HPLC Analysis of Placental 5-HT and 5-HIAA

Concentrations of 5-HT and 5-HIAA in the cell-free supernatant and placental perfusate were determined using a Shimadzu LC20 Performance HPLC chromatograph (Shimadzu, JP) as described previously [3 (link)]. A Kinetex EVO C18 100 A 150 × 3 mm, particle size 5 μm column (Phenomenex, USA) with a guard column was used. Analytes were eluted with a biphasic mobile phase consisting of A − 3:97 (v/v) methanol:acetic acid (0.1 M, pH 4.5, adjusted with NaOH) and B - methanol. The proportion of B was 0% for 0–8.4 min, then linearly increased to 20% at minute 9.6, held at 20% until minute 22.6, linearly decreased to 0% after minute 23.2 then held at 0% until minute 30. Excitation and emission wavelengths of the fluorescence detector were set for individual compounds: 280/334 nm for 5-HT from 0 to 6.5 min and 276/333 nm for 5-HIAA from 6.5 to 18 min.