INTRODUCTION:
ABROCITINIB
Chemically
N-[3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl]propane-1-sulfonamide.
It is also be spelled as"Cibinqo".1-7 It is available in
the Indian market as tablet dosage form in the brand names Cibinqo.8
Abrocitinib is a medicine used to treat the symptoms of Atopic Dermatitis.
Abrocitinib belongs to a class of drugs called Dermatologics, Janus kinase
Inhibitors. It is Highly soluble in Dimethylsulfoxideas, Water.9
Mode of action Abrocitinib is associated with transient and usually mild
elevations in serum amino transferase levels during therapy, but has not been
linked to cases of clinically apparent acute liver injury.
Literature
survey reveals that only NIR method has been developed for estimation of
Abrocitinib so there is no spectroscopic method available to estimate either
from bulk drug or from the pharmaceutical dosage forms. The present
investigation was proposed to estimate Abrocitinib in bulk and pharmaceutical
dosage form by spectroscopic method which is rather simple, sensitive,
specific, precise, accurate method. The present work was to develop and
validate as per ICH guidelines so the method was developed validated according
to ICH guidelines for accuracy, precision, reproductivity, repeatability and
robustness.10
MATERIALS
AND METHODS:
Materials
and reagents:
A
gift sample of Abrocitinib from Ulterius Pharmaceuticals Private
Limited (Cibinqo200mg) film coated tablet dosage form bought from the local
market. Other chemicals like Dimethylsulfoxideas were bought from SD fine
chemicals, Mumbai, India.
APPARATUS
AND EQIUPMENTS REQUIRED:
1.
UV-Vis double beam spectrophotometer (Model; Shimadzu: 1700S, Japan), Electric
Sonicator, Volumetric flasks (10ml, 50ml, 100ml), Calibrated analytical
pipettes, Electronic digital balance (Techno, Mumbai)
Preparation
of concentration range (Beer’s limit):
Determination
of concentration range which obeys the Lambert and Beer’s law is necessary for
accuracy and reproducibility in Spectrophotometric analysis for quantitative determination
of any drug. For this; Abrocitinib stock solution (100µg/ml) was prepared using
pure drug in Dimethylsulfoxideas. Further dilutions were made using
0.2ml,0.4ml, 0.6ml, 0.8ml, 1.0ml, 1.2ml, 1.4ml, 1.6ml and 1.8ml of above
solution was transferred to a series of 10ml volumetric flasks this gave a
series of concentrations ranging from 2 to 18 µg/ml of Dimethylsulfoxideas. The
final volume was made up to 10ml mark using Dimethylsulfoxideas, sonicated for
5 minutes. The resultant solutions were measured using a double beam
uv-vis-spectrophotometer at wave length of 303.0nm against a reagent blank. A
calibration curve was plotted with concentration against absorbance. From the
graph it was clear that Beer’s law was obeyed in concentration range of 2-14µg/ml
and deviation was observed above these concentrations.
Preparation
of standard calibration curve:
100
mg of pure Abrocitinib drug was dissolved in little quantity of
Dimethylsulfoxideas in a 100 ml volumetric flask, the volume was made up to the
mark using Dimethylsulfoxideas. The solution was sonicated for 10 minutes. This
gave a Abrocitinib solution with concentration of 1mg/ml (1000µg/ml). 10ml of
this solution was further diluted 100ml in volumetric flask using
Dimethylsulfoxide as to obtain a concentration of 100µg/ml. Further dilutions
were made using 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8ml solution was
transferred to a series of 10ml volumetric flasks (to Obtain a series of
concentrations ranging from 2-14 µg/ml of Abrocitinib). The final volume was
made up to 10ml mark using Dimethylsulfoxideas, sonicated for 5 minutes, the
absorbances were measured at of 303.0nm against a reagent blank. A calibration
curve was plotted with concentration against absorbance.
Estimation
of Abrocitinib in tablet dosage forms:
Twenty
tablets were weighed accurately and powdered. The tablet powder equivalent to
100 mg of Abrocitinib was transferred into a 100ml volumetric flask and
dissolved in little quantity of Dimethylsulfoxideas. Then the solution was
sonicated for 30 minutes and filtered using whatman filter paper No#41. The
filtrate so obtained was diluted with Dimethylsulfoxideas to produce 100ml.
Further dilutions were made with Dimethylsulfoxideas to get required
concentrations within Beer’s - Lambert limits. The resultant solutions were
measured at wave length of 303.0nm against a reagent blank. The concentration
of drug was calculated with the help of standard calibration curve.
ANALYTICAL
METHOD VALIDATION:
Validation
of an analytical procedure is the process by which it is established, by
laboratory studies, that the performance characteristics of the procedure meet
the requirements for its intended use. All analytical methods that are intended
to be used for analyzing any clinical samples will need to be validated.
Validation of analytical methods is an essential but time consuming activity
for most analytical development laboratories. It is therefore important to wave
length. Understand the requirements of method validation in more detail and the
options that are available to allow for optimal utilization of analytical
resources in a development laboratory.
RESULTS
AND DISCUSSION:
Results
of determination of beer's limit:
Table:1
showing Beer’s range for Abrocitinib:
|
Sl.No.
|
Concentration
in µg/ml
|
**Absorbance
at λmax 303.0nm
|
|
1.
|
0.0
|
0.000
|
|
2.
|
2
|
0.085
|
|
3.
|
4
|
0.180
|
|
4.
|
6
|
0.270
|
|
5.
|
8
|
0.375
|
|
6.
|
10
|
0.470
|
|
7.
|
12
|
0.550
|
|
8.
|
14
|
0.640
|
|
9.
|
16
|
0.820
|
|
10.
|
18
|
1.000
|
(**
Average of three determinations)
Figure:1
showing Beer’s limit for Abrocitinib pure drug:
Standard
calibration curve of abrocitinib:
Table:2
Showing absorbance of Abrocitinib at various concentrations:
|
Sl.No.
|
Concentration in µg/ml
|
Absorbance at λmax 303.0nm
|
|
1.
|
0.0
|
0.000
|
|
2.
|
2
|
0.085
|
|
3.
|
4
|
0.180
|
|
4.
|
6
|
0.270
|
|
5.
|
8
|
0.375
|
|
6.
|
10
|
0.470
|
|
7.
|
12
|
0.550
|
|
8.
|
14
|
0.640
|
Figure
2: showing standard calibration curve for Abrocitinib:
Results
of determination of absorption maxima:
Figure
3: showing absorption maxima of Abrocitinib at 303.0nm:
Estimation
of abrocitinib intablet dosage form:
Table
3: showing absorption of drug from tablet dosage form:
|
Volume
of stock Solution Used
|
Amount
of drug (label claim) (µg/ml)
|
Absorbance
at 303.0nm
|
Amount
of drug found (µg/ml)
|
Percentage
purity found ±S.D** (%w/w)
|
|
0.2ml
|
2
|
0.1093
|
2.019
|
100.75±0.16
|
|
0.6ml
|
6
|
0.317
|
6.043
|
100.68±1.47
|
|
1.0ml
|
10
|
0.514
|
9.985
|
99.85±0.66
|
|
1.4ml
|
14
|
0.698
|
13.984
|
99.90±0.51
|
(**
Average of three determinations)
DETERMINATION
OF ACCURACY:
Table
4: Accuracy results for Abrocitinib:
|
Brands
|
Initial
amount
(µg/ml)
|
Amount
of pure drug
added
(µg/ml)
|
Amount
recovered
(µg/ml)
|
%
Recovery
±S.D**
|
|
|
10
|
8(80%)
|
8.031
|
100.37±0.147
|
|
CIBINQO
|
10
|
10(100%)
|
9.901
|
98.01±0.231
|
|
|
10
|
12(120%)
|
12.030
|
100.25±0.421
|
(**Average
of six determinations, n=6)
DETERMINATION
OF PRECISION:
Table
5: Precision results for Abrocitinib:
|
Sl.
|
Conc.
in(µg/ml)
|
Inter-day
absorbance
Mean±S.D**
|
%
C.V
|
Intra-day
absorbance
Mean±S.D**
|
%
C.V
|
|
1.
|
8
|
0.417±0.009
|
0.42
|
0.416±0.021
|
0.69
|
|
2.
|
10
|
0.516±0.030
|
0.41
|
0.516±0.025
|
0.30
|
|
3.
|
12
|
0.609±0.023
|
0.67
|
0.612±0.031
|
0.31
|
(**Average
of three determinations, n=3)
DETERMINATION
OF RUGGEDNESS PARAMETERS:
Table:6
Showing Ruggedness parameters:
|
Parameters
|
Laboratory
|
Name
of the instrument
|
Manufacturer
of the
chemicals
used
|
|
Lab. 1
with analyst
I
|
M.M.U.
College of Pharmacy, Ramanagara
|
Shimadzu-(model:
1700S,Japan) double beam UV-Vis spectrophotometer
|
S.D
Fine Chemicals,
Mumbai.
|
|
Lab. 2
with analyst
II
|
Dr. H.L.T.
College of Pharmacy, Kengal, Channapatna
|
Systronic
UV-Vis Double beam spectrophotometer
|
Loba Chemicals,
Mumbai
|
Table:7
Showing Ruggedness results for Abrocitinib:
|
Sl.
No
|
Brand
|
Label
claim(mg)
|
Lab.
1* with analyst I
|
Lab.
2* with analyst II
|
|
Amount
found(mg)
|
%Recovery±S.D**
|
Amount
found(mg)
|
%Recovery±S.D**
|
|
1.
|
CIBINQO
|
10
|
10.09
|
100.9±0.749
|
9.97
|
99.7±0.349
|
(Lab 1*
MMU College of pharmacy, Lab 2* Dr.HLT College of pharmacy,.**Average of six determinations,
n=6)
DETERMINATION
OF ROBUSTNESS:
Table
8 Showing Robustness results for Abrocitinib:
|
Type
|
Sl.
No.
|
Conc.
In
(µg/ml)
|
Change
in temperature
|
Change
in PH
|
|
+50C
|
-50C
|
2drops
of 0.1N NaOH
|
2drops
of 0.1N HCl
|
|
Absorbance
at 303.0nm Mean±S.D**
|
|
Pure
|
1
|
8
|
0.411±0.032
|
0.412±0.022
|
0.410±0.017
|
0.412±0.017
|
|
Drug
|
2
|
10
|
0.521±0.028
|
0.519±0.023
|
0.520±0.016
|
0.521±0.033
|
|
|
3
|
12
|
0.615±0.028
|
0.616±0.032
|
0.615±0.055
|
0.614±0.054
|
(**Average
of three determinations, n=3)
Table:9
showing calibration data for Abrocitinib at 303.0nm:
|
 Parameters
|
Calibration
data at 303.0nm
|
|
λmax
|
303.0nm
|
|
Beer’s
law limit (µg/ ml)
|
2
-14µg/ml
|
|
Molar
Absorptivity
|
1.1161Lmol-1cm-1
|
|
Regression
Equation(Y=a+bc)
|
Y=
0.055X+0.016
|
|
Slope
(b)
|
0.01426
to 0.01472
|
|
Intercept(a)
|
-
0.004520 to 0.0016757
|
|
Correlation
Coefficient (R2)
|
0.998
|
|
Limit
of detection (LOD)
|
1.226µg/ml
|
|
Limit
of quantitation (LOQ)
|
5.226µg/ml
|
Determination
of Beer’s limit:
The
Beer’s limit felled in the range of 2-14µg/ml under given experimental
conditions.
Determination
of absorption maxima:
100
µg/ml stock solution of Abrocitinib was prepared and absorbances were measured
from 200nm to 340nm. The optimum wave length was found to be 303.0nm
Assay:
Marketed
tablets contained Abrocitinib were used for the assay. After extraction, proper
dilution, measurement, the concentration was determined using standard
calibration curve. The amount of drug found in the range of 99.80 – 100.70%w/w
Method
validation:
The
proposed method was validated in accordance to ICH guidelines.
a)
Accuracy: Percentage
of recoveries of Abrocitinib in tablets was found in the range of 99.01 –
100.37% w/w
b)
precision:
The percent coefficient of variations (% C.V) was between 0.34-0.60 for
intra-day and 0.42-0.67 for inter-day absorbances.
c)
Repeatability: Repeatability was determined by analyzing the sample
at the given concentration wavelength for at least six times and it was found
that the variability in the results was not more than 0.5%.
d)
Reproducibility: The standard solution of Abrocitinib by analyst-I and
analyst-II separately. The values obtained were evaluated using F-test and
t-test to verify their reproducibility. Calculated value for t-test was found
to be less than the tabulated (standard) value it can calculated that no
significant difference was observed in the result of analysis.
e)
Ruggedness: Ruggedness of the developed method was determined by
changing the analytical toolssuch as laboratory, instruments, analyst and
chemicals. The result (in terms of %RSD) of six determinations indicated that
there were no significant variations in the data.
f)
Robustness: Robustness of the method was established by slightly
changing the temperature and PH of the reaction mixture. The data so obtained
showed no significant variation in the absorption pattern.
g)
Limit of detection and limit of quantitation: were determined
from the standard deviation of y – intercepts of six calibration curves and
average slope of six calibration curves. LOD and LOQ of Finerenonewas found to
be 1.226µg/ml and 5.226µg/ml respectively.
CONCLUSION:
A
new spectrophotometric method was developed to estimate Abrocitinib in pure and
tablet dosage forms. Dimethylsulfoxide as solutions of Abrocitinib was
estimated by using UV-spectrophotometer (Shimadzu 1700S, Japan) with matched
1cm quartz cell. It showed maximum absorption at the range of 2-14µg/ml at
303.0nm with coefficient of correlation (R2) of 0.998. The method so developed
was validated according to ICH guide lines for accuracy, precision (inter and
intra-day precisions), repeatability, reproducibility, ruggedness, robustness
etc.
The
proposed method was found to be simple, accurate, sensitive, precise,
reproducible and rapid.
This
method can be successfully employed for routine quantitative analysis of
Abrocitinib in bulk and tablet dosage form.
ACKNOWLEDGMENT:
The
authors are thankful to Head of the Department of Chemistry.
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