INTRODUCTION:

Ruxolitinib is chemically (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d] pyrimidin-4-yl) pyrazol-1-yl] propanenitrile. (fig 1.)¹Ruxolitinib is a medication for the treatment of intermediate or high-risk myelofibrosis, a type of myeloproliferative disorder that affects the bone marrow; polycythemia vera (PCV), when there has been an inadequate response to or intolerance of hydroxyurea; and steroid-refractory acute graft-versus-host disease. It was developed and marketed by Incyte Corp in the US under the brand name Jakafi, and by Novartis, under the brand name Jakavi. Ruxolitinib is a janus kinase inhibitor.²

Ruxolitinib is associated with transient and usually mild elevations in serum aminotransferase during therapy and to rare instances of self-limited, clinically apparent idiosyncratic acute liver injury as well as to cases of reactivation of hepatitis B in susceptible individuals³Janus kinases (JAKs), consisting of JAK1, JAK2, JAK3, and Tyk2, are intracellular signaling enzymes that act downstream of key proinflammatory cytokines, and each is known to contribute to the pathogenesis of atopic dermatitis^4-11. Recent studies indicate that JAK mediates signaling underlying sensory nerve fber function and may mediate pruritic efects^12-13. Therefore, JAK inhibitors for topical application (e.g., delgocitinib, tofacitinib, oclatinib, and ruxolitinib) have garnered interest as potential therapeutics for atopic dermatitis^14-17. Ruxolitinib is a selective inhibitor of JAK1 and JAK2 that is approved as an oral formulation for the treatment of patients with myeloproliferative neoplasms, including myelofbrosis, polycythemia vera, and steroid-refractory acute graft-versus-host disease ¹⁸.

In the last two decades, a series of important studies dealing with the dosage of Ruxolitinib (among other tyrosine kinase inhibitors) in biological samples for pharmacokinetics and therapeutic drug monitoring evaluations were published. In all these studies, highly performing procedures of sample treatment and quantitative analysis were developed and described in detail. In these investigations, the quantification of Ruxolitinib was performed by relying upon reversed-phase (RP) liquid chromatography analysis with different detection systems (including fluorescence, mass spectrometry). In all of the cases, assay validation procedures were performed according to international recommendations^19-22.

Fig. 1 Chemical structure of Ruxolitinib

The HPTLC method provides accurate and precise results which are comparable to that of liquid chromatographic method. Reduced sample preparation methods, less analysis time, and small quantity of mobile phase required are some of its advantages over liquid chromatography. Densitometric scanning used in HPTLC for quantitative analysis offers advantage of accuracy, precision, and specificity over conventional methods used in TLC. Stability and degradation samples can also be analyzed using a densitometer. HPTLC has become part of many pharmacopoeial monographs for the estimation of the drug and impurities²³. FDA prescribed methods must have a stability-indicating nature which helps to identify the possible stability issues related to the drug and the degradation pathway.

So, the present study involves development and validation of the stability-indicating high-performance thin-layer chromatographic method for the estimation of Ruxolitinib in tablet formulation²⁴To date, several methods are available in the literature to evaluate the concentration of Ruxolitinib in pharmaceutical forms²⁵ and in plasma samples²⁶, which are mainly based on the use of liquid chromatography such as HPLC coupled to mass spectrometry (LC–MS/MS) along with HPTLC and UV spectroscopy.²⁷

The purpose of this research was to develop and validate a stability-indicating simple, precise, sensitive high performance thin layer chromatography method for Ruxolitinib in its bulk and tablet dosage forms, and to validate it by International Conference on Harmonization (ICH) guidelines.²⁸

MATERIALS AND METHODS:

Instrumentation:

For the chromatographic study, an HPTLC system (CAMAG, Switzerland) with a Linomat V autosampler connected to a nitrogen cylinder, a twin trough chamber (20 10cm), a derivatization chamber, a plate heater, TLC Scanner IV (Camag Muttenz, Switzerland), UV cabinet with dual-wavelength UV lamps, and vision` CATS software was used. All weighing was performed using an electronic analytical balance (Shimadzu AUX-220).

Chemicals and reagents:

HPLC grade water, methanol, and acetonitrile were purchased from Merck (Darmstadt, Germany). Working standard of Ruxolitinib was received as a generous gift from Novartis Pharmaceuticals Pvt. Ltd., India. The purity of working standard was found to be 99.9%. Jakavi® tablets were purchased from local pharmacy shop labelled to contain 20 mg Ruxolitinib.

Chromatographic conditions:

The HPTLC analysis was performed on Pre-coated Silica Gel 60 F₂₅₄ HPTLC plates (20 × 10 cm, layer thickness 0.2 mm. HPTLC plates were prewashed with 10 mL of methanol and activated at 80ºC for 5 min before application of sample. The standard and formulation samples of Ruxolitinib were spotted using a Linomat 5 auto sampler fitted with a 100 µl Hamilton syringe (Hamilton, Bonaduz, Switzerland) and operated with settings of a band length of 3.5 mm; band distance of 7.2 mm; distance from the side of plate of 10mm; and distance from the bottom of the plate of 10mm. The plates were developed to a distance of 70mm in a mobile phase consisting of Chloroform: methanol: Formic acid (8.0:2.0:0.05 v/v/v) and development was carried out in twin trough chamber (20 x 10cm) presaturated with the mobile phase saturation time up to 10 minutes. Ruxolitinib separation by densitometric measurement was found at 236nm.

Preparation of Standard stock solution:

Ruxolitinib standard stock solution was prepared by dissolving 10mg of drug in 10.0ml of methanol to get a concentration of 1000g/ml. Ruxolitinibwas then diluted up to 10ml methanol to get working standard solution 100µg/mlfrom the stock solution, 2, 4, 6, 8 and 10 were applied on the TLC plate, at a distance of 10mm from both the x- and y-axis.

Validation of Analytical method:

The analysis method was validated according to the recommended guidelines of the ICH parameters include linearity²⁹, accuracy³⁰, limit of detection, limit of quantitation, specificity, intraday and interday precision, repeatability of peak area measurement, and repeatability of sample application. For the quantification of Ruxolitinib bulk and marketed formulations, a solvent system that would provide dense and compact spots with appropriate and significantly different Rf values was required. The mobile phase is a mixture of chloroform, methanol, and formic acid (8.0:2.0:0.05 v/v/v). We discovered that this system gave compact spots for Ruxolitinib (Rf value of 0.71±0.02). We prepared a stock solution Ruxolitinib (100μg/ml) in methanol³¹. Different volumes of stock solution as 1, 2,3,4,5, and 6μl were spotted on TLC plate to obtain concentration of 100, 200, 300, 400, 500 and 600ng/band of Ruxolitinib, respectively. The data of peak area versus drug concentration were analyzed by linear least-square regression. This drug responded linearly in the concentration range 200-600ng/band.

Method validation:

Linearity- A feature of the analytical process is the ability to obtain witness results (within a certain range), which are directly proportional to the analytical intensity (quantity) of the sample. Linearity was assessed by observing the visual meaning of the plot in the form of material or analytical function^.32

Specificity- Specificity is the ability of the analytical method to distinguish between the analytes and the other components in the sample matrix. Specificity is the study of the chromatographic method is performed by the separation of the analyte form the other potential components such as impurities, degradants or excipients^.33

Accuracy: It is analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. Accuracy should be assessed using minimum of nine determination over of three concentration levels covering the specified range. The acceptance criteria for accuracy are the relative standard deviation (%RSD) for all recovery values should not be more than 2%.³⁴

Precision- the accuracy of an analysis procedures for detecting proximity of agreement (distribution rate) between a string of measurements taken from multiple samples from the same homogeneous sample below specified conditions. Three levels were considered: repetition, average accuracy and reproducibility^.35

Limit of Detection (LOD) - The limit of detection of an individual analytical procedure is the lowest amount of analyte in the sample which can be detected but not necessarily quantified as an exact value.

LOD = 3.3 σ/S

σ – the standard deviation of X- intercept

S- the slope of the calibration curve³⁶

Limit of Quantification (LOQ) – The quantification limit of an individual analytical procedure is the lowest amount of analyte in the sample which can be quantitatively determined with suitable.

LOQ = 10σ/S

Were, σ- the standard deviation of the response

S- the slope of the calibration curve³⁷

Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Six samples’ solutions were prepared and analyzed under the established conditions and the various analytical parameters like flow rate of mobile phase, mobile phase composition and pH.³⁸

RESULTS AND DISCUSSION:

To develop HPTLC method of analysis for ruxolitinib for routine analysis, selection of mobile phase was carried out on the basis of polarity. A solvent system that would give dense and compact spots with appropriate and significantly different Rf value for ruxolitinibwas desired. The method was validated as per ICH guidelines. The optimised chromatogram is shown in Figure 2.

Figure 2: HPTLC chromatogram of marketed formulation

Linearity:

Linearity of the method was evaluated by constructing calibration curves at six concentration levels. Calibration curves were plotted over a concentration range of 100–600ng/spot. Aliquots of standard working solution of Ruxolitinib were applied to the plate (1, 2, 3, 4, 5, and 6 µL/spot). The calibration curves were developed by plotting peak area versus concentrations (n = 6) with the help of the vision CATS software. The calibration curve for this drug is illustrated in fig.3.

Fig. 3. Calibration curve of Ruxolitinib

Precision:

A study of intra-day and inter-day variation demonstrated the precision of the method. In the intraday studies, three different concentrations of standard stock solution were spotted in triplicate and analyzed.³⁹ The percentage RSD was calculated and is shown in Table 1.

Table No.1: Precision studies

Intraday study			Inter day study
Drug	Concentration in ng/spot	% RSD*	Drug	Concentration in ng/spot	% RSD*
RTB	100	0.78	RTB	100	1.17
	200	0.61		200	0.96
	300	1.02		300	1.01

Accuracy

In order to test the accuracy of the method, recovery studies were conducted by over spotting standard drug solution to reanalysed sample solution at three different levels: 80, 100 and 120%. We spiked 80, 100 and 120 ng/band of Ruxolitinib on synthetic mixtures with a basic concentration of 300ng/band. These areas were noted after development of the plate. The Ruxolitinib concentration was calculated by using regression equation. The results are shown in Table 2.

Table No. 2. Recovery Studies of Ruxolitinib

Level of recovery	Drug	Amount of drug applied ng/spot	% Recovery	S. D.	% RSD*
80 %	RTB	80	100.01	0.95	0.93
100%	RTB	100	99.13	0.54	0.58
120%	RTB	120	99.82	0.98	0.96

Limit of Detection (LOD): 3.3 σ

The formula for calculating LOD is --------

σ = the standard deviation of the response for the lowest concentration in the range

S = the slope of the calibration curve.

Limit of Quantification (LOQ): 10 σ

The quantitation limit (QL) may be expressed as ------

Densitograms were analyzed for interference with Ruxolitinib. Absence of interference peaks in the blanks at the Rf of Ruxolitinib was taken as a confirmation that the method was specific. Standard and sample spectra at the same Rf matched exactly, indicating no other interference at that Rf. The summary of validation parameters of the proposed HPTLC method is given in Table 3.

Table 3: Summary of validation parameters

Parameter	RTB
Linearity range in ng/spot	100 - 600
Regression coefficient	0.998
Intercept (c)	6.282
Slope (m)	1360
Limit of detection in ng/spot	8.19
Limit of quantification in ng/spot	25.06
Retention factor	0.71 ± 0.02

Robustness:

In order to determine the robustness of the method, the analysis was conducted during which mobile phase ratio, dimensions of chamber were altered and the effects on the Rf values and area were noted. The mobile phase ratio and dimensions of the chamber were changed, and the effects on the Rf values and area were observed. In the case of mobile phase ratio, the percentage change in Rf did not exceed 0.4 % and the percentage change in area did not exceed 0.06. In case of chamber change, the fractional change in Rf value was not more than 0.01 % & the fractional change in area was not more than 0.06 %. The method was found to be robust since, the monitored parameters were not significantly affected.

CONCLUSION:

A new HPTLC method has been developed for the identification and quantification of ruxolitinib Low cost, faster speed, and satisfactory precision and accuracy are the main features of this method. Method was successfully validated as per ICH guidelines and statistical analysis proves that method is sensitive, specific, and repeatable. It can be conveniently employed for routine quality control analysis of ruxolitinib as bulk drug in marketed tablets. The method was also applied for the estimation of equilibrium solubility of ruxolitinib in various excipients and diffusion studies. The HPTLC method provides accurate and precise results which are comparable to that of liquid chromatographic method. Reduced sample preparation methods, less analysis time, and small quantity of mobile phase required are some of its advantages over liquid chromatography. Densitometric scanning used in HPTLC for quantitative analysis offers advantage of accuracy, precision, and specificity over conventional methods used in TLC.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Principal, Modern College of Pharmacy, Pune for providing necessary infrastructure for research work.

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Received on 18.05.2022 Modified on 30.09.2022

Research J. Pharm. and Tech 2023; 16(9):4219-4224.

DOI: 10.52711/0974-360X.2023.00690