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 150×4 mm id, 5 μm) 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.