Optimized chromatographic analysis of ergosterol in wild and cultivated mushrooms
Conference Paper
Overview
Overview
abstract
Fundação para a Ciência e a Tecnologia (FCT, Portugal) and COMPETE/QREN/EU for
the financial support of this work (research project PTDC/AGRALI/110062/2009) and to CIMO (strategic
project PEst-OE/AGR/UI0690/2011). J.C.M. Barreira also thanks to FCT, POPH-QREN and FSE for his grant
(SFRH/BPD/72802/2010.
Sterols belong to the unsaponifiable fraction of several matrices, where they can be found as free or
conjugated
structures.
In
the
latter,
the
3β-hydroxyl group is esterified with a fatty acid or a
hydroxycinnamic acid, or glycosylated with a hexose (usually glucose) or a 6-fatty acyl hexose [1].
Ergosterol (an important vitamin D2 precursor), is clearly the main sterol in mushrooms. Typically, the
analysis of individual sterols includes the extraction of lipids, saponification (which might include a
previous acid hydrolysis), extraction of unsaponifiable matter and separation/partial purification of sterols.
The subsequent separation step might be performed by high performance liquid chromatography (HPLC),
which is faster than gas chromatography analysis and operates under milder column temperatures and
non-destructive detection conditions [2]. Herein, an analytical method for ergosterol determination in
cultivated and wild mushrooms was developed using HPLC coupled to ultraviolet detection. The
chromatographic separation was achieved in a Inertsil 100A ODS-3 reverse phase column using an isocratic
elution with acetonitrile:methanol (70:30, v:v) at a flow rate of 1 mL/min. Different extraction
methodologies were tested, using n-hexane, methanol:dichloromethane (75:25, v:v) or
chloroform:methanol (20:10, v:v).
After studying the linearity (11 levels) for ergosterol (tR = 13.0±0.1 min; coefficient of variation, CV =
0.65%), a seven-level
calibration
curve
(y
=
0.6566ϰ
+
0.01098;
R2 = 0.9996) was made using the peak/area
ratio
versus
concentration
of
the
standard
(in
μg/mL).
The
average
of
triplicate
determinations
for
each
level was used. The limit of detection (LOD), calculated as the concentration corresponding to 3.3 times the
standard error
of
the
calibration
curve
divided
by
the
slope,
was
0.3498
μg/mL;
the
limit
of
quantification
(LOQ), calculated using the concentration corresponding to ten times the standard error of the calibration
curve
divided
by
the
slope,
was
1.060
μg/mL.
The
precision of the equipment was evaluated injecting seven
consecutive times the sterols extract of the same mushroom. The optimized method proved to be precise
(CV = 1.25%). Repeatability was assessed by applying the extraction procedure three times to the same
dried mushroom powder. The method proved also to be repeatable (CV = 1.88%). The method accuracy
was evaluated by the standard addition procedure (percentage of recovery). The standard mixture was
added to the samples in three concentration levels (25, 50 and 100 % of the peak/area concentration, each
one in triplicate) before the extraction. The method showed good recovery (above 90%).
Overall, a reproducible and accurate HPLC-UV technique was fully optimized. Chromatographic signals
presented good resolution, indicating the suitability of this methodology to assess sterol profiles in future
studies. Moreover, the chosen extraction and saponification steps do not require complex conditions, being
suitable for routine analysis.
