Food Research Inter ional xxx ( xxxx ) xx

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Food Research International

journal homep age:

Chemical analysis and classification of black pepper (Piper nigrum L.) based on
their country of origin using mass spectrometric methods and chemometrics
Jian Liang a,b,1, Jianghao Sun c,1, Pei Chen c, Jared Frazier b, Virginia Benefield b, Mengliang Zhang b,*
a
Research Center for Traditional Chinese Medicine Resources and Ethnic Minority Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
b
Department of Chemistry, Middle Tennessee State University, Murfreesboro, TN 37132, USA
c
Methods and Application of Food Composition Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Services, United States Department of
Agriculture, Beltsville, MD 20705-2350, USA

ARTICLEINFO ABSTRACT

pepper
Keywords: The current study applied gas chromatography-mass spectrometry (GC–MS), liquid chromatography-mass Black spectrometry (LC-MS), and thermal desorption direct
DART-MS
analysis in real-time mass spectrometry (TD-DART-MS) methods to the analysis of black pepper (Piper nigrum L.) samples from different countries. The black pepper
Chemometrics GC MS
LC-MS powder methods, samples a methanol were extraction analyzed directly procedure by TD-DART-MS was employed without before any the analysis.
–
extraction, Various but for compounds, GC–MS and such LC-MS as

Classification piperamides and terpenes, were detected. Partial least squares-discriminant analysis (PLS-DA) was used to classify black pepper samples based on their origins. Total ion
mass spectrum (TMS) data profiles from GC–MS, LC-MS, and TD-DART-MS methods were constructed and evaluated for the
performance of classification. A cubic- root data transformation was tested in the data preprocessing and found to be
effective for improving the classification rates. The average classification rates of PLS-DA models with GC–MS-cubic-root-
TMS, LC-MS-cubic- root-TMS, and DART-MS-cubic-root-TMS data representations were 94.1 ± 0.6%, 87.7 ± 0.6%, and 97.0 ±
0.3% respectively, for 100-time bootstrapped-Latin-partition cross-validation. This study presents for the first time the
analysis of plant-based food materials by using TD-DART-MS, and it has been demonstrated as a simple and high- throughput
method for classification studies.

1. Introduction
As one of the most widely used spices in the world, black pepper
(Piper nigrum L.) is valued for its distinct biting quality (Sangeeth & Suseela,
2016; Srinivasan, 2007). Black peppers are cultivated mostly in tropical
regions like South India and Vietnam. The chemical properties of black
peppers are highly diverse and dependent on various factors, including
plantation practices and the growing environment (Nair,
2011). These factors are often influenced by the culture of the society, e.
g., farmers’ belief in their methods of farming, and are linked with the growth
origin of the black pepper. Therefore, the market price of black pepper is
determined not only by the physical properties (e.g., size and density) but
also by the origin. For example, pepper cultivated in India demands a higher
price than others because they are known for the superior aroma and
pungency properties (Schulz, Baranska, Quilitzsch, Schütze, & Losing, ¨ 2005).
In the global market, black peppers are mostly

Abbreviations: CDF, the network common document format; CID, collision-induced dissociation; DART-MS, direct analysis in real time mass spectrometry; EIC, extracted ion
chromatograms/chronograms; ESI, electrospray ionization; FIMS, flow-injection mass spectrometry; FTIR, Fourier-transform infrared spectroscopy; GC–MS, gas chromatography mass
spectrometry; LC-MS, liquid chromatography mass spectrometry; MDA, 3,4-methylenedioxyamphetamine; MDMA, 3,4-methylenedioxy-methamphetamine; PLS-DA, partial least
squares-discriminant analysis; RMSEE, root-mean-square error of estimation; SPME, solid-phase microextraction; TIC, total ion current chromatograms/chronograms; TMS, total ion
mass spectrum; VIP, variable importance for the projection. * Corresponding author.
E-mail address: mzhang@mtsu.edu (M. Zhang). 1
Contributed equally to this manuscript.
https://doi.org/10.1016/j.foodres.2020.109877
Received 22 May 2020; Received in revised form 30 October 2020; Accepted 1 November 2020 Available online 10
November 2020
0963-9969/© 2020 Elsevier Ltd. All rights reserved.
produced and exported by several major countries. As of 2019, Vietnam, Differentiating the growth origin of black peppers can help prevent issues
Indonesia, India, and Brazil were the top four producers of black pepper, such as intentional mislabeling of geographical origin to fetch higher market
collectively accounting about 72% of global black pepper production (Sawe, prices. Various methods are available for the differentiation of black pepper
2019). products. The morphological approach based on the “Graph Theory” model
was employed to cluster fifty-one cultivars of black pepper (Mathew,

Please cite this article as: Jian Liang, Food Research International, https://doi.org/10.1016/j.foodres.2020.109877
J. Liang et al.
Mathew, & Kumar, 2001). DNA sequencing techniques such as Simple
Sequence Repeat or microsatellite have been demonstrated to be useful
tools for the differentiation of black peppers of different origins based on
genetic variability (Joy, Prasanth, & Soniya, 2011; Wu, Fan, Hu, Wu, & Hao,
2016). The morphological methods require extensive knowledge and
experience for accurate identification and are subject to human errors. DNA
contamination or degradation before the amplification limits the applications
of the genetic methods. Chemical profiling, coupled with chemometrics,
could be a good alternative for the classification of black pepper. Traditional
analytical techniques, such as Fourier-transform infrared spectroscopy (FTIR)
and gas chromatography with flame ionization detector (GC-FID), have been
used in previous studies (Lafeuille, Fregiere-Salomon, Michelet, & Henry,
2020; Lima, Batista, Jesus, Silva, Araújo, & Santos, 2020; Mengliang Zhang &
de B. Harrington, & Chen, 2015). However, FTIR methods were often used to
detect the adulterated materials such as sawdust, walnut shell, papaya seed
in black pepper, and would not have sufficient resolving power to distinguish
black peppers of different growth origins (Lafeuille et al., 2020; Lima &
Batista, Jesus, Silva, Araújo, & Santos, 2020). Additionally, GC-based methods
are labor- intensive and usually require prolonged sample preparation and
instrumental analysis.
Direct analysis in real-time mass spectrometry (DART-MS) is an emerging
technique that has attracted considerable attention for chemical analysis in
complex matrices because of its simplicity and effectiveness (Barnett, Bailey,
& Zhang, 2019; Dong et al., 2019; Liang, Frazier, Benefield, Chong, & Zhang,
2020). Several studies have reported the use of DART-MS for the analysis of
food materials such as garlic (Block, Dane, Thomas, & Cody, 2010), grapes
(Jastrzembski, Bee, & Sacks, 2017), and spinach leaves (Singh, 2016).
However, the samples had to be extracted by a selected organic solvent or
solid-phase microextraction (SPME) in these studies, diminishing the
advantage of minimal sample preparation in DART-MS methods. Recently, a
thermal desorption heating stage was interfaced with the DART-MS to
expand its analytical capabilities and provide additional volatility information
of chemicals when a desirable temperature gradient was applied (Maric,
Marano, Cody, & Bridge, 2018). The thermal desorption DART-MS (TD- DART-
MS) has been used in a few forensic applications (Bridge & Maric, 2019; Liang
et al., 2020), showing several advantages over the traditional DART-MS, such
as reduced ion suppression effect, less complex mass spectra, and additional
chronological/temperature dimension to DART-MS data. However, this
technique has never been applied to food- related materials.
The objective of the present study was to explore the potential of the TD-
DART-MS technique for the differentiation of black pepper cultivated in the
world-leading producing countries, including Vietnam, Indonesia, India, and
Brazil. The conventional analytical methods such as GC–MS and LC-MS were
also applied, and their results were compared. The respective data was
further processed by chemometric methods, such as partial least squares
discriminant analysis (PLS-DA), and the samples were classified based on their
country origins. Data transformation was evaluated in the data preprocessing
step and demonstrated to improve PLS-DA classification rates by the cross-
validation with 100-time bootstrapped Latin partitions. To the best of our
knowledge, this study presents the first report of using TD-DART-MS to
analyze food materials. TD-DART-MS has been shown as a promising tool for
chemical profiling and food origination studies, which are essential to ensure
food quality and authenticity.
2. Materials and methods
2.1. Materials and reagents
Seventeen authentic black pepper samples were acquired from the library
of McCormick & Company, Inc. (Sparks, MD, USA). Water, acetonitrile,
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methanol, and formic acid were LC-MS grade (Fisher Scientific, Pittsburgh, PA,
USA). 2.2. Instruments and conditions
The GC–MS system consisted of a Shimadzu QP2010S mass spectrometer
with an autosampler (Shimadzu, Kyoto, Japan). The GC injector was set at 280
◦
C, and the analytes were separated on an SHRCI- 5MS, 30 m × 0.25 mm ID
capillary column chemically bonded with 5% diphenyl/95% dimethyl
polysiloxane at 0.1 µm film thickness (Shimadzu Scientific Instruments Inc.
Columbia, MD, USA). A constant helium flow of 1 mL/min was used, of which
a fixed split of approximately 1:50 entered the column. The column
temperature was programmed as follows: 50 ◦C, hold for 2 min, ramp at 15
◦
C/min to 290 ◦C, hold for 10 min. The transfer line and ion source
temperatures were maintained at 290 ◦C and 250 ◦C. The full scan mode was
selected for the mass spectrometer, and the scan range was from m/z 33 to
400.
LC-MS analyses were performed using a Thermo LTQ XL mass
spectrometer with a Dionex™ UltiMate™ 3000 UHPLC system consisting of a
Dionex™ UltiMate™ LPG-3400SD Standard Quaternary Pump, a Dionex™
UltiMate™ Standard Well Plate Autosampler, and a Dionex 3000 column
chamber (Thermo Scientific, San Jose, CA, USA). The separation was carried
out on a reversed-phase C18 column (ZORBAX Eclipse XDB, 250 × 3.0 mm i.d.,
5 µm) with an UltraLine UHPLC In-Line Filter (RESTEK, Bellefonte, PA, USA) in
a column oven at the temperature of 25 ℃. The mobile phase consisted of a
combination of A (0.5% formic acid in water, v/v) and B (acetonitrile). The
binary gradient employed a program with a flow rate of 0.5 mL/min as
followed: 31% B, hold for 3 min, ramp from 31 to 55% B at 30 min, from 55 to
90% B at 40 min, hold to 50 min. The mass spectrometer was operated in
positive ionization mode. The conditions were set as follows: sheath gas at 35
(arbitrary units), auxiliary and sweep gas at 15 (arbitrary units), spray voltage
at 3.5 kV, the capillary temperature at 500 ◦C, the capillary voltage at 10 V,
and tube lens at 55 V. For the full scan experiments, the mass range was from
m/z 50 to 1000. For the data-dependent scan experiment, the most intense
ion was selected to offer their MS2 product ions with a normalization collision
energy at 35%.
For DART-MS experiments in this study, a Thermo LTQ XL MS (Thermo
Scientific, Darmstadt, Germany) was coupled with a DART ion source
(IonSense inc., Saugus, MA, USA). The mass range was set in an m/z range of
50–1000 and operated in positive-ion mode. The DART heater was set at 500
℃. The IonRocket temperature gradient system (Biochromato, San Diego,
CA, USA) was used for the thermal desorption DART-MS (TD-DART-MS)
analysis, and the temperature program was as follows: 30 ℃, held for 1.0
min, increased the temperature to 600 ℃ at a rate of 100 ℃/min, and held
for 1.0 min. A schematic view of the TD- DART-MS setup can be found in Fig.
S1.
2.3. Sample preparation
Twenty milligrams of each dried ground sample was mixed with 10 mL of
methanol in 15-mL centrifuge tubes. All samples were sonicated in the water
bath for 1 h at room temperature. The water in the sonicator was replaced by
fresh deionized water from the water purification system (PureLab Ultra,
ULGA, Buckinghamshire, UK) between different batches to maintain the same
initial temperature for the extraction. The samples were then centrifuged at
5000 g for 15 min, and the supernatant was filtered through a 0.22 µm nylon
syringe filter (Celltreat Scientific Products, Pepperell, MA, USA). The injection
volumes for GC–MS and LC-MS experiments were 1 µL and 20 µL,
respectively. All sample extracts were analyzed in triplicate with a random
block design. For DART- MS, about 2 mg of black pepper powders (BPP) was
loaded onto a disposable copper sample pot, which was then placed on the
thermal desorption device. A glass T-junction tube was placed over the
J. Liang et al.
sample to direct the compounds desorbed from the sample to be analyzed
directly by DART-MS.
2.4. Data analysis
The GC–MS data (QGD files) were converted to the network common
document format (CDF) with the open-source software OpenChrom
Community Edition. For the data collected from LC-MS and DART-MS, the
obtained RAW files were converted to CDF files with the Xcalibur software
“File Converter Tool”. The CDF files were read directly into MATLAB 2018b
(MathWorks Inc., Natick, MA) using the netcdf function. MATLAB was used
for the data preprocessing, and SIMCA 14.1 (MKS Umetrics, Umea, Sweden)
was used for the PLS-DA modeling.
The original data from the GC–MS, LC-MS, and thermal desorption DART-
MS analyses are three-dimensional matrices: time × mass-to- charge ratios
(m/z) × intensity. For future data processing, the data sets were binned by
retention time with a 0.01 min increment and binned by m/z with a 1
increment. One-way total mass spectrum (TMS) data was constructed by
summing across the time dimension of the binned three- dimensional data
matrices. The data transformation with nth root functions has been
demonstrated to effectively inflate the characteristic chemical features with
smaller intensities(Dong et al., 2019). The cubic root function was tested and
applied to TMS data sets in the data preprocessing in this study.
Normalization was used to minimize the effect of variations from sample
concentrations and the instrument sensitivity, and all data were normalized
to unit vector length(Barnett & Zhang, 2018; Wang, Chen, Yu, & Harrington,
2013).
PLS-DA is a classical supervised pattern recognition technique to relate a
matrix X to a vector Y or to a matrix Y (Zhang & Harrington, 2014). In our
study, the independent data matrix X, such as GC–MS, LC- MS, or DART-MS
data, can be transformed to the latent variables for regression with the
dependent binary variable matrix Y, which represents country origins.
3. Results and discussion
3.1. Comparison of GC–MS, LC-MS, and DART-MS data
Chromatographic techniques coupled with mass spectrometry, such as
GC–MS and LC-MS, are widely used in food analysis. Two groups of secondary
metabolites in black pepper tend to be of most interest to the researchers:
piperamides and terpenes. Terpenes are volatile compounds that impart the
unique aroma to black pepper, and they were primarily analyzed by GC based
methods (Jirovetz, Buchbauer, Ngassoum, & Geissler, 2002; Kapoor et al.,
2009; Mengliang Zhang & de B. Harrington, & Chen, 2015). Piperamides are
responsible for the pungency, flavor, and bioactivity of black pepper, and LC-
MS was commonly employed for the determination of piperamides (Chandra
Bose. Kotte, Dubey, & Murali, 2014; Friedman et al., 2008). In this study, the
methanol extracts of the black pepper samples were analyzed by both GC–
MS and LC-MS methods. Also, the BPP samples were analyzed directly by
using TD-DART-MS. The representative total ion current
chromatograms/chronograms (TIC) were constructed by summing across the
m/z dimension as the examples shown in Fig. S2.
For the GC–MS analysis, the chemical components were tentatively
identified based on the comparison of their retention indices and mass
spectra from existing literature and the mass spectral libraries from the
National Institute of Standards Technology. In the previous studies carried
out by GC related methods, the terpenes are the major aroma compounds
responsible for the odor of black pepper samples and their essential oil
products (Jirovetz et al., 2002; Kapoor et al., 2009). Germacrene D, limonene,
β-pinene, α-phellandrene, and β-caryophyllene were identified in our study
(Fig. S2A and Table S1). Several piperamides, such as piperine and its isomers,
were also observed in the GC–MS results. In previous studies, nonpolar
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extraction solvents such as n-hexane or solid-phase microextraction (SPME)
with nonpolar polymer fiber coatings were used (Jirovetz et al., 2002; Orav,
Stulova, Kailas, & Muurisepp, 2004). These extraction solvents were
selectively more effective for extracting the nonpolar and/or volatile
components such as terpenes. Therefore, the piperamides as a group of
relatively polar and less volatile compounds were not detected. In our study,
methanol was used as the extraction solvent so that both GC–MS and LC-MS
methods could analyze the extracts. As a result, the piperine appeared to be
the dominant peak in the GC–MS TIC profiles (e.g., RT 22.6 min) of our BPP
extracts.
More than 20 piperamides and derivatives were observed from the LC-MS
analysis of BPP extracts (Fig. S2B). These compounds have demonstrated
some signature patterns in the ESI-MS full scan mass spectra and MS 2 product
ion spectra. The protonated dimer ions ([2 M+H] +) were commonly detected
in the full scan MS spectrum with the protonated molecular ions ([M+H] +)
(Table S1), and similar results can be found in the literature when the LC-ESI-
Q-TOF-MS instrument was used (Chandra Bose. Kotte, et al., 2014). In
addition, the primary mass spectral fragments of piperamides have been
reported in previous studies: m/z 201 (C12H9O3+), 171 (C11H7O2+), and 143
(C10H7O+) were formed by the cleavage of the carboxamide moiety (Chandra
Bose. Kotte, et al., 2014; Friedman et al., 2008; Liu et al., 2015). Although m/z
171 and 143 were also observed in some of the MS 2 product ion spectra in
our study, they were present at relatively low intensity. The product ion at
m/z 135 was discovered as a major ion, which is consistent with most of the
product ion spectra of piperamides (Chandra Bose. Kotte, et al., 2014; Liu et
al., 2015); however, its structure elucidation and fragmentation pathway
were unclear. It was noticed that the core structure of piperamides was
similar to a group of designer drugs. For instance, the introduction of a
methylenedioxy moiety into amphetamine or methamphetamine leads to
illicit drugs, such as 3,4-methylenedioxyamphetamine (MDA) or 3,4-
methylenedioxy- methamphetamine (MDMA). Moreover, CID fragmentations
for these illicit compounds have been extensively studied, and m/z 135 was
reported as one of the characteristic fragment ions for screening derivatives
of MDA and MDMA (Apollonio, Whittall, Pianca, Kyd, & Maher, 2006; Awad,
DeRuiter, & Clark, 2007; Bijlsma, Sancho, Hernandez, ´ & Niessen, 2011;
Montesano et al., 2013). Based on the existing literature, a general
fragmentation pathway is proposed in Fig. S3 from which m/z 135
corresponds to 3,4-methylenedioxybenzyl cation (C 8H7O2+). It is worth noting
that the stereoisomeric structures of piperamides widely exist in black
pepper extracts. For example, there are eight theoretically possible
piperettine cis–trans isomers (Friedman et al., 2008), but the data used in the
present study did not permit assigning specific structures to each isomer. All
the significant chromatographic peaks were identified as piperamides and its
derivatives, and the terpenes were not observed. Most of the terpenes in the
extracts are nonpolar compounds, so they cannot be ionized efficiently by
ESI. Further, the low levels of the terpenes in the BPP extracts also lead to
difficult detection.
The chronogram from TD-DART-MS analysis of BPP is shown in Fig. S2C.
No chromatographic techniques were involved in this process. The
compounds were desorbed from powder samples by a fast temperature
program (i.e., 100 ◦C /min from 30 to 600 ◦C) based on their volatility, and a
mild separation was achieved. As shown in Fig. 1A, the desorption times for
pellitorine and piperettine isomers (e.g., m/z 224 and 312, respectively) from
the BPP samples analyzed by TD-DART-MS varied by a difference of about 1
min. However, the two peaks in the extracted ion chronogram (EIC)
overlapped. The chromatographic peaks of the two piperettine isomers were
well resolved on the EIC from the LC-MS analysis, and they were also
separated from the pellitorine peak,
J. Liang et al. Food Research International xxx (xxxx) xxx

the need for extraction, filtration, and concentration procedures. The

analytical run time for the DART-MS method was down to 8 min compared
with 50 min for the LC-MS method and 30 min for the GC–MS method. The
DART-MS method is also clean and ecofriendly because no organic solvent
was used for the sample preparation and instrumental analysis.
Other non-chromatographic spectral fingerprinting methods such as flow-
injection mass spectrometry (FIMS) have been used for the differentiation of
food and botanicals (Chen, Sun, & Ford, 2014; Sun & Chen, 2011; Jianghao
Sun et al., 2018); however, the FIMS approach may suffer from the ion
suppression issue that limits its capability to reveal subtle differences
between samples (Geng, Zhang, Harnly, Luthria, & Chen, 2015).

thermal desorption DART-MS (A) and from black pepper extracts analyzed by LC-MS (B).
Note: EIC stands for “extracted ion chronogram” for DART-MS data and “extracted ion
chromatogram” for LC-MS or GC–MS data.

presented in Fig. 1B. The chronological order of the pellitorine and

piperettine isomers’ peaks were reversed between the EIC from TD- DART-MS
and LC-MS because of their different separation mechanisms: chemical
volatility and polarity.
Fig. 2 shows the comparison of MS spectra for piperine collected by
different MS methods. For LC-MS, both protonated molecular ions (m/z 286)
and protonated dimer ions (m/z 571) were found in the full scan mass
spectrum (Fig. 2A). The characteristic ions, including m/z 201 and 135, were
observed in the product ion spectrum (Fig. 2B). More fragments were present
in the EI mass spectrum (Fig. 2C) from GC–MS analysis because of the highly
energetic electrons (70 eV) used in EI. The molecular ions (M +, m/z 285) of
piperine were identified in the EI mass spectrum. The DART-MS spectrum for
piperine was similar to the LC-MS spectrum but had a lower relative
abundance of the protonated dimer ions (m/z 571) (Fig. 2D). Metastable
helium (He*) from DART has an energy of 19.8 eV and ionizes the
atmospheric water first, which then transfers the protons to molecules in the
gas stream to produce mainly
[M+H]+ cations (Cody, Laramee, & Durst, 2005). The different ionization
mechanisms result in distinct MS spectra in Fig. 2. Overall, in comparison with
EI, the DART-MS spectrum has fewer fragments. Compared with ESI, adduct
ions such as alkali metal cations attachment are also rarely observed.

3.2. Overall performance of different methods for BPP analyses

Both GC–MS and LC-MS techniques are effective and powerful for
analyzing selected secondary metabolites in BPP sample extracts. TD- DART-
MS cannot offer the equivalent separation efficiency for the mix of
compounds in the complex matrices compared with other traditional
chromatographic methods, but it provides new opportunities for high-
throughput phytochemical profiling and discriminating between botanical
materials when combined with chemometrics. Unlike the GC–MS and LC-MS
methods that require the time-consuming and labor- intensive sample
preparation, the BPP samples were analyzed directly by TD-DART-MS without

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As described previously, a small number of adduct ions and fragment ions

are formed in the DART-MS spectra, so DART-MS simplifies the interpretation
of mass spectra and provides good sensitivity to analytes by not spreading
the ion current over more data channels (Cody et al., 2005). The formation of
noncovalent bond dimers in ESI and other ionization techniques is believed

Fig. 2. Piperine mass spectra under different instrument conditions: (A) ESI-MS full scan mass spectrum; (B) ESI-MS2 product ion spectrum of the [M + H] + molecular

ion (@CID 35); (C) EI-MS full scan mass spectrum; (D) DART-MS full scan mass spectrum after background subtraction.
to be concentration-dependent (Ding & Anderegg, 1995; Forbes &
Verkouteren, 2019; Stefansson, Sjoberg, ¨ & Markides, 1996). However, the
piperine dimer ions from black pepper samples by thermal desorption DART-
MS were found to be mainly affected by the desorption temperature, and
this is evident from the data presented in Fig. S4. The highest intensity of
dimer ions was observed at around 400 ◦C, while the piperine monomer ions
showed the highest abundance at about 300 ◦C. This temperature
dependence phenomenon for dimer formation was also observed in another
study in which the gas- phase nitrate radical was used to oxidize and ionize
limonene, although the mechanism is unclear (Faxon, Hammes, Le Breton,
Pathak, & Hallquist, 2018).
In conclusion, TD-DART-MS has shown its advantages of high sample
throughput, ease of use, the need for minimum sample preparation, and
environmental sustainability for the BPP analysis in our study. It could be a
valuable tool for quality control and authentication studies of black pepper as
well as possibly other food materials.

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3.3. Classification of black pepper based on origin methods, including PLS-DA, optimize the models based on a least-squares
minimization of error, giving a higher weight to larger signals (Massart,
Total ion mass spectral (TMS) profiles were constructed by summing
Vandeginste, Michotte, & Kaufman, 1988). However, small peaks may
across the time dimension of the original three-dimensional GC–MS, LC- MS,
contain more discriminatory information for the classification. With the cubic
and TD-DART-MS data. TMS data representation combined with
root data transformation in the data preprocessing, it can give greater
chemometrics has been used for different classification and discrimination
importance to smaller peaks while building the PLS-DA models. For BPP
studies, exploiting the advantages of smaller data size, resistance to retention
samples from different countries, their chemical profiles are expected to be
time shift influence, and enhanced inner- and inter- laboratory reproducibility
similar, and identifying the subtle differences could be relatively easier with
(Barnett et al., 2019; Sigman, Williams, Castelbuono, Colca, & Clark, 2008).
the data transformation. As compared to the TMS profiles in Fig. 3, the
Given the complex nature of the chemical components in the BPP samples,
relative intensity of the minor peaks appears more significant after the
large variations of ion and intensity distributions were observed. This lead to
transformation. Major chemical components such as terpenes and
difficulties in classification. Partial least square discriminant analysis (PLS-DA)
piperamides are ubiquitous in BPP samples despite their origin. With the
models were constructed by using the TMS data from GC–MS, LC-MS, and

transformed data, the separation of different classes built on the first two
TD-DART-MS for the classification of BPP samples based on their country latent components of PLS-DA models was much
origin.
Fig. 3 displays the representative TMS of GC–MS, LC-MS, and DART- MS
data and the scores plots for the first two latent components derived from
the corresponding PLS-DA models. As demonstrated, the score plot from GC–
MS TMS data provided a limited classification of the black pepper samples of
different origins. Although the PLS-DA models obtained from LC-MS TMS and
DART-MS TMS data demonstrated better classifications for the samples
investigated here, samples from different countries were not entirely
separated from each other. For example, the black pepper samples from
India were overlapped by the samples from Indonesia from the projections of
the first two components of the PLS- DA model by DART-MS TMS data (Fig.
3F). The cubic root data transformation was applied to the TMS data, and the
transformed mass spectral profiles are shown in Fig. 4. Many chemometric

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improved (Fig. 4B, D, and F). spectra at low intensities but with higher weights for the classification. The
The PLS-DA model performance was evaluated by the root-mean- square average EIC profiles of the top 2 ions from the VIP plot, m/z 264 and 363,
error of estimation (RMSEE) value and cross-validation with 100-time from the TD-DART-MS data for the BPP samples of different countries, are
bootstrapped Latin partitions. The results are presented in Table 1. The errors displayed in Fig. S5. In general, two peaks were observed for both ions at the
(RMSEE values) of PLS-DA models were significantly reduced after the cubic desorption time of
root transformation compared with the original TMS data from different
instrumental techniques. Similarly, the average classification rates of PLS-DA
models by bootstrapped Latin Partitions were improved at least 20% after
cubic root data transformation was applied to the TMS profiles. Among the
six data representations tested here, DART-MS cubic-root TMS was the most
effective in the classification of BPP samples by the PLS-DA method, offering
the minimum RMSEE of 0.075 and the best average classification rate of 97.0
± 0.3%. The results indicate that the PLS-DA classifier successfully classified
BPP samples based on their country origin when DART-MS cubic-root TMS
was used.
The variable importance for the projection (VIP) plot reflects the
significance of the variables both to explain X (the projection) and to
correlate to Y (the classes) in the PLS-DA model. The variables with VIP values

larger than 1 are deemed important (Kirdar, Green, & Rathore, 2008). The
interval of VIP values between 1 and 1.5 is also considered as a gray area,
where the importance level depends on the size of the data set. Therefore, a
VIP value of 1.5 was used as the critical value in our study to evaluate the
significance. In total, 57 significant variables were found. The top 10 variables
were sorted from high to low based on VIP values and are shown in Fig. 5 for
the PLS-DA model constructed by DART-MS-cubic-root-TMS data. As
expected, the most important variables from this plot were not those
common terpenes or piperamides. These ions were present in DART-MS

7
J. Liang et al.
Table 1
RMSEE and average classification rates with 95% confidence intervals of PLS-DA models constructed by different data representations.
RMSEE Average classification ratesa (%)
GC–MS-TMS 0.316 61 ± 1
GC–MS-cubic-root-TMS 0.113 94.1 ± 0.6
LC-MS-TMS 0.303 66 ± 1
LC-MS- cubic-root-TMS 0.193 87.7 ± 0.6
DART-MS-TMS 0.144 75 ± 1
DART-MS-cubic-root-TMS 0.075 97.0 ± 0.3
a
Averages were calculated from 100 × 4 bootstrapped Latin partitions.
Fig. 5. The VIP plot with 95% confidence intervals for the PLS-DA model constructed by DART-MS-cubic-root-TMS data.
3.2 and 4.8 min. However, the relative intensities of the EIC profiles have
shown significant variations for samples from different countries. For
example, similar intensities for EIC of m/z 264 and 363 at 3.2 min were
observed for the samples originating from India. The EIC of m/z 264 has also
shown a much higher intensity than the EIC of m/z 363 at 3.2 min for the
Indonesian black pepper samples. The identification of variables was not
attempted in this study. Correlation and interactions among variables are
taken into account in PLS-DA models, so comprehensive profiles of all
important variables instead of selective variables would be critical for the
success of classification.
4. Conclusion
Black pepper samples were analyzed and classified by GC–MS, LC- MS,
and TD-DART-MS with chemometrics. TD-DART-MS was intro-
duced and evaluated for the direct analysis of food materials for the first
time. It was faster and easier to employ as no solvent extraction and
chromatographic separation was required. Reliable discrimination among
black pepper samples from different geological origins was achieved when
coupled with chemometric strategies such as PLS-DA and cubic-root data
transformation. For example, the average classification rate was 97.0 ± 0.3%
for DART-MS cubic-root TMS data from cross- validation with 100-time
bootstrapped Latin partitions. This study demonstrated a high-throughput
and robust method for the classification of BPP samples without using a long
chromatographic program for full separation of primary chemical
constitutions or solvent extractions before the instrumental analysis.
CRediT authorship contribution statement
Jian Liang: Data curation, Investigation, Writing - review & editing.
Jianghao Sun: Data curation, Formal analysis, Writing - review & editing. Pei
Chen: Supervision, Funding acquisition, Writing - review & editing. Jared
Frazier: Investigation, Writing - review & editing. Virginia Benefield:
Investigation, Writing - review & editing. Mengliang Zhang:
Conceptualization, Supervision, Funding acquisition, Writing - original draft,
Writing - review & editing.
8
Food Research International xxx (xxxx) xxx
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence the
work reported in this paper.
Acknowledgment
This research was supported by Middle Tennessee State University
(MTSU), the Agricultural Research Service of the U.S. Department of
Agriculture, and an Interagency Agreement with the Office of Dietary
Supplements at the National Institutes of Health. Jian Liang is grateful for the
financial support of the National Natural Science Foundation Committee of
China (grant number 81560641) and Science and Technology Research
Projects of Education Department of Jiangxi Province (grant number
GJJ170725). Jared Frazier received support from an MTSU Undergraduate
Research Experience and Creative Activity (URECA) grant.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodres.2020.109877.
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