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

www.jamonline.in 224

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



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



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



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



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



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



.

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):

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3 Mesa, RA (2010). "Ruxolitinib, a

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8 ASCO Annual Meeting 2011: JAK

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Retrieved 2012-01-02.

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A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF RUXOLITINIB IN FORMULATIONS