Stress degradation monitoring of Ofloxacin by HPTLC and Bioautography.
A.D. Paprikar, K.P. Patil, S.S. Ranade, V.Y. Rane, S.H Rao and M.C. Damle*
Department of Pharmaceutical Chemistry, A.I.S.S.M.S. College of Pharmacy, Kennedy Road, Near R.T.O., Pune - 411001
*Corresponding Author E-mail: mcdamle@rediffmail.com
ABSTRACT:
A stability indicating High Performance Thin Layer Chromatographic [HPTLC] method was developed for the study of stability of the drug Ofloxacin. Bioautography was performed subsequently to compare the results by both the methods. The method employed TLC (Thin Layer Chromatography) aluminum plates pre-coated with silica gel 60 F254 as the stationary phase. The samples were spotted on TLC plates (10cmx20cm) as 4 mm bands and developed with a mobile phase consisting of n-butanol AR: ethanol AR: ammonia solution AR (5:5:4 v:v:v). The Rf value for Ofloxacin was 0.53 ± 0.03. The drug was exposed to various stress conditions as per ICH guideline viz. acidic, alkaline, neutral hydrolysis, oxidation and photolysis after which the amount remaining was estimated. Densitometric analysis of Ofloxacin was carried out at 299nm. The linear regression analysis data for the area of the bands resulted in correlation coefficient r2 = 0.9859 in the range of 500–2500 ng / band.
The same TLC plates were used to perform Bioautography in petri plates (10cm diameter) using Mueller Hinton agar as the media and Escherichia coli NCIM 2066 as the organism. The results by both the methods were compared. The drug does not undergo degradation with neutral hydrolysis and oxidative conditions, but gets affected in alkaline hydrolytic and acidic hydrolytic conditions. As the HPTLC method could effectively resolve the drug from its degradation products, it can be employed as a stability-indicating method.
KEYWORDS: Ofloxacin, stability- indicating HPTLC, Bioautography.
1. INTRODUCTION:
Ofloxacin1,2 is (RS)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido [1,2,3-de] -1,4-benzoxazine-6-carboxylic acid, a second generation fluoroquinolone derivative with a broad antibacterial spectrum3. Ofloxacin tablets contain not less than 98.5% and not more than 101.5 % of stated amount of Ofloxacin4 .It is used against Chronic bronchitis, Pneumonia, Urethritis, Cervicitis, Pelvic inflammatory disease, Prostatis, Gonnorhea5. Litrature search revealed many methods reported for the determination of Ofloxacin like HPLC6,7, Colorimetric8,9, Spectrophotometry10-12, spectrofluorometry13. Only one stability indicating HPTLC was found to be reported14. Tonessen et al have reported photostability study of Ofloxacin15.
Bioautography is an analytical technique in which organic compounds are separated by chromatography and identified by studying their effects on micro organisms. Bioautography is a method to study antibacterial activity of a compound localized on a chromatogram16.
HPTLC method reported for simultaneous estimation of Ofloxacin and Satranidazole17 was used to study Ofloxacin after its degradation under stress conditions.
It is active against most of the gram positive organisms like Staphylococcus aureus, S. epidermidis, Streptococcus pneumoniae, Bacillus anthracis, Corynebacterium gram negative like Niesseria, Haemophilus and Enterobacteriaceae family18. E. coli was the organism selected for bioautography.
Fig I: Structure of Ofloxacin
2. MATERIALS AND METHOD:
Working standard of Ofloxacin was provided by M/s. Alkem Ltd., Mumbai, India as gift sample which was used without further purification. Methanol AR, n-butanol AR, ethanol AR, ammonia solution AR were purchased from M/s. Thomas Baker.
Muller Hinton agar M173 500G, Soyabean casein digest medium MJ001-100G, autoclavable petri plates (150ml x 20ml unbreakable, 10cm diameter) were purchased from Hi Media.
Bacteria: E. coli NCIM 2066 was procured from NCIM and maintained on suitable culture media.
Culture media: Culture was developed on Muller Hinton broth 50ml at 26-32°C for 3-4 hrs in shaker incubator at 100 rpm.
2.1 Instrumentation:
Shimadzu Model AY- 120 balance was used for weighing purpose. Chromatographic run of drug was performed on Merck TLC plates precoated with silica gel 60 F254 (10 cm ×10 cm with 250 µm layer thickness) from E. Merck, Germany. The samples were applied onto the plates as a band with 4 mm width using Camag 100 µl sample syringe (Hamilton, Switzerland) with a Linomat 5 applicator (Camag, Switzerland).
Linear ascending development was carried out in a twin trough glass chamber (for 10 x 10 cm). Densitometric scanning was performed using Camag TLC scanner 3 in the range of 500-2500 ng/band operated by winCATS software (V 1.4.2, Camag).
Photostability studies were carried out using Photostability chamber make: Newtronics as per ICH guidelines,
2.2 Selection of Detection Wavelength:
After chromatographic development, the bands were scanned over the range of 200-400 nm. It was observed that the drug showed considerable absorbance at 299 nm. So, 299 nm was selected as the wavelength for detection.
2.3 Method validation:
Linearity:
A stock solution of Ofloxacin was prepared in methanol. Different volumes of stock solutions were spotted on TLC plate to obtain concentration of 200, 500, 1000, 1500, 2000 ng per band of Ofloxacin, respectively. The data of peak area v/s. drug amount were treated by linear regression analysis.
2.4 Forced degradation of Ofloxacin:
Standard stock solution:
Accurately weighed 20mg Ofloxacin was transferred to 50 ml volumetric flask. The drug was dissolved in methanol with shaking and volume was made up to 50ml with methanol to get 0.4mg/ml as standard stock solution. From this solution 1ml was taken into 10ml volumetric flask and volume was made up to 10ml with methanol to get 0.04mg/ml.
Degradation studies were carried according to ICH guidelines. Various conditions used were hydrolysis, photolysis, oxidation and exposure to dry heat. Bioautography was carried out subsequently.
2.4.1 Degradation under acid and alkali catalyzed hydrolytic condition:
Acidic: From stock solution (1000mcg/ml) 45ml was taken into round bottom flask. To this 5ml 1N hydrochloric acid was added. The mixture was refluxed for 3 h at 80°C. Cooled to room temperature and made up the volume upto 50ml with methanol. Appropriate volume of resultant solution was applied on TLC plate and densitograms was developed.
Basic: Sample was prepared in the same manner as mentioned above by using 1N NaOH instead of 1N HCl.
2.4.2 Degradation under neutral hydrolytic condition:
Sample was prepared in the same manner as mentioned above by using 5 ml water instead of 5 ml1N HCl.
2.4.3 Photo degradation studies:
UV light: The drug sample was exposed to UV light to different illumination levels ranging from 200 to 1000 watt hrs/m2. This was performed in photosatbility chamber (Make Newtronics) conforming to ICH standards. After the predetermined exposure, the drug was dissolved in methanol and diluted appropriately.
Fluorescent light: The drug sample was exposed to cool white fluorescent light upto illumination of 1.2 million Lux hrs. The exposed drug was dissolved in methanol and diluted appropriately.
Sunlight: The drug samples were exposed to sunlight for 15 and 30 days separately, dissolved in methanol and diluted appropriately.
2.4.4 Degradation under oxidative condition:
Standard stock solution was pipetted and 5ml hydrogen peroxide solution (30%) was added and volume was made upto 50ml with methanol to get the final concentration of 0.25mg/ml. This was refluxed for 2hrs.
2.4.5 Degradation using dry-heat:
The bulk drug was exposed to a temperature of 70°C for 15 hrs. The sample was dissolved in methanol and diluted appropriately to get 0.25mg/ml with methanol.
Bioautography:
TLC plate (10cm x 20cm) was prewashed with methanol, was dried using current of hot air and then activated by keeping it in hot air oven for 10 min at 70°C. The sample solutions after stress degradation were applied using 100 ml Hamilton syringe. After this the plate was developed in chamber having mobile phase of n-butanol AR, ethanol AR, ammonia solution AR (5:5:4 v:v:v) with saturation. After development the plate was dried and scanned at 299 nm. It was cut such that each strip consisted of a single track.
Table I : Degradation study results of Ofloxacin by HPTLC and Bioautography
|
S.N. |
Conditions |
% of Ofloxacin degraded by |
|
|
HPTLC |
Bioautography |
||
|
1.( a) |
Acidic hydrolysis (1NHCl) |
16.81 % |
No degradation |
|
(b) |
(5NHCl, reflux, 2 hours) |
50.96% |
22% |
|
2. |
Basic (5NNaOH reflux, 2 hours,) |
53.12% |
27% |
|
3. |
Neutral (reflux, 2 hours) |
11.04% |
22% |
|
4. |
Oxidation (30 % H2O2, reflux, 2 hours.) |
1.44% |
No degradation |
|
5. |
Heat ( 70˙ C 12hrs) |
30.31% |
22 % |
|
|
Photolysis |
||
|
6.(a) |
Sunlight (15 days) |
19.22% |
No degradation |
|
(b) |
(30 days) |
33.18% |
No degradation |
|
7. |
Fluorescence( 1.2 million Lux hrs.) |
8% |
No degradation |
|
8. |
UV 1400 watt hours./square meter |
5.97% |
64% |
Using sterile culture media Muller Hinton agar M173 and 24hr old culture of E. coli, having a count of 106 cfu/ml, pour plate technique was done and the media was allowed to solidify. The above cut plate strips were placed on the solidified agar for 20min to allow the absorption of the separated components. The strips were removed from the petri plates using sterile forceps and were placed in incubator at 37oC for 24hrs. The zone of inhibition obtained after incubation was noted using vernier caliper. The extent of degradation was calculated from reduction in zones as compared with standard.
Fig. II: Representative densitogram of Ofloxacin at Rf 0.53
Fig III : Linearity analysis of Ofloxacin
Fig IV: Representative densitogram of degradation products by acid hydrolysis (0.72)
Fig V: Linearity by bioautography 1-blank, 2-600ng, 3- 1000ng, 4-1500ng
Fig VI: Linearity by bioautography with 5-2000ng, 6-2500ng, neutral, heat 12 hrs.
Fig VII: sunlight 30 days (1200ng), 5NNaOH, (1200ng), 5NHCl,(1200ng), blank
Fig VIII: standard(1000,600ng), H2O2 (1200ng), UV1400(1200ng).
3. RESULTS AND DISCUSSION:
3.1 Stress degradation:
The amount of drug remaining after exposing the sample to various stress conditions was determined by both HPTLC and bioautography by extrapolation from respective standard curve. The drug was found to degrade the most under acid/base catalysed hydrolytic conditions. There was significant reduction observed upon exposure to dry heat. Photolysis was studied as effect of UV light, fluorescent light and sunlight. The sunlight exposure duration was measured in days whereas exposure to UV and fluorescent light as per units recommended by ICH. The sample exposed to sunlight had more degradation observed.
Even upon hydrolytic degradation, wherein about 50% of the drug was found to degrade, no other bands for the product of degradation were found to get resolved except the one at Rf 0.72. But the band for Ofloxacin had no interference as confirmed by peak purity check in WinCats software. Upon bioautographic study, there was no zone of inhibition observed at any other Rf than that of Ofloxacin, indicating that the product of degradation does not have antimicrobial activity.
4. CONCLUSION:
HPTLC was found to be more sensitive of the two techniques. The conditions which led to less than 20% degradation of Ofloxacin by HPTLC technique, showed no change in bioautography results. The sample exposed to UV light of 1400 watt hrs/m2 showed significant reduction in zone of inhibition value which was not reflected in HPTLC results. This indicates probable loss of antimicrobial activity without chemical degradation of Ofloxacin.
It can be concluded that no product obtained by degradation of Ofloxacin under different stress conditions had any activity against E. coli.
5. ACKNOWLEDGEMENT:
The authors wish to express their gratitude to M/s. Alkem Laboratories, Mumbai (India), for providing working standard of Ofloxacin. The authors are also thankful to The Principal, AISSMS College of Pharmacy for providing necessary facilities to carry out the research work.
6. REFERENCES:
1. British pharmacopoeia, 2007, 1455 accessed soft copy.
2. United States pharmacopoeia.(USP-NF)The official compendia of standards 2007, Rockville, MD, USP Convention Inc, 2793.
3. http://cat.inist.fr/?aModele=afficheN&cpsidt=14568723 Accessed on 31/04/2009
4. Indian pharmacopoeia, 2007, Volume 3.Government of India ministry of Health and family welfare, published by I.P. commission, Ghaziabad, 1468.
5. http://en.wikipedia.org/wiki/Ofloxacin#Licensed_use Accessed on 24/08/2009.
6. Gandhimathi M, Ravi TK, Shukla N. Validated high performance thin layer chromatography method for simultaneous estimation of ofloxacin and ornidazole in tablet dosage form. Indian J Pharm Sci 2006; 68: 838-40.
7. Auten GM, Preheim LC, Sookprane M, Bittner MJ, Sookprane T, Vibhagool A., High pressure liquid chromatography and microbiological assay of serum ofloxacin levels in adults receiving intravenous and oral therapy for skin infections. Antimicrob Agents Chemother, 1991; 35(12): 2558-2561.
8. Eboka GJ, Aigbavboa SO, Akerele JO(1997). Colorimetric determination of the fluoroquinolones J. Antimicrob. Chemother.46:639-641.
9. Martin A, Palomino JC, Portaels F.Rapid detection of ofloxacin resistance in Mycobacterium tuberculosis by two low-cost colorimetric methods: resazurin and nitrate reductase assays.J Clin Microbiol. 2005 Apr;43(4):1612-6
10. Reddy Y.P. et. al. Spectrophotometric estimation of Ofloxacin in Pure and Pharmaceutical dosage forms. Asian Journal of Chemistry, 2009; 21(3): 2473-2475.
11. Jitendra D Fegade,Harshal P Mehta, Rajesh Y Chaudhari, Vijay R Patil, Simultaneous photometric estimation of Ofloxacin and keterolac tromethamine in ophthalmic dosage form,International journal of chemtech research, April-June 2009, Vol.1,No.2,189-194.
12. Hopkala H, Kowalczuk D (2000). Application of derivative UV sectrophotometry for the determination of ciprofloxacin, norfloxacin and ofloxacin in tablets. Acta Pol. Pharm57(1)3-13
13. Hesham S(2005). Spectrofluorometric, atomic absorption spectrometric and spectropotometric determination of some fluoroquinolones. AmJ. Appl. Sci. 2(3):719-729.
14. Gupta, K. R. Wadodkar, S. G. HPTLC Estimation of Ofloxacin and Tinidazole from Pharmaceutical Dosage Form, Indian Drugs; 41(3): 160-164.
15. Tonnesen H.H. et al. Photoreactivity of biologically active compunds. Pharmazie, 2007; 62(2): 105-111.
16. www.dictionary.reference.com/browse/bioautography. Accessed on 31/04/2009
17. Godge Rahul, Bhat Leena, Vora Asfak, Damle Mrinalini, “A validated High Performance Thin Layer Chromatographic method for simultaneous estimation of Ofloxacin and Satranidazole in pharmaceutical Dosage form.” Journal of Pharmaceutical Research, 2007; 6(4) : 233-235.
18. American society of Health-System Pharmacist, AHFS Drug Information, 2004, 2633-2636.
Received on 04.06.2010 Modified on 11.06.2010
Accepted on 17.06.2010 © RJPT All right reserved
Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1275-1278