Role of Liquid Chromatography–High Resolution Mass Spectrometry in Drug Metabolite Identification and Characterization: A Review

The metabolism of drugs is important in the determination of the pharmacological activity of the drug, the safety and the removal of the drug in the body. In the metabolism process, drugs are enzyme-transformed to produce metabolites of different chemical structure and activity. Such metabolic processes are primarily carried out in the liver and their mediation is by enzyme systems like the cytochrome, P450 [1,6]. Depending on the drug development process, it is imperative to understand drug metabolism since the metabolite can either be therapeutic, less effective or toxic. These metabolites are useful in the identification of the safety of drugs and their pharmacokinetic behavior [2].The conventional analytical methods have been largely utilized in the metabolite analysis, which includes high-performance liquid chromatography (HPLC) and gas chromatography (GC). These techniques are, however, not sensitive and resolute enough to detect metabolites with a low concentration in biological samples [3]. The coding of Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) has transformed the practice of identification of metabolites, giving it the ability to measure mass accurately and with high sensitivity. LC-HRMS allows the researcher to identify metabolites that are not known and their molecular structure of the metabolite by analyzing the fragments and the sophisticated data analyzers [4]. As they have such pros, LC-HRMS has emerged as an important method of analysis in drug metabolism, metabolomics studies, and quality control in pharmaceutical production.

2. Principles of LC-HRMS

2.1 Liquid Chromatography

Liquid chromatography is a separation method which separates the individual compounds in a mixture according to their interaction with the stationary and the mobile phases. LC is applicable in drug metabolism testing to isolate metabolites in a drug/biological sample (plasma, urine, tissue extract, etc.) [5,9].

Contemporary chromatographic analytical like Ultra-High-Performance Liquid Chromatography (UHPLC) is more resolute, faster, and sensitive than the traditional HPLC systems.

Workflow of liquid chromatography.

1. Sample and injection preparation
2. Separating in chromatographic column.
3. Metabolites elution at various retention times.
4. Moving of analytes to mass spectrometer[6].

3.High-Resolution Mass Spectrometry. Mass spectrometry is used to determine the mass-to-charge ratio (m/z) of ions at a high level of precision. The resolving power of HRMS instruments enables differentiation of substances with a very small difference in mass [7].

Common HRMS Analyzer

These analyzers typically provide mass accuracy within 1–5 ppm, enabling determination of molecular formulas of metabolites [8,15].

Drug Metabolism

 Sometimes drug metabolism takes place in two stages.

1. 1. Phase I Reactions

Phase I reactions include functionalization reactions that include

• Oxidation
• Reduction
• Hydrolysis

The reactions serve to introduce functional groups into the drug molecules and are primarily catalyzed by the cytochrome P450 enzymes .

Phase I metabolites are examples of hydroxylated or demethylated drug analogs.

1. 2. Phase II Reactions

 Phase II metabolism involves conjugation reactions which increase the polarization of the metabolites. Typical conjugation reactions are:

• Glucuronidation
• Sulfation
• Acetylation
• Glutathione conjugation

Such reactions enable release of metabolites either in the urine or bile [9,10].

Role of LC-HRMS in Regulatory Guidelines

LC-HRMS Applications on Regulatory Rules.

LC-HRMS plays an important role in ensuring the compliance of the regulatory requirements.

Key Guidelines

ICH M3 (R2) - Metabolite safety.

ICH Q3A/Q3B- detection of impurities.

Guidance- Metabolites in safety testing(MIST) issued by FDA.

LC-HRMS is used to identify the identified metabolites whose concentration exceeds the regulatory limit [10,11].

Ionization Techniques in LC-HRMS.

The ionization is significant in the change of the molecules to the ions.

The Electrospray Ionization (ESI).

Most commonly used

Applicable in case of polar and large molecules.

The formed ions are multiply charged.

Atmospheric Pressure Chemical Ionization (APCI)

Applicable to less polar compounds.

This is further improved in stabilized molecules In thermally sensitive molecules, this is further improved.

Atmospheric Pressure Photoionization (APPI)

Used for non-polar compounds

The sensitivity and detection of metabolites depend on the choice of ionization method. In the technique where a photoionized source of atoms has been impregnated on the plasma to create a sustained discharge within the entire chamber  [1,8].

High-Resolution Mass Analyzers Used in LC–HRMS

LC-HRMS systems have high-resolution mass spectrometry (HRMS) which employs the use of high-resolving power and mass accuracy very sensitive mass analyzers. The most enforced high-resolution types are the Orbitrap, Time-of-Flight (TOF), Quadrupole-Time-of-Flight (Q-TOF), and Fourier Transform Ion Cyclotron Resonance (FT-ICR) equipment.

 Orbitrap analyzer works with the principle of trapping the ions in an electrostatic field, and these ions oscillate around a center electrode; then, the frequency of this oscillation is converted into a mass-to-charge (m/z) value using Fourier Transform giving an extremely high resolution (up to 500,000 or more) and excellent mass accuracy (typically less than 2 ppm). Instead, the TOF analyzer isolates ions in different manners depending on the time that they require to travel through a flight tube following acceleration to the identical kinetic energy; lighter ions arrive at the detector sooner than heavier ones, which provide high resolution (10,000-60,000) and fast data acquisition. Q-TOF A hybrid system is a quadrupole mass filter used with a TOF analyzer allowing the selection of the precursor ion and high-resolution detection, so it is very convenient in tandem MS (MS/MS) as well as structural elucidation studies. FT-ICR is the most sophisticated analyzer, where the ions are allowed to travel in circular motion inside a strong magnetic field and the cyclotron frequencies of the ions are measured and transformed into mass spectra by Fourier Transform, this system is capable of a very high resolution (over 1,000,000) and very high mass accuracy though it is a fairly expensive and complicated system, mainly used in high-level research. In general, these high-resolution analyzers provide the determination of mass, characterization of the structure, and analysis of complex mixtures with high precision, which is why they have become the necessary equipment in the area of metabolomics, proteomics, environmental analysis, and pharmaceutical research.[3,12]

Role of Metabolomics in Drug Metabolite Identification

The metabolomics is a novel research methodology that has acquired an overwhelming impetus in investigating the overall modifications of the metabolism of the biological system. LC-HRMS is front burning in metabolomics because it is highly sensitive and specific in the determination of wide range of endogenous and exogenous metabolites. It enables non-specific and defined metabolite profiling that enables exploration of metabolic pathways in a global way in response to the treatment with a drug [20,21].

The process of identifying unknown metabolites and biomarkers with LC-HRMS will be performed by untargeted metabolomics, and the specific metabolites will be measured by the targeted methods. It is the dual aspect that makes LC-HRMS highly beneficial in the research studies regarding metabolism of drugs and identification of biomarkers [22,8].

5.The Metabolite Identification Strategies with LC-MS.

 There are a number of different strategies of identifying metabolites.

 The protonated metabolites were identified in large amounts. ESI-TOF positive molecules. MS mode and of the deprotonated molecules in the negative ESI-TOF MS mode. The mass error for each proposed metabolite and fragment ion structure was less than 5 ppm. In case of those metabolites where no MS/MS data were available. acquired, identification was performed according to the accurate mass of the. Fragmentation observed in the MS and the molecular ion spectrum. A original metabolite concentrations were less than those of detected metabolites .It is calculated as a percentage of the total MS area by dividing the MS peak location of the metabolite (or parent compound) and all identified metabolites[11,3].

5.1 Accurate Mass Measurement

Mass spectrometry with a high resolution gives accurate mass values, which enable the establishment of the molecular formula of metabolites [1].

5.2 Mass Defect Filtering

 Mass defect filtering is utilized to identify the metabolites according to predictable mass change of metabolic transformations [12]

5.3Extracted Ion Chromatograms (EIC)

IT were obtained by extracting the ion currents of different ionized species into the gas phase. Extracted ion chromatograms (EIC) were determined by extracting ion currents of various ionized species to the gas phase. EIC can selectively monitor ions in chromatographic data of predicted metabolite masses [13,15].

5.4 Tandem Mass Spectrometry (MS/MS)

 MS/MS fragmentation analysis is a structural information used to identify the structure of a metabolite [6].

5.5 Data Processing and Bioinformatics.

The HRMS systems of the modern world are based on the spectral interpretation software and prediction of the metabolites and database matching by the means of the modern software [14].

6.Drug Metabolism Studies using LC-HRMS.

6.1 Metabolite Identification

LC-HRMS finds extensive application in drug metabolism studies in pharmaceutical research to identify and to profile metabolites produced during drug metabolism.

 6.2 Pharmacokinetic Studies

LC-HRMS helps determine: Absorption Distribution Metabolism Excretion (ADME) These are critical parameters used to determine drug pharmacokinetics.

6.3 Toxicological Studies

Drug safety and the avoidance of adverse drug reactions are fundamentally based on the identification of the toxic metabolites [15].

6.4 Metabolomics Research

LC-HRMS has become a very important instrument in the field of metabolomics since it allows an in-depth examination of the endogenous metabolites and the metabolic pathways [16].

6.5 Impurity Profiling

HRMS is applied in quality control of pharmaceuticals to identify trace impurities and degradation products in drug preparations [18].

6.6 Water analysis

An important development in WBE methodology is the combination of high-resolution analytical methods with in vitro metabolic research [19].

Table: LC-HRMS Recurring in Pharmaceutical Research.