A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF RUXOLITINIB IN FORMULATIONS
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Transcript of A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF RUXOLITINIB IN FORMULATIONS
Jamonline / 2(2); 2012 / 223–231 Satyanarayana PVV and Siva Madhavi A
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Research Article
Journal of Atoms and Molecules An International Online JournalAn International Online JournalAn International Online JournalAn International Online Journal ISSN ISSN ISSN ISSN –––– 2277 2277 2277 2277 –––– 1247124712471247
A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF RU XOLITINIB 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 Revised on: 23-04-2012 Accepted on: 28–04–2012
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
Introduction:
Ruxolitinib is a drug for the treatment of
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 COMFORT-
II trials showed significant benefits by
* Corresponding author
PVV Satyanarayana,
Email: [email protected]
Jamonline / 2(2); 2012 / 223–
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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
myelofibrosis based on results of the
COMFORT-I and COMFORT
Figure 1: Stricture of Ruxolitinib
Ruxolitinib has been assigned to pregnancy
category C by the FDA. In animal s
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
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).
–231 Satyanarayana PVV
reducing spleen size, relieving debilitating
symptoms, and improving overall survival. In
November 2011, ruxolitinib was approved by
and Drug Administration for
the treatment of intermediate or high-risk
myelofibrosis based on results of the
I and COMFORT-II Trials
Figure 1: Stricture of Ruxolitinib
Ruxolitinib has been assigned to pregnancy
category C by the FDA. In animal 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
ancy only if the
potential benefit outweighs the potential risk
Working standard of Ruxolitinib was obtained
from well reputed research laboratories.
HPLC grade water, Acetonitrile, THF (tetra
was purchased from E. Merck
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
injector with a 20 µ
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
scanned in the range of 200
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 equip
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),
Analytical column Chromosil 100
250×4.6mm, manual injector rheodyne valve)
with 20µL fixed loop, PEAK LC software
was used.
The mobile phase consisted of Acetonitrile:
water: THF 60:30:10(V/V/V). Injections were
atyanarayana PVV and Siva Madhavi A
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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
R (SI234), manual Rheodyne
injector with a 20 µl loop was used for the
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
e standard solutions of Ruxolitinib were
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 equipment and
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),
Analytical column Chromosil 100-5 C18.
250×4.6mm, manual injector rheodyne valve)
L fixed loop, PEAK LC software
The mobile phase consisted of Acetonitrile:
water: THF 60:30:10(V/V/V). Injections were
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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
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
chromatographic separation the quality of the
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
Mobile phase Acetonitrile: Water : THF (60:30:10 (v/v))
Pump mode Isocratic
PH 5.8
Diluents Mobile phase
Column Zodiac C18 column
(250 X 4.6 mm, 5µ)
Column Temp Ambient
Wavelength 227nm
Injection Volume 20 µl
Flow rate 1 ml/min
Run time 10 minutes
Retention Time 4.28 minutes
Table 1 System suitability parameters
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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
regression analysis. The linear regression
equation was y = -1168+ 3071x (r= 0.9999).
Linearity values can shown in Table: 2
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Level Conc. of Ruxolinitinib in PPM Peak Area
Level 1 25 75423
Level 2 50 148937
Level 3 75 231276
Level 4 100 307470
Level 5 125 384621
Level 6 150 461367
Range 25 ppm to 150 ppm
Slope
Intercept
Correlation Coefficient
3071
-1668
0.9999
Table 2: Linearity results of Ruxolitinib
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.
-100000
0
100000
200000
300000
400000
500000
0 20 40 60 80 100 120 140 160
Area
Concentration
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Sample
Conc.
(in ppm) Injection
No. Peak Areas
RSD
(Acceptance Criteria ≤ 2.0%)
Ruxolitinib 40
1 307470
0.185
2 307638
3 307912
4 308967
5 307438
6 308039
Table 3: Intraday Precision Results for Ruxolitinib.
Sample
Conc.
(in ppm) Injection
No. Peak Areas
RSD
(Acceptance Criteria ≤ 2.0%)
Ruxolitinib 40
1 308621
0.26
2 307096
3 308125
4 308852
5 307026
6 308602
Table 4: Inter day Precision Results for Ruxolitinib.
Limit of Detection and Limit of
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
considered as Limit of Detection and Limit of
Quantification is 0.16 ppm.
Parameter Measured Value
Limit of Quantification 0.16 ppm
Limit of Detection 0.05ppm
Table 5: LOD and LOQ of Ruxolitinib
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Robustness
Typical variations in liquid chromatography
conditions were used to evaluate the
robustness of the assay method. In this study,
the chromatographic parameters monitored
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.
S.NO Parameter Condition Area % Of Change
1 Standard Standard conditions 307470 ……..
2 Mobile phase Acetonitrile : H2O : THF
(65:25:10) 312687 1.7
3 Mobile phase PH 5.9 305039 0.791
4 Wavelength 222 nm 305793 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.
Recovery Conc. of sample
(ppm)
Recovery
(ppm)
% of
recovery
50% 50 49.63 99.26
100% 100 99.67 99.67
150% 150 150.54 100.36
Table 7: recovery results of Ruxolitinib
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.
S.NO Tablet Dosage Sample
conc.
Sample
estimated
% of Drug Estimated
in Tablet
1 Jakafi 10mg 100
ppm
99.12
ppm 99.12%
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
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
[14C]INCB018424, a Selective Janus
Tyrosine Kinase 1/2 Inhibitor, in
Humans". Drug Metabolism and
Disposition38 (11): 2023.
2 Mesa, Ruben A.; Yasothan, Uma;
Kirkpatrick, Peter (2012). "Ruxolitinib".
Nature Reviews Drug Discovery11 (2):
103–4.
3 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.
4 Pardanani, A.; Tefferi, A. (2011).
"Targeting myeloproliferative
neoplasms with JAK inhibitors".
Current Opinion in Hematology18 (2):
1.
5 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-
Jamonline / 2(2); 2012 / 223–231 Satyanarayana PVV and Siva Madhavi A
All rights reserved© 2011 www.jamonline.in 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
Myeloproliferative Neoplasms". New
England Journal of Medicine366 (9):
844–846.
8 ASCO Annual Meeting 2011: JAK
Inhibitor Ruxolitinib Demonstrates
Significant Clinical Benefit in
Myelofibrosis
9 "FDA Approves Incyte's Jakafi(TM)
(ruxolitinib) for Patients with
Myelofibrosis" (Press release). Incyte.
Retrieved 2012-01-02.