A sensitive stability indicating RP-HPLC method for the determination of Ibrutinib - An anti-cancer drug

 

Gujju. Hima Bindu*,  Mukthinuthalapati Mathrusri Annapurna

Department of Pharmaceutical Analysis & Quality Assurance, GITAM Institute of Pharmacy,

GITAM University, Visakhapatnam-530045, India

 

ABSTRACT:

Ibrutinib is used for the treatment of mantle cell lymphoma. Mantle cell lymphoma is a fast growing cancer which begins in the cells of immune system. A new HPLC method has been proposed for the quantification of Ibrutinib. Shimadzu Model HPLC system with Kromosil column (250mm x 4.6 mm, 5m particle size) was used for the study. Mixture of phosphate buffer and acetonitrile (45: 55, v/v) with 1.0 mL/min flow rate are the optimized chromatographic conditions and the system was monitored at 295 nm.  Ibrutinib shows linearity 3.5–2100 μg/ml with regression equationy = 15528x + 251.16 (R² = 0.9999). The LOD and LOQ were found to be 0.6927 μg/ml and 2.1578 μg/ml respectively. Stress studies were performed and the method was validated. Assay of marketed formulations was also conducted the system suitability parameters were within the acceptable limits.

 

 

 

INTRODUCTION:

Ibrutinib is an anti-cancer drug used for the treatment of mantle cell lymphoma. Mantle cell lymphoma is a fast growing cancer which initiates from the cells of immune system. It blocks the abnormal protein that signals cancer cells multiplication and finally stops dispersion of cancer. Ibrutinib is a very small molecule and acts by binding to the protein permanently^1-5. Figure 1 shows the structure of Ibrutinib. Very less analytical methods are available in the literature which include HPLC^6-9, UPLC¹⁰, LC-MS/MS¹¹ methods in rat plasma, human plasma and mouse plama. In this paper a new method was proposed for the quantification of Ibrutinib.

 

 

 

 

Figure 1: Structure of Ibrutinib

 

MATERIALS AND METHODS:

Ibrutinibis available with brand name Imbruvica (Label claim: 140 mg) in India. Acetonitrile, KH₂PO₄, H₂O₂, NaOH, HCl were obtained from Merck (India). Ibrutinib stock solution (1000 μg/ml) was prepared in acetonitrile and dilutions were made with mobile phase and the solutions were filtered through 0.45 μm membrane prior to use.

 

Shimadzu Model HPLC system with Kromosil column (250mm x 4.6 mm,  5m particle size) was used for the study. Mixture of phosphate buffer and acetonitrile (45: 55, v/v) with 1.0 mL/min flow rate was used for the quantification of Ibrutinib and the system was monitored at 295 nm. 

Method validation¹²

Linearity

3.5-210 μg/ml Ibrutinib solutions were prepared on dilution from Ibrutinibstock and diluted with mobile phase.10 µl was injected (n=3) in to the HPLC system and the mean peak area was noted.Calibration curve was plotted by taking concentration of the Ibrutinib solutions on the x-axis and the corresponding mean peak area on the y-axis.

 

Precision, robustness and accuracy         

The intra-day and inter-day precision studies as well as accuracy and robustness were performed at three different concentration levels (35, 70 and 105 µg/mL) and on three different days respectively and %RSD was calculated. The accuracy was calculated at three different levels (50%, 100% and 150%) by the standard addition method. The robustness of the assay method was performed by introducing small changes in the chromatographic conditions such as detection wavelength (290 and 300 nm), composition of mobile phase (40 and 50% of acetonitrile) and flow rate (0.9 and 1.1 mL/min).

 

Specificity¹³

Acidic and alkaline degradations were performed by treating 1 ml of Ibrutinib stock solution with 1 ml of 2N hydrochloric acid and 1 ml of 2N sodium hydroxide refluxed for 30 min at 60⁰C, cooled, neutralized and then diluted prior to use.1 ml of stock solution of Ibrutinib was treated with 1ml of 20% hydrogen peroxide for 30 min at 60⁰C, cooled, and then diluted prior to use.For thermal degradation the standard Ibrutinib drug solution was placedina thermostatat105 °C for 6 hours to study dry heat degradation.The photochemical stability of the Ibrutinib was also conducted by exposing the drug solution to UV Light in UV chamber for 7days or 200 Watt hours/m² in photo stability chamber. Neutralization or hydrolysis was performed by refluxing Ibrutinib solution with water for 6 hours at 60º. All the resultant solutionswerediluted and 10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

 

Assay of Ibrutinib capsules

Assay of Ibrutinib capsules was conducted by taking the capsule contents inside. 100 mg of Ibrutinib was dissolved in mobile phase in a volumetric flask and the contents were sonicated for 30 min and filtered.

 

RESULTS AND DISCUSSION:

A sensitive stability indicating RP-HPLC method has been developed for the assay of Ibrutinib using phosphate buffer and acetonitrile (45: 55, v/v) mixture as mobile phase (Blank: Figure 2A) with 1.0 ml/min flow rate at 295 nm and Ibrutinib was eluted at 2.572 min (Figure 2B). The optimized method was compared with the published methods in Table 1 and that of the optimized chromatographic conditions were given in Table 2.

 

 

Table. 1. Review ofpublished methods of Ibrutinib

 

 

 

 

 

 

 

 

 

Table. 2. Optimized chromatographic conditions for Ibrutinib

 

 

 

Figure 2: Typical chromatograms of [A] Blank [B] Ibrutinib standard  [C] Imbruvica^® (Pharmacyclics) (Label claim: 140 mg) [D] Imbruvica^®(Janssen Biotech)

 

Method validation

Ibrutinib shows linearity 3.5-210 µg/mL (% RSD 0.21-0.48) (Table 3) with linear regression equation y = 15528x + 251.16 (R² = 0.9999) (Figure 3). The LOD and LOQ were found to be 0.6927 μg/ml and 2.1578 μg/ml respectively. The % RSD in precision (Table 4), robustness (Table 5) and accuracy (Table 6) was not more than 2 indicating that the method is precise, accurate and robust.

 

Table. 3. Linearity of Ibrutinib

 

 

Figure 3: Calibration curve of Ibrutinib

 

 

Table. 4. Precision ofIbrutinib

*Mean of three replicates

 

 

 

Table. 5. Robustness of Ibrutinib

*Mean of three replicates

 

Table. 6. Accuracy of Ibrutinib

 

Assay of Ibrutinib capsules

Ibrutinibwas determined in two different marketed productsusing the optimized conditions for the applicability of the method developed. The % recovery was 98.81 and 99.38 (Table 7) and the respective chromatograms obtained were shown in Figure 2C and Figure 2D.

 

Table. 7.Assay of Ibrutinib capsule

*Mean of three replicates

Specificity

Ibrutinib has undergone acidic, alkaline, oxidative, thermal, hydrolysis and photolysis. During all degradations less than 5% of the drug was decomposed. Ibrutinib was eluted at 2.572 min. During acidic, alkaline and oxidative degradation treatment, degradants were eluted at 6.322. 6.300 and 3.837 min respectively. The theoretical plates are above 5000 (more than 2000) and the tailing factor is less than 1.5 (Table 8). The typical chromatograms obtained during the stress degradation studies were shown in Figure 4.

 

 

 

 

 

Table. 8. Specificity of Ibrutinib

 

 

CONCLUSION:

A new sensitive and specific stability indicating RP-HPLC method has been developed for the quantification of Ibrutinib and the method was validated. Ibrutinib drug peak had never interfered with the degradants indicating that the method is specific. The method can be applied for the routine analysis of Ibrutinib capsules.

 

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Accepted on 15.10.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(10): 4587-4591.