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Presentationon

“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

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.

ONDANSETRON HYDROCHLORIDE

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

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.

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

Method Development

• Existing method may not be suitable.

• Unreliable

• Expensive

• Time consuming

• Not Easily automated

• New instrumentation technique may provide improved methods

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.

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

Instrumentation

Pump

Injector

Silica PlateDetector

Mobile Phases

Gradient Controller

•

Materials and MethodReagents 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 .

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.

• 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.

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.

Method Validation

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

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.

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.

Linearity

• Different concentrations ranging from 10-90 ppmwere injected in duplicate, out of which fiveconcentrations (10, 30, 50, 70, 90, ppm) wereselected and each concentration was injected induplicate.

• The mean peak areas were recorded and thecalibration curve

• was established by plotting concentration on x-axisand peak area on y-axis.

• The results show that the Beer’s law is obeyed inthe concentration range of 10-90 ppm.

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).

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

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.

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.

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.

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