Using LC–MS/MS to Analyze D-Amino Acids

Scientists from Florida State University in Tallahassee, Florida recently used Marfey’s reagent (MR) and reversed-phase liquid chromatography–tandem mass spectrometry (LC–MS/MS) to analyze D-amino acids secreted from islets of Langerhans, which are cells that defend against pathogens that enter the body through the skin. Their findings were published in the Journal of Chromatography B (1).

Chain of amino acid or biomolecules called protein - 3d illustration | Image Credit: © Christoph Burgstedt - stock.adobe.com

D-amino acids (D-AAs) are the mirror image of L-amino acids, or left-handed amino acids. While D-AAs play an important role in various biological processes (2). For example, some D-amino acids has been found to stop severe seizures, others are neurotransmitters, and some are complementary to neurotransmitters. D-AAs have been found in various tissues including the brain, kidney, and pancreas. These amino acids are also implicated in several diseases, such as Alzheimer’s, schizophrenia, epilepsy, and diabetes mellitus. Specifically, D-serine (D-Ser), D-alanine (D-Ala), and D-aspartate are involved in neurotransmission within the central nervous system, extending to cell signaling processes within the islets of Langerhans in the endocrine portion of the pancreas. Islets of Langerhans release hormones to maintain blood glucose homeostasis.

Currently, there are no reports or comprehensive assessments of D-AAs in islet secretions. It is important to understand how D-AAs function so proper treatment options can be developed for many of the diseases that D-AAs play a role in. To learn more about the release of these compounds, the scientists evaluated the effectiveness of 1-(9-fluorenyl)-ethyl chloroformate (FLEC reagent) and 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (MR) in separation of D/L-AA enantiomeric pairs in islet-specific buffers. Additionally, they used liquid chromatography–tandem mass spectrometry (LC–MS/MS) to analyze chiral separation. This can be done by using either a chiral stationary phase or by derivatizing analytes with a chiral agent, resulting in the formation of diastereomers which are then separated with an achiral stationary phase

Overall, the scientists found that MR-derivatized D/L AAs showed greater than baseline resolution (R_s ≥ 1.5) of 13 enantiomeric pairs when using a non-linear gradient and an acidic mobile phase system; the FLEC-derivatized AAs, however, exhibited limited resolution on both biphenyl and C18 columns. The optimized MR method yielded highly reproducible separation with retention times of less than 1%RSD. Further, the scientists obtained excellent linearity between the analyte concentrations and response (R² > 0.98), with less than 15% RSF being yielded for all analyte responses. Most of the analytes had limits of detection (LODs) at or below 100 nM, with exception to L-Ala, which had a LOD of 200 nM.

The optimized MR method was then used to quantify D-AAs in secretions of 150 murine islets after incubation in 3- and 20-mM glucose. In both scenarios, D-Ser and D-glutamine were tentatively identified by comparing retention times and quantifier-to-qualifier ion rations, both with standards and from spiking experiments. Both D-amino acides were secreted in low quantities, which did not significantly differ in either low (D-Ser: 44 ± 2 fmol islet^-1h⁻¹; D-Gln: 300 ± 100 fmol islet^-1h⁻¹) or high (D-Ser: 23 ± 1 fmol islet^-1h⁻¹; D-Gln: 120 ± 50 fmol islet^-1h⁻¹) glucose across 3 biological replicates. These findings show the robust nature of the developed method. With this, the scientists said it “can be applied to further examine the release of D-AAs and their potential roles in islet physiology” (1).

References

(1) Ogunkunle, E. O.; Davis, J. J.; Skinner, E. L. Thornham, J.; Roper, M. G. Analysis of D-Amino Acids Secreted from Murine Islets of Langerhans Using Marfey’s Reagent and Reversed Phase LC-MS/MS. J. Chromatogr. B. 2023, 1231, 123928. DOI: 10.1016/j.jchromb.2023.123928

(2) Armstrong, D. W. The Role of D-Amino Acids in Humans. AZoNetwork 2021. https://www.news-medical.net/news/20210304/The-Role-of-D-Amino-Acids-in-Humans.aspx (accessed 2024-5-9)