F 1050 fluorescence spectrophotometer

Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin (RF) in the perfusate of HMP were measured using a high-performance liquid chromatography (HPLC) method. The sample (perfusate) was deproteinized by mixing it with pure methanol, followed by centrifugation and filtration. An aliquot of the resulting deproteinized supernatant was directly injected into the HPLC system. The HPLC system consisted of a GASTORR BG-42 degasser (FLOM Co., Tokyo, Japan), L-7100 pump (Hitachi High-Tech Corporation, Tokyo, Japan), model 234 autoinjector (GILSON Inc., Middleton, WI, USA), ATC-10 column oven (Eicom, Kyoto, Japan), L-7400 UV detector (Hitachi), NOD-10 UV detector (Eicom), and F-1050 fluorescence spectrophotometer (Hitachi) with C18 column for reverse phase HPLC analysis InertSustain AQ-C18 (5 µm) and a guard column E (GL Science, Tokyo, Japan). The HPLC conditions were as follows: ODS column (EICOMPAK SC5-ODS; 3 µm, 150 × 4.6 mm), column oven (40 °C), UV–Vis detector (254 nm), fluorescence detector (excitation 445 nm, emission 530 nm), mobile phase (A. methanol, B. acetic acid buffer. A/B = 35/65, v/v), where the acetic acid buffer was a mixture of 4 M sodium acetate (20 mL) and 50% acetic acid (10 mL) in 1 L of deionized water; flow (0.7 mL/min), and injection volume (25 µL). FMN, FAD, and RF contents were expressed as mmol/L.