Metabolite identification applications

QUANTITATION OF DRUG CANDIDATE WITH SIMULTANEOUS METABOLITE IDENTIFICATION APPLICATION... 

While metabolite identification remains a lower throughput effort in most cases, it is a very important support procedure for new drug discovery. A thorough review of metabolite identification is beyond the scope of this chapter, so I will provide a brief overview and refer the reader to several recent reviews that will provide a more complete picture.176-184 Ma et al.176 reviewed the application of MS for metabolite identification this comprehensive review describes how different types of MS equipment can be used for metabolite identification. 

One recent advance in MS hardware that has been found to be useful for metabolite identification studies is the Orbitrap. This MS has a mass resolution of 30,000 to 100,000 (two models). For many applications, 30,000 mass resolution capability is sufficient. While only a few current literature references cite the Orbitrap MS for metabolite identification, it is safe to predict that the Orbitrap will be the subject of many references in the future. Two references related to its use for metabolite identification are Peterman et al.190 and Lim et al.182 Lim s group related an an impressive example of the use of high mass resolution to differentiate a metabolite from a co-eluting isobaric matrix component, as shown in Figure 7.14. 

Besides, High Resolution/Accurate Mass TOF and Q-TOF spectrometers (Figure 4b) allow full detection at low ppb amounts of drugs and their known and unknown metabolites, and they are fully implemented in Life Science applications like Metabolites Identification (MetID), and Drug Discovery. 

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-... 

Vastly reduced solvent consumption for micro-separation techniques has advantages in that it gives superior solvent suppression when protonated solvents are used [88]. Reduced solvent volumes also make the use of fully deuterated solvents more attractive, eliminating the need for solvent suppression [87]. A low-volume capillary probe with a 7 pi cell volume (1.5 pi active) is commercially available and its application to metabolite identification has been reported [89]. 

Ma, S., Chowdhury, S.K. and Alton, K.B. (2006) Application of Mass Spectrometry for Metabolite Identification. Current Drug Metabolism, 7, 503-523. 

Ma SG, Chowdhury SK, Alton KB. Application of mass spectrometry for metabolite identification. Current Drug Metabolism 7, 503-523, 2006. 

Castro-Perez JM. Current and future trends in the application of HPLC-MS to metabolite-identification studies. Drug Discovery Today 12, 249-256, 2007. 

Hop, C. E. C. A. (2004). Applications of quadrupole-time-of-flight mass spectrometry to facilitate metabolite identification. Am. Pharm. Rev. 7 76-79. 

Kerns and co-workers demonstrated the application of LC/MS and LC/MS/MS standard method approaches in preclinical development for the metabolite identification of buspirone, a widely used anxiolytic drug (Kerns et al., 1997). The success of this method relies on the performance of the LC/MS interface and the ability to generate abundant ions that correspond to the molecular weight of the drug and drug metabolites. The production of abundant molecular ions is an ideal situation for molecular weight confirmation because virtually all the ion current is consolidated into an adduct of the molecular ion (i.e., [M+H]+, [M+NH3]+). 

In this study, the LC/MS/MS analysis is <3 min, with injections occurring every 3.5 min. The previous HPLC method with fluorescence detection had a cycle time of 25 min. Thus, many opportunities exist with LC/MS/MS quantitative analyses to optimize chromatographic separations for speed. Similar to standard approaches described for open-access or metabolite identification, standard or generic methods may be usable with this application (Dear et al., 1998). 

A recent innovation is the commercial availabihty of linear two-dimensional ion traps [54], The linear ion trap (LIT) is found to be less prone to space-charging effects, enabling a higher number of ions to be accumulated, which results in enhanced sensitivity. In the conunercial instrument, the linear ion trap is the third quadrapole in a triple-quadrapole arrangement, i.e., (J-q o -LlT. In that setup, it can be used to accumulate product ions generated by CID in a LINAC colhsion cell, providing enhanced sensitivity and lack of low-mass cut-off. Further stages of MS-MS can be performed in the linear ion-trap, which then has similar features as the three-dimensional ion-trap. Early reports described the application of the hnear ion trap in metabolite identification and quantitative bioanalysis [55-56],... 

R. Ramanathan, D.L. McKenzie, M. Tugnait, K. Siebenaler, Application of semi-automated metabolite identification software in the drug discovery process for rapid identification of metabolites and the cytochrome P450 enzymes responsible for theirformation, J. Pharm. Biomed. Anal., 28 (2002) 945. 

Thermospray LC/MS has been extensively used for the study of sulfonylurea herbicides (1-2). These compounds are thermally labile and can not be successfully analyzed by conventional GC/MS. Early applications of thermospray LC/MS included metabolite identification and product chemistry studies. We have recently evaluated the use of thermospray LC/MS for multi-sulfonylurea residue analysis in crops and have found the technique to meet the criteria for multiresidue methods. LC/MS offers both chromatographic separation and universal mass selectivity. Our study included optimization of the thermospray ionization and LC conditions to eliminate interferences and maximize sensitivity for trace level analysis. The target detection levels were SO ppb in crops. Selectivity of the LC/MS technique simplified sample extraction and minimized sample clean up, which saved time and optimized recovery. Average recovery for these compounds in crop was above 85%. 

Other monovalent elements (F, Cl, Br, and I) are counted as hydrogens, trivalent elements (P) are counted as nitrogen, and tetravalent elements (Si) are included with carbon. For chemically possible formulae, r+ db> — 1.5. Odd-electron ions (M+ ) will have an integer value and even-electron ions will have 0.5 r + db more than expected, so round up to next lowest integer.32,33 By way of example, Kind and Fiehn139 have described an integrated application of accurate mass data to metabolite identification, constrained by isotope abundance information and valence rules, in addition to the KI (Section 9.10.4.3.2). 

---