High Performance Liquid Chromatography/Atmospheric Pressure Ionization/Tandem Mass Spectrometry (HPLC/API/MS/MS) in Drug Metabolism and Toxicology

Abstract

Studies of the metabolic fate of drugs and other xenobiotics in living systems may be divided into three broad areas: (1) elucidation of biotransformation pathways through identification of circulatory and excretory metabolites (qualitative studies); (2) determination of pharmacokinetics of the parent drug and/or its primary metabolites (quantitative studies); and (3) identification of chemically-reactive metabolites, which play a key role as mediators of drug-induced toxicities (mechanistic studies). Mass spectrometry has been regarded as one of the most important analytical tools in studies of drug metabolism, pharmacokinetics and biochemical toxicology. With the commercial introduction of new ionization methods such as those based on atmospheric pressure ionization (API) techniques and the combination of liquid chromatography-mass spectrometry (LC-MS), it has now become a truly indispensable technique in pharmaceutical research. Triple stage quadrupole and ion trap mass spectrometers are presently used for this purpose, because of their sensitivity and selectivity. API-TOF mass spectrometry has also been very attractive due to its enhanced full-scan sensitivity, scan speed, improved resolution and ability to measure the accurate masses for protonated molecules and fragment ions. This review aims to survey the utility of mass spectrometry in drug metabolism and toxicology and to highlight novel applications and future trends in this field.

Analytical Methods for Quantification of Drug Metabolites in Biological Samples

“…For the feasibility to successfully monitor the drug metabolism, suitable bioanalytical methods have to be developed and validated. Studies of metabolic fate of drugs in living systems may be divided into three areas: 1) elucidation of biotransformation pathways, 2) determination of pharmacokinetics of the parent drug and/or its primary metabolites and 3) identification of chemically-reactive metabolites that are important in drug-induced toxicity [7].…”

“…A short overview of analytical strategies for identification of metabolites will be provided. More information regarding metabolite identification can be found in following review articles [7,[23][24][25][26][27]. The selection of suitable LC-MS instrumentation is needed for qualitative evaluation of metabolites.…”

“…While soft API interfaces, in particular electrospray, produce molecular ions with minimal fragmentation, high energy sources like EI mostly generate fragment ions. API techniques are most widely used for metabolite detection, identification and quantification [7,28] due to the ability to operate at atmospheric pressure, good compatibility with reversed phase chromatography and generation of intact molecule ions at very high sensitivity. All three API techniques: electrospay ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) are complementary.…”

“…Ions in solution are emitted into gas phase without application of heat making ESI suitable for analysis of thermo labile compounds. Many parameters, such as analyte and solution characteristics: pKa, analyte concentration, other electrolytes in solution, dielectric constant of the solvent, affect the ion formation process [7]. The effects of several mobile phase additives on the ionization efficiency have been reviewed [30] and will be discussed later (section 8).…”

“…Certain classes of compound such as heavily halogenated analoges and highly aromatic compounds will run readily on APCI while giving no or a weak response on ESI [7]. APCI like ESI produces ions based on the API strategy, but thought a completely different process.…”