Over the last 5 years, multiple LC-MS-MS methods have been developed to quantitate small-molecule biomarkers such as citrulline, arginine, and kynurenine. Since these biomarkers are highly polar and very low molecular weight (&lt;250 amu), they are typically not amenable to the conventional reverse-phase chromatography used in traditional LC-MS-MS quantitation approaches. Therefore methods based on a HILIC (hydrophilic interaction liquid chromatography) approach have been developed. These biomarker quantitation methods have assisted drug discovery projects in a variety of stages. In early discovery, screening biomarker levels across multiple tumor cells lines has helped identify the optimal cell lines for syngeneic models, and provided orthogonal correlation with enzymatic expression western blot data. In later stage projects, these methods have quantified the effect of tool and novel drug compounds being investigated, in both cellular and <i>in vitro assays</i>. For development compounds, citrulline quantitation has helped identify optimal dosing schedules to mitigate gut toxicity. These methods have been compatible with a wide variety of sample matrices, including plasma, tumor lysates, cell incubation media, and homogenized animal feed.<br/><br/>The most inclusive method can be used to quantitate eight biomarkers simultaneously: arginine, citrulline, glutamine, methionine, and tryptophan at 1 - 1024 µM; ornithine at 4 - 1024 µM; leucine at 1 - 256 µM; and kynurenine at 0.04 - 10.24 µM. These ranges cover the levels most typically seen within the biological samples tested. Measuring accuracy and precision across six validation sequences, all standards and controls showed &lt;15% bias; average standard bias at each compound level was &lt;10% and average QC bias at each compound level was &lt;12%. Maximum %CV for all compound levels was 8.1%. This HILIC LC-MS-MS method is robust, reliable, and high-throughput at 3.5 minutes per sample run.

Biomarker quantitation by HILIC LC-MS-MS

256th National Meeting (2018)

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