Metabolites are the end products of all chemical or biochemical processes within the system. Metabolism is the process of elimination of foreign and undesirable compounds (xenobiotics) from the body. Metabolites identification is the observation, identification and quantitation of these metabolites on a system basis.
Even though metabolism happens in various body organs such as heart, lung, kidney and intestine; nevertheless liver is the major site of metabolism in the body. Hence, more focus has been levied on liver metabolism. Metabolism in liver occurs in two phases: Phase-I and Phase-II. Phase I metabolism can be termed as functionalisation reaction. Here, a new functional group (hydroxyl) will be added to the test entity or an existing functional group (demethylation) will be exposed. Phase-I reactions convert a drug to a more polar active/ inactive metabolite. Phase-II metabolism can be termed as conjugation reaction. These reactions (Phase-II), increases the
water solubility of a drug by adding up a polar moiety (Glucuronate, Sulfate, acetate). Phase-II reactions convert a parent drug/ phase-I metabolite to a more polar active/ inactive metabolites by conjugation of subgroups (-OH, -SH, -NH2). Drugs metabolised by phase-II reactions are excreted renally.
Various liver preparations exists such as microsomes, S9 fractions, cytosolic fractions and hepatocytes.
- Microsomes are easy to prepare and can be stored for longer periods at -80oC. These fractions have enriched P450, FMO and UGT’s. Microsomes are majorly used to study phase-I metabolism reactions and few phase-II metabolism reactions such as glucuronidation.
- S9 fractions are same as microsomes, but also contain cytosolic enzymes (SULT, GST, XO, ADHs, and NATs). P450 activity is ~5 fold lesser in S9 fractions than microsomes. Compounds that are seen to be metabolising fast in microsomes, might actually metabolise slower in S9 fractions, if major route of metabolism is by phase-I reaction.
- Cytosolic fractions are used to study few phase-II metabolism reactions such as sulfonation, glutathione conjugation and acetylation.
Hepatocytes are the gold standard for drug metabolism studies. These contain all the enzymes/ transporters and cofactors responsible for drug metabolism. Even though, it is a costly affair to use these preparations, nevertheless the information obtained is unmatched.
Metabolite identification provides information on the site that needs to be blocked or modified in order to improve metabolic properties of the molecule. This is also termed as soft spot identification. However, it is not always true to block or modify the sites, as there exist active metabolites. Metabolites identification helps in identifying the major metabolite based on abundance and also the biotransformation pathway. In vitro metabolite profiling across species provides information on extent of metabolism and also the selection of right species for toxicity studies.
Mass spectrometer (MS) has emerged as an ideal technique for the identification of almost all structurally diverse metabolites. Detection in mass spectrometer is based on m/z ratio. MS/MS provides distinctive fragment patterns which in turn help in providing the structural information. Given the superb speed, sensitivity, and selectivity, MS has become the method of choice for metabolism and metabolite identification studies in drug discovery and development.
SCIEX Hybrid QTRAP System
QTRAP combines the capabilities of a triple quadrupole mass spectrometer and ion trap technology on a single platform. These systems possess tandem in time (QTRAP) and tandem in space functionalities (QQQ). The sensitive and selective performance of an ion trap and the performance of a triple quad in single platform.
QTRAP Systems enable the unique capabilities provided by this technology by coupling targeted triple quadrupole experiments, such as MRM, precursor ion, and neutral loss scans, with high sensitivity ion trap scan functions, such as enhanced MS (EMS), enhanced product ion (EPI), enhanced resolution (ER), enhanced multiple charge (EMC) and MS/MS/MS (MS3) scan. Having a variety of new scan types and the possibility to combine them in single LC runs enable completely new workflows for many LC/ MS/ MS applications, including sensitive bioanalytical quantitation, pharmacokinetics studies and metabolite identification/ quantification.
The Enhanced Product Ion scan of QTRAP Systems has several advantages over product ion scanning of triple quadrupole mass spectrometers, including:
- Much higher sensitivity of Enhanced Product Ion (EPI) spectra because of ion accumulation in the linear ion trap (LIT).
- More product ion information in a single MS/ MS spectrum because of Collision Energy Spread.
- Lower cycle time due to faster scanning of linear ion trap (20000 da/ sec)
- No inherent low mass cut-off due to fragment generation in the collision cell rather than in the ion trap
- Remarkable immunity of LIT from space charge effects which result in mass shift and incorrect isotopic pattern in the spectrum.
Hybrid Triple Quadrupole Linear Ion Trap (QTRAP) Systems provide a novel workflow for the screening and identification of a multitude of targeted analytes by combining selective MRM detection with a highly sensitive MS/MS scan using Q3 as Linear Ion Trap. In Information Dependent Acquisition (IDA) experiments, the detection of an MRM signal above a specified threshold automatically triggers an Enhanced Product Ion (EPI) scan. These EPI spectra are as sensitive and selective as MRM signals and contain the complete molecular fingerprint because of precursor ion selection in Q1, product ion generation in the collision cell, and ion accumulation in the LIT.
The information saved into a full scan MS/ MS spectrum allows identification with a higher degree of confidence minimising the risk of potential false positive and negative detection. In addition, the improved cycle time for all confirmatory MRM transitions can be used to increase the dwell time of all other MRM transitions to improve S/N, resulting in better reproducibility and accuracy.
Following are different Information dependent acquisition (IDA) workflows in QTRAP system for Metabolite Identification and Quantitation experiments:
- EMS – ER – EPI – MS3 (LIT scan experiments): Best for screening applications in metabolites/ impurities identification (Non-targeted analysis approach)
- NL-ER-EPI-MS3 (QqQ and LIT scan): Screening for structural analogues and GSH conjugate analysis (Targeted analysis approach)
- PI –ER-EPI or PI-ER-EPI (QqQ and LIT scan): Screening for structural analogues and GSH conjugate analysis (Targeted analysis approach)
- MRM-ER-EPI or pMRM-ER-EPI (QqQ and LIT scan): Target analysis and confirmatory analysis (can be used for Quantitative and Qualitative analysis)
Work flow for metabolite identification studies
- Incubation of test entity with various liver preparations, such as microsomes, S9 fractions and hepatocytes. Compound concentration has to be optimised to provide relevant information of the metabolites that even form at relatively low abundance. Ideally compounds can be incubated at 10 µM concentration.
- Protein precipitation has to be used as sample preparation technique. Protein precipitation even though a crude extraction technique, helps in extraction of all the putative metabolites. This is subject to solubility of parent drug in extraction solvent. Ideally, acetonitrile and methanol are the choice of extraction solvents for protein precipitation.
- Supernatants from the centrifuged samples can be injected directly in to mass spectrometer. Alternatively, supernatants can be diluted 1:1 with water and used for analysis. Supernatants can also be concentrated by evaporation under nitrogen gas.
- Samples have to be initially analysed with acquisition method built with the assistance of IDA method wizard (IDA wizard is a specific software feature available in analyst software). IDA wizard helps in combining full scan (EMS, EMC, MRM and NL) functions and dependent scan functions (EPI). Full scan functions help in identifying the parent ion in case of EMS, EMC scan function, parent ion-fragment ion reaction in case of MRM and NL scan functions. Parent ions/ parent-fragment ion reaction detected in full scan function will further trigger the EPI scan function. EPI scan function will provide the complete fragment information of all these full scan masses.
- Metabolites have very similar fragmentation pattern to that of parent drug. However, there exist few fragments that are different from the parent drug. These unique fragment ions will help in proposing the soft spots for metabolites. In this case, soft-spot has to be proposed for di-demethylated metabolite of verapamil. Few fragments such as 165.10 and 150.10 are common in between the parent drug and metabolite. However a unique fragment ion (275.10) that is seen in metabolite does not exist with the parent drug. This fragment ion is a di-demethylated fragment ion of the fragment ion (303.20) seen in parent. This helped in proposing the di-demethylation reaction to a particular portion of the structure. Typically, this is the procedure followed in identifying and proposing the soft – spots for putative metabolites.