Metabolites plasma assays

In addition to the pharmacokinetics of the drug, the first study in man can provide important information about its metabolites. If assay methodology has been developed, metabolites in plasma can be detected and the AUCs and half-lives determined. Further information can be obtained from assaying urine for drug and, if possible, metabolites. Renal clearance can be calculated over time intervals and the ratio of... 

Doyle et al [9] published an article describing two fully automated assays, one for zaprinost and the other for pantoprazole and its sulfone metabolite. Both assays were developed to support pharmacokinetics studies. Plasma or serum (20-200 pL) was placed directly into an auto-sampler, and all subsequent manipulations were performed mechanically [9],... 

As a compound proceeds into first-in-human clinical trials, the assay method reaches an apex in terms of performance. These methods require maximal sensitivity to be able to support dose escalation studies. The selectivity of the method is well established versus matrix components, concomitant medications, and metabolites. The assay is revalidated in the human matrices (plasma, serum, and urine) and will again meet well-established guidelines for inter- and intraday precision and accuracy. If significant changes are made to the method, a comparison of methods study will be conducted to understand the relative accuracy of the methods. After this benchmark, assay requirements, especially limit of quantitation, are usually less demanding and use of the assay becomes more routine, as it is applied again and again to additional clinical studies for pharmacokinetic support. 

In addition to the dosed compound, often one can look for common metabolites when assaying plasma samples from a PK study this step is often referred to as metabolite profiling. In our laboratory we have used the QTrap MS/MS system to provide both quantitative analysis and metabolite profiling when assaying plasma samples. The hardware and software tools that are part of the QTrap MS/MS system are well suited to this purpose [110-114], We have found it useful to use three rules for deciding when to report a possible metabolite. The metabolite reporting rules are as follows ... 

Lau, G.S.N. Critchley, J.A.J.H. The estimation of paracetamol and its major metabolites in both plasma and urine by a single high-performance liquid chromatography assay. J.Pharm.Biomed.Anal., 1994, 12, 1563-1572 [extracted metabolites plasma urine blood LOD 1 ng]... 

Cyr, T.D. Matsui, F. Sears, R.W. Curran, N.M. Lovering, E.G. Liquid chromatographic methods for assay of carbamazepine, 10,11-dihydrocarbamazepine, and related compounds in carbamazepine drug substance and tablets. J.Assoc.OffAnal.Chem., 1987, 70, 836-840 Hartley, R. Lucock, M. Forsythe, W.I. Smithells, R.W. Solid-phase extraction of carbamazepine and two megor metabolites from plasma for analysis by HPLC. J.Liq.Chromatogr., 1987, 10, 2393—2409 [extracted metabolites plasma SPE LOD 50 ng/mL nitrazepam (IS)]... 

Freed, C. R., and Asmus, P. A., 1979, Brain tissue and plasma assay of L-dopa and a-methyldopa metabolites by high-performance liquid chromatography with electrochemical detection,/. Neurochem. 32 163-168. 

Agarwal et al. 1978), the quantification of these specific enzymes may indicate that exposure to endosulfan has occurred. Blood tests, such as decay curves for aminopyrine in plasma, which are semiquantitative indices of liver enzyme induction, have been used successfully in the past to demonstrate enzyme induction in pesticide-exposed workers. Because numerous chemicals found at hazardous waste sites also induce these hepatic enzymes, these measurements are not specific for endosulfan exposure. However, measurements of enzyme activity, together with the detection of the parent compound or its metabolites in tissue or excreta, can be useful indicators of exposure. All of these potential biomarkers require further verification in epidemiological studies. Further studies with focus on the development of methods to separate and measure the estrogenicity of endosulfan in in vitro assays would be valuable since these assays are more sensitive and discriminative than other conventional biomarkers. Preliminary results have been presented by Sonnenschein et al. (1995). 

Some hydroxy metabolites of coplanar PCBs, such as 4-OH and 3,3 4,5 -tet-rachlorobiphenyl, act as antagonists of thyroxin (Chapter 6, Section 6.2.4). They have high affinity for the thyroxin-binding site on transthyretin (TTR) in plasma. Toxic effects include vitamin A deficiency. Biomarker assays for this toxic mechanism include percentage of thyroxin-binding sites to which rodenticide is bound, plasma levels of thyroxin, and plasma levels of vitamin A. 

As part of new drug discovery, the trend toward screening for metabolites in plasma samples being assayed for the dosed compound (NCE) is increasing. This effort is sometimes called metabolite profiling116 and it is important for two reasons (1) for compounds with low bioavailability due to extensive metabolism, metabolites may help medicinal chemists learn to modify the NCE to block... 

Our laboratory used the QTRAP MS/MS system for both quantitative analysis and metabolite profiling when assaying plasma samples. We found it useful to use three rules for deciding when to report a possible metabolite ... 

Additional assays used in early pharmaceutical property profiles usually include plasma protein binding, individual cytochrome P450 assays, stability in the presence of serum, production of metabolites likely to be involved in covalent binding to biomolecules, and interaction with efflux pumps ... 

GG is used extensively for analysis of antidepressants (Orsulak et al, 1989), but HPLC assays and enzyme immunoassays have become more popular in recent years. However, GC has advantages such as economy and ready availability. LCD and NPD generally are the detectors of choice (Coutts and Baker, 1982). NPD is relatively efficient for the analysis of tricyclic antidepressants (TCAs) as derivatization is not necessary, although the secondary, demethylated amines are sometimes derivatized to improve resolution and peak shape (Coutts and Baker, 1982). Acetylation, under aqueous or anhydrous conditions, followed by GC-NPD, has been used extensively for analysis of TCAs and the tetracyclic antidepressant maprotiline in plasma samples (Drebit et al., 1988). O Table 1-1 summarizes GC assays for some commonly prescribed antidepressants and their metabolites. 

B.4.2.2 Nonbenzodlazeptnes GG-NPD was used by Kivisto et al. (1999) to quantitate the nonbenzodiazepine anxiolytic buspirone and its major metabolite, l-(2-pyrimidinyl)-piperazine, using separate extraction methods and separate assays. The hmit of quantification in plasma for both compounds was 0.2 ng/mL, which makes this assay useful for pharmacokinetic studies of this compound (Kivisto et al., 1999). A rapid, simple method for analysis of buspirone in rat brain requiring a single extraction step followed by GC-NPD has also been described (Lai. et al., 1997). 

The use of HPLC to analyze biogenic amines and their acid metabolites is well documented. HPLC assays for classical biogenic amines such as norepinephrine (NE), epinephrine (E), dopamine (DA), and 5-hydroxytryptamine (5-HT, serotonin) and their acid metabolites are based on several physicochemical properties that include a catechol moiety (aryl 1,2-dihydroxy), basicity, easily oxidized nature, and/or native fluorescence characteristics (Anderson, 1985). Based on these characteristics, various types of detector systems can be employed to assay low concentrations of these analytes in various matrices such as plasma, urine, cerebrospinal fluid (CSE), tissue, and dialysate. 

Recently, three papers have reported the determination of risperidone and its active metabolite 9-hydroxyrisperidone using LLE and SPE technologies. The analytical columns used to separate these compounds were C4 or C18 bonded phases of 3 pm or 5 pm particle sizes with UV/VIS detection. Mobile phases consisted of phosphate bufiers (pH 3-4) in acetonitrile. The sample volumes used ranged from 200 pi to 1 ml, with extraction recoveries averaging 90%. The limits of quantitation ranged from 0.5 to 10 ng/ml in human plasma (Nagasaki et al., 1999 Avenso et al., 2000 Titier et al., 2002). A study by Titier showed the simultaneous determination of clozapine, olanzapine, haloperidol, risperidone, and its active metabolites by RP-HPLC in human plasma. The assay involved LLE with a hexane/isoamyl alcohol mixture... 

Maris FA, Dingier E, Niehues S. 1999. High-performance liquid chromatographic assay with fluorescence detection for the routine monitoring of the antidepressant mirtazapine and its demethyl metabolite in human plasma. J Chromatogr B Biomed Sci Appl 721(2) 309-316. 

Murphey LJ, Hachey DL, Vaughan DE, Brown NJ, Morrow JD. 2001. Quantification of BKl-5, the stable bradykinin plasma metabolite in humans, by a highly accurate liquid chromatographic tandem mass spectrometric assay. Anal Biochem 292 87. 

Besides the poor specificity of many of the assays used to determine plasma drug concentrations, another problem which has arisen from these studies has been the length of the "wash-out" period necessary before the patient is given the neuroleptic under investigation. As a result of the prolonged duration of blockade of dopamine receptors in the brain by conventional neuroleptics and their metabolites, it is necessary to allow a wash-out period of several weeks before the patients are subject to a pharmacokinetic study. This raises serious ethical questions. Perhaps with the advent of new imaging techniques it may be possible in the near future actually to determine the rate of disappearance of neuroleptics from the brain of the patient. This may enable the relationship between plasma concentration and clinical response to be accurately determined. 

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