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Jun 3, 2011 - Development and Validation of RP-HPLC and Spectrophotometric. Method for Simultaneous Estimation of Domperidone and. Pantoprazole in ...
International Journal of Chemical and Analytical Science ISSN: 0976-1206 Research Article www.ijcas.info

Development and Validation of RP-HPLC and Spectrophotometric Method for Simultaneous Estimation of Domperidone and Pantoprazole in Pharmaceutical Dosage Forms Sarif Niroush Konari* and Vipin Prakash Dept of Pharmaceutical Analysis, JDT ISLAM College of Pharmacy, Calicut -12. A simple, precise, accurate and rapid HPLC and spectrophotometric method has been developed and validated for determination of pantoprazole and Domperidone simultaneously, in combined dosage form. The mobile phase used was a mixture of solvent A and solvent B (50; 50). Solvent A was acetonitrile, Solvent B HPLC water and 0.4%v/v. Triethylamine, PH is adjusted to 4 with orthophosporic acid. The detection of pantoprazole & domperidone was carried o ut on 236nm u sing PDA dectector and in ternal standard used was etoricoxib, the spectrophotometric method involves the formation and solvation of simultaneous equation at 285nm and 301nm using Acetonitrile and water (2:8) as solvent. The results of the analysis were validated statistically, and by recovery studies. The proposed method can be successfully used to determine the drug content of marketed formulation. Key Words: Pantoprazole- PAN, Domperidone- DOM, Etoricoxib- ETR, Acetonitrile-ACN, Triethylamine-TEA, High performan ce liquid chromatography-HPLC

INTRODUCTION Pantoprazole1 used as anti ulcer drug.itis a newer H+ K+ ATPase inhibitor particularly employedin bleeding ulcer and acute stressulcer. It is chemically 5-(Difluoromethoxy)-2-{(3, 4dimethoxy-2-pyridinyl) methyl) sulfinyl} H- benziimidazole. Domeridone2,3 is antiemetic drug it is chemically 5-chloro-1{1-(3{2,3-dihydro-2-oxo-1H–benzimidazol—1yl)propyl}-4piperidinyl}-1,3-dihydro-2H-benzimidazol-2-one. Literature survey shows that HPLC methods for determination of pantoprazole& domperidone4and it’s individual HPLC method 5 were also reported. Suvery also indicate that spectrophometric6some bioanlytical7,8,9, and HPTLC10method for estimation of pantoprazole and domperidone were also reported but no method is reported for simultaneous estimation of pantoprazole and domperidone and their validation parameter in the pharmaceutical dosage form using RP- phenyl column and by spectrophometrically. The available methods are time consuming, tedious and expensive. The proposed method is rapid, simple, accurate, reproducible and successfully employed in routine analysis of both drugs simultaneously in tablet dosage form.

MATERIALS AND METHODS Water HPLC grade (rankem), Acetonitrile HPLC grade (s, d fine), TriethylamineHPLC grade, Orthophosphoric acid HPLC grade. Double distilled water for spectrophometric work. Instruments used: Dhona balance 200 D, Elico pH meter LI 127, Eutech pH meter, Shim adzu L C-20 Separation is carried out on an isocratic HPLC system with LC20at binary HPLC pump, LC20at PDA detector, LC lab solution software, and RP-Phenyl column, (25×0.046mm i. D.; particle size 5µ ), Sonica Ultrasonic cleaner, Millipore, UV/Vis double beam spectrophotometer models SHIMADZU UV-1700 with 1cm UV matched quartz cells were used. Methods development in HPLC: The following chromatographic conditions were fixed for the simultaneous estimation of pantoprazole and domperidone.

Stationary phase: Phenomenex phenyl hexyl column (250 mm x 4.6mm i.d, 5µm), Mobile phase: Solvent A: water and 0.4%v/v TEA, Solvent. B: Acetonitrile, PH: 4 (adjusted with Orthophosphoric acid). Solvent ratio: 50:50, Detection wavelength: 236nm, Flow rate: 1ml/min, Temperature: Room temperature of 20±2oC, Internal Standard: Etoricoxib (10µg/ml) Preparation of Standard Solution: 1. 10 mg of pantoprazole was taken in a 10 ml standard flask. To this 2 ml of mobile was added for dissolving the drug. Shake it for one min. to get a clear solution and make up the volume to 10 ml with mobile phase solution. 2. 10 mg of domperidone was taken in a 10ml standard flask and diluted with few ml of mobile phase until the sample dissolves completely and make up the volume to 10 ml with mobile phase. 3. The internal standard solution was prepared by taking 10 mg of Etrocoxib in a 10ml standard flask. It is dissolved by adding 3ml of mobile phase, shake it for few minutes to get a clear solution and make up the final volume to 10ml with mobile phase. 4. The final standard solution was prepared in such a way that each standard flask contains 2, 4, 6, 8, 10µg and 4, 8, 12, 16, 20µg of domperidone and pantoprazole and 10µg of etrocoxib (IS). Preparation of Formulation Solutions: Twenty tablet containing 20mg of pantoprazole and 10mg domperidone were weighed and finely powdered. From the powdered tablet, a quantity of powder equivalent to 10mg was weighed. Then was then extracted this was then filtered and finally it is diluted to 1000µg/ml with mobile phase. From this final dilution were prepared contain 4µg/ml of domperidone and 8µg/ml of pantoprozole and 10µg/ml of intern al standard for b rand A. For brand B it was 6 µg/ml of domperidone, and 12µg/ml of pantoprozole & 10µg/ml of intern al standard.

Corresponding Author: Sarif Niroush K onari, Dept of Pharmaceutical Analysis, JDT ISLAM College 37 of Pharmacy, Calicut -12. Received 30-04-2011; Accepted 03-06-2011 August, , 2011

International Journal of Chemical and Analytical Science, 2011, 2 (8), 133-135

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Sarif Niroush Konari and Vipin Prakash: RP-HPLC and Spectrophotometric Method for Estimation of Domperidone and Pantoprazole

Method or Recording of chromatogram: The typical chromatograms of the sample and standard solutions were also recorded. The chromatograms of sample showing the3D image & peak pu rity profile and peak purity curves of pantoprazole, domperidone and etrocoxib (IS) were also shown. This procedu re was repeated using the sample solution. The peak areas were noted and the response factors of the standard and sample solution peaks were calculated. Spectrophotometric Method Development: Simultaneous equation method based on principle that, the total absorbance of the components in a mixture is the sum of individual absorbance, two wave length selected to frame the simultaneous equation method were at 285nm and 301 nm (fig no 7; & fig no: 8) since at these two wave length; ratio of absorptivities of two component are maximum. For calibration curves, stock solutions of domperidone and pantoprazole in the concentration of range of 5 – 15μg/ml and 7–17.5μg/ ml respectively. The absorbance of domperidone and pantoprazole were measured at 285nm and 301nm, calibration curves were plotted. The absoptivities of both the drugs at both the wavelengths were determined. The absorbance and the absorptivity values at the particular wavelength were calculated and substituted in the following equation11, to obtain the concentration. Cdom = (A1ax2 – A2ax1) / (ax2ay1 – ax1ay2). ……… (Equation no;1) Cpan = (A2ay1 – A1ay2) / (ax2ay1 – ax1ay2). Where, Cdom, Cpan are concentration of domperidone and pantoprazole respectively, A1 is the absorbance of sample at 285nm, A2 is the absorbance of sample at 301nm, ax1 is the absorptivity of dom at 285nm and ax2 is the absorptivity of dom at 301nm, ay1 is the absorptivity of pan at 301nm and ay2 is the absorptive of pan at 285nm. Preparation Standard Stock Solution: Standard stock solution for domperidone and pantoprazole were prepared by weighing each10mg of domperidone and pantoprazole were transferred to a 10ml volumetric flask and volume made to 10ml with to get a concentration of 1mg/ml, with acetonitrile and water (2:8) from this solution, an aliquot of 1ml was withdrawn, and it was further diluted to10 ml with water (100µg/ml).

Preparation of calibration curves: For preparation of calibration curves, stock solutions of domperidone and pantoprozole the concentration of range of 5 –15μg/ml and 7.5 –17.5μg/ ml were taken respectively. For domperidone 0.5ml, 0.75ml, 1ml, 1.25ml, 1.5ml were withdrawn from standard stock solution (100µg/ml) and make to 10ml with water so that concentration of these solution contain 5µg/ml, 7.5µg/ml, 10µg/ml, 12.5µg/ml, 15µg/ml. For pantoprozole 0.75ml, 1ml, 1.25ml, 1.5ml, 1.75ml were withdrawn from standard stock solution (100µg/ml) and make to 10ml with water so that concentration of these solution contain 7,5µg/ml, 10µg/ml, 12.5µg/ml, 15µg/ml, 17.5 µg/ml. Preparation of Sample Solution: Twenty Tablets of brand Pantium -D– SR (intas Pharma), Pepmark SRD (unimarck ) label claim30mg of domperidone, and 40mg of pantoprazole were weighed, average weight determined and finely powdered. Appropriate quantity of powder from each tablet equivalent to 30mg of domperidone and 40mg pantoprazole was accurately weighed. The mixture was then extracted with acetonitrile and water. Then extract was filtered through whatman filter no: 41and filtrate was further diluted to get final concentration. 5µg/ml to 17.5µg/ml for both brands. Absorbance of this solution was measured at 285nm, and 301nm and valu es were substituted in the respective equation (no:1 )

RESULTS AND DISCUSSION Estimation of pantoprazole and domperidone in dosage forms by High Performance Liquid Chromatography was carried out using op timized chromatographic conditions. The standard and sample solutions were prepared and chromatograms were recorded. The peak area ratios of standard and sample solutions were calculated. The assay procedu re was repeated for 6 times and mean peak area, mean peak area ratio, mean weight of standard drugs, mean weight of sample taken for assay were calculated. The percentages of individual drugs found in formulations, mean, and standard deviation in formulations were calculated and presented in Table 1. The results of analysis shows that the amount of drugs was in good agreement with the label claim of the formulation.

Table 1: Analysis of Formulation Drug Name

Brand A* Brand B * Mean of Three Replicates Brand A –dompan Brand B -pentab

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Domperidone Pantoprazole Domperidone Pantoprazole

Label Claim Mg/Tablet 10mg 20mg 10mg 20mg

Estimated*Amount Mg/Tablet 9.98 20.09 9.98 20.05

% Label claim* 99.82 100.45 99.98 100.25

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% RSD* 0.22 0.27 0.17 0.14

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Sarif Niroush Konari and Vipin Prakash: RP-HPLC and Spectrophotometric Method for Estimation of Domperidone and Pantoprazole

Validation of the HPLC Method12, 13 The accuracy of the method was determined by recovery experimen ts. The recovery studies were carried out 6 times and the percentage recovery and percentage relative standard deviation of the percentage recovery were calculated and presented in Table 2. The chromatogram of the recovery studies was recorded at two differen t levels and shown in Fig. 5. From th e data obtained, recoveries of standard drugs were found to be accurate and are within the specified limits. The precision of the method was determined by studying repeatability and reproducibility. The response factor of drug peaks and percentage relative standard deviation were calculated. The results revealed that the method developed is reproducible. The standard drug solutions in varying concentrations ranging from 50 to 150% of the targeted level of the assay concen tration containing internal standard were examined by the assay procedu re. The linearity and range for drugs was found to be from 2 to 10µg/ml. for domperidone and 4to 20 µg/ml for pantoprazole The LOD and LOQ of the developed method were determined by analyzing progressively low concentration of the standard solutions using the developed methods. The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio of 3). LOD of domperidone and pantoprazole were found to be 175 and 315ng/ml. The LOQ is the smallest concentration of the analyte, which gives response that can be accurately quantified (signal to noise ratio of 10). The LOQ of domperidone, pantoprozole were found to be 530 and 950ng/ml. The resolution, capacity factor, theoretical plates/meter, peak symmetry was

calculated for the standard solutions and is presented in Table. 1. The values obtained demonstrated the suitability of the system for the analysis of the above drug combination. Table 2: Accura cy (Recovery Studies) of H PLC

Drug pantoprazole Domperidone

% Recovery* 50% 100% leve l level

% RSD* 50% leve l

100% level

99.94

100.11

0.04

0.05

99.80

100.20

0.05

0.02

* Mean of three Replicates

Spectrophotometric Method: The overlain spectra of both the drugs showed that the peaks are well resolved, thus satisfying the criteria for obtaining maximum precision, based on simultaneous equation (fig. no.8). The proposed methods were successfully used to estimate the amount of domperidone and pantoprazole, present in two of the marketed tablet formulations. The assay values for both the tablets were compared with corresponding labeled amounts and the validation parameters of proposed methods (Table no.4). The result of analysis has been validated statistically 14,15 and by recovery studies. The standard deviation was found less than 1% for the assay of tablet. The method was found to be simple, accurate, rapid, precise and econnmical, hence proposed method can employed for routine analysis of tablet in pharmaceutical industries

Table 3: System suitability studies of HPLC

Drug

Rs*

domperidone pantoprazole

10.854

N*

K’*

4889.109

0.000

9570.065

0.684

Tailing factor at (10%level) 1.1295 1.124

HETP 28 25

LODng/ml 175 315

LOQng/ml 530 950

Rs*- resolution, N* –theoretical plates, k*- capacity factor Table.4: Analysis of Commercial Formulation of Spectrophometric FORMULATIONS METHOD pantium- D-SR Domperidone pantoprazole % recovery* 97.84% 98.19% Standard deviat ion 0.1222 0.2730 (SD) Standard error (SE) 0.07055 0.1576 RSD in % 0.12 0.28 pepmark -SRD % recovery* 98.54 97.19 Standard deviat ion 0.3512 0.4314 (SD) Standard error (SE) 0.2028 0.2491 RSD in % 0.30 0.44 *Mean of Three Replicate

Fig. No: 3 typical chromatogram of standard solution of pantoprozole, domperidone and etoricoxib (IS) for brand B

Fig. No: 4: T ypical chromatogram of Sample solution of brand B

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Sarif Niroush Konari and Vipin Prakash: RP-HPLC and Spectrophotometric Method for Estimation of Domperidone and Pantoprazole

Fig. No: 8. over laid UV spectrum of domperidone & pantoprazole

Fig No. 5: chr omatogram of recovery studies 50%level

CONCLUSION HPLC and spectrophotometric methods has been developed for domperidone and pantoprozole in combined dosage form. On comparing both method, developed HPLC method has found more precise, less time consuming , accurate and rapid over spectrophotometric method hence, it can be effectively app lied for routine analysis in research institutions, quality control department in industries, app roved testing labo ratories, bio-pharmaceutics and bio-equivalence

ACKNOWLEDGEMENTS Fig No: 6. UV spectrum of pantoprozole

Pantoprazole and Domperidone were received from gift samples from Alidac, Ahmedabad, India to carry this work.

REFERENCE 1.

Merck Index, 12th Edition, White Hou se Publica tion, USA, pp1205

2.

Merck Index, 12th Edition, White Hou se Publica tion, USA, pp. 578

3.

CIIMS, CMPmedia India P vt Ltd, 2006, pp.70, 76

4.

RA Singh, S Arora, R Kumar, D Kumar, AK AgarwalPharma Rieveiw (2005)126

5.

Sudana., GS and Potadar, Indian journal of pharmaceutical sciences, 54/4(1992) 162-4

6.

P Ravi kumar, and Banu.p, E-Journal Of Chemistry 3/12 (2006)142-145

7.

Ramakriskna,N, vishwottam., KN., and Kotaeshwara,, journal of chromatography B, 882/1-2 (2005)326-329

8.

Deeptijain and sahu R., Preeti, Indian pharmaceutical science, 68/4 (2005)503-505

9.

Ferron Gerald ine., M A bell, M, Un ruh., M, American Journal health system, pharmacy, 60/13( 2003)132-4

10.

DancaAgbaba, novovic D and karljikovic.K, journal planar chromatography (2004), 169-172

Fig No: 7. UV spectrum of domperidone

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journal

134

of

Sarif Niroush Konari and Vipin Prakash: RP-HPLC and Spectrophotometric Method for Estimation of Domperidone and Pantoprazole

11.

BeckettAH and Stenlake JB, pharmaceutical chemistry part2. Fourth ed., CBS publisher and distributor. new delhi 2004,pp.-284-286

12.

ICH Topic Q2A, “Validation of Analytical Procedures”: Text, 6th Nov 1996, www.ich.org.

13.

ICH Topic Q2A, “Validation of Analytical Procedures”: Methodology, 6th Nov 1996, www.ich.org.

14.

Gurmani N, an introduction to biostatics, first ed, MJP publisher, Chennai India, 2004pp80-88.

15.

Charles Henry Brase and Pellillo Brase, understandable stat istics, third ed, dc heath and company, Lexington Toronto Canada, 1987pp390-395.

Source of support: Nil, Conflict of interest: None Declared

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