Talanta 208 (2020) 120397

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Parallel enrichment of polyphenols and phytosterols from Pinot noir grape T

seeds with molecularly imprinted polymers and analysis by capillary high-
performance liquid chromatography electrospray ionisation tandem mass
spectrometry
Shima N.N.S. Hashim1,2, Reinhard I. Boysen1, Yuanzhong Yang, Lachlan J. Schwarz,
Basil Danylec, Milton T.W. Hearn∗
Australian Centre for Research on Separation Science (ACROSS), School of Chemistry,Monash University, Monash, 3800, Victoria, Australia

ARTICLE INFO ABSTRACT

Keywords: This investigation describes an integrated workflow for the parallel extraction and recovery of polyphenols and
Molecularly imprinted polymers phytosterols from Pinot noir grape seeds. Using (E)-resveratrol and stigmasterol as exemplars, the approach
Covalent and non-covalent imprinting employs two different molecular imprinted polymers in tandem for the extraction of these compounds and their
Solid-phase extraction subsequent analysis by capillary high-performance liquid chromatography (capHPLC) interfaced with electro-
Polyphenols
spray ionisation tandem mass spectrometry (ESI MS/MS). Information on the selectivity of the solid-phase ex-
Phytosterols
traction processes was obtained through analysis of the binding behaviour of (E)-resveratrol- and stigmasterol-
imprinted polymers using structurally similar polyphenols or phytosterols with the extent of binding determined
from the capHPLC-ion trap ESI MS/MS data. This study documents with Pinot noir grape seed extracts and
optimised solid-phase extraction protocols that the (E)-resveratrol-templated MIP enabled a very high recovery
(99%) of the health-beneficial polyphenol (E)-resveratrol with co-purification of procyanidin and catechin/
epicatechin. Further, the stigmasteryl-3-O-methacrylate-templated polymer resulted in high recovery (96%) of
the phytosterol stigmasterol with co-purification of campesteryl glycoside. The results also demonstrate that
rapid and high-resolution capHPLC-ESI MS/MS methods can be used as part of the work flow for selectivity
optimisation and monitoring of the performance of MIPs intended for use in the solid-phase extraction of
bioactive molecules with nutraceutical properties from agricultural waste streams.

1. Introduction challenges have hitherto made their separation both costly and labour
intensive. Therefore, the development of simple and robust platform
In the manufacture of plant-derived food products and beverages, methodologies that enable the convenient, rapid and selective enrich-
current processing practices rarely convert all of the available plant ment and isolation of specific bioactives is expected to benefit the food,
mass into valuable products. Often, considerable quantities of by-pro- beverage, and consumer product manufacturing sectors and pharma-
ducts are discarded as waste streams. Many health-beneficial bioactives ceutical industries by producing value-added products for use as food
remain in these high-volume wastes, which are frequently disposed as additives, dietary supplements or as lead compounds in therapeutic
low value stock feed, compost or sent to land fill. The targeted ex- drug discovery.
traction and concentration of these bioactive compounds currently Grape is one of the largest produced fruit crops worldwide with
present many challenges because of their relatively low concentrations, annual production in 2017 of 74.3 million metric tons [[Link]
albeit availability in large volumes, and their structural diversity. These [Link]/faostat/en/#data/QC/] with ca. 80% used for making wines

Abbreviations: ACN, acetonitrile; BPC, base peak chromatogram; ESI, electrospray ionisation; DAD, diode array detector; LC, liquid chromatography; LOD, limit of
detection; LOQ, limit of quantification; MeOH, methanol; MIP, molecularly imprinted polymer; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NIP,
non-imprinted polymer; RP-HPLC, reversed-phase liquid chromatography; SPE, solid-phase extraction
∗
Corresponding author.
E-mail address: [Link]@[Link] (M.T.W. Hearn).
1
These authors contributed equally to the work.
2
Current address: Department of Chemistry Malaysia, Jalan Sultan, 46661, Petaling Jaya, Selangor, Malaysia.

[Link]
Received 5 September 2019; Received in revised form 26 September 2019; Accepted 27 September 2019
Available online 28 September 2019
0039-9140/ © 2019 Published by Elsevier B.V.
S.N.N.S. Hashim, et al. Talanta 208 (2020) 120397

and jams. It has been estimated that 15–20% of this mass, e.g. about Sigma-Aldrich Limited (Sydney, Australia). (E)-resveratrol (99.8%
9–12 million metric tons, represents the waste streams, which could purity) was purchased from AK Scientific (Union City, CA, USA).
potentially be exploited commercially as raw material feedstocks to A mixture of these ten polyphenolic standards was prepared at a
produce a diverse range of by-products [1]. During wine making, these concentration of 0.1 mM in EtOH for each component from the fol-
crude feedstocks (stems, skins and seeds) are generated during de- lowing standard stock solutions. Stock solutions of ( ± )-catechin and
stemming, grape crushing and pressing. Grape by-products have tradi- (E)-resveratrol were prepared in ACN at a concentration of 5 mM. Gallic
tionally been used to generate low value chemicals, e.g., tartaric acid or acid, p-coumaric acid, chlorogenic acid, ferulic acid, rutin, morin and
ethanol, as well as to produce compost. Commercial opportunities as- quercetin stock solutions were prepared in DMSO at a concentration of
sociated with the extraction and purification of health-beneficial com- 10 mM. The caffeic acid stock solution was prepared using a mixture of
pounds from grape sources are increasingly recognised [2] with grape ACN and DMSO (2:1, v/v) at a concentration of 2.5 mM. All laboratory
pressings known to contain valuable amounts of bioactive polyphenol procedures involving samples containing (E)-resveratrol were per-
and phytosterol compounds [3]. Grape skins contain appreciable formed in dim light to avoid photochemical isomerisation of (E)-re-
quantities of polyphenols, in particular flavonols, anthocyanins [4] and sveratrol to (Z)-resveratrol.
resveratrol [5]. Resveratrol, also found in grape seeds [6], has im- The calibration curve for (E)-resveratrol was generated by serial
portant in vitro and in vivo anti-oxidant, anti-inflammatory and anti- dilution of the (E)-resveratrol stock solution with EtOH over the con-
proliferation properties [7–9]. Grape seeds contain about 7% phenolic centration range of 5.0 × 10−1 mM to 1.3 × 10−4 mM. The calibration
compounds and 6% sterols [8,10], as well as phenolic pro-cyanidins curve was calculated by linear regression according to the formula
[11], which elicit a broad spectrum of therapeutic properties [12], in- y = mx + b, where y is the peak area obtained after RP-HPLC, x is the
cluding potent antioxidants of which catechin and epicatechin are the concentration of (E)-resveratrol (mg/L), m is the slope and b is the in-
major components [13]. Phytosterols, such as β-sitosterol, campesterol tersection near to the origin (~0.0). The equation for the resulting
and stigmasterol, have also been isolated from grape seed oil [14,15]. calibration curve was y = 77.1x with a correlation factor (R2) of 0.9.
The polyphenol and phytosterol compositions in grape by-products The limit of detection (LOD) (multiplying the standard deviation (SD)
depend on the grape variety and cultivar, climatic condition, geo- by a factor of 3) and the limit of quantitation (LOQ) (10 times the SD)
graphical origin and degree of grape maturity [16–18]. for (E)-resveratrol were determined to be 1.1 × 10−2 mg/L and
Molecularly imprinted polymers (MIPs) represent a versatile ap- 3.7 × 10−2 mg/L, respectively.
proach for the rapid isolation of specific target compounds from com-
plex matrices. MIPs are synthetic porous materials, designed to contain 2.3. Standards for sterol analysis
complimentary ‘receptor-like’ binding sites capable of recognising a
specific molecular target [19–22]. MIPs can be synthesised in various Commercial standards of sterols, including stigmasterol and cho-
physical formats, including surface coatings, preformed porous struc- lesterol (Sigma Aldrich Limited, Sydney, Australia) and ergosterol
tures (monoliths, membranes) and particles [23–26]. The incorporation (Fluka Chemicals, Melbourne, Australia) were used in these experi-
of MIPs into solid-phase extraction (SPE) procedures, known as MISPE, ments with purity of ≥95%. A series of commercially unavailable
has been used to facilitate separation and analysis of bioactive com- stigmasterol analogue standards (stigmasteryl-3-O-ferulate, stigmas-
pounds from biological feedstocks [27–29]. teryl-3-O-(4-acetoxy-ferulate) and stigmasteryl-3-O-acetate) were che-
In this study, an integrated work-flow for the parallel extraction of mically synthesised in-house at the Centre for Green Chemistry, Monash
health-beneficial compounds from Pinot noir grape seeds is described, University, Melbourne, Australia.
targeting the two compound classes of polyphenols and phytosterols. The calibration curve for stigmasterol was generated by serial di-
Specifically, the molecular recognition properties of two different types lution of a stigmasterol stock solution with ACN/H2O (9:1, v/v) over
of imprinted polymers have been investigated for the selective enrich- the concentration range of 5.0 × 10−1 mM to 1.6 × 10−4 mM. The
ment of polyphenols and phytosterols present in Pinot noir grape seed calibration curve was obtained by linear regression according to the
extracts, namely an optimised (E)-resveratrol-templated MIP polymer formula y = mx + b, resulting in y = 950.1x with a correlation factor
[29–35], in parallel with an optimised stigmasteryl-3-O-methacrylate (R2) of 0.9. The LOD and LOQ for stigmasterol were determined to be
templated MIP polymer [36,37]. Capillary high-performance reversed- 1.2 × 10−3 mg/L and 3.9 × 10−3 mg/L, respectively.
phase liquid chromatography (capRP-HPLC) with electrospray ionisa-
tion tandem mass spectrometry (ESI MS/MS) was then applied for the 2.4. Sample preparations
analysis of the captured target compounds as well as structurally similar
co-purified compounds. Two bunches of Pinot noir grapes (clone MV6, in total 181 g) har-
vested at enological maturity from a Mornington Peninsula winery
2. Materials and methods (Victoria, Australia) and with no signs of damage from pests or abiotic
factors, were manually de-stemmed, crushed and pressed. The juice was
2.1. Reagents discarded whilst the skins and seeds of the grapes were separated for
further analysis.
Reagent grade acetic acid (AcOH) and formic acid (FA) were pur-
chased from Ajax Finechem (Melbourne, Australia). Ethanol (EtOH), 2.5. Grape seeds extraction for polyphenol analysis
methanol (MeOH), acetonitrile (ACN) dimethylsulphoxide (DMSO),
sodium hydroxide (NaOH) and potassium hydroxide (KOH) were pur- The grape seeds (19.6 g) were freeze-dried in a lyophiliser
chased from E. Merck (Melbourne, Australia). All solvents used for the (Labconco, Missouri, USA) at 0.01 mbar for two days. The sample
SPE and chromatographic procedures were HPLC grade. Water was (5.0 g) was crushed using a blender (Kambrook, Victoria, Australia) for
purified using a Cascada™ IX water purification system (Pall Corp., 1 min in an extraction solvent containing a mixture of EtOH/H2O (1:1,
Melbourne, Australia) to a resistivity of 18.2 MΩ cm. v/v, 30 mL). The sample mixture was stirred in the dark for 30 min
using a magnetic stirrer, and then centrifuged (Eppendorf AG
2.2. Standards for polyphenol analysis Centrifuge 581OR, Hamburg, Germany) for 10 min at 4000 rpm at
20 °C. The supernatant was transferred into a volumetric flask and di-
Gallic acid, caffeic acid, p-coumaric acid, ferulic acid, chlorogenic luted with the extraction solvent (50 mL total volume). Aliquots of this
acid, ( ± )-catechin hydrate, rutin hydrate and quercetin hydrate (all extract were injected into the RP-HPLC-ESI-MS/MS system or loaded
hydrates were lyophilised prior to use) and morin were purchased from onto MISPE and NISPE columns. The injection and the MISPE protocol

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S.N.N.S. Hashim, et al. Talanta 208 (2020) 120397

were carried out in duplicate (n = 2). (5 × 2 mL). Both columns were reconditioned in sequence with H2O,
ACN and ACN/H2O (9:1, v/v) (3 × 2 mL).
2.6. Grape seeds extraction for phytosterol analysis
2.9. RP-HPLC analytical conditions
The pellet remaining after the centrifugation and removal of the
polyphenol supernatant was transferred into a vacuum oven to remove RP-HPLC analyses were performed using an Agilent Technologies
the residual solvents and was further dried in a vacuum oven for 1 h. A HPLC 1100 series (Agilent Technologies, Waldbronn, Germany)
saponification/alkaline hydrolysis procedure was employed based on an equipped with a degasser, a binary gradient pump, an auto-sampler
established method (namely ISO 18609-Animal and vegetable fats and oils; with a 900 μL sample loop, a thermostated column compartment and an
Determination of unsaponifiable matter; Method using hexane extraction) 80 Hz UV-DAD. Separations were performed with a double end-capped
with some minor modifications. The sample (5.0 g) was refluxed with an Zorbax Eclipse XDB C18 column (4.6 mm × 150 mm, 5 μm particle size,
ethanolic potassium hydroxide solution (1 M, 50 mL) for 2 h in a round- purchased from Agilent Technologies, Melbourne, Australia) which was
bottom flask. Distilled water (50 mL) was added, and the sample was thermostatically controlled to 20 °C (for polyphenols) and 23 °C (for
filtered prior to the extraction three times with n-hexane (3 × 15 mL). sterols). The UV-DAD was set to 200–600 nm at a spectral acquisition
The combined n-hexane extracts were washed with distilled water rate of 2 nm scans per step. Compounds in the sample were identified
(2 × 50 mL) until neutral according to a pH indicator paper. The n- based on their respective reference standards, by comparing the re-
hexane extracts were dried with anhydrous sodium sulphate and filtered tention times and UV/Vis spectra, using Agilent Technologies
using Whatman No. 1 filter paper. The n-hexane extracts were evapo- ChemStation software (B.02.01 SR1). All mobile phases were filtered
rated to dryness under reduced pressure using a rotary evaporator (Buchi through polypropylene membrane filters (0.2 μm pore size, 47 mm
Rotavapor R-215, Flawil, Switzerland). Removal of the n-hexane yielded diameter) purchased from Pall Corp. (Melbourne, Australia) and de-
a residue (that represents all the substances present in the product which, gassed in an ultrasonic bath (Elmasonic, Singen, Germany) for 30 min.
after saponification and extraction with hexane, are not volatile under
the specified operating conditions), which was dissolved in ACN/H2O 2.10. Polyphenol analysis by RP-HPLC with formic acid (FA)-based eluents
(9:1, v/v, 10 mL). Aliquots of this extract were injected into the RP-HPLC
- LC-ESI ion trap MS/MS system, whilst other aliquots were loaded onto The mobile phase compositions were 10 mM aqueous FA (eluent A)
the MISPE and NISPE columns. and aqueous EtOH (80%, v/v) containing 10 mM FA (eluent B). The
samples were chromatographed using a linear gradient of eluent B:
2.7. MISPE protocol for (E)-resveratrol analysis 0–2 min: 10% (v/v), 2–52 min: 10–70% (v/v), 52–53 min: 10% (v/v).
The injection volume was 5 μL at a flow rate of 0.5 mL/min. Detection
For generation of the resveratrol-selective MISPE polymeric mate- was monitored simultaneously at the wavelength of 280 nm, 320 nm
rial, the relevant MIPs/NIPs were prepared using a noncovalent and 370 nm.
strategy with (E)-resveratrol as template, 4-vinyl pyridine (4VP) as
functional monomer, ethylene glycol dimethacrylate (EGDMA) as cross- 2.11. Phytosterol analysis by RP-HPLC with an acetic acid (AA)-based
linking monomer and 2,2′-azobis(2-methylpropionitrile) (AIBN) as in- isocratic eluent
itiator, employing a bulk polymerisation procedure [32]. MIP and NIP
particles with size distribution of 60–90 μm, obtained after grinding and The phytosterol separation was performed using isocratic elution
sieving, were packed as EtOH slurries (1 g polymer beads per 30 mL with EtOH/H2O (9:1, v/v) containing 0.2% (v/v) AcOH. The injection
EtOH) into polypropylene columns (2.0 cm diameter × 8.7 cm length) volume was 5 μL at a flow rate of 0.5 mL/min. The instrumental system
with fritted polypropylene disks (20 μm pore size). The MISPE and is described in 2.9. Detection of stigmasterol, stigmasterol analogues
NISPE columns were then conditioned (in sequence) with EtOH/AcOH and cholesterol was performed at 210 nm whilst ergosterol was de-
(9:1, v/v), EtOH and EtOH/H2O (15:85, v/v) (3 × 5 mL), respectively. tected at 280 nm.
The supernatant of the grape seed extract (5 mL) was then loaded onto
the MISPE and NISPE columns, washed with EtOH/H2O (9:1, v/v) 2.12. capHPLC-ESI ion trap MS/MS instrumentation
containing 1% (v/v) AcOH (4 × 5 mL) followed by elution with EtOH/
AcOH (9:1, v/v) (10 × 5 mL). Finally, both columns were reconditioned Separation and identification of polyphenols and phytosterols was
for re-use with H2O, EtOH and EtOH/H2O (15:85, v/v) (3 × 5 mL carried out on an Agilent Technologies 1100 capillary LC system
each). In the case of more complex samples, an additional wash step (Agilent Technologies, Palo Alto, CA, USA) that consisted of a binary
with aqueous 0.1 mM NaOH was introduced. pump, a capillary pump with an online micro vacuum degasser, a
thermostated micro well-plate sampler with a 40 μL sample loop, a
2.8. MISPE protocol for stigmasterol analysis thermostated column compartment with a two-position/six-port micro-
valve and a diode-array detector and an Agilent Technologies 1100
For the stigmasterol analysis, the MIPs/NIPs were prepared using a Series LC/MSD-SL ion trap mass spectrometer equipped with an Agilent
covalent imprinting strategy with stigmasteryl-3-O-methacrylate as a Technologies G1607A orthogonal electrospray ionisation (ESI) inter-
post-synthetically cleavable monomer-template conjugate, EGDMA as face (Agilent Technologies, Palo Alto, California, USA). The reversed-
cross-linking monomer and AIBN as initiator, employing a bulk poly- phase LC separations were performed using a double end-capped
merisation procedure [36,37]. MIP/NIP particles with a size distribu- Zorbax XDB capillary C18 column (0.3 mm × 150 mm, 3.5 μm particle
tion of 60–90 μm, obtained after grinding and sieving, were packed as size) from Agilent Technologies (Melbourne, Australia) at 20 °C for
ACN slurries (1 g polymer beads per 30 mL ACN) into polypropylene polyphenols and at 30 °C for phytosterols.
columns (2.0 cm diameter × 8.7 cm length) with fritted polypropylene
disks (20 μm pore size). The MISPE and NISPE columns were then 2.13. Polyphenol and phytosterol analysis with capHPLC-ESI ion trap MS/
conditioned with ACN and ACN/H2O (9:1, v/v) (2 × 2 mL), respec- MS
tively. The residue of the grape seed extract after saponification and
hexane extraction (obtained as described in 2.6), following re-dissolu- The gradient elution conditions employed for the capHPLC-ESI MS/
tion in ACN/H2O (9:1, v/v, 2 mL), was loaded onto the MISPE and MS analyses of the polyphenols and the phytosterols were the same as
NISPE columns, washed with ACN/H2O (9:1, v/v) containing 1% (v/v) employed for the corresponding analytical RP-HPLC analyses except
AcOH (4 × 2 mL) followed by elution with EtOH/AcOH (9:1, v/v) that a flow rate of 2 μL/min and an injection volume of 0.1 μL was

3
S.N.N.S. Hashim, et al. Talanta 208 (2020) 120397

employed. For the stigmasterol analysis, instead of a premixed mobile

phase employed in the isocratic analytical separations, the eluent
EtOH/H2O (97:3, v/v) was mixed using the capillary pump with eluent
A consisting of 0.2% (v/v) AcOH in water and eluent B containing 0.2%
(v/v) AcOH in EtOH (99.9% purity). The outlet of the column was di-
rectly connected to the electrospray source of the ion-trap mass spec-
trometer, and the UV detector bypassed.
Mass detection was performed in the full scan mode for the poly-
phenols in the m/z range from 100 to 1200 using the negative ionisa-
tion mode and for the phytosterols in the m/z range from 100 to 1000
using the positive ionisation mode. The capillary voltage was set to
3.5 kV. Nitrogen was used as the nebulising gas at a pressure of 10 psi.
The drying gas (N2) flow rate was set to 5 L/min at 300 °C. The target
mass, compound stability and trap drive level were set to m/z 600, 80%
Fig. 1. RP-HPLC chromatograms recorded at a wavelength of 320 nm for the
and 100%, respectively. The ion accumulation time was automatically
quantification of (E)-resveratrol (tR = 32.7 min, peak 15) following (a) MIP
adjusted via the Ion Charge Control (ICC) feature of the instrument. The
(blue line, front) and after (b) NIP (pink line, middle) SPE treatment, and (c) in
ICC target was 30,000 units and the maximal accumulation time was the untreated grape seed extract (black line, back). The LC chromatograms were
300 ms. For MS/MS analysis, helium was used as the gas for collision obtained with a Zorbax Eclipse XDB C18 column (4.6 mm × 150 mm, 5 μm
induced dissociation (CID). The instrument was operated using the particle size) at 20 °C. LC conditions: eluent A: 10 mM FA in water, eluent B:
‘smart fragmentation’ mode which allowed the fragmentation ampli- 10 mM FA in EtOH/H2O (4:1, v/v), gradient: 0–2 min to 10% (v/v) B, 2–52 min
tude to be varied from 30 to 200% of the default 1.0 V. All data ac- to 70% (v/v) B, 52–53 min to 10% (v/v) B; flow rate: 0.5 mL/min; 5 μL injec-
quisition and processing were conducted with an Agilent ChemStation tion. (For interpretation of the references to colour in this figure legend, the
(version 4.2) and MSD Trap Control software. reader is referred to the Web version of this article.)

2.14. Data analysis normalized basis). In a comparison to the MISPE binding, the control
NISPE column showed negligible affinity for the target analyte (Fig. 1,
Compound identification was performed using information from the chromatogram b).
literature, including general citations on the mass spectrometry of
polyphenols [38] and sterols [39–41]. The following data bases were
used: ChemSpider ([Link] European MassBank 3.3. Identification of polyphenols from Pinot noir grape seed extracts before
– High Resolution Mass Spectral Database ([Link] MISPE treatment by capHPLC-ESI ion trap MS/MS
MassBank), Lipidomics Gateway ([Link] [42,43]
and MoNA - MassBank of North America ([Link]). Using the negative ionisation MS/MS mode, 18 phenolic compounds
were identified by capHPLC-ESI ion trap MS/MS procedures as depicted
3. Results and discussion in Fig. 2A. Information about the polyphenolic substances in the Pinot
noir grape seed extract including the compound name, retention time,
3.1. Pinot noir grape seed extract for polyphenols analysis precursor ion, product ion(s) prior and after MISPE and NISPE treat-
ment is presented in Table 1.
The Pinot noir grape preparation resulted in 10.8% (g/g) grape seed In this investigation, (E)-resveratrol from Pinot noir grape seed ex-
based on the fresh grape weight. These seeds were freeze-dried to re- tract was assigned as peak 15 based on the presence of a precursor ion
move moisture. The dry matter content of these grape seeds was de- [M-H]ˉ at m/z 227, which fragmented to generate several distinctive
termined to be 48.3% (g/g) on the basis of the mass before and after product ions at m/z 185, 157 and 143. (E)-resveratrol eluted with a
lyophilisation. This value is in agreement with previous publications retention time of 41.5 min with the capHPLC-MS/MS system and with a
which have reported that the seeds from different grape varieties have retention time of 32.7 min with the analytical RP-HPLC system. This
about 38–52% of dry matter content [44]. variation in retention time of 8.8 min is due to differences in the gra-
dient delay volumes of the two instruments, the different column di-
3.2. Analysis of (E)-resveratrol and other polyphenols from Pinot noir grape mensions and the additional delay volume for the UV detector relative
seed extract before and after MISPE by RP-HPLC to the MS detector in the capHPLC-MS/MS system.
Fig. 2A and Table 1 show that flavanols and tannins were the major
Fractions of the Pinot noir grape seed extracts before and after bioactive constituents in the Pinot noir grape seed extract based on the
MISPE were injected into the RP-HPLC system with (E)-resveratrol ion current intensity. The presence of catechin (peak 4) and its ste-
(peak 15) eluting with a retention time of 32.7 min (Fig. 1, chromato- reoisomer epicatechin (peak 8) were identified with the precursor ion
gram a). The amount of (E)-resveratrol was determined to be 11.4 μg/g [M-H]ˉ at m/z 289. Epicatechin-3-O-gallate (peak 9) at a retention time
dry extract based on a linear calibration curve of the (E)-resveratrol of 31.0 min was assigned based on the presence of the precursor ion [M-
standard. In a previous investigation, Sun et al. [5] reported that for H]ˉ at m/z 441. Peak 14 was detected as a precursor ion [M-H]ˉ with m/
Castelão grape seeds the abundance of (E)-resveratrol was 5.8 mg/kg z 605, attributed to the epicatechin-ethyl dimer, whereby two units of
dry seed. Despite a successful determination of (E)-resveratrol in the epicatechin are linked by an ethyl-bridge [5]. This compound frag-
Pinot noir grape seed extract, a poor baseline separation was observed mented to generate several characteristic product ions at m/z 451, 315,
for components present in the grape seed extract before MISPE treat- 289, 245 and 205. The product ion of m/z 451 correlated with a retro-
ment (Fig. 1, chromatogram c). The remarkable advantage of MISPE for Diels-Alder (RDA) fragmentation of this epicatechin-ethyl dimer [45],
rapid sample clean-up and to selectively capture (E)-resveratrol can be whilst the fragment with m/z 315 is consistent with the vinyl-catechin
appreciated from Fig. 1. Reduction of the matrix interferences as well as adduct [46]. The signals at m/z 289, 245 and 205 represented dis-
improvement of the resolution improved the quantification of the (E)- tinctive product ions belonging to the catechin/epicatechin monomer.
resveratrol (peak 15) in the sample. The application of the MIP in SPE The occurrence of flavan-3-ols in Pinot noir grape seeds observed in the
format resulted in the concentration enrichment of (E)-resveratrol to current studies is in agreement with the findings of Kammerer et al.
6.5 mg/L (in the MISPE eluate) from 1.4 mg/L (on a mass and volume [47] for pomace derived from several red grape cultivars (Cabernet

4
S.N.N.S. Hashim, et al. Talanta 208 (2020) 120397

Fig. 2. Base peak chromatogram (BPC) of (A) grape

seed extract and (B) MISPE (blue line, front) and
NISPE (red line, back) eluates from grape seed ex-
tract. Inserted is also a mass spectrum of (E)-resver-
atrol with the characteristic product ions. (E)-resver-
atrol (peak 15) eluted at tR = 41.5 min. The base
peak chromatograms were obtained after RP-HPLC
separation on a Zorbax XDB C18 capillary column
(0.3 mm × 150 mm, 3.5 μm particle size) in the m/z
range of 100–1200 using the negative ionisation
mode by LC-ESI ion trap MS/MS at 20 °C. LC condi-
tions: eluent A: 10 mM FA in water, eluent B: 10 mM
FA in EtOH/H2O (4:1, v/v), gradient: 0–2 min to 10%
(v/v) B, 2–52 min to 70% (v/v) B, 52–53 min to 10%
(v/v) B; flow rate: 2 μL/min; 0.1 μL injection. (For
interpretation of the references to colour in this figure
legend, the reader is referred to the Web version of
this article.)

Mitos, Lemberger, Spätburgunder, and Trollinger) with the seeds reported approach for the concentration of (E)-resveratrol from complex grape
to contain a high level of different catechins and flavanols following seed extracts prior to chromatographic analysis was demonstrated.
extraction with methanol, C18 reversed-phase SPE, RP-HPLC, and Fig. 2B shows that the MIP had a strong affinity for (E)-resveratrol
analysis by diode-array detection and mass spectroscopy. Our ob- (peak 15) based on the analysis of the MISPE eluate in comparison to
servations are also in accord with studies performed by Mane et al. the NISPE eluate. The MISPE method also improved the resolution of
[48], who found using solvent extraction procedures guided by a peak 15 and at the same time allowed discrimination of peak 16 (re-
Doehlert design response surface methodology that Pinot Meunier, then sveratrol glucoside). Large reductions in the ion current intensity as-
Pinot noir followed by Chardonnay grape seeds contain higher amounts sociated with peaks 4, 5 and 8 were also observed after the sample had
of flavanols than the skins, Our results also quantitatively parallel other been processed through the MISPE, with the exception of peak 9. The
findings related to conventional solvent extraction procedures and magnitude of the peak 9 (corresponding to epicatechin-3-O-gallate) ion
analysis by RP-HPLC-DAD methods of phenolic compounds, such as as observed in the BPC chromatogram of the MISPE eluate is consistent
flavan-3-ols, catechin/epicatechins and anthocyanins, from the seeds with epicatechin-3-O-gallate exhibiting binding site competition with
and skins of other red and white grape cultivars (e.g., Vitis vinifera var. (E)-resveratrol. However, there was no apparent enrichment of epica-
Cabernet Sauvignon, var. Chelva, var. Tempranillo and var. Viura) techin-3-O-gallate. The binding of epicatechin-3-O-gallate by the MIP
[11,47–50]. A comparative study by Thorngate et al. of flavanol loca- might be related to the higher concentrations of epicatechin-based
lisation across different grape varieties found that Pinot noir seeds compounds in the Pinot noir grape seed extracts, which compete with
contained higher amounts of flavanols when compared to seeds from a lower affinity with the target molecule for the pyridinyl-functionalities
Cabernet Sauvignon cultivar [51]. in the MIP recognition sites. Since the ring C hydroxyl groups of the
In the current investigation with the Pinot noir grape seed extract, epicatechin monomer are in a similar meta disposition as found in (E)-
various B-type procyanidins were detected with different degrees of resveratrol, this structural feature may account for why the epicatechin-
polymerisation, including the B-type dimer [52] (peaks 2, 5, 7 and 10), 3-O-gallate can bind to the imprinted cavities in the polymer network.
and the gallate dimer (peak 6), trimer (peak 3) and tetramer (peak 11) Presumably, hydrogen bonding interactions between the positively
with the precursor ions [M-H]ˉ at m/z 577, 729, 865 and 1017, re- charged pyridinyl-functionalities (derived from the 4-VP functional
spectively. The procyanidin tetramer ion (m/z 1017) underwent frag- monomers that form the cavities) and the delocalised π-electron system
mentation in the ion trap to generate the procyanidin trimer ion (m/z of the aromatic flavonoid rings, as well as hydrophobic interactions
865) followed by the production of procyanidin gallate dimer ion at m/ between the aromatic ring systems are the main factors responsible for
z 729. This product ion underwent further fragmentation to yield a the binding site recognition of epicatechin-3-O-gallate by the MIP.
product ion with m/z 577 (procyanidin dimer) due to the loss of a
galloyl group (152 mass units). Further fragmentation led to a product
ion with m/z 451, corresponding to the loss of a pyrogallol unit [M-H- 3.5. Binding efficiency of the (E)-resveratrol-templated MIP for Pinot noir
126]ˉ on the C-ring. The product ion at m/z 425 is generated through grape seed extract
the elimination of the B-ring followed by generation of a product ion of
m/z 407 due to the loss of water. A bond cleavage produced an ion In order to test the binding efficiency of the (E)-resveratrol-tem-
corresponding to the monomer of catechin/epicatechin at m/z 289. The plated MIP, 5 mL of Pinot noir grape seed extract was loaded onto a
high occurrences of procyanidin oligomers in the Pinot noir grape seed MISPE and a NISPE column, respectively. Application of the wash sol-
extract corroborate the findings of Prieur et al. [53]. vents enabled the removal of the majority of the (E)-resveratrol, ap-
proximately 10.8 μg/g (94.7%), from the NISPE column (Table 2). In
contrast, the MIP retained a significant amount of (E)-resveratrol
3.4. Identification of polyphenols from the Pinot noir grape seed extracts (10.4 μg/g), with only a small amount of the target molecule (0.9 μg/g)
after MISPE treatment by capHPLC-ESI ion trap MS/MS eluted from the MIP column during the wash stages. As expected, the
MIP also exhibited a greater selectivity for (E)-resveratrol compared to
During the course of this study, the usefulness of the MISPE the NIP. The majority of the target compound was desorbed from the

5
S.N.N.S. Hashim, et al. Talanta 208 (2020) 120397

Table 1
Identification and detected presence (✓) or absence (✗) of various polyphenols in Pinot noir grape seed extract before or after MISPE or NISPE treatment as
determined by LC-ESI negative ion trap MS/MS over the m/z range of 100–1200. Bold entries represent significant enrichment of phenolic compounds by MIPSE
above the ion current intensity level of 0.5 × 105. The chemical structures for multiple peaks are only shown once. ChemSpider IDs and molecular structures were
obtained from [Link] with structural similarity to (E)-Resveratrol highlighted in blue.
Peak No. Identified compound tR (min) Precursor ion [M- Product ion(s), m/z ChemSpider ID/ Structure Pinot noir grape MIP NIP
H]ˉ m/z reference seed extract

1 Gallic acid 12.0 169 125 361 ✓ ✗ ✗

a
2 B-type procyanidin 16.2 577 451, 425, 407, 289 [52] ✓ lc ✗

3 B-type procyanidin trimer 16.9 865 847, 739, 695, 577 ✓ ✗ ✗

4 (+)-Catechin 20.7 289 245, 205, 179 8711, 65929 ✓ ✓ ✗

a
5 B-type procyanidin 21.6 577 451, 425, 407, 289 [52] See above ✓ ✓ ✓
6 B-type procyanidin dimer 23.7 729 652, 577, 355, 289, ✓ ✗ ✗
gallate 245
a
7 B-type procyanidin 24.7 577 451, 425, 407, 289 [52] See above ✓ ✓ ✗
8 (−)-Epicatechin 25.9 289 245, 205, 179 65230, 158494 ✓ ✓ ✗

9 Epicatechin-3-O-gallate 31.0 441 289, 205, 169, 125 58567 ✓ ✓ ✗

10 B-type procyanidin 31.5 577 451, 425, 407, 289 [52] See above ✓ ✓ ✗
11 B-type procyanidin tetramer 31.9 1017 865, 729, 557, 451, ✓ ✗ ✗
425, 407, 289
12 (+)-Astilbin 37.2 449 303, 285, 151 390877 ✓ ✗ ✗
(neoastilbin)

13 Quercetin-3-O- 37.8 477 301, 179, 151 32674249 ✓ ✗ ✗

glucuronide

14 Epicatechin-ethyl dimer 38.8 605 451, 315, 289, 245, ✓ ✗ ✗

205
15 (E)-Resveratrol 41.5 227 185, 157, 143 392875 ✓ ✓ ✗

16 (Z)-Piceid 42.7 389 227 8353968 ✓ ✗ ✗

17 (Z)-Resveratrol 48.1 227 185, 157, 143 1265933 ✓ ✗ ✗

18 Quercetin 49.2 301 179, 151 4444051 ✓ ✗ ✗

lc Phenolic compound present at low concentration.

a
Based on the MS/MS spectra, no further identification was possible.

MIP sorbent upon elution with EtOH/AcOH (9:1, v/v) associated with (E)-resveratrol from the Pinot noir grape seed extracts is depicted in
disruption of hydrogen bonding interactions between the hydroxyl- Fig. 3.
groups from (E)-resveratrol and the basic site of the pyridinyl-func-
tionalities. This imprinting effect, expressed as an imprinting factor, 3.6. Optimisation of sterol extraction methods
was 26. The recovery of (E)-resveratrol after MISPE was determined to
be 99.1%. The performance of MIP and NIP in SPE column to capture The analysis of phytosterols in complex matrices is a challenging

6
S.N.N.S. Hashim, et al. Talanta 208 (2020) 120397

Table 2 This finding indicated that most of the phytosterols was present in the
The amount of (E)-resveratrol (μg/g per g of polymer) recovered from the grape Pinot noir grape seed extract pellet as conjugated esters.
seed extract following MISPE and NISPE treatment. Wash steps were performed
with a mixture of EtOH/H2O (4:1, v/v) containing 1% (v/v) AcOH followed by 3.6.2. Saponification methods
elution steps with after elution with EtOH/AcOH (9:1, v/v).
To circumvent the above constraint, a combination of saponification
(E)-Resveratrol (μg/g) followed by chromatographic fractionation was employed to extract the
phytosterols from the original Pinot noir grape seed extract pellet. The
MISPE NISPE
saponification method has two main purposes; to remove abundant
Before treatment Sample loading (1 × 5 mL) 11.4 11.4 triacylglycerols and other acyl lipids from the crude lipid extract and to
After treatment Wash steps (4 × 5 mL) 0.9 10.8 hydrolyse ester bonds in the conjugated phytosterols. Upon saponifi-
Elution steps (10 × 5 mL) 10.4 0.4 cation, the triacylglycerols are converted to water-soluble compounds
Total wash and elution 11.2 11.2
that can easily be excluded by extraction with a combination of water
Total recovery (%) 99.1 98.2
Imprinting factor (IF = MIP eluate/NIP eluate) 26
and organic solvent.
Both alkaline saponification conditions (1 M ethanolic KOH/NaOH)
and acid hydrolysis (6 M HCl) have previously been employed for sterol
extraction [36]. However, acid hydrolysis is not preferable for the de-
tection of Δ7-phytosterols (e.g., Δ7-stigmasterol) because acidic hydro-
lysis causes decomposition or isomerisation of these compounds after a
short time [55,57]. Alkaline saponifications can be carried out either at
room temperature (cold saponification) or at elevated temperatures
(hot saponification). The unsaponifiable residue contains all non-vola-
tile compounds that have undergone the saponification reaction fol-
lowed by extraction with hexane, i.e., the de-esterified phytosterols,
esterified phytosterols that are resistant to the saponification process,
other naturally occurring non-esterified phytosterols, higher hydro-
carbons and alcohols, as well as other foreign organic matter. The un-
saponifiable residue content can be expressed as a percentage by mass
of the sample before and after the saponification reaction. In the current
study, it was experimentally established that a cold saponification
method required a longer time to complete the reaction, therefore hot
saponification was used which involved refluxing the Pinot noir grape
seed extract pellet at 115 °C for 1 h, 2 h or 18 h with 1 M ethanolic KOH
solution (10 mL) with the unsaponifiable residue content determined to
Fig. 3. Comparison of (E)-resveratrol present in the wash and elution fractions
be 3.8%, 4.3% and 4.4%, respectively. Hot saponification for 2 h was
derived from the grape seed extract after wash with EtOH/H2O (4:1, v/v)
containing 1% (v/v) AcOH followed by elution with EtOH/AcOH (9:1, v/v) found to be optimal conditions to recover the sterols since hot saponi-
from the MISPE and NISPE columns. Measurements were performed in dupli- fication for longer times up to 18 h did not result in an appreciable
cate (n = 2). Error bars indicate the standard error. increase in unsaponifiable residue content.

3.7. Optimisation protocols for stigmasterol analysis

task and requires reliable analytical techniques for the extraction, iso-
lation and quantitative assays [7]. Plant-derived sterols in oil seeds can
In previous investigations [36,37], the stigmasterol MIP was shown
be isolated by solvent extraction followed by saponification and chro-
to have binding affinity towards stigmasterol in a standard mixture
matographic separation [54]. For example, previous research has
containing stigmasterol analogues, cholesterol and ergosterol with
shown that phytosterols can be isolated from sunflower seeds by solvent
ACN/H2O (9:1, v/v) as incubation solvent during static batch-binding
extraction with n-hexane followed by a mixture of chloroform-methanol
as well as dynamic binding experiments. Although some binding of
[55]. In order to establish an alternative procedure for sterol extraction
stigmasterol also occurred in n-hexane with the MIP, the NIP control
without the need to use chlorinated solvents, a series of optimisation
also gave similar results, which ultimately reduced the imprinting effect
experiments involving n-hexane extraction or saponification (hot or
and selectivity. Therefore, a direct loading of the crude Pinot noir grape
cold approach) was carried out using 1.0 g of pelleted Pinot noir grape
seed oil (containing both unsaponified and saponifiable matter such as
seed extract obtained after the polyphenol extractions.
triglycerides and saturated fatty acid esters) in n-hexane onto the MIP
was not possible. For these reasons, the Pinot noir grape seed oil re-
3.6.1. Direct n-hexane solvent extraction sidues after polyphenol extraction were saponified in order to produce a
The Pinot noir grape seed extract pellet was extracted with n-hexane de-esterified phytosterol fraction. This readily dissolved in a mixture of
and sonicated for 30 min. Evaporation of the n-hexane extract resulted ACN/H2O (9:1, v/v) and evaluated by MISPE and subsequent analysis
in the Pinot noir grape seed oil in 7.8% (g/g) yield. This value is con- by reversed-phase LC.
sistent with the work of Crews et al., who reported about 6–20% of oil Analyses of phytosterols usually have been conducted using normal-
extracted from grape seeds of different varieties [16]. However, this oil phase chromatographic methods. By contrast, fewer studies have de-
is incompatible with direct RP-HPLC analysis for phytosterols since scribed sterol analysis by RP-HPLC. In a previous study, Careri et al.
many other hydrophobic substances such as triglycerides are present in [58] reported that the separation of β-sitosterol and stigmasterol could
the sample and they interfere with detection [56]. The oil fraction was be performed using reversed-phase chromatography with a mixture of
further extracted with ACN, sonicated for 30 min and the mixture MeOH, H2O and ACN as mobile phase. Based on the use of the same
centrifuged at 10,000 rpm at 20 °C for 10 min. The supernatant was optimised RP-HPLC parameters, which had been previously developed
evaporated to dryness and redissolved in ACN prior to injection into RP- in the current investigation, an isocratic elution method was employed
HPLC. This ACN extraction method however proved unsuccessful due to as the procedure of choice for the analysis of sterols due to its metho-
the very low amounts of free stigmasterol that could be determined by dological simplicity. The separation of stigmasterol in a complex
RP-HPLC although the supernatants were concentrated up to 20-fold. sample was conducted using a mixture of EtOH/H2O (9:1, v/v)

7
S.N.N.S. Hashim, et al. Talanta 208 (2020) 120397

Fig. 4. RP-HPLC chromatograms recorded at a

wavelength of 210 nm for the quantification of
stigmasterol (tR = 20.3 min, peak 5) in (A) the
elution profile for the grape seed extract after sa-
ponification and (B) the elution profile of the con-
centrated grape seed extract after saponification
followed by MISPE (a, blue line, front) and NISPE
(b, purple line, back) treatments, respectively. The
LC chromatograms were obtained with a Zorbax
Eclipse XDB C18 column (4.6 mm × 150 mm, 5 μm
particle size) at 23 °C using isocratic elution. The
eluent was EtOH/H2O (9:1 v/v) with 0.2% (v/v)
AcOH; flow rate: 0.5 mL/min; 5 μL injection. (For
interpretation of the references to colour in this
figure legend, the reader is referred to the Web
version of this article.)

containing 0.2% (v/v) AcOH as a benign mobile phase. wash solvent was changed to ACN/H2O (9:1, v/v) containing 1% (v/v)
The chromatography of the Pinot noir grape seed extract after sa- AcOH. This new wash solvent composition resulted in the removal of
ponification confirmed the presence of stigmasterol (peak 5) with a the majority of the stigmasterol (16.4 μg/g) from the non-templated
retention time of 20.3 min with detection at 210 nm (Fig. 4A). Based on NISPE column whilst the target compound stigmasterol was pre-
a linear calibration curve for stigmasterol, the amount of this compound dominantly retained by the templated MISPE (Table 3). This finding
was determined to be 18.2 μg/g dry seed extract. The MISPE treatment indicates that weakly acidic conditions are required to elute non-se-
selectively extracted stigmasterol from the Pinot noir grape seed extract lectively bound compounds from the control polymer when dealing
after saponification as evident from Fig. 4B. Nearly quantitative capture with a complex sample. The target stigmasterol (13.1 μg/g) remained
of the stigmasterol by the MIP was observed, e.g. the peak eluting with a captured by the MIP polymer whilst only a small portion of the target
retention time of 6.3 min (as obtained by RP-HPLC), was significantly analyte that was non-selectively bound to the MIP was eluted (4.4 μg/g)
reduce after the extraction (Fig. 4B). Reduction in the peak heights was by these wash conditions. Elution of the stigmasterol-target could then
also observed for the compounds eluting with retention times of 9.7 min be affected through the use of EtOH/AcOH (9:1, v/v). Recovery of
and 11.3 min after MISPE as shown by the subsequent RP-HPLC se- stigmasterol from MISPE was determined to be 96.2%. The overall
paration. The MISPE technique can thus be considered a very attractive performance of MIP versus NIP to capture and release the target stig-
separation and concentration method for stigmasterol in natural sam- masterol in wash and elution steps is illustrated in Fig. 5. This
ples.

3.8. Quantification of stigmasterol before and after MISPE treatment by RP- Table 3
HPLC Recoveries of stigmasterol (μg per g polymer) from the grape seed extract after
saponification, following MIP and NIP in SPE columns. The wash steps were
performed with a mixture of ACN/H2O (9:1, v/v) containing 1% (v/v) AcOH
The Pinot noir grape seed residue after saponification and dissolu-
and elution stages with EtOH/AcOH (9:1, v/v).
tion in a mixture of ACN/H2O (9:1, v/v) was loaded onto the MISPE or
NISPE columns and eluted with the same solvent mixture (ACN/H2O Stigmasterol (μg/g)
(9:1, v/v)) as a sequential wash step, in an attempt to remove non-
MISPE NISPE
selectively bound components whilst keeping the compound of interest
retained on the sorbent. Previously, this same wash solvent mixture was Before treatment Sample loading (1 × 2 mL) 18.2 18.2
evaluated with a series of sterols standards as part of a competitive After treatment Wash steps (4 × 2 mL) 4.4 16.4
binding study. However, when this wash solvent was employed for the Elution steps (5 × 2 mL) 13.1 1.1
Total wash and elution 17.5 17.5
Pinot noir grape seed residue after saponification, the stigmasterol-
Total recovery (%) 96.2 96.2
templated MIP showed reduced affinity for the target compound stig- Imprinting factor (IF = MIP eluate/NIP eluate) 12
masterol in the MISPE format. As an alternative, the composition of the

8
S.N.N.S. Hashim, et al. Talanta 208 (2020) 120397

additional product ion at m/z 297 [M+H–H2O–C7H14)]+, whilst the

loss of the entire C17 side chain together with two hydrogens from the
steroid nucleus [41] resulted in a product ion at m/z 255 [M
+H–H2O–C10H20)]+.
The shorter retention time of about 15.2 min observed for stig-
masterol obtained with the capHPLC-ESI MS system compared to that of
20.3 min obtained with the analytical LC system can be primarily at-
tributed to the difference in the composition of the mobile phase em-
ployed in these two LC separation systems. The capHPLC separations
were performed with a mobile phase consisting of EtOH/H2O (93:7, v/
v), whilst for the analytical LC separations a mobile phase consisting of
EtOH/H2O (90:10, v/v) was used. Moreover, the reduction of 5.1 min of
the retention time can be attributed to the effect of a higher tempera-
ture of 30 °C employed in the capHPLC separations compared to the
23 °C used in the analytical LC system. It should be noted that in con-
trast to the downscaling of the gradient elution method for (E)-resver-
atrol, the difference of gradient delay volumes of the two LC systems
has no bearing here, since isocratic methods were employed for these
separations. Nevertheless, since the detection in the capHPLC system
was performed with UV followed by MS in contrast to the analytical LC
Fig. 5. Comparison of stigmasterol recovery levels (μg/g) in grape seed extract system were only UV detection was performed, the contribution of the
that had undergone saponification after the wash and elution steps from MISPE delay volume between UV detector and MS detector was also included.
and NISPE columns. Measurements were performed in duplicate (n = 2). Error
Several other compounds were also identified as present in the Pinot
bars indicate the standard error expressed by the formula Error = = n ,
noir grape seed residue after saponification (before MISPE treatment),
where σ is the standard deviation and n is the number of measurements.
using the ion trap MS/MS in positive ion mode. Under these conditions,
the typical ionisation of the free forms of phytosterols involves the loss
investigation demonstrated the use of MIP to preconcentrate and se- of a neutral molecule of water, [M+H–H2O]+. Derivatives, for instance
lectively extract stigmasterol from a Pinot noir grape seed residue ex- steryl glucosides, lose glucose and a water molecule, to generate the
tract in an ACN-aqueous matrix resulted in an imprinting factor of 12. corresponding [aglycone+H–H2O]+ product ions; acyl steryl gluco-
sides lose both the fatty acid (FA) bound to the glucose moiety and
3.9. Analysis of stigmasterol before and after MISPE treatment by capLC- water molecule to generate the corresponding [M-FA-Glu+H–H2O]+
ESI ion trap MS/MS product ions and esters lose the fatty acid and water to generate the
corresponding [M-MFatty_acid+H–H2O]+ product ions. Details related to
The peak identity confirmation of the target compound was per- the precursor ion and product ions of the compounds observed in the
formed by capLC-ESI ion trap MS/MS in the positive ionisation mode in sample, together with their identification based on their ChemSpider/
the m/z range from 100 to 1000 based on a previously developed MoNA/HMDB ID and molecular structures are listed in Table 4.
method [58]. The base peak chromatogram (Fig. 6A) showed eight The compound associated with peak 1 with a precursor ion [M
components that could be detected in the Pinot noir grape seed residue +H]+ at m/z 345 could not be identified with a high level of con-
after saponification. The presence of stigmasterol in this sample was fidence. The precursor ion [M+H]+ at m/z 677 was attributed to the
verified using the reference standard. Stigmasterol (peak 5) was iden- presence of β-sitostanol linolenate (C18:3) (peak 2) [59]. This com-
tified based on the presence of the precursor ion [M+H]+ of m/z pound underwent fragmentation under the MS/MS ion trap conditions
413.4. This precursor ion fragmented in the ion trap MS/MS to generate to generate a [M+H–H2O]+ product ion at m/z 399 (β-sitostanol
a product ion m/z 395.5, due to the neutral loss of a molecule of water moiety). The compound related to peak 3 with a precursor ion [M
([M+H–H2O]+) [36]. Further elimination of 98 mass units via the loss +H]+ at m/z 647 could also not be identified with a high level of
of part of the C17 side chain through breakage at the C-20/C-22 bond – confidence.
associated with the presence of the double bond, resulted in an Campesteryl glucoside (peak 4), the only compound that was

Fig. 6. Base peak chromatogram (BPC) of (A) grape

seed extract after saponification and (B) grape seed
extract after saponification followed by MISPE (blue
line, front) and NISPE (red line, back) treatments,
respectively. Inserted is a mass spectrum of stigmas-
terol with the characteristic product ions.
Stigmasterol (peak 5) eluted at tR = 15.2 min. The
base peak chromatograms were obtained after RP-
HPLC separation on a Zorbax XDB C18 capillary
column (0.3 mm × 150 mm, 3.5 μm particle size) in
the m/z range of 100–1000 using the positive ioni-
sation mode by LC-ESI ion trap MS/MS at 30 °C. LC
conditions: isocratic; eluent A: 0.2% AcOH in water
(7%, v/v), eluent B: 0.2% AcOH in EtOH (93%, v/v);
flow rate: 2 μL/min; 0.1 μL injection. (For inter-
pretation of the references to colour in this figure
legend, the reader is referred to the Web version of
this article.)

9
S.N.N.S. Hashim, et al. Talanta 208 (2020) 120397

Table 4
Identification of sterols and other components present in the Pinot noir grape seed extract after saponification following MISPE and NISPE treatment using the
capHPLC-ESI positive ion trap MS/MS over the m/z range of 100–1000. Bold entries represent significant enrichment of sterol compounds above the ion current
intensity of 0.3 × 105.
Peak No. tR (min) Tentative Precursor ion Product ions [M+H]+ ChemSpider ID/ Structure Grape seed extract MIP NIP
Identification [M+H]+ m/z m/z reference after saponification

1 12.3 Unidentified 345 326, 300a, 282, 265 ✓ ✗ ✗

matrix compound
a
2 12.6 β-sitostanol 677 630, 551, 419, 399 , ✓ ✗ ✗
linolenate (C18:3) 339, 303

3 13.4 Unidentified 647 610, 497, 457a, 413, ✓ ✗ ✗

matrix compound 378, 271
4 14.4 Campesteryl-D- 563 544a, 526, 470, 414, 4884485 ✓ ✓ ✗
glucoside 383

5 15.2 Stigmasterol 413 395, 393, 301a, 297, 4444352, ✓ ✓ ✗

283, 255, 189 MoNAFFF00269

6 15.8 22:6 Cholesteryl 697 680, 650, 593a, 423, 24823285 ✓ ✗ ✗

ester 369, 368

7 16.2 Squalene 411 395, 368, 341a, 327, 553635, ✓ ✗ ✗

315, 299, 274, 259, HMDB00256
247, 219,205a 191a,
177a, 163a, 151a, 135
8 17.8 Δ5-Avenasterol 413 395, 331, 301a, 189, 4444703 ✓ ✗ ✗
171, 141

a
Most intense product ion. Abbreviations: MoNA, MassBank of North America; HMBD, Human Metabolome Database.

identified as co-purified with stigmasterol (peak 5), was identified in seed oil derived from different varieties in a range of 0.3–4.5%.
this sample based on the presence of the precursor ion [M+H]+ of m/z The high fidelity of the MIP binding sites in comparison to the NIP
563, which underwent fragmentation in the MS/MS ion trap to produce to capture stigmasterol (peak 5) is depicted in Fig. 6B. Interestingly, the
a distinctive product ion at m/z 383 (campesteryl moiety [aglycone MIP showed a selective binding recognition towards the stigmasterol-
+H–H2O]+) [16]. Although the Pinot noir grape seed pellet was sa- target and enabled discrimination of Δ5-avenasterol. The Δ5-avenasterol
ponified with 1 M ethanolic potassium hydroxide, the steryl glucoside possesses a very similar molecular structure to stigmasterol except it
can still be detected in the unsaponifiable residue. It has previously has a double bond at the C24 position in the side chain. On the other
been suggested that the ether linkage between the sterol hydroxyl- hand, the MIP captured campesteryl-D-glucoside, probably due to
group and the sugar moiety in the steryl glycosides may not necessarily competitive binding although this compound has a larger size relatively
be completely hydrolysed under alkaline conditions [60]. A 22:6 cho- to the target molecule. The reason for this might be that one of the
lesteryl ester (peak 6) was assigned based on the presence of the pre- hydroxyl-groups in campesteryl glucoside participated in the formation
cursor ion [M+H]+ at m/z 697 [61]. This steryl fatty acid ester frag- of hydrogen bond interactions with the residual carboxylic acid groups
mented to generate several product ions including the cholesteryl in the MIP cavities.
moiety ion [M-Mfatty_acid+H–H2O]+ at m/z 369. Peak 7 was identified
as squalene, a triterpene hydrocarbon, which is a precursor of the
4. Conclusions
synthesis of all plant sterols with the precursor ion [M+H]+ at m/z 411
[62].
This study has explored methods for the analysis and quantitation of
Finally, the precursor ion [M+H]+ at m/z 413 and product ions at
resveratrol, procyanidin and epicatechin as well as stigmasterol and
m/z 395 [M+H–H2O]+, were indicative of an Δ5-avenasterol structure
campesteryl glycoside from the same Pinot noir grape seed waste based
(peak 8) [60]. According to Rozenberg et al. [60], stigmasterol, Δ5-
on the use of two different MISPE materials of optimised selectivity in
avenasterol and Δ7-avenasterol possess the same molecular mass,
conjunction with an integrated and parallel solid phase extraction
however the MS2 analysis allowed these compounds to be differ-
protocol and capHPLC-ESI ion trap MS/MS procedures. These methods
entiated. With the assistance of a stigmasterol standard in the present
allowed the identification of non-binding compounds and binding
study, the elution position and fragmentation fingerprint of stigmas-
target compounds, as well as structurally related compounds in the
terol in the sample was readily distinguished from avenasterol. Crews
flow-through and elution fractions with two different MISPE systems.
and co-workers [16] reported that Δ5-avenasterol was present in grape
The use of a fast and reusable MISPE enrichment and separation

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