Development and validation of a HILIC-MS/MS method for the quantitation of fructose in human urine in support of clinical programs.

In a previous publication [1], a 20-minute UPLC®-MS/MS method, employing a surrogate analyte approach, was developed and validated to measure fructose and sorbitol, as mechanistic biomarkers, in human plasma to support first-in-human (FIH) studies. Different from plasma which maintains its homeostasis, urine has no such homeostasis mechanisms [2], therefore it is expected to be able to accommodate more changes. Here we describe the development and validation of a LC-MS/MS method for the quantiation of fructose in human urine to support clinical trials. A hydrophilic interaction chromatography (HILIC) method using an Asahipak NH2P-50 column (Shodex, 4.6 × 250 mm, 5 µm) was developed. Acetone precipitation was utilized to extract fructose from urine. For validation, stable isotope-labeled 13C6-fructose was used as the surrogate analyte for fructose in the preparation of calibration curves. QCs were prepared using both the surrogate analyte (13C6-fructose) and the authentic analyte (fructose). Difficulties were encountered for post-extraction stability experiments especially for authentic fructose QCs at low concentrations. Extensive troubleshooting revealed that fructose's chromatography improved as the column aged. As a result, the response factor of fructose increased over time for low concentration samples, leading to failed post-extraction stability experiments. A column cleaning procedure was implemented to ensure consistency in chromatography performance. The HILIC-MS/MS method was successfully validated and applied to analyze clinical samples with a 91% overall run passing rate.

Development and validation of a quantitative ultra performance LC® hydrophilic interaction liquid chromatography MS/MS method to measure fructose and sorbitol in human plasma.

AIM: Fructose and sorbitol are utilized as biomarkers for nonalcoholic steatohepatitis. Measurement of fructose and sorbitol levels helps understanding disease progression, drug response and underlying mechanism. MATERIALS & METHODS: Stable isotope-labeled fructose and sorbitol were used as surrogate standards and internal standards. Human plasma samples were processed and analyzed by ultra performance LC®-MS/MS via chromatographic separation on a hydrophilic interaction liquid chromatography analytical column without derivatization. Assay was validated with biomarker fit-for-purpose concept. RESULTS: A 12-min ultra performance LC®-MS/MS method was developed and validated to directly measure fructose and sorbitol in human plasma with acceptable intra- and inter-assay precision and accuracy. CONCLUSION: This sensitive, selective, and high-throughput assay with suitable dynamic ranges was successfully applied to clinical studies to provide reliable fructose and sorbitol biomarker data.