BIOMEDICAL CHROMATOGRAPIIY, VOL. 6, 196-197 (1992)

TLC Separation of Certain Tetracycline and Amino Glycopeptide Antibiotics R. Bhushan* and Imran Ali* Dcpartment of Chemistry, Univcrsity of Roorkee, Roorkcc 247 667, India

The separation of tetracycline and amino glycopeptide antibiotics was achieved on silica gel thin layers. Tetracycline antibiotics were resolved on a Co+* (1.0%) impregnated silica gel layer using ethanol:acetic acidzwater (10:6:6, vlvlv) as the mobile phase. Amino glycopeptideantibiotics were separated on an untreated silica gel layer using the mobile phase n-butano1:formic acid: water (6: 5: 7, vlvlv). The spots of these antibiotics were located by exposing the chromatoplate to iodine vapours.

INTRODUCTION

The chemical substances which are produced within certain micro-organisms and interfere with their growth or metabolism are called antibiotics. Antibiotics have achieved a great reputation in biochemistry, molecular biology, genetics, genetic engineering, pharmacology, tissue culture and organic chemistry (Vandamme, 1984). Though a rational classification of antibiotics is very difficult they have been classified into several groups based on their chemical structure or the nature of their activity. Antibiotics belonging to the tetracycline and amno glycopeptide groups have achieved great attention in medicinal science because of their wide application in checking the growth of gram-negative bacterial infections and other sorts of infectious diseases (Vandamme, 1984). Therefore, the separation and identification of various components belonging to each of these two groups become important and require effective and rapid methods. Our earlier studies on the TLC resolution of various compounds (Bhushan and Ah, 1986, 1989, 1990; Bhushan et al., 1989a) on impregnated plates indicate that impregnation of thin layer plates by a variety of reagents provides an improved resolution. The literature on qualitative and quantitative TLC analyses of several antibiotics has been reviewed (Sherma, 1986, 1988, 1990; Sherma and Fried, 1984). Resolution of tetracycline antibiotics on kieselguhr impregnated with EDTA was reported by Gatsonis and Ageloudis (1982). A survey of TLC of those antibiotics which are in therapeutic use has been presented by Kreuzig (1991); it covers papers published after 1980. Tt is evident from the literature that there are no other reports for the TLC resolution of tetracycline and amino glycopeptide classes of antibiotics on impregnated layers. In view of the above, resolution of tetracycline and amino glycopeptide antibiotics was carried out on silica gel layers impregnated with CU+', CO+' and Ni+' ions and new solvent systems were developed. The results are presented in this communication. EXPERIMENTAL The capsules of antibiotics were purchased from various manufacturers viz. TDPL, Rishikash, India (tetracycline-

* Authors to whom correspondence 0269-3879/92/040196-02 $06.00 01992 by John Wiley & Sons, Ltd.

HCl), Cyanamide India Ltd., Bombay, lndia (MinocyclineHCI and Demeclocycline-HCI), Pfizer Ltd., Bombay, India (oxytetracycline-HCl), Maharastra Antibiotics and Pharmaceuticals Ltd., India (doxycycline-HCl), Sarabhai Chemicals, Bombay, India (streptomycin sulphate), Fulford India Ltd., Bombay, lndia (sisomycin sulphate), Aristo Pharmaceuticals Pvt. Ltd., Bombay, lndia (tobramycin sulphate and amikacin sulphate) and Saphire Laboratories, Bombay, India (neomycin sulphate). Silica gel G (10-40 pm), with calcium sulphate (13%) as binder and impurities of chloride, iron and lead (0.02%) each showing a pH 7.0 in 10% aqueous suspension, was obtained from E. Merck, Bombay, India. Thin layer plates (20 cm x 20 cm x 0.5 mm) were prepared by spreading a slurry of silica gel G (50 g) in distilled water (100 mL). The plates were then dried overnight at 60 k 2 "C in an oven. For impregnated plates the slurry was prepared in distilled water (100 mL) containing copper sulphate (CuS04.5 H20), 1.0 g, cobalt sulphate [Cu(SO,), . 7 H20], 1.0 g and nickel chloride (NiC13.6 H20), 1.0 g. The powdered content of the capsules (200 mg) was extracted with absolute ethanol (three portions of 20 mL each). The combined extract was concentrated in vacuum and allowed to crystallize. The mother liquor was decanted and the crystals were washed with a little ether. The standard solution ( 1 0 - 4 ~ of ) each of the antibiotics was prepared in 70% ethanol. The solutions were applied at 500 ng level using a 25 pL Hamilton syringe. The chromatograms for tetracycline antibiotics (Table 1) were developed up to lOcm for 50 min in ethano1:acetic acid :water (10 :6 :6, vlvlv) in a paper-lined rectangular glass chamber which had been pre-equilibrated with the solvent system for 10-15 min at 12 f2 "C. Those for amino glycopep-

Table 1. hRe values of tetracycline antibiotics on untreated and impregnated silica gel layers Impregnated Antibiotics

Untreated

Cof2

Cucz

Nit3

55 65 70 1OT 1. Demeclocycline-HCI 15 60 65 80 2. Minocycline-HCI 75 10T 70 3. Doxycycline-HCI 70 4. Oxytetracycline-HCI 02 07 10T 15T 5. Tetracycline-HCI 20T 50 50 80 T = tailing. 6:6, vlvlv). Solvent system =ethanol :acetic acid :water (10: Solvent front= 10 cm. Developing time = 50 min. Room temperature = 12 k 2 "C. Detection reagent = iodine vapours.

should be addressed. Received 27 December 1991 Accepted 13 January I992

TLC SEPARATION OF CEKTAIN TETRACYCLINE AND AMINO GLYCOPEPTIDE ANTIBIOTICS

Table 2. hRfvalues of amino glycopeptidegroup antibiotics on untreated and impregnated silica gel layers Impregnated Antibiotics

Untreated

CO+~

Cut'

Ni+3

20 20 19T 20T 1. Sisornycin sulphate 25 25T 30T 30T 2. Tobrarnycin sulphate 35 22 20 20 3. Gentarnycin sulphate 30 19T 14T 15T 4. Neornycin sulphate 5. Arnikacin sulphate 55 40T 34T 35T 6. Streptomycin sulphate 45 25 22 22 T = tailing. Solvent system = n-butanol :formic acid :water (6:5 : 7, v/v/v). Solvent front = 10 crn. Developing tirne=70 rnin. Room temperature = 12 k 2 "C. Detection reagent = iodine vapours.

tide antibiotics (Table 2) were developed up to lOcm for 75 min in the solvent system n-butanol :formic acid :water (7:5:6, v/v/v) at 12+2"C for 30 rnin and the spots were located by exposing the chromatoplate to iodine vapours.

RESULTS AND DISCUSSION

The hR, values of tetracycline antibiotics (Table 1)and aminoglycopeptide antibiotics (Table 2) on untreated and impregnated layers are reported. The results are an average of at least three identical runs with a standard deviation of 0.40-0.51 on untreated and impregnated layers. The resolution of these antibiotics was confirmed by calculating the resolution possibilities by the usual method (Bhushan and Ali, 1986) by dividing the distance between two spot centres with the sum of their radii. In order to optimize the separation conditions, various concentrations of the solvent constituents of the ternary solvent mixture and impregnating reagents were used. As a result of extensive experimentation the best solvent systems and impregnating reagents were selected and reported herewith. ~.O%) The tetracycline antibiotics resolved on C O +(1 impregnated silica gel layer using ethanol : acetic acid : water (10:6:6, v/v/v) as the mobile phase. The hR, values of tetracycline antibiotics as shown in Table 1 increased on plates impregnated with all three metal ions; most of these antibiotics showed tailin on untreated plates and plates impregnated with Cu+' and Ni+3ions. The tailing was more with C U +than ~ with Ni+3. Thus the plates impregnated with Cot2 (1.0%) were found to be the best as the spots were very

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compact and there was a complete resolution of five tetracycline antibiotics. The metal ion may be considered to have formed a complex with the antibiotic molecule. The separation of tetracycline was thus influenced by the chromatographic behaviour (adsorption/partition) of the complex so formed. This resulted in complete and clear resolution of tetracycline antibiotics on Co+*(1.0%) impregnated silica gel layers using the solvent system mentioned above. The same solvent system, i.e. ethanol: acetic acid: water (10:6:6, v/v/v) and the silica gel impregnated with C U + ~CO+* , and Ni+3were tried for the resolution of six amino glycopeptide antibiotics; there was tailing of spots (Table 2). Systematic variation in solvent composition did not result in an improved resolution on impregnated plates. It was considered that the antibiotic molecules belonging to the amino glycopeptide class having a large molecular structure containing a glycosidic link with several monosaccharide units do not form stable complexes with the metal ions available in chromatographic systems. Therefore, efforts were made to develop a suitable solvent system without any impregnating reagent and the solvent system n-butanol :formic acid :water (6 :5 :7, v/v/v), optimized systematically, was found to be very good for the resolution of amino glycopeptide antibiotics. Ninhydrin (Claes, 1982), fast violet (Oka, 1984) etc. have been used as detection reagents for antibiotics; these are considered to have certain disadvantages, e.g. they react with the compound on the chromatogram and change its chemical nature, and there is no single reagent which can detect a variety of antibiotics. The application of iodine vapours for locating the spots proved to be very useful, as it provided spots with very sharp boundaries and visualized small traces of antibiotics; the antibiotics on the chromatogram remained chemically unaltered making the compound available for subsequent use, if required. Thus, the reported chromatographic system can be considered as a successful, fast and reliable method for the detection and identification of the antibiotics belonging to the above-mentioned two groups and can be recommended for separation of antibiotics from unknown solutions.

Acknowledgements The authors are thankful to the Council of Scientific and Industrial Research, New Delhi, India for the award of Research Associateship (to IA). Financial assistance from the UGC is also gratefully acknowledged.

REFERENCES Bhushnan, R. and Ali, 1. (1986). J . Liq. Chrornatogr. 9, 3479. Bhushan, R. and Ali, I. (1989). J . Planar Chromatogr. 3, 85. Bhushan, R., Chauhan, R. S., Reena and Ah, I. (1989a). J . Liq. Chromatogr. 7 , 1341. Bhushan, R. and Ali, I. (1990). J. Planar Chromatogr. 3, 85. Bhushan, R., Reena and Chauhan, R. S. (1989b). Biomed. Chromatogr. 3, 46. Claes, P. J. and Vanderhaeghe, J. (1982). J . Chromatogr. 248(3), 483. Gatsonis, C. D. and Ageloudis, C. A. (1982). Pharrnazie 37, 649.

Kreuzig, F. (19911. In Handbook of Thin Layer Chromatography, ed. by Sherma, J. and Fried, B., p. 407. Marcel Dekker, New York. Oka, H., Uno, K., Haroda, K. I., Hayashi, M. and Suzuki, M. (1984). J . Chromatogr. 295(1), 129. Sherrna, J. (1986). Anal. Chem. 58(5), 69R. Sherma, J. (1988). Anal. Chem. 60(12). 74R. Sherrna, J. (1990). Anal. Chem. 62(12), 371R. Sherma. J. and Fried, B. (1984). Anal. Chem. 56(5) 48R. Vandarnrne, E. J. (1984). Biotechnology of lndustrial Antibiotics, pp. 3-35,229-355. Marcel Dekker, New York.

TLC separation of certain tetracycline and amino glycopeptide antibiotics.

The separation of tetracycline and amino glycopeptide antibiotics was achieved on silica gel thin layers. Tetracycline antibiotics were resolved on a ...
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