High performance liquid chromatography for the determination of glucosamine sulfate in human plasma after derivatization with 9-fluorenylmethyl chloroformate.
In this study, we developed a simple, rapid, sensitive, and reliable method for the determination of glucosamine sulfate in human plasma, which was based on derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by reverse-phase HPLC-FLD. For the first time, FMOC-Cl was introduced into derivatization of glucosamine sulfate in human plasma. The amino groups of glucosamine sulfate and vertilmicin sulfate (the internal standard) were trapped with FMOC-Cl to form glucosamine-FMOC-Cl and vertilmicin-FMOC-Cl adducts, which can be very suitable for HPLC-FLD. Precipitation of plasma proteins by acetonitrile was followed by vortex mixing and centrifugation. Chromatographic separation was performed on a C18 column (DIAMONSIL 150 x 4 mm id, 5 microm) with a mobile phase gradient consisting of acetonitrile and water at a flow-rate of 1 mL/min. The retention times of glucosamine-FMOC-Cl and vertilmicin-FMOC-Cl adducts were 8.9 and 21.2 min, respectively. This method was shown to be selective and sensitive for glucosamine sulfate. The limit of detection was 15 ng/mL for glucosamine sulfate in plasma and the linear range was 0.1-10 mg/mL in plasma with a correlation coefficient (r) of 0.9999. The relative standard deviations (RSDs) of intra-day and inter-day assays were 5.2-8.1% and 6.1- 8.5%, respectively. Extraction recoveries of glucosamine sulfate in plasma were greater than 90%. The validated method was successfully applied to the determination of glucosamine sulfate in human plasma samples.
Huang
Tao-min
TM
Department of Pharmacy, Fudan University, Shanghai, PR China.
Deng
Chun-Hui
CH
Chen
Nian-zhu
NZ
Liu
Zhen
Z
Duan
Geng-Li
GL
D001774:Blood Chemical Analysis / Q000379:methods / Q000706:statistics & numerical data; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D019587:Dietary Supplements / Q000032:analysis; D005449:Fluorenes; D005944:Glucosamine / Q000008:administration & dosage / Q000097:blood*; D006801:Humans; D007202:Indicators and Reagents; D012680:Sensitivity and Specificity
D005449:Fluorenes; D007202:Indicators and Reagents; C054007:1-(9-fluorenyl)methyl chloroformate; D005944:Glucosamine