INTRODUCTION:

To develop and validate a simple, cheap, accurate, the rapid Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method is used as per ICH guidelines for estimation of Ruxolitinib in pharmaceutical dosage forms. Ruxolitinib .is use for secretion of inflammatory cytokines results in myeloproliferation, which is use for increased bone marrowfibrosis (BMF), and extramedullary hematopoiesis.¹

The past decade has witnessed considerable progress in the understanding of the cellular and molecular biology of MPNs, and this has recently resulted in the addition of the Janus kinase (JAK) 1 and JAK2 inhibitor ruxolitinib to our therapeutic armamentarium² Janus kinases (JAKs), consisting of JAK1, JAK2, JAK3,and Tyk2, are intracellular signaling enzymes that actdownstream of key proinflammatory cytokines, and each isknown to contribute to the pathogenesis of atopic dermatitis^3-10

Analytical Method validation:

High performance liquid chromatography Definition:^11-15

High Performance Liquid Chromatography which is also known as High Pressure Liquid Chromatography. It is a popular analytical technique used for the separation, identification and quantification of each constituent of mixture. HPLC is an advanced technique of column liquid chromatography. The solvent usually flows through column with the help of gravity but in HPLC technique the solvent will be forced under high pressures up to 400 atmospheres so that sample can be separated into different constituents with the help of difference in relative affinities

HPLC Theory and Principle:^8,9,10

Chromatography is an analytical method that finds wide application for the separation, identification and determination of chemical components in complex mixtures. This technique is based on the separation of components in a mixture (the solute) due to difference in migration rates of the component through a stationary phase by gaseous or liquid mobile phase. HPLC was derived from classical column Chromatography and has found an important place in analytical technique. Most of drugs in multi component dosage forms can be analyses by HPLC method because of the several advantages like rapidity, specificity, accuracy, precision and ease of automation in this method. The principle of separation in normal phase mode and reverse phase mode is adsorption. When mixtures of components are introduced into HPLC column, they travel according to their relative affinities towards the stationary phase. The component which has more affinity towards the adsorbent travels slower.

MATERALS AND METHOD:

Moble phase preparations:

Diluted Orthophosphoric acid: Accurately transfer 2.0ml of orthophosphoric acid into10ml volumetric flask and make volume up to the mark with water and mixed well.

Buffer preparation:

Accurately weigh 6.8gm of potassium dihydrogen phosphate in 1000ml of water and mixed well. Filtered the buffer solution with a 0.45µ PVDF membrane filter.

Buffer pH 3.5:

1000ml of above buffer was taken and pH was adjusted to 3.50 with a diluted ortho phosphoric acid solution (1.0ml):

Mobile phase A – Buffer pH 3.5(100%) line A

Mobile Phase B – Acetonitrile: MeOH (100%) line B 50:50 (V/V)

Methodology:

Selection of mobile phase:

For optimization of mobile phase, several trials were done to select the best mobile phase for the separation of Ruxolitinib with an effective resolution from its degradant impurity and peak symmetry of Ruxolitinib peak. The trials for the selection of mobile phase are shown in table

Standard solution 1:

Weigh accurately about 66mg of Ruxolitinib phosphate standard and transfer into a 50ml volumetric flask. Add 35ml of Diluent and sonicate to dissolve it. Diluent to volume with diluent and mix well.

Standard solution:

Pipette 5.0ml of sample solution 1 and transfer into a 50ml volumetric flask. Dilute to volume with diluent and mix well.

Sample Solution 1:

Carefully weigh 20 Tablets and Determine average weight Take 20 Tablets and crush in mortal pestal and prepared fine powder fine powder. Mix the content homogeneously prior to weighing. Weigh accurately equivalent to about 66mg of Ruxolitinib and transfer into a 50ml volumetric flask (with swirling) containing about 35ml of Diluent, and sonicate for about 30minutes with intermittent shaking. Maintain the temperature of ultrasonic bath at room temperature. Cool to room temperature and diluent to volume with Diluent, mix well

Sample solution:

Pipette 5.0ml of sample solution-1 and transfer into a 50ml volumetric flask. Dilute to volume with diluent and mic well. Filter the solution through 0.45μm PVDF membrane syringe filter. Discard the first 5ml of filtrate.

Chromatographic Conditions:

Column: zorbax SB(Stable bond) phenyl (150×4.6) mm 3.5µm

Wavelength – : 310nm

Column temperature - : 35 ºC

sample cooler temperature : 4^º C

injection volume - : 10µL

Rinsing line : Methanol (100%)

Elution type : Gradient

Flow rate – : 1.0ml/ min

Run time - : 15min

Gradient Program :

Table no 1: Mobile Phase gradient Ratio

Time	Mobile phase A	Mobile phase B
0.0	100	0
2.0	30	70
5.0	30	70
8.0	30	70
10.0	100	0
15.0	100	0

EVALUTION PARAMETERS^16;17

Method Validation:

Authentification of the investigative method is the process that starts by laboratory studies in which the requirements of the performance properties of method are met for the intended analytical application. To the validation of analytical procedures RP-HPLC method developed was validated according to International Conference on Harmonization (ICH) and USP guidelines. Various parameters or criteria are used for the method of validation, such as linearity, accuracy, precision, limit of Quantification (LOQ) and limit of Detection (LOD).

Linearity:

Linearity the method was tested from 80-120% of the targeted level of the assay concentration for analyte. Standard solutions contained 2-12μg/mL Ruxolitinib

Linearity solutions were injected in triplicate. The equations of the calibration curves for Montelukast sodium obtained were y = 02E+06X in the Ruxolitinib determination, the calibration graphs were found to be linear in the aforementioned concentrations with correlation coefficients 0.9998.

Accuracy:

The precision was determined with the help of recovery experiments, by the verification with percentage mean recovery of sample at three different levels (50-150%). 20 blank tablets were powdered and mixed. This powder was then spiked with a quantity of Ruxolitinib corresponding to 50%, 100% and 150% of the labeled claim.

Precision:

System precision:

The value for %RSD (Relative Standard Deviation) was obtained less than 2 by six replicate injections of the standard solution at working concentrations, concerning the peak area for the drug. It designates the adequate reproducibility and hence the accuracy of the system.

Method precision:

The accuracy of procedure was determined by conducting assay of sample with the tests of (I) Repeatability (Intraday precision) and (II) Intermediate precision or ruggedness (Interday precision) completed within 3 successive daysbythree different analysts, at working concentration.

Repeatability (Intraday precision):

The value for %RSD (Relative Standard Deviation) less than 2 for six successive injections of the sample solution from the same homogenous mixture at working concentrations, concerning % assay for understood that the method gives consistently reproducible results the drug which indicate that the method developed is method precise by the test of repeatability.

LIMIT OF DETECTION (LOD):

The detection limit of an specify analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. The limit of detection may be expressed as:

DL = 3.3×σ/S

Where,

DL = The standard deviation of the response

S = The slope of the calibration curve

The estimation of LOD may be carried out in multiple ways. LOD can calculate from the linearity correlation equation. The slope S may be estimated by calibrated curve.

LIMIT OF QULIFICATION (LOQ):

Several approaches for determining the quantitation limit are possible, depending on whether the procedure is non-instrumental or instrumental. LOQ may be expressed as:

QL=10×σ/s

Where,

σ = Response of standard observation

S = The slope of the calibration curve

The slope S may be estimated from the calibration curve of the analyte.The estimate of σ may be carried out in a variety of ways. LOD can calculate from the linearity correlation equation.

RESULT AND DISCUSSION:

Figure no. 1: Diluent Chromatogram of Ruxolitinib Contractions (132μg/ml

Figure no. 2: Standard Chromatogram of Ruxolitinib Contractions (132μg/ml)

Figure no. 3: Sample Chromatogram of Ruxolitinib Contractions (132μg/ml)

Table no. 2: Result of Sample Chromatogram of Ruxolitimb Contractions

Solution	Mean peak area	%RSD
Standard	2349764	0.11
Sample	2292161	0.49

Table No 3: Linearity Data of Ruxolitinib

Concentration of RUXOLITINIB (µg/ml) (x)	Area (y)
25.415	560868
50.829	1122142
81.327	1796699
101.659	2276371
152.488	3338381

Table No 4: Data of regression analysis of Ruxolitinib

Drug

Straight line equation of calibration curve

Correlation coefficient (r)

Ruxolitinib

Y=21936.2900+12577.8888

0.9996

Acceptance Criteria:

The correlation co-efficient (r) value should not be less than 0.9996 over the working range.

Table No 5: Precision Repeatability:

Set No.	Area	% Assay
Set-1	2253624	100.9
Set-2	2252698	100.8
Set-3	2251469	100.7
Set-4	2250469	100.8
Set-5	2235625	100.3
Set-6	2226548	99.8
Mean	2245072.16	100.55
% Standard Deviation	11258.11	0.4230
%RSD	0.5	0.4

Acceptance Criteria: % RSD of values of six sample preparation should not be more than 2.0%

Table No. 6: Inter day Precision

Sr No.	Area	%Assay
Set -1	2256452	101
Set-2	2254896	101
Set-3	2247692	101.2
Set-4	2246859	100.6
Mean	2251474	100.94
% Standard Deviation	4902.11	0.2516
% RSD	0.22	0.25

Acceptance Criteria:

% RSD of values of sample preparation should not be more than 2.0%. Cumulative RSD value of precision and Interday precision should not more than 2.0%

CONCLUSION:

The %RSD value for Ruxolitinib is Well within the limit of acceptance criteria and % cumulative RSD value of precision and interday precision is well within the limit of acceptance criteria that means the method was precise for intended use for tiopronin tablets assay in market formulation

Accuracy:

Table no 7: Accuracy Data

Accuracy level	Test amount	Drug taken	Spiked standard amoount	Total amount recover in	% recovery
50%	132	66	198	197.490	99.74
100%	132	132	264	263.612	99.85
150%	132	264	396	395.164	99.78

Calculations:

% Assay of Ruxolitinib=

AuAs×w150×550×50w2×505×P100×100

96.924%

A_u= Average peak area of Ruxolitinib in sample solution

A_s=Average peak area of Ruxolitinib in standard solution

W1=Standard weight of Ruxolitinib

W2=Sample weight of Ruxolitinib

P= Potency of Ruxolitinib

Acceptance criteria: NLT 90% and NMT 105%

SUMMARY:

Table No. 8: Result Parameters

Sr No	Parameter	Result
1	Specificity	All the peaks were separate and identical
2	Regression equation	Y=21936.2900x+12577.8888
3	correlation coefficients (R)²	0.9996
4	Repeatability (%RSD	0.25%
5	Inter day Precision (%RSD)	0.4%
6	Range (µg/ml)	25.415-152.488 µg/m
7	Assay	96.924%
8	Accuracy (%Recovery)	99.74 -99.85%

CONCLUSION:

Based on the empirical evidence, the present method was strongly claimed about the novelty of the developed method over the reported methods. This is the first stability-indicating method, which is ‘rapid’ because it significantly reduced the total analysis time which is the lowest analysis time required. The method justifies “easy” because the proposed method does not involve use of dual-wavelength, gradient techniques. The present method is “stability indicating” as this has been shown less degradation pattern in stressed conditions and good separation of ruxolitinib among the other degraded peaks. The method considers “validated” because all the results of validation parameters were found within the limits as per the ICH Q2B guidelines. Hence the present developed method can be designate as reliable, validated and highly useful for the routine analytical and quality control study of the ruxolitinib in the tablet dosage form.

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Received on 01.09.2023 Modified on 11.01.2024

Research J. Pharm. and Tech 2024; 17(7):3040-3044.

DOI: 10.52711/0974-360X.2024.00475