Which Methods do Experienced Bioanalytical Testing Labs Implement to Assess Metabolites in Active Drugs

The assessment of metabolites in active drugs is a fundamental component of the drug discovery and development process. Drug metabolites can often be toxic or pharmacologically active. Hence, studying the metabolic fate of drug products becomes even more crucial while evaluating and assessing drug compounds. Evaluating drug metabolites early in the discovery process provides data for choosing drug compounds with desirable safety and pharmacokinetic profiles. Data suggest that increased focus on drug metabolism and pharmacokinetic profiles (DMPK) has reduced the drug attrition rate from 40% to 10% between 1990 and 2000.

Assessing these drug metabolites in complex biological samples needs the development of robust analytical methods. Bioanalytical testing services employ several methods for detecting and quantifying drug metabolites. LC-MS and LC-MS/MS systems have emerged as an ideal option for evaluating active drug metabolites. Due to being inherently sensitive, specific, and speedy, LC-MS/MS systems are widely used in metabolism and pharmacokinetic studies. Moreover, HPLC units can also be coupled with different detector units such as UV detectors. Similar to LC-MS systems, assay development services also use gas chromatography and electrophoretic methods for analyzing active drug metabolites. In this article, we discuss the most used methods to measure active drug metabolites in complex biological matrices.

Liquid chromatography

An efficient chromatographic separation is the basis of robust and accurate metabolite quantification. As mentioned above, liquid chromatography can be coupled with UV or MS detector units. However, for non-MS systems, it is necessary to achieve a baseline resolution. In LC-MS systems, although extensive separation is not required for drug products, enough resolution is needed for certain drugs to avoid MS interferences. Apart from this, reverse phase chromatography and hydrophilic interaction chromatography are also used for analyzing active drug metabolites.

Nevertheless, different drug metabolites such as glucuronides and hydroxylated metabolites may share similar MRM transitions and can be a challenge during LC-MS analysis. Moreover, unstable metabolites can get converted to the parent drug through thermal degradation or in-source dissociation. In addition, liquid chromatography may also face matrix effects through interference with endogenous compounds.


Gas chromatography coupled with mass spectrometry is beneficial in analyzing trace amounts of non-polar, organically extractable, and volatile compounds. For polar compounds, GC-MS requires analyte extraction into a volatile solvent, increasing extensive time for sample preparation. This drawback was the primary reason for the use of LC-MS in assessing active drug metabolites. Nonetheless, GC-MS is still beneficial in the analysis of forensic toxicology and doping studies.

Capillary electrophoresis

Capillary electrophoresis is another technique used in the analysis of active drug metabolites. Capillary electrophoresis provides high efficiency, higher resolution, and rapid sample analysis. Moreover, compared to other HPLC techniques, capillary electrophoresis is simple, reduces the time associated with sample preparation, and saves valuable solvents. Thus, it provides ease of access for method development services. However, capillary electrophoresis offers low sensitivity, and therefore other techniques are usually selected for assessing active drug metabolites.

The road ahead

Current early drug discovery studies are focused on DMPK properties to reduce the drug attrition rate as early as possible during drug development. Active drug metabolites can pose several problems associated with drug-drug interactions, bioavailability, and drug toxicity. Hence, assessing active drug metabolites early in the drug development process will likely increase the success of a drug compound.