In this instalment, we introduce a way of using high-resolution mass spectrometry (HR-MS) systems to characterize the structures
of metabolites. This results in a general workflow for metabolism studies for drug discovery and development.
Drug metabolism (biotransformation) is a process by which lipophilic xenobiotics are converted into hydrophilic metabolites,
to facilitate elimination from the body. Cytochrome P450, uridine 5'-diphosphate glucuronosyltransferase (UGT), and sulphotransferase
(SULT) are commonly involved in metabolic conversions. They catalyze many different reactions including hydroxylation, dealkylation,
epoxidation, reduction, glucuronidation, and sulphation.
In most cases, metabolites are less pharmacologically active and less toxic than their corresponding parent forms. Some compounds,
however, can be metabolized to pharmacologically active metabolites or reactive species that result in prolonged efficacy
or idiosyncratic toxicity, respectively. For example, levodopa exerts its therapeutic effect by converting to its active form,
dopamine (1), and acetaminophen undergoes P450 bioactivation to a highly reactive intermediate N-acetyl-p-benzoquinone imine, which is widely accepted as the primary culprit for acetaminophen-induced acute liver failure (2).
Metabolism is one of the vital determinants of a drug's pharmacokinetic properties. High metabolic liability usually leads
to high clearance and poor bioavailability. The formation of active or toxic metabolites enhances the pharmacological and
toxicological effects. Thus, drug discovery and development scientists have increasingly become aware of the important role
of metabolite profiling. Knowledge of the metabolic soft spots and bioactivation potential in vitro and of the metabolic pathways
in vivo guide chemists in lead optimization from the earliest stages. Such knowledge is similarly valuable in the drug-development
process for selecting a candidate from among several entities equally effective in their therapeutic response and for explaining
pharmacokinetic or pharmacodynamic disconnects.
The primary tasks in metabolism study are the detection of metabolites and determination of the type and site of modifications.
Yet performing these tasks can prove challenging. Biological matrices are complex, containing large excesses of proteins,
lipids, and other endogenous compounds that can interfere with detection. In addition, metabolism often results in a number
of structurally diverse metabolites with relatively low concentrations (nanomolar to micromolar).
Many analytical techniques can be utilized to identify drug metabolites (3,4). Among them, mass spectrometry (MS) has proven
itself pivotal for several decades because it can greatly improve analytical sensitivity, selectivity, and speed. The molecular
weight of each metabolite can be readily obtained from the measured mass-to-charge ratio (m/z) of the protonated or deprotonated molecule in positive or negative ionization mode. If the measured m/z is sufficiently accurate (<5 ppm), the elemental composition of a metabolite can also be definitely derived.
Generally speaking, metabolites share the same nuclear structure as that of the parent drug. Consequently, one can identify
the various types of biotransformation by comparing the molecular weight or elemental composition of the metabolite with that
of the parent. The mass changes of biotransformations most often encountered in metabolism study appear in Table 1. Furthermore,
the detailed fragmentation data generated from the MS–MS experiments provide rich structural information on the metabolites
Table 1: Mass and mass defect shifts for common biotransformations.
Liquid chromatography (LC) offers the capability to separate the analytes from matrix interferences and one another, thus
facilitating detection and structural elucidation. Yet the coupling of LC with MS (LC–MS) was once difficult because of the
need to convert aqueous LC effluent into gas-phase ions. With the development of the atmospheric pressure ionization (API)
techniques of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), analytical laboratories have
routinely used LC–MS since the mid-1980s for rapid determination of drugs and structure elucidation of drug-related materials
directly from biological matrices (8).
Mass analysers, the components of mass spectrometers that sort the ions according to their mass-to-charge ratios, come in
two types: Unit-resolution and high-resolution. Two unit-resolution systems, triple-quadrupole and ion-trap mass spectrometers,
have been the most frequently used in the metabolite identification field during the last decade (8–10).
A triple-quadrupole mass spectrometer can be operated in these scan modes: Full, constant neutral loss (NL), precursor ion
(PI), and product ion. The ion-trap mass spectrometer can perform multiple-stage mass spectrometry (MSn), provide detailed fragmentation pathways, and thus narrow the potential sites of modification (8,11–13). Yet because of
their relatively low resolution, these techniques lack the necessary selectivity to distinguish metabolites from the isobaric
interferences in complex matrices. In addition, these unit-resolution-based techniques are usually performed by experienced
scientists with a strong background in metabolite identification.
Innovations in ion-physics technology have prompted a paradigm shift in the field of drug metabolism, from using low-resolution
triple-quadrupole or ion-trap instruments toward adopting high-resolution (HR) mass spectrometers. Modern HR-MS mass spectrometers
include time-of-flight (TOF) instruments, Fourier transform-based instruments such as orbital trap systems (Thermo Fisher
Scientific), FT-ion cyclotron resonance (FT-ICR), and hybrid instruments such as quadrupole TOF (QTOF), linear ion trap–orbital
trap, and linear ion trap–FT-ICR systems (3,14–17). They all provide high resolution — greater than 10,000 full-width half-maximum
(FWHM) — and accurate-mass capabilities, generally ≤5 ppm for QTOF and ≤2 ppm for orbital traps. Therefore, these instruments
enable discrimination of metabolite ions from nominally isobaric biotransformations or background interferences.
Accurate-mass measurements by MS, MS–MS, or MSn allow for the unequivocal determination of empirical formulae of metabolites and their fragments, providing invaluable information
to aid, postulate, or assign structures to the metabolites (18,19). Along with the development of the state-of-art HR-MS instrumentation,
"intelligent" data acquisition and data mining tools have also emerged, which markedly accelerate and simplify the metabolite
screening and identification process (20–33). For drug metabolism scientists, HR-MS is now the premier analytical tool of
choice in their research.
Owing to its superior sensitivity, resolution of 20,000–40,000 FWHM, mass accuracy of ≤5 ppm, speed of ≤20 spectra/s (without
sacrificing sensitivity or resolution), dynamic range of 4–5 orders of magnitude, and relatively low cost, QTOF-MS has gained
wide acceptance as a highly attractive tool in small-molecule drug metabolism studies. Many applications have demonstrated
the ability of the technique to determine labile spots, elucidate the structures of metabolites in the circulatory system
and excreta and evaluate the bioactivation potential of drugs (24,25,34–42). In addition, QTOF-MS enables simultaneous, exact-mass,
qualitative, and quantitative analysis of multiple analytes using the ful-scan mode in one injection cycle, which meets the
demand for high throughput in a fast-paced, drug research and development (R&D) environment (43–47). QTOF-MS has therefore
become the most often used HR-MS technique in drug discovery and development.
The development of several hybrid orbital trap instruments has expanded the application field of the orbital trap from bimolecular
analysis to small-molecule research (15, 48–51). Hybrid orbital trap systems provide greater mass-resolving power (>100,000
FWHM) than QTOF-MS, and they can perform exact-mass MSn analysis (<2 ppm). Therefore, these systems increase confidence in the identification of biotransformations and localization
of their sites (15,52,53). The orbital trap analyser's relatively slow scan speed (~1 s at 100,000 FWHM, ~0.25 s at 60,000
FWHM, ~0.1 s at 17,500 FWHM) is a drawback, however. When an orbital trap is coupled with an ultrahigh-pressure liquid chromatography
(UHPLC) system, operators usually specify relatively low resolution (<70,000) to ensure sufficient data points across narrow
chromatographic peaks (1–2 s) (54). The detection sensitivity and dynamic range (3 orders of magnitude) of orbital traps are
also lower than those of the QTOF-MS. Furthermore, the orbital trap system exhibits the low-mass-cutoff problem characteristic
of ion-trap mass spectrometers and is often referred as the "one-third rule." To correct this problem (at least in part),
the new generation of orbital trap–based devices use dual collision cells, a collision-induced dissociation (CID) cell and
a high-energy C-trap dissociation (HCD) cell (55). The HCD cell reportedly produces more abundant, low m/z ions, in a similar fashion to a QTOF instrument (56).
FT-ICR-MS can deliver ultra high mass resolution (>750,000 FWHM) with part-per-billion mass accuracy, so it is very useful
for studying macromolecules like proteins with multiple charges (57–60). However, its relatively high purchase price, maintenance
cost, and space requirements limit routine use in small-molecule drug metabolism research at the drug discovery stage.
In this column instalment, we introduce a way of using HR-MS systems to characterize the structures of metabolites. This approach
results in a general workflow for metabolism study with HR-MS in drug discovery and development.