The HPLC Determination of Salicin and Polyphenolic ... - Shimadzu

The HPLC Determination of Salicin and Polyphenolic Glucosides
in White Willow Bark Extract Using
Evaporative Light Scattering Detection
Introduction
Extract from the bark of the White or European Willow, salix alba, has been used as folk
medicine for many years in treating inflammation, pain and fever. The principle component of
the water extract from salix bark is salicin, 2-(hydroxymethyl)phenyl-ß-D-glucopyranoside, a
human-metabolic precursor to salicylic acid. A number of water soluble tannins, polyphenolic
glucosides, are also present [1,2] . Presently, White Willow is used as a “value-added”
constituent in a host of nutraceutical products.
HO
O O
OH
OH
salicin
OH
OH
For this study, a normal-phase HPLC method with complementary detectors, ELSD and
multi-wavelength UV (PDA), reveals salicin and numerous non-chromophoric glucosides in a
single run. Salicin content in White Willow bark extract is also quantified by ELSD.
The Evaporative Light Scattering Detector is a complement to spectroscopic detectors
for HPLC. The ELSD measures photons scattered from semi- and non-volatile particles that
have been dried of mobile phase through evaporation. Because its response is independent
of the light absorbing properties of molecules, it can reveal sample components that UV
detectors miss and provide a more accurate profile of relative component abundance than is
possible with a spectroscopic detector.
Sample Preparation
20mg of commercial White Willow bark powder was added to a flask containing 20mL of
a mixture of methanol and water (80/20 v/v). The mixture was sonicated for 10min., and then
syringe filtered (0.45µm, nylon). Salicin (Aldrich) was identified by authentic injection of the
standard. Experiments using ELSD-LT evaporation temperatures from 28 - 40?C showed no
variation in detector response to the salix constituents, indicating that precise adherence to
drift tube temperature is not critical to detection accuracy and reproducibility.
O
OH
OH
salicylic acid
Method and Conditions
Mobile Phase:
A: acetonitrile B: water
Gradient:
(Time, %B)(0,25)(12,25)(20,100)(22,100)
Column:
Shimadzu Premier Carbohydrate, 5?m, 250x4.6mm
Injection Volume: 5?L
Flow Rate:
1mL/min.
Column Temp: 35?C
Detector Settings Gain = 10; Temp = 32?C; Press. = 400kPa
SSI-HPLC -AN-002

Figure 1: Simultaneous PDA (UV 210, 268nm) and ELSD-LT chromatograms obtained for
commercial White Willow bark extract.
Results and Conclusion
Salicin shows two absorbance maxima in the UV, 210 and 268nm. Figure 1 shows a
comparison of the chromatographic profiles of ELSD with those two wavelengths. Here, the
ELSD-LT reveals a number of non-chromophoric constituents eluting later than the salicin.
Based upon their retention mechanism with this column chemistry, akin to a saccharide series,
these are presumably polyphenolic glucosides [2] .
While salicin may be determined using either detector, the myriad of salix constituents
are revealed by the evaporative light scattering detector alone. As illustrated by the data, a
compromise would be appropriate to balance the detector selectivity and sensitivity with
baseline response when using a UV detector. This is a common situation. Conversely, the
ELSD is immune to changes in solvent bulk properties and also yields better sensitivity toward
the constituent chemicals of salix alba.
SSI-HPLC -AN-002

As seen in the salicin calibration data, Figure 2, the ELSD shows excellent linearity
within the range of standard concentrations used. From the data, the concentration of salicin
in the unknown sample of White Willow bark extract was found to be 177µg/mL. Also based
upon salicin response, the sum total content of later-eluting polyphenolic glucosides was
117µg/mL.
Figure 2: ELSD standard curve for salycin (n=3 for each data point).
Salycin Calibration
7.1
6.9
6.7
Log Area Response
y = 1.6661x + 6.5538
R 2 = 0.9998
6.5
6.3
6.1
5.9
5.7
-0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4
5.5
Log ? g on-column
References
1. Felter, H. W., Lloyd, J. U. King’s American Dispensatory, 234.
2. Hagerman, A. E. Tannin Handbook (1998) 2002.
SSI-HPLC -AN-002

System Configuration*
Part #
Description
228-39000-92 LC-10ADvp Pump (2)
228-35830-91 100?L Semi-Micro Gradient Mixer
228-35359-92 DGU-14A On-Line Degasser
228-41576-32 ELSD-LT Evaporative Light Scattering Detector
228-35555-92 SPD-M10Avp Photodiode Array Detector
228-34350-92 SCL-10Avp System Controller
223-03507-91 PC-55N A/D option board for SCL
228-34611-92 CTO-10ACvp Column Oven
228-39005-92 SIL-10ADvp Autosampler
220-97205-23 Windows® 2000 Data Station and CLASS-VP 7.2 software
*Other options are available. Ask your Sales Representative for a system that’s right for you.
SSI-HPLC -AN-002