[Simultaneous determination of five water-soluble vitamins in human serum by high performance liquid chromatography]

To establish a method for the simultaneous determination of 5 water-soluble vitamins including VB1, VB6, Nicotinic acid, Nicotinamide and Folic acid by high performance liquid chromatography.

Methanol (200 microL) was added to 50 microL human blood sample to precipitate proteins. The sample was frozen (-18 degrees C) for 30 min, and then centrifuged at 8000 r/min for 8 min. The supernatant was subsequently applied to the solid-phase cartridge for further purification. The elute was collected and evaporated to 0.20 mL under nitrogen in a water bath at the temperature of 50 degrees C and finally 50 microL of the sample was injected onto HPLC column for analysis. The five water-soluble vitamins were determined under optimized condition of solid phase extraction and chromatograph.

The optimized condition for solid phase extraction (SPE) was identified as: AGT cleanert ODS C18 column as SPE column and CH3OH (2.4 mL)-H2O (0.6 mL) mixed liquor as eluent, with a flow rate of elution of 0.5 mL/min. The optimized chromatographic condition was identified as, Phenomenex C18 Luna column (250 x 4.6 mm, 5 microm) as analytical column under a temperature of 20 degrees C and 0.05 mol/L potassium dehydrogenates phosphate buffer (pH 6.0) and methanol as gradient elution with mobile phase with a flow rate of 0.9 mL/min. The detection wavelength was 266 nm. The correlation coefficients for the standard curves were greater than 0.999 in the range from 0 to 100 ng (RSD < 5%). The detection limits (S/N 2) ranged from 0.076 to 0.170 microg/mL. The recovery rate ranged from 80.2% to 115.0%. Except for Nicotinamide, the other four vitamins were detected successfully in the blood sample.

This quick and convenient method can be applied to the simultaneous determination of 4 water-soluble vitamins in human serum samples. But the sensitivity for detecting Nicotinamide needs to be improved.