Stability Indicating HPTLC Method Development and Validation for Acebrophylline in Bulk and its Tablet Dosage Form

 

Arun Maruti Kashid¹*, Amey Chandrakant Khilare², Shivanjali Balasaheb Sable²

¹Department of Pharmaceutical Chemistry, Sinhgad Institute of Pharmacy, Narhe, Pune, Maharashtra, India - 41.

² Department of Pharmaceutical Quality Assurance,

Sinhgad Institute of Pharmacy, Narhe, Pune, Maharashtra, India – 41.

 

ABSTRACT:

A simple, precise, accurate stability-indicating method was developed and validated for Acebrophylline in bulk and tablet dosage form. The chromatographic separation was achieved by using Methanol: Acetonitrile: Acetone: Formic acid (4:4:2:0.1 v/v/v/v) as mobile phase and UV detection at 272nm. The retention factor for Acebrophylline was found to be 0.74±0.10. The developed method was validated with respect to linearity, accuracy, precision, limit of detection, limit of quantitation, and robustness as per ICH guidelines. Results were found to be linear in the concentration range of 400-2400ng band ^-1 with a correlation coefficient of 0.9921. The % assay was found to be 102.4±5. The drug was subjected to stress conditions of hydrolysis (acid, base), oxidation, photolysis, and thermal degradation. The developed method can be used for the quantification of drug in the dosage form, bulk drug as well as for routine analysis in quality control laboratories.

 

 

 

INTRODUCTION:

Acebrophylline, chemically known as 4-[(2-amino-3,5-dibromophenyl) methylamino] cyclohexan1-ol; 2-(1,3-dimethyl-2,6-dioxopurin-7-yl)acetic acid, is a compound formed by combining equimolar amounts of theophylline-7-acetic acid, a xanthine derivative that inhibits phospholipase A, and phosphatidylcholine. This combination results in the formation of a salt known as acebrofylline.¹ Acebrofylline acts by reducing the production of potent pro-inflammatory substances such as leukotrienes and tumor necrosis factor. These substances are known to contribute to inflammation, which is a significant factor in airway obstruction, particularly in chronic forms.

 

By inhibiting the synthesis and release of these inflammatory mediators, acebrophylline effectively reduces inflammation, providing relief from airway obstruction.¹High-Performance Thin Layer Chromatography is a versatile technique that can be used for the separation, identification, and quantification of a wide range of compounds, including small molecules, natural products, pharmaceuticals, and food additives. It can be applied to various sample types, such as liquids, solids, and semi-solids.^2-3 According to a thorough literature survey review, there are only a few analytical methodsfor determining Acebrophylline in bulk, Pharmaceutical formulations which include RP-HPLC, HPTLC and UV-Spectroscopic method.^4-8   

 

The objective of this study to describe a simple, rapid, accurate, precise and robust stability indicating HPTLC method for Acebrophylline in bulk and it’s tablet dosage form. The parameters used to validate the method were linearity, precision, accuracy, limit of detection, limit of quantification and robustness.

Drug profile:

Acebrophylline detail information is given in table no. 1

 

Table 1. Drug profile of Acebrophylline

 

MATERIALS AND METHODS:

1. Chemicals and Reagents:

Methanol was used in mobile phase composition and it is also used as a diluent, which was purchased from  HiMedia Laboratories Pvt. Ltd. Mumbai. Other chemicals used were Acetonitrile, Acetone and Formic acid of Analytical grade also purchased from HiMedia Laboratories Pvt. Ltd. Mumbai.

 

2. Instruments: 

High-Performance Thin Layer Chromatography (HPTLC) manufactured by CAMAG was used in the current study. For UV- analysis Double beam UV spectrophotometer model V-630 manufactured by Jasco International Co. Ltd. Japan is used. For functional group detection, FTIR Spectrophotometer 4100 series is used manufactured by Jasco, Japan. For accurate weighing of drug weighing balance model CB-50 is used manufactured by Contech, Pune. For sonication purposes, the UCB-40 Sonicator is used manufactured by Spectra Lab. For drying purposes, a Hot air oven 250 BSS is used and manufactured by Pathak Pvt. Ltd, Mumbai.  

 

3. Selection of optimum condition for the HPTLC process:

In order to view the appropriate peak for the Acebrophylline the mobile phase used was Methanol: ACN: Acetone: Formic acid (4:4:2:0.1). saturation time for the mobile phase is kept for 15 min. Aluminum plates precoated with silica gel 60 F254 (Merck, Ltd. Mumbai.) are used for the application of the sample.

 

4.     Preparation of solutions:

a.     Selection of Wavelength for Determination

The range of 200-400nm was used and a concentration of 1600µg/ml for the determination of the wavelength of the standard solutions of Acebrophylline. ACB showed maximum absorbance at 249nm which further selected for the analysis

b. Preparation of standard solution

20mg of Acebrophylline standard were accurately weighed and transferred into 10mL volumetric flask respectively. About 10mL of methanol was added, sonicated to dissolve. Final concentration of Acebrophylline were made to 2000µg/ml.

 

c. Preparation of Sample solution:

The marketed formulation of Acebrophylline manufactured by Sun Pharma Laboratories Ltd. was available in the local market. The quantity of tablet equivalent to 20mg of Acebrophylline was transferred into a 10mL volumetric flask. About 10mL methanol was added and the solution was sonicated for 5mins with intermittent shaking. The final concentration of Acebrophylline was made to 2000µg/ml.

 

d. Analysis of Marketed Formulation (Tablet):

The sample solution was applied to the TLC plate five times. The plate was developed, scanned in a densitometer and the peak is measured and the % assay was calculated against the peak area of a band of standard solution applied in the same concentration.

 

Forced Degradation Studies¹⁵  

The degradation samples were prepared by transferring 20 mg of Acebrophylline into a 100 ml volumetric flask and diluted to the mark with 0.1 N HCl, 0.1 N NaOH, and 3% H₂O₂ for acidic, basic and oxidative degradation studies respectively. The sample solution was kept at room temperature for 30 min then samples were withdrawn and applied on HPTLC plate. 

 

Method Validation:^9-14

According to ICH Guidelines Method Validation is defined as “established the documented evidence which provides a high degree of assurance that a specific process will consistently produce a product of predetermined specifications and quantity attributes”

 

The developed method was validated as per the ICH guidelines Q2 (R1) for the parameter’s linearity, accuracy, precision, limit of quantitation, limit of detection, and robustness.

 

1. Accuracy:

Accuracy refers to how closely a measured value aligns with the true value. In a highly accurate method, the measured value matches the true value of a known sample.

Accuracy can be determined through three approaches:

·       Comparison to a reference standard.

·       Recovery of the analyte spiked into a blank matrix.

·       Standard addition of the analyte.

 

2.  Precision:

Precision in an analytical method refers to the degree of agreement among a series of measurements obtained from repeated sampling of the same homogeneous material under similar analytical conditions. It can be categorized into three types.

i. Repeatability: This refers to the precision achieved when the method is used by the same analyst under identical operating conditions within a short time period.

ii. Intermediate precision: This category assesses the method's precision by testing it on different days, using different instruments, or involving different analysts, while maintaining the same methodology.

iii. Reproducibility: Reproducibility evaluates the precision of the method through inter-laboratory studies, where different laboratories apply the same methodology to analyse the same sample. According to the guidelines provided by the International Council for Harmonisation (ICH), to ensure repeatability, it is recommended to perform a minimum of 9 determinations within a specified range (such as three concentrations with three replicates each), or a minimum of 6 determinations at 100% of the test concentration.

 

3) Specificity:

Specificity relates to the capacity of an analytical method to accurately distinguish and quantify the target analyte within complex mixtures. It is particularly important when assessing impurities and validating identification tests. During the determination of impurities and the validation of identification tests, it is essential to thoroughly investigate the specificity of the analytical method.

 

4) Limit of Detection (LOD) and Limit of quantification (LOQ):

LOD is the lowest concentration in a sample that can be detected but not necessarily quantified under the stated experimental conditions and LOQ is the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy.

LOD = 3.3*(SD) / S

LOQ = 10* (SD) / S

where,

SD = Standard deviation of response

S = Slope of the calibration curve.

 

 

5) Robustness:

Robustness refers to how sensitive the method is to uncontrolled small changes in parameters such as temperature, pH of solution, mobile phase composition, and flow rate. There should not be interference from a blank with the main peaks Active ingredients peak in test preparation should be spectrally pure.

 

RESULT AND DISCUSSION:

Drug Identification:

Melting point measurement, solubility studies, and UV and IR spectra were used as preliminary work for drug identification, which yielded the following results.

Solubility Study:

A solubility study was carried out with different solvents such as methanol, acetonitrile, ethanol, water, triethylamine, ethyl acetate, chloroform, ether, acetone, and ammonia. Acebrophylline is soluble in methanol, DMSO, ethyl acetate, ethanol, conc. HCl, tetrahydrofuran, acetonitrile, formic acid, water, and acetic acid.

b. Melting point determination:

The melting point for Acebrophylline was evaluated and it was compared with reported standard values which is found to be 213-214°C.

 

UV analysis:

In order to perform UV analysis, a 10 mg quantity of the drug was dissolved in 10 ml of methanol, resulting in a 1000 ppm solution. Then, 1 ml of this solution was withdrawn and diluted to a final volume of 10 ml, creating a 100 ppm solution. Subsequently, 2ml, 4ml, 6ml, and 8ml portions of the 100ppm solution were separately withdrawn and each diluted to a final volume of 10 ml. This yielded 20 ppm, 40 ppm, 60 ppm, and 80 ppm solutions, respectively. The absorbance of each of these solutions will be measured, and the R² value will be calculated. The scanning range will be selected from 200-400 nm. The reported wavelength for Acebrophylline is 272-274 nm and wavelength found will be 272.4 nm.

 

Fig 1: UV Spectrum of Acebrophylline

 

Fig. 2: FT-IR of Acebrophylline          

 

 

Fig. 3 Isobestic Point by HPTLC for Acebrophylline

 

FT-IR Spectroscopy:

The IR spectrum is shown in Fig. 2 and the functional group detected was shown in Table no. 3

 

Table 2: IR frequencies of Acebrophylline functional group

 

In Figure 3, the isobestic point spectra of Acebrophylline were presented. The spectra were obtained by scanning a range of 200-400nm. During the analysis, a significant peak was detected at a wavelength of 249nm.

 

Method Validation:

Calibration curve:

The linear regression data for the calibration curve showed a good linear relationship over the concentration range 400-2400ng/band. A linear regression equation was found to be y = 402.17+2.316*X, r² = 0.992

 

Precision:

The precision of the developed method was represented in terms of the % relative standard deviation(%RSD) of the peak area. The results depicted indicated the high precision of the method is presented in Table 3.

 

Table 3: Precision data for Acebrophylline.

 

Recovery studies:

The recovery studies were executed at 80%, 100%, and 120% of the test concentration as per ICH guidelines. The % recovery of Acebrophylline at all three levels was found to be satisfactory. The amounts of drug added and determined and the % recovery are listed in Table 4.

 

Table 4: Accuracy data of Acebrophylline

LOD and LOQ:

The limit of detection (LOD) of the developed method was found to be 0.23 ng per spot while the limit of quantitation (LOQ) was found to be 0.72 mg per spot for Acebrophylline.

 

Robustness:

Table 5: Robustness of Acebrophylline.

 

The method was found to be unaffected by changing the parameters. It also did not show any significant changes due to alteration of mobile phase composition, and saturation time. The developed method was found to be robust.

 

Overall, the validation parameter for acebrophylline is validated according to ICH guidelines. They are Linearity, Precision, Accuracy, and Robustness. results for all parameters are summarized below in table no. 6

 

Table 6: Summary of validation parameters for Acebrophylline

 

Degradation studies:^16-17

Acebrophylline was subjected to forced degradation studies, where it was exposed to different stress conditions. The resulting chromatograms can be observed in Figure (4-7). The percentage of degradation was determined and documented. It was found that Acebrophylline undergoes decomposition of 3.91%, 6.74%, 4.54%, and 7.55% under acidic, alkaline, oxidative, and Neutral conditions, respectively, producing multiple degradation products. It can be concluded that Acebrophylline demonstrates moderate degradation in oxidative and Neutral conditions.

 

 

Fig.4: Degradation of Acebrophylline in 0.1 N HCl

 

Fig. 5: Degradation of Acebrophylline in 0.1 N NaOH

 

 

Fig. 6: Degradation of Acebrophylline in 3%H₂O₂

 

Fig. 7: Degradation of Acebrophylline in Distilled water. 

 

CONCLUSION:

The developed HPTLC method is precise, specific, sensitive, and accurate. Statistical analysis proved the method is reproducible and selective for the analysis of Acebrophylline in the bulk drug and its tablet dosage form. The developed method is validated as per ICH Guidelines for validation parameters like Linearity, Accuracy, Precision, and Robustness The forced degradation study shows that ACB is more degraded in Neutral, base, and oxidative conditions. Forced degradation studies provide knowledge about possible degradation pathways and degradation products of the active ingredients. This information will help to improve the formulation manufacturing process and determine the storage conditions. The present method can be used for the analysis of stability samples of Acebrophylline.

 

ACKNOWLEDGEMENT:

The authors were thankful to STES, Sinhgad Institute of Pharmacy, Narhe, Pune for providing necessary infrastructural facilities to carry out research work.

 

DISCLOSURE STATEMENT:

No potential conflict of interest was reported by authors.

 

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Received on 19.07.2023       Modified on 13.08.2023

Accepted on 16.09.2023   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2023; 13(4):255-260.