Tetracyclines methods

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. 

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 identification of oxytetracycline in pharmaceutical preparations, USP 28 [1] describes Method II under identification of tetracycline <193> (see Table 1), BP 2003 [4] describes a TLC and color test. 

UV detection, diode-array detector (DAD) and fluorescence have been the detection techniques used, coupled to HPLC for the analysis of OTC. UV detection is set at 355 nm [49-51], 350 nm [40], or at 353 nm [52], Using the diode array detector [49] offers advantages that the target peak can be identified by its retention time and absorption spectrum. Compared to UV detection, fluorescence detection is generally more specific and is less interfered by other compounds in the sample matrix [51]. A HPLC method with electrochemical detection has also been suggested recently. Zhao et al. [53] described HPLC with a coulometric electrode array system for the analysis of OTC, TC, CTC, DC, and methacycline (MC) in ovine milk. An amper-ometric detection coupled with HPLC was developed by Kazemifard and Moore [54] for the determination of tetracyclines in pharmaceutical formulations. 

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. 

OTC can be well separated from TC, DC, and its impurities by means of capillary electrophoresis [25]. However, the use of CE in the analysis of OTC residues is restricted because of the low concentration sensitivity of this technique [28]. HPLC is by far the most widely used method for the analysis of OTC residues in food and fisheries products. Chromatographic analysis of tetracycline including OTC analysis in foods was reviewed by Oka et al. [65] and MacNeil [72]. HPLC methods for the analysis of OTC are summarized in Table 2. 

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. 

Most of the methods in which the paper is treated with a chelating agent are capable of showing a separation of some of the tetracycline drugs from each other and from their respective epimeric forms. They are also capable of revealing the presence of common degradation compounds of these drugs. 

The usual method of detecting chromatographed tetracycline antibiotics involves fuming the paper with ammonia vapor and observing the yellow fluorescence under UV light. As little as 0.2-0.5 pg can be visualized by this technique. 

A number of papers have appeared reporting the HPLC separation of CTC from its isomers and/or other tetracyclines. There is not a consensus of opinion as to the most satisfactory approach thus, it appears that at this time one must still verify the optimal system for a particular instrument. Methods found in the literature for CTC are described in Table 6. EDTA is added to prevent the formation of complexes of the tetracyclines with metallic surfaces. 

Others have described modifications of these methods for various purposes. Hiscox (82) suggested the direct spec-trophotometric assay of CTC in either acid or alkaline solution at various UV wavelengths. The possible contamination of CTC with other tetracycline drugs was addressed by Chicearelli et... 

The tetracycline which may be present is then converted to an anhydro derivative by heating in acid and is measured spectrophotometrically. Feldman et al. (84) developed the alkaline degradation method to measure CTC in fermentation mash and Spock and Katz (85) used this method to determine CTC in animal feed premixes. 

Several qualitative and quantitative immunochemical methods for CAP analysis in biological matrices of animal origin have been described [101,102, 104,105] (see Table 3). Van de Water et al. [ 102] described an ELISA that detected CAP in swine muscle tissue with an IC50 value of 3 ng mL1. This immunoassay was improved and subsequently optimized incorporating the streptavidin-biotin amplification system. There are also several commercially available test kits (see Table 4). RIDASCREEN is a competitive enzyme immunoassay for the quantitative analysis of CAP residues in milk, eggs, and meat in a microtiter plate. The measurement is made photometrically, obtaining a LOD of 100 ng L 1 in meat and eggs and 150 ng L 1 in milk. The test has been also applied to the analysis of tetracyclines. 

Immunochemical methods have been developed and placed on the market to analyze tetracycline residues (see Table 4). Thus, a qualitative EIA has been developed and used to analyze tetracyclines in honey samples with a detection level of 20 pg/kg-1 [96]. A microplate-based indirect ELISA has been developed to analyze tetracyclines using polyclonal antibodies. The assay could measure tetracycline in the range between 0.1 and 6 ng mL L Other tetracycline antibiotics such as chlortetracycline, rolitetracycline, or minocycline are also highly recognized in this assay [98]. Several immunoassay kits are commercially available for the analysis of tetracyclines although, to our knowledge, none of them... 

The use of the Charm II RIA test to analyze tetracycline antibiotics in water (both surface and groundwater) has been reported [84, 97]. This RIA, which was initially developed to analyze tetracycline in serum, urine, and milk, was subsequently adapted to analyze water samples at concentration levels around 1 pg L-1. Thus, samples from hog lagoons, surface water samples, and ground-water samples were tested using the RIA method and the results confirmed by LC-MS. 

Figure 13.18 Replica plating for the selection of bacteria containing a recombinant vector, (a) The method used to transfer colonies of bacteria between plates, (b) Comparison of the distribution of the colonies between the plates enables identification of the colonies containing the recombinant vector. The dotted circles represent colonies that do not grow on tetracycline.

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