Gas chromatography–mass ...
Gas chromatography–mass spectrometry method for determining, Artykuły naukowe, SPME i HS-SPME
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Journal of Chromatography A, 1017 (2003) 151–159
Gas chromatography–mass spectrometry method for determining
the methanol and acetic acid contents of pectin using headspace
solid-phase microextraction and stable isotope dilution
Brett J. Savary
∗
, Alberto Nuñez
Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture,
600 East Mermaid Lane, Wyndmoor, PA 19038, USA
Received 29 January 2003; received in revised form 16 July 2003; accepted 16 July 2003
Abstract
A simple, fast, and direct procedure was developed for the simultaneous determination of the methanol and acetic acid
present as esters in the plant cell wall polysaccharide pectin. After base-hydrolysis of esters and acidification of pectin samples,
headspace solid-phase microextraction (SPME) was performed using a Carboxen-PDMS fiber assembly. Methanol and acetic
acid were separated by gas chromatography with a Chrompak PoraPlot Q capillary column and detected using electron impact
mass spectrometry with selected ion monitoring. Stable deuterated isotopomers (d
3
-methanol and d
3
-acetic acid) were used
as internal standards and for constructing calibration curves, providing accurate and absolute quantification of analytes. The
methanol and acetic acid contents in 1 mg quantities of fruit and vegetable pectins were readily quantified by this procedure.
© 2003 Elsevier B.V. All rights reserved.
Keywords:
Headspace analysis; Stable isotope dilution; Solid-phase microextraction; Pectin; Methanol; Acetic acid; Polysaccharides
1. Introduction
through rhamnose residues)
[3,4]
. In homogalactur-
onans, galacturonic acids are extensively esterified
with methanol at C6-carboxyl groups and variably es-
terified with acetic acetic at C2/C3 hydroxyl groups.
Galacturonic acids in rhamnogalacturonan may be
similarly esterified with acetic acid. The methanol
and acetic acid contents of pectin extracted from
citrus peel is about 12 and 0.2%, respectively, and
about 9 and 5%, respectively, from sugar beet root
[3]
. The contents can vary considerably by species,
tissue type, age, and by extraction and processing
conditions
[5,6]
. The methanol and acetic acid con-
tents are often indicated by degree of esterification
(DE), the percent mole ratio to anhydrogalacturonic
acid determined for a pectin. Specifically, they are
Pectin is a complex of acidic polysaccharides
that form an interpenetrating network in the plant
cell wall
[1]
. They are an important food hydrocol-
loid and traditionally used in gelling and thickening
applications
[2]
. Pectin is composed primarily of
linear homogalacturonan (
-1,2-rhamnose)
chains (the neutral sugar branches are attached
-1,4-galacturonic acid to
∗
Corresponding author. Tel.:
+
1-215-233-6458;
fax:
+
1-215-233-6406.
E-mail address:
bsavary@arserrc.gov (B.J. Savary).
0021-9673/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0021-9673(03)01293-7
-1,4-galacturonic acids)
chains interspersed with branched rhamnogalactur-
onan (
152
B.J. Savary, A. Nuñez / J. Chromatogr. A 1017 (2003) 151–159
represented as degree of methoxylation (DM) and
degree of acetylation (DA), respectively. The DM is a
primary factor influencing the conditions and mech-
anism for gelling by commercial pectins
[2]
. Most
commercial pectins are produced from citrus peel,
and pectins with lower DM are prepared by chemical
treatments
[2]
. Hydrolysis of methylesters using the
enzyme pectin methylesterase has been investigated
as an alternative means to chemical deesterification
and may provide pectins with unique functional prop-
erties
[3,7,8]
. Acetyl esters generally act to inhibit
pectin gelling
[9]
; but treatment with enzyme prepa-
rations containing pectin acetylesterase can be used
to improve gelling properties of pectin extracted from
sugar beet
[10,11]
.
A standard titration method is used for determin-
ing the degree of methyl esterification and anhy-
drogalacturonic acid content of commercial pectins
[12]
, but this is subject to error by contribution of
acetic acid esters and putative non-methyl galactur-
onosyl carboxyl esters
[13]
. Chromatographic and
spectroscopic methods have been reported for de-
termination of pectin DE
[14–18]
. Direct determi-
nation of methanol content of pectin is frequently
cited by colorimetric assay based on oxidation of
methanol released from pectin and derivatization of
the resulting formaldehyde with pentane-2,4-dione
[19]
. Improvements have included enzymatic oxida-
tion of the methanol
[20]
and adaptation to HPLC
with derivatization of formaldehyde by condensation
with 2,4-dinitrophenylhydrazine
[21]
. A GLC–FID
method was developed to measure methyl esters in
plant cell walls and pectin
[22]
using a Carbowax
20 M packed column. A HPLC method based on
an ion-exchange resin column provided separation
of both methanol and acetic acid
[23]
, but suffered
in sensitivity and specificity due to refractive index
detection. An improved HPLC method was reported
recently
[24]
. Although any of these methods can
be used to measure enzymatic hydrolysis by pectin
methylesterase, titration assay is most conveniently
used to determine standard units of enzyme activity
[25]
. Probably, because of the low acetyl content
in commercial pectins, methods for acetic acid de-
termination are less advanced. Colorimetric assays
commonly used are the Hestrin method
[26]
or hy-
droxamic acid reaction
[27]
. A commercial enzyme
assay kit (Boehringer–Mannheim) has been used to
measure acetate released from pectin by chemical or
enzymatic hydrolysis
[11]
. The GLC–FID method
reported for methyl ester content was also proposed
for use in determining acetyl ester content
[22]
. More
recently, GC with a Porapak QS column was used for
enzymatic or chemical treatment of pectin samples
worked up from ion-exchange and solvent extraction
[10]
, but no details were reported for the methodology.
Quantification of a particular analyte by GC analy-
sis requires the use of internal standards. Deuterated
isotopomers of analytes provide an ideal internal
standard when they are used in conjunction with
mass spectrometer detectors, providing direct and
accurate determination of concentration by stable
isotope dilution assay
[28]
. This compensates for
losses during sample workup, thereby reducing error
in determinations, and coupled with the selectivity of
MS in selected ion mode, can provide unequivocal
identity by fragment pattern in the presence of unre-
solved contaminants
[29]
. The recent development of
solid-phase microextraction (SPME) fiber systems fa-
cilitates the selective extraction of individual analytes
from a solution based on their affinity with a specific
fiber adsorbant. SPME was introduced originally for
application in environmental analysis
[30]
, and it has
found wide use in biomedical, forensic, and food
analysis applications
[31,32]
. The SPME technique
uses a polymer-coated fused silica fiber that provides
sample extraction, concentration, and transfer to the
chromatograph in a single step. Headspace sampling
is preferred over direct immersion as it avoids contact
with organic polymers that can degrade fiber perfor-
mance and lifetime, and it eliminates introduction of
non-volatile contaminants. Differences in factors such
as concentration, volatility, and partition equilibria
in headspace sampling for methanol and acetic acid
could possibly introduce bias in quantitative determi-
nations, but such limitations are overcome with stable
isotope-labeled internal standards
[29]
. We report here
an integrated gas chromatography–mass spectrome-
try (GC–MS) method for the simple, fast, direct, and
simultaneous determination of methanol and acetic
acid released from pectin. This method exploits the
availability of: (1) fully deuterated forms of methanol
(d
3
-MeOH) and acetic acid (d
3
-HOAc) for use as in-
ternal isotopomer standards and (2) suitably selective
SPME fibers for headspace capture of methanol and
acetic acid.
B.J. Savary, A. Nuñez / J. Chromatogr. A 1017 (2003) 151–159
153
2. Experimental
at least 10 min between samples. In a control experi-
ment with pectin solutions having no standards added,
no methanol or acetic acid was detected, demonstrat-
ing negligible ester hydrolysis occurred under these
sampling conditions. Two samples were prepared for
each sample point and each run in duplicate (
2.1. Materials and reagents
All chemicals and solvents were of analytical grade
and purchased from Sigma (St. Louis, MO, USA)
and Burdick and Jackson (Muskegon, MI), respec-
tively, unless otherwise indicated. Deuterated stan-
dards were purchased from Aldrich: acetic-d
3
acid-d
(99.9 at.% D) and methyl-d
3
alcohol-d (99.8 at.% D).
Solid-phase microextraction fibers were purchased
from Supelco (Belfont, PA): 75
4).
For pectin analyses, vials received 0.200 ml pectin
(1 mg), 0.200 ml of 1.0 M NaOH, and 0.100 ml
d
3
-standards (2.00
n
=
mol of MeOH and 0.500 or
mol of HOAc). The vials were immediately
capped and heated at 40
◦
C for 1 h, and then placed
on ice. Vials then received 0.5 ml of 0.4 M sulfuric
acid (final pH
m Carboxen-PDMS
m Carbowax-DVB (5–7312). Fibers
were conditioned as recommended by manufacturer
prior to use. HPLC-grade water used as diluent was
sparged with helium. Apple (73.5% anhydrogalac-
turonic acid equivalents, AGA; 9.7% methoxyl) and
citrus pectins (72.1% AGA; 8.1% methoxyl) was pur-
chased from Sigma; methylated lime pectin (Grind-
sted URS: 89.0% AGA; 81.5% methoxyl) was a gift
from Danisco USA (New Century, KS), sugar beet
pectin (Classic RU 301: 65% AGA; 6.2% methoxyl;
3.5% acetyl) a gift from Herbstreith and Fox KG
(Neuenbuerg, Germany). Samples of
Aloe vera
and
onion pectin were gifts from Rose Chau and Marshall
Fishman, USDA-ARS, Wyndmoor).
2.0). Headspace-SPME sampling
was performed as described for calibration samples.
Duplicate samples for each pectin were prepared and
analyzed in triplicate (total analyses,
n
=
6).
<
2.3. Gas chromatography–mass spectroscopy
detection
The GC–MS system consisted of a 5890 Series II
Plus gas chromatograph with a Mass Selective Detec-
tor (Hewlett-Packard, San Fernando, CA) fitted with
a PoraPLOT Q capillary column, 25 ml
×
0
.
25 mm
m (Chrompack, Raritan, NJ)
and a narrow bore (0.75 mm) SPME injection liner
(Supelco). All injections were splitless with the injec-
tor set at 300
◦
C and detector at 250
◦
C, using helium
as carrier gas at 1 ml/min. The oven temperature gra-
dient profile was 40
◦
C (1.6 min) to 250
◦
C (5 min)
at 50
◦
C/min and held at temperature for 5 min. The
detector was set at electron impact ionization mode
(70 eV) with data collected using the selected ion
recording for selected ions at 1.2 scans/s. Methanol
concentrations were calculated by plotting the peak
area ratios (normal to deuterated forms) for base
ion pairs (
m
/
z
29/30
d
) over the indicated range of
concentration ratios. Acetic acid concentrations were
similarly calculated using peak area ratios of base
ion pairs (
m
/
z
43/46
d
) over the indicated range of
concentration ratios.
2.2. Sample preparation and SPME procedure
mol) internal
standards, and varying quantities of unlabelled an-
alytes (0.10–8.0
mol methanol and 0.05–2.0
mol
acetic acid). The final volume was 1 ml and 0.100 M
monobasic sodium phosphate (pH 2.0 with sulfuric
acid). Vials were capped and warmed to 40
◦
Cinan
aluminum block heater, then the needle of the SPME
device was inserted through the septum and the fiber
was exposed to the headspace vapor for 15 min.
Thereafter, the fiber was retracted and immediately
transferred to the GC injection port for sample desorp-
tion. The fiber remained exposed in the injection port
3. Results and discussion
3.1. Headspace sampling by SPME
Carbowax-DVM SPME fiber is recommended by
the manufacturer for applications involving alcohols
0.050
(5–7318) and 65
i.d., film thickness 8
Pectins were treated by dissolution in water,
freezing, and then lyophilization to remove resid-
ual solvents from commercial pectin processing.
Fresh pectin solutions were prepared at 5 mg/ml by
dissolving in water with brief heating (60
◦
C) and
sonication. For calibration samples, vials (4 ml vol-
ume) received sugar beet pectin (1 mg), d
3
-methanol
(2.0
mol) and d
3
-acetic acid (0.500
154
B.J. Savary, A. Nuñez / J. Chromatogr. A 1017 (2003) 151–159
and polar compounds (MW 40–275) and was deter-
mined to be most effective for headspace sampling of
ethanol and other volatile compounds in blood
[34]
.
Carboxen-PDMS fiber was reported as most sensi-
tive for adsorbing small molecules and organic acids
from coffee sample headspace
[35]
, and it was rec-
ommended for analysis of methanol or formic acid in
bodily fluids
[36]
. Both fiber types were compared in
this study for their ability to bind methanol and acetic
acid in sample headspace at ambient and elevated tem-
perature (
Fig. 1
). Carboxen-PDMS proved a much bet-
ter adsorbent than Carbowax-DVB for both analytes.
The sensitivity for acetic acid increased further by a
factor of three with elevated temperature during sam-
pling. Methanol binding was not increased at higher
temperature. Carboxen-PDMS fiber sampling was se-
lected for calibrating standard curves and analysis of
methanol and acetic acid released from pectin samples.
Optimizations such as added salts, increased tempera-
ture, and exposure time have provided increased sen-
sitivity for extracting volatile compounds from sample
headspace by SPME
[31,32,34]
. However, no further
optimization of headspace extraction was attempted
in this study since the system was sufficiently sensi-
tive for the range of methanol and acetic acid contents
possible in 1 mg of pectin.
3.2. GC separation
Although a range of GC columns have been used
for analyses that have included methanol and acetic
acid, generally more polar columns are preferred for
resolving such volatile compounds. Derivatization of
organic acids, such as methylation of the acid group, is
generally recommended for improved elution and sep-
aration, but such manipulations introduce additional
steps in the analytical procedure. A PoraPLOT Q col-
umn provided separation of polar or non-polar volatile
compounds in the range of C
1
–C
7
with little influence
by the polarity or boiling point of the molecule and, in
general, separated compounds primarily by molecule
size without the need for derivatization
[37]
.Weeval-
uated this PoroPLOT Q column to separate a mix-
ture of methanol and acetic acid in the natural and
deuterated forms. Suitable retention times and reso-
lution of methanol and acetic acid were obtained in
a 10 min program. Methanol eluted at about 4.75 min
(approximately 180
◦
C) while acetic acid eluded at
about 6.33 min (250
◦
C), as shown in the total ion cur-
rent chromatogram in
Fig. 2
. A PoroBond Q column
was also evaluated, but it was found unsuitable due to
excessive tailing of the acetic acid peak.
3.3. Ion selective mass detection and
calibration curves
Fig. 1. Headspace sampling for methanol and acetic acid with
Carboxen-PDMS (CXen) and Carbowax-DVB (CWax) SPME
fibers at two temperatures. Samples in 4 ml vials contained 1 mg
pectin in 1 ml volume at pH 2.0 and spiked with 60
g deuterated
Deuterated forms (d
3
) of methanol and acetic
acid are available commercially, inexpensive, and
highly-labeled (99.8 at.% D). Coupled with a mass
selective detector, these isotopomers can be used as
near ideal internal standards to provide direct and ac-
curate quantification by stable isotope dilution assay
[28,29]
. The electron impact spectra for the natural
and deuterated form of methanol and acetic acid are
shown in
Fig. 3
. The base ions (100% relative inten-
sity) are observed at
m
/
z
29 [HCO]
+
and 30 [DCO]
+
for methanol and d
3
-methanol, respectively, and
m
/
z
43 [CH
3
CO]
+
and 46 [CD
3
CO]
+
for acetic acid and
d
3
-acetic acid, respectively. Molecular ions are ob-
served at
m
/
z
32 (25%) and 35 (32%) for methanol
and d
3
-methanol, respectively. Correspondingly, the
molecular ions for both forms of acetic acid are at
m
/
z
60 (51%) and 63 (46%). These sets of ion pairs
(molecular and base) have the relative abundance nec-
essary for use in the stable isotope dilution method
standards.
B.J. Savary, A. Nuñez / J. Chromatogr. A 1017 (2003) 151–159
155
Fig. 2. Total ion chromatogram from GC–MS for separation of methanol and acetic acid mixture. Insert: selected ion chromatogram
overlays for acetic acid (
m
/
z
43, 60) and d
3
-acetic acid (
m
/
z
46, 63).
[28]
and provide two quantitative alternatives for each
analyte, and comparison of analyte determinations
using each can be used to provide a qualitative means
to assess for cross-ion contamination.
The reconstructed ion chromatograms obtained for
the selected ions for the deuterated and natural forms
for both compounds indicated a partial resolution of
the isotopomeric mixture by the PoraPLOT Q col-
umn. This is shown for acetic acid in the insert in
Fig. 2
. Deuterated forms eluted slightly before unla-
belled forms. Because of this partial separation of iso-
topomer pairs, quantification of analytes was based
on peak area ratios determined from reconstructed
ion peak area chromatograms rather than ion intensity
height ratios as described in the stable isotope dilution
method
[28,29]
.
The calibration curves generated for methanol and
acetic acid quantification are plotted for the selected
Fig. 3. Electron impact (70 eV) mass spectra for: (A) methanol; (B) acetic acid; (C) d
3
-methanol; (D) d
3
-acetic acid.
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