This document summarizes a dissertation submitted for a PharmD degree. It describes the development and validation of an analytical method for the quantitative analysis of metoclopramide hydrochloride using HPLC and UV spectroscopy. The objectives are to develop a simple, sensitive, accurate and economic RP-HPLC method and validate it according to ICH guidelines. A literature review provided background on previous related studies and informed the methodology. The method was developed using an HPLC system with a C8 column, gradient elution and UV detection. The method will be validated for accuracy, precision and other parameters and applied to analyze metoclopramide in samples.

1. Dissertation submitted to
Rajiv Gandhi University of Health Sciences, Bangalore, Karnataka
ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR
METOCLOPRAMIDE HYDROCHLORIDE (ANTI EMETIC)”
Under the guidance of
Dr. C.SREEDHAR
M.Pharm, Ph.D.
Professor and Head
Department of Pharmaceutical Analysis
Presented by:
Mr. Meghnath chaudhary
Department of Pharmaceutical Analysis
Karnataka college of Pharmacy
Reg no. 16PA024

2. Chapter no. Particulars
1. INTRODUCTION
2. DRUG PROFILE
3. OBJECTIVE
4. REVIEW OF LITERATURE
5. METHODOLOGY
6. RESULTS
7. DISCUSSION
8. CONCLUSION
9. SUMMARY
10. REFERENCE

3. Pharmaceutical analysis is the branch of chemistry involved in separating,
identifying and determining the relative amounts of the components making up a
sample of matter.
A drug is any substance (other than food that provides nutritional support)
that, when administered via various route to treat or prevent an illness or disease.
Analytical chemistry studies and uses instruments and methods used to separate,
identify, and quantify matter.
Spectroscopy is the branch of science dealing with the study of interaction of
electromagnetic radiation with matter.

4. UV-Spectroscopy
 form of absorption spectroscopy.
 ranges between 200-780 nm.
Principle :
 based on electronic transitions occurring in a molecule.
 valence electron present in a molecule absorb the radiant energy from UV-visible region.
 move from the ground state to the higher energy levels.

5. Instrumentation of UV-Visible Spectroscopy
Components UV region Visible region
Radiation source Deuterium discharge lamp,
Hydrogen discharge lamp,
Mercury arc, Xenon
discharge lamp.
Tungsten Lamp.
Monochromators Prism Monochromator,
Grating Monochromator
Prism Monochromator,
Grating Monochromator
Filters Absorption filters,
Interference filters.
Absorption filters,
Interference filters.
Sample cells Made of quartz or fused
silica.
Made of glass or plastic.
Detectors Photovoltaic cell,
Photomultiplier tube,
Phototube.
Photovoltaic cell,
Photomultiplier tube.
Phototube.

6. APPLICATION
• Detection of Impurities
• Quantitative analysis
• Qualitative analysis
• Dissociation constants of acids and bases.
• Chemical kinetics
• Quantitative analysis of pharmaceutical substances.
• Molecular weight determination
• As HPLC detector

7. CHROMATOGRAPHY
• method of separation of the compound based on their affinity towards
mobile and stationery phase.
• relatively a new technique which was first invented by M.Tswett, a
botanist in 1906 in Warsaw.
• Tswett termed this system of colored bands as the chromatogram and
the method is chromatography after the greek words ‘chroma’ and
‘graphos’ meaning ‘color’ and ‘writing’ respectively.
• Various type of chromatography is available based on their method of
separation.

8. HIGH PERFORMANCE LIQUID CHROMTOGRAPHY

10. Validation
Validation of an analytical method is the process by which it is established, by laboratory
studies, that the performance characteristics of the method meet the requirements for the
intended analytical applications.
Reasons for Validation
There are two important reasons for validating assays in the pharmaceutical
industry. The first, and by for the most important, is that assay validation is an
integral part of the quality-control system. The second is that current good
manufacturing practice regulation requires assay validationa

11. METHOD VALIDATION PARAMETERS
• Accuracy
• Precision
• Specificity
• Detection Limit
• Quantification Limit
• Linearity
• Range
• Ruggedness
• Robustness

12. Drug profile
Metoclopramide is a medication used mostly for stomach and
esophageal problems. It is commonly used to treat and prevent nausea
and vomiting, to help with emptying of the stomach in people with
delayed stomach emptying, and to help with gastro esophageal reflux
disease. It is also used to treat migraine headaches
structure of metoclopramide hydrochloride

13. Molecular formula: C14H23Cl2N3O2
Molecular weight: 336.257 g/mol
Solubility: Solubility at 25 ºC (g/100 mL): 95% ethanol 2.30;
absolute ethanol 1.90; benzene 0.10; chloroform 6.60
Pka: 9.27
Melting point: 147.25 °C
Category: Anti emetics

14. Mechanism of action:
Metoclopramide inhibits gastric smooth muscle relaxation produced by dopamine, therefore increasing
cholinergic response of the gastrointestinal smooth muscle. It accelerates intestinal transit and gastric
emptying by preventing relaxation of gastric body and increasing the phasic activity of antrum.
Simultaneously, this action is accompanied by relaxation of the upper small intestine, resulting in an
improved coordination between the body and antrum of the stomach and the upper small intestine.
Metoclopramide also decreases reflux into the esophagus by increasing the resting pressure of the lower
esophageal sphincter and improves acid clearance from the esophagus by increasing amplitude of
esophageal peristaltic contractions. Metoclopramide's dopamine antagonist action raises the threshold of
activity in the chemoreceptor trigger zone and decreases the input from afferent visceral nerves. Studies
have also shown that high doses of metoclopramide can antagonize 5-hydroxytryptamine (5-HT)
receptors in the peripheral nervous system in animals.

15. Medical use
Nausea
Metoclopramide is commonly used to treat nausea and vomiting associated with conditions such as uraemia,
radiation sickness, cancer and the effects of chemotherapy, labor, infection, and emetogenic drugs. It is also
used in pregnancy as a second choice for treatment of hyperemesis gravidarum (severe nausea and vomiting of
pregnancy).It is also used preventatively by some EMS providers when transporting people who are conscious
and spinally immobilized.
Migraine
In migraine headaches, metoclopramide may be used in combination with paracetamol (acetaminophen) or in
combination with aspirin.
It is also used in gastro esophageal reflux disease

16. OBJECTIVE
A high performance liquid chromatographic instrument is used for the study of RP-HPLC
method for quantitative determination of metoclopramide in pure and drug samples. The
present developed method is rapid, cheaper, time saving and effective than the previous
method. The proposed method will be validated as per ICH guidelines.
The extensive literature survey carried out, reveals that not much work has been done on this
particular drug for its determination in bulk and pharmaceutical dosage form using HPLC and
UV techniques. In view of the need for a suitable method for analysis, attempts are being made
to develop a simple, precise and accurate analytical methods for the estimation of
metoclopramide .

17. Hence there is a need to validate the new methods developed.
The study makes an attempt to establish sensitive, precise and accurate methods for the
estimation of metoclopramide in bulk and pharmaceutical dosage form
•To develop a new simple, sensitive, accurate and economic analytical RP-HPLC method for
estimation of metoclopramide.
• Validating the proposed newly developed methods in accordance with the analytical
parameters mentioned in the IP, USP, BP and ICH guideline.
•To apply the proposed newly developed methods for analysis of these drugs in their bulk and
pharmaceutical dosage form.
•To compare results of various methods.

18. • Supriya Shidhaye et al., have reported development and validation of stability indicating HPLC method for
estimation of Metoclopramide Hydrochloride from a novel formulation using a mobile phase ofacetonitrile:
water (25:75), with 0.06 % triethylamine and pH adjusted to 4 using orthophosphoric acid. The analysis
wascarried out using Hi-Q-Sil C18 column [250 mm x 4.6 mm, 5 μm] at flow rate of 1 ml/min and the UV
detection at 274nm. The method was validated for accuracy, precision, linearity, range, selectivity and
robustness. The linearity of the proposed method was investigated in the range of 0.5–18 μg/ml (r2 =
0.9985).[17]
• Ahmad Khan, et al.,have done validation and application of RP-HPLC method for the quantification of
Metoclopramide hydrochloride in oral formulations prepared for IVIVC studies using mobile phase of
acetonitrile,20mM potassium dihydrogen phosphate buffer solution (pH 3.0 adjusted with orthophosphoric
acid) in the ratio of 40:60. The column used was Waters C18 3.9×300mm μBondapak (RP). The flow rate of
the mobile phase was 2ml/ minute.The detector was set at the wavelength of 275nm. This method validated
in plasma and was found to be linear, withcorrelation coefficient (R2), value of 0.9988, in the range of 48
ng/ml-0.25ng/ml.[18]

19. • Gaikwad S, et al.,have reported RP-HPLC method for the simultaneous determination of metoclopramide
hydrochloride and paracetamol in tablet dosage form. The method was carried out on a HiQsil C8,
(4.6×250mm) column with a mobile phase consisting of acetonitrile:acetate buffer (pH 6.78) (50:50v/v) at a
flow rate of 1ml/min. Detection was carried out at 308 nm. The retention time of paracetamol and
metoclopramide hydrochloride was 3.2 and 5.5 min respectively.[19]
• Dudhane NP,et al.,have done Simultaneous UV Spectrophotometric estimation of Metoclopramide
hydrochloride and Paracetamol in solid dosage form. UV Spectrophotometric method includes Simultaneous
Equation method (Method I), Absorbance Ratio method (Method II) and correction method (Method III), For
development of Method I, wavelengths selected were243.0 nm and 273.5 nm for estimation of
metoclopramide hydrochloride (MET) and paracetamol (PAR)respectively while for Method II, 243.0 nm
λmax for paracetamol, 262.0 nm Isoabsorptive point of Par and Met and309.0 nm for correction method.[20
Review of literature

20. Apparatus and software:
•Agilent 1120 Compact LC HPLC system
• Gradient pump
•Rheodyne injector
•UV variable wavelength detector
•Agilent syringe
• Zorbax Eclipse Plus C8 column 5µm, (4.6x150mm)
•Analytical weighing balance (Shimadzu AUX 220)
•Sonicator (EQUITRON230VAC, 50Hz),
•Vaccum pump
•Filtration kit (TARSONS) and Nylon membrane filter (Merck Millipore)
•Double beam UV Visible spectrophotometer (SHIMADZU-UV 1700)
•The EZ Chrome Elite software-single channel
HPLC METHODOLOGY
Reverse phase HPLC techniques were selected

21. Reagents and Solvents:
•Pharmaceutical formulation tablet (label claim containing 10 mg of metoclopramide hydrochloride)
•HPLC grade Acetonitrile (Merck)
• Ammonium Acetate
Selection of Wavelength:
Selectivity of HPLC method that uses UV detector depends on proper selection of wavelength. The
component shows reasonably good response at 274nm.
Absorption Maxima of metoclopramide hydrochloride

22. S.No Parameters Estimation of metoclopramide hydrochloride
1 Mobile phase optimized Ammonium acetate (buffer):
acetonitrile (45:55v/v, pH 3.7)
2 Stationary phase C18 5µm 250 X 4.6 mm
3 Flow rate (ml/min) 1
4 Run time( min) 6
5 Column Temperature OC 25±1
6 Volume of Injection (µl) 20
7 Detection Wavelength (nm) 274nm
8 Retention time Rt 3.16min for metoclopramide hydrochloride
Optimized chromatographic conditions for estimation of metoclopramide
hydrochloride using RP-HPLC Method

23. RESULT
LINEARITY
Concentration (
µgml-1 )
Area
5 2500971
10 5218585
15 7615822
20 11431030
25 12776416
30 16034244
• By using the working standard, a liquots of 5µg/ml,
10µg/ml, 15µg/ml, 20µg/ml, 25µg/ml, 30µg/ml,
were prepared with acetonitrile and buffer mixture.
• Six dilutions of each of the above mentioned
concentrations were prepared separately and from
these six dilutions, 20µl of each concentration were
injected into the HPLC system.
• Then their chromatogram was recorded
LINEARITY DATA FOR METOCLOPRAMIDE HYDROCHLORIDE

24. Precision
The inter day (between 2 days) and intra day (at the same days: morning and evening) precision were carried
out . The variation of results were calculated and %RSD was determined
Chromatogram showing intraday precision (At morning) Chromatogram showing intraday precision (At afternoon)

25. Chromatogram showing interday precision (day1
Chromatogram showing interday precision (day 2)

26. Morning
Injection(
15µg/ml)
Areas Average Sd Rsd%
1 7784037
7690594 46094.57 0.59
2 7668002
3 7670245
4 7665378
5 7680400
6 7675504
Afternoon
Injection(
15µg/ml)
Areas Average Sd Rsd%
1 7760697
7721837 46998.14 0.6
2 7791690
3 7670772
4 7727592
5 7691525
6 7688746
Day1
Injection(
15µg/ml)
Areas Average SD Rsd%
1 7754104
7685104 84655.31 1.1
2 7798518
3 7717862
4 7592003
5 7601515
6 7646622
Day2
Injection(
15µg/ml)
Areas Average SD Rsd%
1 7683557
7664471 37500.65 0.48
2 7639220
3 7601398
4 7688907
5 7701884
Precision

27. Accuracy
The accuracy for estimation of metoclopramide using acetonitrile was determined by adding known amount of the
analyte. The accuracy was calculated from the test results as the percentage of the analyte recovered by the assay
Chromatogram for 100% accuracy
Chromatogram for 80% accuracy

28. Chromatogram for 120% accuracy
Sl.no Level of
percentage
recovery
Amount
present
(mg/tablet)
Amount of
standard drug
added
Area response Mean SD RSD
%
Total amount
recovery
%
recovery
1 80% 10 8 7572240 7635249 56582.27 0.74 10.02 100.2
7681718
7651790
2 100% 10 10 11404069 11432140 64029.56 0.56 10 100
11505411
11386941
3 120% 10 12 12844734 12893282.33 86104.30 0.61 10.88 100.88
12842415
12992698
Accuracy for metoclopramide hydrochloride

29. Limit of detection (LOD) and Limit of quantification (LOQ)
LOD and LOQ were calculated according to ICH recommendations where the approach is based
on the signal-to-noise ratio. Chromatogram signals obtained with known low concentrations of
analytes was compared with the signals of blank samples. A signal to noise ratio 3:1 and 10:1
was considered for calculating LOD and LOQ respectively.
Table : LOD and LOQ for estimation of metoclopramide hydrochloride.
Name of drug LOD µg/ml LOQ µg/ml
Metoclopramide
hydrochloride 0.1601 0.5337

30. Conclusion
The proposed RP-HPLC method can be used for estimation of metoclopramide
hydrochloride in bulk and in pharmaceutical formulation since it is simple, precise, and
accurate. According to the results obtained, it met all the criteria given on USP and ICH
method of validation .So this method can be used for quantitative estimation, quality
control and quality assurance of metoclopramide hydrochloride in laboratories and
pharmaceutical industry

31. S.No Parameters Estimation of metoclopramide hydrochloride
1 Mobile phase optimized Ammonium acetate (buffer):
acetonitrile (45:55v/v, pH 3.7)
2 Stationary phase C18 5µm 250 X 4.6 mm
3 Flow rate (ml/min) 1
4 Run time( min) 8
5 Column Temperature OC 25±1
6 Volume of Injection (µl) 20
7 Detection Wavelength (nm) 274nm
8 Retention time Rt 3.16min for metoclopramide hydrochloride
Summary

32. 9 λ max 274nm
10 Linear Range(µg/ml) 5-30
11 Correlation Coefficient(r2) 0.9918
12 Limit of Detection (µg/ml) 0.1601
13
Limit of
Quantification(µg/ml)
0.5337
14
Number of Theoretical Plates
per meter
6130
15 Tailing Factor 1.30
16 Capacity Factor 0.0000
17 Signal/Noise ratio 93.68224

33. REFERENCE
• Sharma BK. Instrumental methods of Chemical Analysis. 19th ed. Meerut: Goel
Publishing House; 2000. 1
• Stedman's Medical Dictionary. Retrieved 2014-05-01 – via Drugs.com
• Skoog, Douglas A.; West, Donald M.; Holler, F. James; Crouch, Stanley R.
(2014). Fundamentals of Analytical Chemistry. Belmont: Brooks/Cole, Cengage
Learning. p. 1. ISBN 0-495-55832-X.
• B.K.Sharma. Spectroscopy. 23rd ed. Meerut: Goel Publishing House; 2011:
3,4,11.
• Prepared by Experienced Academicians. A textbook of Pharmaceutical Analysis. 4th ed.
Mehdipatnam: Professional Publications; 1.22-1.26.

34. • V.Shivashankar ,M.Gandimati,T.K.Ravi . RP-HPLC method development and validation
for the analysis of rivaroxaban in pharmaceutical dosage forms.International Journal of
pharmacy and analytical research.Dec 2015;4(4):406-10.
• Prepared by Experienced Academicians. A textbook of Pharmaceutical Analysis. 4th ed.
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• Beckett AH, Stenlake JB. Practical Pharmaceutical Chemistry. 4th ed. New Delhi: CBS
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• Tips on Liquid Chromatography, Waters, www.waters.com.
• https://www.google.co.in/imgres?imgurl=https://image.slidesharecdn.com/classification of
chromatography.