INTRODUCTION:

ONDH is a widely used antiemetic drug, commonly prescribed to prevent nausea and vomiting caused by chemotherapy, radiation therapy, or surgical procedures. Its chemical structure is (RS)-2- [ (3-Fluorophenyl) methylthio ]-3-[(2S)-1-methylpyrrolidin-2-yl] propylamine.

Fig. 1: Chemical structure of ONDH

ONDH appears as a crystalline substance ranging from white to slightly off-white in color, exhibiting a melting point of approximately 129-131°C. It has limited solubility in water but dissolves more readily in methanol. The drug exerts its effect by blocking serotonin 5-HT3 receptors, which is essential in managing nausea and vomiting resulting from chemotherapy, radiation therapy, or surgical interventions. ONDH is commonly administered through oral or intravenous routes, particularly for patients receiving chemotherapy or undergoing surgical procedures. ONDH is effective in managing nausea and vomiting, providing relief for patients undergoing chemotherapy, radiation therapy, or surgery. Common side effects include headache, constipation, dizziness, and fatigue, but serious side effects are rare. It is used to reduce nausea and vomiting brought on by chemotherapy, radiation therapy, or anaesthesia and surgery for cancer¹.

While traditional techniques like UV spectrophotometry and HPLC have been used to analyze ONDH in pharmaceutical formulations, there is limited research on using HPTLC for its quantification. Planar chromatography is a multistage distribution process². HPTLC is a reliable chromatographic method where the stationary phase is applied to a flat surface, setting it apart from column chromatography TLC, a simpler and more cost-effective technique, is often used for identifying and quantifying various drugs, including ONDH.

The introduction of HPTLC into pharmaceutical analysis is a significant advancement, offering the ability to analyze multiple samples at once with reduced solvent usage. HPTLC is an analytical technique based on TLC, which has been improved to allow for quantitative analysis of the compounds. The application of HPTLC is widely regarded and approved, and in order to standardize the test procedures, numerous approaches are being developed³.Thin layer chromatography is the basic planar chromatography method is used for the separate the volatile and non-volatile substances⁴. This approach saves both time and costs, making it a more efficient alternative to methods like HPLC. HPTLC is especially beneficial in routine pharmaceutical quality control due to its speed, affordability, and higher sensitivity compared to UV spectrophotometric methods^5,6.

Literature survey reveals few analytical methods were employed for the estimation of ONDH employing HPTLC method^7-17.

The method undergoes thorough validation in accordance with the guidelines established by the International Council for Harmonisation (ICH)¹⁸, ensuring its accuracy and reliability. This study presents a sensitive, rapid, and precise HPTLC method for analyzing ONDH in pharmaceutical formulations, fully validated and suitable for routine quality control processes. Ethanol is employed as green solvent¹⁹.

MATERIALS AND METHODS:

Chemicals and Reagents:

Ethanol and ethyl acetate, along with Additional analytical chemicals, were sourced from MERCK Chemicals, Mumbai, India.

Instrumentation:

The analysis was carried out using the Camag HPTLC system, which included a semi-automated sample applicator (Linomat IV) for precise sample application. A Hamilton syringe with a 100µL capacity was utilized to apply the sample. For detection and analysis, a Camag TLC Scanner III was used, and the resulting data was processed with winCATs software (version 1.3.4). Chromatographic development occurred in a twin trough chamber, measuring 20 x 10cm.

Experimental:

Analytical method development:

Standard stock solution:

A 10mg sample of Ondansetron hydrochloride (Reference standard with a purity of 99.7% w/w) was carefully weighed, dissolved, and then ethanol was added to adjust the volume to 25mL, leading to a solution with a conc of 1 mg per ml.

Selection of adsorption layer:

Silica Gel 60 F254-coated plates used for HPTLC analysis were utilized for analysis to facilitate effective separation of ONDH. Prior to sample application, the plates were washed with methanol to remove any adsorbed impurities, moisture, and volatile substances. Following the wash, the plates were left to dry at room temperature and subsequently activated by heating in a hot air oven at 100°C for 5minutes to attain optimal performance during the analysis.

Selection of detection wavelength:

A proper dilution of the ONDH stock solution was made using ethanol to achieve a concentration of 10μg/mL. The solution was scanned within the UV range of 200 to 400nm, and the corresponding UV spectrum was recorded to determine the optimal wavelength for detection.

Optimized chromatographic conditions:

· Instrument: Camag HPTLC System

· Sample dispenser: Linomat V, equipped with a Camag 100µl syringe for precise sample delivery

· Chamber: Camag Twin Trough Glass Chamber (20x10cm), ensuring uniform mobile phase distribution

· Scanner: Camag TLC Scanner III, providing accurate and high-resolution detection

· Software: Wincat Software, used for data analysis and documentation

· Stationary phase: HPTLC plates, pre-coated with Silica Gel 60 GF254 on aluminum sheets, providing excellent separation efficiency

· Mobile phase: Ethanol: Ethyl acetate (9:1 v/v), selected based on preliminary trials for best separation

· Development duration: 10minutes, providing adequate migration of the analyte

· Development temperature: Ambient temperature to maintain stability of the sample

· Detection wavelength: 302nm, chosen for maximum absorbance of the analyte

· Rf value: ONDH – 0.62

Analytical method validation:

Linearity:

Various volumes of the ONDH working standard solution were aliquoted into five distinct 10ml containers volumetric flasks were used and the solutions were subjected to dilution with ethanol to achieve the desired final concentrations ranging from 50 to 150ng/spot. From each solution, 1 μl was applied a TLC plate with a pre-coated surface was employed, developed, dried, and then analyzed by scanning at 302 nm. The areas of the peaks were measured, and calibration plots were generated. The linearity densitogram and calibration plot are shown in Figures 3 and 4, while the linearity data is presented in Table 1.

Detection Limit (LOD):

The limit of detection (LOD) for the analytical method indicates the lowest amount of an analyte in a sample that can be detected, though it may not be quantified with precision. The detection limit can be calculated using the following formula:

LOD = (3.3 × σ) / S

Where:

σ represents the standard deviation of the blank,

S is the slope of the calibration curve

Quantitation Limit (LOQ)

The limit of quantitation (LOQ) for the method refers to the lowest concentration of an analyte in a sample that can be measured with acceptable accuracy. The LOQ is calculated using the following formula:

LOQ = (10 × σ) / S

Where:

σ = Standard deviation of the response

S = Slope of the calibration curve

Precision:

Intra-day and inter-day precision (also known as intermediate precision) the studies were conducted by examining the 100% concentration six repetitions on the Within a single day and across multiple days. A 1μl portion of the solution was introduced onto the pre-coated TLC plate and developed. Once developed, the plates were allowed to dry. and analyzed by scanning at 302nm. The peak areas were recorded. Densitogram and precision data are presented in Figures 5 and Table 3.

Accuracy:

The method's accuracy was assessed through recovery studies using the standard addition approach. Accuracy was assessed at 50%, 100%, and 150% of the intended concentration. Densitogram and accuracy data are shown in Figure 6 and Table 4.

Analytical procedure for commercially available formulation:

20 tablets containing 8 mg of ONDH were individually weighed to determine their average weight. A portion of amount Corresponding to 25mg of the drug was subsequently used measured, and 15mL of ethanol was added to the mixture, which was then subjected to Subjected to sonication for 20minutes to facilitate drug extraction. The resulting solution was diluted to a final volume of 25mL with ethanol and thoroughly mixed. The solution was filtered through Whatman filter paper, and the obtained filtrate was collected for subsequent analytical procedures. 1μL aliquot of the prepared solution was applied to the precoated TLC plate and developed. After development, the plates were dried and analyzed at 302nm. The peak area corresponding to the standard was measured, and the drug content was calculated using the following formula.

Amount present = (Peak area of the Sample / Peak area of the Standard) × Cs × DF

Where:

Cs = Concentration of the Standard

DF = Dilution Factor

Robustness:

The method's robustness was assessed by assessing its capacity to remain consistent under varying conditions. stable under minor, deliberately introduced variations in the method parameters. The following factors were modified:

· Detection wavelength (within ±2 nm)

· Composition of the mobile phase(Ethanol: Ethyl acetate – 9.0 ± 0.2 : 1.0 ± 0.2)

· Development distance (7, 8, and 9 cm)

· Saturation time (15, 30, and 45 minutes)

The impact of these changes on the quantification the data were analyzed, and the outcomes of the robustness study are presented in Table 6

Assessment of greeness:

Environmental of optimized HPTLC method for ONDH was assessed using various tools for evaluating environmental sustainability, such as AGREE, GABI, and the Analytical Eco-Scale. The results of these evaluations are shown in Figures 8 and 9.

RESULTS AND DISCUSSION:

UV Spectra of ONDH:

Two absorption maxima were observed at 246 and 302 nm in the UV spectra of ONDH. The λmax of 302nm was chosen for the study. The UV spectrum of ONDH is as follows:

Figure 2: UV Spectra of ONDH

Estimation of ONDH by HPTLC:

In the present work, the estimation of ONDH by HPTLC method is proposed

Optimized HPTLC method:

A standard solution with a concentration of 10μg/mL of ONDH was prepared and used for further analysis. The recorded standard densitogram was shown in Fig.1

Figure 3: Standard densitogram of ONDH in Peak area mode

VALIDATION:

Linearity and Range:

Linearity of ONDH by the proposed method was performed and the corresponding values were tabulated as follows:

Table 1: Linearity of ONDH

S. No.	Conc of ONDH (ng per spot)	Peak area
1.	50	877.6
2.	75	1210.5
3.	100	1639.5
4.	125	2127.2
5.	150	2501.4

Figure 4: Linearity densitogram of ONDH

Figure 5: Calibration graph of ONDH

Table 2: System Suitability Parameters

S. No.	Parameters	Results
1.	Linearity range (ng per spot)	50 – 150
2.	Regression equation	y=16.657x+5.52
3.	Correlation coefficient (r²)	0.9965
4.	Limit of Detection (ng per spot)	0.28
5.	Limit of Quantification (ng per spot)	0.85
6.	Retention time	0.62

Precision:

Intra-day and inter-day variability studies were conducted. The %RSD was determined to be less than 2%, and the findings are summarized as follows:

Table 3: Precision study of ONDH

Repeatability	Concentration (µg per mL)	ONDH
Repeatability	Concentration (µg per mL)	Amount found in µg per mL (Mean ± SD)*	% RSD
Intra-day	100	81.40 ± 0.85	1.044
	100	100.20 ± 1.17	1.437
	100	98.30 ± 0.45	0.553
	100	97.52 ± 0.74	0.909
	100	100.53 ± 1.02	1.253
	100	99.70 ± 0.22	0.270
Inter-day	100	80.76 ± 0.95	1.167
	100	99.96 ± 1.55	1.904
	100	96.54 ± 1.45	1.781
	100	100.20 ± 0.71	0.872
	100	98.54 ± 0.78	0.958
	100	98.77 ± 1.11	1.364

Mean ± SD* = average of six determinations

(a)

(b)

Figure 6: (a and b) : Densitograms showing Precision (Intra and Inter day) for ONDH

Accuracy:

The method accuracy was assessed at concentrations of 50%, 100%, and 150% of the target concentration. The percentage recovery was within the acceptable range (98-102% w/w), and the outcomes are summarized below.

Table 4: Accuracy study of ONDH

Drug	Level (%)	Concentration (µg per mL)		Amount recovered (µg per mL) (Mean ±SD)*	% Recovery
Drug	Level (%)	Sample Conc.	Spiked Standard Conc.	Amount recovered (µg per mL) (Mean ±SD)*	% Recovery
ONDH	50	25	25	50.13 ± 0.75	100.52
	100	25	50	99.86 ± 0.35	99.86
	150	25	125	148.56 ± 1.10	99.04

Mean ± SD* = average of Six determinations

Figure 7: Densitogram showing Accuracy for ONDH

Assay procedure for commercially available formulation:

The assay was conducted using commercially available formulations of ONDH (each containing 8 mg of ONDH). The sample was processed according to the procedure outlined for ONDH analysis The %RSD was determined to be under 2%, and the results are presented below.

Table 5: Assay of ONDH in marketed formulation

Drug	Label claim (mg)	Amount estimated (mg)	% Assay (n=6)
ONDH	8	7.87	98.37
		MEAN	100.5
Mean ± SD* = average of Six determinations		SD	0.33
		%RSD	0.32

Figure 8: Assay densitogram of ONDH in marketed formulation

Robustness:

The robustness of the proposed method was assessed by examining its ability to maintain stability despite minor, deliberate changes in the method parameters. In all tests, variations in conditions did not notably impact the separation or quantification of the drugs being studied. Additionally, the relative standard deviation (RSD) of the peak areas stayed below 2%, confirming the method's robustness.

Assessment of greeness of the established method:

The environmental impact of the developed HPTLC method for ONDH was evaluated using different greenness assessment tools, such as AGREE, GABI, and the Analytical Eco-Scale. The results of this evaluation are presented below:

Figure 9: GABI

Figure 10: AGREE

Table 7: Outcomes of AES calculation

Factors related to AES calculation	No of Pictogram	Sub total
1.Solvents Ethanol (0-10 ml)	2	4
Ethyl acetate	1	2
2. Energy consumption by instrument (HPTLC uses ≤ 0.1 kWh per sample)	0	0
3.Occupational hazard	0	0
4.Wastage and recycling (0-10 mL)	3	3
	Total PP	9
	AES	(100-9)= 91

CONCLUSION:

A novel, environmentally friendly HPTLC method for estimating ONDH has been developed and validated. The results indicate that all validation parameters are within the limits set by ICH guidelines. The method demonstrated sensitivity, accuracy, precision, robustness, and reproducibility for quantifying ONDH in tablet formulations. Green evaluation of the method highlights its compliance with several principles of green analytical chemistry, including the use of safer solvents, low solvent volume (10mL), and reduced chemical waste. The approach also enables simultaneous analysis of multiple samples, which optimizes energy use and reduces the overall environmental footprint. These features make the method not only analytically reliable but also aligned with sustainability requirements. Therefore, the developed HPTLC method is highly suitable for routine application in quality control laboratories for the simultaneous determination of ONDH in pharmaceutical formulations, while supporting eco-friendly and resource-efficient laboratory operations.

ACKNOWLEDGEMENT:

Authors would like to extend their sincere thanks and acknowledge the Management of SRM Institute of Science and Technology for providing the essential facilities that helped in the accomplishment of this work.

CONFLICT OF INTEREST:

The authors declare that they have no conflicts of interest to report.

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Received on 22.04.2025 Revised on 09.09.2025

Accepted on 13.11.2025 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2068-2074.

DOI: 10.52711/0974-360X.2026.00297

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

S. No.	Parameter	Variations	Rf	Peak area ± SD	% RSD
1.	Detection wavelength, ±2 nm	300nm	0.62	1638.56 ± 1.36	0.08
		302nm
		304nm
2.	Mobile phase composition (Ethanol:Ethyl acetate – 9.0±0.2ml:1.0±0.2ml v/v)	8.8:1.2 ml	0.61	1774.12 ± 3.01	1.69
		9.0:1.0 ml
		9.2:0.8 ml
3.	Development distance (7, 8 ,9 cm)	7 cm	0.62	1666.86 ± 1.09	0.06
		8 cm
		9 cm
4.	Time of saturation (15, 30, 45 min)	15 mins	0.62	1725.51 ± 2.01	0.11
		30 mins
		45 mins