Development and Validation of RP-HPLC and UV Spectroscopy Method for the Determination of Diroximel Fumarate in Bulk Drug and Pharmaceutical Dosage Form

 

Jayesh D. Patil, Amitkumar R. Dhankani, Sunil P. Pawar

Department of Quality Assurance, Poojya Sane Guruji Vidya Prasarak Mandal, Shahada,

Dist. Nandurbar, 425409, (MS), India.

*Corresponding Author E-mail: patiljayesh314@gamil.com

 

ABSTRACT:

Two new, simple, specific, precise and accurate UV-spectrophotometric and one reverse phase high performance liquid chromatography methods have been developed for the estimation of Diroximel Fumarate in Pharmaceutical dosage form. First UV-spectrophotometric methods show absorption maxima at 253nm for the Diroximel Fumarate. Percentage mean recovery obtained was 100.00%; coefficient of variance was found to be less than 2% and linearity coefficient was 0.9998. Linear response obtained for Diroximel Fumarate was in the concentration range of 0.5-2.5mg/ml. The limit of detection and limit of quantification for Diroximel was found to be 0.049mg/ml and 0.149mg/ml, respectively, and Second RP-HPLC method, chromatography analysis is carried out using Methanol: 10mm of KH2PO4 buffer solution (pH 3.1) (70:30%, v/v) as the mobile phase and C18 Waters (4.6mm x 150mm), 5.0μ column as stationery phase with detection wavelength of 272nm linearity was obtained in the concentration range of 5-25μg/ml for Diroximel Fumarate. Both UV-spectrophotometric and reverse phase high performance liquid chromatography methods were statistically validated according to I.C.H. Guidelines.

 

KEYWORDS: Diroximel Fumarate, HPLC, UV-Spectrophotometric.

 

 

INTRODUCTION:

Diroximel fumarate is a new drug from the fumarate class formulated to treat various relapsing forms of Multiple sclerosis.1,2 Diroximel fumarate 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 multiple sclerosis.3,5,6

 

Diroximel fumarate relieves the neurological symptoms of relapsing multiple sclerosis 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).7,8,9 Literature survey revealed that only a few analytical methods were reported for the estimation Diroximel Fumarate stability indicating method.10 There is no RP-HPLC and UV-spectroscopic methods for the analysis of Diroximel Fumarate reported.

 

Fig.1: Chemical Structure of Diroximel fumarate

 

Materials and Methods Chemicals:

Diroximel Fumarate was obtained from RSITC Jalgaon, HPLC grade solvents, such as methanol (HPLC grade), 0.05% OPA (HPLC grade), Acetonitrile (HPLC grade) and KH2PO4 were purchased from Merck Ltd., India. Double-distilled water was used during the entire HPLC procedure.

 

Instruments and Equipments:

In this analytical number of analytical instruments are used that is given in table no 1.

 

Table No 1: Instrument Details used during Method Development

 

Name of Instrument

Company Name

1

HPLC Instrument

Agilent Tech. Gradient System With Auto injector (Chemstation 10.1 software)

2

UV Spectrophotometer

Analytical Technologies Limited

3

Column(C8)

Waters C18 (150mmX 4.6mm,5µm) :

4

pH meter

VSI pH meter(VSI 1-B)

5

Balance

WENSAR™ High Resolution Balance.

6

Sonicator

Ultrasonic’s electronic instrument

 

ANALYTICAL METHOD DEVELOPMENT:

To optimize the HPLC parameters, several mobile phase compositions were tried. Satisfactory results were obtained from given chromatographic condition for Diroximel fumarate by HPLC shown in table no 2.

 

Table No.2: Chromatographic conditions (HPLC) details used during method Development

1.

HPLC

Agilent Tech. Gradient System With Auto injector, UV Detector

2.

Software

chemstation 10.1

3.

Column

C18 Waters (4.6mm x 150mm) column

4.

Particle size packing

5mm

5.

Stationary phase

C-18 (Agilent)

7.

Mobile Phase

Methanol: 10 mm of KH2PO4 buffer solution (pH 3.1) (70:30%, v/v)

7.

Detection Wavelength

253 nm

8.

Flow rate

0.8 ml/min

9.

Temperature

Ambient

10.

Sample size

20 ml

11.

Ph

3.1

 

Fig No 2: Optimized Chromatogram of Diroximel fumarate 

 

Table No: 3. System Suitability Parameters

No.

RT[min]

Area

[mV*s]

Theoretical Plates

Asymmetry

1

2.798

628.7930

5926

0.89

 

Preparation of standard solutions

Accurately weight and transfer 5 mg Diroximel fumarate working standard into 10 ml volumetric flask as about diluents Methanol completely and make volume up to the mark with the same solvent to get 500µg/ml standard (stock solution) and 15 min sonicate to dissolve it.

 

Validation of method for analysis of Evogliptin:

The developed method was validated as per ICH guidelines Q2 (R1).12

 

Linearity

The linearity of an analytical method indicates its ability to obtain the response directly proportional to the concentration of the analyte in the sample within a definite range. From the standard stock solution 0.1ml of this solution diluted up to 10ml volumetric flask with diluents was added to make up the volume with methanol in the range of 5, 10, 15, 20, 25 and 30mg/mL for HPLC and 0.5, 1.0, 1.5, 2.0 and 2.5 for UV-spectroscopy.

 

The Result is shown in; Table No 4. and Fig. 3.

 

 

Table No 4. Linearity of Diroximel fumarate

Sr. No

HPLC Method

UV-Spectroscopy Method

Concentration (μg/ml)

Area

Concentration (μg/ml)

Absorbance

1

5

334.1709

0.5

0.100

2

10

627.9622

1.0

0.212

3

15

944.2367

1.5

0.321

4

20

1286.6579

2.0

0.436

5

25

1574.2965

2.5

0.560

 

Fig. 3: Calibration curve of Diroximel fumarate (HPLC)

 

 

UV Spectrophotometric method:

The data obtained in the calibration experiments when subjected to linear regression analysis showed a linear relationship between peak areas and concentrations in the range 0.5-2.5µg/mL for Diroximel Fumarate (Table No: 4) depict the calibration data of Diroximel Fumarate The linear equation for Diroximel Fumarate was y = 0.020X+0.003 where x is the concentration is area of peak. The correlation coefficient was 0.999. The calibration curve of Diroximel Fumarate is depicted in (Fig No.4).  

 

Fig. 4: Calibration curve of Diroximel fumarate (UV-Spectroscopy Method)

 

Accuracy:

The accuracy was determined by Diroximel fumarate (equivalent to 231mg of Diroximel fumarate) (80%, 100 % and 120% of the label claimed, respectively) to quantity equivalent to average weight of marketed Tablets. This powder mixture containing 5mg of Diroximel fumarate were triturated and then subjected to chromatographic analysis using the described method. The resulting mixtures were analyzed in triplicates over three days. The % recovery of added drug was taken as a measure of accuracy by both methods. The Result is shown in; Table No 5.

 

 

 

 

Table No: 5 Results from study of accuracy

METHOD

Level (%)

Mean % Recovery

Standard

Deviation*

%   RSD

UV

Method

80%

98.94

1.50

1.52

100%

99.27

0.65

0.41

120%

99.20

0.41

0.65

 

RP-HPLC Method

80%

99.09

0.27

0.27

100%

97.27

0.019

0.38

120%

98.36

0.21

0.22

 

Analysis of marketed formulation:

Weigh 20 Diroximel Fumarate Capsule and calculated the average weigh 0.3026mg accurately weigh and transfer the sample equivalent to 6.522mg Diroximel Fumarate into 10ml volumetric flask. Add about 10ml of diluent and sonicate to dissolve it completely and make volume up to the mark with diluent. Mix well and filter through 0.45µm filter. Further pipette 0.3ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with diluents. (20µg/ml). The simple chromatogram of test Diroximel Fumarate the amounts of Diroximel Fumarate per Capsule were calculated by extrapolating the value of area from the calibration curve. Analysis procedure was repeated Two times with tablet formulation. Tablet Assay for %Lable claim for %RSD Calculated, The Result is shown in; Table No 6.

 

Table No: 6 Results from study of accuracy for Marketed Product

Assay

Lable Claimed

Amt.Found

RSD

(%)

%Lable Claim

HPLC Method

20.00

1282.6787

1.00

101.24

20.00

1282.717

1.01

100.79

UV Method

1.00

0.1408

1.01

101.35

1.00

0.1411

1.02

101.86

 

Repeatability:

Precision of the system was determined with the sample. Two replicates of sample solution containing Diroximel fumarate were injected and peak areas were measured and taking absorbance for UV method and %RSD was calculated it was repeated for two times result are shown in; Table No 7.

 

Table No 7: Repeatability studies on RP-HPLC and UV-Spectroscopy for Diroximel Fumarate

Method

Concentration (mg/ml)

Amount found (mg)

% Amount found

%

RSD

HPLC

10

21.02

99.22

0.14

UV-Method

2

2.04

102.21

0.41

 

Precision:

Precision of an analytical method is the degree of agreement among Individual test results when the procedure is applied repeatedly to multiple Samplings of a homogenous sample. Precision of an analytical method is usually expressed as standard deviation or relative standard deviation. Also, the results obtained were subjected to one way ANOVA and within-day mean square and between-day mean square was determined and compared using F-test.


Table No 8: Result of Intraday and Inter day Precision studies on RP-HPLC and UV method for Diroximel Fumarate

METHOD

Concn(µg/ml)

Intraday Precision

Interday Precision

Mean± SD

%Amt Found

Mean± SD

%Amt Found

HPLC Method

10

621.62±1.03

97.01

627.87±1.78

98.05

15

946.92±3.85

100.86

943.40±4.32

100.46

20

1250.7±6.54

100.98

1251.42±1.80

101.04

UV Method

2

0.202 ±0.001

99.95

0.2014 ±0.004

99.20

3

0.3090 ±0.08

102.00

0.3063±0.003

101.07

4

0.4006±0.002

99.40

0.4013 ±0.002

99.55

 

Intra-day precision:

 Sample solutions containing 5 mg of Diroximel fumarate three different concentration (10µg/ml, 15µg/ml, 20µg/ml) Diroximel fumarate were analyzed three times on the same day and % R.S.D was calculated. The Result is shown in; Table No 8.

 

Inter-day precision:

Sample solutions containing 5mg of Diroximel fumarate were analyzed three times on different concentration (10µg/ml, 15µg/ml, and 20µg/ml) Diroximel fumarate different days and %R.S.D were calculated. It is usually expressed as standard deviation or relative standard deviation. The Result is shown in; Table No 8.

 

Robustness:

The Robustness of a method is its ability to remain unaffected by small deliberate changes in parameters. To evaluate the robustness of the proposed method, small but deliberate variations in the optimized method parameters were done. The effect of changes in mobile phase composition and flow rate, wavelength on retention time and tailing factor of drug peak was studied.

 

The mobile phase composition was changed in(±0.8 ml/min-1) proportion and the flow rate was varied by (±1ml/min-1), and wavelength change (±0.8 ml/min-1) of optimized chromatographic condition. Robustness parameters were also found satisfactory; hence the analytical method would be concluded.

 

CONCLUSION:

The analytical method described in this paper has two new good accuracy, precision, linearity, stability under different conditions. Here in we proposed a RP-HPLC and UV-spectroscopy method for the quantification of Diroximel fumarate, which was not previously found in the literature. The instrumental conditions were evaluated for validation parameters and were found to be accurate, precise, stable, economical, time saving, and robust. The method provided reasonable resolution and acceptable peak parameters according to ICH guidelines, the validated methods was found environment friendly when compared on a greenness scale as part of ecological safety. The developed method was also tested on self-prepared formulation as it demonstrated no interfering peak of the polymers. Furthermore, the method also detected the analyte in marketed brands which depicts that the validated method can play a significant role in the quantification of Diroximel fumarate. It can be utilized for analysis of dosage forms in the pharmaceutical industry and during stability studies with a single run time of less than 4 min.

 

ACKNOWLEDGEMENT:

The principal of the Poojya Sane Guruji Vidya Prasarak Mandal, Shahada, Dist. Nandurbar (MS) 425 409, is gratefully acknowledged by the authors for providing the essential library resources.

 

REFERENCE:

1.      Gold, R, Arnold, DL, Bar-Or, A, et al. Long-term effects of delayed-release dimethyl fumarate in multiple sclerosis: Interim analysis of ENDORSE, a randomized extension study. Mult Scler. 2017; 23(2): 253–265.

2.      Polman, CH, Reingold, SC, Banwell, B, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol. 2011; 69(2): 292–302.

3.      Gold, R, Kappos, L, Arnold, DL, et al. Placebo-controlled phase 3 study of oral BG-12 for relapsing multiple sclerosis. N Engl J Med. 2012; 367: 1098–1107.

4.      Mehta, D, Miller, C, Arnold, DL, et al. Effects of dimethyl fumarate on lymphocytes in RRMS: Implications for clinical practice. Neurology. 2019; 92(15): e1724–e1738.

5.      Giles, K, Hanna, J, Wu, F, et al. Efficacy of delayed-release dimethyl fumarate in newly diagnosed and other early MS patients, and patients switching from interferon or glatiramer acetate, in routine medical practice: Interim results from Esteem. Mult Scler J. 2017; 23(Suppl 3): 612.

6.      Mehta, D, Miller, C, Arnold, DL, et al. Effects of dimethyl fumarate on lymphocytes in RRMS: Implications for clinical practice. Neurology. 2019; 92(15): e1724–e1738.

7.      Giles, K, Hanna, J, Wu, F, et al. Efficacy of delayed-release dimethyl fumarate in newly diagnosed and other early MS patients, and patients switching from interferon or glatiramer acetate, in routine medical practice: Interim results from Esteem. Mult Scler J. 2017; 23(Suppl 3): 612.

8.      Fox, EJ, Vasquez, A, Grainger, W, et al. Gastrointestinal tolerability of delayed-release dimethyl fumarate in a multicenter, open-label study of patients with relapsing forms of multiple sclerosis. Int J MS Care. 2016; 18(1): 9–18

9.      K, Spinelli, KJ, Stuchiner, T, et al. Three-year clinical outcomes of relapsing multiple sclerosis patients treated with dimethyl fumarate in a United States community health center. Mult Scler. 2018; 24(7): 942–950.

10.   Kiranjyothi, R., Balakrishnan, M. and Chandrasekhar, K.B. Stability Indicating RP-HPLC Method Development and Validation for Estimation of Diroximel Fumarate in bulk and its dosage forms. Research Journal of Pharmacy and Technology. 2021; 14(5): 2603-2607.

11.   Guideline, IHT. Validation of analytical procedures: text and methodology Q2 (R1). In International Conference on Harmonization, Geneva, Switzerland. 2005: 11-12.

 

 

 

 

Received on 20.05.2024       Modified on 02.07.2024

Accepted on 06.08.2024   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2024; 14(3):147-150.

DOI: 10.52711/2231-5675.2024.00026