Jamonline / 2(2); 2012 / 223–231 Research Article

Satyanarayana PVV and Siva Madhavi A

Journal of Atoms and Molecules
An International Online Journal
ISSN – 2277 – 1247

A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF RUXOLITINIB IN FORMULATIONS P.V.V. Satyanarayana*, Alavala Siva Madhavi Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, Andhra Pradesh, India Received on: 11-04-2012 Abstract: A simple, precise and accurate RP-HPLC method was developed and validated for rapid assay of Ruxolitinib in tablet dosage form. Isocratic elution at a flow rate of 1ml/min was employed on a symmetry Chromosil C18 (250x4.6mm, 5µm in particle size) at ambient temperature. The mobile phase consisted of Acetonitrile: Water: Tetra Hydro Furan (THF) 60:30:10% (V/V/V). The UV detection wavelength was 227nm and 20µl sample was injected. The retention time for Ruxolitinib was 4.28min. The percentage RSD for precision and accuracy of the method was found to be less than 2%. The method was validated as per the ICH guidelines. The method was successfully applied for routine analysis of Ruxolitinib in tablet dosage form and bulk drug. Key Words: Ruxolitinib, RP-HPLC, UV detection, recovery, precise, 227 nm Revised on: 23-04-2012 Accepted on: 28–04–2012

Introduction: Ruxolitinib is a drug for the treatment of * Corresponding author PVV Satyanarayana, Email: sivamadhavi@rocketmail.com intermediate or high-risk myelofibrosis, a type of bone marrow cancer. It is also being

investigated for the treatment of other types of cancer and for plaque psoriasis. It is a Janus kinase inhibitor with selectivity for subtypes 1 and 2 of this enzyme.The phase III Controlled Myelofibrosis Study with Oral JAK

Innhibitor-I (COMFORT-I) and COMFORTII trials showed significant benefits by All rights reserved© 2011 www.jamonline.in 223

Jamonline / 2(2); 2012 / 223– –231 reducing spleen size, relieving debilitating symptoms, and improving overall survival. In November 2011, ruxolitinib was approved by the U.S. Food and Drug Administration for the treatment of intermediate or high high-risk myelofibrosis based on results of the

Satyanarayana PVV and Siva Madhavi A atyanarayana Apparatus A Series HPLC system PEAK LC7000 isocratic HPLC with PEAK 7000 delivery system, Rheodyne manual sample injector with switch (77251), Analytical column Chromosil C18. 250×4.6mm, Electronic


balance-DENVER (SI234), manual Rheodyne R injector with a 20 µ loop was used for the µl injection of sample. PEAK LC software was used. UV 2301 SPECTROPHOTOMETER was used to determine the wavelength of maximum absorbance Determination of wavelength of maximum absorbance The standard solutions of Ruxolitinib were e

Figure 1: Stricture of Ruxolitinib Ruxolitinib has been assigned to pregnancy category C by the FDA. In animal s studies, treatment with ruxolitinib resulted in an increase in late resorptions and reduced foetal weights at maternally toxic doses. There are no adequate and well controlled studies of ruxolitinib in pregnant women. Ruxolitinib should be used during pregnancy only if the ancy potential benefit outweighs the potential risk to the developing fetus. Experimental: Materials Working standard of Ruxolitinib was obtained from well reputed research laboratories. HPLC grade water, Acetonitrile, THF (tetra hydro furan) was purchased from E. Merck (Mumbai, India). All rights reserved© 2011

scanned in the range of 200 -400 nm against mobile phase as a blank. Ruxolitinib showed maximum absorbance at 277 nm. So the wavelength selected for the determination of Ruxolitinib was 227 nm. Chromatographic conditions The development and validation of the assay was performed on A Series 200 HPLC system PEAK LC7000 isocratic HPLC with PEAK 7000 delivery system. Rheodyne manual sample injector with switch (77251), equipment equip and

Analytical column Chromosil 100 100-5 C18. 250×4.6mm, manual injector rheodyne valve) with 20µL fixed loop, PEAK LC software L was used. The mobile phase consisted of Acetonitrile: water: THF 60:30:10(V/V/V). Injections were www.jamonline.in 224

Jamonline / 2(2); 2012 / 223–231 carried out using a 20 µl loop at room temperature (20 + 2 °C) and the flow rate was 1 ml/min. Detection was performed at 227 nm with 10min runtime. Standard and sample solutions A 10 mg amount of Ruxolitinib reference substance was accurately weighed and

Satyanarayana PVV and Siva Madhavi A chromatography was monitored by applying the following system suitability tests: capacity factor, tailing factor and theoretical plates. The system suitability method acceptance criteria set in each validation run were: capacity factor >2.0, tailing factor ≤2.0 and theoretical plates >2500. In all cases, the relative standard deviation (R.S.D) for the analytic peak area for two consecutive injections was < 2.0%. A chromatogram obtained from reference substance solution is presented. System suitability parameters were shown in Table.1. Standard chromatogram was given in Figure.2

dissolved in 10 ml mobile phase in a 10 ml volumetric flask to obtain 1000 ppm

concentrated solution. From standard solution by the serial dilution we prepared required concentrations of 100 ppm. A composite of 20 tablets was prepared by grinding them to a fine, uniform size powder. 100mg of Ruxolitinib was accurately weighed and quantitatively transferred into a 100 ml volumetric flask. Approximately, 25 ml mobile phase were added and the solution was sonicated for 15 min. The flask was filled to volume with mobile phase, and mixed. After filtration, an amount of the solution was diluted with mobile phase to a concentration of 100 ppm. Method validation Method validation was performed following ICH specifications for specificity, range of linearity, accuracy, precision and robustness. Results and Discussions: System Suitability Having optimized the efficiency of a

Mobile phase Pump mode PH Diluents

Acetonitrile: Water : THF (60:30:10 (v/v)) Isocratic 5.8 Mobile phase Zodiac C18 column (250 X 4.6 mm, 5µ) Ambient 227nm 20 µl 1 ml/min 10 minutes 4.28 minutes

Column Column Temp Wavelength Injection Volume Flow rate Run time Retention Time

chromatographic separation the quality of the

Table 1 System suitability parameters

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Jamonline / 2(2); 2012 / 223–231

Satyanarayana PVV and Siva Madhavi A

Figure.2 Standard Chromatogram of Ruxolitinib

Range of linearity Standard curves were constructed daily, for three consecutive days, using seven standard concentrations in a range of 25, 50, 75, 100, 125 and 150ppm for Ruxolitinib. The linearity of peak area responses versus concentrations was demonstrated by linear least square All rights reserved© 2011

regression analysis. The linear regression equation was y = -1168+ 3071x (r= 0.9999). Linearity values can shown in Table: 2

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Jamonline / 2(2); 2012 / 223–231 Level Level 1 Level 2 Level 3 Level 4 Level 5 Level 6 Slope Range 25 ppm to 150 ppm Intercept

Satyanarayana PVV and Siva Madhavi A Conc. of Ruxolinitinib in PPM 25 50 75 100 125 150 Peak Area 75423 148937 231276 307470 384621 461367 3071 -1668 0.9999

Correlation Coefficient Table 2: Linearity results of Ruxolitinib

500000 400000 300000 Area 200000 100000 0 0 -100000 Concentration 20 40 60 80 100 120 140 160

Figure 3: Calibration curve of Ruxolitinib Precision To study precision, six replicate standard solutions of Ruxolitinib (100 ppm) were prepared and analyzed using the proposed method. The percent relative standard deviation (% RSD) for peak responses was calculated and it was found to be which is well within the acceptance criteria of not more than 2.0%. Results of intraday and inter day precision studies are shown in Table.3 and Table.4 respectively.

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Jamonline / 2(2); 2012 / 223–231

Satyanarayana PVV and Siva Madhavi A


Conc. (in ppm)

Injection No.

Peak Areas

RSD (Acceptance Criteria ≤ 2.0%)

1 2 Ruxolitinib 40 3 4 5 6

307470 307638 307912 308967 307438 308039 0.185

Table 3: Intraday Precision Results for Ruxolitinib.


Conc. (in ppm)

Injection No.

Peak Areas

RSD (Acceptance Criteria ≤ 2.0%)

1 2 Ruxolitinib 40 3 4 5 6

308621 307096 308125 308852 307026 308602 0.26

Table 4: Inter day Precision Results for Ruxolitinib. Limit of Detection and Limit of considered as Limit of Detection and Limit of Quantification is 0.16 ppm. Parameter Limit of Quantification Limit of Detection Measured Value 0.16 ppm 0.05ppm

Quantification: To determine the Limit of Detection (LOD) sample was dissolved by using Mobile phase and injected until peak was disappeared. After 0.05 ppm dilution Peak was not clearly observed, based on which 0.05ppm is

Table 5: LOD and LOQ of Ruxolitinib All rights reserved© 2011 www.jamonline.in 228

Jamonline / 2(2); 2012 / 223–231 Robustness Typical variations in liquid chromatography conditions were used to evaluate the

Satyanarayana PVV and Siva Madhavi A were retention time, area, capacity factor, tailing factor and theoretical plates. The robustness acceptance criteria set in the validation were the same established on system suitability test describe above.

robustness of the assay method. In this study, the chromatographic parameters monitored

S.NO Parameter 1 2 3 4 Standard Mobile phase Mobile phase PH Wavelength

Condition Standard conditions Acetonitrile : H2O : THF (65:25:10) 5.9 222 nm

Area 307470 312687 305039 305793

% Change …….. 1.7 0.791 0.55


Table 6: Robustness results of Ruxolitinib. Recovery: Recovery test was performed at 3 different concentrations i.e.50ppm, 100ppm, and 150 ppm. Results are given in table.7.

Conc. of sample Recovery (ppm) 50% 100% 150% 50 100 150

Recovery (ppm) 49.63 99.67 150.54

% of recovery 99.26 99.67 100.36

Table 7: recovery results of Ruxolitinib

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Jamonline / 2(2); 2012 / 223–231 . Sample conc. 100 ppm

Satyanarayana PVV and Siva Madhavi A

S.NO Tablet Dosage

Sample estimated 99.12 ppm

% of Drug Estimated in Tablet





Table.8: Formulation Analysis

Conclusion: The proposed method for the assay of Ruxolitinib in tablets or capsules is very simple and rapid. It should be emphasized it is isocratic and the mobile phase do not contain any buffer. The method was validated for specificity, linearity, precision, accuracy and robustness. Although the method could effectively separate the drug from its 4 3

Nature Reviews Drug Discovery11 (2): 103–4. Mesa, RA (2010). "Ruxolitinib, a

selective JAK1 and JAK2 inhibitor for the treatment of myeloproliferative

neoplasms and psoriasis". IDrugs : the investigational drugs journal13 (6): 394–403. Pardanani, A.; Tefferi, A. (2011). "Targeting neoplasms with myeloproliferative JAK inhibitors".

products, further studies should be performed in order to use it to evaluate the stability of pharmaceutical formulations. References: 1 Shilling, A. D.; Nedza, F. M.; Emm, T.; Diamond, S.; McKeever, E.; Punwani, N.; Williams, W.; Arvanitis, A. et al (2010). "Metabolism, Excretion, and Pharmacokinetics of 5

Current Opinion in Hematology18 (2): 1. Harrison, C.; Kiladjian, J. J.; Al-Ali, H. K.; Gisslinger, H.; Waltzman, R.; Stalbovskaya, V.; McQuitty, M.;

Hunter, D. S. et al (2012). "JAK Inhibition with Ruxolitinib versus Best Available Therapy for Myelofibrosis". New England Journal of Medicine366 (9): 787–798. 6 Verstovsek, S.; Mesa, R. A.; Gotlib, J.; Levy, R. S.; Gupta, V.; Dipersio, J. F.; Catalano, J. V.; Deininger, M. et al (2012). "A Double-Blind, Placebo-

[14C]INCB018424, a Selective Janus Tyrosine Humans". Kinase Drug 1/2 Inhibitor, in and


Disposition38 (11): 2023. 2 Mesa, Ruben A.; Yasothan, Uma; Kirkpatrick, Peter (2012). "Ruxolitinib".

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Jamonline / 2(2); 2012 / 223–231 Controlled Trial of Ruxolitinib for Myelofibrosis". New England Journal of Medicine366 (9): 799–807. 7 Tefferi, A. (2012). "Challenges Facing JAK Inhibitor Therapy for

Satyanarayana PVV and Siva Madhavi A

Myeloproliferative Neoplasms". New England Journal of Medicine366 (9): 844–846. 8 ASCO Annual Meeting 2011: JAK Inhibitor Significant Myelofibrosis 9 "FDA Approves Incyte's Jakafi(TM) (ruxolitinib) for Patients with Ruxolitinib Clinical Demonstrates Benefit in

Myelofibrosis" (Press release). Incyte. Retrieved 2012-01-02.

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