New Study Outlines on SFC Technique for Chiral Bioanalysis

A team of scientists from Bristol Myers Squibb, led by Takao Yamaguchi and Satoshi Obika, recently developed a new approach for chiral bioanalysis based around supercritical fluid chromatography–tandem mass spectrometry (SFC–MS/MS). Their findings were published in the Journal of Chromatography A (1).

Laboratory worker scientist laboratory assistant tests chemicals for reactions in test tubes, interfering with different substances and obtaining a visual reaction. Concept of: Science, Chemistry. | Image Credit: © dkHDvideo - stock.adobe.com

A chiral center is an atom that has four different groups bonded to it, which causes it to have a nonsuperimposable mirror image (2). Various pharmaceuticals contain at least one chiral center. These stereoisomers (isomers that differ in spatial arrangement of atoms instead of order of atomic connectivity) contain the same molecular and structural formula. However, they possess different spatial orientations, which can have significant impact on interactions with their intended targets, such as enzymes, transporters, and receptors (3). When a dosing drug is a racemic material, or an equimolar mixture that consists of two optically inactive enantiomers, significant differences in the pharmacokinetic (PK) profile of each enantiomer can impact drug safety or efficacy due to differential PK/pharmacodynamic (PD) properties (4). However, if a dosing material is a pure enantiomer, significant chiral inversion in vivo can lead to its antipode with different PK/PD properties. This can cause different therapeutic and toxicological effects.

According to the researchers, SFC has not been a mainstay of bioanalytical laboratories due to old generations of SFC instrumentation having limitations on robustness and reliability (1). A variant of high-performance liquid chromatography (HPLC), SFC involves a compressible fluid at high density acting as a solvent (5). However, with newer generations of SFC instruments and chiral columns having significant advancements, the technique has new potential for implementation in bioanalytical laboratories. This is especially the case for difficult analysis of chiral separations where traditional normal/reversed phase and polar organic mode chromatography are not adequate or require long run times.

In this study, the scientists used six model chiral compounds to systematically examine key aspects of SFC–tandem mass spectrometry (SFC–MS/MS) and ultra-high performance SFC–MS/MS (UHPSFC–MS/MS) for chiral bioanalysis, such as chiral columns and organic modifiers/additives. Moreover, they utilized 3-sub micron particles and below for regulated bioanalysis, specifically by developing, qualifying, and validating chiral methods focused on the separation of immunomodulatory chiral imide drugs, a class of drugs that contain a glutarimide ring and that are known to be protein degraders. The compounds with different R1 and R2 groups had no additional chiral centers besides the one in the glutarimide ring. This motif, a key structural component in thalidomide, has been extensively studied due to its significant pharmacological properties. Previous research for this compound class typically utilizes chiral high-performance liquid chromatography (HPLC) in normal phase, reversed phase, or polar organic mode to separate the enantiomers of thalidomide and other imide drugs; these can provide valuable insights into their stereochemistry and biological activities.

While traditional HPLC has tremendous chiral separation capability, there is a strong demand to explore modern SFC with more sophisticated chiral supports to achieve better resolution and efficiency; this is especially so with developing immobilized columns and novel chiral stationary phases. Additionally, UHPSFC–MS/MS was shown to offer robust and reliable chiral assays, displaying good sensitivity, peak resolution, and sample throughput, while being well-suited for chiral separation in regulated bioanalysis.

References

(1) Santos, I. C.; Zhang, Z.; Luo, L.; et al. Emerging Applications of Quantitative Supercritical Fluid Chromatography-Tandem Mass Spectrometry for Chiral Bioanalysis. J. Chromatogr. A 2025, 1744, 465727. DOI: 10.1016/j.chroma.2025.465727

(2) Introduction to Chirality and Chiral Centers. Socratic Q&A 2015. https://socratic.org/organic-chemistry-1/r-and-s-configurations/introduction-to-chirality-and-chiral-centers (accessed 2025-2-20)

(3) Chirality and Stereoisomers. LibreTexts Chemistry 2025. https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Supplemental_Modules_(Organic_Chemistry)/Chirality/Chirality_and_Stereoisomers (accesseed 2025-2-24)

(4) What’s a Racemic Mixture? Master Organic Chemistry 2024. https://www.masterorganicchemistry.com/2012/05/23/whats-a-racemic-mixture/ (accessed 2025-2-20)

(5) Supercritical Fluid Chromatography. ScienceDirect 2007. https://www.sciencedirect.com/topics/chemical-engineering/supercritical-fluid-chromatography (accessed 2025-2-20)