109 / 328
Primary and Secondary Evaluation of Method SJW-04
SJW-04:
Simultaneous Determination of the Predominant Hyperforins and Hypericins in St.
John’s wort (Hypericum perforatum L.) by Liquid Chromatograph
Author(s):
Dean E. Gray, George E. Rotiinghaus, H.E. Gene Garrett, and Stephen G. Pallardy,
University of Missouri, Department of Forestry, 203 A-B Natural Resources Building,
Columbia, MO 65211, Veterinary Medical Diagnostic Laboratory, PO Box 6023,
Columbia, MO 65205, University of Missouri, Department of Forestry, 203 A-B Natural
Resources Building, Columbia, MO 65211
S
UMMARY OF
M
ETHOD
:
G
ENERAL
C
OMMENTS
:
This method is used for the simultaneous determination of both hyperforins and hypericins in
leaf/flower with isocratic HPLC and UV-FLD containing high, medium and low concentrations of the
analytes. The method is not applicable to extracts and dietary supplements and flavonoids are not
included into the method. St. John’s wort aerials were extracted in methanol for two hours and diluted
with acetonitrile prior to cleanup with mixed solid phase column. Most impurities which can be
minimized by MSP elute much earlier than the analytes of interest. Hypericin is not shown in
fluorescence detection nor at 590 nm. Quantitation was calculated using forced through origin external
calibration curves. There chromatographic separation is short, although there is slight peak tailing.
P
ROS
/S
TRENGTHS
:
This method has fast chromatographic separation, good recovery for hyperforins, and short isocratic run
(8 minutes). Hyperforins and hypericinsare both within one method and analyze two (2) classes of
compounds as noted in the SMPR. Fluorescence sensitivity increased for hypericin and pseudohypericin
compared with UV absorbanceIsocratic separation.
C
ONS
/W
EAKNESSES
:
The use of in-house prepared mixed solid phase clean-up columns, could lead to variability between
laboratories. Quantitation of pseudohypericin and adhyperforin calculated from hypericin and
hyperforinusing forced through origin calibration curves can lead to errors in calculations due to poor
fitting. Time consuming clean-up (2 h extraction time, MSP = Mixed Solid Phase), 2 detectors are
necessary (UV and FLD), FLD though hypericin could be measured selectively at 588 nm. This method
has very poor precision for hyperforins.
EXPERT REVIEW PANEL VOTE
AND
RECOMMENDATION
MOTION: Not to consider this method for First Action Official Method status.
Mudge, Reif (Unanimous) Motion Passed
ERP PROFILE SUMMARIES
86