Development of a Fluorescent Labeling Reagent for Determination of Proline and Hydroxyproline in Myeloma Patient Plasma by HPLC-FLD.
A fluorescent derivatization reagent, 3,6-dimethoxy-9-phenyl-9H-carbazole-1-sulfonyl chloride (DPCS-Cl), previously designed, synthesized and developed for amino acids labeling, is here employed. It was used as a pre-column derivatization reagent for the determination of proline (Pro) and hydroxyproline (Hyp) with high-performance liquid chromatography. Both of the analytes were labeled with DPCS-Cl at 60°C for 30 min. The optimized concentration of DPCS-Cl was 25 µg mL-1and the molar ratio of analytes to DPCS-Cl was 1:6. Excitation wavelength of 318 nm and emission wavelength of 440 nm were selected for the fluorescence detection. For Pro and Hyp, the linear correlation coefficients were all >0.999; the linear ranges of calibration curve were all 5.0 nmol L-1to 5.0 μmol L-1; the detection limits were 20.0 and 10.0 fmol, respectively; the intra-day and inter-day relative standard deviations were 2.6-3.3% and 3.5-4.6%, respectively. This reagent was applied to the determination of Pro and Hyp in plasma of myeloma patients with its merits of high efficiency of derivatization, stability of the derivatives and high sensitivity.
College of Chemistry and Chemical Engineering, Jiangsu Key Laboratory of Environmental Engineering and Monitoring, Yangzhou University, Yangzhou 225002, China.
Zhenyuan
Z
Teng
College of Chemistry and Chemical Engineering, Jiangsu Key Laboratory of Environmental Engineering and Monitoring, Yangzhou University, Yangzhou 225002, China.
Hongying
H
Lv
College of Chemistry and Chemical Engineering, Jiangsu Key Laboratory of Environmental Engineering and Monitoring, Yangzhou University, Yangzhou 225002, China.
Yaoyao
Y
Zhang
College of Chemistry and Chemical Engineering, Jiangsu Key Laboratory of Environmental Engineering and Monitoring, Yangzhou University, Yangzhou 225002, China.
Yi
Y
Zhang
Clinical Medical College of Yangzhou University, Subei People's Hospital of Jiangsu province, Yangzhou 225002, China guanjun-tt@163.com.
Jun
J
Guan
College of Chemistry and Chemical Engineering, Jiangsu Key Laboratory of Environmental Engineering and Monitoring, Yangzhou University, Yangzhou 225002, China.
Wang
W
Chengyin
D001774:Blood Chemical Analysis / Q000379:methods* / Q000592:standards; D002227:Carbazoles; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006909:Hydroxyproline / Q000097:blood*; D057230:Limit of Detection; D009101:Multiple Myeloma / Q000097:blood*; D011392:Proline / Q000097:blood*; D013050:Spectrometry, Fluorescence
D002227:Carbazoles; D005456:Fluorescent Dyes; C041514:carbazole; D011392:Proline; D006909:Hydroxyproline