Oxytetracycline, assay

The USP 28 [1], European Pharmacopoeia [2], BP 2003 [4], and Indian Pharmacopoeia 1996 [7] use the liquid chromatography method as described above in Section 2.2.2 for the assay of oxytetracycline. The International Pharmacopoeia 1988 [5], Pharmacopoeia of the People s Republic of China [6], Indian Pharmacopoeia [7], and Indonesian Pharmacopoeia [8] use a microbiological method. This methodology will be described in more detail in a later section. 

For assaying oxytetracycline content in injections, tablets, capsules, ointments, and oral suspensions, the United States Pharmacopoeia 28 [1] uses a liquid chromatography method described in the assay under oxytetracycline. For oxytetracycline and Nystatin capsules and for oral suspension, United States Pharmacopoeia 28 [1] uses a microbiological method listed under antibiotics-microbial assays <81>. 

Fernandez-Gonzales et al. [16] described a method for determination of OTC in medicated premixes and feeds by second-derivative synchronous spectrofluorome-try. The assay based on the reaction of oxytetracycline with divalent metal ion (Ca2+) at pH 6-10 to form a yellow complex that can be analyzed by synchronous spectrofluorometry (AX = 115 nm). Rao et al. [17] described a spectrophotometric method for the determination of OTC in pharmaceutical formulations based on the color reaction with uranium, which was detected at 413 nm. 

United States Pharmacopoeia 28 [1] describes a microbiological method under antibiotics-microbial assays for the analysis of OTC and nystatin capsules, OTC and nystatin for oral suspension, OTC HC1 and hydrocortisone ointment, and OTC HC1 and polymyxin B sulfate ointment. The methods are relative rather than absolute, which are based on the determination of the level of oxytetracycline by a microbiological response to a series of standard oxytetracycline concentrations by a... 

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. 

Mutagenesis Tetracycline and oxytetracycline have produced positive mutagenic results in mammalian cell assays in vitro. 

In the period 1992-1994, the drugs and groups of drugs screened by the National Residue Program included antibiotics, sulfonamides, ivermectin, halofuginone, levamisole, and morantel tartrate. The microbial assays applied by FSIS for monitoring antibiotics could detect chiortetracyciine, oxytetracycline, tetracy-... 

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. 

W. Naidong, S. Geelen, E. Roets, and J. Hoogmartens, Assay and purity of oxytetracycline and doxcycline by thin-layer chromatogra-phy-a comparison with liquid chromatography, J. Pharm. Biomed. Anal.,8 m (1990). 

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)... 

Several workers (28-29) reported an increase in the methylene blue reduction time when 0.05-0.5 lU Penicillin were present per milliliter of milk. Similarly, Manokidis et al. (30) noted that Penicillin and Oxytetracycline were responsible for a false positive phosphatase test in pasteurized or partially pasteurized milk while Streptomycin, Erythromycin and Neomycin inhibited the phosphatase test to some extent in partially pasteurized milk, but not in raw milk. Although raw milk containing antibiotic residues was never mistakenly identified as pasteurized, these authors suggested that as a "precaution" antibiotic assays be run in conjunction with the phosphatase test. 

Stehly GR, Gingerich WH, Kiessling CR, Cutting JH, A bridging study for oxytetracycline in the edible fillet of rainbow trout Analysis by a liquid chromatographic method and the official microbial inhibition assay, J. AO AC Int. 1999 82(4) 866-870. 

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. 

A microbiological method (see p. 813) is prescribed by the B,P. although the U,S,P. XV (for oxytetracycline itself) relied on a version of the colorimetric method with ferric chloride (see below). The microbiological determination of potency in the B,P. is augmented by a spectrophoto-metric assay ... 

Pour about 50 ml of the solution above on the dry column and allow it to percolate. Adjust the inactivated extract to pH 4-5 with N hydrochloric acid and transfer 20 ml to a 50-ml graduated flask. Add a known amount of a solution of the oxytetracycline standard equal to the amount estimated to be present in 4 g of the test sample and make up to volume with phosphate buffer solution. From this prepare further final dilutions as required in the assay. 

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