Chlortetracycline, assay

Vadnais, M. L. Kirkwood, R. N. Tempelman, R. J. Sprecher, D. J. Chou, K. Effect of cooling and seminal plasma on the capacitation status of fresh boar sperm as determined using chlortetracycline assay. Anim. Reprod. Sci. 2005, 87, 121-132. 

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 large number of antibiotics, namely chlortetracycline, doxycyline, gentamicin, neomycin, streptomycin, tobramycin and the like may be assayed tubidimetrically with fairly good accuracy. 

WJ Blanchflower, RJ McCracken, AS Haggan, DG Kennedy. Confirmatory assay for the determination of tetracycline, oxytetracycline, chlortetracycline and its isomers in muscle and kidney using liquid chromatography-mass spectrometry. J Chromatogr B 692 351-360, 1997. 

Spectrofluorimetry differs from absorption spectrophotometry in not yielding an absolute scale of values. For this reason it is essential to employ a reference standard for quantitative measurements. For example, some pharmacopoeial tests, such as the test for uniformity of content for digitoxin tablets, employ a spectrofluorimefric assay and comparison wi an ofticial reference standard. Quantitative Spectrofluorimetry has been proposed for a munber of naturally fluorescent compoimds, including ergometrine, riboflavine, tiie catechol-amines, phenothiazines, the barbiturates (at pH 13), and certain antibiotics such as chlortetracycline and oxytetracycline. 

Naidong, W., Hua, S., Roets, E., Hoogmartens, J. Assay and purity control of tetracycline, chlortetracycline and oxytetracycline in animal feeds and premixes by TLC densitometry with fluorescence detection. J. Pharm. Biomed. Anal. 33, 85-93 (2003)... 

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. 

Specificity Study. Compounds used for the specificity study were monensin, lasalocid, narasin, salinomycin, lincomycin, chlortetracycline and roxarsone. The compounds were first dissolved in acetone or acetonetwater (8 2 v/v) at a concentration of 1 mg/mL. Further dilutions were prepared in PBS 7.6. Concentrations ranging from 0 to 10 xg/mL were tested. The assays were performed by using 50 xL of the varying concentrations of the compounds in place of the maduramicin in the standard curve procedure as described above. The concentration of the competing compounds required to exhibit 50% displacement of the antibody binding to the solid support was calculated. 

Ribosomes of Plasmodium knowlesi were isolated and characterized recently by Sherman et al. 21 These ribosomes sedimented in the 80S range and could be dissociated into 60S and 40S subparticles. The ribosomal RNA had a low % G+C of 37% and had sizes of 24.2S and 16.6S.22 The ribosomes demonstrated high activity in poly(IJ)-directed synthesis of polyphenylalanine and were strongly inhibited by 10 4m of nucleocidin, chlortetracycline, ethidium, puromycin, cycloheximide or berenil.23 Similar studies have been also carried out on Plasmodium lophurae, and similar profile of drug sensitivities were demonstrated.24 Most of the well-known antimalarial drugs tested showed no significant inhibitory activity in this in vitro assay. 

DasGupta, S., Mills, C.L., and Fraser, L.R. (1993). Ca -related changes in the capacitation state of human spermatozoa assessed by a chlortetracycline fluorescence assay. J. Reprod. Fertil. 99 135-143. 

Lee, M.A., Trucco, G.S., Bechtol, K.B., Wummer, N., Kopf, G.S., Blasco, L and Storey, B.T. (1987). Capacitation and acrosome reactions in human spermatozoa monitored by a chlortetracycline fluorescence assay. Fert. Steril. 48 649-658. 

Ward, C.R. and Storey, B.T. (1984). Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay. Dev. Biol. 704 287-296. 

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). 

Chlortetracycline peak, tube 52 AT = 1.11 Oxytetracycline peak, tube 30 Af = 0.435 Phases analyzed by assaying against Bacillus megatherium Ref. 

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. 

Suspend a 20 g sample of the fortified feed in 100 ml of acid-acetone mixture, shake for one hour and separate in a centrifuge. Neutralise a 25-ml portion of the supernatant solution with N sodium hydroxide using methyl orange as indicator and make up to 100 ml in a graduated flask with phosphate buffer solution, pH 4-5. Measure 25 ml of this solution into a 50-ml beaker, add 2-5 ml of N sodium hydroxide and boil gently for fifteen minutes. Cool and add 2-5 ml of N hydrochloric acid. Transfer quantitatively to a 50-ml graduated flask and add a known amount of a solution of the chlortetracycline standard equal to the amount estimated to be present in the test sample. Make up to volume with phosphate buffer solution and from this prepare further final dilutions as required in the assay. 

---