Tetracyclines , detection

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. 

Another subclass of proteases attacks internal peptide bonds and Hberates large peptide fragments. Bromelain, a plant protease derived from the stem of the pineapple plant, can even produce detectable semm proteolysis after oral adrninistration (180). Oral therapy with bromelain significantly reduces bmising that stems from obstetrical manipulations (181). Bromelain—pancreatin combinations have been more effective in digestive insufficiency compared to either pancreatin or placebo (182,183). Bromelain may also enhance the activity of antibiotics, especially tetracycline, when adrninistered concurrently (184). 

For detection residue amounts of tetracyclines in dairy products widely used methods FIPLC, immunoaffinity chromatography, kinetic spectrophotometry, which are expensive and complicated. 

Tetracycline (h/ f 35-40) produced blue and doxycycline (h/fj 15-20), chlorotetracycline (h/ f 25-30) and oxytetracycline (h/fj 40-45) produced violet chromatogram zones on a yellow background. The detection limits for all 4 compounds were 2 ng substance per chromatogram zone (X = 550 nm). 

In a recent study, Walters et al. [141] described the occurrence and loss of several PhC from biosolid-soil mixtures exposed at ambient outdoor conditions for 3 years. Some compounds showed no detectable loss over the monitoring period, including diphenhydramine, fluoxetine, thiabendazole and triclosan, while half-life estimates ranging from 182 to 3,466 days were determined for others such as azithromycin, carbamazepine, ciprofloxacin, doxycycline, tetracycline, 4-epitetracycline, gemfibrozil, norfloxacin and triclosan. These findings highlight the potential use of T. versicolor to reduce the impact of biosolids once released to the environment, which could reduce the concentrations of PhC in much shorter periods of treatment. 

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. 

On the other hand, the analgesic phenylbutazone, the antidepressant paroxetine, tetracycline antibiotics doxycycline and chlorotetracycline, the antibiotics tilmico-sin, danofloxacin, enoxacin, nifuroxazide, the (3-blockers betaxolol, carazolol, pindolol, the (3-agonist clenbuterol, barbiturates, the antihypertensive lisinopril, and the drug to treat cancer tamoxifen were never detected. 

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. 

Macrolide antibiotics (clarithromycin, dehydroerythromycin, etc.) and sulfonamides (sulfamethoxazole, sulfadimethoxine, sulfamethazine, and sulfathi-azole) are the most prevalent antibiotics found in the environment with levels around a few micrograms per liter, whereas fluoroquinolones, tetracyclines, and penicillins have been detected in fewer cases and usually at low concentrations (nanograms per liter) [3,20,23,72]. This result is not surprising, since penicillins are easily hydrolyzed and tetracyclines readily precipitate with cations such as calcium and are accumulated in sewage sludge or sediments. Several reviews have reported the environmental occurrence of different antibiotics in aquatic and soil compartments. Some of these data are detailed in Table 1. 

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... 

As occurred with the other antibiotics, commercial immunoassay formats, also available as kits for tetracyclines and penicillins such as the Parallux, the LacTek, or the Charm II, have also been placed on the market for the analysis of sulfonamides (see Table 4). Thus, the Parallux detects sulfamethazine and sulfadimethoxine in raw milk with a LOD of 10 pg L1. The Charm II detects almost all sulfonamides in honey and milk with a LOD in the range from 1 to 10 pg L, whereas LacTek is able to detect sulfamethazine. Moreover, the 5101SULlp and 5101SUDAlp tests reach LOD values for sulfamethazine and sulfadiazine of around 0.2 pg L 1 and they have been applied to the analysis of urine, milk, and plasma. These tests have proved to be efficient as a point of care for on-site applications on farms. Moreover, commercially available antibodies can be found from several sources such as Silver Lake Research, US Biological, Cortex Biochem. Inc., Accurate Chemical Scientific, Fitzgerald Industries International Inc., and Biotrend Chemikalien GmbH. 

We have found only one attempt to use immunoassays to detect sulfonamides in environmental samples. As in the case of penicillins and tetracyclines and also for fluoroquinolones (see below), Campagnolo et al. [84] measured sulfonamides in water samples proximal to a farm in Iowa using a commercial Charm II RIA test, accomplishing a LOD of 5 pg L 1 for sulfamethazine. 

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