An enzyme with high specific affinity for organophosphate and N-methylcarbamate insecticides has been incorporated into a new test for detection of these insecticides at the level of parts per billion (ppb) (commercially available as the Charm Pesticide Test). To measure the extent of insecticide inhibition of the enzyme, a specific bioluminescent substrate is used. The signal is counterproportional to the amount of insecticides. Random sampling of four baby food brands and testing for the cumulative levels of organophosphate and N-methylcarbamate insecticides found carbaryl to be the most common residue. Out of the 155 samples tested there were 132 negative samples (85.2%) and 23 suspected positive samples (14.2%). The suspected positive samples were further analyzed by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). Carbaryl was confirmed in 18 of the samples. One of the samples contained an active metabolite of tetrachlorvinphos and in 3 of the positive samples an insecticide could not be identified by GC/MS. One positive sample was not processed for confirmation due to high fat content.

A rapid determinative method was developed for identification and quantitation of common β-lactam drugs (e.g., amoxicillin, ampicillin, cephapirin, penicillin G, ceftiofur, and cloxacillin) in milk. This method combines the advantages of liquid chromatography (LC) with the selectivity and sensitivity of a microbial receptor assay. This LC-receptorgram procedure identifies and quantitates β-lactams at 10 ppb or lower within 3 h. A simple purification scheme, using solid-phase extraction, gave recoveries ranging from 50% for amoxicillin to 80-90% for other β-lactams. Individual drugs were separated by LC using a reversed-phase C8 column and an isocratic buffer system containing methanol (35%) in phosphate buffer (50 mM, pH 6.0). The Charm II quantitative procedure was used to detect (5-lactams in the LC fractions. In a blind study of raw milk fortified with β-lactams, the coefficients of variance for quantitation of 6 β-lactams at the safe levels ranged from 4.1 to 13%. The confidence level for selectivity was greater than 99%, and for detection of positives at safe levels, it was 95% or greater. Analysis of 6 β-lactams in incurred samples showed that all 6 drugs are depleted to below 10 ppb in less than 48 h. Incurred samples with cephapirin and ceftiofur contained active metabolites, in addition to parent drug, that were detected by the LC-receptorgram. In market milk samples that screened positive in routine testing programs, penicillin G, cephapirin, and ceftiofur were identified by the LC-receptorgram as the most common contaminants. In several samples containing cephapirin or ceftiofur, an active metabolite was detected in addition to the parent drug. Only the parent drug was detected for other β-lactams.

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A determinative method was developed for confirmation, identification, and quantitation of 12 sulfonamide residues and p-aminobenzoic acid (PABA) in milk. This method, termed "HPLC receptorgram," uses liquid chromatography in conjunction with the microbial receptor assay (MRA) to monitor sulfonamides. The MRA for sulfonamides (commercialized as the Charm II test) uses a microcrobial receptor and [3H]sulfamethazine tracer. Sulfonamides in milk bind to the receptor and inhibit the binding of the tracer to the receptor. Milk samples spiked at 10 ppb for each sulfonamide result in at least a 50% decrease in binding on the HPLC receptorgram, with the exception of sulfanilamide and sulfacetamide, which give approximately a 25% decrease. Additionally, milk samples spiked at the minimum detection level of the MRA assay can be confirmed, identified, and quantitated with coefficients of variation (%) of 7.6-24. Analysis of market milk samples found positive by the MRA showed 9 samples containing sulfamethazine, 4 samples containing sulfadimethoxine, 2 samples containing sulfadoxine, 2 samples containing sulfadiazine or sulfathiazole, and 1 sample each containing sulfisoxazole or sulfapyridine. Three samples contained multiple sulfonamide residues. Ten sulfonamides are determined with an isocratic buffer elution system containing 22% acetonitrile mobile phase. p-Aminobenzoic acid (PABA), an interfering sulfonamide analog, sulfanilamide, and sulfacetamide are eluted by using a 10% acetonitrile mobile phase

The Charm Safe-Level β-Lactam Test was evaluated by a U.S. Food and Drug Administration (FDA) test protocol administered by the AOAC–Research Institute. The sensitivity and selectivity of the test were evaluated with >800 negative raw commingled and drug-fortified milk samples by the manufacturer and an independent laboratory. Probit analysis by the independent laboratory determined the following 90% positive levels with 95% confidence: amoxicillin, 5.6 ppb; ampicillin, 8.5 ppb; cephapirin, 13.7 ppb; ceftiofur, 46.2 ppb; and penicillin G, 3.6 ppb. These values were within a range of ±20% of the manufacturer's data. Selection of negative samples met confidence specifications. Ruggedness parameters were studied and defined, and the stability of frozen milk was verified. There were no interferences from somatic cells (1 000 000 somatic cell count/mL) or bacteria (300 000 colony-forming units/mL), or from 27 other non-β-lactam animal drugs. Test performance with raw milk samples containing incurred penicillin, ampicillin, and amoxicillin was consistent with the dose responses determined with fortified milk samples. Incurred cephalosporin in raw milk samples was detected at lower levels than was cephalosporin in fortified milk samples, presumably because of the presence of metabolite, as verified by other test methods. Quality control data support consistency in manufacture between batches and the stability of refrigerated test reagents for up to 1 year. Successful fulfillment of these criteria led to FDA certification of the test when used with a reader in U.S. milk testing programs.