INTRODUCTION:

Ondansetron HCl is a monohydrochloride salt of (±)-1,2,3,9-tetrahydro-9-methyl-3-[(2methyl-1H-imidazol-1yl) methyl] is ondansetron.4-one-carbazol-4H.¹ The molecular formula is C₁₈H₁₉N₃O,² and it is a carbazole derivative having nitrogen and carbon rings. It is structurally related to serotonin.^3,4 The selective 5-HT₃ receptor antagonist ondansetron hydrochloride is used to relieve nausea and vomiting brought on by chemotherapy.^5,6 Nausea and vomiting are among the adverse effects experienced by patients undergoing chemotherapy, radiotherapy, anaesthesia, opioid analgesic therapy, postoperative recovery, or suffering from irritable bowel syndrome. Through decreased appetite, weight loss, decreased social contact, and more serious problems like dehydration, electrolyte imbalance, and extended hospitalization, these symptoms can seriously lower quality of life.⁷ Both central and peripheral nerves are affected by ondansetron. It inhibits the vomiting centre in the medulla oblongata by decreasing vagus nerve activity. It also inhibits the chemoreceptor trigger zone (CTZ) serotonin receptors.⁸ Upon reviewing the literature, it is evident that there are very few analytical techniques available for estimating ondansetron hydrochloride. Spectrophotometric techniques, HPLC, and HPTLC are among them.⁹ The most used technique for analysing it is HPLC because of its noticeable polarity and comparatively poor water solubility.¹⁰^. The ICH guidelines are followed in the validation of the suggested HPLC method. Because of its simplicity, speed, sensitivity, robustness, and novelty, it is the perfect option for high-throughput analysis.¹¹

Fig. 1: Structure of Ondansetron Hydrochloride

MATERIALS AND METHOD:

Every reagent used was of analytical and HPLC quality and Acetonitrile was purchased from Merk chemicals and Ortho phosphoric acid was purchased from Finar chemicals Ahmedabad Ondansetron hydrochloride was supplied as gift sample from Neon Laboratories Ltd., Mumbai.

Chromatographic system and condition:

A Shimadzu LC with a 20AB Prominence liquid Chromatograph pump, SPD20A UV Prominence UV–Vis detector, a C-18 column (Discovery C18 column, 250mm × 4.6mm inner diameter, 5µm), and a hydraulic method with a mobile phase made up of 0.1% orthophosphoric acid solution and Acetonitrile in (75:25 v/v) comprised the equipment used for the HPLC analysis. The mobile phase had a flow rate of 1 ml/min and was degassed using ultrasonication after being vacuum-filtered through a 0.22µm Millipore membrane. 20µL was the injection volume. Aliquots of samples were injected following solvent equilibration to create a stable baseline.

At 310nm, the eluent's absorbance was measured with a detection sensitivity of 0.250 aufs. Every analysis was carried out at room temperature for a runtime of 10 min.

Preparation Standard Solution:

Methanol was used to make standard stock solution I of Ondansetron hydrochloride (1mg/ml). The stock solution I was diluted with methanol to create stock solution II, which contained Ondansetron hydrochloride 100 μg/mL. The concentrations of 4, 8, 12, 16, 20, 24, 28μg/ml were obtained by pipetting aliquots of 0.4, 0.8, 1.2, 1.6, 2.0, 2.4 and 2.8ml from the stock solution II of Ondansetron hydrochloride into a 10ml volumetric flask and diluting it with 0.1% ortho phosphoric acid solution and acetonitrile in 75:25 ratio. After passing through a 0.45μm Millipore membrane filter, the produced calibration standards were examined.

Method development:

Table 1: Chromatographic parameters

Instrument	Shimadzu SPD-20A UV
Column	C18 (4.6 x 250 mm, 5 mm)
Mobile Phase	0.1% ortho phosphoric acid solution and acetonitrile in (75:25)
Flow Rate	1.0ml/min
Detector	Photo Diode Array (PDA)
Detection Wavelength	310nm
Injection Volume	20 µl
Run Time	10min
Pump	Low pressure gradient
Diluent	Mobile phase

Preparation of Sample solution:

Twenty Ondansetron tablets (ONDEM-4) were accurately weighed and finely powdered. An amount of the powder equivalent to 8mg of Ondansetron hydrochloride was transferred to a 25mL volumetric flask, to which 10mL of diluent (0.1% ortho phosphoric acid and acetonitrile in a 75:25 v/v ratio) was added, and the mixture was sonicated to ensure complete dissolution. The volume was then made up to the mark with the same diluent, and further dilution was carried out to obtain a final concentration of 25µg/mL. The resulting solution was filtered through a 0.45µm nylon syringe filter before analysis.

System Suitability:

As per the test protocol, stock solution-II (20µg/ml) of Ondansetron standard was injected into the HPLC system six times. By calculating the percentage RSD of retention times, tailing factor, theoretical plates, and peak areas from six replicate injections, the system suitability parameters were assessed from the obtained standard chromatograms.¹²

Validation:

1. Linearity: The method's linearity was shown across the 4-28µg/ml concentration range. From stock solution-II, aliquots containing concentrations of 4, 8, 12, 16, 20, 24, 28µg/ml. For every concentration level, a volume of 20μl of sample was injected, and the peak area was plotted against the drug concentration to create the calibration curve, this was repeated for the (n=3) times.¹³

2. Accuracy:

The accuracy of the analytical method was assessed by recovery studies using the standard addition method. Known quantities of Ondansetron hydrochloride standard were added to a pre-analyzed sample solution at three concentration levels—80%, 100%, and 120% of the nominal concentration.⁸

The mean percentage recovery ranged from 98.0% to 102.0%, confirming the accuracy of the method and indicating no interference.

3. Precision:

Precision of the analytical method was evaluated at two levels: repeatability and intermediate precision. Repeatability refers to the closeness of agreement between a series of measurements obtained under the same operating conditions over a short interval of time. It was determined by performing six replicate injections of the sample on the same day. Intermediate precision (intra-day and inter-day) was assessed by analyzing three replicates of Ondansetron hydrochloride at a concentration of 20 µg/mL at different time intervals on the same day (intra-day) and on three consecutive days (inter-day) to evaluate the method’s reproducibility under varying conditions.¹⁴

3.1 Intraday Precision: As per the test procedure, a standard solution containing 20μg/ml was made and injected six times on the same day.

3.2 Interday Precision: As per the test procedure, a standard solution containing 20μg/ml was made and injected six times on three days.

4. Limit of Detection and Limit of Quantification:

The lowest analyte concentration in the sample that could be detected under the specified experimental conditions was known as the limit of detection (LOD), and the lowest concentration of the active ingredients in a sample that could be identified with generally recognized precision and accuracy was known as the limit of quantification (LOQ). The ICH recommends that the method be based on the response's standard deviation (SD). The quantification and detection limits were established using slope.¹⁵

LOD was calculated using the following formula:

LOD = 3.3 σ / S,

LOQ was calculated using the following formula:

LOQ = 10 σ / S,

σ = Standard deviation

S= Slope of Calibration curve

Slope is the sensitivity of the calibration curve, representing the change in instrument response per unit change in concentration.

SD is the measure of variability (noise) in the response, usually taken from blank readings, low-concentration samples, or intercepts of multiple calibration curves.

5. Robustness:

The study of aliquots from homogenous lots using various physical parameters, such as flow rate, wavelength, column temperature and mobile phase composition, revealed the robustness of the suggested approach. Although the responses varied, they remained within the assay's defined boundaries.¹⁶

RESULTS AND DISCUSSION:

1 System Suitability:

Table 2: System Suitability results:

Drug name	Ondansetron Hydrochloride
Retention time, min	5.4min
Area	1062439
Tailing factor	1.4315
Asymmetry factor	1.36
Theoretical plates	46483
Peak resolution	12.46

2 Linearity:

Table 3: Linearity results

Sl. No	Concentration(µg/ml)	Retention time(min)	Area
1	0	0	0
2	4	5.4	418158
3	8	5.4	536159
4	12	5.4	785643
5	16	5.4	1016187
6	20	5.4	1282638
7	24	5.4	1575235
8	28	5.4	1823050

Fig. 2: Calibration graph of Ondansetron Hydrochloride

Table 4: Regression analysis of the calibration curves of Ondansetron Hydrochloride

Parameters	Values
Linear range (µg/ml)	4-28
Slope	65247
Correlation Coefficient (R2)	0.9973

The developed HPLC method for Ondansetron Hydrochloride exhibited a clear linear response across the concentration range of 4–28µg/mL. All chromatograms consistently showed a retention time of 5.4 minutes, indicating excellent stability of the system and reproducibility of the analytical conditions. The calibration plot of mean peak area versus concentration produced the regression equation y = 65,247x with a correlation coefficient (R² = 0.9973), confirming a strong linear correlation between concentration and detector response.

The high R² value, along with the low standard deviation (SD) observed for each concentration, demonstrates the precision, accuracy, and reliability of the method. Overall, the results validate that the proposed HPLC procedure is simple, robust, and suitable for routine quantitative determination of Ondansetron Hydrochloride in both bulk drug and pharmaceutical dosage forms.¹⁷

Fig 3: A typical chromatogram of standard Ondansetron HCl

3. Accuracy

The accuracy of the developed HPLC method for Ondansetron Hydrochloride was assessed by the standard addition method at three concentration levels—80%, 100%, and 120%. The mean recovery values ranged from 98.00% to 101.04%, which are within the ICH-recommended range of 98–102%, confirming the method’s accuracy. The close correlation between the added and recovered amounts indicates that the method is free from interference and suitable for routine analysis of Ondansetron Hydrochloride in bulk and dosage forms. The minimal variation among recovery results further confirms the precision and robustness of the analytical method.¹⁸ (Table-5).

4. Precision:

4.1 Intraday Precision:

Table 6: Intraday precision of Ondansetron hydrochloride

Sl. No.	Concentration (µg/ml)	Rt	Area
1	20	5.4	1243685
2	20	5.4	1241563
3	20	5.4	1264289
4	20	5.4	1253108
5	20	5.4	1234437
6	20	5.4	1240673
			%RSD=0.81

The intraday precision of the developed HPLC method for Ondansetron Hydrochloride was determined by six replicate injections of a 20 µg/mL solution on the same day. The retention time remained constant at 5.7 min, and the calculated %RSD (0.81) was well within the ICH-recommended limit of ≤2%, confirming excellent repeatability and system stability.¹⁹

4.2 Interday Precision:

Table 7: Interday precision of Ondansetron hydrochloride

Sl. No	Concentration (µg/ml)	Rt	Area (n=3)
1	20	5.4	1234574
2	20	5.4	1230452
3	20	5.4	1253178
4	20	5.3	1242017
5	20	5.3	1223326
6	20	5.4	1230562
			%RSD=0.85

The interday precision of the developed HPLC method for Ondansetron Hydrochloride was evaluated by analyzing a 20µg/mL solution over three consecutive days. The retention time remained consistent (5.6–5.7 min), and the calculated %RSD was 0.85, which is well within the ICH-recommended limit of ≤2%. These results confirm the reproducibility and stability of the method over different days, indicating its suitability for routine quality control analysis of Ondansetron Hydrochloride in bulk and dosage forms.¹⁹

5. Limit of Detection and Limit of Quantification:

LOD was calculated using the following formula:

LOD = 3.3 σ / S,

LOD = 1.1µg/ml

LOQ was calculated using the following formula:

LOQ = 10 σ / S,

LOQ = 3.5µg/ml

The sensitivity of the developed HPLC method for Ondansetron Hydrochloride was evaluated by determining the Limit of Detection (LOD) and Limit of Quantification (LOQ) based on the standard deviation of the response (σ) and the slope (S) of the calibration curve. The calculated LOD and LOQ were 1.1µg/mL and 3.5µg/mL, respectively. These low values indicate the high sensitivity of the method, allowing reliable detection and quantification of even small amounts of Ondansetron Hydrochloride. The results confirm that the developed method is suitable for routine analytical applications where accurate and sensitive quantification is required, consistent with ICH Q2(R1) guidelines.

6. Robustness:

Table 8: Robustness of Ondansetron hydrochloride

Physical Parameters	Variation	USP Plate Count	USP Tailing
Composition of mobile phase	20% less	4263	1.612
	*Actual	6214	1.458
	20%more	5663	1.364
	0.8ml/min	5944	1.721
Changes in the flow rate	*1ml/min	6101	1.464
Changes in the flow rate	1.4ml/min	4813	1.581
Changes in wavelength	246nm	4312	1.321
	*310nm	6419	1.435
	211nm	5138	1.721
Changes in the Column temperature(C⁰)	25	4182	1.733
	*37	6489	1.412
	32	4753	1.489

The robustness of the developed HPLC method for Ondansetron Hydrochloride was evaluated by making deliberate variations in key chromatographic parameters such as mobile phase composition, flow rate, wavelength, and column temperature. The results (Table 8) showed that minor changes in these conditions produced no significant effect on the USP plate count or tailing factor, which remained within acceptable limits (plate count > 4000; tailing factor < 2.0).

These observations confirm that the method maintains consistent system performance, peak symmetry, and resolution under varied analytical conditions. Hence, the developed HPLC method is considered robust, stable, and reliable for routine analysis of Ondansetron Hydrochloride in both bulk and pharmaceutical dosage forms.¹⁹

CONCLUSION:

It was determined that the suggested RP-HPLC approach was appropriate for estimation of ondansetron HCl. Acceptable findings for system compatibility, specificity, linearity range, precision, accuracy, LOD, LOQ, and robustness were obtained during the method's successful validation in accordance with ICH recommendations. Therefore, this approach has great significance in pharmaceutical quality control and can be used successfully for the analysis of ondansetron hydrochloride in pharmaceutical dosage forms.

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Received on 25.11.2025 Revised on 19.12.2025

Accepted on 09.01.2026 Published on 27.01.2026

Available online from February 02, 2026

Asian Journal of Pharmaceutical Analysis. 2026; 16(1):29-33.

DOI: 10.52711/2231-5675.2026.00005

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

Concentration of the sample (μg/ ml)	Concentration of drug added (μg/ ml)	Percent of spiked	Percent of spiked	Recovered amount	Percent recovery
10	8	80%	80%	17.64	98%
10	10	100%	100%	20.19	100.95%
10	12	120%	120%	22.24	101.04%