Development and Validation of HPTLC Method for Simultaneous Estimation of Omeprazole and Ondansetron in Tablet Dosage Form

Zarna Dedania¹*, Ronak Dedania¹, G Vidhyasagar¹, Bhavna Patel², Chetan Ramolia³ and Vaishali Karkhanis⁴

¹Department of Pharmaceutical Analysis, Veerayatan Institute of Pharmacy, Kutch

²Ramanbhai Patel College of Pharmacy, Changa, Gujarat India

³CRO, Veeda, Ahmedabad, Gujarat India

⁴A.R. College of Pharmacy, Vallabh Vidhyanagar

*Corresponding Author E-mail: zaroo229@yahoo.co.in

ABSTRACT

A simple, precise, sensitive, rapid and reproducible HPTLC method for the simultaneous estimation of the omeprazole and ondansetron in tablets was developed and validated. This method involves separation of the components by HPTLC on precoated silica gel G₆₀F254 plate with solvent system of methanol: ethyl acetate: toluene(2:6:2 % v/v) and detection was carried out densitometrically using a UV detector at 280 nm in absorbance mode. This system was found to give compact spots for omeprazole (Rf value of 0.68±0.02) and for ondansetron (Rf value of 0.17±0.02). Linearity was found to be in the range of 1000-3500 ng/spot and 1000-3500 ng/spot for omeprazole and ondansetron respectively. The limit of detection and limit of quantification for omeprazole were 123.42 and 374.01 ng/spot and for ondansetron were 134.63 and 407.97 ng/spot, respectively. The method was found to be beneficial for the routine analysis of combined dosage form.

KEYWORDS: Simultaneous Estimation; HPTLC; Omeprazole; Ondansetron;

INTRODUCTION:

Omeprazole is chemically 5-methoxy-2-[[(4-methoxy-3, 5-dimethyl-2-pyridinyl) methyl] sulfinyl ]-1H-benzimidazole and it is used as proton pump inhibitor and in the treatment of gastro-oesophageal reflux disease (GERD), peptic ulcer and Zollinger-Ellison syndrome ¹. Ondansetron is chemically (±) 1, 2, 3, 9-tetrahydro-9-methyl-3-[(2-methyl-1H-imidazol-1-yl) methyl ]-4H-carbazol-4-one, monohydrochloride, dehydrate and it is used as selective 5-HT₃receptor antagonist and used in the management of nausea and vomiting induced by cytotoxic chemotherapy and radio therapy and also post operative nausea and vomiting. Literature survey reveals that RP-HPLC in formulation ^{2, 3} and in plasma and blood ^{4, 5, 6}, HPTLC ⁷, spectrophotometry ⁸ are available for the determination of omeprazole and spectrometric ^{9, 10} in formulation, RPHPLC in formulation¹¹ and blood ¹²for determination of ondansetron. The review of the literature revealed that no HPTLC method has so far been reported for the combination of omeprazole and ondansetron. So an attempt has been made to estimation of omeprazole and ondansetron simultaneously by HPTLC.

MATERIALS AND METHODS:

DORON-O containing 10 mg/tab of omeprazole and 4 mg/tab of ondansetron, which are manufactured and marketed by BestoChem Formulation (I) Ltd., Delhi was estimated. Reference standard of omeprazole and ondansetron were kindly supplied as a gift sample by BestoChem Formulation (I) Ltd., Delhi. All chemicals and reagents used were of analytical grade and were purchased from E. Merck (India) Ltd., Mumbai. A Camag HPTLC system comprising of Camag Linomat V semi automatic sample applicator, Hamilton Syringe, Camag Twin trough chamber, Camag TLC Scanner-3, Camag WinCATS Software and stationary phase precoated silica gel G60F₂₅₄ and ultrasonicator were used.

Preparation of standard and sample solution:

Standard stock solution mixture of omeprazole and ondansetron were prepared in methanol of 500 ng/μl and 500 ng/μl. Aliquots of standard solution having concentration ranging from 1000-3500 ng/spot and 1000-3500 ng/spot (2, 3, 4, 5, 6 and 7 μ1) of omeprazole and ondansetron were applied on the TLC plate. The TLC plate was dried, developed and analysed.

Table 1: Method Validation Parameters for Proposed Method

Parameters	Omeprazole	Ondansetron
Linearity range (ng/spot)	1000-3500	1000-3500
Correlation coefficient	0.9925	0.9932
Slope	2.7387	2.5107
Intercept	5216.6	3679.8
Limit of Detection (ng/spot)	123.42	134.63
Limit of Quantitation (ng/spot)	374.01	407.97
Repeatability (RSD)	0.91	1.07

Table 2: Precision Data for Omeprazole and Ondansetron:

Drug

Concentration

(µg/spot)

Intra day (n=3)

% RSD

Inter day (n=3)

% RSD

Omeprazole

2000

2500

3000

100.28 ± 0.79

100.10 ± 0.49

101.04 ± 0.09

100.45 ± 0.76

99.72 ± 0.81

101.23 ± 1.09

Ondansetron

2000

2500

3000

100.32 ± 0.26

101.28 ± 0.11

100.14 ± 0.32

101.68 ± 0.15

101.42 ± 0.45

9.75 ± 0.86

Twenty tablets were weighed, powdered, transferred into a volumetric flask and extracted with methanol. The extract was filtered through Whatmann filter paper No 41 and residue was washed with methanol. Aliquots of 3 µl (3000 ng/spot of omeprazole and 1200 ng/spot of ondansetron) were applied on the precoated silica gel G60 F₂₅₄ TLC plate.

Chromatoghaphic condition:

The TLC plates were pre-washed with methanol and activated by keeping at 115° for about 30 min. The samples were spotted in the form of bands of width 6 mm at 4 mm interval under stream of nitrogen gas with Hamilton microlitre syringe on the precoated silica gel G₆₀ F₂₅₄ plate (10×10 cm), slit dimension was kept at 5×0.45 mm, respectively. The mobile phase used was methanol: ethyl acetate: toluene(2:6:2 v/v/v), chamber and plate saturation time of 30 min, migration distance allowed was 70 mm, linear ascending development was carried out in 10×10 cm, twin trough glass chamber. Subsequent to the development TLC plates were dried in a current of air and then scanned between 200 and 400 nm using Camag TLC scanner using Win CATS software. Both components show reasonably good response at 280 nm and densitometric scanning was performed at 280 nm. The overlain spectra are shown in Fig. 1.

Fig 1: Overlain spectrum of Omeprazole (OME) and Ondansetron (OND) at 280nm

Fig 2: Chromatogram of standard solution containing Omeprazole and Ondansetron using mobile phase as Methanol: Ethyl Acetate: Toluene (2:6:2 %v/v)

Aliquots of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 µl standanrd solution of omeprazole and ondansetron (500 µg/ ml) were applied on TLC plate. TLC plate was air dried, developed and analysed.

Validation of Method:

The developed methods was validated in terms of linearity, accuracy, limit of detection, limit of quantitation, intra day and inter day precision, repeatability of measurement as well as repeatability of sample application.

Analysis of the marketed formulation:

Three µl of sample solution of marketed formulation was spotted on the same plate followed by development scanning. The analysis was repeated in triplicate. The content of the drug was calculated from the peak area recorded.

RESULTS AND DISCUSSION

The method was validated for linearity, accuracy, limit of detection, limit of quantification, inter-day and intra-day assay precision, repeatability of measurement and repeatability of sample application and validation parameter shown in Table 1. The mobile phase consisted methanol: ethyl acetate: toluene (2:6:2 v/v/v), gave R_f values of 0.68 ± 0.02 for omeprazole and 0.17 ± 0.02 for ondansetron shown in Fig 2. A good linear relationship was obtained over the concentration range of 1000-3500 ng/spot of omeprazole and 1000-3500 ng/spot of ondansetron, respectively. The linear regression data showed a regression co-efficient of 0.9925 for omeprazole and 0.9932 for ondansetron. The limit of detection was found to be 123.42 and 134.63 ng/spot for omeprazole and ondansetron respectively. The limit of quantitation was found to be 374.01 and 407.97 ng/spot for omeprazole and ondansetron respectively. The 3D view of all tracks of omeprazole and ondansetron at 280nm with Tablet formulation shown in Fig 3

Table 3: Recovery Study for Omeprazole and Ondansetron:

Label claim Mg/tablet

Amount added %

Total amount added (mg)

Amount recovered * (mg) ± SD

% Recovery ± SD

Omeprazole

100

150

5.07 ± 0.11

10.13 ± 0.07

14.98 ± 0.14

101.4 ± 2.22

101.36 ± 0.70

99.90 ± 0.99

Ondansetron

100

150

1.98 ± 0.07

3.98 ± 0.08

6.09 ± 0.05

99.33 ± 3.88

99.51 ± 1.75

101.6 ± 0.82

* Each value indicate mean of three determination

Table 4: Analysis of Omeprazole and Ondansetron:

Label claim (mg/tablet)	% of Drug found *	% RSD
Omeprazole 10	100.58 ± 0.18	0.18
Ondansetron 4	99.45 ± 0.45	0.45

* Each value is mean of three determinations

Fig 3: 3D view of all tracks of Omeprazole (OME) and Ondansetron (OND) at 280nm with Tablet formulation

The intra and inter day precision are given in Table 2. Repeatability of sample application was assessed % RSD for measurement of peak area of omeprazole and ondansetron were found to be 0.91 and 1.07 respectively. To confirm specificity of proposed method, the solution of the formulation was spoted on the TLC plate, developed and scanned. It was observed that the excipient present in the formulation did not interfere with the peaks of omeprazole and ondansetron.

Recovery study of drugs were carried out for the accuracy parameter. The recovery of added standard was studied at three different levels viz. 50%, 100% and 150% for omeprazole and ondansetron, of the estimated amount of drug. Percentage Recovery was found to be within the limits as listed in Table 3. The assay value for the marketed formulation was found to be within limit as listed in Table 4. The low RSD value indicates precision and suitability of the method for routine analysis of omeprazole and ondansetron in pharmaceutical dosage forms.

Hence the developed HPTLC technique is simple, precise, specific and accurate, statistical analysis proved that the method is repeatable and selective for simultaneous analysis of omeprazole and ondansetron as bulk drugs and in pharmaceutical dosage forms without any interference from the excipients.

ACKNOWLEDGMENTS:

The authors are grateful to Institute of science and technology for advanced studies and research (ISTAR) and A. R. college of Pharmacy Vallabh vidhyanagar, India, for providing the facilities to carry the experiment and BestoChem Formulation (I) Ltd., Delhi for providing gift samples of omeprazole and ondansetron.

REFERENCES:

1. James EF and Kathlean P. Martindale. The extra Pharmacopoeia.1232-2.

2. Alexandre S, Almeci LW, Cleder A, Cristiane C, Lisians B, Ricardo C and Simone GC. Determination of Omeprazole in bulk and injectable by LC. J. of AOAC. 2003; 86(3): 501-504.

3. Gangadhar S, Rajendra kumar GS, Mamidi and Rao, N.V.S. RP-HPLC assay of Omeprazole in plasma. Indian Drugs. 1997; 34(2): 99-101.

4. Balian JD, Sukhova N, Harris JW, Hewett J, Pickle L, Golestein JA, Woosley RL and Flockart DA. The hydroxylation of omeprazole correlates with S-mephenytoin metabolism. Cli. Pharma Thera.1995; 57: 662-629.

5. Karol MD, Granneman GR and Alexander K. Determination of lansoprazole and five metabolites in plasma by HPLC J. of Chromato B 1995; 668: 182-186.

6. Cairns AM, Chiou RH, Rogers JD and Demetriades JL. Enantioselective HPLC determination of omeprazole in human plasma, J. of Chromato B 1995; 666: 323-328.

7. Ray S and De Prabir kumar. HPTLC and TLC method for rapid quantification and identification of Omeprazole. Indian Drugs 1994; 31(11): 543-547.

8. Sastry CSP, Naidu, PY and Murthy, SSN. Spectrometric determination of Omeprazole in pharmaceutical dosage form. Indian Drugs 1996; 33(12): 607-610.

9. Siva T, Rao and Sastry CSD. Spectrometric determination of Amiodarone and Ondansetron in Pharmaceutical dosage form with citric acid- acetic acid anhydrate reagent. Indian J. Pharma Sci 2002; 64(5): 482- 484.

10. Rajasekaran A., Krishnaveni A., Maalathi G. and Jayalair B. Spectrometric determination of Ondansetron Hydrochloride in Pharmaceutical preparation. Indian Drugs 1999; 36(2): 127-129.

11. Stewart JT, Venkateshwaran TG and King, DT. HPLC determination of ondansetron-atropine and ondansetron-glycopyrrolate mixtures in 0.9% sodium chloride injection. J. of Liq Chrom 1995; 18: 2647-2659.

12. Colthup PV, Felgate JL and Scully NL. Determination of ondansetron pharmacokinetics in the young and elderly. J. of Pharma Sci 1991; 80: 868-871.

Received on 04.07.2009 Modified on 09.08.2009

Asian J. Research Chem. 2(4):Oct.-Dec. 2009 page 574-576