Metabohte analysis

Mercke P, Kappers IF, Verstappen FWA, Vorst O, Dicke M, Bouwmeester HJ (2004) Combined transcript and metabohte analysis reveals genes involved in spider mite induced volatile formation in cucumber plants. Plant Physiol 135 2012-2024... 

The half-life of 18F (109 min) is sufficiently long to carry out complex synthesis procedures, apply long PET imaging protocols, and carry out metabohte analysis. Furthermore, it is possible to produce the radiopharmaceutical in a laboratory and transport it to a distant site only equipped with an imaging device. These kinds of satellite PET centers have boomed all around the world and permitted the fast expansion of PET as an everyday clinical tool in certain pathologies (mainly in oncological diseases). 

Liquid Ghromatography/Mass Spectrometry. Increased use of Hquid chromatography/mass spectrometry (Ic/ms) for stmctural identification and trace analysis has become apparent. Thermospray Ic/ms has been used to identify by-products in phenyl isocyanate precolumn derivatization reactions (74). Five compounds resulting from the reaction of phenyUsocyanate and the reaction medium were identified two from a reaction between phenyl isocyanate and methanol, two from the reaction between phenyl isocyanate and water, and one from the polymerisation of phenyl isocyanate. There were also two reports of derivatisation to enhance either the response or stmctural information from thermospray Ic/ms for linoleic acid hpoxygenase metabohtes (75) and for cortisol (76). 

The development of methods of analysis of tria2ines and thek hydroxy metabohtes in humic soil samples with combined chromatographic and ms techniques has been described (78). A two-way approach was used for separating interfering humic substances and for performing stmctural elucidation of the herbicide traces. Humic samples were extracted by supercritical fluid extraction and analy2ed by both hplc/particle beam ms and a new ms/ms method. The new ms /ms unit was of the tandem sector field-time-of-flight/ms type. 

The typical procedure for analysis of cannabinoids from plasma, urine or oral fluids includes prehminary steps such as a SPE for enhancement of the analytes and for minimizing interfering effects of the matrices. Because the metaboHtes in humans are often conjugated, an anterior hydrolysis of these conjugates either with chemicals like sodium hydroxide or with enzymes [103] is recommended. 

As with urine, saliva (spumm) is easy to collect. The levels of protein and lipids in saliva or spumm are low (compared to blood samples). These matrices are viscous, which is why extraction efficiency of xenobioties amoimts to only 5 to 9%. By acidifying the samples, extraction efficiencies are improved as the samples are clarified, and proteinaceous material and cellular debris are precipitated and removed. Some xenobioties and their metabohtes are expressed in hair. Hair is an ideal matrix for extraction of analytes to nonpolar phases, especially when the parent xenobioties are extensively metabolized and often nondetectable in other tissues (parent molecules of xenobioties are usually less polar than metabolites). Hair is a popular target for forensic purposes and to monitor drug compliance and abuse. Human milk may be an indicator of exposure of a newborn to compounds to which the mother has been previously exposed. The main components of human milk are water (88%), proteins (3%), lipids (3%), and carbohydrates in the form of lactose (6%). At present, increasing attention is devoted to the determination of xenobioties in breath. This matrix, however, contains only volatile substances, whose analysis is not related to PLC applications. 

The analysis of codeine, morphine, 6-monoacetylmorphine (6-MAM, a metabohte of heroin), and cocaine is important for many toxicology labs to determine illicit drug use. When analyzing opiates in urine samples, frequently the matrix chosen for drug screening, the conjugated metabolites must be hydrolyzed however, this process can break down 6-MAM (Christophersen et al., 1987). These compounds can be derivatized to increase sensitivity, and both BCD and NPD are used for these assays. Derivatizations used include reaction with N-methyl-N-trimethylsilyltrifluoroacetamide followed by GC-FID (Lin et al., 1994) or with N,0-bis(trimethylsilyl)trifluoroacetamide (Christophersen et al., 1987 Lee and Lee, 1991), PFPA (Christophersen et al., 1987), or heptafluorobutyric anhydride (HFBA) followed by GC-ECD. All these methods show good sensitivity and selectivity. 

Pharmacokinetic data reported thus far show dose-proportional exposure with a short half-life of ca. 2 h. Exposure in patients receiving 250 mg/day of CP-724714 reportedly exceeds the plasma levels required for efficacy in precHnical tiunor xenograft models (50 to 60% TGI). However, robust efficacy (stasis or minor regression) in mouse models was only achieved at doses resulting in plasma exposures 40% higher than those observed in humans. From a kinetic standpoint, it appears that within 3 h, plasma concentrations in hiunans of CP-724714 fall below the levels required for pErbB knockdown in mouse models. This analysis, of course, neglects free fraction differences that may exist between species and the differential levels of active metabohtes. Enrollment in this trial continues at the 250 mg/tid dosing level. 

MS can measure the ratio between molar fractions of mass isotopomers. The ratio between two mass isotopomer pools of masses nti and m2 is defined in the present work as intensity ratio Jmi/m2- K identical with a mass spectral intensity ratio. If more than two mass isotopomer pools are assessed, their relative ratios, normalized to the sum, are named mass isotopomer distribution. The mass distribution of a compound can be thus obtained from the analysis of ions, which contain the intact carbon skeleton of the analyte. In the area of me-tabohc flux analysis, mass distributions of various metaboHtes have been assessed by MS. The major method used is GC/MS, whereby the analytes are deriva-tized into forms with desired physico-chemical properties such as increased volatihty, thermal stabiHty and suitable MS properties [62]. The mass of the formed derivate must be sufficiently high (usually above 175 apparent mass units) to avoid background interference [48]. To obtain the mass distribution of a compound, ions with the entire carbon skeleton of the analyte have to be present. For accurate quantification of the mass distribution of such ions, they should occur in high abundance and preferably be unique species, thus being formed by only one fragmentation pathway. 

Yu, Y, T.L. Wade, J. Eang, S. McDonald, and J.M. Brooks. 1995. Gas chromatographic-mass spectrometric analysis of polycychc aromatic hydrocarbon metabohtes in Antarctic hsh (Notothenia gibberifrons) injected with Diesel Euel Arctic. Arch. Environ. Contam. Toxicol. 29 241-246. 

Blood samples should be collected in sufficient volume for analysis of parent drug and active metabohte(s), if any. The sampling times should be such that it should be able to capture the C and T during the max max absorption period. Sampling should be carried out for at least three terminal elimination half-hves for both parent drug and active metabolite(s). Whole blood, plasma or serum, whichever is appropriate for the analytes, should be harvested promptly and samples should be frozen at 20 C or -70 C to maintain sample stability. 

Typically, a PK study is composed of three phases, namely the in-life phase, bioanalysis, and data analysis. The in-life phase includes administering the compound to animals or humans and collecting samples from an appropriate matrix of interest such as blood or urine at predetermined time intervals for bioanalysis. The bioanalytical phase involves analysis of a drug and/or its metabohte(s) concentration in blood, plasma, serum, or urine. This analysis typically involves sample extraction and detection of analytes via LC-MS/MS. The third phase is data analysis using noncompartmental or compartmental PK computational methods. 

Two main types of analysis are required (a) qualitative determination of the presence of elements and (b) quantitative determination of the amount of elements or species of interest contained in pharmaceutical products. Most analyses for pharmaceutical applications involve separation steps combined with ICP-MS, such as HPLC-ICP-MS or gel electrophoresis and the analysis of gel blots by LA-ICP-MS. Phosphorylated proteins (e.g., (J-casein) have been measured by LA-ICP-MS with a detection hmit of 16 pmol. HPLC-ICP-MS has been employed for the identification and quantification of metabohtes of bradykinin in human and rat plasma. ... 

Beverly, M. et al. Liquid chromatography electrospray ionization mass spectrometry analysis of the ocular metabohtes from a short interfering RNA duplex. J. Chromatogr. B. 2006, 835, 62-70. 

Figure 11-17 shows the results of automated, unattended MRM and subsequent MS/MS analysis of buspirone, one of the four substrates to be rapidly metabolized by liver microsomes. A total of 42 MRMs were monitored in this particular MS/MS analysis. The MRMs pre-selected represented the common phase I oxidative metabohtes, including mono- and d-hydroxylation, A-oxide formation, and A-dealkylation, to name a few. For 21 of the MRM transitions, the mass corresponding to the expected oxidative metabolite was added to the mass of the precursor ion (e.g., buspirone MW = 386, mono-hydroxylated buspirone MW = 402), keeping the mass of the product ion of the MRM pair unchanged. For the remaining 21 MRM transitions, the mass of the expected... 

A variety of neutral degradation products of the chloracetanilide herbicide alachlor was identified [63, 115]. However, the ionic metabohtes such as the oxoethanesulfonic acid derivative appear to be of more significance, as they are readily leached to groundwater. WMe alachlor itself is amenable to GC-MS, its ionic metabolites are not. Initially, GC-MS, LC-UV-DAD, and fast-atom bombardment MS-MS were applied in the analysis and identification of such metabolites [119]. Subsequently, the potential of LC-ESI-MS in this area was recognized [120]. Both oxanilic acid and oxoethanesulfonic acid metabohtes of alachlor, acetochlor and metolachlor were identified in snrface water and gronnd water, and subsequently determined with detection limits at the 0.01-pg/l level using off-hne SPE in combination with LC-MS [120]. 

Both in vitro and in vivo metabohsm has to be studied. Most of the results reviewed are from in vitro studies. The metabolites are generated by the use of recombinant enzymes, e.g., related to the cytochrome P450 (CYP) complex, by the use of subcellular fractions, e.g., microsomes, cytosols, or S9 fractions, or by the use of hepatocytes or liver slices. In the case of in vivo metabolism studies, the metabohtes are generated in living animals or humans, and analysis is performed in urine, plasma, bile, and/or faeces. 

The product-ion MS-MS spectrum of S9788 revealed a fragment ion at m/z 341, due to loss of the metabolically stable di(4-fluoropheityl)ethylamino part of the molecule [30]. This can be apphed in the neutral-loss analysis mode to screen biological samples for metabohtes of S9788, as demonstrated in the analysis of a rat bile sample in Figure 10.6. Next to the already known metabolites (i) through (v), three new minor metabohtes were found (A, B, and C). 

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