Volume 5, No. July 7, 2024
p ISSN 2723-6927-e ISSN 2723-4339
Stability and
Development of the Ruxolitinib Estimation Method
Using RP-HPLC
Dila Wahyu
Utami1*, Supriyadi2, Iswandi3
Setia Budi University Surakarta, Indonesia1*23
Email: [email protected]
ABSTRACT
Ruxolitinib is a Janus Kinase 1 and 2 inhibitor drug that
was designated by the Food and Drug Administration (FDA) in 2011 as a treatment
for myelofibrosis with moderate to high risk. Ruxolitinib
has instability to light that can cause a decrease in levels. The aim of this
research is to obtain a selective, accurate and precise analytical method to
determine levels and determine the stability of ruxolitinib.
Determination of optimum RP-HPLC conditions from the development of analytical
methods with variations in mobile phase composition, flow rate, buffer
concentration and buffer pH. The new analytical
method determines ruxolitinib levels by RP-HPLC using
a C18 Hypersil ODS column, wavelength 310 nm, mobile
phase composition acetonitrile: H2O: citrate buffer (75:20:5), injection volume
20 μL, 0.10 M citrate buffer and pH 5.8.
Calibration curve between ruxolitinib concentrations
in the range of 1000-1800 μg/mL, correlation
coefficient 0.9997. Accuracy 99.65%, repeatability precision 0.17%, interday precision 0.36%, LOD 26.48 μg/ml,
and LOQ 88.26 μg/ml. The decrease in ruxolitinib levels after exposure to acids was 82.56%,
bases were 88.01%, light was 69.06%, temperature was 75.29%. So
it was concluded that the method resulting from method development and
validation met the validation criteria, determining ruxolitinib
levels showed exposure to unstable acids, bases, temperature and light.
Keywords: ruxolitinib, method development, method validation,
stability, degradation, concentration
INTRODUCTION
Ruxolitinib belongs to the class of Janus Kinase (JAK) 1 and JAK 2 inhibitor drugs
which function to activate Signal Transducers and Transcription Activators
(STATs) which results in the activation of the JAK STAT signal and has an
impact on cell differentiation, proliferation and survival (Gerson et al.,
2018). In 2011 the Food and Drug
Administration (FDA) designated ruxolitinib as a treatment for moderate to high
risk myelofibrosis (Mascarenhas &
Hoffman, 2012).
The types
of myelofibrosis included in ruxolitinib treatment include primary
myelofibrosis (PMF), post-polycythemia vera myelofibrosis (post-PV MF) and
essential thrombocythemia myelofibrosis (post-ET MF) (Altomare &
Kessler, 2019). The active substance ruxolitinib is
reported to have instability to light which is characterized by changes in the
color of the active substance (Salmonson &
Hemmings, 2012). Information on the stability of a
medicinal product is important to know because drug instability can result in a
decrease in the levels of active substances and it is feared that toxic
products will arise during decomposition such as degradant products (Aashigari et al.,
2019).
Drug degradation can also be
influenced by the environment of the drug product and drug formulation which
influence the mechanism and speed of degradation (Loftsson, 2014b). The degradation pathway has several pathways, namely, hydrolysis,
oxidation, isomerization, photochemical degradation, decarboxylation,
dehydration and polymerization pathways (Loftsson, 2014a). Testing the stability of an active substance or medicinal
product uses forced degradation, namely using forced degradation of acids,
bases, temperature and light. The results of the degradation test until the
degraded product reaches approximately 20% by determining the content (Lakka, Narasimha S. Kuppan, 2020).
Until now,
ruxolitinib levels have been determined using HPLC instruments. The use of HPLC
has the advantage of good analysis speed, resolution and sensitivity (Suzanne Suzanne
Nielsen, 2017). Previous research used
tetrahydrofuran (THF) and phosphate mobile phases as buffers (Satyanarayana &
Madhavi, 2012)(Charlier et al.,
2019)(Biswal et al.,
2019). Tetrahydrofuran has toxic properties
and the use of phosphate buffers can cause abrasive effects on pump seals. Use
of phosphate buffers exceeding pH 7 can shorten column life (Agrahari et al.,
2013).
Based on
this, to overcome the weaknesses of the previous method, the method for
determining ruxolitinib levels and the HPLC method
for stability testing were used. Method development and validation of HPLC
methods with variations in mobile phase, flow rate, buffer concentration,
buffer pH. The results of development and validation
will be used if they meet the ICH validation criteria, linearity with a square
correlation coefficient ≥ 0.98, precision RSD value < 2.0% and
accuracy of percent return value 98-102% (GHT ICH, 2005).
RESEARCH METHODS
Material
Ruxolitinib was obtained from Dingmin
Pharmaceutical of South Korea.
Preparation of
citrate buffer
Sodium
citrate weighed 2.94 grams and citric acid weighed 2.10 grams each dissolved in
100 ml and stirred until it reached pH 5.8.
Standard ruxolitinib preparation
100
mg of ruxolitinib standard was dissolved in methanol in a 50 ml volumetric
flask. Take 5 ml of the solution and put it in a 10 ml measuring flask and add
methanol to the tera mark.
Wavelength Search
Ruxolitinib solutions with
three different concentrations were examined in the UV region in the range of
200-400 nm and using methanol as a blank on a UV-Vis spectrophotometer.
Sample Solution
Preparation
10
mg of ruxolitinib powder was weighed and dissolved in
a 10 ml volumetric flask with methanol solvent.
Chromatographic
Conditions
The
method used was a C18 Hypersil ODS column 250mm x 4.6 mm particle size 5μm
with isocratic acetonitrile: H2O: citrate buffer pH 5.8 (75:20:5). Flow rate
1.0 ml/min, wavelength 310 nm and injection volume 20μl.
Linearity
The
ruxolitinib standard solution was prepared in five
concentration series from the standard ruxolitinib
stock solution in the range of 1000-1800 μg/ml
and then the values were processed to obtain the linear
regression equation and correlation coefficient.
Accuracy
Determination
of accuracy using different drug additions of 80%, 100% and 120%. An 80%
concentration was prepared by dissolving 8 mg ruxolitinib
in methanol in a 10 ml volumetric flask. The 100% test solution was prepared by
dissolving 10 mg ruxolitinib in methanol in a 10 ml
volumetric flask. The 120% test solution was prepared by dissolving 12 mg ruxolitinib in methanol in a 10 ml volumetric flask. Values
are calculated to obtain an assessed recovery percentage.
Precision
Precision
testing is determined using repeatability and medium precision methods.
Replication was carried out six times with a concentration of 1000 μg/ml. Intermediate precision was performed on
different days and values were tested against RSD values
(RSD < 2.0%).
LOD and LOQ
LOD
and LOQ tests are determined based on a calibration curve with several test
solution concentrations, namely 1000, 1200, 1400, 1600 and 1800 μg/ml. Values are created from the
calibration curve to obtain the values of σ (standard
deviation of response) and S (slope of the calibration curve) which are
estimated from the analyte regression line.
Robustness
The
endurance test used deliberate variations with varying changes in flow rate
(0.8 ml/minute and 1.2 ml/minute) and wavelength (308 and 312 nm). Retested
values are percent and RSD (RSD < 2.0%).
Forced
degradation of acids
The
forced acid degradation test used 1000 ppm ruxolitinib solution with the
addition of 1 ml of 0.1 M HCl, refluxed for 60 minutes at 70�C and reduced to
room temperature. Processing degradation test data to obtain the percent
reduction/loss of ruxolitinib levels by dividing the sample area after exposure
by the area before exposure multiplied by 100%. LOD and LOQ testing is
determined based on a calibration curve with several test solution
concentrations, namely 1000, 1200, 1400, 1600 and 1800 μg/ml. Values
are created from the calibration curve to obtain the values
of σ (standard deviation of response) and S (slope of the
calibration curve) which are estimated from the analyte regression line.
Robustness
The
endurance test used deliberate variations with varying changes in flow rate
(0.8 ml/minute and 1.2 ml/minute) and wavelength (308 and 312 nm). Retested
values are percent and RSD (RSD < 2.0%).
Forced
degradation of acids
The
forced acid degradation test used 1000 ppm ruxolitinib solution with the
addition of 1 ml of 0.1 M HCl, refluxed for 60 minutes at 70�C and reduced to
room temperature. Processing degradation test data to obtain the percent
reduction/loss in ruxolitinib levels by dividing the sample area after exposure
by the area before exposure multiplied by 100%.
Forced
degradation of bases
The
forced base degradation test used 1000 ppm ruxolitinib solution with the
addition of 1 ml of 0.1 M NaOH, refluxed for 60 minutes at 70�C and reduced to
room temperature. Processing degradation test data to obtain the percent
reduction/loss in ruxolitinib levels by dividing the sample area after exposure
by the area before exposure multiplied by 100%.
Temperature
forced degradation
Temperature
forced degradation test using 1000 ppm ruxolitinib solution and kept in an oven
at 105�C for 60 minutes. Processing degradation test data to obtain the percent
reduction/loss in ruxolitinib levels by dividing the sample area after exposure
by the area before exposure multiplied by 100%.
Forced
degradation of light
The
forced degradation test uses a light ruxolitinib solution of 1000 ppm and is
exposed to a UV chamber for 60 minutes or 200 watt
hour/m2 in a photo stability chamber. Processing degradation test data to
obtain the percent reduction/loss in ruxolitinib levels by dividing the sample
area after exposure by the area before exposure multiplied by 100%.
RESULTS AND DISCUSSION
Wavelength
Selection
Determination of
the wavelength used is 2.5 ppm, 5 ppm and 10 ppm in the wavelength area of
200 � 400 nm (Table 1). At several different concentrations it
shows the same wavelength, namely 310 nm.
Table 1. Determination of
wavelengths with different concentrations.
|
Ruxolitinib Concentration (ppm) |
Wave Length (nm) |
Absorbance |
|
2.5 5 10 |
310 |
0.71317 |
|
310 |
0.43316 |
|
|
310 |
0.25037 |
Chromatographic
Conditions
Several
variations were used to obtain the optimal method, mobile phase composition,
flow rate, buffer concentration and pH. Variations in
flow rate (0.8 ml/minute, 1.0 ml/minute and 1.2 ml/minute), citrate buffer
concentration (0.05 M, 0.10 M and 0.15 M) and buffer pH (3.8
; 4.8 and 5.8).
Results of the
development of a method for determining ruxolitinib levels using a C18 Hypersil
ODS column, flow rate 1.0 ml/minute, mobile phase composition acetonitrile:
H2O: citrate buffer (75:20:5), injection volume 20 μl, wavelength 310 nm,
buffer concentration 0.10 M and pH 5.8. Determination of ruxolitinib levels
showed a retention time of 3,348 minutes. The tailings factor result is 1, the
theoretical plate is 4483.6 and the HETP is 0.0557.
Linearity
The ruxolitinib
linearity test uses five graded ruxolitinib series test solutions, namely
ruxolitinib standard stock solutions of 1000, 1200, 1400, 1600 and 1800 ppm.
The results of the ruxolitinib linearity test curve can be seen in (Figure 1),
showing the linear regression results of y = 25.711x + 4923.4, R� value of
0.9995, r value of 0.9997.
Figure 1. Graph of linearity test results
Accuracy
The accuracy test
used three concentrations with three repetitions, each concentration having a
percent return of 80%, a concentration of 99.46%, a percent return of 100% of
99.65% and 120%, a concentration of 99.65% with an average of 99.65% .
Precision
The repeatability
precision test results obtained an RSD value of 0.17% and the medium precision
test obtained an RSD value of 0.36%.
LOD and LOQ
The LOD test
results were 26.48 ppm and LOQ was 88.26 ppm.
Robustness
The results of
the resistance test can be seen in (Table 2) and this variation is expected to
return the assessed percentage and the %RSD is not much different from before
the variation was given.
Table 2. Robustness Test Results
|
Variation |
Recovery (%) |
RSD (%) |
|
Flow rate 0.8 ml/min Flow rate 1.0 ml/min Flow rate 1.2 ml/min λ 308 nm λ 310 nm λ 312 nm |
99.78 |
0.01 |
|
101.17 |
0.02 |
|
|
99.01 100.82 101.17 101.18 |
0.01 0.01 0.02 0.01 |
Forced
degradation of acids
The results of
the forced acid degradation test resulted in a reduction in ruxolitinib levels
of 82.56% and the chromatogram image can be seen in (figure 2).
Figure 2. Chromatogram of forced acid
degradation test
Forced
degradation of bases
The results of
the forced base degradation test resulted in a reduction in ruxolitinib levels
of 88.01% and the chromatogram image can be seen in (figure 3).
Figure 3. Chromatogram of forced base
degradation test
Temperature
forced degradation
The results of
the forced temperature degradation test resulted in a reduction in ruxolitinib
levels of 75.29% and the chromatogram image can be seen in (figure 4).
Figure 4. Forced temperature
degradation test chromatogram
Forced
degradation of light
The results of
the force of light degradation test resulted in a reduction in ruxolitinib
levels of 69.06% and the chromatogram image can be seen in (figure 5).
Figure 5. Forced light degradation
test chromatogram
Discussion
The
development of a method for determining ruxolitinib levels using an HPLC
instrument begins with determining the wavelength of ruxolitinib, namely a
wavelength of 310 nm. variations in chromatography test conditions to obtain a
good and efficient test method. A C18 Hypersil ODS column was used, wavelength
310 nm, mobile phase consisting of acetonitrile: H2O: citrate buffer (75:20:5),
injection volume 20 μl, flow rate 1ml/minute, citrate buffer concentration
0.10 M and pH 5 .8 obtained ruxolitinib retention time of 3,348 minutes. And
giving a theoretical plate value of 4483.6 (>2000), HETP value of 0.0557 and
a tailings factor of 1 (< 2) which shows that this value is included in the
requirements(Moldoveanu & David, 2013).
The
method is validated for testing to determine the validity of the method
obtained. The linearity test of the method was determined using several
concentrations and produced a correlation coefficient close to 1, thus
indicating the method(GHT ICH, 2005). Accuracy and precision validation tests provide an average
percent return value in the range of 98-102%, thus indicating an accurate
method and a repeatability value expressed in % RSD of no more than 2.0%
indicating an acceptable method(GH ICH, 2022). Limit of detection (LOD) and limit of quantitation (LOQ)
indicate the sensitivity of the developed method. Providing a variety of
methods to determine the accuracy of the method is indicated by a % RSD result
of no more than 2.0% which can be interpreted as a strong method and does not
provide a significant influence(GH ICH, 2022).
The
results of the ruxolitinib stability test showed that light exposure caused the
highest reduction in levels compared to other exposures. This decrease in
levels can form new forms of decomposers according to the exposure given.
CONCLUSION
The ruxolitinib
analysis method uses HPLC, namely using a C18 hypersil ODS column, at a
wavelength of 310 nm, mobile phase composition acetonitrile: H2O: citrate
buffer (75: 20: 5), injection volume 20 μl, buffer concentration 0.10 M
and pH 5, 8. This method meets the method validation criteria according to ICH.
Ruxolitinib with the influence of acid reduces levels by 82.56%, the influence
of base by 88.01%, the influence of temperature by 75.29% and the influence of
light by 69.06%.
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(2024) |
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