Does anyone have a method for the determination of hypercin and hyperforin by HPLC?
The samples were eluted isocratically using a mobile phase consisting of acetonitrile and 0.1% v/v phosphoric acid (90:10, v/v) at a 1.5 ml/min flow rate with UV (273 nm) detection.
Reference;
Development of a simple, rapid and reproducible HPLC assay for the simultaneous determination of hypericins and stabilized hyperforin in commercial St. John's Wort preparations, Gerlie C de los Reyes,
Robert T Koda, Journal of Pharmaceutical and Biomedical Analysis, Volume 26, Issues 5–6, December 2001, Pages 959–965
Gudluck
:)
Hi salma
Good seperation of different constituents was obtained by 0.3% phosphoric acid (A); acetonitrile (B) and methanol (C). The gradient elution was: 0-12 min 100-80% A; 0-15% B; 0-5% C; 12-20 min 10%A; 75% B, 15% C; 20-27 min 5%A; 80%B, 15%C; 27-30 min 85%A and 15% B. The flow rate was 1 ml/min and the detection wave were 270 nm for the most components (hyperforine and derivatives) and 590 nm for hypericin and derivatives. You can see Hosni et al., 2011 Biochemical systematics and ecology:39:43-50.
In addition to what Robin has said, the chemical profiles of the aliquots obtained may be monitored by using TLC analysis by running the two samples collected alongside authentic samples of hypericin and hyperforin in an appropriate solvent system.
HPLC assay of hypericin, pseudohypericin and hyperforin
Liquid chromatographic analysis of hypericin and pseudohypericin was performed on a Shimadzu HPLC system. Hypericins were eluted isocratically, at 30 °C, on a reversed-phase Hypersil C18 column protected by a guard column of the same material and quantified by fluorescence detection at 322/593 nm (ex/em). The mobile phase was prepared by mixing 95 volumes of methanol with 5 vol. of phopshate buffer solution (pH 2.2). For the preparation of the buffer solution, 2.5 g of KH2PO4 was dissolved in 950 ml double distilled water, adjusted to a pH of 2.2 with concentrated phosphoric acid and filled up to 1000 ml with double distilled water. The mobile phase was filtered before use and was delivered isocratically at a flow-rate of 1 ml/min.
Hyperforin was chromatographed on the same HPLC system furnished with a spectrophotometric detector SPD 10AV at 276 nm, connected in series with fluorescence detection, as per conditions for hypericins. All the analytes were quantified using peak heights.
J Chromatogr A. 2005 Nov 4;1093(1-2):1-10.
Gioti EM, Skalkos DC, Fiamegos YC, Stalikas CD.
Many thanks all , maria i don't have fluoresecent detector , can i do this for hypercin by using uv detection
You can try at 236 nm and/or at 592 nm. Please, find in attachment 2 files.
A reversed-phase HPLC method was developed and validated for the simultaneous determination of hypericins and stabilized hyperforin in St. John's wort extract. The sample solution was prepared by extraction of the finely powdered extract with methanol-water (80:20, v/v) containing 5% HP-beta-cyclodextrin, and adjusted to pH 2.5 with orthophosphoric acid. Diluted extract solutions, maintained at 0 degrees C, were injected into a C18 column. The samples were eluted isocratically using a mobile phase consisting of acetonitrile and 0.3% v/v phosphoric acid (90:10, v/v) at a 1.5 ml/min flow rate with simultaneous fluorescence (315/590 nm, excitation/emission) and UV (273 nm) detection.
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