Tetracyclines cleanup

An on-line concentration, isolation, and Hquid chromatographic separation method for the analysis of trace organics in natural waters has been described (63). Concentration and isolation are accompHshed with two precolumns connected in series the first acts as a filter for removal of interferences the second actually concentrates target solutes. The technique is appHcable even if no selective sorbent is available for the specific analyte of interest. Detection limits of less than 0.1 ppb were achieved for polar herbicides (qv) in the chlorotriazine and phenylurea classes. A novel method for deterrnination of tetracyclines in animal tissues and fluids was developed with sample extraction and cleanup based on tendency of tetracyclines to chelate with divalent metal ions (64). The metal chelate affinity precolumn was connected on-line to reversed-phase hplc column, and detection limits for several different tetracyclines in a variety of matrices were in the 10—50 ppb range. 

A number of methods have been described for determination of tetracycline (chlortetracycline, tetracycline, and oxytetracycline) residues in tissues of food-producing animals (53-62), fish (63), eggs (64), and honey (65,66). Most of these methods use reversed-phase HPLC for determination. However, one uses TLC with UV densitometry ( ) and one uses GLC ( ), and one uses a direct mass spectrometric method CAD MIKE spectrometry (collisionally activated decomposition mass-analyzed ion kinetic spectrometry) for oxytetracycline in milk and meat (62). Several use solid-phase extraction in the cleanup procedure using XAD-2 resin (56,58) or Cj g cartridges... 

Apart from improving extractions, ion-pairing techniques can also improve liquid-liquid partition cleanup. Examples of effective ion-pairing cleanup procedures have been described in the analysis of tetracycline (60) and penicillin (68) residues in milk using tetrabutylammonium reagent, the resulting ion pairs turned out to be fairly lipophilic and readily extractable with organic solvents. 

The four-plate test was initially based on the German Hemmstoff-test with an additional plate of Sarcina lulea at pH 8.0, designed for the detection of lower levels of macrolides, and a fourth plate of Escherichia coli at pH 7.2 for the detection of sulfonamides (74,75). The modified version adopted by the European Community for screening carcasses is based on three plates with Bacillus subtilis BGA at pH values of 6.0, 8.0, and 7.2 with added trimethoprim, respectively, and a fourth plate with Micrococcus luteus NCTC 8340 at pH 8.0 (74). This test as described elsewhere (76) is intended to detect residues of -lactams, tetracyclines, aminoglycosides, sulfonamides, and macrolides in muscle tissue of slaughtered animals, without any prior extraction or cleanup. 

Due to their extremely polar character, tetracyclines bind with proteins to form conjugates that are difficult to extract from biological matrices. Use of dilute mineral acids is of great help in dissociating tetracyclines from proteins, but once in aqueous solution, their exfraction into volatile organic solvents for further concentration and cleanup is hampered by the unfavorable partition coefficients. Most of these antibiotics are photosensitive compounds, whereas all of them show poor stability under strong acidic and alkaline conditions with reversible formation of the 4-epi-tetracyclines in weakly acidic conditions (pH 3), and anhydro-tetracyclines in strong acidic conditions (below pH 2). 

When analyzing liquid samples such as milk for tetracycline residues, a pretreatment centrifugation step for fat removal is usually required (278-281). Dilution of liquid samples, prior to cleanup, with acetate buffer (212), phosphate... 

Liquid-liquid partitioning cleanup is generally directed to removal of the matrix constitutents from the aqueous extract into organic immiscible solvents (14, 298, 299, 302, 308). Unfortunately, tetracyclines cannot be quantitatively recovered into organic immiscible solvents at any pH value because of their high polarity. However, recoveries higher than 85% were reported when tetrabutylammonium ions were employed in the ion-pair extraction of oxytetracycline and tetracycline into dichloromethane at pH 8.2 (297). 

Following sample extraction and cleanup, tetracycline antibiotics may be separated by thin-layer or liquid chromatographic procedures, and quantified by spectrophotometric, fluorometric, or mass spectrometric detection systems. Direct spectrophotometric and capillary electrophoretic methods have also been described (Table 29.8). 

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. 

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. 

Antibiotics The AOAC has listed methods for sulfamethazine residues in swine tissues with determination either by GC-MS or GC-ECD of methylated derivatives and for sulfamethazine (and for the class of sulfonamides) in milk with determination by HPLC-UV. There is an AOAC method for the class of sulfonamide antimicrobials in animal tissues using solvent extraction and liquid partitioning with determination by TLC and fluorimetric scanning. For analysis of tetracyclines, AOAC describes methods based on buffer extraction from tissue samples and SPE (Cis) cleanup, or metal chelate affinity binding from milk samples, with determination in both cases by HPLC-UV. USDA/FSIS methods include (1) a method (similar to the AOAC GC-MS method for sulfamethazine) for confirmation of sulfonamide residues in edible tissues using solvent extraction and multiple liquid partitioning with determination of the methylated derivatives by GC-MS (2) methods for determination and confirmation of chloramphenicol in muscle by solvent extraction, liquid partitioning, and determination of the trimethylsilane (TMS) derivative by GC-ECD and GC-MS, respectively and (3) a method for determination of the beta-lactam antibiotic amoxicillin by aqueous extraction, cleanup by tricarboxylic acid precipitation, and ether extraction and formation of a fluorescent derivative for determination by LC. 

Residual tetracyclines in animal tissues were determined by solid-phase extraction on a Cig SPE column followed by TLC on an EDTA-treated silica gel plate and UV densitometry (Ikai et al., 1987). Seven tetracyclines were separated on silica gel layers impregnated with buffered EDTA and a mobile phase of dichloromethane-methanol-water (59 35 6) by Naidong et al. (1989). Residues of tetracyclines along with aminoglycosides, macrolides, and chlomamphenicol were screened in poultry meat by cellulose and silica gel TLC following extraction and cleanup on Cig SPE columns (Vega et al., 1990). 

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