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RESEARCH TOPIC – Detection of Quinolones on Thin Layer Chromatographic Plates Using Sigma- and Piacceptors in 1,4-Dioxane

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Detection of Quinolones on Thin Layer Chromatographic Plates Using Sigma- and Piacceptors in 1,4-Dioxane



The use of sigma- and pi-acceptors for the detection of some quinolones on thin layer chromatographic plates was investigated. Thin layer chromatographic plates, coated with silica gel were air-dried and stored in an oven at llO°C for 30 minutes. Before use, the plates were allowed to cool to room temperature. Each of the drug samples (Reference, ciprom, Ofloxacin) was spotted twice on a pair of coated plates. he plates were then placed vertically in a development tank containing a solvent system and allowed to stand undisturbed until the solvent front reached 15cm from the origin. The plates were then removed, air-dried and sprayed with an appropriate locating reagent (iodine or chloranilic acid) and examined for colour formationlstability. In another set of experiments, the plates were counter-sprayed with dimethyl formamide (DMF) and re-examined for reaction. The results showed that the
chromogens fonned from iodine and chloranilic acid were reddish brown and violet respectively. The reddish brown colour chinged to yellow meanwhile the violet colour disappeared after six and 20 minutes, respectively. On counter-spraying with DMF, the reddish brown colour changed to yellow and there was no increase in intensity in the reference drug, but there was, in the test drugs. However, the intensity of the violet colour was generally increased. There were no significant difference~( p0.05 for each) in the Rfv alues of iodine and chloranilic acid chromog-e ns of all the drugs. Sigma- and pi-acceptors can be applied in qualitative detection of quinolones.




Sigma- and pi-acceptors are aromatic systems containing electron-withdrawing substituents [I]. Thus, acceptor complexation is based on the reaction of some drugs (nelectron donors) with sigma-acceptors such as iodine, or with pi-acceptors such as 7,7,8,8-tetracyanoquinodimethane; 2,3-dichloro- 5,6-dicyano1-, 4-benzoquinone; tetracyanoethylene; 2,3,5,6-tetrabromo-14-benzoquinone (bromanil); and 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil)
[2]. These compounds readily form complexes with donor molecules giving rise to chromogens thus making them versatile in pharmaceutical and clinical analysis.

The colour formed with such donor molecules has been reported to have intensities proportional to concentrations of donor-receptor complexes [3]. Sigma- and pi-acceptors have been found to be very useful as detecting reagents in drugs and chemicals possessing nitrogen and other donor molecules [2-61. Research workers have detected Padrenergic blocking agents [4], antidepressant drugs [6],alkaloids [7], cehalosporins [8], diuretics and oral hypoglycaemic
drugs [9] as well as penicillins [lo,! 11 using acceptor complexation.

Ciprofloxacin and ofloxacin are both 4-quinolones containing a carboxylic acid moiety in the 3 position of the basic ring structure, a fluorine substituent at position 6, and a piperazine moiety at position 7 [12]. These drugs Shu/ Mukd Ogbodol Madukd Ezeunala are potent bactericidal agents against E.. coli and variousspecies of Salmonella, Shigella, Enterobacter, Campylobacter, and Neisseria [13]. They are generally well tolerated [14] and microbial resistance to their action does not develop rapidly [15]. The importance of these drugs espccially in the third world countries can be estimated by the high success rates recorded in their use in some high morbidity and mortality ailments.

Attempts at quantitative detection of ciprofloxacin in our laboratories by charge transfer complexation using a spectrophotometer have failed principally because of the inability of complexes to remain stable enough for detection. Furthermore, this method is cumbersome and expensive, especially in least and less developed countries. Thus, the use of iodine and chloranilic acid for the qualitative analysis of ciprofloxacin and ofloxacin on thin layer  Chromatographic plates is hereby discussed.



Detection of Quinolones on Thin Layer Chromatographic Plates Using Sigma- and Piacceptors in 1,4-Dioxane


Materials and Methods


The following reagents were collected from commercial sources and used as supplied: chloranilic acid, hexane, iodine (BDH); ammonia, silica gel G-1 (Merck); sulphuric acid, chloroform (May and Baker); ciprofloxacin, [email protected] (ICI); ofloxacin (Hoeschst).



Preparation of suspension

One tablet each of the different drug samples was crushed to a fine powder in a clean porcelain mortar. A quantity (100mg) of each powder was transferred into a 50ml volumetric flask. A lOml volume of ammonia was added to each of these flasks and stirred with a glass rod until dissolution was achieved. I Oml of chloroform placed in a separating hnnel ws added to the above preparation and shaken for 30 seconds and the chloroform layer transferred into a beaker and kept for analysis.

Preparation of locating reagents

A 50ml volume of l,4-dioxane was placed in each of two volumetric flasks. A 0.5g quantity of each detecting reagent (iodine and chloranilic acid) was  A acurately weighed and transferred into each flask and dissolved by stirring. The solution was made up to volume with 1,4- dioxane to give 0.5%(w/v) solution of iodine and chloranilic acid, respectively. Preparation of thin layer chromatographic (TLC) plates Forty grams of silica gel (G-1) were added to 80ml of
acetone in a clean beaker and mixed to form a pourable slurry. The slurry was spread over clean dried 20x 10 TLC glass plates to a thickness of 250mm using a kenso spreader (model CJK-520). The coated plates were airdried at room temperature and stored in an oven at 1 1 0 ‘ ~ for 30 minutes and allowed to cool to room temperature just before use.

Preparation of solvent system

The most suitable solvent system observed from trial runs was composed of chlorofoim, ethyl acetate, hexane, and, water in the ratio of 1:3:0.5:1 respectively. This was thoroughly mixed and transferred into a development tank. ,The tank was allowed to stand undisturbed for two hours to obtain equilibrium.


Method of detection


Each of the drug samples was spotted twice on the same pair of activated plates using glass capillary tubes. The plates were then placed vertically in the tank and allowed to stand undisturbed until the solvent front reached 15cm from the origin. The plates were then removed, air dried and sprayed with appropriate locating reagent and examined for colour formation and stability after which they were countersprayed with DMF and re-examined for reaction.
The experiment was carried out five times for comparison and accuracy. The results of colour reactions and their corresponding Rf values were recorded.


The Table summarizes the colours U. the chromogens formed, their reactions after counterspraying with DMF and their respective Rf values. The chloranilic acid gave a violet colour with the drugs (w4ich disappeared after 20 minutes).

The colour intensities of the ciprofloxacin and ofloxacin were greater than that of ciprodD(a brand of – ciprofloxacin). On the other hand, iodine gave a reddishbrown colour with the drugs which changed to yellow after six minutes. T e intensity of the reddish brown colour was more in the reference drug than in the ciproe and ofloxacin.

On counterspraying with DMF, the intensity of the violet colour was generally increased and it lasted for 24 hours. However, DMF did not affect the yellow colour of iodine in the reference drug but increased it in the test drugs. There were no significant differences (p>0.05 for each) between the Rf values of iodine and chloranilic acid chromogens of all the drugs. The limits of detection for the drugs (reference ciprofloxacin, ciproa, ofloxacin) were 0.8, 1.2, and 1.6 mglml, respectively.


Detection of Quinolones on Thin Layer Chromatographic Plates Using Sigma- and Piacceptors in 1,4-Dioxane


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