CHARM II test

Results showed a total of 2.8% of the samples (n 2972) to be inhibitor positive by the Delvotest SP test further examination identified 1.7% as -lactam antibiotics, and 1.1 % as sulfonamides and dapsone. The percentage of chloramphenicol suspicious samples determined by the Charm II test was amazingly high however, tests for confirmation were not available and contamination of the samples by residues of the chloramphenicol-based preservative azidiol could not be excluded with certainty. Low concentrations of streptomycins were also detected in 5.7% of the samples (n 1221), but the MRL was not exceeded. Macrolide and tetracycline residues were not found in significant levels. Model trials with commercially applied yoghurt cultures confirmed how important the compliance to MRLs can be to dairy industry compared to antibiotic-free milk, a pH of 5.0 was reached with a delay of 15 min in the case of contamination with cloxacillin 30 min in the case of penicillin, spiramycin, and tylosin and 45 min in the case of oxytetracycline contamination. 

More versatile than the growth-inhibition assays and potentially applicable to determining the presence of different antibiotic residues in different matrices are the microbial receptor CHARM I and II test assays (19, 20). The Charm I test, developed exclusively for -lactams in milk, constitutes the first rapid test recognized by The Association of Official Analytical Chemists (AOAC) with a test time of 15 min (19). The speed and sensitivity of this test permitted testing of milk tankers before they unloaded at the processing plant (21). In 1984-1985, the CHARM I test was further developed to test for antibiotics beyond -lactams to include tetracyclines, sulfonamides, aminoglycosides, chloramphenicol, novobiocin, and macrolides. The extended version has been referred to as CHARM II test. 

The CHARM II test is a general screening and identification test for members of different groups of antibacterial residues in milk in a rapid 15 min procedure (22-24). The test has been also applied to the analysis of other animal products, but a simple 30 min extraction with an aqueous buffer is usually required for tissue and eggs. 

The CHARM II test for tissues is relatively fast, easy to perform, and requires limited laboratory equipment. However, for antibacterials with established tolerance levels, it can serve only as a screening test because the results are not quantitative and therefore should be supported by additional quantitative chemical methods. The microbial receptor assay, with its broad-spectrum capability, can enhance any existing monitoring system as a first-line monitoring test or as a confirmation for any program using microbial inhibition tests. 

In 1989, an experimental study designed by the International Dairy Federation in 53 laboratories of 22 different countries to achieve deeper insight into state of proficiency of routinely applied tests showed that the most frequently used microbial inhibitor screening tests were the disc assays with Bacillus stearo-tliermopliilus, Delvotest-P, Brilliant black reduction test, acidification test, CHARM II test, and the Penzyme test (32), Currently available microbial inhibitor tests for screening of residual antibacterials in milk and milk products are presented in Table 27.1. 

Apart from those methods, a variety of test assays primarily derived from modifications of tire existing fast milk testing procedures have been also developed and widely used for testing animal tissues, eggs, and honey for antibacterial residues. Sound examples are the CHARM II test and the CHARM farm test (CFT), which have been successfully used since 1987 in many animal-derived products and mattices such as swine and cattle tissues, eggs, and honey. 

Antibacterial German three-plate test EEC four-plate test CHARM II test New Dutch kidney test STOP CAST FAST CHARM farm test ATP test... 

A radioimmunoassay test for TCs Charm II was compared with HPLC determination of OTC residues in milk samples. There was a significant difference between test methods with respect to the presence of OTC at the FDA safe concentrations. Using the HPLC test results as the standard with which Charm II test results were compared, 47 false presumptive-violative test results were obtained. These samples contained less than 30 yug/L of OTC, as evaluated by HPLC (40). 

The HPLC-receptorgram assay combined the advantages of HPLC separation with the multiresidue detection of the Charm II tests. The procedure was tested for identification and quantitation of the most common veterinary drugs at regulatory levels or lower. It was validated for 40 individual drugs from seven antibiotic families 10 /3-lactams, 13 sulphonamides, 8 tetracyclines, 4 macrolides, 3 amphenicols, and other miscellaneous antimicrobials. This procedure combined a simple aqueous extraction and SPE with HPLC fractionation of individual drugs. Final identification and quantitation was achieved with the Charm II test. A drug contaminant could be identified in less then 3 hours (50). 

WA Moats, KL Anderson, JE Rushing, DP Wesen. Comparison of a radioimmunoassay (Charm-II) test with high-performance liquid chromatography for detection of oxytetracycline residues in milk samples from lactating cattle. Amer J Vet Res 56 795-800, 1995. 

Carlsson, A., Bjorck, L. 1992. Liquid chromatography verification of tetracycline residues in milk and influence of milk fat lipolysis on the detection of antibiotic residues by microbial assays and the Charm II test. J. Food Prot. 55, 374-378. 

The range of kits currently available for the Charm II system and their applications are given in Table 5.5. The test manufacturer claims that the Charm II tests are capable of detecting compounds belonging to the antimicrobial class at or below their defined MRLs (or USFDA tolerances, as indicated) within the relevant matrix, including, milk, urine, serum, animal tissue, honey, and other substances at concentrations of interest to regulatory agencies. 

TABLE 5.5 Range of Charm II Test Kits and Their Applications Currently Available for Antibiotic Residue Screening from Charm Sciences Inc. 

Charm II Tests for Antibiotics Test Kits Available (number of individual compounds for which detection concentration are quoted) Application(s) Sample Preparation Time (minutes) Assay Time (minutes)... 

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. 

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. 

The Charm II 6600/7600 system is the only commercial immunoassay test available for the detection of several macrolides in different matrices (see Table 4). The LOD of erythromycin in milk is 40 pg L 1 and 100 pg L 1 in tissues. 

This method combines the advantages of liquid chromatography with the selective and sensitive receptor assay Charm H-Q test. Milk sample previously precipitated (with Mcllvaine buffer) and preconcentrated on a C8 cartridge was fractionated using an LC system. The fractions were collected according to the retention times and peak widths, and they were assayed directly with Charm II-Q test with small modifications. This method was suited for the AMO, AMP, PenG, CLO, CEF, and CEP residues in the milk samples and after small modifications for CFD, TIC, and NAF. A simple purification scheme gave recoveries from 50% for AMO to 80-90% for other /3-lactams (95). 

Tetracyclines Charm II RIA test Hog lagoon Surface water Groundwater 1.00 l-20d 348... 

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