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Sphinxsai.comInternational Journal of PharmTech Research
CODEN (USA): IJPRIF ISSN : 0974-4304
Vol. 3, No.2, pp1169-1173, April-June 2011
Development and Validation of Densitometric
Method for Metronidazole and Tetracycline
hydrochloride in capsule Dosage form
S. Sharma1, M. C. Sharma*
1Department of Chemistry Chodhary Dilip Singh Kanya Mahavidyalya, Bhind (M.P) India
*School of Pharmacy, Devi Ahilya Vishwavidyalaya, Indore (M.P) 452001, India
*Corres.author: [email protected]
Abstract: A new, simple, precise, and accurate HPTLC method for simultaneous quantitation of Metronidazole (MET)
and and Tetracycline hydrochloride (TET) as the bulk drug and in capsule dosage forms have been developed.
Chromatographic separation of the drugs was performed on aluminium plates precoated with silica gel 60 F254 as the
stationary phase and the solvent system consisted of benzene: ethyl acetate: toluene: methanol: glacial acetic acid
(9.5:2.0:5.0:1.5:0.5 v/v/v/v/v),Densitometric evaluation of the separated zones was performed at 254 nm. The two drugs
were satisfactorily resolved with Rf values of 0.43 ± 0.02 and 0.74± 0.02 for MET and TET, and 0.29 Ofloxacin (OF)
respectively. The accuracy and reliability of the method was assessed by evaluation of linearity (600-2400ng spot-1 for
MET and 600-1800ng spot-1 for TET).Ofloxacin (OF) was used as an internal standard. Linearity was observed over the
concentration range of 600-2400 ng.band-1. The linearity of the calibration plots was confirmed by the high value of the
correlation coefficients (r2 = 0.9998 for MET and 0.9991 for TET). Hence it can be applied for routine quality control
analysis of Metronidazole and Tetracycline hydrochloride in capsule Dosage form.
Keywords: Metronidazole, Tetracycline hydrochloride, Ofloxacin, Densitometric.
dosage form5-9. The proposed method overcomes manydifficulties of tracing out lowest determination and Chemically Metronidazole (MET) is 2-(2-methyl-5- quantification of related substances the products. Also nitro-1H-imidazole-1y1) ethanol, acts by causing loss the affirmative points are; less instrument set up time of helical structure of DNA, strand breakage and by mean of simple isocratic elution which results into a accompanying the impairment of DNA’s function and negligible noise as compare to gradient methods.
used in the trichomoniasis, amoebiasis, girardiasis1,2while Tetracycline hydrochloride (TET) is (4s, 4as, Materials and methods
5as, 6s, 12as)-dimethylamino-1,4, 4as, 5a, 6, 11, 12a-octa hydro-10, 12, 12a-pentahydroxy-6-methyl-1,11- Toluene, acetonitrile, methanol and formic acid used dioxo naphthacene-2-carboxamide hydrochloride were of analytical grade. All dilutions were performed which acts by inhibiting subsequent binding of amino in standard volumetric flasks. The commercially acyl transfer RNA to ribosomes resulting in available capsules; Meklin (Label claim: MET termination of peptide chain growth and used to 400mg, TET 333mg) manufactured by Bennett S.T.D., U.T.I. infection, traveller’s diarrhea3,4. Author Pharmaceuticals, Baroda, Gujarat, India was procured of the article and his research team has developed a from local market. Acetonitrile and Phosphate buffer HPTLC Method development different pharmaceutical AR grade was obtained from Merck Limited, India.
M. C. Sharma et al /Int.J. PharmTech Res.2011,3(2)
1.0 mL of Ofloxacin (OF), internal standard stock Chromatographic separation of drug was performed on solution in 50.0 mL volumetric flask.
Merck TLC plate pre-coated with silica gel 60 F254 (10 cm ×10 cm with 250 mm layer thickness) from E.
Preparation of sample solution
Merck, Germany. The samples were applied onto the Twenty tablets were Meklin (Label claim: MET plates as a band with 6 mm width using Camag 100 μl 400mg, TET 333mg) weighed and average weight was sample syringe with a Linomat 5 applicator. Linear calculated. These tablets were crushed, powdered and ascending development was carried out in a twin taken in a 10 mL volumetric flask weight equivalent to trough glass chamber (for 10 x 10 cm). Densitometric one tablet and dissolved in minimum amount scanning was performed using Camag TLC scanner 3 acetonitrile. To this flask 1.0 mL of stock\ solution of in the range of 600-3000 ng/spot and operated by win internal standard was added and diluted up to the mark CATS software (V 1.4.2, Camag). The plates were with methanol and sonicated for 30min. This solution developed in a twin through chamber previously was then filter through Whatman no. 41.The filtrate saturated for 30 min with mobile phase, benzene: ethyl was collected in the flask and used as sample solution.
acetate: toluene: methanol: glacial acetic acid(9.5:2.0:5.0:1.5:0.5 v/v/v/v/v), for a distance of 8 cm.
Selection of Detection Wavelength
The spots on the air dried plate were scanned with a After chromatographic development bands were scanner III at 283 nm using the deuterium source. The scanned over the range of 200-400 nm. It was plates were prewashed by methanol and activated at observed that the drug showed considerable 120oC for 5 min prior to chromatography. A constant application rate of 0.1 was employed and spacebetween two bands was 5 mm. The slit dimension was Method Validation10-12
kept at 5mm, 0.45mm and 10 mm/s scanning speed Linearity
was employed. The monochromatic bandwidth was set Seven different concentrations of mixture of MET and at 20 nm, each track was scanned thrice and baseline TET were prepared from stock solution of MET and correction was used. The optimized chamber TET in the range of 600 to 2400.50 μg/mL and 600to saturation time for mobile phase was 20 min at 1800 μg/mL respectively, in methanol to obtain desire room temperature (35 oC) at relative humidity of 60% linearity range. 10 μL of each solution was applied to a . The length of chromatogram run was 8 cm.
plate (i.e. 0.5-2.5 μg/spot for MET and 0.75-3.75 Subsequent to the development; TLC plates were dried μg/spot for TET) by sample applicator and the plate in current of air with the help of air dryer in wooden was developed. The detector response to the different chamber with adequate ventilation. The flow rate in laboratory was maintained unidirectional (laminarflow, towards exhaust). The solution was sonicated Limit of detection (LOD) and Limit of
for 15min. the extracts were filled through Whatmann quantification (LOQ):
filter paper No. 41 and residue washed thoroughly with The LOD and LOQ were calculated using following methanol. The extracts and washings were transferred equations as per International conference on to 25ml volumetric flask and volume was made up to 25ml with methanol. Required dilutions were made to Where σ = standard deviation of the response and S =the standard deviation of y intercept of regression Preparation of Standard Stock Solutions
50 mg of each drug MET and TET were weighedseparately and dissolved in 20 ml of methanol and then Precision
volume was made up to 50 ml so as to get the Precision of the method was verified by repeatability concentration 1 mg mL-1. From each of these solutions and intermediate precision studies. Repeatability 1ml of solution was pipette out and transferred to 10 studies were performed by analysis of three different ml volumetric flasks and volume was made up to the concentrations (600- 2400 ng.band-1) of the drug, each mark using methanol so as to get the concentration 100 concentration injected six times on the same day.
μg mL-1. The stock solution was stored at 2–8 0C Intermediate precision of the method was checked by repeating studies on three different days.
Preparation of working solution
Robustness of the method
Further, the mixture of working solution was prepared Small changes in the mobile phase composition (± 0.1 by diluting 50 mL of MET and 12 mL of TET with mL for each component) were made and the effects on M. C. Sharma et al /Int.J. PharmTech Res.2011,3(2)
the results were examined. Time from spotting to Recovery experiments were carried out to check for chromatography and from chromatography to scanning the presence of positive or negative interferences from excipients present in the formulation, and to study theaccuracy and precision of the method. Recovery Accuracy
experiment was performed by the standard addition Accuracy was determined by recovery studies. It was method. The recovery of the added standard was studied at three different levels viz 110%, 120% and 120% of the standard drugs to the pre-analysed 130% of the estimated amount of the drug.
marketed sample of MET and TET. Threedeterminations were performed at each level.
Method precision (repeatability) –
The precision of the instrument was checked by
Tablet formulation Assay
repeatedly injecting (n= 6) mixed standard solution of 50 μL of sample solution was spotted along with same concentration of working solution (.5-2.5 μg/spot forMET and 0.75-3.75 μg/spot for TET) on to the plate Intermediate precision (reproducibility) –
under the optimized chromatographic conditions. The The intraday and interday precision of the proposed procedure was repeated seven times. The peak area method was determined by analyzing mixed standard ratio values of MET and TET to the internal standard solution of MET and TET at concentration 600, 1800, were calculated. The densitometric responses from the 2400 ng/spot; 700, 1400, 1800 ng/spot three times on standard and sample were used to calculate the the same day and on three different days. The results are reported in terms of relative standard deviation.
Table 1 Regression Analysis of Calibration Graph for MET and TET
Table 2 -Recovery Study
M. C. Sharma et al /Int.J. PharmTech Res.2011,3(2)
Table 3 Result of Assay of Tablet Formulation
Results and Discussion
precoated on aluminium sheet. The mobile phasecomprises benzene: ethyl acetate: toluene: methanol: To optimize the HPTLC parameters, several mobile glacial acetic acid (9.5:2.0:5.0:1.5:0.5 v/v/v/v/v), phase compositions were tried. Satisfactory which gives good separation between Ofloxacin (R separations for AT, RA and AS were obtained with mobile phase consisting of benzene: ethyl acetate: was observed over the concentration range of 600- 2400 ng.band-1. The linearity of the calibration plots (9.5:2.0:5.0:1.5:0.5 v/v/v/v/v), Quantification was was confirmed by the high value of the correlation achieved with UV detection at 283 nm based on peak coefficients (r2 = 0.9998 for MET and 0.9991 for area. Better resolution of the peaks with clear baseline TET). The LOD and LOQ for a signal to noise ratio of separation was found. Small changes in the mobile 8:1 and 12:1 was found to be 81 and 97 ng.band-1 for phase composition, the effects on the results were MET and TET which indicates the method has examined. Mobile phases having different composition sufficient sensitivity. There was no indication of like Chloroform: Methanol: Toluene: Acetic acid (8.1: degradation in solutions of MET and TET as revealed 1:1:0.1 v/v/v/v), were tried and chromatograms were by peak purity data and from the value of RSD (< 2%) run. The plates were prewashed by methanol and for peak areas of bands of solution stored at different activated at 110oC for 5, 10, 15 min respectively prior times. In Conclusion proposed HPTLC method was validated as per ICH guidelines. The standard chromatography and from chromatography to scanning deviation, %RSD and standard error calculated for the was varied from 0, 20, 40 and 60 minutes. Robustness method are low, indicating high degree of precision of of the method was done at three different the methods. The results of the recovery studies concentration levels 600, 1200, 2400ng spot-1and 600, performed show the high degree of accuracy of the 1400, 1800ng spot-1 for MET and TET, respectively.
proposed methods. The proposed method is highly The method was a normal phase HPTLC method. It accurate, selective and precise hence can be used for a makes use of a silica gel 60F254 stationary phase routine quality-control analysis and quantitative M. C. Sharma et al /Int.J. PharmTech Res.2011,3(2)
simultaneous determination of MET and TET in Acknowledgement
We are grateful to referee for given valuable suggestions.
6. Sharma, M. C.; Sharma, S.; Kohli, D. V.Annals of 1. Nagulwar, V.; Upadhye, P.A.; Mehta, N.K.; 7. Sharma, M. C.; Sharma, S.; Kohli, D. V.; Sharma, Upadhye, K.;Bakhe, S.;Deshpandey, S.;Dixit, G.
A. D. Der Pharma Chemica. 2010, 2(1), 121-126 Indian J. Pharma . Sci. 2005, 67(2), 258-260.
8. Sharma, M. C.; Sharma, S.; Kohli, D. V.; Sharma, 2. Patel, P.U.; Suhajia, B.N.; Patel, M.M. Indian A. D. Der Pharmacia Lettre. 2010, 2 (1) 489-494.
9. Sharma,S.;Sharma,M.C. Indian Drugs .47(11),68- 3. Indian Pharmacopoeia, Vol. 2, Ministry of Health and Family Welfare, Government of India, New 10. ICH, Q2A Validation of Analytical Procedure: 4. Mitscher, L.A. Antibiotics and antimicrobial Harmonization, Geneva, October 1994.
agents, In; Foye’s Principle of Medicinal 11. ICH, Q2B Validation of Analytical Procedure: Chemistry, 5th Edn., Lippincott Williams and Wilkins, Philadelphia P.A., 2003, p.p.857.
5. Sharma, M. C.; Sharma, S.; Kohli, D. V.; Sharma, 12. ICH Guidance on Analytical Method Validation, A. D. Archives of Applied Science Research.
in: Proceedings of the International Convention on Quality for the Pharmaceutical Industry, Toronto,Canada, September 2002.
NOUVEAUTÉS 2e semestre 2008 FRANCAIS JEUNESSE Mikaël Ollivier : Frères de sang, Thierry Magnier, 2008, ISBN 978-2-84420-434-9 Rick Riordan : Le sort des titans, Albin Michel, 2008, ISBN : 978-2226183316 Rick Riordan : La mer des monstres. Percy Jackson T. 2, Albin Michel, 2008, ISBN : 978-2226186089 Anne Robillard : Les chevaliers d'Emeraude (7 tomes) Michel Lafon, 2007 -