Immunochemical assays, metabolites

The amount of total residues is generally determined by study with radiolabeled drugs and is expressed as the parent drug equivalent in milligrams per kilogram of the food. Bound metabolites can be measured as the difference between the total and extractable residue. Microbiological assays measure the parent molecule and its bioactive metabolites immunochemical assays measure the parent molecule and closely chemically related metabolites. 

Dry reagent chemistries have been described for the analysis of a variety of blood constituents. These include metabolites, enzymes, electrolytes, hormones, and therapeutic drugs. A partial list is presented in Table 3. With the exception of electrolytes, nearly all analyses depend on enzyme-mediated chemistries and that includes immunochemical assays. A brief survey of element structures will illustrate how physical functions and chemical reactions used in conventional multistep procedures are integrated in the construction of dry reagent test devices. These examples will illustrate how reactions in dry reagent elements can be compartmentalized and how end produas are shunted to other compartments for further reaction. In its final form, each element provides a complete analytical procedure. 

Most biological samples contain complex mixtures of arachidonate metabolites, usually present at exceedingly low concentrations. This circumstance calls for some caution on the researcher s part. For example it is not advisable to measure eicosanoid levels in unfractionated samples, even when using specific immunochemical assays, because the chemical structures of the eicosanoids present... 

Not all arachidonate metabolites have favorable absorbance properties, however. When this is the case, two additional techniques of high sensitivity may be used. The eicosanoids eluting from the HPLC column may be measured with the aid of an immunochemical assay (e.g., radioimmunoassay or enzyme-linked immunoassay).Alternatively, these compounds may be chemically coupled to a chromophore before HPLC purification, and measured by ultraviolet or by fluorescence spectroscopy. Direct analysis of the HPLC eluant by mass spectrometry (HPLC/MS) is also feasible, but its use has been hampered so far by high costs... 

A PFIA, commercialized by Abbot, is one of the most common immunochemical methods used in clinical laboratories to analyze salicylic acid (SA) in serum. The assay also recognizes gentisic acid (GA) but is insensitive to salicylamide, salicyluric acid, and conjugates of S A and of its metabolites [187]. 

It has been recently reported (109) that use of both Penase and lactamase II hydrolysis and screening assays prior to chromatographic analysis can tentatively classify -lactams into three subgroups the first group includes a ceftiofur metabolite represented by desfuroyl-ceftiofur-cysteine the second, cephapirin and the third, penicillin G, ampicillin, amoxicillin, and cloxacillin. In this approach, portions of aqueous extracts of tissues are treated separately with Penase and lactamase II, and results are compared with those of untreated samples and positive controls. Bioactive ceftiofur metabolites are present, provided that the extracts retain inhibitory activity after Penase treatment but lose activity after lactamase II treatment and are positive in response to the immunochemical Lac-Tek-Cef test but negative to the Lac-Tek-Bl test (113). This approach can eliminate a large number of negative samples and, therefore, increases the efficiency of the assay. 

Mycotoxins, toxic metabolites of some fungi, can be assayed by immunochemical techniques to determine concentration in animal feed and foodstuffs. Some of the analytes assayed in kits and the detection limits are listed in Table 4 (45). These assays are especially advantageous for routine analysis of large samples of foodstuffs (45,46). 

Acetaminophen (paracetamol) is a commonly used analgesic which is hepatotoxic at high doses in humans and in laboratory animals. Toxicity is believed to be mediated by the reactive metabolite N-acetyl-p-benzoquinone imine which binds to protein thiols as 3-(cystein-S-yl)acetaminophen adducts. Ultrasensitive immimoassays for 3-(with parallel elevations in serum adducts and serum levels of the liver-specific transaminase ALT. This suggested that the serum adducts were of hepatic origin and could be monitored as a biomarker of acetaminophen toxicity. Analysis of serum samples from acetaminophen overdose patients demonstrated a positive correlation between immunochemically detectable serum adducts and hepatotoxicity. 

Drugs of abuse, such as cocaine, cannabinoids, and amphetamines, were detected in hair by immunoassay followed by GC-MS for confirmation and quantitation [118]. Immunochemical screening was done with the Siemens EMIT 11 Plus assays for opiates, amphetamines and ecstasy, and Microgenics CEDIA assays for cocaine. All samples with positive results in the screening test for any drug and/or metabolite were analyzed by GC-MS for confirmation and quantitation. The cutoff value for cocaine was 0.20 ng mg . ... 

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