Marker metabolites

The in vitro approaches feature human liver microsome incubations that contain drug candidates at a range of concentrations that span the anticipated maximum steady state plasma levels. The microsomal incubations also contain a specific probe substrate where the concentration closely approximates that Km value for the reaction under investigation. Quantitative analysis of the specific marker metabolites and internal standards using MRM provides a simple assessment of the potential inhibitory effects drug candidates have on the metabolism of specific CYP probe substrates. 

Nineteen outbred male rats were dosed intraperitoneally once with 200 mg/kg benzo[a]pyrene in sunflower oil (Likhachev et al. 1993). Concentrations of benzo[a]pyrene-7,8-diol, a marker metabolite of bioactivation of benzo[a]pyrene. and 3-hydroxy-benzo[a]pyrene, a marker metabolite of deactivation, were measured daily in the urine and feces for 15 days. Levels of these metabolites were correlated with tumor latency. Another group of 10 rats was dosed intraperitoneally once with 15 mg/kg benzo[a]pyrene in sunflower oil and urine was collected for 3 days. Five rats were killed on day 3 and the other 5 were killed on day 8. Liver DNA concentrations of... 

The metabolism of albendazole has been well studied (/,5). The parent compound is short-lived with the major metabolites being generated by a combination of oxidative and hydrolytic processes (Figure 2). One of the major metabolites, albendazole 2-aminosulfone, has been designated as the marker metabolite for which an approved regulatory method has been established in liver, the target tissue. 

Plasma. Albendazole (ABZ), albendazole-sulfone (ABZ-S02), albendazole 2-aminosulfone (ABZ-NH2, marker metabolite) and albendazole-sulfoxide (ABZ-SO), were extracted from the plasma using a perchloric acid precipitation method followed by a solid phase extraction procedure. The residue levels were quantitated using a normal or reversed phase HPLC method with fluorescence detection. 

For tissue residue monitoring, the FDA uses the concept of the Rm value which is defined as the ratio of the concentration of the designated marker metabolite to that of the total residue. 

The substantial differences (from single dose ABZ) observed in the plasma and tissue residue profiles in this study, tend to indicate that the quantitative metabolic profile may be altered in the case of the sustained-release of albendazole. If the established Rm = 0.2 factor is applied to the marker metabolite concentration of 651 ppb observed at 0-day withdrawal in this study, a total residue of 3255 ppb (651x5) would be indicated. However, considering the facts that... 

Because nitrofuran metabolites are very small molecules, the marker metabolites are not highly specific. This is especially the case for SEM. However, the presence of tissue-bound metabolites is more specific, because this indicates the administration of nitrofurans. Therefore, analytical methods are described focusing on bound residues. ... 

An example of the low selectivity of SEM as a marker metabolite for nitrofurazone is the false non-compliant findings caused by the use of azodicarbonamide (ADC,... 

Nitrofurans are banned substances within the EU and in some other countries because of their mutagenic and geno-toxic characteristics. Nitrofuran metabolites are still found, primarily in aquaculture products originating from Southeast Asia, with SEM (the metabolite of nitrofurazone) having the highest incidence. Methods for detecting residues of nitrofurans aim for protein-bound metabolites that may persist in tissues for considerable periods after treatment. Methods are reported for the detection and identification of nitrofuran metabolites in many different food products. The main difficulty in nitrofuran metabolite analysis is the low selectivity of SEM as a marker metabolite of nitrofurazone. Several other possible sources of SEM have been identified and investigated, the most important of which is the use of... 

Bendall J, Semicarbazide is non-specific as a marker metabolite to reveal nitrofurazone abuse as it can form under Hof-mann conditions. Food Addit. Contam. 2009 26 47-56. 

So far, evidence for abnormal peripheral (Elliott 1992) or central (Horton 1992) monoamine function in depression is equivocal, and no consistent biochemical markers have emerged to provide a firm link between the two (Table 20.2). One widely cited finding is that subjects who have attempted violent suicide form a neurochemically distinct group because the concentration of the 5-HT metabolite, 5-HIAA, in their CSF is lower than normal, suggesting that a deficit in 5-HT release is associated with suicide... 

It is appropriate at this stage to evaluate procedures that will be used to assess the effectiveness of bioremediation, which have been discussed in Chapter 13. These may include (a) use of radiolabeled substrates (although these will not generally be permitted in field operations) and the application of C-labeled substrates, (b) evaluation of the occurrence of metabolites, and (c) evaluation of markers such as specific enzymes. 

The researeh on dehydroepiandrosterone (DHEA) is limited beeause of the laek of radiolabeled metabolites. Robinzon et al. [126] showed that, using pig liver mierosomes, the radiolabeled metabolites of DHEA can be prepared in stable, pure form for bioehemical smdies. They utilized pig liver microsomal (PLM) fractions to prepare pH]-labeled 7a-hydroxy-DHEA (7a-OH-DHEA), 7[3-hydroxy-DHEA (7P-OH-DHEA), and 7-oxo-DHEA substrates from 50 pM [1,2,6,7-3H]DHEA. The metabolites were separated by silica gel PLC plates using ethyl aeetate-hexane-gla-eial aeetic acid (18 8 , v/v) as the mobile phase, extracted with ethyl aeetate, and dried under a stream of nitrogen. The purity of markers was determined with the use of TLC and GC/MS. 

Typically, neurotoxic effects of drugs on monoamine neurons have been assessed from reductions in brain levels of monoamines and their metabolites, decreases in the maximal activity of synthetic enzymes activity, and decreases in the active uptake carrier. In the present study, the traditional markers described above have been used, including the measurement of the content of monoamines and their metabolites in brain at several different timepoints following drug administration. Since reports in the literature have documented that MDMA and MDA can inhibit the activity of tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin synthesis (Stone et al. 1986 Stone et al. 1987). it is unclear whether MDMA-induced reductions in the content of serotonin and its metabolite 5-hydroxyin-doleacetic acid (5-HlAA) may be due to suppressed neurotransmission in otherwise structurally intact serotonin neurons or may represent the eonsequenee of the destruction of serotonin neurons and terminals. 

However, there is no general requirement that enforcement methods need to monitor all metabolites of an active ingredient. The primary purpose of enforcement methods is to detect violations of good agricultural practice. For this purpose, residue levels found in samples from the market (so-called Market Basket Surveys) have to be compared with MRLs, which are derived from residue concentrations found in supervised trials. It is not necessary for this comparison to be based on the total pesticide residue. Most often the choice of a single compound (e.g., parent or primary metabolite) as a marker of the total pesticide residue is more feasible. Method development and the later method application are much easier in that case. Only for intake calculation purposes, e.g., when the daily intake of pesticide residues (calculated from the results... 

Several extraction techniques have also been described that use enzymatic or chemical reactions to improve extraction efficiency. A technique that has been used to increase the overall recovery of the marker residue is enzymatic hydrolysis to convert specific phase II metabolites (glucuronides or sulfates) back into the parent residue. Cooper etal used a glucuronidase to increase 10-fold the concentration of chloramphenicol residues in incurred tissue. As an example of a chemical reaction, Moghaddam et al. used Raney nickel to reduce thioether bonds between benomyl and polar cellular components, and as a result achieved a substantially improved recovery over conventional solvent extraction. In choosing to use either of these approaches, thorough characterization of the metabolism in the tissue sample must be available. 

Specificity is a measure of how selectively the analytical method measures the marker compound in the presence of other compounds. The descriptors used to establish specificity differ depending upon the guideline (see Table 3), but the purpose behind them is the same. In all cases, the method must be demonstrated to have no interference from several (at least five) confrol animals that represent variation in sex, age, and breed. Further, incurred residue samples or authentic metabolite standards must demonstrate no interference with the marker residue detection. The method must be tested with other approved dmgs for the target species to show that no interference exists if these compounds are also present. 

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