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

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 208; p. 114462
• Main Authors: Li, Fumin, Xing, Gang, Cousineau, Christopher, Clemens, Sara, Mofikoya, Melissa, Kim, Moo-Young, Zhang, Jenny (Yanhua), Zhang, Yizhong, Raha, Nancy
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 20.01.2022
• Subjects: Aged; Chromatography, Liquid; Clinical; Fructose; HILIC-MS/MS; Humans; Hydrophobic and Hydrophilic Interactions; Quantitation; Reproducibility of Results; Tandem Mass Spectrometry; Urine; Validation; Urine; Validation; Clinical; Quantitation; Fructose; HILIC-MS/MS
• ISSN: 0731-7085; 1873-264X; 1873-264X
• DOI: 10.1016/j.jpba.2021.114462

•A HILIC-MS/MS method was successfully developed, validated, and employed to measure fructose in human urine samples.•Significant chromatography challenges arose when fructose improved over time and 13C6-fructose remained relatively constant.•It was critical to maintain consistent chromatographic performance for both surrogate and authentic analytes. 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.