G Model

CHROMB-19774; No. of Pages 8 ARTICLE IN PRESS

Journal of Chromatography B, xxx (2015) xxxxxx

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Simultaneous determination of capsaicin and dihydrocapsaicin for

vegetable oil adulteration by immunoafnity chromatography
cleanup coupled with LCMS/MS
Fei Ma a,b,c,d,e,,1 , Qingqing Yang a,b,c,d,1 , Bertrand Matthus f , Peiwu Li a,b,c,d,e, ,
Qi Zhang a,b,c,d, , Liangxiao Zhang a,b,c,e
a
Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China
b
Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan 430062, China
c
Key Laboratory of Detection for Mycotoxins, Ministry of Agriculture, Wuhan 430062, China
d
Laboratory of Quality & Safety Risk Assessment for Oilseeds Products (Wuhan), Ministry of Agriculture, Wuhan 430062, China
e
Quality Inspection & Test Center for Oilseeds Products, Ministry of Agriculture, Wuhan 430062, China
f
Federal Research Institute of Nutrition and Food, Max Rubner-Institut, Detmold 32756, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Capsaicin and dihydrocapsaicin were selected as adulteration markers to authenticate vegetable
Received 30 July 2015 oils. In this study, a method of immunoafnity chromatography (IAC) combined with liquid
Received in revised form chromatographytandem mass spectrometry was established for the determination of capsaicin and
18 November 2015
dihydrocapsaicin in vegetable oils. In this method, immunosorbents were obtained by covalently coupling
Accepted 10 December 2015
highly specic capsaicinoid polyclonal antibodieswith CNBr-activated Sepharose 4B, and then packed into
Available online xxx
a polyethylene column. In this paper, the major parameters affecting IAC extraction efciency, including
loading, washing and eluting conditions, were also investigated. The IAC column displayed high selec-
Chemical compounds studied in this article:
Capsaicin (Pubchem CID: 1548943)
tivity for capsaicin and dihydrocapsaicin with the maximum capacity of 240 ng. The limit of detection
Dihydrocapsaicin (Pubchem CID: 107982) (LOD) and limit of quantication (LOQ) for capsaicin were calculated as 0.02 and 0.08 g kg1 , and for
Capsaicin-d3 (Pubchem CID: 57369257 dihydrocapsaicin were 0.03 and 0.10 g kg1 . The recoveries of capsaicin and dihydrocapsaicin in oil
Dihydrocapsaicin-d3 (Pubchem CID: samples were in the range of 87.395.2% with the relative standard deviation (RSD) of less than 6.1%. The
71316141) results indicated that capsaicinoid compounds could not be found in edible vegetable oils. Therefore, the
proposed method is simple, reliable and adequate for routine monitoring of capsaicinoid compounds in
Keywords:
vegetable oils and has an excellent potential for detection of adulteration with inedible waste oil.
Capsaicinoids
Immunoafnity chromatography 2015 Elsevier B.V. All rights reserved.
Isotope dilution internal standard
LCMS/MS
Inedible waste oil

1. Introduction inedible oils are generally contaminated by hazardous compounds

Waste oil, consisting of cooking oil collected from food wastes,
waste cooking oils recycled from drains or grease traps, residual
oils extracted from animal fats, deep frying oils, and other inedible
oils, have caused serious food safety scandals in developing coun-
tries when they are illegally sold and used as edible oils [1]. These
Corresponding authors at: Oil Crops Research Institute, Chinese Academy of
Agricultural Sciences, Wuhan 430062, China. Fax: +86 27 86812862.
E-mail addresses: mafeicpu@163.com (F. Ma), peiwuli@oilcrops.cn (P. Li),
Zhangqi01@caas.cn (Q. Zhang).
1
These authors contributed equally to this study.
such as condiments, heavy metal ions, bio-toxins, polycyclic
aromatic hydrocarbons (PAH), 3-monochloropropane-1,2-diol (3-
MCPD) and its esters, phthalates, dioxins and polychlorinated
biphenyls (PCBs) [29] or degradation products of oxidation pro-
cess. In the illicit rening industry, the complete removal of
contaminants or additives is difcult. Parts of them still remain
in recycled used oils though they are treated by degumminrg,
neutralizing, washing, drying, bleaching, ltering and deodorizing
processes [10]. As a result, those oils can adversely impact human
health due to oxidation products and other hazardous materials
from the draining and reprocessing processes [11].

http://dx.doi.org/10.1016/j.jchromb.2015.12.017
1570-0232/ 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: F. Ma, et al., Simultaneous determination of capsaicin and dihydrocapsaicin for
vegetable oil adulteration by immunoafnity chromatography cleanup coupled with LCMS/MS, J. Chromatogr. B (2015),
http://dx.doi.org/10.1016/j.jchromb.2015.12.017
G Model
CHROMB-19774; No. of Pages 8 ARTICLE IN PRESS
2 F. Ma et al. / J. Chromatogr. B xxx (2015) xxxxxx
Generally, sensory evaluation was used to detect adulteration
of inedible waste oils. However, sensory analysis depends on the
experience of analysts, and the subjective judgment may cause false
negative results. In this case, many analytical methods were devel-
oped to detect and quantify the adulterants. As rapid analytical
methods, NMR, NIR and Raman could characterize oil samples in the
whole spectrum [1214]. Recently, the metabolite proles of fatty
acids, triacylglycerols and volatile components were used to ana-
lyze inedible oils and detect potential adulteration [1517]. During
data processing, the optimized model needs many standards and
possible adulterated samples and might not be effective for samples
out of the training set. Compared with the preceding methods, liq-
uid chromatographytandem mass spectrometry (LCMS/MS) is a
promising approach to assess adulteration and quality of vegetable
oils since it satises routine analysis requirements for rapid deter-
mination and identication of contaminants or additives, which can
be used as marker compounds [18,19].
With the sensory attributes of pungency, aroma and color,
hot peppers are popular food additives and have been consumed
in large quantities throughout China and the world. The most
abundant capsaicinoids are capsaicin and dihydrocapsaicin (the
molecular structures of these chemicals are present in Fig. 1), which
constitute nearly 90% of the total pungency of pepper fruits [20].
Previous researches indicated that capsaicin and dihydrocapsaicin
were lipophilic and stable during the rening process of inedible
waste oils with high boiling points. Therefore these compounds
could be selected as specic biomarkers to detect and authenticate
edible vegetable oils [21].
Considering the complexity of lipid matrix and low concentra-
tion of capsaicinoids in inedible oils, the analytical methods usually
use thin layer chromatography (TLC) or solid phase extraction
(SPE) to extract and enrich the analytes [2224]. The procedures
generally consist of liquidliquid extraction, saponication and
neutralization, which provide non-specic retention and are time-
consuming, laborious, low sensitivity and consuming large volumes
of organic solvents. Immunoafnity chromatography (IAC) is a sep-
aration method which takes advantage of the specic interaction
between antibodies and antigens. It is a simple and selective tech-
nique to purify analytes without tedious pretreatment and can save
organic solvents during pretreatment [25,26]. Many reports have
been found for the determination of toxins, pesticide residues, vet-
erinary drugs and illegal additives using IAC cleanup [2729]. To the
best of our knowledge, IAC cleanup has not been used to determine
capsaicin and dihydrocapsaicin in combination with LCMS/MS.
The aim of this study was to establish IAC using poly-
clonal antibodies (pAb) covalently immobilized on CNBr-activated
Sepharose-4B for simultaneous determination of capsaicin and
dihydrocapsaicin in vegetable oils, which were further quantied
by LCMS/MS. The extraction conditions of the IAC column for
capsaicinoid compounds were optimized and the IAC column was
characterized in terms of binding capacity, extraction recovery and
reproducibility. Under the optimized conditions, the analytes in
lipid matrix were successfully quantied and used to detect oils
adulterated with inedible waste oil.
2. Experimental
2.1. Reagents and materials
CNBr-activated Sepharose 4B was purchased from GE Health-
care (Uppsala, Sweden). Capsaicin and dihydrocapsaicin standards
were obtained from SigmaAldrich (St. Louis, MO, USA). Capsaicin-
d3 and dihydrocapsaicin-d3 standards were obtained from Toronto
Research Chemicals (North York, Canada). Formic acid, methanol
(MeOH) and acetonitrile of HPLC grade were from SigmaAldrich
Please cite this article in press as: F. Ma, et al., Simultaneous determination of capsaicin and dihydrocapsaicin for
vegetable oil adulteration by immunoafnity chromatography cleanup coupled with LCMS/MS, J. Chromatogr. B (2015),
http://dx.doi.org/10.1016/j.jchromb.2015.12.017
(Shanghai, China). Sodium acetate (NaAc), sodium chloride (NaCl),
sodium dihydrogen phosphate, disodium hydrogen phosphate,
glacial acetic acid, tris(hydroxymethyl) aminomethane and sodium
azide (NaN3 ) were from Sinopharm Chemical Reagent (Shanghai,
China). Hydrochloric acid (HCl), caprylic acid, hexane, ammonium
sulfate, sodium bicarbonate and potassium dihydrogen phos-
phate were obtained from Kermel Chemicals (Tianjin, China). All
other chemicals and organic solvents were of analytical reagent
grade. Ultra-pure water (18 m) was obtained from a Milli-Q
water purication system (Milford, MA, USA). Nonspecic rabbit
immunoglobulins were produced in our laboratory.
The stock solutions of capsaicin, dihydrocapsaicin, capsaicin-d3
and dihydrocapsaicin-d3 were separately prepared by accurately
weighing 1 mg of the reference materials and dissolving them in
20 mL methanol. A series of standard solutions were prepared by
diluting the stock solutions with methanol. All standard solutions
were sealed with paralm, covered with aluminum foil, and stored
in the dark at 4 C until use.
2.2. Instrumentation
A Thermo lyophilizer (Savant, England) was used to freeze
dry antibodies. A shaker (Chinese Academy of Sciences Scientic
Instrument, Wuhan) was used to prepare the immunosorbents.
Centrifugation of the vegetable samples was performed on a
centrifuge (Hitachi CF 16RX), and a homogenizer (IKA Labora-
tory Equipment, Germany) was used for sample preparation.
Sample analysis was performed in selected reaction monitoring
(SRM) mode on the Accela HPLC system coupled to TSQ Quan-
tum Ultra EMR (Thermo Fisher Scientic, USA). Separation was
performed at 35 C on a Thermo Scientic C18 column (Hyper-
sil Gold, 100 mm 2.1 mm, 3.0 m). Mobile phase A consisted of
0.1% aqueous formic acid and mobile phase B acetonitrile. A lin-
ear gradient elution program was applied as follows: initial B was
linearly increased from 33.5% to 83.5% in 10 min, held for 1 min,
and returned to 33.5% B in 3 min, which was held for 4 additional
minutes for re-equilibration, giving a total run time of 18 min. The
mobile phase ow rate was 250 L min1 , and an aliquot of 10 L
sample was injected into the HPLC system. The mass spectra were
obtained by Thermo TSQ Quantum Ultra EMR triple quadrupole
(Thermo Scientic, USA) coupled with electrospray interface (ESI).
The MS/MS conditions were set as follows: spray voltage, 3.5 kV
in positive mode; capillary temperature, 350 C; sheath gas pres-
sure (N2 ), 30 units; auxiliary gas pressure (N2 ), 5 units; collision
gas (Ar), 1.5 mTorr; scan time, 0.1s. As shown in Table 1, two
parent-to-product transitions for each analyte were simultane-
ously monitored in SRM mode.
All the experiments were conducted in triplicate, and the aver-
age values standard deviations (SD) were reported. LCMS/MS
data were processed using Xcalibur 2.0.7 SP1. Statistical analyses
were performed with the @Risk 5.5.1 software package (Palisade,
Australia).
2.3. Production and purication of polyclonal antibodies
Polyclonal antibodies (pAb) against capsaicinoids were pro-
duced according to the procedure described in our previous study
[30]. As illustrated in Fig. 2, the hydroxyl group of capsaicin
molecules was changed to the ester group under bromination reac-
tion, and then the esters were alkaline hydrolyzed to the carboxyl
group which was covalently coupled to the carrier protein (bovine
serum albumin, BSA) using the modied active ester method.
The capsaicin-BSA conjugate was dialyzed and then used as an
immunogen to immunize New Zealand white rabbits for polyclonal
antibody production. The obtained antiserum was further puried
according to the modied saturated ammonium sulfate caprylic
G Model
CHROMB-19774; No. of Pages 8 ARTICLE IN PRESS
F. Ma et al. / J. Chromatogr. B xxx (2015) xxxxxx 3

Fig. 1. Molecular structures of capsaicin and dihydrocapsaicin and their stable isotope labeled internal standards selected as the biomarkers for oil detection and authenti-
cation.

Table 1
LCMS/MS parameters for the determination of capsaicinoid compounds.

Compound Parent ion (m/z) Product ion (m/z) Collision energy (eV) Tube lens voltage (V)

Capsaicin 306.1 136.9a 18 126

306.1 122.0 18 126

Dihydrocapsaicin 308.2 136.9a 18 136

308.2 122.0 36 136

Capsaicin-D3 309.1 140.2a 25 126

309.1 182.3 15 125

Dihydrocapsaicin-D3 311.1 140.2a 15 136

311.1 184.3 15 136
a
The most abundant product ion selected for the quantitative analysis.

Fig. 2. Synthesis scheme of the capsaicinoid-protein conjugate.

acidammonium sulfate method [25]. The antiserum was diluted dried and stored at 20 C until use. The cross-reactivity (CR) was
with an equal volume of saline, and then the saturated ammonium calculated as the following equation,
sulfate solution (pH 7.4) was gently added under magnetic stirring
at 4 C for 2 h. After the turbid solution centrifuged at 12,000 rpm
IC50 of capsaicin
for 30 min, the precipitate was dissolved in 1/8 antiserum volume CR% = 100% (1)
IC50 of analytes
of phosphate buffer solution (PBS) (0.01 M, pH 7.4) and dialyzed
against PBS for 48 h. The puried antibody solution was then freeze-
where IC50 is the concentration at which 50% of the antibody is
bound to the analytes and CR were calculated for capsaicin and
dihydrocapsaicin, respectively.

Please cite this article in press as: F. Ma, et al., Simultaneous determination of capsaicin and dihydrocapsaicin for
vegetable oil adulteration by immunoafnity chromatography cleanup coupled with LCMS/MS, J. Chromatogr. B (2015),
http://dx.doi.org/10.1016/j.jchromb.2015.12.017
G Model
CHROMB-19774; No. of Pages 8 ARTICLE IN PRESS
4 F. Ma et al. / J. Chromatogr. B xxx (2015) xxxxxx
2.3.1. Preparation of the IAC column
Immunosorbents were prepared following the manufacturers
instructions and related literature [25]. Briey, 0.3 g CNBr-activated
Sepharose 4B was swelled and washed with 40 mL 1 mM HCl for
15 min, and then mixed with 4 mL coupling buffer (0.1 M NaHCO3 ,
0.5 M NaCl, pH 8.3) containing 10.2 mg puried antibody. The cou-
pling solution reacted for 3 h in a shaker at room temperature, and
then it was transferred to the column and the unbound antibody
was washed with the coupling buffer of at least 5 times the medium
volume. The active gel groups were capped with the blocking buffer
(0.1 M TrisHCl, pH 8.0) at 4 C for 2 h without shaking. To remove
excess uncoupled immunosorbents, the gel was washed with high
and low pH buffer solutions (0.1 M HAc-NaAc buffer, pH 4.0, con-
taining 0.5 M NaCl, and 0.1 M TrisHCl buffer, pH 8.0, containing
0.5 M NaCl) of at least 5 times the medium volume for three cycles.
Finally, the gel was equilibrated with 0.01 M PBS and stored in
0.01 M PBS containing 0.01% NaN3 at 4 C before use.
2.4. Sample preparation
For the development and validation of the method, at least 1.0 kg
vegetable oil samples, including maize oil (5), sesame oil (5), sun-
ower oil (5), soybean oil (5), olive oil (7), rapeseed oil (7) and
blend oil (5) were collected from supermarkets. Waste oil sam-
ples (9) were collected from local restaurant grease traps. The
detailed information of samples including type, place of produc-
tion, rening step were added in Supplementary material Table 1S.
All oil samples were stored at 25 C before use. Each oil sample
was homogenized by thoroughly stirring with a mechanical mor-
tar and then stored in the dark at 4 C until analysis. The oil samples,
in which no detectable amount of capsaicinoids was conrmed by
LCMS/MS, were used as the blank sample (soybean oil). Then, the
blank samples spiked with standard solutions of capsaicin, dihydro-
capsaicin, capsaicin-d3 and dihydrocapsaicin-d3 prior to extraction
were used as inedible waste oils.
5.0 g oil sample spiked with 200 L of internal standards
(capsaicin-d3 and dihydrocapsaicin-d3 ) was placed in a 25 mL
polypropylene-capped centrifuge tube. Subsequently, it was mixed
with 5 mL methanol and vortexed for 2 min before being cen-
trifuged at 4500 rpm for 5 min at 4 C. The upper layer was
transferred and ltered through an organic membrane (0.45 m).
2.5. IAC extraction
For IAC cleanup, 0.5 mL of the organic extracts was diluted to
5 mL with PBS (pH 7.4) and then passed through the IAC column
with a ow rate of 1 mL min1 to remove the interference resulted
from the lipid matrix. The column was washed with water (10 mL)
and eluted with 1 mL of methanol. The eluting solvent was evapo-
rated to dryness under a gentle stream of nitrogen at 40 C. Finally,
the residue was reconstituted with 200 L of acetonitrile/H2 O
(50:50, v/v) and ltered through a PTEE lter (0.22 m) before
LCMS/MS.
3. Results and discussion
3.1. Antibody characterization
With the purpose of IAC cleanup considered, the characteriza-
tion of the antibody plays an important role in the preparation
of the IAC column for a single analyte or for an analyte and its
derivatives. The antibody against capsaicin showed a high titer
(1/128,000), showing a cross-reactivity of 95% and 88% for capsaicin
and dihydrocapsaicin, respectively. Thus, the antibody was suitable
Please cite this article in press as: F. Ma, et al., Simultaneous determination of capsaicin and dihydrocapsaicin for
vegetable oil adulteration by immunoafnity chromatography cleanup coupled with LCMS/MS, J. Chromatogr. B (2015),
http://dx.doi.org/10.1016/j.jchromb.2015.12.017
for establishing a single IAC column which could simultaneously
capture capsaicin and dihydrocapsaicin.
3.2. Preparation of the IAC column
The absorbents supporting the IAC column have a signicant
role in the covalent reaction with the antibody and purica-
tion of the sample matrix. The basic properties of the supporting
absorbents are biochemical inertness, mechanical and chemical
stability, porosity and uniformity in particle size, and covalent
reaction with the antibody under mild conditions. Compared
with the supporting absorbents for immobilization procedures,
agarose-based supports are the most common absorbents used
for preparation of the IAC column, especially for off-line purica-
tion [25,31]. CNBr-activated Sepharose 4B, which could be easily
coupled to the antibodies with the activated group of the parti-
cle surface, is chemically and mechanically inert, mild derivative,
water stable and hydrophilic and retains the bio-specic activity of
the antibody. In the present study, it was selected as the supporting
absorbents for IAC extraction. Sepharose 4B of (0.3 g) was dissolved
in 4 mL gel, and then 10.2 mg of pAb was covalently reacted with
the gel. From the data of UVvis spectrometry, 8.8 mg of pAb was
immobilized on the gel. According to the ratio of the antibody cou-
pled with the solid support gel to the initial antibody, the coupling
efciency of the capsaicinoid antibody on the immunosorbents was
86.3%. Finally, 0.2 mL of the immunosorbents were transferred into
a 1 mL polyethylene column, and stored in 0.01 M PBS containing
0.01% NaN3 at 4 C.
3.3. Optimization of IAC conditions
The afnity interaction between antigens and antibodies is
generally thought to be a comprehensive combination of various
non-covalent bonding mechanisms including electrostatic attrac-
tion force, van der Waals attraction force, hydrogen bonding, and
hydrophobicity. The characteristic properties of the loading, wash-
ing, and eluting solutions, such as polarity, pH value and ionic
strength, have important effects on the afnity interaction of IAC.
To obtain high extraction efciency and avoid irreversibly change
of antibody afnities, the properties of the loading, washing and
elution conditions were investigated and optimized according to
the previous study [25]. All fractions in the loading, washing and
eluting steps were collected respectively, and the analytes were
determined by LCMS/MS.
3.3.1. Loading condition
To achieve satisfactory loading procedure, different percent-
ages of MeOH-PBS (pH 7.4) mixture (0:100, 5:95, 10:90, 15:85,
20:80, 30:70, v/v) was investigated and compared. As shown in
Fig. 3a, when the concentration of MeOH was less than 5%, the
recovery increased with the increasing MeOH concentration. When
the MeOH concentration was further increased from 5%, the recov-
ery decreased to less than 78.7%. The recoveries were affected by
the solubility of capsaicinoids in organic solvents. The results indi-
cated that the percentage of organic solvents was important for
the equilibrium extraction process of analyte dissolution and anti-
body desaturation. Therefore, pH 7.4 PBS containing 5% MeOH was
selected as the loading solution.
The volumes of the loading medium (1, 2, 5, 8, 10, and 15 mL)
inuencing the recoveries were also studied (Fig. 3b). The results
showed that with the increase of the loading solvent volume from
1 mL to 5 mL, the extraction recoveries of capsaicin and dihydro-
capsaicin gradually increased from 63.2% to 94.4% and from 66.8%
to 96.2%, respectively. Further increase of the loading solvent did
G Model
CHROMB-19774; No. of Pages 8 ARTICLE IN PRESS
F. Ma et al. / J. Chromatogr. B xxx (2015) xxxxxx 5

Fig. 3. The inuence of several parameters on IAC extraction efciency: (a) loading solvent, (b) loading volume, (c) washing solvent, and (d) eluting solvent. The oil sample
solutions with capsaicin and dihydrocapsaicin spiked at 2.0 g kg1 .

Fig. 4. The amount of capsaicin and dihydrocapsaicin retained in the IAC column
with continuous sample loading.

not signicantly improve the recoveries, thus 5 mL was chosen as

the loading volume in the following experiments.

3.3.2. Washing condition

To avoid matrix effects and poor chromatographic separation,
the oil- and fat-based interfering substances which were retained at
the sol-gel column should be removed as much as possible. The fol-
Fig. 5. LCMS/MS chromatograms of the blank vegetable oils with capsaicin and
lowing washing media were tested: PBST (Tween 20/PBS, 0.5:99.5,
dihydrocapsaicin spiked at 0.5 g kg1 , respectively.
v/v), MeOHwater (10:90, v/v), PBS, and water. In Fig. 3c, the exper-
imental results indicate no signicant differences between these
washing mediums except PBST. When nonionic detergent-Tween 3.3.3. Eluting condition
20 was added, the nonspecic hydrophobic interactions between To dissociate the analytes from the antibodyantigen complex,
the detergent and the lipophilic capsaicinoid compounds in the elution solutions were selected as organic aqueous solvents (MeOH
aqueous system resulted in low recoveries. Consequently, 10 mL was widely applied), which were successfully applied in most
water was adopted to minimize nonspecic binding and improve off-line IAC procedures [2529]. In this study, different percent-
sensitivity of LCMS/MS analysis. ages of waterMeOH (20:80, 15:85, 10:90, 5:95, 0:100, v/v) were

Please cite this article in press as: F. Ma, et al., Simultaneous determination of capsaicin and dihydrocapsaicin for
vegetable oil adulteration by immunoafnity chromatography cleanup coupled with LCMS/MS, J. Chromatogr. B (2015),
http://dx.doi.org/10.1016/j.jchromb.2015.12.017
G Model
CHROMB-19774; No. of Pages 8 ARTICLE IN PRESS
6 F. Ma et al. / J. Chromatogr. B xxx (2015) xxxxxx

Table 2
Recoveries and precisions for the determination of capsaicinoid compounds in oil samples.

Analyte Accuracy, mean recoverya (%), n = 3 Intra-day (RSD%, n = 6) Inter-day (RSD%, n = 3)

1
1 g kg1 5 g kg1 10 g kg1 1 g kg1 5 g kg1 10 g kg1 1 g kg1 5 g kg1 10 g kg

Capsaicin 87.3 94.1 95.2 3.5 4.1 3.8 3.0 2.7 4.2
Dihydrocapsaicin 90.4 94.2 94.3 2.6 4.3 4.2 6.1 4.6 4.1
a
Recoveries are calculated as mean value of triplicate analysis.

Table 3 3.4. Method validation

Results for the determination of capsaicin and dihydrocapsaicin in oil samples.

Sample Number of samples Capsaicin a Dihydrocapsaicin a 3.4.1. Linearity

(g kg1 ) (g kg1 ) Calibration curves were constructed using the ratio of the
Waste oil 1 9 8.4 0.5 3.4 0.3 chromatographic peaks of the analyte to its internal stan-
Waste oil 2 4.4 0.3 11.4 1.1 dard, at different levels in the range of 0.2050.0 g kg1 . To
Waste oil 3 9.7 0.7 26.7 1.9 calibrate the effect of ion suppression or enhancement, capsaicin-
Waste oil 4 2.8 0.1 9.5 0.7
d3 and dihydrocapsaicin-d3 were used as internal standards
Waste oil 5 19.5 0.7 10.8 0.9
Waste oil 6 26.7 1.9 9.8 0.7 for capsaicin and dihydrocapsaicin, respectively. The linearity
Waste oil 7 17.5 4.9 8.6 0.2 of the afore-mentioned ranges was established by analyzing
Waste oil 8 41.2 6.4 19.9 2.8 seven calibration levels (0.20 g kg1 , 0.50 g kg1 , 1.00 g kg1 ,
Waste oil 9 45.2 2.1 16.8 0.9
5.00 g kg1 , 10.00 g kg1 , 20.00 g kg1 , and 50.00 g kg1 ) in
Maize oil 5 N.D.b N.D.
Sesame oil 5 N.D. N.D. three replicates. Good linearity was obtained for each analyte
Sunower oil 5 N.D. N.D. throughout the concentration range, and the calibration equations
Soybean oil 5 N.D. N.D. were calculated as: y = 0.28435 0.00113 with the correlation
Olive oil 7 N.D. N.D. coefcient (r2 ) of 0.9998 for capsaicin and y = 0.21286 0.01895
Rapeseed oil 7 N.D. N.D.
with the correlation coefcient (r2 ) of 0.9992 for dihydrocapsaicin,
Blend oil 5 N.D. N.D.
respectively.
a
Values represent the mean of the triplicate analyses the standard deviation.
b
N.D., not detected.
3.4.2. Limit of detection (LOD) and limit of quantication (LOQ)
Blank samples were selected and spiked with mixed-standard
solutions, and then prepared and analyzed under the optimized
conditions. The LOD was dened as the lowest detectable concen-
evaluated. As shown in Fig. 3d, the best recovery was obtained tration with a signal-to-noise ratio of at least 3, whereas the LOQ
when using 1 mL MeOH. Elution media with lower percentage of was dened as the lowest quantiable concentration with a signal-
methanol may cause less damage to the antibodies and can increase to-noise ratio of at least 10. The LODs and LOQs were calculated to
the reusability of the IAC column. During the evaporation step, be 0.02 and 0.08 g kg1 for capsaicin and 0.03 and 0.10 g kg1
water contained in the eluate makes the cleanup procedure more for dihydrocapsaicin. The results indicated that this method was
time-consuming. sensitive to the capsaicinoid compound in vegetable oils. In Fig. 5,
the LCMS/MS chromatograms were present for the vegetable oil
spiked with 0.5 g kg1 of capsaicin and 0.5 g kg1 of dihydrocap-
3.3.4. Reconstitution medium saicin.
A tailing peak was obtained when MeOH was used as the
reconstitution medium because the analytes have an ionic char- 3.4.3. Accuracy and precision
acter. The reconstitution medium including different percentages The accuracy of the proposed method was studied in terms
of MeOH/H2 O, acetonitrile/H2 O, acetonitrile/PBS (50:50, 40:60, of trueness (systematic error) and expressed as the mean recov-
30:70, 20:80, 10:90, 5:95, v/v) and ACN were evaluated. The results ery. Firstly, blank oil samples were spiked with capsaicin and
showed that acetonitrile/PBS (50:50, v/v) and acetonitrile/H2 O dihydrocapsaicin at different concentrations ranging from 1.0 to
(50:50, v/v) gave larger MS/MS signal than other solutions including 10.0 g kg1 , and the analyte concentrations in crude oil sam-
aqueous MeOH. The ionization suppression of the analytes could ples were calculated based on the calibration curves. Then, the
been caused by the ions from PBS during the electrospray process, oil samples were spiked at different concentrations, and the con-
which would worsen the sensitivity of the MS/MS and contaminate centrations were also calculated in the method mentioned above.
ion source. Thus acetonitrile/H2 O (50:50, v/v) was optimized as the Finally, the recoveries were obtained by comparing the calculated
reconstitution medium in the following experiments. spiked concentration with the corresponding spiked values. In
Table 2, the results indicated that the recoveries of the samples
spiked with different concentrations of capsaicin and dihydro-
capsaicin ranged from 87.3% to 95.2% and from 90.4% to 94.3%,
3.3.5. Binding capacity respectively.
Under the optimal IAC conditions for capsaicinoids described The reproducibility of the method was determined by the intra-
above, the column capacity was evaluated by loading 30 mL and inter-day precision detection of blank vegetable oils spiked
standard solution mixed with capsaicin and dihydrocapsaicin with capsaicin and dihydrocapsaicin at three different concentra-
(10 ng mL1 ). As shown in Fig. 4, the results indicated that the max- tions. The intra-day precisions were determined by six parallel
imum binding capacity of IAC was 240 ng for the total amount of extractions of the sample solution at different concentration levels
capsaicin and dihydrocapsaicin. After the loading volume exceeded within one day, and the inter-day precisions were determined by
the maximum capacity, the amount of analytes remaining on the three parallel extractions of the sample solution at different con-
IAC column attened out, which illustrated the saturation of cap- centration levels for three consecutive days. The results showed
saicinoid binding sites. that the intra- and inter-day RSDs were less than 4.3% and 6.1%,

Please cite this article in press as: F. Ma, et al., Simultaneous determination of capsaicin and dihydrocapsaicin for
vegetable oil adulteration by immunoafnity chromatography cleanup coupled with LCMS/MS, J. Chromatogr. B (2015),
http://dx.doi.org/10.1016/j.jchromb.2015.12.017
G Model
CHROMB-19774; No. of Pages 8 ARTICLE IN PRESS
F. Ma et al. / J. Chromatogr. B xxx (2015) xxxxxx
respectively (Table 2), indicating that satisfactory reproducibility
was achieved by the method.
3.4.4. Matrix effect
The matrix of the complex samples, as well as other variables
such as sample preparation, chromatography and mass spectrom-
etry, could diminish or enhance the ion intensity of the analyte
and affect reproducibility and accuracy of the analytic method. In
addition to optimizing sample preparation and purication, the co-
eluting stable isotope labeled internal standard (SIL-IS) plays an
important role in solving this problem [32]. When added to the
sample at the beginning of the extraction, the SIL-IS experienced all
changes experienced by the analyte, including the ion suppression
effects affecting the ESI response. It was found that when SIL-ISs
were used for analysis, the precision of the slopes of the calibration
standards prepared in ve different lots of vegetable oils was within
the range 1.33.5%, irrespective of the HPLC-MS interface utilized.
The results indicated that the selectivity of the immunoafnity
chromatography method was acceptable for routine analysis of the
capsaicinoid compounds in vegetable oils.
3.5. Application immunoafnity chromatography cleanup
coupled with LCMS/MS for oil adulteration
Under the optimized conditions, the proposed method was
applied to determination of the capsaicinoid compounds in oil sam-
ples including inedible waste oil, maize oil, sesame oil, sunower
oil, soybean oil, olive oil, rapeseed oil, and blend oil. As present in
Table 3, capsaicinoids compounds were found in waste oils, and
the overall ranges of analytes were 2.845.2 g kg1 for capsaicin
and 3.426.7 g kg1 for dihydrocapsaicin. The results also showed
that no capsaicinoid compounds were detected in the vegetable oils
from the market. As expected, capsaicin and dihydrocapsaicin were
only found in waste oil samples, which could act as the putative
biomarkers to detect and authenticate edible vegetable oils.
4. Conclusions
To the best of our knowledge, this is the rst time the anti-
body was generated against capsaicinoid compounds and applied
to IAC cleanup. In this study, a simple, sensitive and accurate IAC-
LCMS/MS method was established for determination of capsaicin
and dihydrocapsaicin in vegetable oil samples. By covalently cou-
pling the highly specic capsaicinoid pAb with CNBr-activated
Sepharose 4B and packing the immunosorbents into a polyethylene
column, the IAC column was utilized for extraction and purica-
tion. Satisfactory results were obtained with regard to sensitivity,
accuracy and precision, and the method was effective in remov-
ing potential interference from vegetable oils since no interference
was observed for quantication of capsaicinoid compounds. The
results also showed that no capsaicinoid compounds were detected
in the vegetable oils from market, and capsaicin and dihydro-
capsaicin were only found in inedible waste oils. Therefore, the
IAC-LCMS/MS method was adequate for routine monitoring of
capsaicinoid compounds in vegetable oil samples, and provided a
practical tool for detection of adulteration with inedible waste oil.
Acknowledgements
This work was supported by the Project of National Science &
Technology Pillar Plan (2012BAK08B03), the National Natural Sci-
ence Foundation of China (31201447), Deutscher Akademischer
Austausch Dienst (DAAD) and the National Major Project for Agro-
product Quality & Safety Risk Assessment (No. GJFP2015006).
Please cite this article in press as: F. Ma, et al., Simultaneous determination of capsaicin and dihydrocapsaicin for
vegetable oil adulteration by immunoafnity chromatography cleanup coupled with LCMS/MS, J. Chromatogr. B (2015),
http://dx.doi.org/10.1016/j.jchromb.2015.12.017
7
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.jchromb.2015.
12.017.
References
[1] F. Lu, X. Wu, China food safety hits the gutter, Food Control 41 (2014)
134138.
[2] M. Abalos, J. Parera, E. Abad, J. Rivera, PCDD/Fs and DL-PCBs in feeding fats
obtained as co-products or by-products derived from the food chain,
Chemosphere 71 (2008) 11151126.
[3] P. Felizardo, M.J. Neiva Correia, I. Raposo, J.F. Mendes, R. Berkemeier, J.M.
Bordado, Production of biodiesel from waste frying oils, Waste Manage. 26
(2006) 487494.
[4] G. Hageman, R. Kikken, F. Ten Hoor, J. Kleinjans, Assessment of mutagenic
activity of repeatedly used deep-frying fats, Mutat. Res. 204 (1988) 593604.
[5] C.J. Jacob, C. Lok, K. Morley, D.A. Powell, Government management of two
media-facilitated crises involving dioxin contamination of food, Public
Underst. Sci. 20 (2011) 261269.
[6] Y. Guo, Z. Zhang, L. Liu, Y. Li, N. Ren, K. Kannan, Occurrence and proles of
phthalates in foodstuffs from China and their implications for human
exposure, J. Agric. Food Chem. 60 (2012) 69136919.
[7] R. Weihaar, Fatty acid esters of 3-MCPD: overview of occurrence and
exposure estimates, Eur. J. Lipid Sci. Tech. 113 (2011) 304308.
[8] X.F. Leong, A. Aishah, U. Nor Aini, S. Das, K. Jaarin, Heated palm oil causes rise
in blood pressure and cardiac changes in heart muscle in experimental rats,
Arch. Med. Res. 39 (2008) 567572.
[9] M. Grootveld, C.J.L. Silwood, P. Addis, A. Claxson, B.B. Serra, M. Viana, Health
effects of oxidized heated oil, Foodservice Res. Int. 13 (2001) 4155.
[10] W.M. Cao, X.H. Sun, F.X. Chen, B. Xue, W. Chen, Q. Jin, X. Wang, Prospect on
discerning technology of swill-cooked dirty oil, China Oils Fats 37 (2012) 15.
[11] H. Esterbauer, Cytotoxicity and genotoxicity of lipid-oxidation products, Am.
J. Clini. Nutr. 57 (1993) 779S785S.
[12] M.T. Osorio, S.A. Haughey, C.T. Elliott, A. Koidis, Evaluation of methodologies
to determine vegetable oil species present in oil mixtures: proposition of an
approach to meet the EU legislation demands for correct vegetable oils
labelling, Food Res. Int. 60 (2014) 6675.
[13] E. Cubero-Leon, R. Penalver, A. Maquet, Review on metabolomics for food
authentication, Food Res. Int. 60 (2014) 95107.
[14] C.A. Nunes, Vibrational spectroscopy and chemometrics to assess
authenticity, adulteration and intrinsic quality parameters of edible oils and
fats, Food Res. Int. 60 (2014) 255261.
[15] L. Zhang, P. Li, X. Sun, W. Hu, X. Wang, Q. Zhang, X. Ding, Untargeted fatty acid
proles based on the selected ion monitoring mode, Anal. Chim. Acta 839
(2014) 4450.
[16] R. Salghi, W. Armbruster, W. Schwack, Detection of argan oil adulteration
with vegetable oils by high-performance liquid chromatography-evaporative
light scattering detection, Food Chem. 153 (2014) 387392.
[17] S. Mildner-Szkudlarz, H.H. Jelen, R. Zawirska-Wojtasiak, E. Wasowicz,
Application of headspacesolid phase microextraction and multivariate
analysis for plant oils differentiation, Food Chem. 83 (2003) 515522.
[18] D.S. Wishart, Metabolomics: applications to food science and nutrition
research, Trends Food Sci. Technol. 19 (2008) 482493.
[19] F. Puiggros, R. Sola, C. Blade, M.-J. Salvado, L. Arola, Nutritional biomarkers
and foodomic methodologies for qualitative and quantitative analysis of
bioactive ingredients in dietary intervention studies, J. Chromatogr. A 1218
(2011) 73997414.
[20] S.F. Kosuge, M. Furata, Studies on the pungent principle of capsicum. Part XIV:
chemical constitution of the pungent principle, Agric. Biol. Chem. 34 (1970)
248256.
[21] L. Ang, J. Jin, S. Wang, X. Wang, Y. Tian, J. Chen, A novel method for the
identication of illegal cooking oil (1): detection of three capsaicinoids with
liquid chromatographymass spectrometry, Se Pu 30 (2012) 10941099.
[22] G.F. Barbero, A. Liazid, M. Palma, C.G. Barroso, Fast determination of
capsaicinoids from peppers by high-performance liquid chromatography
using a reversed phase monolithic column, Food Chem. 107 (2008)
12761282.
[23] A. Pena-Alvarez, E. Ramirez-Maya, L.A. Alvarado-Suarez, Analysis of capsaicin
and dihydrocapsaicin in peppers and pepper sauces by solid phase
microextractiongas chromatographymass spectrometry, J. Chromatogr. A
1216 (2009) 28432847.
[24] G.F. Barbero, A. Liazid, M. Palma, C.G. Barroso, Ultrasound-assisted extraction
of capsaicinoids from peppers, Talanta 75 (2008) 13321337.
[25] Y.R. Wang, Q. Zhang, P.W. Li, W. Zhang, Y. Li, X.X. Ding, Selective sample
cleanup by immunoafnity chromatography for determination of fenvalerate,
J. Chromatogr. B 879 (2011) 35313537.
[26] F. Ma, R. Chen, P. Li, Q. Zhang, W. Zhang, X. Hu, Preparation of an
immunoafnity column with amino-silica gel microparticles and its
application in sample cleanup for aatoxin detection in agri-products,
Molecules 18 (2013) 22222235.
[27] F.A. Esteve-Turrillas, J.V. Mercader, C. Agullo, A. Abad-Somovilla, A.
Abad-Fuentes, Development of immunoafnity columns for pyraclostrobin
G Model
CHROMB-19774; No. of Pages 8 ARTICLE IN PRESS
8 F. Ma et al. / J. Chromatogr. B xxx (2015) xxxxxx

extraction from fruit juices and analysis by liquid chromatography with UV
detection, J. Chromatogr. A 1218 (2011) 49024909.
[28] H.Z. Senyuva, J. Gilbert, Immunoafnity column clean-up techniques in food
analysis: a review, J. Chromatogr. B 878 (2010) 115132.
[29] M. Gasparini, M. Curatolo, W. Assini, E. Bozzoni, N. Tognoli, G. Dusi,
Conrmatory method for the determination of nandrolone and trenbolone in
urine samples using immunoafnity cleanup and liquid
chromatographytandem mass spectrometry, J. Chromatogr. A 1216 (2009)
80598066.
[30] P.W. Li, F. Ma, Q. Zhang, Q.Q. Yang, L.X. Zhang, X.X. Ding, W. Zhang, The design
and synthesis of capsaincin hapten and its application in polyclonal
antibodies, China Patent Int. Cl., CN103951577A, 2014.07.30.
[31] J. Xie, T. Peng, J.L. He, Y. Shao, C.L. Fan, Y. Chen, W.X. Jiang, M. Chen, Q. Wang,
X.Y. Pei, S.Y. Ding, H.Y. Jiang, Preparation and characterization of an
immunoafnity column for the selective extraction of aatoxin B1 in 13 kinds
of foodstuffs, J. Chromatogr. B 998 (2015) 5056.
[32] A.K. Hewavitharana, Matrix matching in liquid chromatographymass
spectrometry with stable isotope labelled internal standardsis it necessary?
J. Chromatogr. A 1218 (2011) 359361.

Please cite this article in press as: F. Ma, et al., Simultaneous determination of capsaicin and dihydrocapsaicin for
vegetable oil adulteration by immunoafnity chromatography cleanup coupled with LCMS/MS, J. Chromatogr. B (2015),
http://dx.doi.org/10.1016/j.jchromb.2015.12.017