**3. Metabolite profiling by LC-MS**

Mass spectrometry plays an important role in the metabolite profiling of drugs during drug discovery and development. Quality and productivity of the metabolite identification process is improved by the availability of high-resolution (HR) MS instrumentation with superior accuracy and new data acquisition methods and data mining techniques. Hence, HPLC coupled with the high-resolution MS is the analytical tool of choice for metabolite profiling studies [71].

Drug metabolites can be categorized into expected metabolites and unexpected metabolites. Expected metabolites are those produced by common biotransformation reactions and are predictable, unexpected metabolites are those produced by uncommon reactions and are not easily predictable. Mass shift values from the parent drug can be used to calculate the molecular masses (*m/z* values) of expected metabolites. Acquisition of full-scan MS data using MS instrument, followed by extracted ion chromatography (EIC) of the ions can be used to accomplish the detection of expected metabolites by LC/MS [72, 73]. The most difficult task in drug metabolism studies is the detection and structural elucidation of very low levels of unexpected metabolites in the presence of endogenous interfering components [74–76].

Time-of-flight (TOF) and Fourier transform Orbitrap (Thermo Fisher Scientific) are the most commonly used high-resolution mass spectrometers in metabolite profiling of drugs. The principle involved in the TOF mass analyzers is ions of different *m/z* values having different velocities when accelerated by the same kinetic energy in the field-free flight tube. The time required for the ions to travel through the flight tube is proportional to the square root of their *m/z* values. The *m/z* value of each ion is determined by measuring the time taken for the ion to arrive at the detector. The resolution of the instrument is dependent on the capacity of the instrument to generate and maintain a focused ion beam through the ionization and acceleration region. The resolving power of the instrument is improved by utilizing reflectrons (reflecting ion mirrors), which decreases the spreading of kinetic energy among ions accelerated from the accelerator [77]. The ion saturation problem in the TOF instruments is effectively resolved by improvements in the ion detection technologies, for example, the use of segmented multichannel plates or analog-to-digital converters in place of time-to-digital converters. Modern TOF instruments offer good accuracy (~2–5 ppm), required resolution (~30,000) at full-width half maximum [FWHM]), and fast scan speed (20–50 spectra/second) [78, 79].

The Orbitrap mass analyzer consists of two electrodes, one is an outer barrellike electrode and other is a coaxial inner spindle-like electrode. A static electric field is applied between the outer and inner electrodes. Around the central spindle

#### In vitro *Metabolic Stability of Drugs and Applications of LC-MS in Metabolite Profiling DOI: http://dx.doi.org/10.5772/intechopen.99762*

electrode, ions are radially trapped, rotate about the inner electrode, and harmonically oscillated along the central electrode with a frequency characteristic to its mass to charge ratio (*m/z* value). An image current is produced on split outer electrodes due to the axial motion of the ions around the inner electrode. The mass spectrum is generated by fast Fourier transformation of the image current to convert the time-domain signal into a frequency, and then into an *m/z* spectrum [80, 81]. The Orbitrap MS offers good resolving power (~30,000–240,000) and mass accuracy (<3 ppm). The resolution of the Orbitrap instrument depends upon the scan speed; to achieve higher resolution, longer total cycle times are required. This is a limiting factor for the Orbitrap device to couple with the UPLC instruments in which peak width is only a few seconds. The most popular HRMS instrument configurations used for metabolite profiling are Q-TOF (quadrupole – time of flight) and LTQ (linear trap quadrupole) -Orbitrap, because of their high resolution and mass accuracy characteristics. A Q-TOF instrument consists of quadrupole, collision cell, and TOF mass analyzer. Q-TOF is obtained by switching the last quadrupole in the TQMS (triple quadrupole mass spectrometer) with TOF mass analyzer. It provides fast data acquisition, high sensitivity and resolution, and accurate mass on both MS and MS/MS modes, thus proven to be a powerful tool in metabolite profiling studies. LTQ (linear trap quadrupole) -Orbitrap is a hybrid mass spectrometer that combines a linear ion trap with Orbitrap MS. Accurate mass measurements are possible on multiple stages of fragmentation for structural elucidation of metabolites and MSn (multi stage mass spectrometry) experiments can be performed simultaneously with ion trap detection while continuing mass measurements with Orbitrap. QExactive™ hybrid quadrupole - Orbitrap mass spectrometer combines quadrupole precursor ion selection with high resolution, accurate mass Orbitrap detection. It is compatible with fast chromatography techniques because of its high scan speed (~12 Hz) and spectral multiplexing capabilities; hence fit for high-throughput metabolite profiling. Current progress in HRMS leads to the development of various new data acquisition and data mining techniques for the rapid identification of drug metabolites.

#### **3.1 Data acquisition methods for HRMS drug metabolite identification**

Data acquisition methods used for metabolite identification include: ion intensity-dependent acquisition, accurate-mass inclusion list-dependent acquisition, isotope pattern-dependent acquisition, pseudo neutral loss-dependent acquisition, and mass defect-dependent acquisition [77].

#### *3.1.1 Ion intensity-dependent acquisition*

In this method, an ion intensity threshold is used to trigger the MS/MS acquisition of ions. Prior knowledge of the *m/z* values of the precursor ions is not required for this generic method. This method is very effective for *in vitro* metabolite profiling. Fifteen metabolites of nefazodone were identified from human liver microsomal incubations by applying intensity-dependent MS/MS acquisition of the three most intense ions from a single LC–MS/MS run [82]. This method is not suitable for complex biological samples because of matrix interferences. It is very difficult to acquire MS/MS spectra of trace level metabolites using this method because highintensity endogenous ions are mainly selected for MS/MS or MSn acquisition.

#### *3.1.2 Accurate-mass inclusion list-dependent acquisition*

This method uses a list of accurate masses of predicted or expected metabolites to trigger MS/MS acquisition of preset metabolite ions. Data analysis is performed

by the software in real time to determine any mass in the list is detected in the full scan. If any ion is detected within a certain mass tolerance window and above a particular intensity threshold, the software will switch to MS/MS mode automatically and obtain the product ion (MS/MS) spectrum. This method increases the chance of getting MS/MS spectra for low level metabolites present in the complex biological samples. Moreover, in a single LC–MS/MS run both full-scan MS and MS/MS spectra of predicted metabolites will be obtained [83]. By using this approach and different post-acquisition data mining techniques, a total of 58*in vitro* metabolites of carvedilol were detected from human liver microsomal incubations [84].

On the other hand, preparation of an accurate mass inclusion list for every compound is time intensive and not suitable for high-throughput metabolite profiling during drug discovery. Besides, many of the major metabolites are generated by rearrangement, ring scission, or ring contraction and are hence difficult to predict.

#### *3.1.3 Isotope pattern-dependent acquisition*

Molecules containing elements like Cl and Br can be easily identified by their unique isotopic patterns in the mass spectra. Assume that during biotransformation these halogens remain intact and their unique isotopic pattern is used as a selective trigger for MS/MS acquisition of metabolite ions. It assists in the easy identification of metabolites and provides MS/MS spectra for structural analysis. The software is programmed such that any ion detected with a unique isotopic pattern (e.g., Cl-containing compounds: ion pairs with *m/z* difference of 1.99705 Da and an intensity ratio of 3:1; Br-containing compounds: ion pairs with *m/z* difference of 1.99795 Da and intensity ratio of 1:1) in the full scan MS would be automatically followed by an MS/MS experiment for rapid identification of metabolites. This approach has demonstrated to be very effective in metabolite profiling of a rat bile sample collected following a single oral dose (30 mg/kg) of a 14C-bromine containing compound on a quadrupole time-of-flight mass spectrometer. Over 30 metabolites were detected with their MS/MS spectra automatically obtained in the same LC–MS/MS run [41]. Isotope pattern-dependent acquisition is also applicable to compounds containing synthetically incorporated isotopes (e.g.,2 H-, 13C-,15N-, 18O-, etc.) or radiolabeled compound (14C-) with a distinct 12C/14C isotopic pattern. Glutathione (GSH) trapped reactive metabolites from microsomal incubations are detected by this approach with a linear ion trap mass spectrometer [85]. Lim et al. also applied this approach for simultaneous detection and structural elucidation of GSH conjugates generated from human liver microsomal incubations by using LTQ/Orbitrap in a single run [86]. This approach is compound dependent and not suitable for various metabolites.

#### *3.1.4 Pseudo neutral loss-dependent acquisition*

This approach is based on neutral loss, which is a trigger for MS/MS acquisition. It is useful for the detection of metabolites, which shows neutral losses due to collision-induced dissociation. This approach consists of two full scans, one scan is at low collision energy (i.e., 5 eV) followed by a second scan with higher collision energy ramping (i.e., 20–40 eV), and spectra will be monitored for characteristic *m/z* differences of ion-pairs (neutral loss) between successive low and high collision energy full-scan MS. When such neutral losses are detected within a certain mass tolerance window, precursor ions will be identified from the low collision energy data and the instrument switches to MS/MS mode automatically to get the product ion spectra of those specific ions [87]. This approach is mainly useful for the phase II metabolites detection and characterization (e.g., neutral losses of 79.9568 for

sulfate conjugates, 129.0426 for glutathione conjugates, and 176.0321 for glucuronide conjugates, etc.). The limitation of this approach is that it is unable to record MS/MS spectrum of metabolites with unpredictable fragmentation.
