Chlortetracycline methods

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... 

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... 

The incidence of tetracycline residues in turkeys has been also studied recently (13). During meat inspection, suspicious flocks were identified by fluorescence detection in bones. A total of 85 flocks showed significant fluorescence in bones. When bones, liver, kidney, and muscle of these animals were analyzed for tetracyclines, residues of tetracycline, chlortetracycline, oxytetracycline, or doxycycline were found in 83 flocks. These results suggest that the detection of fluorescence in bones might be a useful screening method for tetracycline residues in poultry carcasses. 

Several microbiological methods have been reported for analyzing residues within the groups of sulfonamides, tetracyclines, macrolides, and -lactams in fish tissues however, references for these groups of compounds are limited to sulfadiazine and trimethoprim, oxytetracydine and chlortetracycline, erythromycin, and ampicillin, respectively. 

Key to the successful production of generic antisera for the tetracycline antibiotics has been the synthesis of the hapten 4-hydrazino-4-dedimethylamino-tetracycline and its subsequent conjugation to protein (73). The immunochemical method developed from this sera could detect tetracycline, chlortetracycline, and oxytetracycline residues in meat and milk, with adequate sensitivity (19). 

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... 

JD MacNeil, VK Martz, GO Korsrud, CDC Salisbury, H Oka, RL Epstein, CJ Barnes. Chlortetracycline, oxytetracycline, and tetracycline in edible animal tissues, liquid chromatographic method—collaborative study. J AOAC Int 79 405 -417, 1996. 

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for maduramicin in poultry feed. The assay utilized polyclonal anti-maduramicin antibody raised in rabbits, maduramicin monoamide with 1,6-hexane diamine-conjugated ovalbumin as the coating antigen, horseradish peroxidase conjugated goat anti-rabbit IgG and 2,2 azinobis(3-ethylbenzthiazoline) sulfonic acid (ABTS) for quantitation. Standard curves ranging from 0 to 80 ng/mL maduramicin were constructed. The assay did not cross-react with monensin, lasalocid, salinomycin, lincomycin, narasin, chlortetracycline or roxarsone. Broiler feed fortified at 4 to 7 ppm maduramicin were shown to be quantifiable by ELISA at an average recovery of 98.1%. This ELISA method for maduramicin in poultry feed is comparable to the established HPLC-F method. 

White, C.R. Moats, W.A. Kotula, K.L. Optimization of a liquid chromatographic method for determination of oxytetracycline, tetracycline, and chlortetracycline in milk. J.AOAC Int., 1993, 76, 549-... 

Goto et al. " described a simple, rapid, and simultaneous analysis method for oxytetracycline, tetracycline, chlortetracycline, penicillin G, ampicillin, and nafcillin in meat using electrospray ionization tandem mass spectrometry. The samples were homogenized with water followed by a centrifugal ultrafiltration after addition of internal standards (demeclocycline, penicillin G-ds, ampicillin-fi 5, and... 

AO AC, Chlortetracycline, oxytetracycline and tetracycline in edible animal tissues, 995.09, in Official Methods of Analysis, 18th ed. (revised), AO AC International, Gaithersburg, MD (available at http //www.eoma.aoac.org/gate way/readFile. asp id=995 09. pdf accessed 3/24/10). 

Cinquina A, Longo F, Anastasi G, et al., Validation of a high performance liquid chromatography method for the determination of oxytetracycline, tetracycline, chlortetracycline and doxycycline in bovine milk and muscle, J. Chromatogr. A 2003 987 227-233. 

Tetracyclines including doxycycline (DC) and oxytetracycline (OTC) and degradation products, like 4-epitetracycline (ETC), chlortetracycline (CTC), 4-epianhydro-tetracycline (EATC), anhydro-tetracycline (ATC), can also be separated on silica gel HPTLC plates (Merck), that have been predeveloped with a saturated aqueous Na2EDTA solution, dried at room temperature for Ih and activated at 130°C for 2h (47). In this method, 100 nl of the reference solutions (10 mg/ml methanol) were applied. The following solvents are used ... 

For the assay and purity control of chlortetracycline and demeclocycline a densitometric method has been published (55). With the solvent dichloromethane/methanol/water (60 35 5) for CTC and (59 35 6) for DMC and silica gel layers (Macherey-Nagel), previously sprayed with 10% Na2EDTA, adjusted to pH 8.0, all major impurities of CTC or DMC are separated from the main components and from each other. The less important anhydro derivatives of CTC or DMCTC were better separated on RP2 (20 X 20 cm) and RP 8 (10 x 10 cm, Merck both) with a mobile phase of methanol/acetoni-trile/0.5 M oxalic acid pH 2.0 (1 1 6) and methanoI/acetonitrile/0.5 m oxalic acid 2.0 (1 1 4), respectively. The coefficient of variation for the determination of the main component is 2%. The correlation with HPLC-resuIts is excellent (r = 0.9999). 

Masawat and Slater have presented the FIA method for determination of tetracyclines (tetracycline, chlortetracycline, and oxytetracycline) in chicken and shrimp meats and pharmaceutical samples [52]. The electrochemical oxidation of analytes was performed using a gold screen-printed electrode and good detection limits were achieved, 0.96,0.58, and 0.35)imoll for tetracycline, chlortetracycline, and oxytetracycUne, respectively. Moreover, some advantages of the screen-printed electrodes were highlighted such as potential for miniaturization, reliability, portability, low cost, simplicity of construction, and operation. 

A microbiological assay is the method of choice (see p. 813) but spectro-photometric methods are also applicable. One spectrophotometric method is based on the formation of a yellow colour with an absorption maximum at 380 m//, when tetracycline is dissolved in 0-25N sodium hydroxide, another on the orange-brown colour (maximum absorption 490 m//) formed on mixing a dilute hydrochloric acid solution of the sample with ferric chloride solution. The former method which is described below is also applicable to oxytetracycline but not to chlortetracycline while the ferric chloride reaction, which is given in detail under oxytetracycline, applies to all three. 

The colour produced with chlortetracycline, under the conditions described above, fades rapidly and for the determination of tetracycline in mixtures with chlortetracycline containing up to 20 per cent of the latter antibiotic the above method may be applied directly. However, for mixtures of the two antibiotics in which chlortetracycline is the major constituent, the extinction due to the chlortetracycline, with an absorption maximum at 345 m//, must be taken into account. In this case the tetracycline will give a shelf at 380 m/ superimposed on the chlortetracycline band and since the height of this shelf depends on the concentration of tetracycline, small amounts of tetracycline may be determined using a standard curve prepared with mixtures of the two antibiotics of known concentration. 

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