Tetracycline epimers

They are approximately in agreement with values reported in literature, but the spectrum there was obtained with a HA-100 (23). The nmr shifts appear to be general regardless of solvent. Similar shifts are obtained in pyridine, trifluoroacetic acid and dimethyl sulfoxide. Particularly notable is the substantial downfield shift in the resoncance due to the C 4 proton when the tetracyclines epime-rize at C 4. This is especially convenient as both isomers are usually available and can be distinguished easily in this way. NMR was used to monitor the epimerization of TC since the dimethylamino resonance of TC and its C 4 epimer differ by 0.1 ppm (24). Formation of anhydrotetracycline could also be readily detected through NMR (23). 

Biosensors based on microbial immobilization have also been used for food applications, e.g., the inexpensive and rapid high-throughput bacterial biosensor developed by Virolainen et al. for rapid detection of tetracyclines and their 4-epimer derivatives in poultry meat [188, 189]. 

Detection is made in UV light. The yellow fluorescence of tetracyclines and their epimers in UV light is greatly enhanced by exposing the paper to ammonia vapours. They may also be detected by Ehrlich s reagent or by starch-iodine following N-chlorination (137). 

In acidic conditions the tetracyclines undergo epimerisation at carbon atom 4 to form an equilibrium mixture of tetracycline and the epimer, 4-epi-tetracycline (Scheme 4.7). The 4-epi-tetracycline is toxic and its content in medicines is restricted to not more than 3%. The epimerisation follows the kinetics of a first-order reversible reaction (see equation (4.24)). The degradation rate is pH-dependent (maximum epimerisation occurring... 

Another instability leading to a dramatic decrease of antibacterial action, to which all clinically used tetracyclines are subject, is epimerization of the natural 4-a-dimethy-lamino group A to the p-epimer B (Eq. 6.11). Under acidic conditions a 1 2 equilibrium is established in solution within a day. This occurs in a variety of solvents, especially acetic acid. Anions also tend to support this process. Divalent ions that chelate tetracyclines, particularly Ca2+, facilitate the reversal of the epimerization from the epi to the natural isomer. 

The stability of various classes at different pH values makes it less than ideal to extract some antibiotics under the same conditions. Tetracyclines are more stable at pH 3-4 than at pH > 5, and changes in pH can lead to the formation of their epimers and even further degradation. Macrolides and f)-lactams are stable at neutral or slightly basic conditions (i.e., pH > 8 or 8.5). Erythromycin degrades to erythromycin-H2O with loss of one H2O molecule at low pH " and is often measured as erythromycin-H20 at m/z 716 in environmental samples but not in food. The... 

On the basis of existing knowledge, novel quantitative approaches for the analysis of tetracyclines should involve extraction under mild acidic conditions, incorporate metalchelating agents, and investigate polymeric SPE sorbents. In addition, they should be able to detect and quantify OTC, TC, CTC, DC, and their associated 4-epimers at concentrations that are fit for their intended purpose. If confirmation is required, LC-MS/MS is recommended for its selectivity. 

The nitroimidazoles, sulfonamides, and tetracyclines all present analytical challenges because of metabolism and/or chemical degradation. In the case of the nitroimidazoles, this is further complicated by the relatively low requirements for detection. Method development therefore has to take into account both metabolites as additional target compounds and low detection limits. Sulfonamide analysis has to take into account the potential for conversion of N -acetyl metabolites back to the parent compound. In contrast, in the analysis of honey, deconjugation is regarded as necessary to accurately determine sulfonamide concentrations. The facile, reversible formation of epimers is of particular concern in the analysis of those tetracyclines that can epimerize in the 4 position. Protein and metal binding are other issues that have to be overcome for successful tetracycline residue determination. 

Cherlet M, ScheUcens M, Croubels S, De Backer P, Quantitative multi-residue analysis of tetracyclines and their 4-epimers in pig tissues by high-performance liquid chromatography combined with positive ion electrospray ionization mass spectrometry, Anal. Chim. Acta 2003 492 199-213. 

It must be established that the analyte remains stable throughout the analysis. There have been some reports in the literature of conversion of the analytes to apparent metabolites or degradation products, such as in the case of tetracyclines, and this can certainly confuse the interpretation of the analytical results. This has led some jurisdictions to amend MRLs to take account of this phenomenon. For example, in the EU, the MRL for the various tetracyclines is expressed as the sum of the parent tetracycline and the 4-epimer of the veterinary drug. ... 

---