foods

Article
HPLC-Based Chemometric Analysis for
Coffee Adulteration
Wai Lok Cheah and Mingchih Fang *
Department of Food Science, College of Life Science, National Taiwan Ocean University, No 2, Beining Rd.,
Keelung City 20224, Taiwan; [email protected]
* Correspondence: [email protected]; Tel.: +88-62-2462-2192 (ext. 5123)




Received: 2 June 2020; Accepted: 30 June 2020; Published: 4 July 2020


Abstract: Coffee is one of the top ten most adulterated foods. Coffee adulterations are mainly
performed by mixing other low-value materials into coffee beans after roasting and grinding,
such as spent coffee grounds, maize, soybeans and other grain products. The detection of
adulterated coffee by high performance liquid chromatography (HPLC) is recognized as a targeted
analytical method, which carbohydrates and other phenolic compounds are usually used as markers.
However, the accurate qualitation and quantitation of HPLC analyses are time consuming. This study
developed a chemometric analysis or called non-targeted analysis for coffee adulteration. The HPLC
chromatograms were obtained by direct injection of liquid coffee into HPLC without sample
preparation and the identification of target analytes. The distinction between coffee and adulterated
coffee was achieved by statistical method. The HPLC-based chemometric provided more characteristic
information (separated compounds) compared to photospectroscopy chemometric which only provide
information of functional groups. In this study, green Arabica coffee beans, soybeans and green
mung beans were roasted in industrial coffee bean roaster and then ground. Spent coffee ground
was dried. Coffee and adulterants were mixed at different ratio before conducting HPLC analysis.
Principal component analysis (PCA) toward HPLC data (retention time and peak intensity) was able
to separate coffee from adulterated coffee. The detection limit of this method was 5%. Two models
were built based on PCA data as well. The first model was used to differentiate coffee sample from
adulterated coffee. The second model was designed to identify the specific adulterants mixed in the
adulterated coffee. Various parameters such as sensitivity (SE), specificity (SP), reliability rate (RLR),
positive likelihood (+LR) and negative likelihood (−LR) were applied to evaluate the performances
of the designed models. The results showed that PCA-based models were able to discriminate pure
coffee from adulterated sample (coffee beans adulterated with 5%–60% of soybeans, green mung
beans or spent coffee grounds). The SE, SP, RLR, +LR and −LR for the first model were 0.875, 0.938,
0.813, 14.1 and 0.133, respectively. In the second model, it can correctly distinguish the adulterated
coffee from the pure coffee. However, it had only about a 30% chance to correctly determine the
specific adulterant out of three designed adulterants mixed into coffee. The SE, RLR and −LR were
0.333, 0.333 and 0.667, respectively, for the second model. Therefore, HPLC-based chemometric
analysis was able to detect coffee adulteration. It was very reliable on the discrimination of coffee
from adulterated coffee. However, it may need more work to tell discern which kind adulterant in
the adulterated coffee.

Keywords: coffee; adulteration; chemometrics; PCA; food fraud

Foods 2020, 9, 880; doi:10.3390/foods9070880 www.mdpi.com/journal/foods

Foods 2020, 9, 880 2 of 11

1. Introduction
The coffee bean is one of the most widely traded agricultural commodities and its consumption
has increased rapidly [1]. Many gourmets say what you choose to drink in the morning tells what
kind of person you are. The methods of coffee roasting, brewing and drinking are currently hyped
up to an art in certain specialty coffees. Animal-passed coffee beans may be one of the most poetic
examples. Based on the Database of United States Pharmacopeia Convention (USP) [2], coffee is the
top ten adulterated food products due to its high commercial value and the shortage of coffee beans
worldwide. Coffee adulteration may be performed by changing the quality of beans or adding other
low-cost coffee and non-coffee materials, such as spent coffee grounds, corn, barley, maize, soybeans
and other grains which bear a resemblance to coffee beans in term of color, particle size and texture [3,4].
Roasted soybeans have been found to be a good adulterant of coffee, because their color, flavor and
aroma are similar to coffee. The making process has even be patented [5]. The contamination of
low-priced robusta species in claimed 100% Arabica coffees is another means of coffee fraud. There are
also cases of coffee products being recalled due to presence of undeclared drug contents, namely,
sildenafil and tadalafil [6]. In order to protect consumers and ensure food safety, the authenticity
assessment of coffee products is a positive way of approaching the problem of coffee fraud.
Several analytical methods have been proposed to identify the adulteration of ground roasted coffee,
including gas chromatography-mass spectrometry (GC-MS) [3], high-performance liquid chromatography
(HPLC) [7–9], ultraviolet-visible spectrophotometry (UV-Vis) [10], ultra-performance liquid chromatography
(UPLC) [11], Fourier-transform infrared spectroscopy (FTIR) [12] and voltammetric electronic tongue [13].
The above methods apply either targeted- or non-targeted analysis methods. Targeted analysis methods
are broadly used to detect food contaminations—as well adulterations—which focus on detecting one
or more compounds in a specimen. Specific targeted methods are generally complicated but can detect
analytes in part per trillion (ppt) levels in complex matrices [14]. However, most of the adulterations are
unknown additive compounds, so the targeted analysis is not always effective. Non-targeted methods
such as chemometric analyses are the combination of emerging analytical methods and statistic software
to detect food adulterations [15]. Non-targeted analysis treats food data as fingerprints. These indicate
authenticity and provide early warning information for adulterated food. Therefore, the development of
an HPLC-based non-targeted analysis to detect coffee adulteration is paramount.
In a review of the literature, detecting coffee adulteration by using liquid chromatography belongs
to a target analysis method that analyzes compounds (carbohydrates) in coffee and adulterated coffee to
accomplish the distinction [7–9,11]. In contrast, spectroscopy methods have evolved as non-target analytical
methods to detect coffee adulteration. For example, one study used ultraviolet-visible (UV-Vis) spectroscopy
and successive projection algorithms (SPA) to construct a linear discriminant analysis (LDA) model for the
identification of adulterants in ground roasted coffee [10]. In many coffee researches, coffee beans were
roasted in convection ovens in laboratories to the desired color and intensity. The heating/roasting effect of
a conventional oven was slow and inhomogeneous. Hence, the roasting time was long—40 min on average
for a batch [12,16], while a commercial coffee bean roaster only requires 12–15 min. In order to simulate
the commercial roasting method and enhance the representativeness of the sampling, industrial roaster
was used in the study. In addition, many chemometric spectroscopy methods for coffee adulteration have
been conducted on the powdered form of coffee [12], or extracted by solvents such as CDCl3 for nuclear
magnetic resonance (NMR) analysis [17]. However, coffee is normally consumed in liquid form, and hot
water is the only solvent. In this study, commercial roasting and brewing methods were applied for coffee
sample preparation. Non-targeted analysis, coupled with HPLC was developed for the discrimination
among roasted coffee, common adulterants and mixtures of coffee and adulterants. The statistical method
used in this research was principal component analysis (PCA). Two models were developed in order to
evaluate the ability of the method to detect coffee adulteration, as well as to identify the adulterants in the
adulterated sample.
Foods 2020, 9, x FOR PEER REVIEW 3 of 11
Foods 2020, 9, 880 3 of 11

2. Materials and Methods

2. Materials and Methods
2.1. Coffee and Adulterants
2.1. Coffee
Greenand Adulterants
Arabica coffee beans (Chanchamayo, Peru) were purchased from Ho Hsin Beans Trading
Co., Green
Ltd (Taichung,
Arabica coffee Taiwan). Soybeans and Peru)
beans (Chanchamayo, green were
mung beans were
purchased from obtained
Ho Hsin Beans from Trading
a local
supermarket.
Co., Ltd (Taichung,SpentTaiwan).
coffee grounds
Soybeans were
and supplied
green mung by beans
chain 85
were°Cobtained
Bakery Cafefromshop
a localinsupermarket.
Taiwan and
kept frozen
Spent coffee(−12 °C) until
grounds wereuse.supplied by chain 85 ◦ C Bakery Cafe shop in Taiwan and kept frozen
(−12 ◦ C) until use.
2.2. Roasting
2.2. Roasting
Spent coffee grounds were defrosted at room temperature for 18 h, and then dried in a
convectioncoffee
Spent oven grounds
(Model were defrosted
DK-500DT, at room
Bioman, Newtemperature for 18 h, at
Taipei, Taiwan) and then
100 °Cdried
for 5inh.a Inconvection
order to
oven (Model DK-500DT, Bioman, New Taipei,coffee Taiwan) at 100 ◦ C for
simulate the roasting methods of commercial in this study, an 5 h. In order to simulate
industrial-grade the
800-N coffee
roasting
bean roastermethods
(YangofChia
commercial
Machine coffee in this
Works, study, an
Taichung, industrial-grade
Taiwan) was used.800-NCoffeecoffee
beansbean(500roaster (Yang
g), soybeans
Chia
(500 g)Machine
and greenWorks,
mung Taichung,
beans (500 Taiwan) waseach
g) were used.subjected
Coffee beans
to the(500 g), soybeans
coffee bean roaster (500atg)180
and°C.
green
The
mung beans (500
temperature wentg)down
were each
to the subjected
turningtopoint
the coffee bean up
and went roaster
to 160 °C ◦in
at 180 C. 8The temperature
min, which was went
the
down to the turning point and went up to 160 ◦ C in 8 min, which was the dehydration process of coffee
dehydration process of coffee beans and adulterants. The first cracking sound happened in the coffee
beans ◦ C.
beans and adulterants.
at 11.5 min and 195 The°C.
first cracking
The sound happened
other materials in the coffee
did not crackle. Coffeebeans
beans,at 11.5 min and
soybeans and195green
The
mung other materials
beans did not crackle.
were removed Coffee beans,
to an air-cooling soybeans
chamber andthe
when green mung beans
temperature were
rose removed
to 210 to an
°C, 230 °C
air-cooling chamber when the temperature rose to 210 ◦ C, 230 ◦ C and 240 ◦ C, respectively. The roasting
and 240 °C, respectively. The roasting times were also different for each sample: 11–14 min for
times
roastingwere alsobeans,
coffee different for min
16–18 eachforsample: 11–14
roasting the min for roasting
soybeans and 16–17 coffee
min beans, 16–18 min
for roasting for roasting
the green mung
the soybeans
beans. and 16–17
The roasting min for was
end-point roasting the greenby
determined mung beans. The roasting
the appearance in color end-point
to the desiredwas determined
brown. The
by the appearance
roasting in color
curves of coffee to the
beans, desiredand
soybeans brown.
green The roasting
mung beanscurves of coffee
are shown beans,
in Figure 1. soybeans and
Samples were
green
grinded mung beans are
by electric shown
grinder in Figure
(Model 600,1.Yang
Samples
Chia were grinded
Machine Works,by electric
Taichung,grinder (Model
Taiwan) after600, Yang
roasting
Chia Machine Works,
and submitted to colorTaichung,
evaluation.Taiwan) after roasting and submitted to color evaluation.

Figure 1. Roasting curves of green Arabica coffee

coffee beans,
beans, soybeans
soybeans and
and green
green mung
mung beans.
beans.

2.3.
2.3. Color
Color Evaluation
Evaluation
AA tristimulus colorimeter
tristimulus colorimeter (TC-1800
(TC-1800 MK-II,
MK-II, Tokyo
Tokyo Denshoku
Denshoku Co.,Co., Tokyo,
Tokyo, Japan)
Japan) was
was used
used to
to
measure the luminosity (CIELAB L*) of the samples, which was the most relevant
measure the luminosity (CIELAB L*) of the samples, which was the most relevant parameter above parameter above
a*
a* and
and b*
b* for
for roasted
roasted coffee
coffee beans
beans [18].
[18]. It
It was
was also
also successfully
successfully employed
employed asas aa reference
reference for
for roasting
roasting
degree
degree [16]. Comparing the value of luminosity, the degree of coffee-roasting can be classified into
[16]. Comparing the value of luminosity, the degree of coffee-roasting can be classified into
three
three groups:
groups: light (23.5 <<L*
light (23.5 L*<<25.0),
25.0),medium
medium (21.0
(21.0 < L*
< L* < 23.5)
< 23.5) andand dark
dark < L*<<L*
(19.0
(19.0 < 21.0)
21.0) [16]. [16].
The
The measurement
measurement of luminosity
of luminosity waswas performed
performed in three
in three replicates.
replicates.
Foods 2020, 9, 880 4 of 11

2.4. Experimental Design

Coffee beans, soybeans and green mung beans were each from three roasting batches (for three
replicates). Spent coffee grounds were obtained from three different dates. Ten pure materials of each
batch including coffee beans, soybeans, green mung beans and spent coffee grounds were prepared as
pure samples—in total 4 × 10 × 3 = 120 samples (4 materials, 10 replicates, 3 batches). The adulterated
samples were prepared by intentionally mixing coffee beans and adulterants at different ratios (coffee:
adulterants, 95:5, 90:10, 80:20, 60:40, 40:60% w/w) in every batch (3 × 3 × 5 = 45, 3 batches, 3 adulterants,
5 concentrations). Cross-batch mixtures were also prepared (3 × 5 = 15, 3 adulterant cross-batches,
5 concentrations). In total, 60 adulterated samples were prepared. All 180 samples were submitted to
HPLC analysis.

2.5. Chromatograms Acquisition of Brewed Coffee and Adulterated Coffee

A Shimadzu LC-2040C Plus (Shimadzu Co., Kyoto, Japan) equipped with an Agilent Zorbax
SB-Phenyl C18 column (150 mm × 4.6 mm, 5 µm) and a PDA (photodiode-array) detector was used
in the measurement. The mobile phases were pure water (solvent A) and methanol (solvent B).
The gradient elution was operated as flowing: 0–4 min: 2% B, 4–8 min: 4%–10% B, 8–13 min: 10%–20%
B, 13–20 min: 20%–35% B, 20–27 min: 35%–90% B, 27–27.5 min: 90%–2% B, 27.5–30 min: 2% B.
The injection volume and flow rate were 20 µL and 1 mL min−1 , respectively. The PDA was set at scan
range between 200 and 800 nm; the chromatogram was monitored at 254 nm.
Coffee and adulterated coffee samples were well-brewed in order to simulate commercially brewed
coffee. The water temperature was controlled in the range of 91–94 ◦ C. Two grams each of ground
coffee beans and adulterated coffee samples were brewed with 25 mL water for 10 min. Each sample
was centrifuged at 1000 × g for 5 min and then filtered with No. 1 filter paper. The filtrate was passed
through a 0.2-µm PTFE filter and then transferred into a 1.5 mL vial for analysis.

2.6. Statistical Analysis

SPSS version 22 (Statistical Product and Service Solutions, IBM Co., New York, NY, USA) was used
to evaluate the color measurements between the samples of coffee and adulterated coffee in order to
ensure uniformities among batches, adulterants and mixtures. An independent t-test was used in this
analysis. Principle component analysis (PCA) with normalized HPLC chromatogram (retention time
alignment of peaks) data was applied to distinguish adulterated coffee from roasted coffee samples.
For PCA analysis, data metrices were constructed (each row corresponded to a sample; each column
represented peak intensity at a given retention time) and handled by MATLAB software version 2018a
(MathWorks, Natick, MA, USA) with the Classification Learner software application. Two models
were built to us the PCA data. The first model was built with 180 samples (30 for pure coffee, 30 for
pure soybeans, 30 for pure green mung beans, 30 for pure spent coffee grounds, and 60 for adulterated
coffee) aimed on the authentication of coffee samples. The second model was built with 150 samples
(180 samples from the first model, excluding 30 samples of pure coffee) to test the ability to identify the
adulterants. In the model experiments, 60% of the samples were randomly selected as training set; the
others were treated as the test set.
The performances of the studied models were characterized by evaluating the quality of figure of
merit (FOM). Figure of merit correspond to numeric parameters such as specificity (SP), sensitivity
(SE) and reliability rate (RLR) [19]. The definition of the specificity (SP) was the ratio of true-negative
samples (TN) to the sum of false-positive samples (FP) and the total number of known-negative samples
(equation 1). It provided a measure of how well the model can predict samples of the class of controls.
The sensitivity (SE) defined as the ratio of true positive samples (TP) to the sum of false-negative
samples (FN) and the total number of known positive samples (equation 2). It provided a measure of
Foods 2020, 9, 880 5 of 11

how well the model correctly identify samples of a given class [20]. The reliability rate (RLR) provided
an overview of the trueness of the model and its definition was shown in equation 3 [12].

TN
Specificity (SP) = (1)
(TN + FP)

TP
Sensitivity (SE) = (2)
(TP + FN )
FN FP
   
RLR = 1 − + = SP + SE − 1 (3)
TP + FN TN + FP
In the first model, the TP was defined as the adulterated sample and was successfully detected
by the system. The TN was the pure coffee sample or other pure adulterants and was successfully
detected. The FP was the pure coffee sample but was misclassified as adulterated sample. The FN
was the adulterated sample but was misclassified as pure coffee or other adulterants. In the second
model, the TN was the pure adulterant and successfully detected. The FP was the pure adulterant
and misclassified as adulterated coffee. TP was defined as the adulterated sample and the specific
adulterant was successfully identified. The FN was the specific adulterant in the adulterated coffee
which was classified as the other adulterant in the adulterated coffee or the pure adulterant.
The positive likelihood ratio (+LR) and the negative likelihood ratio (−LR) were also applied to
evaluate the performance of the studied models. The definitions of +LR and −LR are listed in equation
4 and equation 5 [21]. A bigger value of +LR gave the stronger confidence of the positive result, while
a smaller value of −LR presented the higher value of the testing results [22].

SE
+ LR = (4)
1 − SP
1 − SE
− LR = (5)
SP

3. Results and Discussion

3.1. Coffee Roasting and Color Measurement

The color measurement was performed to evaluate whether the appearance of the roasted
adulterants and the coffee were similar after roasting and grinding. Table 1 shows the L* value of
roasted coffee (ground) was significantly different from roasted soybeans, green mung beans and
spent coffee grounds (p < 0.05) and spent coffee grounds was the darkest one (L* was the lowest
18.0). Similar studies of roasted coffees and adulterants in convection ovens with precisely controlled
temperatures, such as 240 ◦ C 11 min for coffee, 240 ◦ C 30 min for corn and 250 ◦ C 28 min for barley
resulted in color similarity [16]. In this study, soybeans and green mung beans required higher roasting
temperatures and time to reach the desired color. The industrial-grade coffee bean roaster continually
increased the temperature over time during the roasting and it was not easy to control, compared to
convection oven-roasting in which there is a constant temperature during the whole roasting procedure.
We tried to roast the adulterants at higher temperatures and for longer times. In these overheated
roasting conditions, a similar color was obtained, but the flavor was burnt. Therefore, we decided
to roast the adulterants in coffee-bean way. The final temperature was slightly increased to 230 ◦ C
and 240 ◦ C for soybeans and green mung beans, respectively. The maximum temperature of the
commercial roaster for coffee was 240 ◦ C. The roasting time for adulterants was extended to around
18 min as shown in Figure 1. The condition resulted in good-smelling soybeans and green mung beans;
the soybean especially smelled like coffee. Though the color of adulterants and coffee was different,
there was no significant difference among all adulterated coffees in which coffee was mixed with 20%
adulterants (p > 0.05).
Foods 2020, 9, x FOR PEER REVIEW 6 of 11
Foods 2020, 9, 880 6 of 11

Table 1. L* values of coffee beans, adulterants and adulterated coffee beans.

Table 1. L* values of coffee beans, adulterants andMeasurement
Luminosity adulterated coffee
Resultbeans.
Analyte L* (Mean ± SD,
p-Value (t-Test
Luminosity of Coffee and
Measurement Other Analytes)
Result
Analyte n = 3)
Coffee beans SD, n = 3)
20.8 ±±0.8
L* (Mean p-Value (t-Test of Coffee and Other Analytes)
Soybeans
Coffee beans 27.5 ± 0.4
20.8 ± 0.8 0.00
Green mung
Soybeans beans 30.5 ± 1.2
27.5 ± 0.4 0.00 0.00
Spentmung
Green coffeebeans
grounds 18.0 ± 0.2
30.5 ± 1.2 0.00 0.00
Coffeecoffee
Spent beansgrounds
+ soybeans * 21.4 ± 0.7
18.0 ± 0.2 0.10 0.00
Coffee
Coffeebeans
beans + soybeans
+ green mung *beans * 21.5 ± 0.6
21.4 ± 0.7 0.07 0.10
Coffee beans++green
Coffeebeans spentmung
coffeebeans * *
grounds 21.5
20.5 ± 0.6
± 0.4 0.28 0.07
Coffee beans + spent coffee grounds * roasted20.5
* 20% ± 0.4 in coffee beans.
adulterant 0.28
* 20% roasted adulterant in coffee beans.
3.2. HPLC Chromatograms
3.2. HPLC Chromatograms
Applications of HPLC for the detection of adulterated coffee were mainly based on targeted
methods.Applications of HPLC
Analytes such for the detectionmonosaccharides,
as oligosaccharides, of adulterated coffee were mainly
trigonelline, based
and/or on targeted
nicotinic acid
methods. Analytes such as oligosaccharides, monosaccharides, trigonelline, and/or
were applied as markers to identify adulterants in coffee [7–9,11]. In this study, HPLC with nicotinic acidnon-
were
applied as markers to identify adulterants in coffee [7–9,11]. In this study, HPLC
targeted analysis was developed to differentiate coffee adulteration. HPLC analysis was directly with non-targeted
analysis was
performed afterdeveloped to differentiate
sample extraction and thecoffee adulteration.
generated HPLC was
chromatogram analysis wasas
utilized directly performed
a “fingerprint”.
Figure 2 shows the HPLC chromatograms of pure coffee, pure adulterants and adulteratedFigure
after sample extraction and the generated chromatogram was utilized as a “fingerprint”. coffee 2
shows the20%
containing HPLC chromatograms
soybeans. of pure
Coffee beans coffee,2A),
(Figure puresoybeans
adulterants and adulterated
(Figure 2B) and green coffee
mung containing
beans
20% soybeans. Coffee beans (Figure 2A), soybeans (Figure 2B) and green mung
(Figure 2C) showed very distinct chromatograms because each material presented their own special beans (Figure 2C)
showed very distinct chromatograms because each material presented their own
compounds, such as caffeine, tannic acid, linoleic acid, nicotinic acid, chlorogenic acids and special compounds,
such as caffeine,
trigonelline tannic[23].
in coffee acid,Soybeans
linoleic acid, nicotinic isoflavones
contained acid, chlorogenic
[24]. acids
Greenandmung
trigonelline
beans incontained
coffee [23].
Soybeans contained isoflavones [24]. Green mung beans contained polyphenols, polysaccharides
polyphenols, polysaccharides and peptides [25]. However, the chromatograms of coffee, spent coffee and
peptides [25]. However, the chromatograms of coffee, spent coffee grounds (Figure 2D)
grounds (Figure 2D) and adulterated coffee beans (Figure 2E) showed high similarities. Chemometric and adulterated
coffee beans
approaches were(Figure 2E) showed
employed high similarities.
to discriminate chromatograms Chemometric approaches
generated from differentwere employed
adulterants andto
discriminate chromatograms
adulterated coffee beans. generated from different adulterants and adulterated coffee beans.

Figure
Figure2.2.HPLC
HPLC chromatograms
chromatograms (overlap
(overlapofof
three) ofof
three) (A) coffee
(A) beans,
coffee (B)
beans, soybeans,
(B) (C)
soybeans, green
(C) mung
green mung
beans, (D)
beans, (D) spent coffee
spent coffeegrounds
groundsand
and(E)
(E)adulterated
adulteratedcoffee
coffeebeans
beanscontaining
containing20%
20%soybeans.
soybeans.
 The PCA values of adulterated coffees were disorderly around pure coffee. The 60% adulterated
coffee obtained values close to zero in PC1 and close to adulterants as well. The various mixing ratios
(5%, 10%, 20%, 40% and 60%) are displayed in different colors in Figure 3. The results indicated that
it was possible to detect coffee adulteration with various adulterants in admixture as low as 5% (w/w).
This
Foodsstudy
2020, 9,was
880 able to use HPLC chromatograms for chemometric analysis. However, in Figure72, ofit
11
is clear that overlapped HPLC chromatograms did not perfectly match each other. In many
chemometric applications, the spectra—especially the most adopt FTIR spectra—are almost identical
3.3. Principal Component Analysis (PCA)
to each other in the same test group. The uses of HPLC chromatograms as fingerprints for
chemometric analysis scatter
The PCA-scores is powerful becausefrom
plot derived HPLC chromatograms
normalized provide more distinct
HPLC chromatograms is shownandindetailed
Figure 3.
information,
Pure coffee, due to the peaks
soybeans, greenrepresented
mung beans toand
different
spentcompounds.
coffee groundsWhile
areusing FTIR for chemometric
well-separated. The coffee
analysis,
sample was the more
peakstoonly representofdifferent
the positive PC1 and functional groups.
the others were Figure
more 4 shows
to negative ofthe
PC1. FTIR
Thespectra of
successful
roasted coffee
separation beanscoffee,
among and soybeans.
soybeanTheyand were
greensimilar
mungin beancertain
wasdegree; compared
as expected, to Figure
because each2A,B the
material
chromatograms
contained different of compounds
coffee and and soybean werea very
presented different.
specific However,
chromatogram. Thethe system between
separation stability coffee
and
chromatogram
and spent coffee reproducibility
grounds was very of HPLC
goodwas not as
as well. good aselements
Detailed spectrophotometric methodindicated
from PCA analysis (e.g., FTIR).
that
In thissmall
some study, the appeared
peaks detection between
limit of 20–25
adulterants
min ininHPLC
coffeechromatograms
was set at 5%. of Compared
spent coffeetogrounds
many
chemometric analyses with
(Figure S1) contributed mostIRto
spectra, the detectionbetween
the discrimination limit was below
spent 1% grounds
coffee [26]. and coffee.

PCA-scoresscatter
Figure3.3.PCA-scores
Figure scatterplot
plotofofcoffee
coffeebeans
beansinincomparison
comparisontotosoybeans,
soybeans,green
greenmung
mungbeans,
beans,spent
spent
coffeegrounds
coffee groundsand
andadulterated
adulteratedcoffee
coffeecontaining
containingdifferent
differentadulterants
adulterantsinindifferent
differentmixing
mixingratios
ratios(5%,
(5%,
10%,20%,
10%, 20%,40%
40%and
and60%).
60%).

The PCA values of adulterated coffees were disorderly around pure coffee. The 60% adulterated
coffee obtained values close to zero in PC1 and close to adulterants as well. The various mixing ratios
(5%, 10%, 20%, 40% and 60%) are displayed in different colors in Figure 3. The results indicated that it
was possible to detect coffee adulteration with various adulterants in admixture as low as 5% (w/w).
This study was able to use HPLC chromatograms for chemometric analysis. However, in Figure 2, it is
clear that overlapped HPLC chromatograms did not perfectly match each other. In many chemometric
applications, the spectra—especially the most adopt FTIR spectra—are almost identical to each other
in the same test group. The uses of HPLC chromatograms as fingerprints for chemometric analysis is
powerful because HPLC chromatograms provide more distinct and detailed information, due to the
peaks represented to different compounds. While using FTIR for chemometric analysis, the peaks only
represent different functional groups. Figure 4 shows the FTIR spectra of roasted coffee beans and
soybeans. They were similar in certain degree; compared to Figure 2A,B the chromatograms of coffee
and soybean were very different. However, the system stability and chromatogram reproducibility of
HPLC was not as good as spectrophotometric method (e.g., FTIR). In this study, the detection limit
of adulterants in coffee was set at 5%. Compared to many chemometric analyses with IR spectra,
the detection limit was below 1% [26].
 Foods 2020, 9, x FOR PEER REVIEW 8 of 11
Foods 2020, 9, 880 8 of 11
Foods 2020, 9, x FOR PEER REVIEW 8 of 11

Figure 4. FTIR (ATR, Ge) spectra of roasted coffee beans and soybeans.

4. FTIR (ATR, Ge) spectra of roasted coffee

Figure 4.
Figure coffee beans
beans and
and soybeans.
soybeans.
3.4. Discriminatory Power of Models Built
3.4. Discriminatory Power of Models Built
Two models Power
3.4. Discriminatory were built in this
of Models study. The first model was designed to distinguish pure coffee
Built
formTwo adulterated coffee. The second
models were built in this study. modelThe wasfirst
designed
modelto wasidentify the adulterants
designed to distinguish thatpure
existcoffee
in the
Two models were built in this study. The first model was designed to distinguish pure coffee
adulterated
form coffee.
adulterated A total
coffee. Theofsecond
180 sample
modelsetswas were divided
designed into twothe
to identify groups including
adulterants thata exist
training set
in the
form adulterated coffee. The second model was designed to identify the adulterants that exist in the
(60% data) and
adulterated a test
coffee. set (40%
A total of 180 of data).
sampleWe used
sets werethedivided
trainingintodatatwosetgroups
to calculate the parameters
including a training and
set
adulterated coffee. A total of 180 sample sets were divided into two groups including a training set
to generate
(60% theamodels
data) and test set for
(40%theoffirst and
data). We second
used themodels.
trainingThedata
testset
settowas used tothe
calculate test the classification
parameters and to
(60% data) and a test set (40% of data). We used the training data set to calculate the parameters and
results ofthe
generate themodels
models.for A the
confusion
first and matrix
second was used toThe
models. illustrate
test setthe testused
was results.
to test the classification
to generate the models for the first and second models. The test set was used to test the classification
resultsInofthe
thefirst model,
models. A the classesmatrix
confusion 1–5 were wascoffee,
used tosoybean,
illustrate green mung
the test bean, spent coffee ground
results.
results of the models. A confusion matrix was used to illustrate the test results.
and Inadulterated coffee.
the first model, the Of 18 pure
classes coffee
1–5 were samples
coffee, soybean, (true class
green mung1), 2bean,
samples
spent were
coffee classified
ground and as
In the first model, the classes 1–5 were coffee, soybean, green mung bean, spent coffee ground
adulteratedcoffee.
adulterated coffee.Of Of1836pure
adulterated coffees (true class 1),
coffee samples 5), 22 samples
samples werewere classified
classified asasadulterated
pure coffee.
and adulterated coffee. Of 18 pure coffee samples (true class 1), 2 samples were classified as
The training
coffee. set contained
Of 36 adulterated 2 false-positive
coffees (true class(FP)
5), 2samples
samples(classifying pure as
were classified coffee
pureascoffee.
adulterated coffee),
The training
adulterated coffee. Of 36 adulterated coffees (true class 5), 2 samples were classified as pure coffee.
70 contained
set true negative (TN) samples
2 false-positive (classifying
(FP) unadulterated
samples (classifying pure sample
coffeecorrectly), 2 false-negative
as adulterated (FN)
coffee), 70 true
The training set contained 2 false-positive (FP) samples (classifying pure coffee as adulterated coffee),
samples (TN)
negative (classifying
samples adulterated
(classifying coffee as pure coffee)
unadulterated sampleandcorrectly),
34 true positive (TP) samples
2 false-negative (FN)(classifying
samples
70 true negative (TN) samples (classifying unadulterated sample correctly), 2 false-negative (FN)
adulteratedadulterated
(classifying coffee correctly)
coffee (Figure
as pure 5A).
coffee)These
and parameters
34 true positive were(TP)
used to calculate
samples the discriminatory
(classifying adulterated
samples (classifying adulterated coffee as pure coffee) and 34 true positive (TP) samples (classifying
powercorrectly)
coffee and are summarized
(Figure 5A). Thesein Table 2. Then, data
parameters werefrom
used the test set were
to calculate applied to the power
the discriminatory established
and
adulterated coffee correctly) (Figure 5A). These parameters were used to calculate the discriminatory
firstsummarized
are model. The results
in Tableare2. shown in Figure
Then, data from5B.theThree FPwere
test set and three FN to
applied were
theobtained.
established Thefirst
firstmodel.
model
power and are summarized in Table 2. Then, data from the test set were applied to the established
wasresults
The able toarecorrectly
shown indistinguish
Figure 5B. 21 adulterated
Three FP and threesamples
FN werefromobtained.
24 adulterated
The firstcoffees.
model However,
was able to3
first model. The results are shown in Figure 5B. Three FP and three FN were obtained. The first model
pure coffees
correctly were recognized
distinguish 21 adulterated as adulterated
samples from coffees, and 3 adulterated
24 adulterated coffees were
coffees. However, 3 purerecognized
coffees were as
was able to correctly distinguish 21 adulterated samples from 24 adulterated coffees. However, 3
pure coffees.
recognized as adulterated coffees, and 3 adulterated coffees were recognized as pure coffees.
pure coffees were recognized as adulterated coffees, and 3 adulterated coffees were recognized as
pure coffees.

Figure5.5.Confusion
Figure Confusionmatrix
matrixofofthe
thefirst
firstmodel
modelthat
thatused
usedtotodiscriminate
discriminatecoffee
coffeefrom
fromadulterated
adulteratedcoffee
coffee
for (A) training set and (B) test set (class 1—coffee;
for A) training set and B) test set (class 1—coffee; class 2—soybeans;
2—soybeans; class 3—green mung beans;class
class 3—green mung beans; class
Figure 5. Confusion matrix of the first model that used to discriminate coffee from adulterated coffee
4—spent
4—spentcoffee
coffeegrounds;
grounds;class
class5—adulterated
5—adulteratedcoffee
coffeebeans).
beans);
for A) training set and B) test set (class 1—coffee; class 2—soybeans; class 3—green mung beans; class
4—spent coffee grounds; class 5—adulterated coffee beans);
Foods 2020, 9, 880 9 of 11

Table 2. Estimated figure of merit (FOM) of the first and second model.

SP SE RLR +LR −LR

First model
Training set 0.972 0.944 0.916 33.7 0.0576
Test set 0.938 0.875 0.813 14.1 0.133
Second model
Training set 1.00 0.583 0.583 – 0.417
Test set 1.00 0.333 0.333 – 0.667
SP—specificity; SE—sensitivity; RLR—reliability rate; +LR—positive ratio likelihood; −LR—negative
Foodsratio
2020,likelihood.
9, x FOR PEER REVIEW 9 of 11

The second
The second model
model was
was designed
designed for for distinguishing
distinguishing which
which kindkind ofof adulterants
adulterants were
were in in the
the
adulterated coffees.
adulterated coffees. The
The results
results are
are shown
shown in inFigure
Figure6.6. In
In the
thetraining
trainingset,
set,thethepure
puresoybeans,
soybeans,green
green
mung beans and spent coffee grounds were correctly classified, while the
mung beans and spent coffee grounds were correctly classified, while the adulterated coffees wereadulterated coffees were
partially classified. For example, in 12 soybean-adulterated coffees (class 4), 2 were
partially classified. For example, in 12 soybean-adulterated coffees (class 4), 2 were classified as classified as green-
mung-bean-adulterated and one
green-mung-bean-adulterated and wasoneclassified as spent-coffee-grounds-adulterated
was classified as spent-coffee-grounds-adulterated (Figure(Figure
6A). In6A).
the
test set of the second model, all 36 pure adulterants (12 for each) were distinguished
In the test set of the second model, all 36 pure adulterants (12 for each) were distinguished the from the from
adulterated coffee.
adulterated coffee. However,
However, the the second
second model
modelwas wasnot
notable
abletotoidentify
identifythe theadulterants
adulterants correctly
correctly in
the adulterated coffees. For example, in 8 green-mung-bean-adulterated coffees
in the adulterated coffees. For example, in 8 green-mung-bean-adulterated coffees (Figure 6B, (Figure 6B, class 5),
only 5),
class 2 were
onlycorrectly identified
2 were correctly as green-mung-bean-adulterated,
identified as green-mung-bean-adulterated, one was one misclassified as soybean-
was misclassified as
adulterated and 5 were classified as spent-coffee-grounds-adulterated. In the
soybean-adulterated and 5 were classified as spent-coffee-grounds-adulterated. In the second model,second model, the FN
samples
the were many
FN samples wereand hadand
many a highhadchance
a highofchance
misjudging the adulterants
of misjudging existed in the
the adulterants adulterated
existed in the
coffee.
adulterated coffee.

Figure 6.6. Confusion

Figure Confusion matrix
matrix of
of second
second model
model that
that used
used toto identify
identify the
the adulterants
adulterants existed
existed in
in the
the
adulterated
adulteratedcoffee
coffeefor
for(A)
(A)training
trainingset
setand
and(B)
(B)test
testset
set(class
(class1—soybeans;
1—soybeans;classclass 2—green
2—green mung
mung beans;
beans;
class
class3—spent
3—spentcoffee
coffeegrounds;
grounds;class
class4—adulterated
4—adulteratedcoffeecoffeecontaining
containingsoybeans;
soybeans;class
class5—adulterated
5—adulterated
coffee
coffee containing green mung beans; class 6—adulterated coffee containing spentcoffee
containing green mung beans; class 6—adulterated coffee containing spent coffeegrounds).
grounds);

Figure
Figure of merit(FOM)
of merit (FOM)was was used used to characterize
to characterize the performances
the performances of the of the studied
studied models.models.
Statistic
Statistic equations were applied (Equation 1–5) for the calculation of SP, SE,
equations were applied (Equation 1–5) for the calculation of SP, SE, RLR, +LR and −LR. In first model, RLR, +LR and −LR.
In
SP (0.938) and SE (0.875) of the test set were satisfied. The reliability rate (RLR) was 0.813 provedwas
first model, SP (0.938) and SE (0.875) of the test set were satisfied. The reliability rate (RLR) the
0.813 proved
trueness themodel
of first trueness[12].ofPositive
first model [12]. Positive
likelihood ratio (+LR)likelihood
was used ratio (+LR) was
to evaluate used to
whether theevaluate
positive
whether
result wasthecorrectly
positivetested.
result The
washigh correctly tested.
+LR value (>10) high +LR
Thestrongly value (>10)
indicated strongly
the positive indicated
result the
tested was
positive tested was correct [22]. The obtained +LR
correct [22]. The obtained +LR of the first model were 33.7 and 14.1 for training set and test for
result of the first model were 33.7 and 14.1 set,
training set and test set, respectively (Table 2). On the other hand, the −LR values
respectively (Table 2). On the other hand, the −LR values were low (0.0576 for training set and 0.133 were low (0.0576 for
training set and
for test set) 0.133 there
indicated for testwas set)strongly
indicated there was
evidence strongly
to prove evidence
the TN result to prove
was the TN
detected result was
correctly [22].
detected correctly [22]. Thus, the performance of first model was
Thus, the performance of first model was good. In the second model, lower SE values good. In the second model, lower SE
were
observed—0.583 and 0.333 for the training and test sets, respectively. The lower SE values indicated
that the ability of the model to detect the positive sample was low. This model performed 100% in
prediction of TN samples and 0 case of FP sample. The obtained SPs were 1.00 for either training or
test set. Therefore, the RLRs of the second model were same as the SEs. However, the second model’s
SP value was satisfied (means the second model detected the pure adulterants correctly), but the aim
Foods 2020, 9, 880 10 of 11

values were observed—0.583 and 0.333 for the training and test sets, respectively. The lower SE values
indicated that the ability of the model to detect the positive sample was low. This model performed
100% in prediction of TN samples and 0 case of FP sample. The obtained SPs were 1.00 for either
training or test set. Therefore, the RLRs of the second model were same as the SEs. However, the second
model’s SP value was satisfied (means the second model detected the pure adulterants correctly),
but the aim of this model was to identify the adulterant mixed in the adulterated coffee (positive
sample). Therefore, the SE value was more important and relevant to the ability of the model. On the
other hand, the −LR values were 0.417 and 0.667 for training set and test set, respectively, which
represented no evidence to prove that the true negative result was correct [22]—even if the SP value
was 1.0. This indicated that the −LR was better than the SP for explaining the performance of the
model. Thus, the second model was only suitable for the discrimination of pure coffee and adulterated
coffee, while the ability of identifying adulterants existed in the adulterated coffee was low.

4. Conclusions
HPLC-based chemometric analysis was performed and able to distinguish coffee from adulterated
coffee with detection limit around 5%. This study simulated commercial roasting conditions in sample
preparation and liquid sample was obtained by coffee brewing. The chemometric analysis applied
chromatograms (information of compounds) instead of photospectra (information of functional groups)
studied in most chemometric analysis. The experimental model was able to separate pure coffee
and adulterated coffees. However, the performance of telling what kind of adulterants existed in the
adulterated coffee still needs to be improved.

Supplementary Materials: The following are available online at http://www.mdpi.com/2304-8158/9/7/880/s1,

Figure S1: HPLC chromatogram of coffee and spent coffee grounds sample for 20–25 min of retention time.
Author Contributions: Investigation, W.L.C.; methodology, W.L.C.; project administration, M.F.; writing—original
draft, W.L.C.; writing—review & editing, M.F. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by MOST, Grant Number 107–2320-B-019–001.
Conflicts of Interest: The authors declare no conflicts of interest.

References
1. International Coffee Organization. Coffee Market Report: November 2019. Available online: http:
//www.ico.org/documents/cy2019-20/cmr-1119-e.pdf (accessed on 9 February 2020).
2. Song, H.Y.; Jang, H.W.; Debnath, T.; Lee, K.G. Food Chemicals Codex, 8th ed.; United States Pharmacopeia:
Rockville, MD, USA, 2013.
3. Oliveira, R.C.; Oliveira, L.S.; Franca, A.S.; Augusti, R. Evaluation of the potential of SPME-GC-MS and
chemometrics to detect adulteration of ground roasted coffee with roasted barley. J. Food Compos. Anal. 2009,
22, 257–261. [CrossRef]
4. Toci, A.T.; Farah, A.; Pezza, H.R.; Pezza, L. Coffee Adulteration: More than Two Decades of Research.
Crit. Rev. Anal. Chem. 2015, 46, 83–92. [CrossRef] [PubMed]
5. Hullah, W.; Trail, A.W.; Mills, D.; Cringle, J.; Drive, P.; Albrecht, S.; Ave, M. Instant Coffee Substitute from
Soybeans and Method of Making. U.S. Patent H673, 1989.
6. FDA Recalls. Market Withdrawals, & Safety Alerts. 2019. Available online: https:
//www.fda.gov/safety/recalls-market-withdrawals-safety-alerts/brian-richardson-dba-tha-pink-issues-
voluntary-nationwide-recall-kopi-jantan-tradisional-natural (accessed on 19 March 2020).
7. Domingues, D.S.; Pauli, E.D.; de Abreu, J.E.; Massura, F.W.; Cristiano, V.; Santos, M.J.; Nixdorf, S.L.
Detection of roasted and ground coffee adulteration by HPLC by amperometric and by post-column
derivatization UV–Vis detection. Food Chem. 2014, 146, 353–362. [CrossRef]
8. Pauli, E.D.; Barbieri, F.; Garcia, P.S.; Madeira, T.B.; Acquaro, V.R.; Scarminio, I.S.; Da Camara, C.A.P.;
Nixdorf, S.L. Detection of ground roasted coffee adulteration with roasted soybean and wheat. Food Res. Int.
2014, 61, 112–119. [CrossRef]
Foods 2020, 9, 880 11 of 11

9. Song, H.Y.; Jang, H.W.; Debnath, T.; Lee, K.-G. Analytical method to detect adulteration of ground roasted
coffee. Int. J. Food Sci. Technol. 2018, 54, 256–262. [CrossRef]
10. Barbosa, M.F.; Souto, U.T.D.C.P.; Dantas, H.V.; de Pontes, A.S.; Lyra, W.D.S.; Diniz, P.H.G.D.; Araujo, M.C.U.;
Da Silva, E.C. Identification of adulteration in ground roasted coffees using UV–Vis spectroscopy and
SPA-LDA. LWT 2015, 63, 1037–1041. [CrossRef]
11. Cai, T.; Ting, H.; Jin-Lan, Z. Novel identification strategy for ground coffee adulteration based on UPLC–HRMS
oligosaccharide profiling. Food Chem. 2016, 190, 1046–1049. [CrossRef]
12. Reis, N.; Botelho, B.; Franca, A.S.; Oliveira, L.S. Simultaneous Detection of Multiple Adulterants in Ground
Roasted Coffee by ATR-FTIR Spectroscopy and Data Fusion. Food Anal. Methods 2017, 10, 2700–2709.
[CrossRef]
13. De Morais, T.C.B.; Rodrigues, D.R.; Souto, U.T.D.C.P.; Lemos, S. A simple voltammetric electronic tongue for
the analysis of coffee adulterations. Food Chem. 2019, 273, 31–38. [CrossRef]
14. Kaufmann, A.; Butcher, P.; Maden, K.; Walker, S.; Widmer, M. Reliability of veterinary drug residue
confirmation: High resolution mass spectrometry versus tandem mass spectrometry. Anal. Chim. Acta 2015,
856, 54–67. [CrossRef]
15. Kaufmann, A. Combining UHPLC and high-resolution MS: A viable approach for the analysis of complex
samples? TrAC Trends Anal. Chem. 2014, 63, 113–128. [CrossRef]
16. Reis, N.; Franca, A.S.; Oliveira, L.S. Performance of diffuse reflectance infrared Fourier transform spectroscopy
and chemometrics for detection of multiple adulterants in roasted and ground coffee. LWT 2013, 53, 395–401.
[CrossRef]
17. Okaru, A.O.; Scharinger, A.; de Rezende, T.R.; Teipel, J.C.; Kuballa, T.; Walch, S.G.; Lachenmeier, D.W.
Validation of a Quantitative Proton Nuclear Magnetic Resonance Spectroscopic Screening Method for Coffee
Quality and Authenticity (NMR Coffee Screener). Foods 2020, 9, 47. [CrossRef] [PubMed]
18. Mendonça, J.C.; Franca, A.S.; Oliveira, L.S. Physical characterization of non-defective and defective Arabica
and Robusta coffees before and after roasting. J. Food Eng. 2009, 92, 474–479. [CrossRef]
19. Olivieri, A.C. Analytical Figures of Merit: From Univariate to Multiway Calibration. Chem. Rev. 2014, 114,
5358–5378. [CrossRef]
20. Szymanska, E.; Saccenti, E.; Smilde, A.K.; Westerhuis, J.A. Double-check: Validation of diagnostic statistics
for PLS-DA models in metabolomics studies. Metabolomics 2011, 8, 3–16. [CrossRef]
21. Baratloo, A.; Safari, S.; Elfil, M.; Negida, A. Evidence Based Emergency Medicine Part 3: Positive and
Negative Likelihood Ratios of Diagnostic Tests. Emergency 2015, 3, 170–171.
22. Sloane, P.D.; Slatt, L.M.; Ebell, M.H.; Jacques, L.B.; Smith, M.A. Essentials of Family Medicine, 5th ed.; Lippincott
Williams & Wilkins: Baltimore, MD, USA, 2008; p. 44.
23. Patay-Éva, B.; Bencsik, T.; Papp, N. Phytochemical overview and medicinal importance of Coffea species
from the past until now. Asian Pac. J. Trop. Med. 2016, 9, 1127–1135. [CrossRef]
24. Ciabotti, S.; Silva, A.C.B.B.; Juhasz, A.C.P.; Mendonça, C.D.; Tavano, O.L.; Mandarino, J.M.G.;
Gonçalves, C.A.A. Chemical composition, protein profile, and isoflavones content in soybean genotypes
with different seed coat colors. Int. Food Res. J. 2016, 23, 621–629.
25. Hou, D.; Yousaf, L.; Xue, Y.; Hu, J.; Wu, J.; Hu, X.; Feng, N.; Shen, Q. Mung Bean (Vigna radiata L.): Bioactive
Polyphenols, Polysaccharides, Peptides, and Health Benefits. Nutrients 2019, 11, 1238. [CrossRef]
26. Zou, H.; Zhang, W.; Feng, Y.; Liang, B. Simultaneous determination of melamine and dicyandiamide in milk
by UV spectroscopy coupled with chemometrics. Anal. Methods 2014, 6, 5865–5871. [CrossRef]

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