INTRODUCTION:

Lenalidomide is a derivative of thalidomide with better biological activity. Lenalidomide chemically it is 3-(4-Amino-1, 3-dihydro-1-oxo-2H-isoindol-2-yl)-2, 6-piperidinedione is represented in figure 1.

Figure 1: Chemical Structure of Lenalidomide

The chemical formula is C13H13N3O3 and molecular weight is 259.261g/mol. Lenalidomide (LND) is an oral immunomodulatory drug with anti-angiogenic and anti-neoplastic properties. It fundamentally takes after thalidomide however has an improved danger profile and progressively strong immunomodulatory action^1,2. LND showed wonderful clinical movement in treatment of numerous myeloma malady^3-7 by means of a various pathways system 8-11The system of activity of lenalidomide stays to be completely portrayed, anyway it has been exhibited that lenalidomide represses the outflow of cyclooxygenase-2 (COX-2), yet not COX-1, in vitro. In vivo it initiates tumor cell apoptosis legitimately and in a roundabout way by restraint of bone marrow stromal cell support, by hostile to angiogenic and against osteo clastogenic impacts, and by immunomodulatory action. LND shows direct pharmacokinetics in sound subjects just as in patients with ordinary renal capacity. The plasma introduction of LND is portion corresponding and it doesn't aggregate with numerous dosages. Around 66% of orally directed LND is disposed of as unaltered in pee, likely through glomerular filtration and dynamic rounded emission. The mean terminal half-life (t1/2) is 3 to 4 hours^12,

Experimental Work:

Materials and Methods: The instrument used for developing method for HPLC was Shimadzu LC-2010 HT equipped with a degassed unit, low pressure gradient bunit; pump unit, ultra fast auto sampler, and UV-Visible detector integrated with LC solutions software and C-18 column (GL science). UV–Visible spectrophotometer nicolet evolution integrated with vision pro software. Digital electronic balance- Mettler Toledo, pH meter- Global Digital and Ultra sonicator- Citizen.

Reagent and solutions:

Lenalidomide active pharmaceutical ingredient was procured from API industry and marketed formulation was procured from local pharmacy store. All solvents are in HPLC grade Distilled water procured from Martin Synge pharma science pvt Ltd, Methanol, Acetonitrile and Dipotassium hydrogen phosphate procured from Avantor Performance materials Ltd. Potassium Dihydrogen Phosphate procured from Thermo Fischer scientific pvt Ltd.

Preparation of Mobile Phase:

Preparation of Phosphate (pH 3.6): weigh accurately 1.625 gr of potassium dihydrogen phosphate and 0.3 gr of di potassium hydrogen phosphate transferred in 1000 capacity measuring cylinder and diluted with HPLC Grade water up to 550ml.

Preparation of 1000 ml Mobile Phase Composition: The 450 ml Acetonitrile and 550 ml phosphate buffer was filtered separately through 0.45 micron pore size membrane filter using vacuum filtration assembly. The filtered 450 ml acetonitrile transferred in 1000 ml measuring cylinder and make up to 550 ml with phosphate buffer.

Selection of Analytical wavelength:

To fix the for analysis the prepared stock solution was scanned in ultraviolet spectroscopy over the range of 200-400nm from resultant spectrum wavelength at 242 nm was chosen in this maximum absorption drug occurs. This wave length considered for analysis. The UV spectra of lenalidomide was shown in figure 2.

Figure 2: UV Spectra of Lenalidomide

Analytical methodology:

Assay of Marketed Lenalidomide Formulation:

Standard Preparation:

Accurately weighed 50mg of Lenalidomide Standard in to 50ml volumetric flask and diluted with methanol. Pipette out 0.5ml from above solution into 10ml volumetric flask and made up to mark with methanol and sonicate well. The resulting solution concentration was 50µg/ml.

Sample Preparation:

Weigh 10 capsules contents and determine the average weight. Accurately weigh and transfer quantity of capsule contents equivalent to about 10mg of LND sample transfer into 10ml volumetric flask dissolved and diluted up to the mark with methanol. Pipette out 0.5ml from above solution into 10ml volumetric flask and made up to mark with methanol and sonicate well.

RESULTS AND DISCUSSION:

Method Development:

Optimized Method:

The trials were performed for the method development and the method was developed by using Phosphate buffer: Acetonitrile (55:45) v/v as mobile phase and retention time was found at 2.5 min. The number of theoretical plates and tailing factor for lenalidomide was observed 16199.817 (NLT 2000) and 1.128 (NMT 2).

Optimized Chromatographic Conditions:

Column: C18 (250 X 4.6 mm ,5µ)

Mobile phase: Phosphate buffer: Acetonitrile (55:45) v/v

Flow rate: 1 ml/min

Temperature: 25°C

Injection volume: 20µL/min

Detection Wave length: 242nm

The optimized condition chromatogram was shown in figure 3.

Figure 3: Optimized Chromatogram of Lenalidomide

Method Validation:

Linearity: The different concentration varies from 25 µg/ml to 150µg/ml were prepared. Chromatograms were recorded by injecting 20µl from each concentration of the solution. Regression analysis was done on the peak areas of the drug (y) v/s concentration (x). The linearity ranges of lenalidomide were 25-150µg/mL and correlation coefficient was found be 0.99. Linearity graph was shown in figure 4 and overlaid linearity chromatogram was shown in figure 5.

Figure 4: Linearity Graph of Lenalidomide

Figure 5: Linearity Overlaid Chromatogram

System Suitability:

Prepared 100 µg/ml samples from the standard stock solution and injected six replicates. The system suitability data is shown in table 1. The system suitability parameters are within the acceptance limits, so system suitability is passed.

Table 1: System Suitability Data of Lenalidomide

S. NO.	Rt	Peak Area	Theoretical Plates	Tailing factor	Resolution
1	2.595	5611647	16118.25	1.08	5.821
2	2.590	5616705	16231.78	1.28	6.515
3	2.592	5617795	16282.58	1.35	7.564
4	2.592	5618547	16565.45	1.38	6.809
5	2.593	5621330	16832.45	1.45	7.825
6	2.593	5616350	16215.36	1.22	6.415

Accuracy:

Accuracy was estimated by using a known amount of standard lenalidomide added to measured quantity of sample. By calculating peak area ratio the amount of lenalidomide was estimated. By the use of three different concentrations equivalent to 50,100 and 150% of the active ingredient accuracy was evaluated by system suitability parameters. The accuracy data is shown in table 2.

Method Precision (Repeatability):

The analysis of drug was carried out six times on the same day to find the repeatability of the sample by using 100 µg/ml and % RSD was calculated for the resultant peak area. The repeatability results shown in table 3.

Table 2: Accuracy Data of Lenalidomide

Level (%)	Sample conc. (µg/mL)	Standard Conc. (µg/mL	Total conc. (µg/mL)	Peak area	Amount recovered (µg/mL	Mean % Recovery ± S.D	% RSD
50%	50	25	75	4195451	74.69	100.12 ± 0.86	0.8
				4198585	74.75
				4259585	75.83
100%	50	50	100	5636525	100.35	100.15 ± 0.23	0.2
				5611647	99.90
				5629663	100.22
150%	50	75	125	6987684	124.40	100.03 ± 0.49	0.4
				7027984	125.12
				7055984	125.62

Table 6: Degradation Studies Data of Lenalidomide

S.No.	Type of Stress condition	% Degradation
1	Acid degradation	No Degradation
2	Base degradation	6.878
3	Oxidation degradation	6.907
4	Photolytic degradation	No Degradation
5	Thermal degradation	6.950

CONCLUSION:

This present work is precise and validated for the estimation of Lenalidomide. The retention time of leanalidomide was found to be 2.5 min. The lenalidomide % Recovery was obtained as 100.02%. LOD and LOQ values obtained from regression equations of lenalidomide 0.058 and 0.174 respectively. The retention time was decreased and that run time was decreased, so the method developed was economical and accurate. The results of various degradation methods ,which were conducted following ICH guidelines. LND was found to be more stable under the acidic and photolytic stress conditions than base, oxidative and thermal stress conditions.

ACKNOWLEDGEMENT:

The authors are thankful to Ramanbhai Patel College of Pharmacy, CHARUSAT for providing support and facilities.

CONFLICTS OF INTEREST:

The authors declare that there are no conflicts of interest.

REFERENCES:

1. Kastritis E, Dimopoulos MA. The evolving role of lenalidomide in the treatment of hematologic malignancies. Expert Opin Pharmacother. 2007; 8: 497–509.

2. Yogeshwar Reddy M, Ramesh V, Kista Reddy Ch, Venugopal N, Saravanana G, Suryanarayana M.V, Shyam B, Sunder, Surendranath Reddy R, Raju G. Validated Chromatographic Method for the Determination of Process Related Genotoxic Impurity in Lansoprazole. Asian J. Research Chem.2011; 4(6): 965-967.

3. Tariman JD. Lenalidomide: a new agent for patients with relapsed or refractory multiple myeloma. Clin J Oncol Nursing. 2007; 11: 569–574.

4. Preeti Tiwari, Rakesh K Patel. Quantification of Quercetin and Rutin in Arjunarishta Prepared by Traditional and Modern Methods by Validated HPTLC Densitometry. Asian J. Research Chem.2011; 4(6): 1019-1024.

5. Shah SR, Tran TM. Lenalidomide in myelodysplastic syndrome and multiple myeloma. Drugs. 2007; 67: 1869– 1881.

6. Falco P, Cavallo F, Larocca A, Liberati AM, Musto P, Boccadoro M, Palumbo A. Lenalidomide and its role in the management of multiple myeloma. Expert Rev Anticancer Ther. 2008; 8: 865–874.

7. Hideshima T, Richardson PG, Anderson KC. Current therapeutic uses of lenalidomide in multiple myeloma. Expert Opin Invest Drugs. 2006; 15: 171–179.

8. Bhushan Pingale, Ravi Tiwari. Development and Validation of an RP-HPLC Method for the Estimation of Bupropion Hydrochloride. Research J. Pharm. and Tech.2011; 4(9): 1480-1482.

9. Anand Babu K, Kumudhavalli M.V, Sainath Reddy K, Jaykar B. Stability Indicating UV-Spectrophotometric Determination of Nevirapine in Pharmaceutical Dosage Form. Research J. Pharm. and Tech.2011; 4(9): 1386-1388.

10. Mohit D, Bauskar, Prafulla Sonawane, Santosh Y, Nandedkar, Rajendra D. Wagh, Tanvir Shaikh, Vaibhav Jagtap. Development and Validation of Reverse Phase Liquid Chromatographic Methods for the Determination of Acebrophylline in Capsule Form. Research J. Pharm. and Tech.2011; 4(10): 1542-1546..

11. Kalyankar T.M, Kakde R.B. Reversed-Phase Liquid Chromatographic Method for Simultaneous Determination of Artesunate and Mefloquine in Pharmaceutical Preparations. Research J. Pharm. and Tech.2011; 4(10): 1563-1566.

12. Sudha T, Shanmugasundram P. Development and Validation of RP-HPLC and HPTLC Chromatographic Methods of Analysis for the Quantitative Estimation of Raltegravir Potassium in Pharmaceutical Dosage Form. Research J. Pharm. and Tech.2011; 4(11): 1746-1750.

Received on 09.06.2020 Modified on 10.09.2020

Research J. Pharm. and Tech. 2021; 14(8):4281-4286.

DOI: 10.52711/0974-360X.2021.00744

S. No.	Concentration (µg/ml)	Peak Area
1	100	5636324
2	100	5609017
3	100	5618658
4	100	5624648
5	100	5642311
6	100	5635179

S. No.	Concentration (µg/ml)	Intraday (Peak Area)		Interday (Peak Area)
S. No.	Concentration (µg/ml)	Fore Noon	After Noon	Day-1	Day-2
1	100	5656122	5611647	5656122	5622618
2	100	5629141	5619119	5629141	5632419
3	100	5621674	5636525	5621674	5625127
Mean		5635645.66	5622430.33	5635645.66	5626721.33
Standard Deviation		18121.78	12765.28	18121.78	5091.29
% RSD		0.32	0.23	0.32	0.09

S.No.	Peak area
S.No.	standard	sample
1	2784562	2799555
2	2714412	2698845
3	2834314	2779526
4	2803133	2780651
5	2802145	2764428
Mean	2787713.20	2764601.00
S.D	44723.25	38813.72
% RSD	1.604	1.404
% Purity	99.17