Advanced Chromatographic Resolution of Chiral Compounds, Part V: APIs

Active pharmaceutical ingredients, or APIs, are the biologically active substances responsible for the therapeutic effect of medicines. In modern pharmaceutical development, more than half of the APIs currently synthesized and marketed possess one or more chiral centers, meaning that they would exist as enantiomers if not properly separated.

In biological systems, receptors, enzymes, and transporters exhibit strict stereoselectivity. As a result, enantiomeric pairs of a drug molecule often display profoundly distinct pharmacodynamic, pharmacokinetic, and toxicological pathways. While one enantiomer (the eutomer) delivers the intended therapeutic effect, its mirror-image counterpoint (the distomer) can be inactive, less effective, or potentially toxic.

For these reasons, the strict separation and determination of enantiomers are critical throughout drug discovery, development, and quality control. Regulatory bodies worldwide, including the US FDA and ICH, strictly dictate that chiral impurities in APIs must be profiled, quantified, and limited (typically to less than 0.15%) to ensure safety, efficacy, and batch-to-batch consistency.

In this article, we present application notes that cover five target APIs, each representing a distinct analytical challenge: Nomifensine, Atorvastatin calcium, Orforglipron, Lubabegron fumarate, and Relebactam.

• Nomifensine is an antidepressant, characterized by an N-methylated tetrahydroisoquinoline skeleton with a single chiral center, where the secondary and tertiary amine functions impart strong basicity to the molecule. Under ordinary HPLC conditions, these basic centers interact strongly with residual silanol groups on the silica support, causing severe peak tailing, loss of resolution, and unpredictable retention times.
• Atorvastatin calcium is a well-established HMG-CoA reductase inhibitor, containing two stereogenic centers on its dihydroxypentanoic acid side chain, forming four distinct stereoisomers: the therapeutic (3R, 5R)-form, its enantiomer (3S, 5S) which the application note focuses on, and two diastereomeric pairs.
• Orforglipron is a novel, non-peptide small-molecule GLP-1 receptor agonist containing a highly crowded, sterically hindered environment surrounded by multiple hydrogen-bonding domains. Achieving high enantiomeric excess requires an analytical method that can disrupt strong non-specific interactions to target the dense chiral core.
• Lubabegron fumarate contains a single chiral center located on the β-carbon of its hydroxypropylamine side chain. The primary challenge lies in baseline-resolving the active (2S)-enantiomer from its antipode while avoiding interference from the geometric trans isomers of the fumarate counterion.
• Relebactam features a highly strained, bridged azabicyclic core containing multiple chiral centers. Its exceptional polarity and permanent charge distribution make retention on conventional reversed-phase columns highly problematic, requiring a chiral stationary phase capable of multi-modal interactions.