Stability Indicating TLC–Densitometric Method for Determination of Alcaftadine in Presence of its Degradation Products and Dosage form Preservatives

 

Aya T. Soudi¹*, Ola G. Hussein², Eman S. Elzanfaly¹, Hala E. Zaazaa¹, Mohamed Abdelkawy²

¹Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt.

²Pharmaceutical Chemistry Department, Faculty of Pharmaceutical Science and Pharmaceutical Industries, Future University in Egypt, Cairo, Egypt.

 

ABSTRACT:

Stability indicating methods are essential to determine the stability of newly developed dosage forms. In this work a simple stability-indicating thin-layer chromatographic (TLC) method was developed and validated to determine Alcaftadine in presence of its main degradation products and preservative benzalkonium chloride in bulk powder and in pharmaceutical formulations. Separation was developed on TLC aluminum plates precoated with silica gel 60F-254 as a stationary phase using chloroform: methanol: ammonia (5:5:0.1, by volume) as a developing system. Densitometric scanning was carried out in the absorbance mode at 282nm. Alcaftadine was subjected to oxidation, acid and alkaline hydrolysis. It was observed that the drug is susceptible to oxidation only. Method validation was carried out according to ICH guidelines and the proposed method was successfully applied to analyze alcaftadine in pharmaceutical dosage form with no interference from dosage form additives or preservatives. Linearity range was found to be 0.50 - 12.00µg/band with a good linear relationship r of 0.9998. Results obtained by the proposed TLC-densitometric method were statistically compared with those obtained by a reported spectrophotometric method and no differences were found.

 

 

 

INTRODUCTION:

TLC is a popular simple separation technique which can be used as stability indicating method for many drugs [1-7]. TLC automation was used for precise application of the samples, computer-controlled evaluation and quantification of the developed chromatograms. So, it can be considered as reliable for quality control and quantitative drug testing [8,9].

 

A Stability-indicating assay method is a validated quantitative analytical method that can detect any chemical, physical or microbiological changes of the drug substance and drug product.

 

 

They must be specific so that the content of active ingredients and degradation products can be accurately measured without interference [10,11]. The purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light, and to establish a re-test period for the drug substance or a shelf life for the drug product and recommended storage conditions [12-14]. The ICH guideline indicates that stress testing is designed to help determine the intrinsic stability of the molecule by establishing degradation pathway in order to identify the likely degradation products and to validate the stability indicating power of the analytical procedure used [15,16].

 

Alcaftadine (ALC) is H1 histamine receptor antagonist indicated for the prevention of itching associated with allergic conjunctivitis [17,18]. It is a broad-spectrum antihistamine known to display high affinity toward histamine H1 and H2 receptors and a lower affinity for H4 receptors [19]. It also exhibits modulatory action on immune cell recruitment and mast cell stabilizing effects. It is chemically known as 6, 11-dihydro-11-(1-methyl-4-piperidinylidene)-5H-imidazo [2, 1-b] benzazepine-3-carboxaldehyde [20] (Figure 1).

 

Figure 1. Chemical structure of Alcaftadine.

 

Benzalkonium chloride (BZCL) is benzyl-dimethyl-tridecyl-azanium chloride [21]. It is found in up to 70% of eye drops and is still the most common preservative used in ophthalmic solutions at a concentration of 0.001–0.1% [22].

 

Alcaftadine is not an official drug. A literature survey on ALC revealed that several analytical methods were reported for the analysis of ALC including high performance liquid chromatography (HPLC) [23], spectrophotometry [24] and UPLC/MS method [25]. Till date, to best of our knowledge no stability indicating TLC-Densitometric method has been reported for determination of ALC in presence of its degradation products and BZCL preservative.

 

The aim of this work is to develop and validate a simple and selective stability indicating TLC-Densitometric method for determination of ALC either in bulk powder or in pharmaceutical formulation in the presence of its degradation products and BZCL.

 

EXPERIMENTAL:

Instruments:

Precoated silica gel TLC aluminum plates 60F-254; 20 cm × 20 cm with 0.2 mm layer thickness, Fluka (Buchs, Switzerland) were used and the samples were applied using a CAMAG Linomat 5, autosampler (Muttens, Switzerland) with a Camag 100µl sample syringe (Muttens, Switzerland) at an application rate of 10µl/s as a band of 3 mm width. CAMAG TLC densitometric Scanner 3S/N 130319 in the reflectance absorbance mode was used for densitometric scanning with a speed of 20 mm/s, the slit dimension kept at 6.00 mm ×0.30 mm and space between two bands was 10.5 mm. The scanner was operated by WINCATS software (Muttens, Switzerland). Visualization of plates was done using a UV lamp with a wavelength of 254 nm.

 

MATERIALS AND REAGENTS:

Standard Samples:

Pure standard alcaftadine was purchased from Sigma-Aldrich Co., Cairo, Egypt. Its purity was found to be 99.8% according to the reported method [24].

 

Pharmaceutical Dosage form:

Lastacaft^™ ophthalmic solution (Batch Number 99860) was purchased from the Gulf market; each one mL is labeled to contain 2.50mg alcaftadine and 0.005% BZCL as preservative, manufactured by Allergan sales, LLC Waco, TX 76712, USA.

 

Chemicals and Reagents:

All solvents and chemicals used throughout this work were of analytical grade and were used without further purifications.

 

Methanol (grade ≥ 98%) and chloroform (grade ≥ 99%) were obtained from Sigma-Aldrich, Egypt. Hydrogen peroxide (30%) was obtained from EL-Nasr Pharmaceutical Chemical Co. Abu-Zabaal, Cairo, Egypt.

 

Preparation of degradation products:

Acid and base hydrolysis were tried by separately refluxing 25.00mg of ALC powder in 25mL of 5N HCl for acid hydrolysis and 25.00mg of ALC powder in 25 mL of 5N NaOH for base hydrolysis in a round bottom flask at 80ºC for 1, 3, 5, 10, 24, 36, 48, 72 hours. Each solution was spotted on TLC plates after neutralization along with standard ALC to detect any changes.

 

Oxidation was carried out by dissolving 25.00mg of ALC powder in 25mL of methanolic solution of hydrogen peroxide (10%, by volume). The solution was kept for 4 hr at room temperature. The solutions were spotted on TLC plates every 1 hr along with standard ALC to confirm complete degradation.

 

Standard Solutions:

Standard stock solution of ALC was prepared in methanol at concentration of 2.00mg/mL.

 

Stock solutions in methanol of oxidative degradation products of ALC derived from complete degradation of 1.00mg/mL of ALC.

 

Procedures:

Chromatographic conditions:

Chromatographic separation was performed using precoated silica gel 60 F-254 TLC aluminum plates (20×20 cm). Samples were applied in the form of bands (3mm Length, 10.5mm spacing, and 15mm from the bottom edge of the plate). Linear ascending development was performed in a chromatographic tank previously saturated with chloroform: methanol: ammonia (5:5:0.1, by volume) for 15 mins at room temperature to a distance of about 80mm. The developed plates were air dried and then scanned at 282nm.

 

Construction of the calibration curve:

Aliquots equivalent to 0.50-12.00mg of ALC were separately transferred from their respective standard solution (2.00mg/mL) into a series of 10mL measuring flasks and the volume of each flask was completed to mark with methanol. Ten microliters of each solution were applied in triplicate on TLC plates in the form of bands and then analyzed under the previously mentioned chromatographic conditions. The area under the peak was recorded and calibration curve relating the integrated area under peak versus the corresponding concentration as µg/ band was then constructed, from which the regression equation was computed.

 

Application to pharmaceutical formulation:

Two mL of Lastacaft^™ ophthalmic solution was transferred into 10mL volumetric flask and volume was completed to the mark with methanol. Ten microliter from the previously prepared solution were spotted on TLC plate. Development and quantification were performed under the previously mentioned chromatographic conditions.

 

RESULTS AND DISCUSSION:

A simple, sensitive and selective TLC stability indicating assay method was developed and validated to determine ALC in bulk powder and pharmaceutical formulation. The method determines ALC either in presence of its oxidative degradation products or eye drop preservative BZCL.

 

Stress studies were performed on the bulk drug by examining the factors suggested by ICH guidelines Q1A (R2). It was observed that ALC is stable to acid and base hydrolysis but susceptible to oxidation only with the production of three degradation products (Figure 2). According to the chemical structure it can be noticed that the drug will undergo oxidation through nitrogen present in the imidazole ring as it will gain oxygen and will be converted into N-oxide, also the methyl group attached to the nitrogen in the benzene ring will undergo dealkylation and finally the last part expected to undergo oxidation is the aldehyde group which will be converted into carboxylic acid (Figure 1).

 

Figure 2: TLC chromatogram of (a) 1^st ALC Degrade (Rf 0.128), (b) 2^nd ALC Degrade (Rf 0.20), (c) ALC (Rf 0.58), and (d) 3^rd ALC Degrade (Rf 0.79); mobile phase consists of chloroform: methanol: ammonia (5:5:0.1, by volume).

 

Method Development and Optimization:

Optimization was conducted to achieve satisfactory chromatographic resolution and to improve detection of ALC degradation products.

 

Different experimental condition, such as developing system composition, band dimension and slit dimension, were optimized to provide accurate, precise and reproducible chromatographic separation. Different developing systems were tried such as chloroform: methanol (9:1, by volume), chloroform: methanol (1:9, by volume), chloroform: methanol (5:5, by volume) and chloroform: methanol: ammonia (5:5:0.1, by volume). Using the first two systems bad resolution was obtained between the separated components with tailed peaks. Changing the ratio to (5:5, by volume) greatly enhances the chromatographic resolution among the separated peaks but with slight tailing. Addition of ammonia solution improves the shape of the peaks, hence chloroform: methanol: ammonia (5:5:0.1, by volume) is the developing system of choice.

 

Different band dimensions were tested in order to obtain sharp and symmetrical separated peaks. The optimum band width is 3 mm with 10.5 mm. Maximum sensitivity with minimum noise was obtained at 282nm.

 

Under all the previously stated chromatographic conditions, good separation with acceptable system suitability parameters was achieved (Table 1).

Table 1. System Suitability Testing Parameters of TLC-Densitometric Method

Parameters	(a) 1st ALC Degrade	(b) 2nd ALC Degrade	(c) ALC	(c) 3rd ALC Degrade	[26] References value
Symmetry factor	0.87	1.00	0.87	1.00	=1 for typical symmetric peak
Resolution (RS)	---------------	1.80	5.83.	3.66	> 1
Capacity factor (K')	6.81	4.00	0.72	0.26	⬆️ K’ ⬇️ RF
Selectivity factor (α)	---------------	1.70	5.55	2.76	˃1

^a Average of three determinations.

 

Table 2: Validation Results of the determination of ALC by the TLC-Densitometric Method.

Parameters	TLC-Densitometric Method
	ALC
Linearity Range	0.50-12.00 µg/band
Slope	1108.3
Intercept	1365.1
Correlation coefficient	0.9998
Accuracya	99.97±0.17
Precision(±%RSD)
Repeatabilityb	1.05±
Intermediate precisionc	±1.12
LODd	0.10µg/band
LOQd	0.45 µg/band
Robustness	100.16

^aThe accuracy (n=3), average % recovery of three concentrations (4.00,6.00,8.00µg/band).

^bThe intraday precision (n = 9), average of three concentrations (0.50,3.00,7.00µg/band) repeated three times within the day.

^cThe interday precision (n = 9), average of three concentrations (0.50,3.00,7.00µg/band) repeated three times on three consecutive day.

^dLOD practically by visual inspection and LOQ by signal to noise ratio.

 

Table 3: Determination of ALC in Lastacaft^™ ophthalmic solution by the proposed TLC Densitometric Method and Application of Standard Addition Technique.

Lastacaft™ ophthalmic solution B.N. 99860	% Recovery ± SD	Standard Addition Technique
		Claimed amount taken(µg/band)	Standard added (µg/band)	Total found (µg/band)a	%Recovery of addeda
ALC	99.72± 0.90	5.00 5.00 5.00 5.00	0 1.50 3.00 7.00	4.98 6.48 8.09 11.98	0 101.75 102.06 99.81
Mean ± SD					101.20 ± 1.19

^aAverage of three determinations.

 

 

Method validation:

Method validation was conducted according to International Conference on Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines [27].

 

Under optimum chromatographic conditions, linearity of the developed method was evaluated by measuring the integrated area under the peak of different concentrations of ALC, and then plotting calibration graphs relating the peak area against the corresponding concentration. The obtained regression equation and linearity range are listed in Table 2.

 

Accuracy of the method was evaluated by calculating percentage recovery of 3 replicates of 3 different concentrations covering the linearity range (Table 2). Accuracy was further assured by applying the standard addition technique on Lastacaft^™ ophthalmic solution where good recoveries were obtained revealing no interference from excipients (Table 3).

 

Precision was assessed by three replicate determination of three different concentration of ALC in the same day (repeatability) and on three successive days (intermediate precision). The obtained results (% RSD) revealed precision of the method as shown in Table 2.

 

Specificity was ascertained by achieving good separation between ALC and its degradation products and BZCL preservative as shown in Figures 2 and 3, respectively.

 

Figure 3. TLC chromatogram of (a) ALC (Rf 0.58), (b) Benzalkonium Chloride (Rf 0.95); mobile phase consists of chloroform: methanol: ammonia (5:5:0.1, by volume).

 

Robustness of the proposed method was evaluated by deliberate small changes in the studied chromatographic conditions such as developing system composition (e.g. chloroform amount ±1%, methanol amount ± 1%) and the chromatographic tank saturation time ± 5 min. No significant changes in Rf values, peak areas or symmetry of the peaks were found. Results revealed that the method is robust as shown in Table 4.

 

Table 4: Robustness of the Proposed TLC Densitometric Method.

Robustness parameter		Ta	K'a	Rsb	% Assayc
Developing system amount
a) Chloroform	40.00 + 0.40mL 40.00 - 0.40 mL	0.90 0.85	0.72 0.75	5.82 5.80	100.38 99.51
b) Methanol	40.00 + 0.40 mL 40.00 - 0.40 mL	0.89 0.86	0.74 0.71	5.86 5.85	101.21 99.19
Duration of saturation of chromatographic tank	20.00 + 5.00 min 20.00 - 5.00 min	0.87 0.88	0.72 0.74	5.83 5.81	100.05 100.62

^aTailing factor and capacity factor determined for individual peaks.

^bResolution factor determined between each drug peak and the previous one.

^c% Assay calculated from the regression equation.

 

Application on Lastacaft^™ ophthalmic solution:

The suggested densitometric method was successfully applied to determine ALC in Lastacaft^™ ophthalmic solution (Figure 4). The results are satisfactory and with good agreement with the labeled amounts (Table 3). Upon applying the standard addition technique, no interference due to excipients is observed indicating accurate results as shown in Table 3.

 

Results were statistically compared with those obtained by the reported method with no significant differences between the proposed method and reported one confirming good accuracy and precision of the proposed TLC-Densitometric method (Table 5).

 

Figure 4. TLC chromatogram of Lastacaft^™ ophthalmic solution containing ALC (Rf 0.59); mobile phase consists of chloroform: methanol: ammonia (5:5:0.1, by volume).

 

Table 5. Statistical Analysis of the proposed TLC-Densitometric Method and the Reported Method for Determination of ALC in Lastacaft^™ ophthalmic solution [24].

Parameter	TLC-Densitometric Method	Reported Method [24]
	ALC	ALC
Mean	100.35	99.93
S. D	0.90	1.21
N	3	3
Variance	0.81	1.46
Student t-test	0.65 (2.770*)
f-value	1.80 (4.296*)

*Theoretical values

 

CONCLUSION:

A simple, sensitive, accurate, and selective TLC-Densitometric method was suggested for the determination of ALC either in bulk powder or in pharmaceutical formulation in the presence of its degradation products and dosage form preservative benzalkonium chloride. The developed TLC-Densitometric method is more convenient, less time consuming, and economic stability indicating method compared to previously published methods.

 

The suggested method can be used for routine analysis of the studied drug either in bulk powder or in pharmaceutical dosage form without any preliminary separation steps.

 

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Received on 16.03.2020           Modified on 01.05.2020

Accepted on 13.06.2020         © RJPT All right reserved