Estimation of Satranidazole in Bulk and Tablet Dosage Form by RP-HPLC

Journal of Pharmacy Research Vol.3.Issue 11.November 2010

Vishwanath Agrahari et al. / Journal of Pharmacy Research 2010, 3(11),2747-2749

2747-2749

Research ArticleISSN: 0974-6943 Available online through

www.jpronline.info

*Corresponding author.Vishwanath AgrahariCollege of Pharmaceutical Sciences,RKGIT, Ghaziabad-201003, UP, IndiaTel.: + 91-9871229177E-mail:[email protected]

INTRODUCTIONSatranidazole (CG-10213-Go), a novel nitroimidazole possessing a C-N link-age at C

2 of the imidazole ring has been examined (during reduction), for its

ability to damage DNA. The drug produces extensive DNA damage character-ized by helix destabilization and strand breakage. Its comparison with other 2-and 5-nitroimidazoles indicates it may be more active towards anaerobes thanmany 5-nitroimidazoles. It is due to its relatively high redox potential whichmay make it more resistant to inactivation by oxygen [1]. It is recentlyintroduced as an anti-protozoal agent in tablet dosage form. It is a highlypotent, well-tolerated, and clinically useful agent against common protozoa.It is rapidly absorbed and exhibits higher plasma and liver concentration thanmetronidazole. Satranidazole (SAT) is not included in any officialpharmacopoeias such as IP, USP and BP. Chemically, it is 3-(1-methyl-5-nitroimidazol-2-yl)-1-(methylsulfonyl) imidazolidin-2-one (m.f.C

8H

11N

5O

5S;

m.w. 289.26) [2] Fig.1.Literature survey revealed an electron-capture gas chro-matographic assays in blood[3], spectrophotometric methods[4-7],HPTLC[8,9] and HPLC [10-13] methods in pharmaceutical dosage form forestimation of Satranidazole individually as well as in combination withOfloxacin. An attempt has been made to develop a new RP-HPLC method forits estimation in pharmaceutical dosage form with good accuracy, precisionand simplicity. The method was validated according to the ICH guidelines [14-15].

2.0. MATERIALS AND METHODS2.1. ChemicalsAcetonitrile and methanol used were of HPLC grade from E. Merck, India.HPLC grade water was obtained using millipore water purification system.Working standard of satranidazole with potency of 99.67 % was obtainedfrom Alkem Laboratories Limited, Baddi. Other chemicals were analyticalgrade of above 99% purity. All volumetric-ware was pre-calibrated by themanufacturer (Borosil) and was of grade A. HPLC grade water was obtainedusing millipore water purification system. Commercial tablets containingSatranidazole (Satrogyl-300mg) were procured from the local chemist shop.

2.2. InstrumentationAgilent 1200 Series HPLC System, consisting of degasser-G1322A, quater-nary pump-G1311A, column oven- G1316A, and variable wavelength detec-tor- G1314B was used for analysis. The analytical column Eclipse XDB-C18(5µm; 150x4.6mm) was used along with guard column. The software AgilentChemstation ran on HP computer operated with Windows 2003 professional

Estimation of satranidazole in bulk and tablet dosage form by RP-HPLCVishwanath Agrahari*1, Meenakshi Bajpai 1, Sanju Nanda 2, G.N.Singh3, Robin Kumar3

1College of Pharmaceutical Sciences, RKGIT, Ghaziabad-201003, Uttar Pradesh, India2Department of Pharmaceutical Sciences, M.D. University, Rohtak-124001, Haryana, India3Indian Pharmacopoeia Commission, CIPL Campus, Sector-23, Raj Nagar, Ghaziabad-201002 Uttar Pradesh, India

Received on: 15-06-2010; Revised on: 18-08-2010; Accepted on:13-09-2010

ABSTRACTA simple, precise, rapid and accurate reverse phase HPLC method was developed for the estimation of satranidazole in tablet dosage form. The separation wasachieved by using an Agilent 1200 Series HPLC System consisting of analytical column Eclipse XDB-C18 (5µm; 150x4.6mm) along with guard column and variablewavelength detector- G1314B was used for analysis. The mobile phase consisting of water:acetonitrile in the ratio of 80:20 (v/v) was used. The flow rate was 1.0mL/min and the effluents were monitored at 318 nm. The retention time was 6.139 min. The detector response was linear in the concentration of 5-70 mcg/mL.The respective linear regression equation being y = 39.742x + 45.939.The limit of detection and limit of quantification was 0.034µg/ml and 0.106µg/ml.,respectively. The percentage assay of satranidazole was 99.9%. The method was validated by determining its accuracy, precision and system suitability. The resultsof the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate, which is useful for the routine determination of satranidazole in bulkdrug and in its pharmaceutical dosage form.

Key words: Satranidazole, RP-HPLC, Validation, System suitability tests.

used for this method. A rheodyne injector with 20 µl loop was used for injectingthe sample. Shimadzu balance, AY-120 was used for weighing purpose in thismethod.

2.3. Chromatographic conditionsThe analysis was carried out with UV detection at 318 nm using a 20 µlinjection volume. Assay was performed using a C18 reversed-phase columneluted with Acetonitrile and water (20:80, %v/v) at a flow rate of 1.0 ml/ min.Chromatography was carried out at ambient temperature. The solvents weremixed, filtered through a membrane filter of 0.45 micron pore and degassed inultrasonic bath prior to use.

2.4. Standard solution preparationStandard stock solutions were prepared by dissolving 10 mg of satranidazoleworking standard in 8.0 ml of mobile phase and diluting to 10.0 ml with thesame to obtain concentration of 1000 µg/ml. It was filtered through a .22 µmembrane filter. The stock solution was protected from light using aluminiumfoil and stored for 1 week at 40C and was found to be stable during this period.

2.5. Procedure for analysis of tablet formulation20 Tablets of the product under study were weighed, crushed and mixed in amortar and pestle for 20 min. A portion of powder equivalent to the weight of100.00mg was accurately weighed and transferred to a dry 100 ml A-gradevolumetric flask and 80 ml mobile phase was added. The volumetric flask wassonicated for 20 min to effect complete dissolution of satranidazole and madeup to the volume with mobile phase. Suitable aliquots of solution were filteredthrough a 0.45 µm nylon filter. This was further diluted with mobile phase toyield concentration of satranidazole in the range of linearity (40ppm). Eachof standard and test preparation was injected into the chromatograph and theresponses recorded.

3.0. METHOD VALIDATION3.1. LinearityA series of standard curves were prepared over a concentration range of 5 - 70µg/ml by diluting the standard stock solution of SAT (1mg/ml) in mobile phase.

Fig.1.3-(1-Methyl-5-nitroimidazol-2-yl)-1-(methylsulfonyl) imidazolidin-2-one



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4.4. Limit of detection and LOQThe LOD was calculated to be .034µg/ml and the LOQ was calculated to be.106µg/ml.

y = 39.742x + 45.939 R2 = 0.9986

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30 40 50 60 70 80

Concentration (µg/ml)

Area (mAU x S)

Fig. 2 Linearity curve of Satranidazole

Fig.3 HPLC Chromatogram of Standard Satranidazole

Table1: Precision of developed method at working level (n=6)

Injection no. % Assay

1 99.482 100.103 99.234 99.115 99.856 100.35Mean 99.68SD 0.48RSD 0.48

Table 2: Recovery studies of Satranidazole

Amount (%) Theoretical Conc. Found Recovery RSDof drug added content (µg/ml ) (µg/ml ) ± SD * (%) (%)to analyte

80 72 71.93 ± 0.245 99.8 0.12100 80 80.02 ± 0.340 100.5 0.14120 88 87.95 ± 0.315 99.9 0.13

*n=3

Table 3: Ruggedness Analysis

Analyst 1 Analyst 2Sample % Assay Sample % Assay

1 100.04 1 99.892 100.22 2 100.063 99.84 3 100.454 100.35 4 99.785 100.67 5 100.076 100.41 6 99.49Mean 100.25 Mean 99.95SD .289 SD .476RSD .29 RSD .48

The data from peak area versus drug concentration plots were treated bylinear least square regression analysis and r2 was found 0.994. The standardcurves were evaluated for intra-day and inter-day reproducibility. Each experi-ment was repeated in triplicate.

3.2. PrecisionPrecision was measured in accordance with ICH recommendations. The preci-sion study was carried out by injecting sample preparation of 40µg/ml concen-tration six times.

3.3. AccuracyRecovery studies by the standard addition method were performed with a viewto justify the accuracy of the proposed method. Previously analyzed samplesSAT (40 µg/ml) were spiked with known amount of standard so as to get threedifferent levels (80%, 100% and 120%) and the mixtures were analyzed by theproposed method. The experiment was performed in triplicate. Recovery(%), RSD (%) were calculated for each concentration.

3.4. Limit of detection and limit of QuantitationIn order to estimate the limit of detection (LOD) and limit of quantitation(LOQ) values, the blank sample was injected six times and the peak area ofthis blank was calculated as noise level. The LOD was calculated as three timesthe noise level (S/N = 3:1) while ten times the noise level gave the LOQ (S/N=10:1).

3.5. RuggednessThe ruggedness of the method was demonstrated by analysis of the samples asfor precision study by a second analyst. The RSD of the two sets of dataindicates the ruggedness of the method. Further, the t-test was performed onthe data and the difference was found to be not significant.

3.6. RobustnessThe robustness of the method was determined to assess the effect of small butdeliberate changes of the chromatographic conditions on the determinationof SAT. The different variations are in flow rates by ± 0.1 mL/min, in wave-length by ± 2 nm and in temperature by ± 5 °C. The concentration of thesolution analyzed was 40 µg/mL.

3.7. System suitability testsThe chromatographic systems used for analyses must pass the system suitabil-ity limits before sample analysis can commence. The capacity factor (K),injection repeatability, tailing factor (T), theoretical plate number (N) andresolution (Rs) for the principal peak were the parameters tested on a 40 µg/mL sample of SAT to assist the accuracy and precision of the developed HPLCsystem.

3.8. SpecificityThe specificity of the method was tested by chromatographing a mixture ofcommonly used tablet excipients, for example starch, microcrystalline cellu-lose, lactose, talc, magnesium stearate, colloidal silicon dioxide, sodium starchglycollate and comparing the chromatogram with that obtained from a mix-ture of drug and the same additives.

4.0. RESULTS4.1. LinearityPeak area versus drug concentration was plotted to construct a standard curvefor SAT and linearity was shown in concentration range of 5 µg/ml to 70µg/ml. The polynomial regression for the calibration plots showed good linearrelationship with coefficient of correlation, r = 0.9986; slope = 39.742 andintercept = 45.939 over the concentration range studied. Fig.2

4.2. PrecisionThe % assay for tablet was calculated and % RSD was found to be 0.48%.whichproved that the method was precise, as depicted in Table 1.

4.3. AccuracyThe % recovery was calculated for triplicate samples and for all levels andmean recovery was calculated. The mean recovery was well within the accep-tance limit hence the method was accurate, as depicted in Table 2.



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

4.5. RuggednessThe % assay and RSD for samples prepared by second analyst was calculatedand found within limit. Then RSD of analyst 1 and analyst 2 was calculated andfound within limit. This proved that the method is rugged, as depicted in Table3.

4.6. RobustnessThe results of the analysis (% RSD ranged from 0.078 to 1.286 %) of thesamples under the conditions of the above variations indicated the nature ofrobustness of the method.

4.7. System suitability testsThe results of the system suitability tests assure the adequacy of the proposedHPLC method for routine analysis of SAT. The capacity factor (k) was foundto be 2.405, indicating that the SAT peak is well resolved with respect to thevoid volume. The RSD of six consecutive injections performed under theprecision test (Table 1) was found to be 0.48% and thus shows good injectionrepeatability. The tailing factor (T) for SAT peak was found to be 0.9, reflect-ing good peak symmetry. The theoretical plate number (N) was found to be8466, thus demonstrating good column efficiency.

4.8. SpecificityThe chromatograms obtained showed separation of the analyte from theexcipients was complete, i.e. there was no interference from the excipientsunder the chromatographic conditions used for the analysis. No interferenceof the placebo mixtures with the peak of satranidazole was observed.

REFERENCES1. Zahoor A, Knight R C, Whitty P, Edwards DL, Satranidazole: mechanism of action on DNA and

structure -activity correlations, Journal of Antimicrobial Chemotherapy, 18, 1986, 17-25.2. http://sci-toys.com/scichem/jqp016/41841.html.3. Bhatia SC, Shanbhag VD, Electron-capture gas chromatographic assays of 5-nitroimidazole

class of antimicrobials in blood, Journal of Chromatography B: Biomedical Sciences and Appli-cations, 305, 1984, 325-334.

4. Mruthyunjayaswamy BHM, Patil SMM, Raju SA, Spectrophotometric methods for the estima-tion of Satranidazole in pharmaceutical formulations, Indian J Pharm Sci. 63, 2001, 433-436.

5. Raju SA, Shobha M, Manjunath S, Spectrophotometric determination of satranidazole in bulk drugand formulations, Asian J Chem., 14 (1), 2002, 520- 522.

6. Wankhede SB, Prakash A, Chitlange SS, Simultaneous spectrophotometric estimation ofsatranidazole in tablet dosage form, Research J. Pharm. and Tech. 1(4), 2008, 441-443.

7. Wankhede SB, Prakash A, Chitlange SS, Simultaneous spectrophotometric estimation of ofloxacinand satranidazole in tablet dosage form, Asian J. Research Chem.1 (1), 2008, 9-11.

8. Lalla J, Hamrapurkar P, Anu R, Wadhwa T, High-performance thin-layer chromatographicdetermination of satranidazole in its dosage form, Journal of Planar Chromatography -ModernTLC, 16(6), 2003,447-450.

9. Patel MB, Patel KM, Patel GS, Suhagia BN, Prajapati AM, Development and validation of astability-indicating hptlc-densitometric method for Satranidazole, Journal of Liquid Chromatog-raphy & Related Technologies, 30(16), 2007, 2459 – 2471.

10. Natarajan S and Raman B., HPLC determination of satranidazole in bulk and pharmaceuticaldosage forms, Asian J Chem. 2008; 20(3): 1833-1840.

11. Prashant B, Sanjay A, Mrinalini D, Rahul G, A Validated RP-HPLC method for simultaneousestimation of ofloxacin and satranidazole from tablets, Journal of Pharmaceutical Research, 7(2),2008,70-72.

12. Shinde RS, Suvarna IB, Namdev SP, Suman BY, Ashok MB, Simultaneous estimation of satranidazoleand ofloxacin in tablet dosage form by high performance liquid chromatography, E-Journal ofChemistry, 7(1), 2010, 198-202.

13. Boopathy D, Prakash M, Mathew B, Kumar SS, Perumal P, Method development and validationof simultaneous determination of Ofloxacin and Satranidazole in pharmaceutical dosage form byRP-HPLC, Der Pharmacia Letter, 2(2), 2010, 22-26.

14. General Chapter 1225, Validation of compendial methods, USP 30, National Formulary 25,Rockville,Md., USA, The United States Pharmacopeial Convention, Inc., (2007).

15. ICH Harmonised Tripartite Guideline- Validation of Analytical Procedures: Text and Methodol-ogy Q2 (R1) Nov 2005.

5.0. DISCUSSIONSatranidazole, a weak acid, is sparingly soluble in water. The final decision onmobile phase composition and flow rate was made on the basis of peak shape,peak area, tailing factor, baseline drift, ease of preparation, use of readilyavailable cost-effective solvents and time required for analysis. Initial trialexperiments were conducted, with a view to select a suitable solvent systemfor the accurate estimation of the drug. These included methanol–water,methanol–acetonitrile-water and acetonitrile–water in different ratio. Flowrates between 0.5 and 1.2ml/min were studied. A mobile phase system compris-ing of acetonitrile-water (20:80 % v/v) was found to be optimum and a flowrate of 1.0 ml/min gave an optimal peak shape and was selected. The samesolvent mixture was used for the extraction of the drug from the formulationcontaining excipients. No internal standard was used because no extraction orseparation step was involved. The solvents were mixed, filtered through amembrane filter of 0.45 micron pore and degassed in ultrasonic bath prior touse. Using a reversed-phase C18 column, the retention times for satranidazolewas observed to be 6.13 min. Total time of analysis was kept 8.0 min. Themaximum absorption of satranidazole was detected at 318 nm and this wave-length was chosen for the analysis. (Fig3) The developed method was linearshowing the coefficient of correlation of 0.9986. % RSD of accuracy study for

three levels (80, 100 and 120 %) showed below 2.0% and precision was foundto be 0.48. The method was also found to be robust as there was no significantchange in the peak area, peak shape and retention time of SAT. Furthermore,the low values of LOD and LOQ indicate that the method can be employedover a wide concentration range for linearity. The system suitability testsperformed verified the resolution, column efficiency and repeatability of thechromatographic system

6.0. CONCLUSIONThe HPLC method developed is accurate, precise, reproducible and specific.The method is linear over a wide range, economical and utilizes a mobile phasewhich can be easily prepared. All these factors make this method suitable forquantification of SAT in bulk drugs and in pharmaceutical dosage forms. Themethod developed was then subjected to validation as per ICH guidelines andshowed that method is linear, precise, accurate and rugged.

ACKNOWLEDGEMENTThe authors are thankful to M/s Alkem Laboratories Limited, Baddi, India, forthe gift of SAT.

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Estimation of Satranidazole in Bulk and Tablet Dosage Form by RP-HPLC