The assay of pterostilbene in spikedmatrices by liquid chromatography tandemmass spectrometry and isotope dilution method

Article abstract of DOI:10.1002/jms.1718

Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene) is an active component found in several plant species, exhibiting important pharmacological properties. A new and reliable method of assaying this phyto compound in various matrices is presented; the a

Full text of DOI:10.1002/jms.1718

Research Article
Received: 19 October 2009
Accepted: 18 January 2010
Published online in Wiley Interscience: 2 March 2010
(www.interscience.com) DOI 10.1002/jms.1718
The assay of pterostilbene in spiked matrices
by liquid chromatography tandem mass
spectrometry and isotope dilution method
Fabio Mazzotti,a Leonardo Di Donna,a Hicham Benabdelkamel,a
Bartolo Gabriele,b Anna Napolia and Giovanni Sindonaa∗
Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene) is an active component found in several plant species, exhibiting
important pharmacological properties. A new and reliable method of assaying this phyto compound in various matrices is
presented; the assay is based on (1) the selectivity of liquid chromatography (LC) hyphenated with electrospray ionisation (ESI),
(2) the specificity of a two-step mass spectrometric analysis (MS/MS) and (3) the accuracy of the isotope dilution method. The
labelled analogue may be conveniently synthesised in a few steps. The sensitivity of the method is confirmed by the very low
limit of detection (LOD) and limit of quantitation (LOQ) values achieved in the assay of pterostilbene in two distinct fortified
c
matrices, and is further supported by the observed accuracy values. Copyright ꢀ 2010 John Wiley & Sons, Ltd.
Keywords: pterostilbene; ESI-MS/MS; isotope dilution method; multiple reaction monitoring; phytoalexin
Introduction
problems arising from calibration procedure, sample preparation
and matrix effects.[15–18]
Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene), (1, chart),
a dimethylether analogue of resveratrol, is a naturally occurring
phytoalexin identified in several plant species, exhibiting im-
portant pharmacological properties.[1] It belongs to a group of
phenolic compounds known as stilbenes, found in deerberry and
rabbiteye blueberries[2] and several varieties of grapes[3,4]; it has
also been identified in the leaves of Vitis vinifera,[5] in Pterocarpus
marsupium[6] and in the Guibourtia Tessmanii plant,[7] used in tra-
ditional medicine. In particular, the extract of P. marsupium, used
in Ayurvedic medicine, is known for its antiglycemic effect. It is
a strong antioxidant, and is reported to scavenge 1,1-diphenyl-2-
picryl-hydrazyl (DPPH) free radicals and to inhibit the oxidation
of citronellal and lipid peroxidation in rat liver microsomes.[8]
Pterostilbene is cytotoxic against a number of cancer cell lines,
including human breast cancer and murine lymphoid neoplasma
cells[9,10]; moreover, it has been demonstrated that it might re-
duce cholesterol levels.[11,12] Animal studies have shown that
this natural compound can lower blood glucose and may be a
potent anti-diabetic agent.[6] The quantitation of pterostilbene
in biological fluids is currently performed by high performance
liquid chromatography (HPLC) coupled to fluorimetric detection
(FL).[13] Furthermore, pterostilbene has been successfully assayed,
together with other phytoalexins, in grapevine leaf extract and
berries using the HPLC-FL methodology.[3,14]
Experimental
Chemicals
Pterostilbene (97% purity) was used for the assay. ACS-grade
solvents and reagents (see below) were used for synthesis, while
HPLC-grade MeOH and H2O were used for analyses. All the
chemicals were purchased from Sigma–Aldrich, St Louis, MO.
In this work, we present a rapid method of determination
of pterostilbene in blueberry juice and human serum by liquid
chromatography mass spectrometry (LC-MS) under multiple
reaction monitoring (MRM) condition and isotope dilution. The
hexadeutero analogue of pterostilbene (2, chart) is used as
the labelled internal standard. Isotope dilution represents an
extremely accurate method of quantitation of analytes in complex
natural and biological matrices by mass spectrometry and is
best suited to improve precision and accuracy by reducing the
Ł
`
Correspondence to: Giovanni Sindona, Dipartimento di Chimica, Universita
della Calabria, Via P. Bucci cubo 12/C, I-87036 Arcavacata di Rende (CS), Italy.
E-mail: sindona@unical.it
`
a
Dipartimento di Chimica, Universita della Calabria, cubo 12/C, I-87036
Arcavacata di Rende (CS), Italy
`
b
Dipartimento di Scienze Farmaceutiche, Universita della Calabria, cubo 12/C,
I-87036 Arcavacata di Rende (CS), Italy
c
J. Mass. Spectrom. 2010, 45, 358–363
Copyright ꢀ 2010 John Wiley & Sons, Ltd.

Assay of pterostilbene by ESI-MS/MS
Scheme 1. Synthesis of compound 2.
Synthesis of d6-pterostilbene
1H NMR (300 MHz, CDCl3): δH 7.39 (2H, m, J D 8.6 Hz, H-20, H-60
), δH 7.02 (1H, distorted d, J D 16.5 Hz, H-β), δH 6.87 (1H, distorted
d, J D 16.5, H-α), δH 6.82 (2H, m, J D 8.6 Hz, H-2, H-6), δH 6.63 (2H,
m, J D 8.6 Hz, H-30, H-50), δH 6.37 (1H, m, J D 2.1 Hz, H-4), δH 5 (1H,
br s, OH).
3,5-Dimethoxybenzaldehayde (4, Scheme 1): 3,5-dihydroxyben-
zaldehyde (3) (1 g, 7.2 mmol) and anhydrous K2CO3 (20 g,
145 mmol) were stirred in dry acetone (50 ml) and 7 ml (97 mmol)
of CD3I were then added. The mixture was heated under reflux for
45 min, then cooled to room temperature, filtered and evaporated
to dryness. The residue dissolved in CH2Cl2 was washed twice
with water, dried over anhydrous Na2SO4, filtered and purified by
flash chromatography (silica gel, hexane/ethyl acetate 4 : 1, v/v)
affording 4 with 93% yield.
4-(tert-Butyldiphenylsilyloxy)-benzyltriphenylphosphonium
bromide 9 (3 g, 11.9 mmol), synthesised as reported[19] was
treated at ꢁ78 ŽC, in anhydrous tetrahydroufan (35 ml), with
n-butyllithuim (2.5 M, 3 ml). The dialdehyde 4 (1.5 g, 9.3 mmol),
dissolved in 5 ml of tetrahydrofuran, was then added. The
crude product purified by flash chromatography (silica gel,
hexane/ethylacetate 8 : 2, v/v) yielded 90% of a mixture of the E/Z
isomers (10a, 10b).
Trans-pterostilbene-d6 (2): To 1.2 g (2.45 mmol) of (10a,b)
dissolved in 20 ml of anhydrous tetrahydrofuran, 3.4 ml of
tetrabutylammonium fluoride (1 M) were added. The pale yellow
solution, stirred for 45 min and poured into water, was extracted
with dichloromethane. The organic phase, dried on anhydrous
sodium sulfate, was evaporated to dryness to afford the E/Z trans-
pterostilbene-d6 isomeric mixture. The latter, after separation by
flash chromatography (silica gel, hexane/ethyl acetate 9.5 : 0.5,
v/v), yielded 70%, based on 4, of pure trans-pterostilbene-d6
(2,Scheme 1).[19]
HRESI-MS: m/z 263.1553 [M C H]C calculated for C16H11D6 O3
263.1548.
Sample preparation
Blueberry juice: 12.5 µl of a solution (4 µg/ml) of the internal
standard (2) was added to 1 ml of juice, spiked with 1 and diluted
1 : 1 with water; the resulting solution was passed through a
0.22-µm HPLC filter and used for subsequent analysis.
Plasma: 12.5 µl of a solution (4 µg/ml) of the internal standard
(2) was added to 1 ml of human plasma, spiked with 1; the mixture
was treated with 1.5 ml of cold acetonitrile (in order to precipitate
proteins) and centrifuged at 8000 rpm for 5 min. The supernatant,
transferred to an Eppendorf tube, was evaporated to dryness
under nitrogen gas, reconstituted with 0.5 ml of HPLC mobile
phase (see below), passed through a 0.22-µm HPLC filter and used
for the assay.[13]
Mass spectrometry
The LC-MS analysis was carried out on a triple-quadrupole mass
spectrometer LC 320 (Varian Inc., Palo Alto, CA), equipped with
an ESI source interfaced with an HPLC Prostar 210 instrument
(Varian Inc.). The chromatographic analysis was performed using
a Discovery C18 column (75 ð 2.1 mm, Supelco, Bellefonte, PA),
injecting a volume of 20 µl. The flow rate was 0.25 ml/min using
the following eluants and linear gradients: solvent A (H2O, 0.025%
ammonium hydroxide), solvent B (MeOH); from 25% B to 98% B
The isotopic distribution, checked by high-resolution mass
spectrometry, was d5 D 2% and d6 D 98%.
The structure of deuterated pterostilbene 2 was confirmed by
MS and 1H nuclear magnetic resonance (NMR).
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Copyright ꢀ 2010 John Wiley & Sons, Ltd.
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F. Mazzotti et al.
in 7 min; 2 min at 98% B isocratic; from 98% B to 25% B in 2 min.
A re-equilibration time of 3 min was used after each analysis. The
instrument parameters of needle, shield and capillary voltages
were set to ꢁ4.5 kV, 600 V and 70 V, respectively; The drying
(N2) pressure and temperature were set to 20 psi and 250 ŽC,
respectively, while the nebulising gas (air) pressure, the housing
temperature and the electron multiplier voltage were set to 45 psi,
60 ŽC and 1350 V, respectively. The dwell time was 0.200 s/scan,
and the quadrupole resolution was set using a mass peak width
of 0.8 amu. The collision gas pressure (Ar) was fixed to 2 mTorr,
and the collision energies were set to 30 eV for the transitions
m/z 255 ! m/z 197 and m/z 261 ! m/z 197 and 18 eV for the
transitions m/z 255 ! m/z 240 and m/z 261 ! m/z 243.
High-resolution electrospray ionisation (HR-ESI) experiments
were carried out on a hybrid Q-Star Pulsar-i (MSD Sciex Applied
Biosystem, Toronto, Canada) mass spectrometer equipped with an
ion spray (IS) ionisation source. Samples were introduced by direct
infusion (3 µl/min) of the sample containing the analyte (5 ppm),
dissolvedinasolutionof0.1%aceticacid, acetonitrile/water50 : 50
at the optimum IS voltage of 4800 V. The source nitrogen (GS1)
and the curtain gas flows were set at pressures of 20 and 25 psi,
respectively, whereas the first declustering potential (DP1), the
focusing potential and the second declustering potential (DP2)
were kept at 50, 220 and 10 V relative to ground, respectively.
Nuclear magnetic resonance
Figure 1. Partial ESI (C) MS/MS spectra of compounds 1 (A) and 2 (B).
The NMR spectra were recorded using an AC 300 spectrometer
(Bruker BioSpin GmbH, Rheinstetten, Germany) on samples
dissolved in CDCl3; chemical shifts were measured in ppm and
coupling constants (J) in hertz.
positive (C) and negative (ꢁ) modes were checked. As expected,
the ESI (C) MS/MS spectrum performed on the [M C H]C derived
from unlabelled compound 1 provides a wealth of information,
displayingacomplexfragmentationpattern,whosefirstdiagnostic
species is represented by the unimolecular elimination of methyl
radical to give the ion at m/z 242 (Fig. 1(A)).
Analytical Parameters
The limit of detection (LOD) and the limit of quantitation (LOQ)
for each matrix were calculated by applying Eqns (1) and (2),
according to the directives of International Union of Pure and
Applied Chemistry (IUPAC) and the American Chemical Society’s
Committee on Environmental Analytical Chemistry.
The number of peaks (m/z 224 ! m/z 227) arising from
competitive and consecutive hydrogen and methyl radical losses
from the ion at m/z 242 (Fig. 1(A)) represents the major drawback
for a straightforward application of the MRM quantitation method.
This observation is supported by the chemistry of the [MCH]C
of the d6 reference compound 2 (Fig. 1(B)). It can be easily
observed, in fact, that the elimination of d3-methyl radical from
the precursor species at m/z 263 competes with an extensive H/D
isomerisation, giving rise to the elimination of CHD2 and CH2D
SLOD D SRB C 3σRB
SLOQ D SRB C 10σRB
(1)
(2)
SLOD is the signal at the limit of detection, SLOQ is the signal at
the limit of quantitation, SRB is the signal of the matrices without
analyte (blank) and σRB is the standard deviation for same matrices
calculated on seven measurements. The values of the unknown
concentrations were obtained from the appropriate calibration
curves.
ž
radicals, besides the formation of [(M ꢁ CD3) C H)]C species at
m/z 245. The fragmentation pattern is even more complex for the
other consecutive and competitive reaction paths taken by the
reactive species.
The ESI (ꢁ) MS/MS spectrum of the [M ꢁ H]ꢁ deprotonated
ion of the unlabelled compound 1 shows only a few diagnostic
fragment ions at m/z 240, 239, 225, 224 and 197 (Scheme 2 and
Fig. 2(A)).
Results and Discussion
The gas-phase chemistry of the labelled isomer 2 shows the
same fragmentation pattern as that of of compound 1 (Fig. 2(B)).
The deprotonated species at m/z 261 undergoes competitive
and consecutive radical and neutral losses not involving H/D
isomerisation. The elimination of CH3Ð (CD3Ð) radicals gives rise to
a resonance-stabilised distonic radical anion.[21] The consecutive
elimination of the second methyl group likely leads to the species
suggested in Scheme 2, which further fragments through carbon
monoxide elimination to give the species at m/z 197, which
is common to both labelled and unlabelled pterostilbene. This
Tandem mass analysis provides the specificity that allows unam-
biguous correlation between parent and product ions; moreover,
the good to excellent signal-to-noise (S/N) ratio confers the
methodology the sensitivity to perform trace analysis in complex
mixtures through the well-established MRM technique. Atmo-
spheric pressure chemical ionisation tandem mass spectrometry
(APCI-MS/MS) was recently applied to recognise the product of
combinatorial synthesis of stilbenoids.[20]
Inthecaseunderinvestigation, ESImethodwaschosentoionise
the species eluted in the first chromatographic stage, and both
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Copyright ꢀ 2010 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2010, 45, 358–363

Assay of pterostilbene by ESI-MS/MS
Table 1. Analytical parameters of standard solutiona
Standard
solution
(ng/ml)
Average
area
ratio
Area
ratio
Standard
deviation
RSD%
8.40
0.10000
0.11630
0.11593
0.23820
0.23797
0.24047
0.43060
0.42763
0.44237
1.18421
1.11022
1.21136
2.35510
2.35294
2.41568
4.55239
4.52085
4.61905
5
0.111
0.239
0.434
1.169
2.375
4.564
0.009
0.001
0.008
0.052
0.036
0.050
10
20
50
100
200
0.58
1.80
4.48
1.50
1.10
Scheme 2. Fragmentation pattern of protonated compound 1.
fact clearly shows that no hydrogen/deuterium isomerisation
competes with the observed fragmentation pathways.
The assay of analyte 1 was, therefore, performed under MRM
condition in the negative ion mode selecting the transitions
with the highest ion current. Accordingly, the reaction pathways
m/z 255 ! m/z 240 for 1 and m/z 261 ! m/z 243 for the
labelled internal standard were chosen. In addition, the transitions
m/z 255 ! m/z 197 for 1 and m/z 261 ! m/z 197 for 2 were used
to confirm the chromatographic elution of the analytes.
a To each sample of the calibration curve (y D 1.3191x ꢁ 0.2974;
R2 D 0.9982)wasaddedafixedquantityofinternalstandard(50 ng/ml).
built using triplicate samples of six standard solutions at different
concentrations of pterostilbene ranging from 5 to 200 ng/l, and
fixed concentration (50 ng/ml) of the internal standard (Table 1).
Theassayoftheanalyteinthespikedmatriceswasperformedby
means of a calibration curve (y D 1.3191x ꢁ 0.2974; R2 D 0.9982)
Figure 2. ESI (ꢁ) MS/MS spectra of compound 1 (A) and 2 (B).
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F. Mazzotti et al.
Figure 3. HPLC-MRM chromatogram of compounds 1 and 2.
Figure 3 shows an MRM chromatogram of the standard solution
at 50 ng/ml of 1 and 50 ng/ml of 2.
Table 2. Analytical parameters of precision and accuracy
Spiked
concentration
(ng/ml)
Calculated
concentration
(ng/ml)
The standard solutions were proven to be stable for more than
30 days at ꢁ20 ŽC (freezing–thaw cycle) and in sunlight for more
than 48 h.
RSD%
(average)
Accuracy%
Juice
4
The new protocol has been applied to spiked samples of
blueberryjuiceandhumanplasma. Therelativestandarddeviation
(RSD%) value was in all cases under 7%, thus showing a
good repeatability of the measurements. The accuracy of the
method was determined from samples prepared by adding known
amounts of the analyte to blank matrices. In the three examined
points (Table 2), which represent the boundaries of the calibration
curve, the accuracy values ranged from 92% to 112%.
4.5 š 0.6
16.0 š 0.5
146.8 š 4.7
13.3
3.0
112.5
109.6
97.9
15
150
4.7
Plasma
4
4.2 š 0.5
13.9 š 0.9
152.3 š 6.1
11.9
6.4
105.0
92.6
15
150
4.0
101.3
The standard deviation (RSD%) was calculated over three
measurements.
The calculated analytical parameters confirmed the appropri-
ateness of the proposed approach. The values of LOD and LOQ
were 1.5 and 3.9 ng/ml, respectively, for blueberry juice and 2.2
and 3.8 ng/ml for human plasma, suggesting that the method is
suitable for evaluating very low amounts of pterostilbene in differ-
ent matrices. The values of recovery are quantitative for both the
juice and the plasma.[13] In particular, in the case of the juice, the
samples are directly injected into the instrument, after dilution;
finally, the reproducibility is, in all cases, below 10% (Table 3).
Conclusions
Pterostilbene (1) exhibits a wide spectrum of biological functions.
Assessment procedures for the determination of this natural
drug in natural matrices are based on the conventional use of
chromatographic techniques linked to fluorescence detection.
We now propose a different analytical protocol centred on the
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J. Mass. Spectrom. 2010, 45, 358–363

Assay of pterostilbene by ESI-MS/MS
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LOQ (ng/ml)
LOD (ng/ml)
Recovery
3.9
1.5
Reproducibility (RSD%)
9.2
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98%
Plasma
LOQ (ng/ml)
LOD (ng/ml)
Recovery
4.1
2.2
Reproducibility (RSD%)
8.5
98%
The reproducibility of the measurements was obtained by extracting
each sample three times over a period of 1 week.
specificity of tandem mass spectrometry and on the reliability of
the isotope dilution method. The procedure here presented allows
the determination of the analyte with excellent specificity because
of the application of MRM methods, and excellent LOQ and LOD
parameters because of the use of the labelled internal standard 2.
Acknowledgements
This work is part of the Italian national project MIUR-FIRB
RBIP06XMR 004. H.B. thanks the University of Calabria for a PhD
grant.
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J. Mass. Spectrom. 2010, 45, 358–363
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