1. INTRODUCTION:

According to numerous recommendations for national clinical practice, alectinib is the recommended initial treatment for individuals with advanced non-small-cell lung cancer (NSCLC) that is positive for anaplastic lymphoma kinase (ALK).¹^,² The IUPAC name of Alectinib is 9-ethyl-6,6-dimethyl-8-(4-morpholin-4-ylpiperidin-1-yl)-11-oxo-5H-benzo[b]carbazole-3-carbonitrile and chemical formula is C₃₀H₃₄N₄O₂ with the molecular weight 482.62g/molas shown in Figure 1.³^,4

Non-small-cell lung cancer (NSCLC) that is anaplastic lymphoma kinase (ALK)-positive (5% of patients with advanced NSCLC) is a unique subtype of lung cancer.⁴^,⁵ Advanced ALK-positive NSCLC patients tend to be younger than patients with other kinds of lung cancer, to smoke less or never, and to present with the disease at an advanced stage.⁶^,⁷^,⁸

Blood film analysis revealed morphological results such as spheroacanthocytes, moderate-marked acanthocytes, and moderate schistocytes in one case.^9,10It has also been used for tumors that produce the ALK fusion protein, including as anaplastic large cell lymphoma, inflammatory myofibroblastic tumors, and neuroblastoma.^11,12It is used primarily to treat non-small cell lung cancer (NSCLC) that expresses the fusion protein ALK-EML4 (microtubule-associated protein-like in echinoderm 4), which promotes the growth of NSCLC cells.¹³ALK inhibition reduces the viability of cancer cells by preventing the phosphorylation and subsequent downstream activation of STAT3 and AKT.¹³^,¹⁴^,15

Based on a survey of the literature, only few methods of Alectinib estimation in LC MS/ MS have been published.^16,17,18Literature survey reveals that one HPLC method, however, a thorough literature hunt failed to turn up any estimating ways for alectinib in medicinal or API in capsule dosage form.^19-22In order to estimate the quantum of alectinib in medicinal capsule form, a simple, precise, accurate, and provident HPTLC system has been developed and validated in agreement with ICH conditions.^23,24

Fig. 1: Chemical structure of alectinib

2. MATERIAL AND METHODS:

2.1 Instrumentations:

For the application and detection of spots, respectively, the HPTLC system was used, which included the Camag Linomat 5 sample applicator, Camag Hamilton Bonaduz microlitre syringe (100µl), The Camag TLC scanner 4 and the UV chamber with dual-wavelength UV lamps are managed by the vision CATS software (CAMAG). Drug chromatographic separations were carried out using pre-coated HPTLC plates with silica gel 60 F254 (10 × 20cm, 250µm thickness) from Sigma Aldrich. The development of the chromatographic technique was done in a CAMAG twin-trough developing chamber.

2.2 Materials and reagents:

Reference standards for alectinib with a determined potency of 99.5% have been obtained from the Central Drug Testing Laboratory in Mumbai. Alecensa® Hard capsules 150mg (Roche) were purchased from the local market whereas analytical grades of toluene, glacial acetic acid, methanol, and ethyl acetate (HPLC grade) were purchased from Finar Chemicals (Ahmedabad, India). We bought silica gel 60 F254 plates from Sigma-Aldrich in Mumbai, India.

2.3 Selection of wavelength:

The alectinib standard solution (100PPM) was scanned within the scope of 200.0 to 400.0nm using the CAMAG TLC Scanner 4. alectinib showed maximum absorbance at 340nm as shown in Figure 2. Hence, 340nm was selected for the examination of alectinib.

2.4 Preparation of standard solution (100 ppm):

10mg of alectinib reference standard was dissolved in 100mL of methanol to make a final concentration of 100 µg/mL (100 ppm).

2.5 Examination of the marketed formulation:

To determine the concentration of alectinib in capsule dosage form (150mg per capsule), the content of 20 tablets were weighed precisely and average weight was determined. An accurately weighed powder, equal to ten milligrams of alectinib was weighed and transferred into a 50milliliter volumetric flask containing 30ml methanol and 15 minutes of sonication and volume was makeup precisely on target with methanol. The solution is Go through Whatman filter paper number 4. Further dilutions were made to get a concentration (100μg/ml).

2.6 Chromatographic conditions:

Prior to chromatography, the Plate was activated at 110 °C for 20 minutes. 20ml during the mobility phase was placed within the development tank, which was covered with a lid, to saturate the chamber. The assembly was left aside at room temperature for 45 minutes. The samples appeared as 8mm long, elongated narrow bands. To prevent edge effects, the application positions X and Y were fixed at 8mm and 20mm, respectively. Twenty millimetres separated the two bands. Using a nitrogen aspirator, bands were administered at a constant rate of 5nL/s.

In a Camag glass chamber with two troughs saturated in conjunction with the mobile phase made composed of toluene, methanol, ethyl acetate, and glacial acetic acid [6:2:2:1 v/v/v/v] for 45min, the chromatogram was developed linearly ascendingly with the run maintained at 70mm. After the development, the HPTLC plates were dried in a wooden room with sufficient ventilation using an air dryer and a stream of air. The separated components were subjected to a Spectro densitometric examination utilising a Camag TLC Scanner 4 in the reflectance-absorbance mode at 340nm and a deuterium lamp as the radiation's source. The sensitivity was left in auto mode, and the slit size was 6.0mm x 0.3mm. It scanned at a speed of 100nm/s. By employing vision CATS (CAMAG) software for peak area measurement and data processing, the evaluation was accomplished by linear regression within the peak region response against the amount of medication.

2.7 Method optimization:

Using silica gel F254 plates as a stationary phase, alectinib's molecular structure and solubility data were taken into consideration. distinct mobile phase with various compositions of solvents like methanol and toluene. Finally, symmetrical peak shape and acceptable SST parameters were found in conjunction with the mobile phase comprising of Toluene: Methanol: Ethyl acetate: 0.1% Glacial acetic acid (6:2:2:0.1% v/v/v/v).

2.7 Method validation:

The validation of developed method was performed for the specificity, linearity, precision, accuracy, and recovery, limit of quantitation (LOQ), limit of detection (LOD) and robustness as per ICH Q2 (R1) guidelines.

2.7.1 Specificity:

The method's specificity was demonstrated by the examination of alectinib standard solutions. The band for alectinib was verified by comparing the standard's band spectrum and RF value. The spectrum was examined at peak apex (M), peak start (S), and peak end (E) of the band to determine the peak purity of alectinib.

2.7.2 Calibration curve:

The linearity of alectinib was determined within the range of concentration of 100-1500ng/band, by applying seven different concentrations of reference standard solutions of alectinib in triplicates. The linearity of alectinib was illustrated in Figure 6 by plotting the linearity graph, which shows peak regions vs concentrations.

2.7.3 Precision:

Precision of the developed method was verified by performing repeatability and intermediate precision. Peak area measured and calculated the %RSD. The data given in the table 2 and table 3.

2.7.4 Sensitivity:

The lowest amount of analyte in a sample that can be detected but may not be quantified as an exact value under experimental conditions is known as the limit of detection (LOD), and the lowest amount that can be detected and quantified with appropriate precision, accuracy, and reproducibility is known as the limit of quantification (LOQ). These terms are used to express sensitivity. Using the following equation, the LOD and LOQ were determined based on the slope of the calibration curve and the standard deviation of the regression lines: LOD = 3.3×σ/S

LOQ=10×σ/S

where S is the calibration curve's slope and σ is the regression line's standard deviation. Table 1 provides a summary of the findings.

2.7.5 Accuracy:

These experiments can be performed by the recovery test. The method recovery was assessed using known amounts of standard preparation at three different concentration level i.e. 110, 120, 130. From three set, each concentration in duplicated were made and injected. The drug reference standard was included in the formulation (pre-analyzed sample) at levels of 110, 120, 130. The % recovery and % mean recovery for the drug was calculated. Results shown in table 5.

2.7.6 Robustness:

The small variation in method parameters like the mobile phase volume, saturation time and distance travelled by solvent were evaluated. The Rf and standard deviation of peak area were calculated for each parameter and %RSD was determined as less than 2.0 and the table displays the outcomes table 6.

3. RESULTS AND DISCUSSION:

By comparing the alectinib spectra at peak start (S), peak apex (M), and peak end (E), the peak purity was ascertained. The peak purity and correlation values were found to be within the acceptance limits, which confirms that there are no interferences with respect to blank and excipient in the quantitation of alectinib in sample solution and that the approach is specific.

Alectinib has linearity in the 100–1500 ng/band range, and a 0.999 correlation coefficient was discovered. The regression equation obtained was Y=6E-05x + 0.002. The linearity graph was plotted by taking the concentration of the drug on the X-axis and corresponding peak areas on the Y-axis as shown in Fig 6.

The percentage RSD values for repeatability and intermediate precision for the suggested approach were determined to be within the allowed limits according to ICH rules, indicating that it was exact. The precision and intermediate precision data of alectinib are depicted in Table 3 and 4 respectively.

The average percentage of recovery of alectinib sample was discovered to be 99.71% which is within the acceptance limit of 95-105 %, which proves the method to be accurate and suitable for routine analysis of alectinib in pharmaceutical bulk dosage form. The accuracy data of alectinib is summarized in Table 5.

The lower LOQ and LOD values for alectinib indicate that the technique that was created is sensitive. The RF value and % RSD of peak areas for alectinib were found to be within the specified limits.

The robustness of the suggested approach is demonstrated by the low percentage RSD values and unaffected RF values even after purposeful modifications to the method parameters. The results of robustness studies of alectinib are listed in Table 6.

The examination of the alectinib pharmaceutical bulk dosage form possessing a strength of 150 mg showed successful agreement with the label claim as shown in Table 7. Mean % recovery was discovered to be 100.0 Hence, It suggests that alectinib in pharmaceutical bulk dose form can be routinely analyzed using the suggested technology, which is exact and accurate.

Fig. 2: UV spectrum of alectinib

Fig. 3: Three Dimensional densitogram of alectinib

Fig. 4: TLC Visualization

Table 1: Regression analysis data

Parameters	HPTLC method
Linearity range (ng/band)	100-1500ng/band
Regression equation	Y=6E-05x + 0.002
Detection wavelength (nm)	340
Correlation coefficient (R²)	0.999
Limit of detection (ng/band)	3.3ng/band
Limit of quantification (ng/band)	9.87ng/band

Fig. 5: Caliberation curve of alectinib

Table 2: Linearity data for alectinib

Concentration (ng/band)	Peak Area
10	0.00254
50	0.00542
75	0.006965
100	0.00851
125	0.01004
150	0.01171

Table 3: Precision data for alectinib. (Repeatability)

Sr. No	Concentration ng/band	AREA
1	100	0.00778
2	100	0.00775
3	100	0.00773
4	100	0.00771
5	100	0.00772
	MEAN	0.007738
	SD	2.77489E-05
	%RSD	0.358605245

Table 4: Intermediate precision data for alectinib

Oncentration ng/band

Intraday Precision

Interday Precision

Area ± SD

%RSD

Area ± SD

%RSD

100

150

0.005096±0.00004794

0.0065322±0.00001664

0.008177±0.00002866

0.940347

0.25521

0.348298

0.005193±0.00002697

0.006826±0.00003155

0.008278±0.00003553

0.53194

0.46130

0.43977

Table 5: Accuracy data for alectinib

% Level	Amount Found	Amount Found (%)	% Recovery	Mean % recovery	SD of % recovery	%RSD of % recovery
100	99.3658913	99.36%	99.37
100	99.5632634	99.56%	99.56	99.42	0.120782312	0.12148159
100	99.3439611	99.34%	99.34
110	110.747683	110.74%	100.68
110	110.528381	110.52%	100.48	100.68	0.199365766	0.1980198
110	110.966985	110.96%	100.88
120	119.300474	119.30%	99.42
120	119.190823	119.19%	99.33	99.42	0.091375976	0.09191176
120	119.410125	119.41%	99.51
130	129.114254	129.11%	99.32
130	129.16908	129.16%	99.36	99.32	0.042173527	0.04246285
130	129.059428	129.05%	99.28

Table 6: Robustness studies

Sr. No	Parameters	Ratio	Mean %	SD	%RSD
1	Changes chamber saturation time (45min 5± min)	40	101.28	0.434	0.428
1	Changes chamber saturation time (45min 5± min)	50	101.65	0.461	0.453
2	Changes in mobile phase volume (20ml ± 2ml)	22	101.53	0.263	0.260
2	Changes in mobile phase volume (20ml ± 2ml)	18	101.63	0.064	0.0637
3	Changes in distance travelled by solvent (70min ± 5min)	75	102.48	0.320	0.312
3	Changes in distance travelled by solvent (70min ± 5min)	65	102.50	0.156	0.152

Table 7. Analysis of alectinib in capsule dosage form

Formulation name	Label claim (mg)	Amount found in Containing 150 mg	% Label claim
	150	150.8	100.54
Alecensa	150	149.9	99.94
	150	150.2	100.1
Mean			100.1933333
SD			0.310698139
%RSD			0.310098615

5. CONCLUSION:

The suggested HPTLC technique was found successfully validated for parameters such as specificity, linearity, system suitability, precision, assay, accuracy and LOD, LOQ and robustness according to ICH guidelines. The process was discovered to be easy, rapid, economical and precise. All validation parameters were within the acceptance limits. Hence this technique that can be applied for routine analysis and quality control of alectinib in capsule dosage in pharmaceutical industry. Statistical analysis proved that the approach was repeatable and selective for the examination of alectinib without any interference from the excipients.

6. ACKNOWLEDGMENT:

The authors are grateful to Central drug testing laboratory (CDTL), Mumbai for supplying the sample, standard of alectinib and also providing the requirementsfor the project. We are also thankful to Principal and faculty from Gahlot institute of pharmacy, koparkhairane, Navi Mumbai, for providing all necessary facilities.

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Received on 23.08.2023 Modified on 19.02.2024

Research J. Pharm. and Tech 2024; 17(8):3620-3624.

DOI: 10.52711/0974-360X.2024.00565