Figure 2. HPLC profile of the retained material (RM) of disposable C-18 reversed-phase cartridges on a reversed-phase column.

(a), The RM loaded onto the column was eluted with a linear gradient of acetonitrile (ACN) and collected in 50 fractions of 2 ml each. Aliquots (20 μl) of each fraction were evaporated to dryness, dissolved in the physiological saline (pH 7.3) and applied to the bioassay. The bioactive fractions were indicated by the horizontal bar. (b), HPLC profile of the bioactive fractions (in panel a) on a cation-exchange column. Elution was performed in a linear gradient of NaCl in PB (pH 6.8) and eluents were collected in 1-ml fractions. The bioactivity was detected in the flow through fractions (horizontal bar). (c), Final purification of the bioactive substance (0.21 μmoles) by HPLC using a reversed-phase column. Isocratic elution with 36% ACN at a flow rate of 0.3 ml/min. (d), Bioactivity of the isolate on spontaneous contractions of the isolated female vagina. The upward arrow indicates application of an aliquot (2.5 × 10⁻¹¹ M) of the isolate.