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According to INAMethod 106.000—HPLC analysis to deter-
mine the levels of these phenolic compounds in echinacea—
samples are extracted into an ethanol:watermixturewith shak-
ing. The analysis is performed on aC18 reversed phase column
using amobile phase of acetonitrile:dilute phosphoric acid. The
caffeic acid derivatives are detected based on their UV
absorbance at 330nm and quantitated by comparison to an
external standard solution of chlorogenic acid.We used the
Pinnacle II
™
C18 column to analyze a capsule of echinacea and
achieved resolution of four keymarker compounds (Figure 3).
GCAnalysis of FattyAcids inSawPalmetto
Saw palmetto, or Serenoa repens, has been used to treat pros-
trate enlargement and lower urinary tract symptoms. The par-
tially dried, ripe fruits of this plant are typically extractedwith
ethanol, carbon dioxide or hexane to collect the active ingredi-
ents. Saw palmetto contains awide range of compounds,
including free fatty acids, free fatty alcohols, andmonoglyc-
erides. The free fatty acids, whichmake up 70-95% of the puri-
fied lipophilic extract, often are used as themarker compounds
for this herbal product.
TheUSPmonograph for saw palmetto includes a procedure for
the quantitation of fatty acids byGC. To perform this analysis,
the samples are pulverized to a powder, saponified, and deriva-
tizedwith boron trifluoride inmethanol. The test standard
includes 11 fatty acidmethyl esters (FAMEs) and an internal
standard (IS), and the chromatographic separation is performed
on a polar Carbowax
®
column. The analysis of free fatty acids
as theirmethyl esters on anRtx
®
-Wax capillaryGC column
shows excellent resolution of all of the FAMEswhen analyzed
according to theUSPprocedure (Figure 4).
Summary
An increasing number of nutraceutical ingredients and products
are becoming a part of our diet. Because of this, we need accu-
rate quantitation of the active ingredients ormarker compounds
in both rawmaterials and finished products. Restek has awide
range of HPLC andGC columns to analyze these complex sys-
tems for the nutraceutical and food and beverage industries.
Restek columns offer excellent lot-to-lot reproducibility and
must meet stringent quality assurance criteria before they are
sent to customers. For HPLC procedures, Pinnacle II
™
reversed
phase columns offer excellent peak shapes for the active ingre-
dients in awide range of botanical compounds. TheRtx
®
-Wax
capillary column is an excellent choice for theGC analysis of
derivatized fatty acids. TheCrossbond
®
technology used to cre-
ate this column results in long column lifetimes and repro-
ducible results.
AnRtx
®
-Wax capillaryGC column achieves excellent resolution of the FAMEs in a saw palmetto standard.
Figure 4
References
1. USPQualityDemonstration Program for Dietary Supplements,
Draft 2.0 (2000), TheUSPharmacopoeia, Rockville,MD.
2. Barnes, JulianG. andWinter, Greg. TheNewYorkTimes,
May 27, 2001.
3. TheUSPharmacopoeia, Rockville,MD.
References not available fromRestek.
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GC_FF00538
Column
Rtx
®
-Wax
Catalog #: 12423
Dimensions: 30m, 0.25mm, 0.25µm
Conditions
Oven temp.: 120°C (hold 3min.) to 220°C
at 20°C/min. (hold 12min.)
Inj./det. temp.: 250°C/300°C
Carrier gas: helium
Linear velocity: 1mL/min. 34cm/sec.
Split ratio: 100:1
Sample
Saw palmetto standard
Inj.: 1µL
Conc.: see peak list
Peak list
1. methyl caproate (C6:0), 0.4mg/mL
2. methyl caprylate (C8:0), 0.4mg/mL
3. methyl nonanoate (C9:0), 2.0mg/mL
4. methyl caprate (C10:0), 0.4mg/mL
5. methyl laurate (C12:0), 5.0mg/mL
6. methyl myristate (C14:0), 2.0mg/mL
7. methyl palmitate (C16:0), 2.0mg/mL
8. methyl palmitoleate (C16:1), 0.4mg/mL
9. methyl stearate (C18:0), 0.4mg/mL
10. methyl oleate (C18:1), 5.0mg/mL
11. methyl linoleate (C18:2), 1.0mg/mL
12. methyl linolenate (C18:3), 0.4mg/mL
