Quantitative sphingosine measurement as a surrogate for total ceramide concentration—preclinical and potential translational applications

Abstract

Biomarkers are an increasingly important constituent of the drug development process, offering the potential of increased efficiency through reduced compound attrition and earlier proof of mechanism and/or efficacy. Assays developed for compound screening that can be directly translated for clinical trials are especially valuable, but their successful adoption requires a careful balance between assay performance and implementation costs. One such 'fit-for-purpose' biomarker assay, the indirect measurement of pharmacological modulation of sphingolipid biosynthesis and disposition, is presented here. Among sphingolipids, numerous ceramide species are readily detectable in different lipoprotein fractions of mammalian plasma, but their parallel quantification can be prohibitively expensive and time consuming. Ceramides differ in their fatty acid moiety, which is readily removed by hydrolysis, yielding a common sphingosine derivative, the measurement of which serves as an indicator of total ceramide. When followed by liquid chromatography tandem mass spectrometry (LC/MS/MS) for detection, robust analyte quantification becomes relatively straightforward. The practical utility of a method developed to be fit for the purpose of rapidly and quantitatively measuring treatment-induced variations in total ceramide from hamster plasma and individual lipoprotein fractions is described. With a linear calibration range from 0.003 to 33.4 microm sphingosine, precision and accuracy error in plasma-based quality controls spiked with ceramides was less than 15%. The specificity of the assay for ceramides was also assessed. The simplicity of the method would allow for its potential translation to other preclinical species, as well as for clinical applications in later-stage drug development.

Development and validation of LC-MS/MS method for determination of very long acyl chain (C22:0 and C24:0) ceramides in human plasma

“…including thin-layer chromatography (TLC) [24], high performance liquid chromatography (HPLC)[12], immunochemical methodologies [25], enzymatic assay [13], tandem mass spectrometry[22], liquid chromatography–tandem mass spectrometry (LC-MS/MS) [11,14–17,21,26], gas chromatography[18,19], and gas chromatography-mass spectrometry [18,19,23]. While assays for Cer(22:0) and Cer(24:0)have previously been reported [14,16,26], these types of assays, however, are not well-suited for clinical application because matrix-based quality control (QC) samples are not used to monitor assay performance and robustness, and sample throughput tends to be low. Here, we describe a high-throughput method for determination of Cer(22:0) and Cer(24:0) that is validated according to the Food and Drug Administration (FDA) bioanalytical method validation guidance [27] and the principles of fit-for-purpose assay method development for biomarker measurement [28,29].…”