in method like Flow rate at ±0.2 ml/min and temperature by ±5°C.

Assay:

Assay of the marketed formulation was carried out by injecting sample corresponding to equivalent weight into HPLC system to calculate the percentage purity.

Forced Degradation studies:

Degradation studies is carried out on the sample by using Acid, Alkaline, Oxidative, Thermal studies, Photolytic and Thermal degradations. The sample was stressed to these conditions and peak was evaluated for peak purity indicating the above method can be used successfully separate the degradation products from pure active ingredients.

Degradation in acidic condition:

Degradation in acidic medium is carried out by weighing 125 micro grams of Diroximel Fumarate in 10ml Volumetric flask. and the contents was dissolved in 3ml of 0.5N HCL and heated on a water bath at 85°C Neutralise the sample with 0.5N NaOH and injected into HPLC system at different time intervals of 0, 2, 4, 6, 10, 12, 24 hrs.

Degradation in Basic condition:

Degradation in Alkaline medium was Performed by 0.5N NaOH by weighing 125 micrograms in 10ml volumetric flask.it was dissolved in 3ml of 0.5N NaOH and it was kept aside. Neutralise the sample with 0.5N HCL and injected into HPLC system at different time intervals of 0, 2, 4, 6, 10, 12, 24 hrs.

Oxidative degradation:

Oxidative Degradation is performed by Weighing 125 micrograms of Diroximel Fumarate which was transferred into volumetric flask. The contents are dissolved in 5ml of 3% H₂O₂ and kept aside for 1 day and injected into HPLC System at different time intervals of 0, 2, 4, 6, 10, 12, 24 hrs.

UV-degradation:

UV Degradation is performed by weighing 125 micrograms of Diroximel fumarate accurately and kept in UV chamber at 275nm and injected into HPLC system at different time intervals of 0, 2, 4, 6, 10, 12, 24 hrs.

RESULTS AND DISCUSSION:

System Suitability:

Standard solutions of Diroximel Fumarate working standard was prepared as per procedure and were injected six times into the HPLC system. The system suitability parameters were evaluated from standard Chromatograms obtained by calculating the %RSD of retention time, tailing factor, theoretical plates and peak areas from five replicate injections are within range and results were shown in table 1.

Table No. 1 System suitability parameters

S. No	STD Area	Theoretical Plates	Tailing Factor	RT
1	10709826	3815	1.77	2.390
2	10516939	4246	1.77	2.389
3	10556998	4094	1.76	2.390
4	10622063	3887	1.75	2.389
5	10883852	3947	1.71	2.389
6	10333888	4008	1.70	2.391
Avg	10603928		1.74	2.39
SD	185791		0.03	0.001
%RSD	1.752		1.765	0.034

Fig.2 System suitability Chromatogram

Linearity:

To demonstrate the linearity of assay method, 5 standard solutions with concentrations of about 50μg/mL to 150 μg/mL of Diroximel Fumarate were injected. Calibration curve plotted between peak area versus respective concentration which was shown in figure 3 Slope obtained was 835393 Y-Intercept was 60000 and Correlation Co-efficient was found to be 0.999.

Table No.2 Results of Linearity

Linearity Level (%)	Concentration (μg/mL)	Area
50	62.5	5281217
75	93.75	7921825
100	125	10562433
125	156.25	12903041
150	187.5	15843650

Fig .3 Calibration curve of Diroximel Fumarate

Accuracy:

Diroximel Fumarate spiked standard concentrations (50 μg/mL, 100μg/mL, 150μg/mL) at all the three levels were analyzed for percentage recoveries and the results were presented in table 3. The mean percentage recoveries of three levels (3 samples from each concentration were injected) was found to be 100.08%. The accuracy results were within the accepted limits from 98.0% to 102.0% which proves that the method was found to be accurate. Good recovery results obtained for the developed method indicates that this method can be used for regular quality control assay test for Diroximel Fumarate.

Precision:

The precision of a method determines the closeness of agreement between a series of measurements of the same sample. The intraday and interday precisions were carried out 6 times at concentration of 10μg/mL and the %RSD were found to be 0.7 and 0.7, respectively. The precision result (table 4) was within the accepted limits of ≤ 2 % RSD which proves that the method was precise.

LOD and LOQ:

LOD is a limit test parameter and it is a test to determine whether the analyte concentration was present within the specification limit or not. LOQ is a parameter for quantitative assay used particularly for determination of impurities or degradation products as it used for minimum concentrations of analytes in sample. The LOD and LOQ were found to be 6.67 and 22.24 respectively which proves the method was sensitive.

Table No.3 Accuracy data

Sample No.	Spiked Level	Sample Weight (mg)	Sample Area	µg/ml added	µg/ml found	% Recovery	% Mean Recovery
1	50	9.245	5301055	6.23	6.25	100.30	100.15
2	50	9.245	5288206	6.23	6.23	100.05
3	50	9.245	5182224	6.23	6.11	98.05
4	50	9.245	5343124	6.23	6.30	101.09
5	50	9.245	5272227	6.23	6.21	99.75
6	50	9.245	5372227	6.23	6.33	101.64
7	100	18.49	10608765	12.46	12.51	100.36	99.82
8	100	18.49	10556161	12.46	12.44	99.86
9	100	18.49	10490284	12.46	12.37	99.24
10	150	27.735	15950992	18.69	18.80	100.60	99.94
11	150	27.735	16073603	18.69	18.95	101.37
12	150	27.735	15855434	18.69	18.69	100.00
13	150	27.735	15801278	18.69	18.63	99.65
14	150	27.735	15853267	18.69	18.69	99.98
15	150	27.735	15542577	18.69	18.32	98.02

Table No.4 Precision da

Intraday precision

S. No	Sample Weight	Sample Area	% Assay
1	18.49	10537857	99.39
2	18.49	10492882	98.96
3	18.49	10494555	98.98
4	18.49	10676745	100.70
5	18.49	10648596	100.43
6	18.49	10479237	98.83
Avarage Assay:			99.55
STD			0.81
% RSD			0.82

Interday precision

S. No	Sample Weight	Sample Area	% Assay
1	18.49	10561216	99.61
2	18.49	10581125	99.80
3	18.49	10569306	99.68
4	18.49	10682469	100.75
5	18.49	10672469	100.66
6	18.49	10452469	98.58
Avarage Assay:			99.85
STD			0.80
% RSD			0.80

Robustness:

Robustness of the method was performed by changing flow rate (± 0.2mL/min) and change in temperature (± 5⁰C). The results were summarized in table 5. It was observed that even in minor changes of method conditions there was no marked changes in the results demonstrate that the HPLC method developed was robust. The robustness results were within the accepted limits of ≤ 2 % RSD.

Table No.5 Robustness Data

S. No	Condition	Peak area	% Assay
1	0.8 ml/min	10630112	100.26
2	1 ml/min	10591131	99.89
3	1.2 ml/min	10652150	100.47
4	25 °C	10679545	100.72
5	30°C	10531211	99.33
6	35 °C	10582877	99.81

Assay of Marketed Formulation:

Sample solution was injected separately 6 times from the same sample individually into the system and chromatograms were recorded and %RSD was reported from the calculated percentage purity values.

Table No.6 Assay of Formulation

Sample No	%Assay
1	100.68
2	100.40
3.	99.63
4.	99.08
5.	98.94
6.	99.95
AVG	99.78
StdDev	0.70
%RSD	0.7

Forced Degradation studies

Acid Degradation studies

Table No 7:Percentage of various degradation studies

S. No	Nature of the Sample	Sample Weight	Sample Area	% Assay	% of Degradation
1	Acid	18.49	9524683	89.83	10.17
2	Base	18.49	9629742	90.82	9.18
3	Peroxide	18.49	9734801	91.81	8.19
4	Heat	18.49	9555185	90.12	9.88
5	UV	18.49	9661216	91.12	8.88

CONCLUSION:

The developed method was validated as per ICH guidelines. All the validation parameters were found to be well within the acceptance criteria. We concluded that the method is accurate, precise, linear and robust. The developed method can be successfully applied for the analysis of Diroximel fumarate in bulk and pharmaceutical dosage form in quality control laboratories.

CONFLICT OF INTEREST:

None.

REFERENCES:

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Received on 14.01.2020 Modified on 04.05.2020

Research J. Pharm. and Tech. 2021; 14(5):2603-2607.

DOI: 10.52711/0974-360X.2021.00458