Tetracyclines chromatography

For detection residue amounts of tetracyclines in dairy products widely used methods FIPLC, immunoaffinity chromatography, kinetic spectrophotometry, which are expensive and complicated. 

The contents of the tube were then acidified to pH 2 by the addition of sulfuric acid and centrifuged. Examination of the supernatant liquid by paper chromatography employing the methods of 8ohonos et al. Antibiotics Annual (1953-4, page 49), demonstrates the presence of 7-chloro-6-demethyltetracycline, 7-chlorotetracycline and tetracycline. 

Two identification tests for oxytetracycline hydrochloride are given in the USP 28 [1], one being an ultraviolet absorption test and the other a color test. European Pharmacopoeia [2], British Pharmacopoeia (BP) 2003 [4], International Pharmacopoeia [5], and Pharmacopoeia of the People s Republic of China [6] described a thin-layer chromatography and color tests for identification of oxytetracycline hydrochloride and oxytetracycline dihydrate. For identification of oxytetracycline calcium, USP 28 [1] used Method II under identification of tetracycline <193>, whilst BP 2003 [4] described a TLC, color test, and calcium test as the method of identification. 

For confirmatory assay, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is becoming more frequently used in the analysis of OTC owing to its high sensitivity and ability. Electrospray ionization (ESI) [55-57] and atmospheric pressure chemical ionization (APCI) [41] methods combined with tandem mass spectrometry are favored because of their higher sensitivity and better reproducibility. Hamscher et al. [58] developed a method for the determination of persistent TC residues in soil fertilized with manure by HPLC tandem mass spectrometry, MS-MS, and confirmation by MS-MS-MS. Zhu et al. [59] developed an LC-tandem mass spectrometry for the analysis of common tetracyclines in water. The detection limit for oxytetracycline was 0.21 pg/L. Lykkeberg et al. [60] used LC-MS/MS for determination of oxytetracycline and its impurities EOTC, TC, ETC, ADOTC, oc-AOTC, and /i-AOTC. 

Some of the early reports on the chromatography of the tetracycline antibiotics prior to 1957 are of limited value. Fischbach and Levine (43) described a continuous ascending technique and Berti and Cima (44) reported an ascending method using aqueous sodium arsenite as the mobile solvent. Other authors(45,46) reported descending techniques and bioautographic means for locating the zones of activity. 

All of these methods fail to show the presence of the epimeric form of the tetracyclines and in most instances streaking of the spots is a problem. A basic improvement in the paper chromatography of these antibiotics was achieved by Selzer and Wright (47) and Kelly and Bryske (48) when they reported methods for the pretreatment of the paper with com-plexing agents to bind the metallic ions which may be present. 

Other chromatographic techniques that have been applied to the tetracyclines, including CTC, involve low pressure column chromatography. Ascione et al. (64) developed a semiautomated system whereby sample solutions are automatically injected onto a column of diatomaceous earth mixed with... 

Vander Heyden, Y., Luypaert, K., Hartmann, C., Massart, D. L., Hoogmartens, J., and De Beer, J. (1995). Ruggedness tests on the high-performance liquid chromatography assay of the United States Pharmacopeia XXII for tetracycline hydrochloride. A comparison of experimental designs and statistical interpretations. Anal. Chim. Acta 312, 245-262. 

Saevels, J. (1998). Separation of tetracycline and its related substances comparison of liquid chromatography, capillary electrophoresis and capillary electrochromatography Biomed. Chromatogr. 12, 149 — 150. 

Yang S, Cha J, Carlson K (2005) Simultaneous extraction and analysis of 11 tetracycline and sulphonamide antibiotics in influent and effluent domestic wastewater by solid-phase extraction and liquid chromatography-electrospray ionization tandem mass spectrometry. J Chromatogr A 1097 40-53... 

Takeba, K., Kanzaki, M., Murakami, F., Matsumoto, M. 1984. Simplified analytical method for tetracycline residues in honey by high performance liquid chromatography. Kenkyu Nenpo -Tokyo-toritsu Eisei Kenkyusho. 35 187-91. 

Moats, W. A. 198. Determination of Tetracycline Antibiotics in Tissues and Blood Serum of Cattle and Swine by High Performance Liquid Chromatography. J. Chromatog. (In Press)... 

FSIS laboratories also use chemical techniques and instrumentation to identify select antibiotic residues. The tetracyclines of interest are identified by thin layer chromatography. Sulfonamides are detected and quantified by fluorescence thin lay chromatography and confirmed by gas chromatography/mass spectrometry. Amoxicillin and gentamycin are identified and/or quantified by high pressure liquid chromatography. Similar techniques are used to identify ionophores and other antimicrobials of interest. 

Hamscher G., S. Sczesny, H. Hdper, and H. Nau (2002). Determination of persistent tetracycline residues in soil fertilized with liquid manure by high-performance liquid chromatography with electron spray ionization tandem mass spectrometry. Analytical Cheminstry 74 1509-1518. 

Lindsey M.E., M. Meyer, and E.M. Thurman (2001). Analysis of trace levels of sulfonamide and tetracycline antimicrobials in groundwater and surface water using solid-phase extraction and liquid chromatography/mass spectrometry. Analytical Chemistry 73 4640-4646. 

Elimination of coextracted materials and concentration of tetracyclines have also been accomplished using mixed-phase extraction membranes with both re-versed-phase and cation-exchange properties (294,295), or solid-phase extraction columns packed with cation-exchange materials such as CM-Sephadex C-25 (301), aromatic sulfonic acid (310), and carboxylic acid (283, 300). For the same purpose, metal chelate affinity chromatography has also been employed. In this technique, the tetracyclines are specifically absorbed on the column sorbent by chelation with copper ions bound to small chelating Sepharose fast flow column (278-281, 294-296). 

Ultrafiltration (278, 279) and immunoaffinity chromatography (282) have also been described for removal of matrix components from milk extracts, while online trace enrichment has been reported for isolation/purification of tetracycline, oxytetracycline, demeclocycline, and chlortetracycline residues from animal tissues and egg constituents (305). The latter technique involves trapping of the analytes onto a metal chelate affinity preconcentration column (Anagel-TSK Chelate-5PW), rinsing of coextracted materials to waste, and finally flushing of the concentrated analytes onto the analytical column. 

Coupling of liquid chromatography with mass spectrometry provides unequivocal online spectrometric identification of tetracycline antibiotics in animal-derived foods. Typical applications of mass spectrometry in confirming tetracycline residues in edible animal products describe coupling of liquid chromatography with mass spectrometry via particle-beam (280), electrospray (292), or atmospheric pressure chemical ionization (307), using negative-ion detection interfaces. 

In a different approach, Cooper et al. (305) developed an online metal chelate affinity chromatography-liquid chromatographic method for the determination of oxytetracycline, tetracycline, demeclocycline, and chlortetracycline residues in animal tissues and egg. According to this method, a 2 g blended egg or thinly sliced tissue is homogenized with citrate buffer pH 5 (pH 4, for chicken... 

RJ McCracken, W John, WJ Blanchflower, SA Haggan, DG Kennedy. Simultaneous determination of oxytetracycline, tetracycline and chlortetracycline in animal tissues using liquid chromatography, postcolumn derivatization with aluminum, and fluorescence detection. Analyst 120 1763-1766, 1995. 

MC Carson, W Breslyn. Simultaneous determination of multiple tetracycline residues in milk by metal chelate affinity chromatography—collaborative study. J AOAC Int 79 29-42, 1996. 

S Croubels, KEI Vanoosthuyze, CH VanPeteghem. Use of metal chelate affinity chromatography and membrane-based ion-exchange as cleanup procedure for trace residue analysis of tetracyclines in animal tissues and egg. J Chromatogr B 690 173-179, 1997. 

MC Carson, MA Ngoh, SW Hadley. Confirmation of multiple tetracycline residues in milk and oxytetracycline in shrimp by liquid chromatography-particle beam mass spectrometry. J Chromatogr B 712 113-128, 1998. 

G Stubbings, JA Tarbin, G Shearer. Online metal chelate affinity chromatography cleanup for the high-performance liquid chromatographic determination of tetracycline antibiotics in animal tissues. J Chromatogr B 679 137-145, 1996. 

J Sokol, E Matisova. Determination of tetracycline antibiotics in animal tissues of food producing animals by high-performance liquid chromatography using solid-phase extraction. J Chromatogr A 669 75-80,1994. 

H Oka, Y Ikai, J Hayakawa, K Masuda, KI Harada, M Suzuki. Improvement of chemical analysis of antibiotics. 19. Determination of tetracycline antibiotics in milk by liquid chromatography and thin-layer chromatography fast atom bombardment mass spectrometry. J AOAC Int 77 891-895, 1994. 

WJ Blanchflower, RJ McCracken, AS Haggan, DG Kennedy. Confirmatory assay for the determination of tetracycline, oxytetracycline, chlortetracycline and its isomers in muscle and kidney using liquid chromatography-mass spectrometry. J Chromatogr B 692 351-360, 1997. 

WA Moats, R Harik-khan. Rapid determination of tetracycline antibiotics in milk and tissues using ion-pairing high-performance liquid chromatography. ACS Symp Ser 636 85-95, 1996. 

Tricyclazole and tetracycline have been determined by gas chromatography - mass spectrometry [247]. Persistent tetracycline residues have been determined in soil by high-performance liquid chromatography with electrospray ionisation tandem mass spectrometry [248]. 

Paper partition chromatographic methods have been widely applied to the analysis of tetracyclines (128, 129). Pharmaceutical aqueous suspensions for oral use are acidified with HC1 and diluted with methanol. Crystalline formulations are dissolved only in methanol. A paper chromatographic method for TC determination in pharmaceutical preparations is based on the complexation of the antibiotic with a mixture of urea and disodium edetate on paper at pH 7.4. Urea helped in the separation of degradation products and led to the formation of well defined spots (130). Samples from fermentations must be acidified with oxalic acid to liberate TC from the mycelium. TC in filtrates may be precipitated in saturated solution of sodium tetraphenyl borate, precipitate dissolved in ethyl or butyl acetate and applied for paper chromatography. Various solvent systems and hRp values for paper chromatography are given in Table 4. 

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