INTRODUCTION:

Acalabrutinib is a Bruton tyrosine kinase inhibitor used for the treatment of chronic lymphocytic leukemia, mantle cell lymphoma and small lymphocytic lymphoma^1-2. Acalabrutinib (CAS 1420477-60-6) is a secondary carboxamide with molecular weight 465.5 and molecular formula C₂₆H₂₃N₇O₂. Acalabrutinib is chemically 4-[8-Amino-3-[(2S)-1-(1-oxo-2-butyn-1-yl)-2-pyrrolidinyl] imidazo [1,5-a] pyrazin-1-yl]-N-2-pyridinylbenzamide. FDA had approved Acalabrutinib for oral administration in 2017. Mantle cell lymphoma is a rare yet aggressive type of B-cell non-Hodgkin lymphoma with poor prognosis. Bruton tyrosine kinase (BTK) is a signalling molecule of the B-cell antigen receptor and cytokine receptor pathways. Such BTK signaling causes the activation of pathways necessary for B-cell proliferation, trafficking, chemotaxis, and adhesion. Acalabrutinib inhibits BTK-mediated activation of downstream signaling proteins CD86 and CD69, which ultimately inhibits malignant B-cell proliferation and survival. Acalabrutinib is mainly metabolized by CYP3A enzymes and ACP-5862 is identified as the major active metabolite in plasma.

Figure 1: Chemical structure of Acalabrutinib

Acalabrutinib and its impurities were estimated by different analytical techniques using UPLC-MS/MS³, LC-MS⁴, LC-MS/MS^5-6, HPLC^7-14, etc in pharmaceutical formulations as well as biological fluids. In the present study the authors have developed a new stability indicating RP-HPLC method for the determination of Acalabrutinib in capsules and the method was validated.

EXPERIMENTAL:

Preparation of mobile phase and diluent

1.5417 grams of Ammonium acetate was accurately weighed and transferred in to a 1000 ml volumetric flask and about 500 ml of diluent was added to dissolve and then pH was adjusted to 4.0 with glacial acetic acid and finally the volume was made up to volume with HPLC grade water. The diluent solution was prepared mixing water and Acetonitrile in 50:50 (v/v) ratio and was sonicated and filtered. A mixture of 0.02M of Ammonium acetate (pH 4.0) solution and Acetonitrile was used as mobile phase for the entire study.

Preparation of stock solution

25 mg of Acalabrutinib was weighed accurately and transferred in to a 25 mL volumetric flask (1000 µg/mL) and dissolved in HPLC grade Acetonitrile, sonicated and filtered before use. The stock solution (1000 µg/mL) was further diluted with the diluent as per the requirement for the linearity, precision, accuracy, robustness and other studies and all the solutions were filtered through 0.45 μm membrane filter before injection.

Instrumentation and optimised chromatographic conditions

Shimadzu HPLC Model no. iSeries 2050C 3D with Lab solutions Software with PDA detector and Shim-pack GIST C18 column (250 x 4.6 mm., 5 m) were used for the present study. Mobile phase mixture consisting of Ammonium acetate buffer (pH adjusted to 4.0 using glacial acetic acid) and Acetonitrile (50: 50, v/v) (Isocratic mode) with flow rate 1.0 mL/min were the optimised chromatographic conditions (Detection wavelength 285 nm).

Method validation¹⁵

Linearity, Precision, Accuracy and Robustness studies

0.2-150 µg/mL Acalabrutinib solutions were prepared from the Acalabrutinib stock solution and diluted with the diluent (Water: Acetonitrile (50:50, v/v) and injected (n = 3) into the HPLC system and the peak area was noted from the respective chromatograms. Finally, the mean peak area (n=3) was calculated and a calibration curve was drawn by plotting the Acalabrutinib concentration on the x-axis and the corresponding mean peak area on the y-axis. The LOD and LOQ were calculated from the S/N ratio. Intraday (100 µg/mL) and interday precision (10, 20 and 40 µg/mL) studies were performed on the same day and on three different days and the peak area of the chromatograms were recorded during the study from which the mean peak area (n = 3) was calculated. The percentage relative standard deviation was also calculated from the mean peak area and the standard deviation.

Accuracy study was performed by standard addition method i. e. spiking of Acalabrutinib capsule formulation (20 µg/mL) solution with 50%, 100%, 150% of Acalabrutinib API and these solutions were injected (n=3) in to HPLC system and the chromatograms were recorded and the peak areas were noted from which the mean peak area and the % RSD were calculated from the linear regression equation. The robustness of the method was proved by incorporating a very small changes in the optimized chromatographic conditions such as flow rate (± 0.1 mL/min; 1.1 and 0.9 mL/min), mobile phase composition (0.02M of Ammonium acetate (pH adjusted to 4.0 with glacial acetic acid) and Acetonitrile (± 2 % v/v; 52: 48 and 48: 52), detection wavelength (± 5 nm; 280 and 290 nm) and pH (± 0.1 unit; 3.9 and 4.1).

Stress degradation studies¹⁶

Stress degradation or stress degradation studies were performed to determine the stability of Acalabrutinib (100 µg/mL) towards acidic hydrolysis, basic hydrolysis, oxidation and thermal degradation. The specificity of the method was determined from the stability studies and therefore Acalabrutinib was exposed to different stress conditions as given below.

Acidic degradation was performed by treating 1.0 ml of stock solution of Acalabrutinib (100 µg/mL) solution with 1.0 mL of 2N HCl solution for 3 hours on heating at 70^⸰C followed by neutralization with 1.0 mL 2N sodium hydroxide solution, made up to volume with the diluent and then 10 µl of the resultant solution was injected in to the HPLC system. Thermal degradation was performed by heating the Acalabrutinib (100 µg/mL) solution at 70ºC for 3 hours and then cooled, diluted with mobile phase and 10 μl of the resulting solution was injected in to the HPLC system. Oxidative degradation was performed by heating Acalabrutinib (100 µg/mL) solution with 1.0 mL 30% hydrogen peroxide solution in dark room for 3 hours and then the stressed sample was diluted with mobile phase and then 10 µl of the resulting solution was injected in to the HPLC system. Alkaline degradation was performed by treating 1.0 ml of stock solution of Acalabrutinib (100 µg/mL) solution with 1.0 mL of 2N NaOH solution for 3 hours on heating at 70^⸰C followed by neutralization with 1.0 mL 2N HCl solution, made up to volume with the diluent and then 10 µl of the resultant solution was injected in to the HPLC system.

Assay of Acalabrutinib capsules

Acalabrutinib was obtained as gift sample from Natco Pharma (India). Acalabrutinib is available as capsules in India with different brand names Calquence (Astrazeneca), Acanrunat (Natco Pharma), Acaya (Zydus), Acaone (MSN Laboratories), Acaluxem (Everest Pharmacuticals Ltd) etc. with label claim 100 mg. Twenty capsules of two different brands of Acalabrutinib capsules were procured and the contents of the capsules were powdered and powder equivalent to 25 mg of Acalabrutinib was accurately weighed and transferred in to two different volumetric flasks and extracted with HPLC grade Acetonitrile and the mixture was sonicated, and filtered. The resultant solutions were diluted with the diluent as per the requirement and 100 µg/mL of each of the marketed formulation solution was injected (n=3) in to the HPLC system and the peak area was noted from the respective chromatograms and then the mean peak area was calculated. The percentage of purity of Acalabrutinib was calculated from the linear regression equation for the marketed formulations.

RESULTS AND DISCUSSION:

A new stability indicating RP-HPLC method has been developed and validated for the determination of Acalabrutinib in capsules. Shimadzu HPLC Model no. iSeries 2050C 3D with Lab solutions Software with PDA detector and Shim-pack GIST C18 column (250 x 4.6 mm., 5mm) were used for the present study. A mixture of 0.02M of Ammonium acetate (pH adjusted to 4.0 with glacial acetic acid) and Acetonitrile (50: 50, v/v) was used as mobile phase with flow rate 1.0 mL/min were the optimised chromatographic conditions (Detection wavelength 285 nm). The injection volume was 10 µl and the run time was 10 min (Column temperature 40ºC and sample cooler 25ºC). A mixture of water: Acetonitrile (50:50, v/v) was used as diluent. Some of the important parameters of the previously published analytical methods were discussed in detail in Table 1.

Method validation

Acalabrutinib has shown linearity over the concentration range 0.2-150 mg/ml (Table 2) with linear regression equation, y = 18135x + 1732.4 (R² = 0.9998). The LOQ and LOD were found to be 0.1932 mg/ml and 0.06354 respectively. The typical chromatogram obtained for the blank and Acalabrutinib API were shown in Figure 2. The calibration was shown in Figure 3. The % RSD was found to be 0.0102 (Intraday precision) and 0.0128-0.1149 (Inter-day precision) (Table 3) in precision studies which is less than 2.0 indicating that the method is precise. The % recovery in accuracy studies was found to be 99.37-99.85% (Table 4) and % RSD was (0.0724-0.9251) less than 2% indicating that the method is accurate. The % RSD in robustness study was found to be 0.0033-0.0710 which was less than 2% indicating that the method is robust (Table 5).

Table 1: Literature survey of Acalabrutinib

Mobile phase (v/v)	Column	Linearity (µg/mL)	Comment	Ref
0.1% Formic acid (pH 3.2): Acetonitrile (15: 85)	Zorbax RRHD Eclipse Plus C18	0.001-3.0	UPLC-MS/MS Ponatinib (Internal standard)	3
Mobile phase A: 10 mM Ammonium acetate buffer (pH 3.80) Mobile phase B: Methanol: Acetonitrile (90:10)	Shimpak C8	-	LC-Q-TOF-MS and NMR (Gradient mode) (18 degradation products)	4
Acetonitrile: 10 mM Ammonium formate in 0.1% Formic acid buffer (65: 35)	Zorbax Eclipse XDB-C₁₈	0.005-1.6	LC-MS/MS (Human plasma)	5
0.1% aq. Formic acid: Methanol (50:50)	Agilent Zorbax C18 and Chromanik C18	-	LC-MS/MS and NMR	6
Methanol: Acetonitrile: 0.1% Ortho phosphoric acid (45:35:20)	KNAUER Eurospher II C18	0.05-3.0	RP-HPLC (Plasma) Nifedipine (Internal standard)	7
Water: Methanol (60: 40)	Zodiasil C18	25-150	RP-HPLC	8
25 mM Ammonium acetate (pH 4.0): Acetonitrile (50: 50)	Gemini LC C18	5-20	RP-HPLC	9
Methanol: 0.02M Phosphate buffer (pH 3.6) (45:55)	Develosil ODS HG-5 RP C18	12-28	RP-HPLC	10
Phosphate buffer (pH 6.4): Methanol (80:20)	Zorbax XDB-C18	-	RP-HPLC (Impurities)	11
0.1% Potassium di hydrogen ortho phosphate: Acetonitrile (70:30)	BDS C18	12.5-75	RP-HPLC	12
0.1% Ortho phosphoric acid: Methanol (50:50)	Kromosil C18	25-150	RP-HPLC	13
0.1% Ethylene diamine in ethyl tert-butyl ether: Ethanol (60:40)	Chiralpak IA (Chiral chromatography)	-	RP-HPLC	14
0.02M of Ammonium acetate (pH adjusted to 4.0 with glacial acetic acid): Acetonitrile (50: 50)	Shim-pack GIST C18	0.2-150	Stability indicating RP-HPLC	Present work

Figure 2: Typical chromatograms of A) Blank B) Acalabrutinib (100 µg/mL) (API) (Rt: 3.605 min) C) Acalabrutinib capsules (Label claim: 100 mg) (100 µg/mL) (Rt: 3.605 min)

Table 2: Linearity of Acalabrutinib

Conc. (µg/mL)	*Mean peak area
0	0
0.2	3719
0.5	9364
1	18654
5	91782
10	183325
20	365982
40	726984
50	934965
80	1449851
100	1793744
120	2157841
150	2744982

*Mean of three replicates

Figure 3: Calibration curve of Acalabrutinib

Table 3: Precision studies of Acalabrutinib

Intraday precision study
Conc. (µg/mL)	Mean peak area		Statistical analysis *Mean peak area ± SD (% RSD)
100	1793744		1793851.3333 ± 182.1688 (0.0102)
100	1793957
100	1794132
100	1793602
100	1793867
100	1793806
Interday precision study
Conc. (µg/mL)	Day 1	Day 2		Day 3	Statistical analysis *Mean peak area ± SD (% RSD)
10	183325	183731		183627	183561 ± 210.8933 (0.1149)
20	365982	365924		365889	365931.6667 ± 46.9716 (0.0128)
40	726984	726821		726786	726863.6667 ± 105.6709 (0.0145)

*Mean of three replicates

Table 4: Accuracy study of Acalabrutinib

Spiked Conc. (µg/mL)	*Conc. obtained (µg/mL) ± SD (%RSD)	% Recovery
10 (50%)	9.98 ± 0.0072 (0.0724)	99.80
20 (100%)	19.97 ± 0.1789 (0.8957)	99.85
30 (150%)	29.81 ± 0.2758 (0.9251)	99.37

*Mean of three replicates

Table 5: Robustness study of Acalabrutinib

Parameter	Condition	*Mean peak area	*Statistical Analysis Mean ± SD (% RSD)**
Flow rate (± 0.1 mL/min)	1.1 1.0 0.9	1796578	1797797 ± 1276.4313 (0.0710)
		1797689
		1799124
Detection wavelength (± 5 nm)	280 285 290	1797601	1797667.33 ± 58.5861 (0.0033)
		1797689
		1797712
Mobile phase composition (v/v) 0.02M Ammonium acetate: Acetonitrile (pH 4.0) (± 2 %)	48:52 50:50 52:48	1797325	1797504.33 ± 182.0586 (0.0101)
		1797689
		1797499
pH (± 0.1 unit)	3.9 4.0 4.1	1796591	1797216 ± 564.5609 (0.0314)
		1797689
		1797368

*Mean of three replicates

Assay of Acalabrutinib capsules

Two different brands of Acalabrutinib tablets with label claim 50 mg were chosen and the proposed optimized RP-HPLC method was applied for the quantification of Acalabrutinib and the percentage of purity was calculated using the linear regression equation and the percentage of purity was found to be 99.68-99.93 (Table 6). The representative chromatogram obtained for the tablet formulation was shown in Figure 2C.

Table 6: Assay of Acalabrutinib tablets

S. No.	Brand name	Label claim (mg)	*Observed amount (%w/w)	% Recovery*
1	Brand I	100	99.68	99.68
2	Brand II	100	99.93	99.93

*Mean of three replicates

Stress degradation studies

Acalabrutinib was eluted at 3.605 min with theoretical plates 2786 (> 2000) and tailing factor 1.093 (< 1.5). During the acidic degradation Acalabrutinib has shown 13.82% degradation and Acalabrutinib was eluted at 3.605 min with theoretical plates 2645 and tailing factor 1.034 with extra peaks at 2.208, 2.315 and 2.411 min with resolution greater than 4.185 (> 2%). During the alkaline degradation with the conditions, 2N NaOH / 70ºC/3 hrs Acalabrutinib has totally (97.37%) undergone degradation indicating that Acalabrutinib is highly sensitive towards alkaline conditions and therefore the experiment was repeated by exposing the drug to alkaline conditions only for 5 mins without heating and there by only shown 0.89% degradation was observed. During the oxidative degradation Acalabrutinib has shown 7.47% degradation and Acalabrutinib was eluted at 3.605 min with theoretical plates 2835 and tailing factor 1.054. During the thermal degradation Acalabrutinib has shown only 0.22% degradation and Acalabrutinib was eluted at 3.605 min with theoretical plates 2912 and tailing factor 1.037 with extra peaks at 2.219 and 2.912 min with resolution greater than 2.088 (> 2%). The method is specific as Acalabrutinib peak was clearly eluted even in presence of the degradants during all the stress degradation studies. The system suitability parameters were within the acceptable criteria (Table 7) and the resultant chromatograms obtained during the stability studies were shown in Figure 3.

Acidic degradation (3 hrs)

Thermal degradation (3 hrs)

Oxidative degradation (3 hrs)

Alkaline degradation (3 hrs)

Alkaline degradation (5 mins)

Figure 3: Representative chromatograms of Acalabrutinib (100 µg/mL) during stress degradation studies

Table 7: Stress degradation studies of Acalabrutinib

Stress condition	R_t (min)	Mean peak area	% Recovery	% Drug degradation	Theoretical Plates (>2000)	Tailing factor (<1.5)	Resolution (>2)
Standard drug	3.605	1797689	100	-	2786	1.093	-
Acidic degradation 2N HCl / 70ºC /3 hrs	3.605 2.208 2.315 2.411	1549272	86.18	13.82	2645	1.034	4.185
Alkaline degradation 2N NaOH/70ºC /3 hrs	3.605 2.229 2.325 2.400 3.008	47218	2.63	97.37	2792	1.093	2.356
Alkaline degradation 2N NaOH (5 min)	3.605 2.219 2.912	1781663	99.11	0.89	2467	1.022	-
Oxidation degradation H₂O₂/ 70ºC /3 hrs	3.605	1663417	92.53	7.47	2835	1.054	-
Thermal degradation 70ºC /3 hrs	3.605 2.219 2.912	1793744	99.78	0.22	2912	1.037	2.088

*Mean of three replicates

CONCLUSION:

Acalabrutinib is an anti-cancer drug and a a new stability indicating RP-HPLC method has been developed and validated as per ICH guidelines. The method is simple and specific and there is no interference of the degradants and the system suitability parameters were within the acceptable criteria. Acalabrutinib is found to be very sensitive towards alkaline hydrolysis. The proposed method can be applied for the quantification of Acalabrutinib in pharmaceutical formulations (Capsules).

ACKNOWLEDGEMENT:

The authors are grateful to Natco Pharma (India) for providing the gift samples of Acalabrutinib. The authors declare no conflict of interest.

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Received on 04.12.2024 Revised on 12.02.2025

Accepted on 08.04.2025 Published on 02.05.2025

Available online from May 07, 2025

Research J. Pharmacy and Technology. 2025;18(5):2349-2355.

DOI: 10.52711/0974-360X.2025.00336

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

GITAM School of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, India.