1. Rapid determination of free prolyl dipeptides and 4-hydroxyproline in urine using flow-gated capillary electrophoresis
- Author
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Maojun Gong, Ning Zhang, and Qingfu Zhu
- Subjects
Adult ,Male ,0301 basic medicine ,Urine ,Sensitivity and Specificity ,01 natural sciences ,Biochemistry ,Analytical Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Hydroxyproline ,Capillary electrophoresis ,Nipecotic acid ,Humans ,Proline ,Derivatization ,Detection limit ,Chromatography ,010401 analytical chemistry ,Electrophoresis, Capillary ,Reproducibility of Results ,Dipeptides ,0104 chemical sciences ,030104 developmental biology ,chemistry ,Standard addition - Abstract
Unhydrolyzed prolyl hydroxyproline (Pro-Hyp) and total 4-hydroxyproline (Hyp) in urine have been suggested as disease biomarkers for bone turnover and osteoporosis. Here, a rapid method was developed to accurately and selectively determine free prolyl compounds in unhydrolyzed urine samples. Urine samples were treated with o-phthalaldehyde to block primary amines followed by selective fluorogenic derivatization of secondary amines using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) at room temperature. The derivatized mixture was then directly analyzed and quantitated on a flow-gated capillary electrophoresis system. Six prolyl compounds: Pro-Hyp, Pro-Pro, Pro-Gly, Pro-Leu, Hyp, and Pro in unhydrolyzed urine samples were separated in 30 s, which was 60-fold faster than the reported HPLC method, using the separation buffer (pH 9.2) composed of tetraborate, cholate, and deoxycholate at 40 mM each. The limits of detection were ~ 20 nM for the dipeptides and ~ 60 nM for Hyp and Pro. The levels of these prolyl compounds in fresh urine samples were determined by using the one-point standard addition method with nipecotic acid as the internal standard. The present protocol was significantly simplified compared with reported techniques, which could improve accuracy and analytical speed. This method is potentially useful in the determination of prolyl dipeptides and Hyp in biological fluids. Graphical abstract Rapid quantitative analysis of prolyl dipeptides in urine using flow-gated capillary electrophoresis coupled with laser-induced fluorescence detection.
- Published
- 2017