Method Development and Validation of Alectinib Drug by RP-HPLC in Bulk and Pharmaceutical Dosage Form

Prashanthi. Y¹, Tentu Nageswara Rao*², Yellapu Srinivas³

¹Department of Biochemistry, Acharya Nagarjuna University, Andhra Pradesh, India.

²Department of Chemistry, Mahatma Gandhi University, Nalgonda, Telangana, India.

³Mother Degree & P.G College, Tuni, A.P, India.

*Corresponding Author E-mail: tnraochemistry@gmail.com

ABSTRACT:

In this study we developed and validated an isocratic Reversed Phase High Performance Liquid Chromatography method with PDA detection for the estimation of Alectinib drug. The method was selective and is capable of detecting at traces levels in the drug substance. The method was validated on an Inertia ODS C18 analytical column (4.6 X 150 mm, 5 µm), mobile phase consisting of Methanol: phosphate buffer at pH 3 in the ratio of 70:30% v/v. The flow rate was at 1 ml/min and the column was maintained at ambient temperature condition. The detector using photo diode array was set at a wavelength of 265 nm. Calibration plot was linear in concentration range of 10 – 50 ppm levels with the correlation coefficient (r2) of 0.999.The intra and inter-day precision% RSD values were 0.48 and 0.51 % respectively. The Limit of Detection and Limit of Quantification values were found to be 0.77 µg/mland 2.35 µg/ml respectively. The method was validated in terms of system precision, method precision, linearity, accuracy, limit of detection, quantification and robustness. Due to its speed and accuracy, this method can be used for estimation and analysis of Alectinib drug in Active Pharmaceutical Ingredients and Pharmaceuticals.

KEYWORDS: Alectinib, Alecensa, Reversed Phase High Performance Liquid Chromatography, Estimation, Photo Diode Array detector

INTRODUCTION:

Lung cancer is the leading cause of cancer deaths worldwide, generally presenting at diagnosis with locally advanced or metastatic diseases. Non-small cell lung cancer (NSCLC), accounts for 85% of the lung cancer cases and has usually reached an advance stage by the time of diagnosis^{1, 2}.

Anaplastic lymphoma kinase (ALK) is one of the receptor tyrosine kinase (RTK) belonging to the insulin receptor family³.

Genetic alterations in ALK are implicated in the pathogenesis of several human cancers. ALK can be aberrantly activated by mutation, gene amplification or chromosomal rearrangement, leading to the expression of a potent oncogenic driver⁴.

Today, several structurally distinct small molecules have been developed as second generation ALK inhibitors, including Ceritinib and Alectinib drugs⁵. Alectinib is an orally active drug that blocks the activity of ALK, and is used to treat the non-small cell lung cancer⁶ It is approved in Japan in July 2014 for the treatment of ALK fusion gene Positive, unrespectable advance or recurrent non-small cell lung cancer⁷,⁸. Alectinib was approved by the US FDA in December 2015 to treat patients with advanced ALK positive NSCLC whose disease worsened after, or who could not tolerate, treatment with Crizotinib. Patients appear to tolerate Alectinib better than Crizotinib, with fewer patients needing dose reduction, interruption, or withdrawal to manage side effects, Dr. Thomas said (M.D., of the Thoracic and Gastrointestinal Oncology Branch in NCI Center for Cancer Research).

Alectinib is white powder or white to yellow powder with lumps in physical state. It is freely soluble in dehydrated alcohols, and in propylene glycol. Alectinib is insoluble in aqueous buffer at pH 7.5.

Alectinib is an orally active anti-cancer agent. It is a Tyrosine kinase inhibitor. It is approved by FDA (December 11, 2015) for patients with metastatic non-small cell lung cancer (NSCLC) with mutations in the ALK gene.

Dr. Thomas (M.D. National Cancer Research) said that the Both Alectinib and Crizotinib are the inhibitors of ALK and used to treat NSCLC. Although Alectinib has several advantages over Crizotinib, based on studies conducted.

Being a very novel and recently synthesized drug, there are few references for Alectinib. Since, there is no reference, in the present literature, concerning analysis of the Alectinib drug in the pharmaceuticals. The aims of this study were firstly to develop and optimize the RP-HPLC conditions for the estimation and analysis of Alectinib drug in pharma and validate the method, then to apply this method in Active Pharmaceuticals Ingredients and Pharmaceuticals for routine analysis.

MATERIALS AND METHODS:

Chemicals and reagents:

The pure drug of Alectinib was the gift from MSN Laboratories Limited (Hyderabad, India). Acetonitrile, methanol and water were of HPLC grade, Potassium di-hydrogen orthophosphate Analytical reagent was purchased from Merck (Germany). 0.22 µ nylon filters HPLC grade purchased from advance lab (India). 0.45 µ filter papers were purchased from Millipore (Moscheim Cedex, France).

Equipment:

A Waters HPLC system (USA) equipped with auto sampler, separation module 2695 and a 996- photo diode array detector was used for the development and validation studies. For the data acquisition and analysis Empower version-2 software system was employed. An ultra-sonic water bath sonicator model SE60US, Enertech company (USA), digital PH meter (AD 102U, ADWA) digital weighing balance ER 200A from Ascocet (USA) and UV double beam spectrometer (UV 3000+ UV win software) (India) were used in the study.

Chromatographic conditions:

Alectinib drug was separated on Inertsil ODS C18 (4.6 X 150 mm) 5 µm particle size analytical column (USA). The samples were eluted in a mobile phase of Methanol and Phosphate buffer solution pH 3 in the ratio of (70:30% v/v) at 1 ml/min flow rate. Auto sampler and column temperatures were set at ambient condition. The injection volume was used 10 µl and the PDA detector was set at wavelength of 265 nm. The run time was 8.0 minutes.

Preparation of mobile phase:

The mobile phase used under the chromatographic conditions was prepared by the mixing of methanol and phosphate buffer solution pH 3 in the ratio of 70: 30 % v/v. Before mixing the solvents, phosphate buffer solution at pH 3 was prepared by the adding of Ortho-phosphoric acid. Then this mobile phase mixture was degassed by using ultra sonic water bath for 5 min. Finally the resulting mixture was filtered through 0.45 µ filter under vacuum filtration system.

Preparation of diluent:

Mobile phase was used as diluent.

Preparation of sample solution of Alectinib:

Accurately10 tablets were weighed and crushed in mortar and pestle and weight equivalent to10mg of alectinib (marketed formulation) sample into a10mL clean dry volumetric flask and about 7mL of mobile phase was added and sonicated to dissolve it completely and made volume up to the mark with the same solvent. Further 1ml of above stock solution was pipetted into a 10 mL volumetric flask and diluted up to the mark with mobile phase.

RESULTS AND DISCUSSIONS:

Method Development:

Here we established an easy and reproducible method for the determination of Alectinib using Reversed Phase High Performance Liquid Chromatography method. This method is very cheap and environmentally friendly in usage.

Chemical structure of Alectinib shown in Figure No. 1. We selected the Inertia C18 ODS 4.6 X 150 mm, 5 µm analytical columns. The selection of column is based up on the solubility and polarity nature of the Alectinib.C18 column was used for the elution of Alectinib due to its high hydrophilic nature.

In our study we used Methanol: Phosphate buffer pH 3, in the ratio of 70:30% v/v. Good degree of separation was achieved when an acidic aqueous mobile phase was run against the non-polar C18 column. More percentage of Methanol (70%) used in mobile phase which helps in dissolution of Alectinib drug.Aqueous mobile phase was set at pH 3 because pH controls the elution properties by controlling the ionization characteristics of the Alectinib. pH also decreases the retention time, and improved the resolution, tailing factor and helped in good peak separation. We tried with below pH 2 of aqueous mobile phase; here we found that cleavage of silica linkage of C18 column. We also tried with above pH 8, at this condition dissolution of silica takes place. So we confirmed and carried out total analysis with pH 3 aqueous mobile phase. Flow rate was selected as 1 ml/min by considering the column back pressure. Detection wavelength was selected as 265 nm to detect Alectinib. All the spectra were extracted from the photo diode array in the selected chromatographic conditions. For the evaluation of the LOD and LOQ, International Conference of Harmonization Guidelines (ICH guidelines Q2 (R1)[11] was fallowed. According to the guidelines Signal-to-Noise ratio is 3. Our results were matched with these guidelines.

Method validation:

The HPLC developed method was validated for specificity, linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ) and system suitable parameters with the ICH guidelines Q2 (R1)¹⁰.

Percentage Assay study:

20 µLof the sample and standard solutions of Alectinib were injected in triplicate into the chromatographic system. Peak area for each injection was measured. The % assay was calculated by comparing the peak area of standard and sample chromatogram by using the formula.

The obtained results are shown in the Table 1.

Table No. 1: Results of percentage of assay

Drug	Alectinib capsule label claim (mg)	Amount found (mg)	% label claim ± % RSD (n=3)
Alectinib	10	9.924	99.24 ± 0.15

n = number of determination, % RSD = Relative Standard Deviation

System suitability:

At first the HPLC system was optimized as per the chromatographic conditions. 20 µL of standard and sample solution of Alectinib drug were injected in triplicate into the HPLC system. To ascertain the system suitability for the proposed method, the parameters such as retention time, theoretical plates, resolution and tailing factors were calculated. The obtained results are in accordance with ICH guidelines and demonstrating the good performance efficiency. The system suitability results are listed in Table 2.

Table 2: Results of System suitability results

Compound name	USP resolution	Theoretical plates	USP tailing	retention time
Alectinib	6.0	2468	1.2	1.9 min.

Specificity:

The ability of an analytical method to unequivocally asses the analyte in the presence of other components like impurities can be verified by evaluating the specificity.

The specificity of the method was confirmed by injecting the blank solution, standard and sample solution of the Alectinib drug. Specificity proved no interference of impurity or any endogenous peak in the retention time of Alectinib drug peak. The resulted chromatograms for blank, standard and sample solution were shown in the Figure No.2, 3 and 4 respectively.

Linearity:

Prepared 10, 20 30, 40 and 50 µg/ml concentration levels of calibration standard solutions of Alectinib and injected into the chromatographic system. A linear regression was used to plot the calibration graph of peak area (on Y- axis) versus concentration (on X-axis) of Alectinib. Each peak area was used to calculate the correlation coefficient (r²)^8,9. The results for linearity were presented in the Table No.3.

Table No.3: Results of linearity, LOD and LOQ studies.

Parameters	Values (µg/ml)
Regression equation	Y = 899996x-211546
r²	0.999
LOQ	2.35 µg/ml
LOD	0.77 µg/ml

r²= correlation coefficient, LOQ = Limit of Quantification, LOD = Limit of Detection

Accuracy (recovery):

The accuracy of the method was determined by calculating the percentage of recovery values of Alectinib by the method of standard addition. Known amount of standard solution of Alectinib at 50, 100 and 150 % were added to pre quantified, sample solution of Alectinib and injected into the chromatographic system. Each standard solution was prepared in triplicate and analyzed. The peak area of each level was used to calculate the percentage of recovery. The results were in accordance with ICH guidelines. According to ICH guidelines percentage of recovery should be in the range of 98 – 102 %. The obtained results were summarized in the Table No.4.

Table No 4: Recovery data for Alectinib drug

Added (mg) (n=3)	Recovered (mg)	% recovery	% RSD
5	4.99	99.99	0.50
	4.97	99.48
	4.94	98.98
10	9.96	99.66	0.51
	9.99	99.99
	9.90	99.03
15	14.99	99.97	0.96
	14.73	98.24
	14.76	98.40

n = number of determination, % RSD = Relative Standard Deviation

Limit of detection (LOD) and limit of quantification (LOQ):

Limit of detection (LOD) and limit of quantification (LOQ) were calculated as 3.3 X SD/S and 10 X SD/S respectively as per ICH guidelines. Where SD is the standard deviation response (Y-intercept) and S is the slope of the calibration curve. The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio 3). The LOQ is the smallest concentration of analyte which gives the response that can be accurately quantified (signal to noise ratio 10). The calculated LOD and LOQ values were shown in the T able No.3.

Precision:

Method precision for Alectinib drug solution was checked by repeatability. We prepared six injection samples of same concentration of 100 µg/ml of Alectinib drug solution and injected into the chromatographic system.The peak area of each injection was used to calculate the % RSD.For evaluating the intermediate precision we analyzed six injections of 100 µg/ml concentration of Alectinib drug on different days by different columns of same dimensions by different analysts. Each injection area was used to calculate the % RSD. From the data obtained in Table No.5 and 6. The developed method was found to be precise.

Table No. 5: Repeatability study data for Alectinib drug

Drug	Mean peak area (n=6)	SD	%RSD
Alectinib	3161990	15396	0.48

n – Number of determination, SD - Standard Deviation, % RSD - Relative Standard Deviation

Table No.6: Reproducibity data for Alectinib drug

Reproducibility	% RSD
Intra-day precision
Instrument 1 (n =6)
Pa ratio (n =6)	0.48
Rt ratio (n =6)	0.3
Inter-day precision
Instrument 2 (n =6)
Pa ratio (n =6)	0.51
Rt ratio (n =6)	0.1

n = number of determination, % RSD = Relative Standard Deviation, Pa = peak area, Rt = retention time

CONCLUSION:

A fast, accurate and an isocratic RP-HPLC method for the estimation of Alectinib drug in the pharmaceutical dosage form was established and validated as per ICH guidelines. Linearity was achieved for Alectinib in the range of 10 – 50 µg/ml with correlation coefficient (r²) 0.999. The % recovery was achieved in the range of 98 – 102 % which was within the acceptance criteria. The % RSD was less than 2 % which proved the precision of the developed method. The advantage of this method is suitable retention time and high sensitivity. The effect of flow rate and organic composition of mobile phase on system suitability parameters were investigated to obtain the optimum chromatographic conditions. The obtained results suggested that the developed method was validated for precision, accuracy, linearity, LOD and LOQ, and robustness. Hence this method can be used for the routine analysis of Alectinib drug in pharmaceuticals.

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Received on 25.08.2018 Accepted on 29.09.2018

Asian J. Pharm. Ana. 2018; 8(4): 186-190.

DOI: 10.5958/2231-5675.2018.00034.0

Assay %	=	Sample area	X	Dilution sample	X	P	X	Avg.wt.	X	100
		Standard Area		Dilution of standard		100		Lc