Analytical method development and validation for the quantitative analysis of Ibrutinib in API form and marketed pharmaceutical dosage form by using RP-HPLC

Full text

Text extracted from the article PDF for reading and search. Tables, figures and equations are in the PDF, which remains the authoritative version.

Introduction

Ibrutinib is a member of the class of acrylamides that is (3R)-3-[4-amino-3-(4-phenoxyphenyl) pyrazolo [3, 4-d] pyrimidin-1- yl]piperidine in which the piperidine nitrogen is replaced by an acryloyl group. A selective and covalent inhibitor of the enzyme Bruton's tyrosine kinase, it is used for treatment of B-cell malignancies. It has a role as an EC 2.7.10.2 (non-specific protein-tyrosine kinase) inhibitor and an antineoplastic agent. It is a pyrazolopyrimidine, an aromatic amine, aromatic ether, a member of acrylamides, an N-acylpiperidine and a tertiary carboxamide.Ibrutinib1 is a small molecule that acts as an irreversible potent inhibitor of Burton's tyrosine kinase. It is designated as a targeted covalent drug and it presents a very promising activity in B cell malignancies. Ibrutinib was developed by Pharmacyclics Inc and in November 2013 was FDA-approved for the treatment of mantle cell lymphoma. Later, in February 2014, Ibrutinib was approved for the treatment of chronic lymphocytic leukemia and it is also indicated for the treatment of patients with Waldenström's Macroglobulinemia. Ibrutinib2 has also been approved by the EMA for the treatment of chronic lymphocytic leukemia and mantle cell lymphoma. Ibrutinib was approved for use in chronic graft versus host disease (cGVHD) in August 2017 which was later approved to be used in children, making it the first FDA-approved cGVHD treatment for kids 1 year and older after failure of one or more lines of systemic therapy. Ibrutinib [3] is a Kinase Inhibitor. The mechanism of action of Ibrutinib is as a Protein Kinase Inhibitor. The IUPAC Name of Ibrutinib is 1- [(3R)-3-[4-amino-3-(4-phenoxyphenyl) pyrazolo [3, 4-d] pyrimidin-1-yl] piperidin-1-yl] prop-2-en-1-one. The Chemical Structure of Ibrutinib is as follows Fig 1: Chemical Structure of Ibrutinib

Materials and Methods

Instruments Used Table 1: Instruments used S.No Instruments and Glass wares Model 1 HPLC WATERS Alliance 2695 separation module, Software: Empower 2, 996 PDA detector. 2 pH meter LabIndia

7 Digital ultra sonicator Labman

Chemicals Used Table 2: Chemicals used S. No. Chemical Brand Names

3 Acetonitrile for HPLC Merck

HPLC Method Development Preparation of Standard Solution 7ml of Methanol and sonicate4 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 [27].

Mobile Phase Optimization Initially the mobile phase tried was Methanol and Methanol: Water with varying proportions. Finally, the mobile phase5was 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 [6] 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 the solution by vacuum filtration [7] 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.

Diluent Preparation The Mobile phase was used as the diluent [8].

Method Validation Parameters System Suitability solution)

Procedure The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within Specificity Preparation of Standard Solution solution)

Further pipette 0.1ml of the aboveIbrutinib stock solutions diluents.

Preparation of Sample Solution Weight 10 mg equivalent weight ofIbrutinib sample into a 10mL clean dry volumetric flask and add about 7mL of Diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. Further pipette 0.1ml of Ibrutinib 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 assay [9, 10] by using formula:

Linearity and Range solution)

Preparation of Level – I (6ppm of Ibrutinib)

Preparation of Level – II (8ppm of Ibrutinib)

Preparation of Level – III (10ppm of Ibrutinib) ultrasonicator [11].

Preparation of Level – IV (12ppm of Ibrutinib)

Preparation of Level – V (14ppm of Ibrutinib)

Procedure Inject each level into the chromatographic system12 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.

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

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

Procedure Analyst 1 Analyst 2 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 Ibrutinib and calculate the individual recovery and mean recovery values.

Limit of Detection and Limit of Quantification (LOD & LOQ)

Preparation of 0.95µg/ml Solution (For LOD) solution)

Further pipette 0.0095ml of the above Ibrutinib stock solutions into a 10ml volumetric flask and dilute up to the mark with diluents.

Preparation of 2.9µg/ml Solution (For LOQ) solution) diluents.

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 Accurately weigh and transfer 10 mg of Ibrutinibworking 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 chromatograms 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: Optimized Chromatographic Condition Method Validation The method [13] was validated by following the ICH and USP guidelines for system suitability, linearity, accuracy, precision, specificity and limit of detection (LOD).

System Suitability The system suitability [14] was checked by analyzing the repeatability of retention time, tailing factor and theoretical plates of the column.

Table 3: Results of System Suitability for Ibrutinib S. No. Peak Name RT Area (µV*sec) Height (µV) USP Plate Count USP Tailing Std. Dev. 2355.764 Specificity The ICH documents define specificity [15] 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 to measure accurately quantitates Ibrutinib in drug product.

The % purity [16] of Ibrutinib pharmaceutical dosage form was found to be 99.40%.

Linearity To evaluate the linearity [17] of this method, five standard solutions covering the range of 6 to 14μg/ml were analyzed using the method. The plot of peak area ratio of drug and against respective concentration of the drug was found to be linear in the range as shown in figure -3. The regression equation [18] was found to be Y= 185008X-16179 and the coefficient of determination R2 of the standard curve was found to be 0.9996.

Chromatographic Data for Linearity Study Table 4: Data for Linearity of Ibrutinib Concentration g/ml Average Peak Area 6 1078475 8 1461129 10 1808358 12 2211573 14 2593778 Fig 3: Linearity Curve of Ibrutinib Linearity Plot The plot of Concentration (x) versus the Average Peak Area (y) data of Ibrutinib is a straight line. Y = mx + c Slope (m) = 18500 Intercept (c) = 16179 Correlation Coefficient (r) = 0.999 Validation Criteria: The response linearity is verified if the Correlation Coefficient [19] 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 [20] 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 [21]. Repeatability: Obtained Six (6) replicates of 100% accuracy solution as per experimental conditions. Recorded the peak areas and calculated % RSD [22].

Table 5: Results of Repeatability for Ibrutinib S. No. Peak Name Retention time Area(µV*sec) Height (µV) USP Plate Count USP Tailing Std. Dev 3120.433 Intermediate Precision Analyst-1 Table 6: Results of Intermediate Precision for Ibrutinib S. No. Peak Name RT Area (µV*sec) Height (µV) USP Plate Count USP Tailing Std. Dev. 6463.466 Analyst 2 Table 7: Results of Intermediate precision Analyst 2 for Ibrutinib S.No. Peak Name RT Area (µV*sec) Height (µV) USPP late count USP Tailing Std.Dev. 3501.766 Accuracy: As per the ICH guideline, accuracy is inferred once linearity, specificity and precision of the method are established. This approach was followed to establish the accuracy [23] of the method and the method was found to be accurate.

Table 8: The Accuracy Results for Ibrutinib %Concentration (at specification Level) Area Amount Added (ppm) Amount Found (ppm) % Recovery Mean Recovery 50% 109068.3 5 5.021 100.420% 100.72% 100% 202187 10 10.054 100.540% 150% 297032.3 15 15.181 101.206% Limit of Detection The detection limit [24] of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. LOD= 3.3 × σ / s Where σ = Standard deviation of the response S = Slope of the calibration curve Result = 0.95µg/ml Quantitation Limit The quantitation limit [25] 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 = 2.9µg/ml LOD 0.95925344 LOQ 2.906828605 SE of Intercept = Excel Function (Data Analysis → Regression)

SD of Intercept = SE of Intercept * √N LOD = 3.3 * (SD of Intercept/Slope)

LOQ = 10 * (SD of Intercept/Slope)

Robustness The robustness [26] 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 Ibrutinib. The method is robust only in less flow condition. The standard of Ibrutinibwas injected by changing the conditions of chromatography. There was no significant change in the parameters like resolution, tailing factor, asymmetric factor, and plate count.

Summary and Conclusion

The analytical method was developed by studying different parameters.

First of all, maximum absorbance was found to be at 235nm and the peak purity was excellent.

Injection volume was selected to be 10µl which gave a good peak area.

The column used for study was Symmetry ODS C18 (4.6×250mm, 5µm) because it was giving good peak. Ambient temperature was found to be suitable for the nature of drug solution. The flow rate was fixed at 1.0ml/min because of good peak area and satisfactory retention time. Mobile phase is Methanol: Phosphate Buffer pH-3.6 in the ratio of 35:65 v/v was fixed due to good symmetrical peak. So this mobile phase was used for the proposed study. Methanol was selected because of maximum extraction sonication time was fixed to be 10min at which all the drug particles were completely soluble and showed good recovery. Run time was selected to be 8min because analyze gave peak around 2.276 and also to reduce the total run time. The percent recovery was found to be 98.0-102 was linear and precise over the same range. Both system and method precision was found to be accurate and well within range. The analytical method was found linearity over the range of 6-14ppm of the Ibrutinib target concentration. The analytical passed both robustness and ruggedness tests. On both cases, relative standard deviation was well satisfactory.