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Abstract: The specific determination of L-hydroxyproline (Hyp) can serve as a potential indicator for early clinical diagnosis of liver fibrosis. In this work, an integrated strategy based on 4-plex stable isotope labeling derivatization combined with dummy magnetic molecularly imprinted polymers (QSILD-DMMIPs) was developed for specific extraction and rapid determination of Hyp in human serum by ultra high performance liquid chromatography tandem mass spectrometry. A new series of QSILD reagents d0/d1/d2/d3-6-N-methyl-rhodamine 6G-N-hydroxysuccinimidyl formate (d0/d1/d2/d3-MRSF) were designed, synthesized and applied for the high-throughput labeling of Hyp in serum samples. The structural analogue derivative of Hyp with 6-N-ethyl-rhodamine 6G-N-hydroxysuccinimidyl formate (ERSF-Hyp) was synthesized and used as a novel dummy template to prepare DMMIPs. The DMMIPs were well characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), fourier transform infrared spectroscopy (FTIR), Brunner Emmet Teller (BET) measurements, thermogravimetric analysis (TGA), X-ray diffraction (XRD), zeta potential and adsorption experiments. All d0/d1/d2/d3-MRSF-Hyp derivatives were conveniently and specifically adsorbed by DMMIPs in magnetic dispersive solid phase extraction procedure before injection. Method validation results including linearity (0.2-100 ng mL-1), limits of detection and quantitation (0.05 and 0.2 ng mL-1), accuracy, precision, stability, matrix effect and derivatization efficiency were satisfactory. The analytical performances benefited from efficient integration of QSILD and specific DMMIPs extraction. The proposed strategy was successfully applied for Hyp determination in human serum of liver fibrosis patients and healthy controls, which was of great significance to early diagnosis
Template and target information: L-hydroxyproline
Author keywords: Hydroxyproline, Liver fibrosis biomarker, Multiplexed labeling derivatization, Surface imprinting technique, magnetic separation, Triple quadrupole mass spectrometry