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Analysis of caffeine, theobromine and


theophylline in coffee by near infrared
spectroscopy (NIRS) compared to high-
performance liquid chromatography (HPLC)
coupled to mass spectrom...

Article in Analytica Chimica Acta May 2005


DOI: 10.1016/j.aca.2005.01.064

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Analytica Chimica Acta 538 (2005) 195203

Analysis of caffeine, theobromine and theophylline in coffee by near


infrared spectroscopy (NIRS) compared to high-performance liquid
chromatography (HPLC) coupled to mass spectrometry
C.W. Huck , W. Guggenbichler, G.K. Bonn
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 52a, 6020-Innsbruck, Austria

Received 5 October 2004; received in revised form 24 January 2005; accepted 26 January 2005
Available online 2 March 2005

Abstract

Coffee is one of the most important raw materials within the international trade, for which highest quality is demanded. Due to the high
number of samples to be analysed, new analytical techniques providing fast and reliable data about the quality are essential. Therefore, we
established a new analytical method based on near infrared spectroscopy (NIRS) for the quantitation of the three main alkaloids caffeine
(Caf), theobromine (Tbr) and theophylline (Tph) in roasted coffee after discrimination of the rough green beans into Arabic and Robusta.
This validated method was compared to the most commonly used liquid chromatography (LC) connected to UV and mass spectrometric (MS)
detection. In this course non-porous silica-C18 showed to enable the analysis of the three alkaloids with high robustness within 2.5 min,
compared to 20 min employing porous silica-C18 as a stationary phase. As analysis time plays an important role in choosing a reference
method for the calibration of the NIR-spectrometer, the non-porous silica-C18 phase offers a very fast method. Coupling of the optimised
LC method to a mass spectrometer (MS) via an electrospray ionisation (ESI) interface not only allowed to identify Caf, Tbr and Tph by their
characteristic fragmentation pattern using collisionally induced dissociation (CID), but also to quantitate the content of the three analytes,
which was found to be 6% higher compared to UV-detection. The validated LCUV method was chosen as a reference method for the
calibration of the NIRS system. Analysis of 83 liquid coffee extracts in random order resulted for Caf and Tbr in values for S.E.E. (standard
error of estimation) of 0.34, 0.40 g/100 g, S.E.P. (standard error of prediction) of 0.07 and 0.10 g/100 g with correlation coefficients of 0.86
and 0.85 in a concentration range between 0.10 and 4.13 g/100 g. Compared to LC the lower limit of detection (LOD) of the NIRS-method is
found at 0.05 g/100 g compared to 0.2440.60 ng/100 g in LC, which makes it impossible to analyse Tph by NIRS. Therefore, main focus is
put on the NIRS analysis of caffeine and theobromine. Nevertheless, NIRS offers a serious alternative to LC for the coffee producing industry
because of the short analysis time of a few seconds and a guaranteed high sample throughput.
2005 Elsevier B.V. All rights reserved.

Keywords: Coffee; Caffeine; Theobromine; Theophylline; Liquid chromatography; Electrospray ionisation mass spectrometry; Near infrared spectroscopy

1. Introduction prize for one cup, there is only a difference of a few cent be-
tween a high and a low coffee quality. In order to ensure high
Coffee is the second important raw material within the in- quality both for the coffee processor and the consumer, there-
ternational trade, the most important foreign exchange sup- fore, a need exists for objective, rapid, sensitive and selective
plier for many agricultural oriented countries, an attractive analytical techniques.
source for tax yield, and the most popular drink. Quality is Near infrared spectroscopy (NIRS) has been in use for
the premise to run a good coffee business, which means that food analysis since 1938 [1]. The main developments on
the highest quality is just good enough. Calculating of the NIRS of coffee including references have been reviewed by
Schulz in 2004 [2]. This review includes the description of
Corresponding author. Tel.: +43 512 507 5195; fax: +43 512 507 2965. first successful attempts to apply NIRS for the authentication
E-mail address: christian.w.huck@uibk.ac.at (C.W. Huck). of the two different coffee bean varieties Arabica and Ro-

0003-2670/$ see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2005.01.064
196 C.W. Huck et al. / Analytica Chimica Acta 538 (2005) 195203

busta. Generally, Arabica coffee beans are viewed supe- guish between Arabica and Robusta. An NIRS-method for the
rior to Robusta and are therefore the more expensive of the rapid quantitative determination of the caffeine, theobromine
two. Therefore the identification of adulterations and misla- and theophylline content in liquid coffee is described. The
belling is very important with special regard to the consumer advantages and limitations of this method compared to liq-
protection. It also includes the description of a similar NIR uid chromatography (LC) using UV- and mass spectrometric
method for the simultaneous determination of dry matter con- (MS) detection are shown and discussed.
tent in green Robusta coffees and mixtures of roast coffees,
lyophilized powders, air-dried coffee beverages and instant
coffee. It is mentioned that classical methods to discriminate 2. Experimental
the coffee varieties, which are mainly related to the individual
profile of mineral elements, volatile compounds, or sulphur 2.1. Materials and reagents
containing substances, are very time-consuming, the devel-
oped NIRS calibrations allow to perform the characterisation Acetonitrile (ACN, analytical reagent-grade), methanol
in a comparatively short time. In comparison to FTIR spectra (MeOH, analytical reagent-grade) were purchased from
obtained from the same sample set, the NIR technique was Fluka (Buchs, Switzerland), acetic acid (HOAc, analyti-
found to be superior. This may be related to the fact, that cal reagent-grade) from Merck (Darmstadt, Germany). Caf-
FTIR is more sensitive to the inhomogeneity of the individ- feine, theophylline and theobromine (analytical reagent-
ual coffee samples, because stretching and deformation vi- grade) were from Sigma-Aldrich (Deisenhofen, Germany).
brations are directly used and not their overtones or combina- Bidistilled water purified by a NanoPure-unit (Barnstead,
tion vibrations. Another approach is the combination of near- Boston, MA, USA) was used. Standard stock solutions of caf-
and mid-infrared spectroscopy for lyophilized coffee vari- feine, theophylline and theobromine were prepared in 0.1%
etal identification [3]. Schulz also reported on the successful HOAc/ACN = 98/2 (v/v) with concentrations of 1 mg/ml and
NIRS analysis of caffeine and theobromine in tea (Camellia stored at 18 C for several weeks. The working solutions
sinensis) [4]. Caffeine analysis in tablet form down to a limit were prepared by dilution with 0.1% HOAC and stored in
of quantification of 13.7 g/100 g was found to be more flexible a refrigerator for 2 days. All coffee samples were provided
and faster than other techniques such as liquid chromatogra- by Praxmarer-Kaffeevetrieb GesmbH & Co KG (Vols, Tirol,
phy (LC) [5]. For the rapid determination of caffeine content Austria).
in soft drinks the use of FTIR-ATR without the use of any
organic solvent [6] as well as analysis by flow injection are de- 2.2. Liquid chromatography coupled to UV-detection
scribed in detail [7,8]. Other spectroscopic techniques for the (LCUV)
determination comprise ultraviolet-spectroscopy [9], NMR-
spectroscopy [10] and mass-spectroscopy [11]. For routine The LC system consisted of a low-pressure gradient pump
analysis of caffeine, theobromine and theophylline mostly (Model 616, Waters, Milford, MA, USA), a controller (Model
liquid chromatography (LC) [12] in the reversed-phase (RP) 600S, Waters), a column heater (Model TC 1900, ICI, Welsh-
mode using porous alkylated (C18) silica phases combined pool, Australia), a helium degassing system, an autosam-
with UV-detection are used, requiring analyses times down to pler (Model 717 plus, Waters) and a photo diode array
three minutes [1316]. An alternative separation can also be detector (DAD, Model 996, Waters) with a 10 mm path-
achieved by ion chromatography, with cation-offering more length flowcell. Data was recorded on a computer-based data
precise and accurate results [17] than anion exchange. For the system (Millenium32 , Version 3.05.01, Waters). The used
determination in complex biological samples the coupling of non-porous stationary phase was Kovasil MS-C18 (1.5 "m,
the LC system to mass spectrometry (MS) via electrospray 33 mm 4.6 mm i.d., Uetikon, Switzerland), the porous one
ionisation (ESI) [1821], atmospheric pressure chemical ion- was Nucleosil 100-5 C18 (5 "m, 100 A, 250 mm 4.0 mm
isation (APCI) [22] and fast atom bombardment (FAB) [23] i.d., Macherey-Nagel, Dueren, Germany). The mobile phase
are used. Separation based on electrophoresis comprise cap- was 0.1% HOAc/ACN = 98/2 (v/v) and the flow rate was
illary zone electrophoresis (CZE) with !-cyclodextrins [24], 1 ml/min. UV-detection was accomplished at 280 nm. The
carbon nanotubes [25], puffers like borax [26] added to the volume injected was 10 "l.
electrolyte, or micellar electrokinetic chromatography [27],
or capillary electrochromatography (CEC) [28]. 2.3. Liquid chromatography coupled to electrospray
Caffeine (Caf), theobromine (Tbr) and theophylline (Tph) ionisation quadrupole ion trap mass spectrometry
raise cholesteryl ester transfer protein activity levels before (LCESIMS/MS)
LDL cholesterol in normolipidaemic subjects [29], increase
serum lipid transfer protein levels in humans [30], which is For LCESIMS/MS experiments a low-pressure gradi-
important due to an calculated caffeine intake ranging be- ent micropump (Model Rheos 2000, Flux, Karlskoga, Swe-
tween 92 and 146 mg/day [31]. den), a degasser (Model DG-301, Phenomenex, Torrance,
In this work we present a qualitative model for the cluster CA, USA), a microinjector (Model CC00030, Valco, Hous-
analysis of ground coffee samples, which allows one to distin- ton, TX, USA) with a 20 "l internal loop connected to a
C.W. Huck et al. / Analytica Chimica Acta 538 (2005) 195203 197

quadrupole ion trap mass spectrometer (Model LCQ, Finni- a coffee grinder for 4 min. Five grams of each sample was
gan, San Jose, CA, USA) were used. The following param- placed into a round-bottom flask and 100 ml of mobile phase
eters were applied in all experiments: positive ion mode; 0.1% HOAc/ACN = 98/2 (v/v) was added with following re-
source voltage, 4.5 kV; source current, 2 "A; sheath gas fluxing for 30 min. Thereafter, the suspension was filtrated
flow rate, 79.64 (Finnigan units; nitrogen); capillary volt- through a cellulose filter into a volumetric flask and the vol-
age, 21.81 V; temperature of the heated capillary, 270 C; ume brought to 100 ml. Prior to injection into the LC system
tube lens offset, 17.43 V; first octapole offset, 0.81 V; sec- the solution was passed through a 0.50 "m membrane filter
ond octapole offset, 6.08 V; inter octapole lens, 9.88 V. (Millipore, Billerica, MA, USA). Samples were either mea-
For LC a Kovasil MS-C18 (1.5 "m, 33 mm 4.6 mm i.d., sured directly after production, or they were stored in a freezer
Uetikon) was used. The mobile phase consisted of 0.1% at 20 C for not longer than 2 days.
HOAc/ACN = 98/2 (v/v). The flow rate was 1 ml/min. The
volume injected was 10 "l.
3. Results and discussion
2.4. Near infrared reflectance spectroscopy (NIRS)
Prior to quantitative analysis by NIRS a robust, reliable
249 NIR spectra of 83 samples were recorded with a and rapid liquid chromatographic (LC) reference method has
scanning FT-NIR-spectrometer (Buchi, Uzwil, Switzerland) to be established.
in random order over a wavelength range from 4008 to
9996 cm1 with a resolution of 12 cm1 in the reflectance 3.1. Liquid chromatography coupled to UV-detection
or transflectance mode by fiber optics with 10 scans for one (LCUV)
average spectrum to equilibrate inhomogeneities. Chemo-
metrical software Nircal 4.21 (Buchi) was used for creating Fig. 1a shows the separation of a theobromine (Tbr),
a model, i.e., selection of spectra and wavelengths, mathe- theophylline (Tph), caffeine (Caf) standard mixture using
matical pretreatment and statistical analysis performing clus- a non-porous silica-C18 phase (1.5 "m, 33 mm 4.6 mm
ter analysis and partial least squares regression (PLS) [32]. i.d.). Compared to porous silica-C18 (5 "m, 100 A,
Seventy percent of samples were used for calibration, 30% 250 mm 4.0 mm i.d., Fig. 1c) the interaction between the
for validation. The content of caffeine and theobromine was analytes and the stationary phase is much weaker due to
equally distributed over the whole concentration range in both the fact that the total surface area is a function of particle
the learning- and test-set. The spectra were evaluated in the size, which results in analyses times <2.5 min compared to
wavelength range between 4500 and 9996 cm1 . The opti- 20 min with the porous phase (Fig. 1c), which is important
mum number of factors used for the individual prediction when choosing the reference method to calibrate the NIR-
was determined by cross-validation [1]. The selection of the spectrometer. Injection of a liquid coffee extract (Fig. 1b)
best regression model is based on the following values calcu- gave retention factors k of 0.94, 1.69 and 5.22, peak widths
lated for validation purposes: (1) Standard error of estimation at half height b0.5 of 0.068, 0.075, 0.12 min, for Tbr, Tph
(S.E.E.), the standard deviation of the differences between
LCUV or LCESIMS and NIRS-results in the calibration
set. (2) Standard error of prediction (S.E.P.), the counterpart
for the test-set samples. They should be as small as possible.
(3) BIAS, the average deviation between the predicted values
and the actual values, in the calibration-set, should be close
to zero. (4) The correlation coefficient (R2 ) should approach
1 [33].

2.5. Sample preparation

2.5.1. Ground coffee samples


83 cultivars of green coffee samples originating from dif-
ferent geographical regions all over the world were delivered
by Praxmarer-Kaffeevertrieb GesmbH&CoKG (Vols, Tirol,
Austria). Samples were analysed without any further pretreat-
ment after thermostating (23 C) in a rotor with fibre optics
in the reflectance mode.
Fig. 1. RPLCUV of a theobromine, theophylline, caffeine standard (a and
c) and an aqueous coffee extract (b and d). Stationary phase, Kovasil MS-
2.5.2. Liquid coffee samples C18 (1.5 "m, 33 mm 4.6 mm i.d.) (a and b); Nucleosil 100-5 C18 (5 "m,
Eighty-three samples of roasted Arabica and Robusta cof- 250 mm 4.0 mm i.d.) (c and d). Chromatographic conditions, see
100 A,
fee beans from different geographical origin were crushed in Section 2.
198 C.W. Huck et al. / Analytica Chimica Acta 538 (2005) 195203

Table 1
Reproducibility of retention times, calibration parameters and detection limits of caffeine, theophylline and theobromine using (a) non-porous and (b) porous
silica-C18 as a stationary phase for LCUV
Alkaloid Retention time (min)a Calibration parametersb Detection limit amount (pmol)

Mean value CVc (%) a (105 ) b (105 ) Correlation coefficient LODd LOQe

a b a b a b a b a b a b a b
Caffeine 2.48 27.91 1.06 1.06 26.1 28.7 2.14 23.2 0.99 0.99 4.27 10.3 12.8 33.2
Theophylline 1.04 14.22 0.74 0.39 24.7 38.1 7.64 0.15 0.99 0.99 2.94 6.84 9.99 20.2
Theobromine 0.75 9.22 0.58 0.56 24.5 32.6 2.62 0.56 0.99 0.99 1.72 3.76 5.55 11.4
a Retention time of 20 replicate injections, concentration range 0.420 "g/ml.
b Linear regression equation: Y = aX + b; Y, signal area; X, alkaloids concentration ("g/ml); a, slope; b, intercept on Y axis.
c CV = coefficient of variation.
d Lower limit of detection, as signal-to-noise (S/N) of 3/1.
e Lower limit of quantitation, as signal-to-noise (S/N) of 9/1.

Table 2
Reproducibility of the LCUV analysis of caffeine, theophylline and theobromine
Alkaloid Concentration ("g/ml) Coefficient of variationa (%) Recoveryb (%)
Intra-day precision Inter-day precisionc
Caffeine 0.4 1.86 4.63 98.1
20 1.94 4.98 96.3
Theophylline 0.4 1.02 2.03 78.9
20 1.07 2.09 74.6
Theobromine 0.4 0.75 1.25 94.7
20 0.73 1.26 93.8
a Retention time of 20 replicate injections.
b Determined by spiking of a coffee extract with 0.4 and 20 "g/ml.
c Determined by 50 injections during 5 days.

and Caf, respectively, a resolution Rs of 2.1 between Tbr and was less than 4.98%. Finally, spiking of a coffee extract with
Tph, compared to k = 0.82, 1.36 and 5.41, b0.5 = 0.074, 0.13, standards gave recoveries between 74.6% for Tph and 98.1%
0.95 min and Rs = 2.6 with the porous phase (Fig. 1d). Repro- for Caf (Table 2).
ducibility of the method applying both phases was determined
with regard to retention times of single standards of each alka- 3.2. Liquid chromatography coupled to electrospray
loid. Twenty replicate injections gave highly consistent reten- ionisation quadrupole ion trap mass spectrometry
tion times for which coefficient of variation (CV) were <1.06 (LCESIMS)
on both phases (Table 1). The six-point calibration curves
of each alkaloid were obtained by linear regression analy- To evaluate the content of Tbr, Tph, Caf in several cof-
sis and revealed high linearity with correlation coefficients fee extracts by mass spectrometric (MS) detection, the op-
>0.99 for both porous/non-porous particles. The lower limit timised LC method applying the non-porous phase due to
of detection (LOD), established as signal-to-noise (S/N) of 3 the shorter retention times was hyphenated to mass spec-
with CV less than 7.1% for six replicates, were between 1.72 trometry (MS) via an electrospray ionisation (ESI) interface.
and 4.27 pmol using the non-porous, and between 3.76 and Under these soft ESI conditions all three analytes could effec-
10.3 pmol using the porous phase (Table 1). Determination tively be transformed into abundant molecular ions [M + H]+
of the limit of quantitation (LOQ), established as signal-to- m/z 181.2 and 195.2, respectively (Table 3). Comparing the
noise (S/N) of 9 showed a similar tendency: between 5.55 and LCESIMS mass spectra of the raw standards and the peaks
12.8 pmol in case of the non-porous and between 11.4 and of interest in the mass chromatogram ensured that the com-
33.2 pmol for the porous (Table 1). Due to the high perfor-
mance at short retention times further optimisation was done Table 3
Mass spectrometric and MS/MS fragmentation pattern of caffeine, theo-
with the non-porous phase. Precision assessments were gen- phylline and theobromine
erally carried out with concentrations being at the start and
Alkaloid [M + H]+ MS data (MS/MS
the end of the calibration curves (0.4 and 20 "g/ml). Intra-day fragmentation pattern)
precision was determined by analysis of 20 replicate injec-
tions. The intra-day CV was less than 1.94% (Table 2). The Caffeine 195.2 181.2, 151.2, 138.0
Theophylline 181.2 167.2, 153.2, 123.5
inter-day precision was assessed by measuring the analytes at
Theobromine 181.2 167.2, 153.2, 107.5
indicated concentrations for five different days, and the CV
C.W. Huck et al. / Analytica Chimica Acta 538 (2005) 195203 199

Table 4
Reproducibility of retention times, calibration parameters and detection limits of caffeine, theophylline and theobromine using non-porous silica-C18 as a
stationary phase for LCESIMS
Alkaloid Retention time (min)a Calibration parametersb Detection limit amount (pmol)

Mean value CVc (%) a (106 ) b (106 ) Correlation coefficient LODd LOQe
Caffeine 3.21 0.86 22.4 14.1 0.97 3.33 9.99
Theophylline 1.21 0.65 20.2 7.23 0.98 2.29 6.93
Theobromine 0.81 0.71 23.6 6.18 0.98 1.35 4.12
a Retention time of 20 replicate injections, concentration range 0.420 "g/ml.
b Linear regression equation: Y = aX + b; Y, signal area; X, alkaloids concentration ("g/ml); a, slope; b, intercept on Y axis.
c CV = coefficient of variation.
d Lower limit of detection, as signal-to-noise (S/N) of 3/1.
e Lower limit of quantitation, as signal-to-noise (S/N) of 9/1.

Table 5
Reproducibility of the LCESIMS method for caffeine, theophylline and theobromine
Alkaloid Concentration ("g/ml) Coefficient of variationa (%) Recoveryb (%)
Intra-day precision Inter-day precisionc
Caffeine 0.4 5.86 10.6 88.3
20 6.54 10.8 89.7
Theophylline 0.4 7.23 8.65 86.5
20 7.65 8.86 86.4
Theobromine 0.4 6.49 9.56 87.3
20 7.12 9.90 86.9
a Retention time of 20 replicate injections.
b Determined by spiking of a coffee extract with 0.4 and 20 "g/ml.
c Determined by 50 injections during 5 days.

pounds were no artifacts. Tbr, Tph and Caf were tracked limit of quantitation (LOQ), established as signal-to-noise
from a selected ion trace (Fig. 2a) and their identity was (S/N) of 9 was found between 4.12 and 9.99 pmol. Precision
confirmed by extracted mass spectra depicted in Fig. 2b and assessments were carried out as described in the previous
c. Collisionally induced dissociation (CID) was used to ob- chapter. Intra-day precision resulted in CV between 5.86 and
tain fragment ions of structural relevance in liquid coffee ex- 7.12%, inter-day precision between 8.65 and 10.8%, recov-
tracts. Fragment-ion spectra were used as fingerprints for ery in the range 86.489.7% (Table 5). These validation data
fast identifying target compounds, which is shown in Fig. 2d were comparable to those obtained by UV-detection with a
and e. For this the three analytes were fragmented into their slight difference in recoveries.
daughter ions (Table 3). Products of m/z 195.2 and 181.1 As expected for the quantification of Tbr, Tph and Caf in
were generated at 25% collision energy. The fragmentation liquid extracts we got 6% higher values from mass spectro-
pathway was characterised by the loss of a methyl group metric than from UV-detection, which must be ascribed to
and further fragmentation of the ring system. Reproducibil- the higher selectivity of the mass spectrometer.
ity of the method was evaluated. Twenty replicate injections
of the standards gave highly consistent retention times with a 3.3. Near infrared reflectance spectroscopy (NIRS)
maximum CV of 0.86% (Table 4). The six-point calibration
curves were obtained by linear regression analysis and re- 3.3.1. Cluster analysis
vealed high linearity with correlation coefficients R2 0.97 The total of 83 cultivars of green coffee beans, originat-
(Table 4). The lower limit of detection (LOD), established at ing from different geographical regions, were put in a rotor
a signal-to-noise (S/N) of 3 with CV less than 8.4% for six and analysed using fibre optics. Spectra were recorded three-
replicates, were between 1.35 and 3.33 pmol (Table 4). The fold in random order over a wavelength range from 4008

Table 6
Calibration and prediction results obtained for 83 coffee samples by applying NIRS in the reflectance mode
Alkaloid Reference measurements NIRS calibration

Range (g/100 g) Mean S.D. Regression equation S.E.E. (g/100 g) S.E.P. (g/100 g) BIAS R2
Caffeine 0.954.13 2.37 0.59 Y = 0.7489X + 0.5940 0.34 0.40 5.9 1015 0.86
Theobromine 0.100.67 0.31 0.12 Y = 0.7161X + 0.0928 0.07 0.10 5.9 1015 0.85
200 C.W. Huck et al. / Analytica Chimica Acta 538 (2005) 195203

the CH2 (5742 cm1 ), and the CH3 overtone (5808 cm1 ).
Five primary factors were necessary in order to reach the best
calibration model. Discriminating power is mainly due to wa-
ter (6877, 5102 and 4440 cm1 ) and lipids (1170, 5797, 5666,
4305 and 4261 cm1 ) although the water peaks exhibit a sig-
nificant shift from the maxima of pure water, Arabica coffees
being richer in fatty acids than Robusta. Three-dimensional
display after principal component analysis (PCA) and cal-
culation of Mahalanobis-distances in a factor plot (Fig. 4)
shows that Arabica can be distinguished from Robusta with-
out any outlier spectrum. The maximal spectra residual for
calibration was 2.61 104 and the resulting Q-value 0.025.
Thus out of the 25 prediction samples, 22 were correctly clas-
sified, which corresponds to an overall success rate of 88%.
This cluster analysis was tested to possess high reprodro-
ducibility and stability over a time period of approximately
1 year.

3.3.2. Partial least squares regression (PLS)


A model for the quantitative analysis was established for
Caf and Tbr but not for Tph, due to its content below the
detection limit of 0.05 g/100 g of the NIR-method. For the
quantitative analysis of Tbr and Caf LCUV applying the
non-porous silica-C18 was used as a reference method. After
optimisation of the temperature to 23 C, as a rise would in-
duce a broadening in the fundamental vibrational bands [34],
and the optical thin layer to 1 mm spectra of 83 extracted
coffee samples were recorded in the transflection mode with
Fig. 2. RPLCESIMS and MS/MS of an aqueous coffee extract: (a) TIC, the NIR-spectrometer. Fig. 5a shows six original spectra of a
total ion current; (b) selected ion trace, m/z = 180.7181.7; (c) selected ion liquid Arabica Costa Rica coffee sample. Mathematical pre-
trace, m/z = 194.2196.2; (d) CID of caffeine (relative collision energy 25%); treatment and statistical analysis were carried out by perform-
(e) CID of theobromine (relative collision energy 25%). Chromatographic ing partial least squares regression (PLS). For recording the
conditions see Section 2.
reference data each of the 83 extracts was determined in a
random order three-fold in the case of caffeine and two-fold
to 9996 cm1 with a resolution of 12 cm1 in the reflectance in the case of theobromine in separate analysis. All recorded
mode. Ten scans were used for one average spectrum to equi- spectra were normalised (i.e. ordinate-values are stretched
librate inhomogeneities. Seventy percent of the spectra were between zero and one) and transformed to their first deriva-
randomly put into a learning-set and 30% into a validation- tive (Taylor 3 points) before calculating in the linear PLS
set. The 58 calibration samples contained 32 Arabica and 26 model, which also allowed to identify characteristic absorp-
Robusta sorts. For their discrimination a wavelength range tion bands. Fig. 5b shows the pretreated spectra obtained by
from 4500 to 9996 cm1 was used, as important absorption calculation from the original spectra in Fig. 5a. The most
bands are included in this area. The stirring velocity of the intensive band in the spectrum belonged to the vibration of
rotor containing a ground coffee sample during measurement the 2nd overtone of the carbonyl group (5352 cm1 ), fol-
was found to have an important influence on the reflectance. lowed by the C H stretch and C H deformation vibration,
This influence is depicted in Fig. 3a. Also the influence of the OH vibration of water (4440 cm1 ) and the CH2 over-
the applied contact pressure of the fibre optics onto the cof- tone (5742 cm1 ). The vibration of the carbonyl group, the
fee beans during stirring has a major effect (Fig. 3b). In C H and CH2 vibrations are caused by ingredients such as
the following, all measurements were carried out applying proteins, lipids, volatile and non-volatile acids, chlorogenic
the same stirring velocity and contact pressure. All recorded acids, alkaloids and some aroma compounds. Theobromine
spectra were transformed to their first derivative (Taylor 3 has one methyl group less than caffeine and showed differ-
points) before calculating the cluster model (Fig. 3c). The ences compared to caffeine at 7353 cm1 (C H str. + C H
most intensive band in the spectrum belonged to the vibra- def.), 5865 cm1 (C H str., first overtone) and 4383 cm1
tion of the 2nd overtone of the carbonyl group (5352 cm1 ), (C H str. + C H def.). Normalisation allowed to minimise
followed by the C H stretch and C H deformation vibra- the baseline shift [35]. Five primary factors were necessary in
tion (7212 cm1 ), the OH vibration of water (4440 cm1 ), order to reach the best calibration equation. The multivariate
C.W. Huck et al. / Analytica Chimica Acta 538 (2005) 195203 201

Fig. 3. NIRS-spectra of rough Arabica beans obtained by (a) applying different stirring velocities (n = 9) and (b) different pressure; (c) first derivative of
NIR-spectra recorded from 58 ground Arabica coffee samples.

statistical method PLS is used as a full-spectrum method.


Therefore the information of the whole spectral range can
be used for the calibration. In the course of model optimisa-
tion the best statistical results were obtained when spectral
information in the interval between 4500 and 9996 cm1 was
used for calculating PLS. Other data pretreatments, e.g., cal-
culation of the 2nd derivative resulted in worse statistical
parameters compared to performing 1st derivative spectra.
The robustness of the NIRS model is high, which is demon-
strated in the similarity of the results for S.E.E. and S.E.P.:
Fig. 4. Factor plot of 249 spectra of Arabica and Robusta ground cof-
fee beans. Conditions: 1st derivative Taylor 3 points; wavenumber range, 0.34 and 0.40 g/100 g for Caf, 0.07 and 0.10 g/100 g for Tbr
45009996 cm1 ; scans, 10; temperature, 23 C. (Table 6). Accuracy is expressed in the BIAS. The values
202 C.W. Huck et al. / Analytica Chimica Acta 538 (2005) 195203

Fig. 5. NIRS-spectra of liquid Arabica Costa Rica coffee samples (six measurements): (a) original spectra, (b) pretreated spectra (normalisation and calculation
of its 1st derivative Taylor 3 points); wavenumber range, 45009996 cm1 ; optical thin layer thickness, 1 mm; scans, 10; temperature, 23 C.

are 5.9 1015 for Caf and Tbr (Table 6). So the LCUV in mind that here the whole extract is analysed. Validation
results agree with NIRS on average. Calculation of the re- of extracts showed that the robustness and reproducibility
gression equation for Caf (Fig. 6) and Tbr (Fig. 7) resulted of the NIRS model for the determination of Caf and Tbr is
in high correlation coefficients of 0.86 and 0.85 for Caf and high. The model can be used to predict the content of Caf and
Tbr (Table 6). Compared to the LCUV and LCMS method Tbr in liquid extracts. It has to be noticed that the NIRS is
the values for R2 are 13% smaller, but it has to be kept an analytical method that is never fully even though the first
calibration model functions satisfactorily. It is necessary to
collect additional samples especially when their matrix prop-

Fig. 6. Predicted (NIRS) vs. true property (HPLC) for the determina- Fig. 7. Predicted (NIRS) vs. true property (HPLC) for the determination
tion of percentage caffeine in liquid coffee extracts (n = 249). R2 = 0.86; of percentage theobromine in liquid coffee extracts (n = 166). R2 = 0.85;
S.E.P. = 0.40. S.E.P. = 0.10.
C.W. Huck et al. / Analytica Chimica Acta 538 (2005) 195203 203

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