A Validated HPTLC Method for Determination of Ondansetron in Combination with Omeprazole or Rabeprazole in Solid Dosage Form

Literature survey revealed HPLC5,6 in human plasma, visible spectrophotometric method7 for ondansetron in solid dosage form. For omeprazole methods reported are HPLC-MS and HPLC-UV in biological component dosage forms. J Pharm Biomed Anal 2001;25(1):77-84. Indian Pharmacopoeia. Vol. I, New Delhi: The Controller of 9. Publications; 1996. p. 469. British Pharmacopoeia Vol I, London: The British Pharmacopoeia 10. Commission; 2002. p. 1123. United State Pharmacopoeia National Formulary. Vol. 2, Rockville, MD: 11. United States Pharmacopoeial Convention, Inc., 2005. p. 1364. United State Pharmacopoeia Vol. 24, Supplement 1. Rockville, MD: 12. United States Pharmacopoeial Convention, Inc.; 2000. p. 621. Bhanu R, Kulkarni S, Kadam A. Simultaneous estimation of gliclazide 3. and metformin in pharmaceutical dosage by reverse phase HPLC. Indian Drugs 2006;43(1):16-20. Bretnall AE, Clarke GS. Chromatographic method of analysis of 4. metformin hydrochloride. In: Brittain HG, editor. Analytical ProÞ les of drug substances and excipients. Vol. 25. New York: Academic Press; 1998. p. 243-58. Charles BG, Jascoben NW, Ravenscroft PJ. Rapid liquid 5. chromatographic determination of metformin in plasma and urine. Clin Chem 1981;27(3):434-6. Lad NR, Bhoir SI, Bhoir IC, Sundaresan M. Concurrent assay of 6. metformin and glimepiride in tablet using RP-HPLC with wavelength programming. Indian J Pharm Sci 2003;65(6):650-3. Yuen KH, Peh KK. Simple HPLC method for the determination of 7. metformin in human plasma. J Chromator B 1998;710(1-2):243-6. Vasudevan M, Ravi J, Ravisankar S, Suresh B. Ion-pair liquid 8. chromatography technique for the estimation of metformin in its multi Accepted 18 June 2008 Revised 20 December 2007 Received 6 June 2007 Indian J. Pharm. Sci., 2008, 70 (3): 383-386

Ondansetron combination with proton pump inhibitors has recently been introduced in market for the treatment of peptic ulcer, gastroesophagal reflux disease (GERD) and to prevent nausea.Ondansetron antagonizes 5HT-3 receptor both peripherally as well as centrally and block the initiation of the reflux, so is used as antiematic.Ondansetron is official in USP 1 .Omeprazole and rabeprazole are benzimidazole proton pump inhibitors, which suppress gastric acid ß uids 8,9 , capillary electrophoresis 10 , HPLC employing electrochemical and coulometric detection 11 , TLC 12 and spectrophotometry 13 .For rabeprazole method reported are HPLC for detection in blood plasma 14,15 spectrophotometric methods 16 , and LC-MS/MS 17 method.As no analytical method has so far been indicated for the ondansetron combinations with proton pump inhibitors, an attempt has been made to estimate them simultaneously by HPTLC.
Working standards of ondansetron, omeprazole and rabeprazole (25 mg each) were weighed and diluted with methanol to get the Þ nal concentration 0.05 µg/ µl for omeprazole and 0.02 µg/µl for ondansetron and 0.05 µg/µl for rabeprazole and 0.015 µg/µl for ondansetron.For ondansetron combination with rabeprazole, contents of twenty capsules was crushed to fine powder, quantity equivalent to 20 mg rabeprazole (6 mg ondansetron) was weighed accurately and transfer to 10 ml volumetric ß ask and for ondansetron combination with omeprazole, twenty tablets were crushed to Þ ne powder, weight equivalent to 10 mg of omeprazole (4 mg of ondansetron) was transferred to 10 ml volumetric ß ask.Then to each ß ask about 5ml of methanol was added and sonicated for 15 min, Þ nally volume was made to mark with methanol.The extracts were Þ ltered through Whatman filter paper 41 and required dilutions were made to get the final concentration containing 0.05µg/µl rabeprazole, 0.015 µg/µl ondansetron and 0.05 µg/ µl omeprazole, 0.02 µg/µl ondansetron and 6 µl of standard and sample were applied as 5 mm band on the TLC plate.
TLC plates were prewashed with methanol and activated prior to use.The chromatographic conditions maintained were: Precoated Silica gel 60 F 254 (20×10 cm) aluminum sheets as stationary phase.Dichloromethane:methanol (9:1 V/V) as mobile phase for both the ondansetron combinations.Samples were applied as bands 5 mm width at 14.1 mm intervals using Camag linomate V semiautomatic sample applicator and migration distance allowed was 80 mm, drying of plate done for 3 min at 60 0 temperatures.The plates were scanned at 309 nm for omeprazole and ondansetron and 294 nm for rabeprazole and ondansetron combination with Camag TLC scanner III, using Camag Win CATS software (Þ g. 1).detection for omeprazole, ondansetron and rabeprazole, ondansetron was found to be 99 ng/spot, 39.9 ng/spot, and 90.3 ng/spot, 54.2 ng/spot, respectively.The limit of quantification for omeprazole, ondansetron and rabeprazole, ondansetron was found to be 302.8ng/ spot, 121.1 ng/spot and 273.9 ng/spot, 164.4 ng/spot, respectively.
The linear regression data (n=6, Table 1) showed a good linear relationship over a concentration range 100 to 500 ng/spot for omeprazole, ondansetron and rabeprazole.Repeatability of method was determined by 6 times spotting 10 µl of standard drug solution on TLC plate, measurement of peak areas was performed and from the peak areas the % RSD was determined.For omeprazole and ondansetron % RSD was found to be 0.35 and 0.40, respectively and for rabeprazole and ondansetron % RSD was 0.19 and 0.45, respectively.Repeatability of measurement was determined by spotting 10µl of standard drug solution on TLC plate, after development the separated spots were scanned six times without changing position and % RSD for measurement of peak areas of omeprazole and ondansetron was found to be 0.64 and 0.80, respectively and for rabeprazole and ondansetron % RSD was 0.31 and 0.42, respectively.
For calibration curve, 0.1, 0.2, 0.3, 0.4 and 0.5 µg/ µl standard solution of omeprazole, rabeprazole and ondansetron were applied on TLC plate.The TLC plates were dried, developed and analyzed as described earlier.
Filtered solutions (6 µl) of the marketed formulations were spotted on to the plate followed by development scanning.The analysis was repeated six times, the spot was resolved into two peaks in the chromatogram of drug samples.The contents were calculated from the peak areas of standards and samples recorded.
A solvent system that would give dense and compact spots with appropriate and signiÞ cantly different R f values was desired for quantiÞ cation of ondansetron combinations.The mobile phase consisting of dichloromethane: methanol (9:1 V/V) gave R f value of 0.42±0.02,0.54±0.03,respectively for ondansetron and omeprazole while for ondansetron and rabeprazole, 0.41± 0.02 and 0.51±0.02,respectively (Þ g. 2).
The developed method was validated in terms of linearity and range, limit of detection, limit of quantiÞ cation, recovery study, inter days study, intra day study and study by different analysts.The limit of   The assay value for the marketed formulation was found to be within the limits as listed in Table 2.The low RSD value indicates suitability of the method for routine analysis of omeprazole, ondansetron and rabeprazole, ondansetron in pharmaceutical dosage form.Recovery studies were carried out to study accuracy and precision of the method.These studies were carried out at three levels i.e. multiple level recovery studies.To the powder formulations the pure standard drug were added at 80%, 100% and 120% levels, dilutions were made and analyzed by the method, the % recovery was calculated by using formula, % recovery = (T-A)/S × 100 where, T is total amount of the drug estimated, A is the amount of drug contributed by tablet powder and S is the amount of pure drug added.The results of recovery studies for both the ondansetron combinations were found to be around 99-100%, indicating that the method is free from interference from excipients.
The ruggedness of the method was evaluated by studying analyst to analyst, intra day and inter days variations and the % RSD was calculated, that was found to be within range.From the above results it can be concluded that the HPTLC method is accurate, precise, speciÞ c and reproducible and can be used for routine analysis of ondansetron combinations with proton pump inhibitors in solid dosage form.
There is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action because there has been an alarming increase in the incidence of new and re-emerging infectious diseases.Another big concern is the development of resistance to the antibiotics in current clinical use [1][2][3][4][5][6] .
Terminalia catappa L. belongs to the family Combretaceae.It is a tree and extensively planted in tropical India and Burma.The bark is rich in tannins, fruits are in ascorbic acid and seeds contain oil.The fruit is bitter, acrid, astringent and aphrodisiac.The leaves are maturant and emollient; the juice of leaves is used in the preparation of ointment for scabies, leprosy and other cutaneous diseases.The fruit is useful in bronchitis and bowels.The root bark is given in dysentery and diarrhea 7 .The fruits are used as antidiabetic, roots show antimicrobial activity.Manilkara zapota L. belongs to the family Sapotaceae.The plant is evergreen, glabrous tree with a milky juice.It is cultivated throughout India.The seeds are aperients, diuretic, tonic and febrifuge.The bark is antibiotic, astringent and febrifuge.It is used as tonic and the decoction is given in diarrhea and peludism 7 .Piper betel L. belongs to the family Piperaceae.This family usually contains herbs or shrubs often with swollen nodes, usually aromatic.The leaf is pungent, bitter, sweetish, acrid, heating, carminative, stomachic, anthelmintic, tonic, aphrodisiac and laxative.The leaves are useful in cough, foul smell in the mouth, ozoena, bronchitis, elephantiasis of the leg; improves appetite, it improves taste and appetite, tonic to the brain, heart, liver, strengthens the teeth, lessens

Fig. 1 :
Fig. 1: UV spectra of ondansetron in combinations UV spectra of ondansetron in combinations with (a) omeprazole and (b) rabeprazole, respectively

TABLE 1 : VALIDATION PARAMETERS
*Average of 6 determinations