Determination of methoxamine in human plasma and urine by a validated HPLC-fluorescence detection method

A sensitive HPLC method for the determination of methoxamine in human plasma and urine is described. The method is based on derivatization of methoxamine with 4-chloro-7-nitrobenzofurazan in borate buffer of pH 9.5 to yield a yellow, fluorescent product. Isocratic HPLC separation was achieved on an Inertsil C18 column (250 x4.6 mm id, 5 microm particle size) using the mobile phase methanol-water (60+40, v/v) at a flow rate of 1.2 mL/min. Fluorescence detection was used at excitation and emission wavelengths of 458 and 521 nm, respectively. The assay was linear over the concentration ranges of 10-250 and 20-300 ng/mL for plasma and urine, respectively. The LOD values were 3.3 and 6.8 ng/mL and the LOQ values were 10 and 20 ng/mL for plasma and urine, respectively. The extraction recoveries were more than 97.10%. After strict validation, the method indicated good performance in terms of linearity, sensitivity, precision, accuracy (recovery), robustness, and system suitability, and it was successfully applied to the determination of methoxamine in human plasma and urine.