Tetracyclines metal chelate affinity

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. 

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. 

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... 

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. 

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. 

Residues of oxytetracycline, tetracycline, and chlortetracyline in bovine milk were determined and confirmed after centrifugation of the milk, filtration over a 25 kDa cut-off filter, and SPE on a C,8 cartridge. Methanol-oxalic acid-acetonitrile was used as mobile phase. Four ions for each tetracycline from the negative-ion mass spectra were used for confirmation at 100 ng/ml level [97]. Carson et al. [98] reported the determination of tetracycline residues in milk and oxytetracycline residues in shrimp. Off-line metal-chelate affinity chromatography on Cu " -loaded chelating Sepharose in combination with SPE on polymeric ENVI-ChromP material was used for sample pretieatment. LC is performed using a PLRP-s polymeric material and 5 mmol/1 oxahc acid in the mobile phase. The method is vahdated with samples spiked at 30 ng/ml in milk and 100 ng/g in shrimps. 

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. 

The tetracyclines have an avidity for divalent metals similar to that of glycine (III) but they have a greater affinity for the triva-lent metals with which they form 3 1 drug-metal chelates. Therapeutically active tetracyclines form 2 1 complexes with cupric, nickel and zinc ions while inactive analogues form only 1 I complexes. 

Tetracyclines have the tendency to form chelates with bivalent metal ions. As a consequence of their affinity to calcium, tetracyclines tend to accumulate in the bones of treated animals. Although their chelates with calcium show considerable stability, tetracyclines can be extracted from bones containing these drugs and, therefore, may be present in soups and meals when bones from treated animals are cooked (80, 81). The extractability of chlortetracycline from bone tissue is strongly pH-dependent, being higher at low pH values. This can be easily explained by the dependence of the dissociation constant of the chelate from the pH value. 

As tetracyclines have moderate to high lipophilic properties, the poor bioavailability associated with oral administration is somewhat surprising. Papich and Riviere suggest that causes may be multifactorial. As zwitterions, they are mainly ionized at pHs within GIT liquor. Moreover, feed reduces bioavailability, and tetracyclines chelate with polyvalent cations. Oxytetracycline absorption has been shown, experimentally, to be reduced by feed, dairy products, Ca +, Mg +, Al +, and Fe + ions and antacids. Even though doxycycline has a similar structure, affinity for metals is different from that of oxytetracycline with greater affinity for zinc and less for calcium. 

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