HPLC method development and validation of cytotoxic agent phenyl-heptatriyne in Bidens pilosa with ultrasonic-assisted cloud point extraction and preconcentration

Extraction and pre-concentration of a bioactive marker compound, phenyl-1,3,5-heptatriyne from Bidens pilosa, prior to HPLC has been demonstrated using both organic and ecofriendly solvents. Non-ionic surfactants, viz. Triton X-100, Triton X-114 and Genapol X-80, were used for extraction. No back-extraction or liquid chromatographic steps were required to remove the target phytochemical from the surfactant-rich extractant phase. The optimized cloud point extraction procedure has been shown to be a potentially useful methodology for the preconcentration of the target analyte, with a preconcentration factor of 4-99. Moreover, the method is simple, sensitive, rapid and consumes lesser solvent than traditional methods. An isocratic chromatographic separation and quantitation was accomplished on a C(18) column with acetonitrile-acidified aqueous as mobile phase at a flow rate of 1.0 mL/min, UV detection at 254 nm and specificity with photo diode-array detector (PDA) and MS. Under the optimum experimental conditions recovery was satisfactory (99.18-100.33%) without interference from the surfactant. The method seems to be reliable with intraday precision and interday precision below 2.0%. Good linearity was obtained in the working range from 7.5 to 30 mu g/mL with correlation coefficient > 0.99. The limits of detection and quantitation were 1.84 and 6.13 mu g/mL, respectively. The method was validated following international guidelines and successfully applied for quantitative assays of cytotoxic compound phenyl-1,3,5-heptatriyne in Bidens pilosa. Copyright (C) 2010 John Wiley & Sons, Ltd.