RP-HPLC Method Development and Validation for the Quantitative Determination of Ruxolitinib in Pure Form and Marketed Pharmaceutical Dosage Form

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Introduction

Ruxolitinib [1] is a pyrazole substituted at position 1 by a 2-cyano-1-cyclopentylethyl group and at position 3 by a pyrrolo [2, 3- d] pyrimidin-4-yl group. Used as the phosphate salt for the treatment of patients with intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia Vera myelofibrosis and post-essential thrombocythemia myelofibrosis. It has a role as an antineoplastic agent and an EC 2.7.10.2 (non-specific protein-tyrosine kinase) inhibitor. It is a nitrile, a pyrrolopyrimidine and a member of pyrazoles. Ruxolitinib [2] is a Kinase Inhibitor and Janus Kinase Inhibitor. The mechanism of action of Ruxolitinib is as a Janus Kinase Inhibitor. Ruxolitinib, formerly known as INCB018424 or INC424, is an anticancer drug and a Janus kinase (JAK) inhibitor. It is a potent and selective inhibitor of JAK1 and JAK2, which are tyrosine kinases involved in cytokine signalling and hematopoiesis. Myeloproliferative neoplasms, such as myelofibrosis and polycythemia Vera, are often characterized by aberrant activation of the JAK-STAT pathway, leading to abnormal blood cell counts and thrombotic complications. By inhibiting JAK1 and JAK2, Ruxolitinib [3] works to block the dysregulated cell signalling pathways and prevents abnormal blood cell proliferation. Due to a large number of patients with myeloproliferative neoplasms who have JAK2 mutations, Ruxolitinib was the first ATP-competitive inhibitor of JAK1 and JAK2 ever developed. Ruxolitinib is an antineoplastic agent that inhibits cell proliferation, induces apoptosis of malignant cells, and reduces pro-inflammatory cytokine plasma levels by inhibiting JAK-induced phosphorylation of signal transducer and activator of transcription (STAT). Inhibition of STAT3 phosphorylation, which is used as a marker of JAK activity, by Ruxolitinib is achieved at two hours after dosing which returned to near baseline by 10 hours in patients with myelofibrosis and polycythemia vera. In clinical trials, Ruxolitinib reduced splenomegaly and improved symptoms of myelofibrosis. In a mouse model of myeloproliferative neoplasms, administration of Ruxolitinib was associated with prolonged survival. Ruxolitinib inhibits both mutant and wild-type JAK2; however, JAK2V617F mutation, which is often seen in approximately 50% of patients with myelofibrosis, was shown to reduce Ruxolitinib sensitivity, which may also be associated with possible resistance to JAK inhibitor treatment. The IUPAC Name of Ruxolitinib is (3R)-3-cyclo pentyl-3-[4-(7H-pyrrolo [2, 3-d] pyrimidin-4-yl) pyrazol-1-yl] propanenitrile. The Chemical Structure of Ruxolitinib is shown in fig-1.

Fig 1: Chemical Structure of Ruxolitinib Experimental Materials Ruxolitinib API and Marketed Formulation were provided by Synpharma Research Lab, Dilsuknagar, Hyderabad. HPLC grade acetonitrile and phosphoric acid, Dipotassium hydrogen orthophosphate, Potassium dihydrogen orthophosphate, Sodium hydroxide, Hydrochloric acid, 3% Hydrogen Peroxide were purchased from SD fine-Chem ltd; Mumbai. HPLC grade water was obtained from SD fine-Chem ltd; Mumbai.

Apparatus and Chromatographic Conditions Analysis were performed using a Waters HPLC with Empower2 Software with Isocratic with UV-Visible Detector was provided from Synpharma Research Lab, Dilsuknagar, Hyderabad. This Instrument is connected to a Symmetry ODS C18 (4.6×250mm, 5µm) supplied with an isocratic pump and an auto-sampling device. The experiments were operated at ambient temperature. The mobile phase4 was composed of a mixture of Methanol and Phosphate Buffer (35:65%) v/v. The mobile phase flow-rate was 1.0 mL/min. The detection was performed using a UV detector at wavelength 235 nm.

HPLC Method Development Preparation of Standard Solution 7ml of Methanol and sonicate to dissolve and removal of air completely and make volume up to the mark with the same Methanol.

Procedure Inject the samples by changing the chromatographic conditions and record the chromatograms, note the conditions of proper peak elution for performing validation parameters as per ICH guidelines [30].

Mobile Phase Optimization Initially the mobile phase tried was Methanol and Methanol: Water with varying proportions. Finally, the mobile phase was optimized to Methanol: Phosphate Buffer in proportion 35:65% v/v.

Optimization of Column The method was performed with various C18 columns like, X- bridge column, Xterra, and C18 column. Symmetry ODS C18 (4.6 x 250mm, 5m) was found to be ideal as it gave good peak shape and resolution [5] at 1ml/min flow.

Preparation of Buffer and Mobile Phase Preparation of Potassium dihydrogen Phosphate (KH2PO4) buffer (pH-3.6)

Dissolve 6.8043 of potassium dihydrogen phosphate in 1000 ml HPLC water and adjust the pH 3.6 with diluted orthophosphoric acid. Filter and sonicate [6] the solution by vacuum filtration and ultra-sonication.

Preparation of Mobile Phase Accurately measured 350 ml (35%) of Methanol, 650 ml of Phosphate buffer (65%) were mixed and degassed in digital ultra sonicater for 15 minutes and then filtered through 0.45 µ filter under vacuum filtration [7].

Diluent Preparation The Mobile phase was used as the diluent.

Method Validation Parameters System Suitability solution) diluents.

Procedure The standard solution was injected for five times and measured the area for all five injections in HPLC [8]. The %RSD for the area of five replicate injections was found to be within the specified limits.

Specificity Preparation of Standard Solution solution) diluents.

Preparation of Sample Solution Weight 10 mg equivalent weight of Ruxolitinib sample into a 10mL clean dry volumetric flask and add about 7mL of Diluent [9] and sonicate to dissolve it completely and make volume up to the mark with the same solvent. Further pipette 0.1ml of Ruxolitinib above stock solution into a 10ml volumetric flask and dilute up to the mark with diluent.

Procedure Inject the three replicate injections of standard and sample solutions and calculate the assay10-11 by using formula:

Preparation of Level – I (6ppm of Ruxolitinib) sonicate [12] the solution for bubble entrapment using Preparation of Level – II (8ppm of Ruxolitinib)

Preparation of Level – III (10ppm of Ruxolitinib)

Preparation of Level – IV (12ppm of Ruxolitinib)

Preparation of Level – V (14ppm of Ruxolitinib)

Procedure Inject each level into the chromatographic system13 and measure the peak area.

Plot a graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and calculate the correlation coefficient [14].

Precision Repeatability Preparation of Ruxolitinib Product Solution for Precision solution) diluents.

The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD15 for the area of five replicate injections was found to be within the specified limits.

To evaluate the intermediate precision (also known as Ruggedness [16]) of the method, Precision was performed on different days by maintaining same conditions.

Procedure Analyst 1 The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for the area of six replicate injections was found to be within the specified limits.

Analyst 2 The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for the area of six replicate injections was found to be within the specified limits [17].

Accuracy solution) diluents.

Procedure Inject the Three replicate injections of individual concentrations (50%, 100%, 150%) were made under the optimized conditions. Recorded the chromatograms and measured the peak responses. Calculate the Amount found and Amount added for Ruxolitinib and calculate the individual recovery and mean recovery values [18].

Robustness The analysis was performed in different conditions to find the variability of test results. The following conditions are checked for variation of results.

For preparation of Standard Solution solution) diluents.

Effect of Variation of Flow Conditions The sample was analyzed at 0.9 ml/min and 1.1 ml/min instead of 1ml/min, remaining conditions are same. 10µl of the above sample was injected and chromatograms19 were recorded.

Effect of Variation of Mobile Phase Organic Composition The sample was analyzed by variation of mobile phase i.e. Methanol: Phosphate Buffer was taken in the ratio and 40:60, 30:70 instead (35:65), remaining conditions are same. 10µl of the above sample was injected and chromatograms were recorded.

Results and Discussion

Method Development Optimized Chromatographic Conditions:

Mobile phase ratio: Methanol: Phosphate Buffer (35:65) V/V Column: Symmetry ODS C18 (4.6×250mm, 5µm) Column temperature: Ambient Wavelength: 235nm Flow rate: 1ml/min Injection volume: 10µl Run time: 8min Fig 2: Chromatogram of Optimized Chromatographic Condition Method Validation Method validation [20-22] is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability and consistency of analytical results; it is an integral part of any good analytical practice.

System Suitability Table 1: Results of System Suitability for Ruxolitinib S.No. Peak Name RT Area (µV*sec) Height (µV) USP Plate Count USP Tailing Mean 1654667 Std. Dev. 2355.764 % RSD 0.142371 Specificity The ICH documents define specificity as the ability to assess unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products, and matrix components. Analytical method was tested for specificity [23] to measure accurately quantitates Ruxolitinib in drug product.

Standard area Dilution of standard Weight of sample 100 Label claim The % purity of Ruxolitinib in pharmaceutical dosage form was found to be 99.86%.

Linearity Chromatographic Data for Linearity Study:

Table 2: Data for Linearity of Ruxolitinib Concentration g/ml Average Peak Area 6 1078475 8 1461129 10 1808358 12 2211573 14 2593778 Fig 3: Linearity Curve of Ruxolitinib Linearity Plot The plot of Concentration (x) versus the Average Peak Area (y) data of Ruxolitinib is a straight line.

Y = mx + c Slope (m) = 185008 Intercept (c) = 16179 Correlation Coefficient (r) = 0.999 Validation Criteria: The response linearity [24] is verified if the Correlation Coefficient is 0.99 or greater. Conclusion: Correlation Coefficient (r) is 0.99, and the intercept is 0.16179. These values meet the validation criteria.

Precision The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Repeatability: Obtained Five (5) replicates of 100% accuracy solution as per experimental conditions. Recorded the peak areas and calculated % RSD [25].

Table 3: Results of Repeatability for Ruxolitinib S. No. Peak Name Retention time Area (µV*sec) Height (µV)

The Intermediate Precision26 consists of two methods:- Intra Day: In Intra Day process, the 50%, 100% and 150% concentration are injected at different intervals of time in same day.

Inter Day: In Inter Day process, the 50%, 100% and 150% concentration are injected at same intervals of time in different days.

Table 4: Results of Intra-Assay & Inter-Assay Conc. of Ruxolitinib (API)

Observed Conc. of Ruxolitinib (µg/ml) by the Proposed Method Intra-Day Inter-Day Mean (n=6) % RSD Mean (n=6) % RSD Observations: The intra & inter day variation of the method was carried out for standard deviation & % RSD (% RSD < 2%) within a day & day to day variations for Ruxolitinib revealed that the proposed method is precise. Accuracy: Accuracy [27] at different concentrations (50%, 100%, and 150%) was prepared and the % recovery was calculated.

Table 5: The Accuracy Results for Ruxolitinib %Concentration (at Specification Level)

LOD = 3.3 × σ / s Where σ = Standard deviation of the response S = Slope of the calibration curve Result = 1.2µg/ml Quantitation Limit The quantitation limit29 of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined.

LOQ = 10×σ/S Where σ = Standard deviation of the response S = Slope of the calibration curve Result = 3.6µg/ml Robustness The robustness was performed for the flow rate variations from 0.9 ml/min to 1.1ml/min and mobile phase ratio variation from more organic phase to less organic phase ratio for Ruxolitinib. The method is robust only in less flow condition. The standard of Ruxolitinib was injected by changing the conditions of chromatography. There was no significant change in the parameters like resolution, tailing factor, asymmetric factor, and plate count.

Table 6: Result of Method Robustness Test Change in Parameter % RSD Flow (1.1 ml/min) 0.68 Flow (0.9 ml/min) 0.39 Temperature (270C) 0.54 Temperature (230C) 0.63 Wavelength of Detection (280 nm) 0.91 Wavelength of detection (270 nm) 0.93 Acceptance Criteria: The tailing factor should be less than 2.0 and the number of theoretical plates (N) should be more than 2000.

Estimation of Ruxolitinib in TABLET Dosage Form Jakavi 20mg Tablet Twenty tablets were taken and the I.P. method was followed to determine the average weight. Above weighed tablets were finally powdered and triturated well. A quantity of powder equivalent to 10 mg of drug were transferred to 10 ml volumetric flask, and 8 ml of mobile phase was added and solution was sonicated for 15 minutes, there after volume was made up to 10 ml with same solvent. Then 1ml of the above solution was diluted to 10 ml with HPLC grade methanol. The solution was filtered through a membrane filter (0.45 µm) and sonicated to degas. From this stock solution (1.0 ml) was transferred to five different 10 ml volumetric flasks and volume was made up to 10 ml with same solvent system. The solution prepared was injected in five replicates into the HPLC system and the observations were recorded. A duplicate injection of the standard solution was also injected into the HPLC system and the peak areas were recorded. The data are shown in Table-7.

AT = Peak Area of Ruxolitinib obtained with test preparation AS = Peak Area of Ruxolitinib obtained with standard preparation WS = Weight of working standard taken in mg WT = Weight of sample taken in mg DS = Dilution of Standard solution DT = Dilution of sample solution P = Percentage purity of working standard Results obtained are tabulated below:

Table 7: Assay of Ruxolitinib Tablets Brand Name of Capsules Labelled amount of Drug (mg)

Mean (±SD) amount (mg) found by the proposed method (n=5)

Assay + % RSD Jakavi 20mg Tablet (Novartis)

Result & Discussion: The %Purity of Jakavi 20mg Tablet containing Ruxolitinib was found to be 99.476 % (± 0.347).

Summary and Conclusion

To develop a precise, linear, specific & suitable stability indicating RP-HPLC method for analysis of Ruxolitinib, different chromatographic conditions were applied & the results observed are presented in previous chapters. Isocratic elution is simple, requires only one pump & flat baseline separation for easy and reproducible results. So, it was preferred for the current study over gradient elution. In case of RP-HPLC various columns are available, but here Symmetry ODS C18 (4.6×250mm, 5µm) column was preferred because using this column peak shape, resolution and absorbance were good. Mobile phase & diluent for preparation of various samples were finalized after studying the solubility of API in different solvents of our disposal (methanol, acetonitrile, water, 0.1N NaOH, 0.1NHCl). Ruxolitinib was found to be Very slightly soluble in water, sparingly soluble in methanol, slightly soluble in methylene chloride, very slightly soluble in acetonitrile, freely soluble in acetone, in anhydrous ethanol. Using these solvents with appropriate composition newer methods can be developed and validated. Detection wavelength was selected after scanning the standard solution of drug over 200 to 400nm. From the U.V spectrum of Ruxolitinib it is evident that most of the HPLC work can be accomplished in the wavelength range of 235 nm conveniently. Further, a flow rate of 1.0 ml/min & an injection volume of 10µl were found to be the best analysis. The result shows the developed method is yet another suitable method for assay which can help in the analysis of Ruxolitinib in different formulations.