HPLC Analysis of Diazepam and Metabolites in Serum on Discovery^® C18 after SPE using Discovery^® DSC-8

sample preparation

SPE (Solid Phase Extraction)

sample/matrix

goat serum spiked with diazepam and metabolites at 0.5 μg/mL and diluted 1:1 in 25 mM ammonium formate, pH 7.1

SPE well plate

Discovery DSC-8 96-well SPE Plate, 100 mg/well (575627-U)

condition

1 mL methanol followed by 1 mL DI water.

sample addition

1 mL spiked goat serum

washing

1 mL 25% methanol in 25 mM ammonium formate, pH 2.75

elution

1 mL 60% methanol in 25 mM ammonium formate, pH 2.75

elution

Evaporate eluate with nitrogen purge (30 °C; ~15 min.). Reconstitute in 200 μL mobile phase.

column

Discovery C18, 5 cm x 4.6 mm I.D., 5 μm particles (504947)

mobile phase

[A] Methanol; [B]10 mM ammonium acetate, pH 4.5;(45:55, A:B)

flow rate

1.5 mL/min

column temp.

35 °C

detector

UV, 240 nm

injection

25 μL