rehbein, 2007

Prévia do material em texto

Jens Rehbein
Benjamin Dietrich
Marc David Grynbaum
Petra Hentschel
Karsten Holtin
Maximilian Kuehnle
Paul Schuler
Marc Bayer
Klaus Albert
Institute of Organic Chemistry,
University of Tuebingen,
Tuebingen, Germany
Original Paper
Characterization of bixin by LC-MS and LC-NMR
An overview upon modern analytical techniques for the isolation, separation, and
structural identification of the essential bioactive carotenoid bixin is given. Isola-
tion from biological matrices is performed by matrix solid phase dispersion (MSPD).
The extract is separated with shape-selective C30 columns. Structural assignment of
the separated compounds is done by online LC-MS and capillary HPLC-NMR.
Keywords: Bixin stereoisomers / Capillary LC-NMR / Carotenoids / LC-MS /
Received: March 1, 2007; revised: May 5, 2007; accepted: May 6, 2007
DOI 10.1002/jssc.200700089
1 Introduction
Carotenoids provide plants and animals with bright col-
ors and show strong antioxidative effects and are very
effective radical scavengers [1, 2]. Different all-E/Z stereo-
isomers exist and may differ in their biological activity
[3, 4]. Figure 1 shows the structures of the main stereo-
isomers of the investigated carotenoid bixin. Bixin is the
major carotenoid of the seeds of the plant Annatto or
Urucum. Its name is derived from the systematic name of
the plant, Bixa orellana. In Central America and the Carib-
bean, the seed itself or a ground powder is used as a spice
and as dye for dishes. Bixin (E160b) is also widely used for
coloring food, e. g., cheese, butter, snacks, and deserts.
Brazil is the main producer and exporter of Annatto [5–
7]. Other than in former publications the isomers of
bixin were investigated with both HPLC-MS and capillary
HPLC-NMR, leading to the 1H chemical shifts of the major
isomers in aceton-d6.
2 Experimental
2.1 Materials
D2O (Uvasol, 99.8%), acetone (LiChrosolv), ACN (LiChro-
solv), and chloroform (LiChrosolv) were purchased from
Merck (Darmstadt, Germany). ACN-d3 and acetone-d6
were obtained fromDeutero (Katellaun, Germany).
2.2 Sample preparation
A spare and rapid extraction technique is matrix solid
phase dispersion (MSPD) [8, 9]. As carotenoids are very
sensitive to both light and air, an optimized combination
of analytical sample preparation, separation, and detec-
tion techniques has to be used. MSPD, a very gentle and
expeditious extraction technique for solid and viscous
samples, was used for the extraction of bixin. It is conven-
ient to work with, decreases solvent use by up to 98%,
and reduces sample turnaround time by 90% compared
to conventional extraction techniques like liquid–liquid
extraction [10]. Another advantage of MSPD is the higher
enrichment of the analytes, which is very important for
successful NMR measurements. Here, the biological
matrix is ground together with a 5 lm C18 stationary
phase material in a mortar. The C18 material exhibits
effective extraction capabilities as well as matrix protec-
tion probabilities, thus no oxidative damage of the air-
and UV-sensitive carotenoids can occur. The mixture of
the biological matrix and the C18 phase is transferred to
an extraction cartridge, impurities are washed off, and
the target carotenoids are eluted. In this case, 10 mL of
deionized water was used to wash the cartouche and the
target compounds were eluted with 4 mL of CHCl3. After
evaporation of CHCl3 under a nitrogen stream, the
extract was stored at –308C until it was analyzed. For iso-
merization, a modified method after Zechmeister was
used [11]. Usually the isomerization is carried out in hex-
ane, but this was not applicable here, because bixin did
not show a good solvability in this solvent. The isomeriza-
tion was then carried out directly in acetone, therefore it
was dissolved in 2 mL of acetone, two drops of 2% I2 in
hexane were added and exposed to UV radiation for
20 min, after that it was evaporated under a nitrogen
stream again. Before analysis, the extract was redissolved
in 1 mL of ACN or ACN-d6 for HPLC-NMR analysis, sterile
filtered, and the filtrate was washed three times with
330 lL of ACN.
Correspondence: Professor Dr. Klaus Albert, Institute of Organ-
ic Chemistry, University of Tuebingen, Auf der Morgenstelle 18,
D-72076 Tuebingen, Germany
E-mail: klaus.albert@uni-tuebingen.de
Fax: +49-7071-29-5875
Abbreviation: MSPD,matrix solid phase dispersion
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2382 J. Rehbein et al. J. Sep. Sci. 2007, 30, 2382–2390
J. Sep. Sci. 2007, 30, 2382–2390 Liquid Chromatography 2383
2.3 HPLC separation
HPLC and HPLC-MS analyses were carried out on an
HP1100 system (Agilent Technologies, Waldbronn, Ger-
many) using a UV detector (DAD) monitoring at 450 nm.
The separations were performed on a 25064.6 mm2
ProntoSil C30 stainless steel column (Bischoff, Leonberg,
Germany). The average pore diameter was 200 � and the
particle size was 3 lm. The chromatographic separation
utilized an isocratic elution program using a mixture of
acetone and H2O (acetone/H2O = 92:8 v/v), injecting 20 lL
into the system.
Capillary-HPLC and capillary-HPLC-NMR were carried
out on a system consisting of a ternary modular capillary
HPLC pump (Waters, Milford, MA, USA) equipped with an
on-column (150 lm id) Bischoff Lambda 1010 UV detec-
tor operating at 450 nm (Bischoff Chromatography) and
a Vici Cheminert 1004-.1 (Vici AG, Schenkon, Switzer-
land) injection valve (500 nL internal loop).
The capillary separation column was packed on site,
using the slurry packing procedure with Bischoff Pronto-
Sil C30 (3 lm, 200 �, 15 cm6250 lm) and equipped with
end fittings consisting of 2SR1 filter screens in zero-dead-
volume unions ZU1C (ViciAG, Schenkon, Switzerland).
The chromatographic separation utilized an isocratic
elutionmethodwithmixtures of acetone-d6 and D2O (ace-
tone-d6/D2O = 92:8 v/v), injecting 500 nL of the sample
into the system. UV detection was carried out on a capil-
lary (150 lm) at 450 nm. All transfer capillaries consisted
of fused silica with a dimension of (50 lm id/360 lmod).
2.4 HPLC-MS coupling
MS was performed on a Bruker Esquire 3000plus LC-
MS(n)-system (Bruker Daltonics, Bremen, Germany)
equipped with an APCI interface and an IT. The HPLC
APCI/MS coupling was accomplished using an HP1100
system (Agilent Technologies) using a 25064.6 mm2
ProntoSil C30 stainless steel column (Bischoff), and 20 lL
of sample was injected into the system. The detection
was performed using APCI in the positive ionization
mode. The voltage of the corona needle was set to 3.5 kV.
Nitrogen was used as a drying gas as well as a carrier gas
at the flow rate of 5 L/min with a nebulizer pressure of
70 psi. The ionization chamber temperature was set to
3008C and the dry gas temperature was held at 2508C.
The compound stability was set to 80% and the trap drive
level to 70%. The chromatographic conditions were the
same as in the analytical separation described above.
APCI/MS allows the unambiguous identification and
assignment of different carotenoids in positive as well as
in negative ionization mode [12–14], but not between
different isomers of the same carotenoid.
2.5 Capillary HPLC-NMR coupling
In addition to the structural information derived from
MS, the extraction of stereochemical information upon
the configuration of the different isomers is only possi-
ble by NMR spectroscopy [15].
Carotenoids are extremely sensitive to air and UV
exposure. For the unambiguous assignment of natural
carotenoid stereoisomers, the closed-loop LC-NMR tech-
nique shows severe advantages in comparison to the pro-
cedure of offline isolation and identification. Very often,
the application of the offline technique results in carote-
noid isomerization and degradation. Because only few
amounts of sample are available after the extraction
process, the miniaturizationof the hyphenation of HPLC
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Figure 1. Structures of the main stereoisomers of bixin.
2384 J. Rehbein et al. J. Sep. Sci. 2007, 30, 2382–2390
with NMR employing capillaries was a breakthrough in
carotenoid research. Figure 2 shows the instrumental
setup for capillary HPLC-NMR capillary coupling
employed in our laboratory. Because fully deuterated sol-
vents are used, a splitless Waters capillary pump is con-
nected to a self-packed separation capillary (250 lm id)
via a peak parking valve for stopped-flow measurements
and an injection valve. Due to the stray field of the
employed unshielded 14.1 T cryomagnet (AMX 600, 9.4
Tesla, Bruker BioSpin, Rheinstetten, Germany), the capil-
lary HPLC instrument is located at a distance from 3 m to
the magnet using a 3 m fused silica transfer capillary
(50 lm id) to connect it to the NMR probe head. Despite
the overall length of the transfer capillary of 3 m, a peak
broadening of only 1% is observed at flow rates of 5 lL/
min.
In collaboration with Professor Andrew Webb, our
group in Tuebingen is engaged in the construction of sol-
enoidal NMR microprobes. Figure 3 shows the picture of
a homebuilt double-resonant solenoidal NMR micro-
probe. The probe body together with the electronic sup-
ply was built by the mechanic and electronic shop of the
“Chemisches Zentral-Institut”. The NMR detection coil
(length of 4 mm, active volume 2 lL) consists of an eight-
turn solenoidal 200 lm Cu wire directly attached to a
horizontally positioned Duran capillary (od 1.5 mm, id
0.8 mm) tapering at both ends to vertically positioned
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Figure 2. Experimental setup for capillary HPLC-NMR coupling.
Figure 3. Homebuilt double-resonant solenoidal NMR
microprobe.
J. Sep. Sci. 2007, 30, 2382–2390 Liquid Chromatography 2385
capillaries (360 lm od, 100 lm id). The NMR microcoil is
positioned in a container filled with perfluorotributyl-
amine (FC 43) for matching the susceptibility differences
between copper and air. Figure 4 shows the 1H NMR spec-
trum (ARX 400, 9.4 Tesla, Bruker BioSpin) of 10 mM
sucrose in D2O/ACN-d3 proving the excellent NMR resolu-
tion and sensitivity of the current development.
3 Results and discussion
Unlike most of the known natural carotenoids, where
the major isomer is the all-E form, the major isomer of
bixin is the 99-Z bixin. The chromatographic analysis of
the extract showed that it is by far the dominant species.
Zechmeister [11] has already described diverse tech-
niques available for the (E/Z)-isomerization and numer-
ous examples for the preparation and isolation of (Z)-con-
figured isomers. In this case this method, slightly modi-
fied, was used to prepare the all-E isomer. The time of
exposure to the UV-light was varied. It turned out that
after an isomerization time of 20 min, an approximately
uniform concentration of the 99-Z and the all-E are
obtained (Fig. 5). Both isomers were then investigated by
LC-MS (APCI) leading to nearly identical MS-spectra (Figs.
6a and b) making it difficult to distinguish between
them. In both spectra, the [M + H]+molecule peak with m/
z = 395 is dominant; furthermore, the fragment ions
[M + H – H2O]+ with m/z = 377 and [M + H – HOCH3]+ with
m/z = 363 can be observed.
Routine NMR detection is performed with rotating
5 mmNMR techniques inserting in so-called “double-sad-
dle Helmholtz coils” with an internal diameter of 7 mm
(Fig. 7). The continuous acquisition of chromatographic
separations is possible with dedicated continuous-flow
NMR probes with detection volumes between 30 and
120 lL. Here the radio frequency detection coil is directly
attached to the glass tube of the detection “bubble cell”
improving the filling factor, being the ratio sample vol-
ume/detection volume. The NMR registration of capillary
NMR separations can be performed with the help of hori-
zontally positioned “solenoidal” microcoils. According
to theoretical considerations of Hoult [16], a three-fold
increase in sensitivity can be achieved in comparison to
the application of double-saddle Helmholtz coils. The
development of NMRmicrocoils was pioneered by Profes-
sor Andrew Webb [17]. It has successfully been employed
not only for online coupled capillary HPLC-NMR meas-
urements [17–19], but also for stopped-flow measure-
ments [20]. Mass-limited samples can also be inserted
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Figure 4. 1H-NMR spectrum (400 MHz) of 10 mM sucrose, D2O/ACN-d3 (90:10), 1 transient.
2386 J. Rehbein et al. J. Sep. Sci. 2007, 30, 2382–2390
i 2007WILEY-VCH Verlag GmbH &Co. KGaA,Weinheim www.jss-journal.com
Figure 5. HPLC separation of bixin isomers on a C30 phase.
Figure 6. Mass spectra (APCI) of bixin isomers (a) 99-Z, (b) all-E, and (c) unknown isomer.
J. Sep. Sci. 2007, 30, 2382–2390 Liquid Chromatography 2387
into the NMR probe by syringe flow injection. Microcoil
flow-through LC-NMR experiments were then performed
leading to stopped-flow 1-D and 2-D 1H NMR spectra (Figs.
8–10). Various peaks in the chemical shift region of the
protons of the conjugated double bounds could not be
assigned. NMR spectra in between the chromatographic
peaks were then recorded to identify probable impurities
leading to spectra showing signals exactly at the chemi-
cal shift of the unassigned peaks, in the spectra marked
with V. Figure 9 depicts the 1H-1H-COSY spectrum of 99-Z
bixin. All together five spin systems can be determined
by cross peaks, the spin system 7/8, 79/89, 10/11/12, 109/119/
129, and the largest one 14/15/159/149. In the 1H-1H-COSY
spectrum of all-E bixin (Fig. 10) the same couplings can
be observed, although all chemical shifts of the analo-
gous protons on both sides of the chain fall together. Fig-
ure 8 depicts the 1H NMR spectrum of 99-Z bixin from 5.7
to 8.1 ppm, the peaks from the protons at C-89 show a
chemical shift of 7.94 ppm and the one from the protons
at C-79 one of 5.92 ppm. In comparison with the spectrum
of all-E bixin, these peaks fall together with their ana-
logues on the other side of the chain in the case of the
protons at C-89 or overlap in the case of the protons at C-
79. With the information from the 1H-1H-COSY spectra, all
chemical shift and 3J(H/H) coupling constants could be
determined. The derivation of the position of the Z dou-
ble bound can be based very reliable on the observed
chemical shift differences Dd between Z and all-E iso-
mers. In this case, the isomerization shifts show the
same relative magnitude and algebraic sign as described
in literature, but differ in value [21]. This might be due to
the solvent, for it was already reported that solvents
other than CDCl3can affect the isomerization shift signif-
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Figure 7. NMR probes (a) routine 5 mm tube setup, (b) HPLC flow cell, and (c) capillary HPLC flow cell with solenoidal setup.
Table 1. Chemical shifts and chemical shift differences of
the bixin isomers
C atom 99-cis-bixin d
(ppm)
all-trans-bixin d
(ppm)
Dd
(ppm)
7 5.88 (15.5 Hz) 5.88 (15.5 Hz) –
8 7.35 (15.5 Hz) 7.35 (15.5 Hz) –
9 – – –
10 6.64 (11.5 Hz) 6.64 –
11 6.78 (15.0 Hz;
11.5 Hz)
6.78 –
12 6.62 (15.0 Hz) 6.62 –
13 – – –
14 6.47 (10.6 Hz) 6.47 –
15 6.83 (14.2 Hz;
10.6 Hz)
6.83 –
159 6.83 (14.2 Hz;
10.6 Hz)
6.83 –
149 6.47 (10.6 Hz) 6.47 –
139 – – –
129 6.53 (15.0 Hz) 6.62 –0.09
119 6.98 (15.0 Hz;
11.5 Hz)
6.78 0.2
109 6.47 (11.5 Hz) 6.64 –0.17
99 – – –
89 7.94 (15.5 Hz) 7.35 (15.5 Hz) 0.59
79 5.92 (15.5 Hz) 5.89 (15.5 Hz) 0.03
2388 J. Rehbein et al. J. Sep. Sci. 2007, 30, 2382–2390
icantly [21]. It seems that the only proton affected by the
chemical difference at the chain ends is the proton at C-
79,all other protons show, in case of the all-E bixin the
same chemical shifts. The strong influence of one single
Z double bond in case of 99-Z bixin leads to different
chemical shifts at C-79, C-89, C-109, C-119, and C-129. All
chemical shifts, coupling constants, and chemical shift
differences are listed in Table 1. The small peak at
46.1 min shows practically the same MS fragmentation
pattern, showing that another isomer of bixin was
formed (Fig. 6c). Unfortunately, the concentration of this
peak was too small to be investigated by LC-NMR, show-
ing themajor disadvantage of this setup.
4 Concluding remarks
Especially for carotenoids, where the mass fragmenta-
tion pattern is not significant, the hyphenation of capil-
lary HPLC with flow-through NMR is irreplaceable. It
offers the possibility to determine the structure of the
isomers even with small concentrations available, as it is
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Figure 8. Stopped-flow 1H NMR
spectra (600 MHz) of 99-Z- and
all-E-bixin.
J. Sep. Sci. 2007, 30, 2382–2390 Liquid Chromatography 2389
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Figure 9. Stopped-flow 1H-1H-COSY
NMR spectrum (600 MHz) of 99-Z-
bixin.
Figure 10. Stopped-flow 1H-1H-COSY
NMR spectrum (600 MHz) of all-E-
bixin.
2390 J. Rehbein et al. J. Sep. Sci. 2007, 30, 2382–2390
the case when using natural sources. In stopped-flow
mode it is even possible to record 2-D NMR spectra, pro-
viding even more information and the possibility to elu-
cidate the structure of totally unknown components.
The scientific collaboration with Professor Dr. Andrew Webb
(Penn State University, USA) is gratefully acknowledged. The con-
tinuous excellent collaboration with Eberhard Braun and Walter
Schaal together with their coworkers of the mechanic and elec-
tronic shop of the “Chemisches Zentral-Institut of the Faculty of
Chemistry and Pharmacy” is gratefully acknowledged.
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