INTRODUCTION:

Oxetacaine (also known as Oxethazaine) is a potent local anesthetics which is administered through oral or topical route¹and chemically it is 2,2'-((2-hydroxyethyl)imino)bis(N-alpha,alpha-dimethylphenethyl)-N-methylacetamide² (Fig. 1). It is white crystalline powder having a melting point of about 104 ⁰C. The drug is practically insoluble in water³ and methanol soluble⁴. Oxetacaine inhibits gastrin release and secondarily reduces gastric juice secretion. Thus, Oxetacaine provides relief from conditions such as vomiting, epigastric pain, abdominal distention, heartburn, nausea, epigastric discomfort and superfluous urge to defecate, which are linked with various other gastrointestinal disorders⁵.

Fig. 1: Chemical structure of Oxetacaine

Oxetacaine is mainly used for indicative relief from Gastero-oesophageal reflux and throbbing related to haemorrhoids⁵. Adverse drug reactions of Oxetacaine are hyper sensitivity, anorexia, dryness of mouth, constipation etc.,⁵. A very few analytical techniques^6-11 have been documented for the estimation of Oxetacaine in bulk and pharmaceutical formulations which includes UV and RP-HPLC methods individually and in combination. To our knowledge no article related to HPTLC method has been reported in literature¹² for the estimation of Oxetacaine.

Planar chromatography is a multistage distribution process. It is a form of liquid chromatography in which the stationary phase is supported on a planar surface rather than a column¹³. Thin layer chromatography (TLC) is a simple, cost-effective and easy chromatographic technique that has been used in identification and quantification of various drugs as well as biological samples of interest¹⁴. Introducing HPTLC into pharmaceutical analysis represents a major step in terms of quality assurance¹⁵ and owing to the wide spread use of HPTLC in routine analysis¹⁶. The major advantage of HPTLC is that several samples can be run simultaneously using a small quantity of mobile phase unlike HPLC, thus lowering the analysis time and cost per analysis¹⁷. HPTLC method is more efficient and sensitive than UV-Spectrometric method¹⁸. Confirmation of the applicability of the developed method was validated according to the International Conference on Harmonization (ICH)¹⁹.

The preferred procedure is based on the direct estimation of Oxetacaine with a high scope of accuracy and precision²⁰. The present study describes a sensitive, rapid, precise and economical HPTLC method for the assessment of Oxetacaine in pharmaceutical dosage form and validation of method as per ICH guidelines²¹.

MATERIALS AND METHODS:

Chemicals and reagents:

Water for analysis is prepared from Milli-Q-water system. Methanol, glacial acetic acid and all the other analytical reagents employed for the study were procured from MERCK chemicals Mumbai, India.

Instrumentation:

Camag HPTLC system operated with a semi-automated sample applicator (Linomat IV), 100µL capacity Hamilton syringe, HPTLC Camag Scanner III, winCATs software (version 1.3.4). Development was conceded in a twin trough chamber (dimension - 20 x 10) and remicentrifuge models (C30) were used for the study.

Experimental:

Analytical Method Development:

Standard stock solution:

10mg of Oxetacaine (Reference standard containing 99.7% w/w pure Oxetacaine) was weighed, dissolved and diluted to 10mL with methanol to yield a final concentration of 1mg mL^-1 (1000µg mL^-1).

Selection of Adsorption layer:

Precoated HPTLC plates were employed in the study. Silica Gel 60 GF₂₅₄was selected as the adsorption layers for effective separation of the drug. TLC plates were pre-washed before use to remove the adsorbed impurities, water vapors and other volatile substance with the aid of methanol. The plates were permitted to dry at room temperature and then activated by placing in hot air oven at 100⁰C for 5 min prior to the sample application.

Selection of detection wavelength:

Oxetacaine standard stock solution was suitability diluted to obtain a concentration of 10µg mL^-1 using methanol, which was scanned in the UV region from 400-200nm and the UV spectra was recorded, which was shown in Fig. 2.

Optimized Chromatographic conditions:

After various trials, the chromatographic conditions for HPTLC were optimized.

· Instrument employed : Camag

· Sample Applicator : Linomat V equipped with Camag 100 µL syringe

· Chamber : (20 x 10) Camag Twin Trough glass chamber

· Scanner : Camag TLC scanner III

· Software : winCATs

· Stationary phase : Precoated HPTLC plates (Merck) (Aluminium sheets

· Coated with Silica gel 60 F₂₅₄)

· Mobile phase : Methanol: Water: Glacial acetic acid (8 : 1.8 : 0.2 v/v/v)

· Development Time : 15 min

· Development Temp : Ambient

· Injection volume : 1 µL

· Detection : 220 nm

· R_f value : 0.61

ANALYTICAL METHOD VALIDATION:

As per the ICH guidelines of Q2(R1), the developed method was validated in terms of linearity, specificity, precision, accuracy and robustness.

Linearity:

Appropriate aliquots of Oxetacaine standard stock solution was taken in five different 10 mL volumetric flasks and diluted up to the mark with methanol to obtain final concentrations ranging from 80 - 120 ng spot^-1.

1 µL from the above linearity solutions were spotted on the pre-washed and activated HPTLC plate. After development, the plate was dried and scanned at 220 nm. The peak area values were used to construct the calibration curve.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

Limit of detection (LOD) and Limit of quantification (LOQ) values were calculated employing the slope and intercept values of the linear regression line, thereby the sensitivity of the developed method was established.

LOD = 3.3 S.D of Response/ Average of slope

LOQ = 10 S.D of Response/ Average of slope

Precision:

The intra-day and inter-day precision studies (intermediate precision) were carried out by estimating the corresponding responses 3 times on the same day and on 3 different days for three different concentrations of 80, 100 and 120µg mL^-1 respectively.

1µL of the above diluted solutions were spotted on the selected HPTLC plate and chromatographic development was carried out. The plates were dried and scanned at 220nm and their peak areas were recorded.

Accuracy:

Accuracy of the method was performed by recovery studies using standard addition method. Accuracy was performed at 80%, 100% and 120% levels of target concentration.

Assay procedure for suspension:

5mL of syrup (Sucrafil O Gel - containing 10mg of Oxetacaine BP, manufactured by Fourrts (India) Laboratories Pvt. Ltd.,) was pipetted out into 10 mL standard flask. 5mL of methanol was added and the sample solution was extracted for 20 min under sonication. The solution was diluted to the mark with methanol and mixed well. The solution was filtered through Whatmann No.1 filter paper. The resultant solution was suitably diluted for further analysis.

1µL of the diluted solution was spotted on the precoated HPTLC plate. The plate was developed, dried and scanned at 220 nm. The sample peak area was recorded. The amount of drug present in the selected formulation was calculated using the following formula:

Amount present in synthetic mixture =

Peak area of Sample/Peak area of Std x Cs x D_F

where, Cs - Concentration of Standard

D_F - Dilution Factor

RESULTS AND DISCUSSION:

A HPTLC method was developed with the main aim of estimating Oxetacaine in pharmaceutical formulations.

UV scanning from 400-200 nm shows that 220 nm is the suitable wavelength for the detection of Oxetacaine (Fig. 2). The mobile phase of Methanol: Water: Glacial acetic acid (8 : 1.8 : 0.2 v/v/v) was selected, as the combination showed good peak symmetry, minimum tailing and a R_f value of 0.61 for Oxetacaine (Fig. 3).

Fig. 2: UV Spectra of Oxetacaine

Figure 3: Standard densitogram of Oxetacaine in Peak area mode

Oxetacaine showed linearity in the concentration range of 80 - 120 ng spot^-1 with regression coefficient of 0.9988 (Table 1). LOD and LOQ values were found to be 0.30 ng spot^-1 and 0.91 ng spot^-1, respectively. The results were tabulated in Table 2 and the linearity plot was shown in Fig. 4 respectively.

Table 1: Linearity data of Oxetacaine

S. No.	Conc. of Oxetacaine (ng spot^-1)	Peak Area
1.	80	2297
2.	90	2517
3.	100	2729
4.	110	2957
5.	120	3211

Table 2: System suitability parameters

Parameters	Results
Parameters	Oxetacaine
Linearity range (ng spot^-1)	80-120
Regression equation	y=22.68x+474.2
Correlation coefficient (r²)	0.9988
LOD (ng spot^-1)	0.30
LOQ (ng spot^-1)	0.91
Retention time	0.61

Figure 4: Linearity plot of Oxetacaine

The intra-day and inter-day precision studies (intermediate precision) were carried out. The % Relative Standard Deviation (RSD) was found to be less that 2 % as per ICH guidelines and the results were tabulated in Table 3.

Table 3: Precision study of Oxetacaine

Repeatability	Concentration (µg mL^-1)	Oxetacaine
Repeatability	Concentration (µg mL^-1)	Amount found in µg mL^-1 (Mean± SD)*	% RSD
Intra-day	80	81.40 ± 0.85	1.04
	100	100.20 ± 1.17	1.17
	120	120.90 ± 1.32	1.09
Inter-day	80	80.76 ± 0.95	1.17
	100	99.96 ± 1.65	1.65
	120	121.73 ± 0.80	0.65

Mean ± SD* = average of six determinations

Specificity of the method was confirmed by spotting the processed sample solution on the HPTLC plate, developed and scanned. A symmetrical peak was obtained amidst the presence of common excipients which confirms the specificity of the developed method.

Accuracy of the method was ascertained by performing recovery studies. Standard addition method was employed to check the recovery of the drug at three different concentration levels of 80 %, 100 % and 120 % and the mean recovery was calculated. The percentage recovery value was found to be between 100.78 - 101.30 %w/w (Table 4), which was well within the acceptance criteria of ICH guidelines.

Table 4: Recovery results of Oxetacaine

Drug	Levels (%)	Conc. (µg mL^-1)	Recovered Amount (µg mL^-1) (Mean ± SD)*	% Recovery
Oxetacaine	80	80	80.63 ± 0.75	100.78
	100	100	100.86 ± 0.35	100.86
	120	120	121.56 ± 1.10	101.30

Mean ± SD* = average of six determinations

Sample analysis was performed in marketed formulation as described in the procedure under “Assay procedure for suspension” to estimate the content of Oxetacaine in suspensions. The sample densitogram was represented in Fig. 5 and the % RSD was found to be 0.32 (Table 5).

Table 5: Assay of Oxetacaine in marketed formulation

Drug	Label claim (mg)	Amount estimated (mg)	% Assay (n=6)
Oxetacaine	10	09.87	98.7
		Average	100.50
		SD	0.33
		% RSD	0.32

Figure 5: Sample densitogram of Oxetacaine in marketed formulation in Peak area mode

CONCLUSION:

The newly developed HPTLC method for the estimation of Oxetacaine was developed and validated. The obtained results emphasizes that all the validation parameters were performed as per ICH guidelines and were found within the prescribed limits. The current developed method was established to be simple, rapid, precise, sensitive, accurate and robust for the assessment of Oxetacaine in suspension. Hence, the developed HPTLC method for Oxetacaine can be suggested to be released into routine QC laboratory for its quantification in different pharmaceutical formulations.

ACKNOWLEDGEMENT:

The authors are highly thankful and we acknowledge the management of SRM Institute of Science and Technology, for providing the facilities required for completing the work successfully.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 05.08.2020 Modified on 13.11.2020

Research J. Pharm. and Tech 2021; 14(11):5668-5672.

DOI: 10.52711/0974-360X.2021.00985