The document presents a method development and validation for the quantitative estimation of ondansetron hydrochloride using high performance thin layer chromatography (HPTLC). An HPTLC method was developed using a methanol and triethylamine-glacial acetic acid mobile phase to separate ondansetron hydrochloride. The method was validated in terms of linearity, accuracy, precision, specificity, limit of detection, limit of quantitation, ruggedness and robustness. The developed and validated HPTLC method was successfully applied for the estimation of ondansetron hydrochloride in bulk drug samples and tablet dosage forms.

1. Presentation
on
“Method Development and validation for the
Quantitative Estimation of ONDANSETRON
HYDROCHLORIDE by HPTLC in Bulk Drug &
Tablet Dosage Form”
By: LOVE KUMAR
M.Sc Biotechnology

2. What is Drug?
• Drug is substance meant for cure, mitigation
prevention or treatment of disease.
• A drug is a substance which may have
medicinal, intoxicating, performance
enhancing or other effects when taken or put
into a human body.

3. ONDANSETRON HYDROCHLORIDE
• Ondansetron hydrochloride is a serotonin 5-
HT-3 receptor antagonist used mainly as an
antiemetic to treat nausea and vomiting

4. Mechanism of action
• It effects on both peripheral and central nerves system by
reducing the activity of the vagus nerve which deactivate the
vomiting centre in the medulla oblongota and also block
serotonin receptors in the chemoreceptor trigger zone
• It also has little effect on vomiting caused by motion sickness.

5. ondansetron hydrochloride
• 1. Nature : Amorphous
• 2. Colour : White
• 3. Odour : Odourless
• 4. Taste : Bitter
• 5. Melting point : 1200 C
• 6. Solubility : freely soluble in methanol

6. Method Development
• Existing method may not be suitable.
• Unreliable
• Expensive
• Time consuming
• Not Easily automated
• New instrumentation technique may provide
improved methods

7. CHROMATOGRAPHY
• The term ‘Chromatography’ covers those
processes aimed at the separation of the
various species of a mixture on the basis of
their distribution characteristics between a
stationary and a mobile phase.

8. Different modes of chromatography are as
follows:
• Normal Phase Chromatography
• Reversed Phase Chromatography
• Reversed Phase – Ion pair Chromatography
• Ion Chromatography
• Ion-Exchange Chromatography
• Affinity Chromatography
• Size Exclusion Chromatography

9. Instrumentation
Pump
Injector
Silica Plate
Detector
Gradient
Controller
Mobile Phases
•

10. Materials and Method
Reagents and Chemicals:
• Sample and working standards of ondansetron hydrochloride
were obtained from Sun Pharma Pvt Ltd, HPLC grade
methanol was purchased from E.Merck and ammonium acetate
of analytical reagent grade was purchased from Sisco Research
Laboratories.
Instrumentation:
• Uv –vis spectrophotometer is of backman DU640B
• Pump - water 600controller pump
• Auto sampler – water 717 plus
• Uv detector – water 486
• Software – millinium 32 .

11. Chromatographic Conditions
• The elution of ondansateron hydrochloride was obtained by running
HPTLC in isocratic mode using methanol and tiriethylamine –
glacial acetic acid in a ratio of 9.5:0.5 v/v, flowrate was maintained
at 1.0 ml per minute with run time of 10 minutes.
• The Rf for ondansateron hydrochloride was obtained at 7.7 and
detection was performed at 309 nm.
• Mobile phase was previously filtered through what mann filter paper
no 41.

12. • Preparation of Standard Stock solution
• Stock solution (1000 ppm) of ondansateron hydrochloride was
prepared by dissolving 100 mg of Eslicarbazepine Acetate
standard in methanol in a 100 ml volumetric flask, the volume
was made up to the mark with methanol.
• This stock solution was further diluted to obtain desired
concentrations.
• Preparation of Sample solution:
• Weighed 20 tablets of ondansateron hydrochloride (labelled
claim 400 mg of ondansateron hydrochloride ) and average
weight was calculated.
• The tablets were crushed to get homogenous powder and a
quantity equivalent to 100 mg was weighed in a volumetric flask.
• The powder was then allowed to dissolve in methanol by
sonication. Make up the volume to the mark with methanol and
filter the solution through 0.2 μm filters.
• The filtrate was diluted to obtain desired concentrations.

13. Method Development:
Selection of wavelength
• Appropriate dilutions of ondansateron hydrochloride was
prepared from the stock solution,which were scanned over a
range of 200-400 nm and the UV spectrum was .
• The maximum absorbance was found at 309 nm.

14. Method Validation
• Linearity
• Sensitivity
• Precision
• Accuracy
• Limit of Detection (LOD)
• Limit of Quantitation (LOQ)
• Ruggedness
• Robustness
• Stability
• System Suitability

15. Precision
• Precision of an analytical procedure referred to degree of
scatterness between a series of observations obtained from
multiple sampling of same homogenous sample in given
conditions.
• The term intraday precision (repeatability) refers to the use of
analytical procedure within same laboratory conditions over a
short period of time by same analyst and same instrument
where as inter day precision (intermediate precision) refers to
the use of analytical procedure in same laboratory conditions
on different days by different analysts.
• Six injections of ondansateron hydrochloride 50 ppm were
applied twice after a short interval of time on the same day, the
procedure was repeated on two consecutive days by different
analysts and % RSD was reported.

16. Accuracy
• Accuracy is the closeness of the test results to that of the true
value which can be determined in terms of percent recovery.
• The recovery study was performed at three levels mainly
80%, 100% and 120% by adding known amount of pure drug
in to the 50ppm sample solution.
• The results of recovery studies was tabulated and found
satisfactory.

17. Linearity
• Different concentrations ranging from 10-90 ppm
were injected in duplicate, out of which five
concentrations (10, 30, 50, 70, 90, ppm) were
selected and each concentration was injected in
duplicate.
• The mean peak areas were recorded and the
calibration curve
• was established by plotting concentration on x-axis
and peak area on y-axis.
• The results show that the Beer’s law is obeyed in
the concentration range of 10-90 ppm.

18. Range
• Range is the minimum and maximum concentration of the
sample at which the analytical procedure gives reproducible
results.
• Range can be determined by linearity, accuracy and precision
studies.
• The method was found acceptable across wide range of
concentration (10-90 ppm).

19. Specificity
• The retention time of the sample solution of ondansateron
hydrochloride tablet was found to be 6.0 minutes, which is
similar to that of the standard solution of Eslicarbazepine
Acetate.
• This indicates that there is no drug-excipient interference and
the drug is properly resolved by this particular method

20. Robustness
• Robustness determines the reproducibility of the test result
with small and deliberate variations in the method parameters.
• The experiment was carried out by slightly changing the ratio
of methanol (50±2 ml) in the mobile phase, column oven
temperature (25.0 ± 0.2oC)and flow rate (1.0±0.1 ml), The
effectiveness of the deliberate variations was observed on
retention time of peak.
• The statistical data gives no significant variations in the above
parameters indicating that the method is robust.

21. Analysis of Pharmaceutical API & Dosage form
• The proposed method was successfully applied for the
estimation of ondansateron hydrochloride in bulk drug and
tablet dosage form.
• The assay results were compiled and chromatogram found
satisfactory and show there is no interference of the tablet
matrix with the drug.
• Low % RSD shows that this methodcan be easily applied for
the estimation of in Eslicarbazepine Acetate in bulk drug and
tablet dosage form.

22. Conclusion
A high performance liquid chromatography method
for the quantitative estimation of in bulk drug and
tablet dosage form has been developed as per the
requirement of present era.
Statistical result and low % RSD values indicate that
the method is precise, accurate, robust, specific and
can be used as a wide range of concentration.

23. THANK
YOU