Phase-II-metabolites

The complementary nature of APCI and ESI, APCI for less polar compounds (Phase I metabolites) and ESI for more polar compounds (Phase II metabolites). 

Sidelmann UG, U Braumann, M Hofmann, M Spraul, JC Lindon, JK Nicholson, SH Hansen (1997) Directly coupled 800 MHz HPLC-NMR spectroscopy of urine and its application to the identification of the major phase II metabolites of tolfenamic acid. Anal Chem 69 607-612. 

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. 

Many drugs have functional groups that can be metabolized by the addition of water. The major functional groups involved are esters, amides, and epoxides. Several phase II metabolites such as sulfates and glucuronides, which will be discussed in Chapter 7, can also be hydrolyzed back to the parent drug. 

Phase II drug metabolites are often the final form in which a xenobiotic is solubilized for release from the body, and many display significant biological activity. Synthesis of purified phase II metabolites, therefore, is a requirement of the drug development process. 

Keski-Hynnilae, H., Kurkela, M., Elovaara, E., Antonio, L., Magdalou, J., Luukkanen, L., Taskinen, J., and Kostiainen, R. (2002). Comparison of electrospray, atmospheric pressure chemical ionization, and atmospheric pressure photoionization in the identification of apomorphine, dobutamine, and entacapone phase II metabolites in biological samples. Anal. Chem. 74, 3449-3457. 

EtG and EtS are minor phase II metabolites of alcohol and their presence in a biological sample unequivocally attests exposure to this substance. They can be measured in serum [7] and urine [9,11-13] even after ethanol has been eliminated. Recently, the determination of EtG in hair has been proposed as a sensitive and specific marker of chronic ethanol abuse [8,10], The determination of EtG in postmortem blood enables the discrimination between ethanol postmortem formation and alcohol intake before death [17],... 

Hupka et al. [29] developed a method for the determination of morphine and its phase II metabolites, morphine-3-beta-D-glucuronide and morphine-6-beta-D-glucuronide in the blood of heroin victims. The method is based on immunoaffinity SPE, RP-HPLC isocratic separation (mobile phase 90% lOmmol KH2PO4, 2mmol 1-heptanesulfonic acid, adjusted to pH 2.5 with H3PO4 and 10% acetonitrile flow rate 1.5 mL/min), and laser-induced native fluorescence detection. 

Favretto et al. [33] proposed a procedure for the analysis of BUP and nor BUP based on RP-HPLC conpled with ion trap MS nsing an ESI ion source. The use of the ion trap allowed to obtain intense prodnct ions nnder MS-MS conditions, thus achieving better selectivity of detection with respect to LC-ESI-MS-MS methods with triple qnadrupoles where effective fragmentation in the collision cell was fonnd difficnlt to obtain. However, this method does not inclnde phase II metabolites of BUP, which can be only indirectly determined after enzymatic hydrolysis. 

Murota K and Terao J. 2005. Quercetin appears in the lymph of unanesthetized rats as its phase II metabolites after administered into the stomach. FEBS Lett 579 5343-5346. 

Shirley, M. A., Wheelan, P., Howell, S. R., and Murphy, R. C. (1997). Oxidative metabolism of a rexinoid and rapid phase II metabolite identification by mass spectrometry. Drug Metab. Dispos. 25 1144-1149. 

Meyer, K., Fobker, M., Christians, U., Erren, M., Sewing, K. F., Assmann, G., and Benninghoven, A. (1996). Characterization of glucuronidated phase II metabolites of the immunosuppressant cyclosporine in urine of transplant patients using time-of-flight secondary-ion mass spectrometry. Drug Metab. Dispos. 24 1151-1154. 

Five UHPLC methods have already been described to improve speed, resolution, and sensitivity of HPLC assays for the quantification of tamoxifen phase I as well as phase II metabolites. These methods exclusively enabled, within run times of... 

Poon GK et al (1993) Analysis of phase I and phase II metabolites of tamoxifen in breast cancer patients. Drug Metab Dispos 21 1119-1124... 

The biotransformations are implemented as rules based on the presence and absence of substructures. The rules act on the original query molecule to produce a first generation of metabolites and the process is repeated on the generated metabolites to produce a second generation and so on. No further metabolites are generated from a Phase II metabolite because this is regarded as hydrophilic and excretable. The overall process can be terminated when a set number of metabolites have been produced. No indication of the relative importance of the metabolites appears to be given. 

Hydrolyses They are of minor significance. Here amidases and glycosidases are important enzymes. Intestinal bacteria with hydrolytic activity split mainly phase II metabolites in the large intestine, with the result that absorbable catabolites then enter the enterohepatic circulation. 

In some cases. Phase I metabolites may not be detected, owing to their instability or high chemical reactivity. The latter type are often electrophilic substances, called reactive intermediates, which frequently react non-enzymically as well as enzymically with conjugating nucleophiles to produce a Phase II metabolite. A common example of this type is the oxidative biotransformation of an aromatic ring and conjugation of the resulting arene oxide (epoxide) with the tripeptide glutathione. Detection of metabolites derived from this pathway often points to the formation of precursor reactive electrophilic Phase I metabolites, whose existence is nonetheless only inferred. 

The generation of Phase-I metabolites can be considered as a preparation for the Phase-II metabolism. The detection and identification of Phase-I metabolites is important, because possibly toxic metabolites are formed. Some metabolites are even more active than the dmg itself, either in the action the dmg was administered for or in toxic side effects. Administration of a prodrag with more favourable properties is sometimes performed. The prodrag is rapidly transformed in the actual active substance. Moreover, in quite a number of cases the analytical strategy is directed at identification of the Phase-I metabohtes, even after chemical or enzymatic deconjugation of the Phase-II metabolites. 

TOF and Q-TOF instraments are freqnently applied in metabolism studies. The identity of the epothilone B metabolites fonnd by precnrsor-ion analysis (Figure 10.5, Ch. 10.4.2) was confirmed using accnrate-mass determination on a LC-TOF-MS instmment [29]. Some other examples are the characterization of metabolites of moclobemide and remikiren [32], the identification of ketobemidone Phase-I and Phase-II metabolites [33], and the identification of in vitro metabolites of ethoxidine [34]. The nse of a five-channel multiplexed ESI interface (four chaimels for parallel LC-MS and one channel for lock-mass componnd infusion) on a Q-TOF instrument was recently described to speed np metabolite identification and to enhance the efficient nse of the costly instrument [35]. 

Neutral-loss and precursor-ion analysis modes are regularly apphed in combination with LC-MS for the detection and/or characterization of Phase-II metabolites [44, 71, 73-74]. Accurate-mass neutral-loss analysis on a Q-TOF instrament was apphed to screen for glutathione conjugates [74]. 

Xenobiotics thus are cleared from the body in a variety of forms unchanged, as Phase I metabolites, as Phase II metabolites, or products of sequential Phase I and Phase II transformations. Conjugates are the predominant excreted form for most foreign compounds (2). 

Thus, the role of MRPs at the BBB is only partially known. They play a major role in the elimination of amphipatic anions (many of them being phase II metabolites) from the endothelial compartment. This may also be their main physiological function. Based on the limited knowledge about their physiological substrates, one can only speculate about their role in disease processes at the level of the BBB. 

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