Metabolite identification mass measurements

Liquid chromatography—mass spectroscopy has become one of the most powerful analytical techniques in the drug discovery and development proeess. LC tandem MS is obviously the technique of choice for the identification of metabolites as illustrated in this chapter. Exact mass measurements and elemental composition assignment are essential for the characterization of the metabolites. Accurate mass measurements of the product ions, formed in an MS/MS experiment, greatly facilitate the structure elucidation of the metabolites. With the ever evolving technological advances in mass spectrometry and separation science, LC MS will continue to play an important role in metabolite identification in the future. 

Castiglioni S, Zuccato E, Crisci E, Chiabrando C, Fanelly R, Bagnati R (2006) Identification and measurement of illicit drugs and their metabolites in urban wastewater by liquid chromatography-tandem mass spectrometry. Anal Chem 78 8421-8429... 

The Use of Cone-Voltage Fragmentation in Conjunction with High-Accuracy Mass Measurements and LC-MS for Metabolite Identification... 

Clarke, N., Cox, K., Rindgen, D. and Korfmacher, W., The Role of Accurate Mass MS Measurement in Metabolite Identification, American Society for Mass Spectrometry 2000 Conference Abstract, Long Beach, CA, USA, 2000. 

In tandem MS mode, because the product ions are recorded with the same TOF mass analyzers as in full scan mode, the same high resolution and mass accuracy is obtained. Isolation of the precursor ion can be performed either at unit mass resolution or at 2-3 m/z units for multiply charged ions. Accurate mass measurements of the elemental composition of product ions greatly facilitate spectra interpretation and the main applications are peptide analysis and metabolite identification using electrospray iomzation [68]. In TOF mass analyzers accurate mass determination can be affected by various parameters such as (i) ion intensities, (ii) room temperature or (iii) detector dead time. Interestingly, the mass spectrum can be recalibrated post-acquisition using the mass of a known ion (lock mass). The lock mass can be a cluster ion in full scan mode or the residual precursor ion in the product ion mode. For LC-MS analysis a dual spray (LockSpray) source has been described, which allows the continuous introduction of a reference analyte into the mass spectrometer for improved accurate mass measurements [69]. The versatile precursor ion scan, another specific feature of the triple quadrupole, is maintained in the QqTOF instrument. However, in pre-... 

Advances in high resolution mass analyzers (TOF, FT-ICR, orbitrap) have greatly improved the detection and identification of metabolites based on accurate mass measurements. In single MS mode accurate mass determination is mainly used to differentiate between isobaric ions. Combined with LC-MS, it allows the detection of predicted metabolites by performing extracted ion current profiles... 

Kantharaj, E., Ehmer, P. B., Tuytelaars, A., Van Vlaslaer, A., Mackie, C., and Gilissen, R. A. (2005b). Simultaneous measurement of metabolic stability and metabolite identification of 7-methoxymethylthiazolo[3,2-a]pyrimidin-5-one derivatives in human liver microsomes using liquid chromatography/ion-trap mass spectrometry. Rapid Commun. Mass Spectrom. 19 1069-1074. 

MS/MS modes of operation. Additionally, when looking at unknown samples such as the case of metabolite identification, having exact mass measurements and high resolution helps to decipher simple and complicated biotransformations. 

Pike, A., Naegele, E., and Bilsborough, S. (2007). Discovery and identification of low level drug metabolites using a novel searching method combined with exact mass measurement. In Proceedings of the 55th ASMS Conference on Mass Spectrometry and Allied Topics, Indianapolis, IN. 

Superior sensitivity, efficiency, and specificity have made high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS), the predominant analytical technique for characterization and quantitative analysis of metabolites (Kostiainen et al., 2003 Ma et al., 2006 Prakash et al., 2007). Ion trap, triple-quadrupole, and quadmpole time-of-flight (Q-TOF) mass spectrometers are routinely used to profile and characterize metabolites in plasma and excreta (Ma et al., 2006). The combination of scan types and features available on mass spectrometers of different design (product ion, MS", neutral loss, precursor ion scans, accurate mass measurements) allows identification and characterization of putative and unexpected metabolites with or without little prior knowledge of biotransformation pathways of a given dmg molecule. 

In LC-MS analysis, identification of lipids requires the use of accurate mass measurements for the determination of elemental composition, combined with MS" experiments to get information of the molecular fragments. In addition, in LC-MS, one metabolite may produce multiple peaks due to the presence of isotopes, adducts, and neutral loss fragments. Therefore, ion annotation is crucial in order to recognize a group of ions likely to originate from the same... 

To avoid the expensive use of deuterated solvents for H/D exchange experiments, Tolonen et al. [21] have described the postcolumn infusion of D2O to facilitate the LC-MS detection and identification of labile protons in a column eluant. Whilst acknowledging the potential limitations with respect to a reduced level of exchange, and hence sensitivity, compared to the use of deuterated mobile-phase solvents, they optimized the column effluent flow rate (via a splitting connector) with the infused D2O flow rate to enable the very useful determination of up to four labile protons. The method was exemplified by the differentiation of hydroxylated metabolites of the alkaloidal drugs imipramine and omeprazole (Figure 13.5) from the N-oxide and sulfone metabolites, respectively [21]. This was a differentiation that could not be achieved by high-resolution mass measurements. 

A. Deroussent, M. Re, H. Hoellinger, E. Vanquelef, O. Duval, M. Sonnier, T. Cresteil, In vitro metabolism of ethoxidine by human CYPlAl and rat microsomes identification of metabolites by LC—ESI-MS—AdS and accurate mass measurements by TOF-MS, Rapid Commun. Mass Spectrom., 18 (2004) 474. 

Orthogonal extraction, improvements in TOF/MS resolution, and improvements in electronics have created a new application for TOF/MS in accurate mass measurements [70,71]. Accurate mass applications have traditionally been associated with high-resolution magnetic sector and FTMS systems. The ability to provide accurate mass measurements (+5 ppm) with more cost-effective instrumentation has created potential opportunities in the pharmaceutical industry. Such applications can offer improved selectivity for single-stage instrumentation and metabolite or impurity/degradation product identification, particularly when standard compounds are not available, such as in early drug discovery. 

Harvey DJ, Martin BR, Paton WDM (1979) Identification and measurement of cannabinoids and their in vivo metabolites in liver by gas chromatography-mass spectrometry. In Nahas GG, Paton WDM (eds) Marijuana biological effects. Pergamon Press, Oxford, pp 45-62... 

The total amount excreted in urine up to time t C4ex ) can be estimated by measuring the total volume of urine and the compound concentration in urine, while the entire excreted amount (Aex J can be estimated using a similar method with the confirmation that no more compound can be detected in the last portion of urine collection. Early PK studies usually do not collect urine that requires use of metabolic cages. Renal CL usually is estimated when a compound proceeds to a lead stage. In a routine metabolite identification or mass balance study, urine is collected and can be used for renal CL estimation. 

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