Metabolites quantitation

Estimating the amount of a metabolite when an authentic reference standard is not available is still a challenge. Yu et al.191 described a procedure that uses the results of an in vitro metabolite identification based on a test compound that produces 14C-labelled metabolites essentially the 14C-labelled metabolites are used to provide a correction factor for the MS response when assaying samples that contain the same metabolite in a study that did not use the 14C-labelled test compound. Flop192 described another novel approach for metabolite quantitation based on the observation that the MS responses for most compounds are very similar to responses from nanospray ESI. Valaskovic et al.193 also reported equimolar MS responses for multiple compounds when the flow rate to the nanospray ESI source was set to about 10 nl/min. It is too soon to know whether these intriguing findings can be readily applied to discovery metabolite identification studies. 

Regulatory requirements in the field of drug residues analysis are limited, in most cases, to the identification of only the major metabolites. Quantitative selection of major over minor metabolites is certainly devoid of rational biological ground, since several studies have shown that the toxic metabolites are usually transitory and often present in small quantities. However, isolation and identification of all these metabolites are difficult and proper assessment of the toxicity of drug residues is still a real challenge. 

The application of the twin ion technique [257] is also of importance in metabolism studies. The doubly labelled steroids [4- C+ 7-l- Ho.44]-androstenedione and [4- C + 7/3- Ho.42]-testosterone, were incubated with human placental microsomes and the resulting metabolites quantitated by counting C and identified by GC-MS [258]. The identified metabolites 17/8,19-dihydroxyandrost-4-en-3-one, 19-hydroxyandrost-4-en-3,17-dione, 17/8-hydroxy-3-oxo-androst-4-en-3-one, 3,17-dioxoandrost-4-en-19-al, oestradiol-17/3 and oestrone were easily recognisable from the double sets of relevant ions in their spectra due to the mixture of hydrogen and deuterium substitution at C-7. Hence the presence of the aromatizing enzymes in the placental preparation and the intermediates in oestrogen biosynthesis were confirmed. 

Tests that identify an unknown amino acid or metabolite. Quantitative Tests... 

Immunoassay. THC is extensively metabolized to a large number of compounds, most of which are inactive. The principal urinary metabolite is U -nor-A -tetrahydro-cannabinol-9" carboxylic acid (THC-COOH) and its glucuronide conjugate (see Figure 34-30). Immunoassays designed to screen urine samples for marijuana use measure this and other THC metabolites. These assays are calibrated with THC-COOH, but because of cross-reactivity with many other THC metabolites, quantitative results based on them are 1.5 to 8 times greater than the actual concentration of THC-COOH as determined by GC-MS. Therefore... 

No quantitative data were located regarding absorption in humans after inhalation exposure to DNPs. A metabolite of 2,4-DNP, 2-amino-4-nitrophenol, was commonly detected by the Derrien test in the urine of workmen (women were generally not employed in dangerous processes) exposed via inhalation to vapor and airborne dust of 2,4-DNP and by direct contact of the skin with the solid chemical in the munitions industry in France (Perkins 1919). Exposure may have occurred by the dermal and possibly oral routes, as well as by inhalation. In addition, examination of the blood, unspecified organs, and urine of workmen in this industry who died from exposure to 2,4-DNP revealed the presence of 2,4-DNP and its metabolites quantitative data were not provided (Perkins 1919). Despite its limitations, the study provides some evidence of absorption from inhalation exposure. 

Wright, P., Miao, Z., and Shilliday, B., Metabolite quantitation Detector technology and MIST implications, Bioanalysis, 1(4), 831, 2009. 

Metabolite quantitation in humans and two species of preclinical animals at Cmax in plasma is shown in Figure 16.11. UPLC—AMS separated and quantified fractions of variable width that depended on metabolites expected from the animal species. UV absorbance (dashed lines in the figure) monitored resolution and provided a marker near 8 min for one metabolite spiked into the plasma samples before separation. AMS data is plotted as the fraction of the total eluted isotope signal in the trace on both linear and log scales for 18 compounds that produced peaks in one or more of the three species. Multiple metabolic differences are noted not only between the human and the animals but also between the two animal species. Specifically, the metabolites included as a spike, M , is disproportionately found in humans at more than 10% of the total content and only minimally, but distinctly, in species 2. Chromatographic conditions broadened the UV metabolite peak in species 1 with resolution also lost in leaving only a hint of the metabolite presence. M is the only other metabolite exceeding 10% of total in human plasma in this... 

Current Approaches for Metabolite Quantitation in the Absence of Synthetic Standards... 

Metabolite quantitation plays an important role in drug discovery and development as the level of metabolites may potentially impact the efficacy and safety of a drug (Baillie et al., 2002 Leclercq et al., 2009 Smith and Obach, 2006). Even for metabolites with no biological activity at the clinical relevant dose, the amount of a specific metabolite formed often reveals a major metabolic pathway that may allow medicinal chemists to block some labile sites on the molecule or soft spots to metabolically stabilize the compound. The recent U.S. Food and Drug Administration (FDA) guidance on Safety Testing of Drug Metabolites further put forward the requirements for... 

CURRENT APPROACHES FOR METABOLITE QUANTITATION IN THE ABSENCE OF SYNTHETIC STANDARDS... 

Although all of the above methods have shown some success in standard-free metabolite quantitation, there is a strong demand to continue to explore analytical methodologies that offer improved specificity and sensitivity for the quantitation of metabolites in the absence of synthetic standards of the metabolites or radio-labeled parent compound. A new analytical methodology that offers a high level of specificity and sensitivity and has demonstrated initial success and good potential for standard-free metabolite quantitation is nanospray. This ehapter will review the principles, initial applications, and future directions of nanospray for standard-free metabolite quantitation. 

FIGURE 17.4 Schematic diagram of the calibration approach used in standard-free metabolite quantitation. [Reprinted from Valaskovic et al. (2006) with permission.]... 

Josephs JL, Luk CE, Grubb M, Yang Y, Zhang H, Cai H, Langish R, Shipkova P, Sanders M, Humphreys WG. An integrated approach to in vitro and in vivo metabolite quantitation based on high resolution full scan MS Data. Oral presentation. Presented at the 57th Annual Conference of Am Soc Mass Spectrom, Philadelphia, Jmre 2009. 

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