A derivatization procedure for the simultaneous analysis of iminosugars and other low molecular weight carbohydrates by GC-MS in mulberry (Morus sp.)

Article abstract of DOI:10.1016/j.foodchem.2010.10.097

Different derivatization procedures were assayed to simultaneously analyse iminosugars such as deoxynojirimycin (DNJ) or fagomine and other carbohydrates of low molecular weight by gas chromatography coupled to mass spectrometry (GC-MS) in Morus sp. Both oximation + trimethylsilylation and oximation + acetylation allowed the separation of target compounds, whereas trimethylsilyl (TMS) and acetylated derivatives showed several coelutions. Nevertheless, oximation + acetylation were discarded for giving inaccurate results for ketoses due to their incomplete derivatization. Different conditions for the conversion into trimethylsilyl oximes (TMSO) were assayed, the best results being achieved using hexamethyldisilazane with trifluoroacetic acid as silylation agent. Contents of iminosugars (DNJ, fagomine and pipecolic acid derivatives) and other carbohydrates such as mono and disaccharides, myo-inositol and galactinol isomers in mulberry extracts (fruits, leaves and branches) were determined by GC-MS using the TMSO procedure.

Full text of DOI:10.1016/j.foodchem.2010.10.097

Food Chemistry 126 (2011) 353–359
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
A derivatization procedure for the simultaneous analysis of iminosugars and
other low molecular weight carbohydrates by GC–MS in mulberry (Morus sp.)
⇑
S. Rodríguez-Sánchez, O. Hernández-Hernández, A.I. Ruiz-Matute, M.L. Sanz
Instituto de Química Orgánica General (CSIC), Juan de la Cierva, 3 28006 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Different derivatization procedures were assayed to simultaneously analyse iminosugars such as deoxy-
nojirimycin (DNJ) or fagomine and other carbohydrates of low molecular weight by gas chromatography
coupled to mass spectrometry (GC–MS) in Morus sp. Both oximation + trimethylsilylation and oxima-
tion + acetylation allowed the separation of target compounds, whereas trimethylsilyl (TMS) and acety-
lated derivatives showed several coelutions. Nevertheless, oximation + acetylation were discarded for
giving inaccurate results for ketoses due to their incomplete derivatization. Different conditions for the
conversion into trimethylsilyl oximes (TMSO) were assayed, the best results being achieved using hexam-
ethyldisilazane with trifluoroacetic acid as silylation agent. Contents of iminosugars (DNJ, fagomine and
pipecolic acid derivatives) and other carbohydrates such as mono and disaccharides, myo-inositol and
galactinol isomers in mulberry extracts (fruits, leaves and branches) were determined by GC–MS using
the TMSO procedure.
Received 12 November 2009
Received in revised form 25 June 2010
Accepted 23 October 2010
Keywords:
Deoxynojirimycin (DNJ)
Fagomine
Pipecolic acid
Iminosugars
Carbohydrates
Acetylated oximes
Trimethylsilyl oximes
GC–MS
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Gupta & Vankar, 2009); however, there is a high interest in obtain-
ing these compounds from natural sources. Different extraction
Polyhydroxyalkaloids (also known as iminosugars) are hydrox-
ylated derivatives of piperidine, pyrrolidine, indolizidine, pyrrolizi-
dine and nortropane (Molyneux, Gardner, James, & Colegate, 2002),
which have been detected in a high number of plant families (Legu-
minosae, Moraceae, Hyacinthaceae, Campanulaceae, etc.) and micro-
organism genus (Streptomyces, Bacillus, etc.) (Watson, Fleet, Asano,
Molyneux, & Nash, 2001). Different pharmacological and ecological
activities have been attributed to these compounds, the inhibitory
effect against glycosidase being the most studied, which makes
them potential drug candidates for the treatment of several dis-
eases (i.e. diabetes, cancer, AIDS, and viral infections) (Yokoyama,
Ejiri, Miyazawa, Yamaguchi, & Hirai, 2007).
procedures based on the use of polar solvents such as ethanol,
methanol, hot water (Molyneux et al., 2002) or acidulated water
(Kim et al., 2003) have been carried out. However, all these proce-
dures are not selective for the target compounds and the extraction
of interferences such as other low molecular weight carbohydrates
takes also place, a purification step being necessary. Therefore, the
use of analytical methods which allow the determination of imino-
sugars and interferences to control their extraction is of crucial
importance.
HPLC has been used for the analysis of polyhydroxylated alka-
loids; however, the poor resolution achieved by this technique
and the extreme hydrophilicity and low solubility of these com-
pounds in non-hydroxylic organic solvents make difficult the
selection of the most suitable solvent system-column packing
combination (Molyneux et al., 2002) and require the search of dif-
ferent alternatives. Moreover, the lack of chromophores in these
chemical structures limits their detection. The use of HILIC systems
coupled to evaporative light scattering detectors (Kimura et al.,
2004) or amino columns coupled to MS detectors (Chen, George,
Weir, & Leapheart, 1990; Nash, et al., 1988) have, in some measure,
reduced these problems. Furthermore, different methods using di-
rect ESI MS analysis have been proposed for rapid detection of
polyhydroxyalkaloids (Egan, et al., 1999).
Deoxynojirimycin (1,5-dideoxy-1,5-imino-
polyhydroxyalkaloid typical of the six-membered ring piperidine
group. It has been shown to be a potent -glucosidase inhibitor,
although DNJ does not inhibit -amylases (Hughes & Rudge,
D-glucitol; DNJ) is a
a
a
1994). Its presence has been described in leaves and roots of Morus
sp., Hyacinthus orientalis bulbs and larvae of Bombyx mori (Watson
et al., 2001) in which its content has been used as antihyperglyce-
mic quality criterion (Kim et al., 2003).
Many reports have been published about the chemical synthesis
of iminosugars (Ferreira, Botuha, Chemla, & Perez-Luna, 2009;
However, GC–MS combines the good resolution of GC and the
structural information obtained by MS. Both trimethylsilyl (TMS),
⇑
Corresponding author. Tel.: +34 915622900x212; fax: +34 915644853.
E-mail address: mlsanz@iqog.csic.es (M.L. Sanz).
0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.10.097

354
S. Rodríguez-Sánchez et al. / Food Chemistry 126 (2011) 353–359
(Molyneux, 1993) and acetylated (Magalhães, Santos, Magalhães, &
Nogueira, 2002) derivatives have been used to transform imino-
sugars into volatile compounds. Nevertheless, the appearance of
multiple peaks for each reducing carbohydrate is one the main dis-
advantages (Molyneux, 1993), the development of new procedures
to analyse iminosugars free from interferences being necessary.
Therefore, the aim of this work was to develop a derivatization
procedure for the simultaneous analysis of iminosugars (mainly
DNJ and fagomine) and other carbohydrates of low molecular
weight, and validate its application to extracts of mulberry leaves,
fruits and branches, which are known to be a natural source of
these compounds.
ane and 200 lL of Milli-Q water. One microliter of the hexane
upper layer was injected into the GC.
2.4.1.2. Acetylation. Acetylation was carried out following the pro-
cedure described by Magalhães et al. (2002). Samples (10 mg) were
treated with acetic anhydride (0.7 mL) in pyridine (0.5 mL) for 24 h
at room temperature. After reaction, samples were centrifuged at
8000 rpm for 10 min, and 1
the GC injection port.
lL of supernatants was injected into
2.4.1.3. Oximation + Acetylation. Oximes were obtained by addition
of 350 L of a solution of 2.5% hydroxylamine chloride in pyridine
l
after 30 min at 75 °C according to Brobst and Lott (1966). Then,
they were acetylated with acetic anhydride (0.45 mL) for 5 h. After
reaction, samples were centrifuged at 8000 rpm for 10 min, and
2. Materials and methods
1 lL of supernatants was injected into the GC injection port.
2.1. Standards
Analytical standards of fructose, glucose, galactose, ribose, su-
crose, myo-inositol, galactinol (O-a-D-galactopyranosyl-(1-1)-L-
myo-inositol), pipecolic acid (2-piperidinecarboxylic acid), nipecot-
ic acid (3-piperidinecarboxylic acid), isonipecotic acid (4-piperi-
2.4.1.4. Oximation + Silylation. Taking into account the problems
found for iminosugar derivatization, different preparation proce-
dures were followed for the formation of TMSO. Extracts were
dried by two different ways:
dinecarboxylic acid),
a
-methyl-L-proline (2-methylpyrrolidine-2-
carboxylic acid) and phenyl-b-
D
-glucoside were obtained from Sig-
(i) under vacuum, with and without acetone addition to help
water removal and
(ii) freeze-drying: freeze dried samples were also treated by two
different procedures: exposing them to atmospheric mois-
ture prior to derivatization according to Kite and Hughes
(1997) and in close vials without moisture exposition.
ma Chemical Co. (St. Louis, US). DNJ was purchased from Toronto
Research Chemicals Inc. (Ontario, Canada). Glyceryl-b-galactoside
was obtained by transglycosidation reaction of lactose treated with
b-galactosidase (Cardelle-Cobas, Martínez-Villaluenga, Sanz,
Montilla, 2009). This product was kindly gifted by Dr. Montilla
(CSIC, Spain).
&
Oximes were formed as indicated above for acetylated oximes
using a solution of 2.5% hydroxylamine chloride in pyridine,
whereas TMS derivatives were obtained by four different
procedures:
2.2. Samples
Samples of Morus alba leaves, M1L and M2L were obtained from
Tarancón (Cuenca, Spain) and from Madrid (Spain), respectively.
Leaves (M3L), fruits (M3F) and branches (M3B) of Morus alba were
collected from Hoyo de Pinares (Avila, Spain). Samples of leaves
(M4L), fruits (M4F) and branches (M4B) from Morus nigra were ob-
tained from Madrid (Spain).
(i) 300
(MSTFA) and 3
for 1 h (Molyneux et al., 2002);
lL
of
N-methyl-N-trimethylsilyltrifluoroacetamide
lL of trimethylchlorosilane (TMCS) at 60 °C
(ii) 90
10
l
l
L of bis(trimethylsilyl)trifluoroacetamide (BSTFA) and
L of trimethylchlorosilane (TMCS) at room temperature
for 1 h (Knapp, 1979);
2.3. Carbohydrate extraction
(iii) 100 L of trimethylsilylimidazole (TMSI) and 100 lL of tri-
l
methylchlorosilane (TMCS) at room temperature for
30 min (Troyano et al.,1991);
(iv) 350 lL of hexamethyldisylazane (HMDS) and 35 lL of triflu-
The extraction of carbohydrates from mulberry samples was
carried out using acidulated water, previously suggested by other
authors (Kim et al., 2003), as it has been described that the pres-
ence of acid may suppress the decomposition of some pyrrolidines
(Molyneux et al., 2002). One gram of samples was ground before
extraction in 10 mL of acidulated water with 0.1% HCl (v/v) for
24 h. Samples were filtrated through Whatman No. 4 paper and
kept at À20 °C until analyses.
oroacetic acid (TFA) at 45 °C for 30 min (Brobst & Lott, 1966).
After reaction, samples were centrifuged at 8000 rpm for
10 min and kept at 4 °C for 24 h before analysis. 1 lL of superna-
tants was injected into the GC injection port.
2.4.2. GC–MS analysis
2.4. Carbohydrate analysis
GC–MS analyses of derivatised samples were carried out in a
Hewlett–Packard 7890A gas chromatograph coupled to a 5975C
quadrupole mass detector (both from Agilent, Palo Alto, CA, USA),
2.4.1. Derivatization procedures
Except for those derivatization procedures specifically indi-
cated, 1 mL of extracts were mixed with 0.5 mL of a 70% ethanolic
using He at ꢀ1 mL minÀ1 as carrier gas.
A
30 m  0.25 mm
i.d. Â 0.25
lm film thickness TRB-1 (crosslinked methyl silicone)
solution of phenyl-b-L
-glucoside (1 mg mLÀ1) employed as internal
from Teknokroma (Barcelona, Spain) was used. Oven temperature
program was optimised, the best conditions being: from 100 °C
to 200 °C at 15 °C minÀ1, kept at this temperature for 15 min and
finally programmed to 300 °C at 15 °C minÀ1 and kept at this tem-
perature for 10 min. Injector temperature was 300 °C and injec-
tions were made in the split mode with a split ratio 1:20. Mass
spectrometer was operating in electronic impact (EI) mode at
70 eV, scanning the 35–650 m/z range. Interface and source tem-
perature were 280 °C and 230 °C, respectively. Acquisition was
standard and dried under vacuum at 38–40 °C.
2.4.1.1. Silylation. Trimethylsilyl (TMS) ethers were prepared
according to Troyano, Olano, Fernández-Díaz, Sanz, and Martí-
nez-Castro (1991). Hundred microliters of anhydrous pyridine,
100
chlorosilane (TMCS) were added to the evaporated samples.
Extraction of the TMS ethers was carried out using 100 L of hex-
lL of trimethylsilylimidazole (TMSI) and 100 lL of trimethyl-
l

S. Rodríguez-Sánchez et al. / Food Chemistry 126 (2011) 353–359
355
done using a HP ChemStation software (Hewlett–Packard, Palo
Alto, CA, USA).
afford a peak for each tautomer), leading to overlapped peaks.
Although TMS derivatization has been the most common proce-
dure used to analyse iminosugars (Molyneux, 1993), and DNJ could
easily be quantified using this procedure in mulberry extract, fago-
mine coeluted with one of the five tautomeric peaks of ribose. In
the acetylated sample, DNJ had the same retention time as glucose,
making also its quantification difficult.
Linear retention indices (IT) were calculated from the retention
times of TMSO derivatives and suitable n-alkanes.
Quantitative values were calculated using the internal standard
method. Response factors were determined by injecting standards
of different concentration containing internal standard (phenyl-b-
D
-glucoside) by triplicate. LOD was calculated as three times the S/
Therefore, a different derivatization procedure for the simulta-
neous analysis of sugars, iminosugars and inositols was assayed.
Fig. 1C shows the GC–MS profile of carbohydrates in mulberry
M1L, previously submitted to the oximation + acetylation derivati-
zation as indicated in materials and methods. By using this deriv-
atization method reducing aldoses are converted into per-
acetylated aldononitriles (PAAN), giving rise to a single peak.
Non-reducing sugars, inositols and iminosugars gave rise to a sin-
gle derivative corresponding to the per-acetylated compound.
Reducing ketoses should form the per-acetylated oximes (with
syn (E) and anti (Z) isomers) giving rise to two different peaks.
However, derivatization of these carbohydrates was not complete,
and up to five different peaks could appear for each ketose.
The effect of acetylation time of glucose, fructose, myo-inositol
and DNJ, previously submitted to the oximation step, was evalu-
ated by sampling the reaction mixture every hour by triplicate.
Fig. 2 shows the behaviour of these standards during acetylation.
Although previous studies suggested 24 h at 20 °C for carrying
the acetylation of iminosugars out (Magalhães et al., 2002), incuba-
tion for 5 h was enough for the complete derivatization of glucose,
myo-inositol and DNJ. Nevertheless, acetylation of fructose was
variable during the 24 h period studied. Therefore, this derivatiza-
tion procedure was discarded for mulberry samples, where fruc-
tose is one of the main sugars.
N (signal-to-noise) ratio while LOQ was considered ten times this
ratio according to Foley and Dorsey (1984). Accuracy was esti-
mated by carrying out recovery assays by the addition of known
amounts of glucose, DNJ and myo-inositol standards to a Morus
alba extract; the three carbohydrates were measured in both
unspiked and spiked samples and recoveries were calculated. Pre-
cision was calculated from the results obtained of a standard mix-
ture and a Morus alba extract previously derivatised and analysed
in five different days.
3. Results and discussion
3.1. Derivatization assays
Figs. 1A and B show the GC–MS profiles of acetyl and TMS deriv-
atives of mulberry M1L extract. Derivatised sugars (ribose, glucose,
fructose and sucrose), myo-inositol, galactinol and DNJ were iden-
tified by comparison of their retention times and mass spectra with
the corresponding derivatised standards. Fagomine was identified
by its characteristic m/z fragment ions according to Malgalhães
et al., (2002) for acetylated derivatives (m/z: 43 (100), 98 (29),
140 (92), 182 (57), 196 (11), 242 (3)) and according to the fragmen-
tation suggested by Molyneux, et al., (2002) for TMS derivatives of
alkaloids (Table 1). However, up to five peaks can appear per
reducing sugar (both methods keep the sugar ring intact and can
Fig. 1D shows the GC–MS profile of carbohydrates in mulberry
M1 extract previously submitted to the oximation + trimethylsily-
3
Abundance
4
Abundance
2+3
4+5
3600000
3200000
2800000
2400000
2000000
1600000
1200000
800000
4
8
18000000
14000000
10000000
6000000
2000000
B
A
6
6
8
9
5
1
1+2
1
7
7
9
400000
min
6.00 10.00 14.00 18.00 22.00 26.00 30.00
min
6.00 10.00 14.00 18.00 22.00 26.00 30.00
Abundance
Abundance
5
3
7
4
6
6
4800000
4200000
3600000
3000000
2400000
1800000
1200000
600000
D
8
4
3
C
1400000
1200000
1000000
800000
600000
400000
200000
3
3
8
II
1
4+5
3
I
III
5
9
9
7
1
2
2
min
min
6.00
12.00
18.00
24.00
30.00
6.00 10.00 14.00 18.00 22.00 26.00 30.00
Fig. 1. GC profiles of mulberry leaves extract converted into their (A) acetylated, (B) trimethylsilyl, (C) acetylated oxime and (D) trimethylsilyl oxime derivatives. (1) Ribose,
(2) fagomine, (3) fructose, (4) glucose, (5) galactose, (6) myo-inositol, (7) DNJ, (8) phenyl-b-glucopyranoside (i.s.), (9) galactinol.

356
S. Rodríguez-Sánchez et al. / Food Chemistry 126 (2011) 353–359
Table 1
Linear retention indices (IT) and MS data of TMS derivatives of polyhydroxy alkaloids found in mulberry.
Polyhydroxyalkaloid
Pipecolic acid
Structure
IT
Mass spectrum
1250a
1371b
56 (7)*, 75 (7), 84 (100), 103 (4), 186 (6)a
73 (24), 156 (100), 147 (4), 230 (4) 258 (0.5)b
N
H
COOH
Methylpyrrolidine carboxylic acid
N.C.**
1458a
1506b
1532a
75 (28), 84 (100), 103 (3), 186 (7), 201 (0.3)a
73 (56), 147 (17), 156 (100), 230 (6), 258 (6)b
73 (27), 82 (100), 156 (5), 172 (80), 289 (0.1)a
cis-5-Hydroxypipecolic acid
HO
N
H
COOH
COOH
Hydroxypipecolic acid with a methyl group
trans-5-Hydroxypipecolic acid
N.C.
1565a
1578a
73 (100), 142 (87), 147 (29), 186 (66), 288 (9), 303 (1)a
73 (88), 82 (100), 156 (4), 172 (82), 289 (0.3)a
HO
N
H
Fagomine
1698a
73 (91), 129 (27), 144 (100), 170 (29), 260 (67), 348 (2)a
OH
OH
N
H
CH2OH
OH
DNJ
1940a
1854b
73 (100), 144 (47), 217 (82), 258 (34), 348 (52), 436 (1)a
OH
73 (100), 147 (26), 216 (32), 258 (6), 348 (8), 420 (87) 508 (0.7)b
HO
N
H
CH2OH
*
m/z Values (Relative abundances in brackets).
**
N.C. not confirmed.
Data corresponding to the compound with only hydroxyl groups derivatised.
a
b
Data corresponding to the compound with both hydroxyl and imino groups derivatised.
lation procedure. Both reducing aldoses and ketoses showed two
peaks from the syn (E) and anti (Z) isomers whereas non-reducing
sugars and inositols gave rise to one peak. However, partial deriv-
atization of iminosugars was the major drawback of this reaction:
while the hydroxyl groups were readily silylated, the reaction for
the amino group depended on the compound and on the derivati-
zation conditions, resulting in one or two peaks for each
iminosugar.
Therefore, different conditions were assayed trying to achieve a
complete derivatization of iminosugars. Residual moisture and
derivatization reagents appear to be important in controlling the
extent of trimethylsilylation of these alkaloids.
Concerning residual moisture, previous studies have shown that
a complete silylation of the hydroxyl groups of hydroxypipecolic
acids occurs without silylation of the amino group when samples
are first freeze-dried and then exposed to atmospheric moisture be-
fore derivatization (Kite & Hughes, 1997). When this behaviour was
assayed in a triplicate analysis of pipecolic acid and DNJ, with and
withoutexposingsamplestoatmosphericmoistureafterfreeze-dry-
ing, no differences were shown between both treatments. Moreover,
pipecolic acid and DNJ solutions were also evaporated under vac-
uum before derivatization treatment to compare the effect and no
notable differences were observed. Similar results were also ob-
served in those samples treated with and without acetone before
the complete evaporation under vacuum (data not shown).
Regarding derivatization, four different approaches as indicated
in materials and methods (Section 2.4.1.4) (MSTFA, BSTFA, TMSI/
TMCS and HMDS) were followed after treatment with 2.5% hydrox-
ylamine chloride in pyridine for pipecolic acid (Fig. 3A) and DNJ
(Fig. 3B):
(i) MSTFA gave rise to the formation of two silyl derivatives for
both pipecolic acid and DNJ differing in the presence of
derivatization in the amino group. On the contrary, Moly-
neux et al. (2002) stated the formation of only one single
derivative for polyhydroxy alkaloids (i.e.: swainsonine, cast-
anospermine) when using MSTFA at 60 °C.
(ii) The use of BSTFA afforded a single peak corresponding to the
non-derivatised imino group for DNJ. However, the extent of
derivatization was non-reproducible for pipecolic acid giv-
ing randomly rise to one or two peaks; formation of these
peaks was impossible to control.

S. Rodríguez-Sánchez et al. / Food Chemistry 126 (2011) 353–359
357
(iii) After derivatization with TMSI and TMCS, DNJ with the non-
derivatised imino group was the only peak formed. How-
ever, pipecolic acid was not derivatised under these condi-
tions (data not shown).
A/Api
1.50
(iv) Best results were obtained using HMDS and TFA: for both
DNJ and pipecolic acid all the hydroxyl groups being deriva-
tised, whereas for pipecolic acid only traces of the deriva-
tised imino group were detected. These derivatives are
sufficiently volatile for analysis (Molyneux et al., 2002) and
the formation of a single peak is more appropriate to avoid
coelutions and quantification problems. Therefore, the use
of HMDS and TFA was selected for further analyses.
1.25
1.00
0.75
0.50
0.25
0.00
Derivatised samples were kept at 4 °C at least for 24 h till their
analyses, this period being of time also critical. Their stability was
also controlled.
0
5
23
24
3.2. Qualitative analysis
Time (h)
Fig. 4 shows close-up views of the GC profile of the mulberry ex-
tract M1L previously submitted to the oximation + silylation deriv-
atization procedure. No coelution was observed for DNJ and
Fig. 2. Optimization of acetylation time of glucose (j), fructose (.), myo-inositol
(d) and DNJ (N).
A
MSTFA
BSTFA
HMDS
5.2
4.0
4.4
4.8
Time (min)
B
MSTFA
BSTFA
TMSI/TMCS
HMDS
8.0
9.0
10.0
11.0
12.0
Time (min)
Fig. 3. GC–MS profiles of TMSO of pipecolic acid (A) and DNJ (B) obtained using MSTFA, BSTFA, TMSI/TMCS and HMDS. (1) monosilylated derivatives, (2) disilylated
derivatives.

358
S. Rodríguez-Sánchez et al. / Food Chemistry 126 (2011) 353–359
at IT = 1458 (peak 4) and IT = 1506 (peak 6) were also observed.
Abundance
2000000
I
Their mass spectra were similar to that of monosilylated pipecolic
acid and disilylated pipecolic acid (see Table 1). Their GC retention
times were compared with those of available standard compounds
having a similar structure to that of pipecolic acid (methylproline,
nipecotic and isonipecotic acids) with negative results. Taking into
account the similarity among their mass spectra, these peaks could
correspond to a methylated pyrrolidine carboxylic isomer.
Two peaks with mass spectra corresponding to 5-hydroxypipe-
colic acid (Table 1) with IT of 1532 (peak 7) and 1578 (peak 9) could
be assigned to cis and trans isomers, respectively, according to Kite
and Hughes (1997). This compound had been previously described
in mulberry roots (Suzuki & Kohno, 1987). A compound with a IT of
1565 (peak 8) was also identified as a derivative of pipecolic acid:
although its chemical structure could not be confirmed, its mass
spectrum (Table 1) is compatible with a hydroxypipecolic acid
structure with an additional methyl group.
2
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
7
5
9
3
8
10
1
6
4
4.50
5.00
5.50
15
6.00
6.50
7.00
7.50
min
16
II
Abundance
1600000
18+19
17+18
20
1400000
1200000
1000000
800000
600000
400000
200000
14
17
11
13
Different acids such as glyceric (IT = 1333, peak 3), malic
(IT = 1490, peak 5), citric (IT = 1808, peak 12), quinic (IT = 1877,
peak 13), gluconic (IT = 2115, peak 19) acids and the gluconic lac-
tone (IT = 1899, peak 14), were also detected. Moreover, a peak
with IT = 2784, and a characteristic m/z ion at 337 (peak 22) was
identified as a glyceryl-b-galactoside by comparison with previous
data (Cardelle-Cobas et al., 2009).
12
min
9.00
10.00
11.00
12.00
13.00
14.00
15.00
Abundance
Galactinol (IT = 3907, peak 23) was identified by comparing
with the commercial standard. Other peaks (peaks 24 of Fig. 4C)
which showed a similar mass spectrum were identified as isomers
of galactinol, whereas some disaccharides were also detected in
the samples (peaks 25 of Fig. 4C).
III
21
23
1200000
1000000
800000
600000
400000
200000
25
24
24
22
24
3.3. Validation of the method
min
24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00
The range of linearity of MS response was checked by employ-
ing calibration curves in the range from 0.013 to 1.3 mg mLÀ1 of
standards previously converted to their TMSO. Response factors
relative to phenyl-b-D-glucoside varied from 0.75 of DNJ to 2.1 of
myo-inositol and galactinol.
Fig. 4. Close-up views of GC profile of mulberry leaves (Fig. 1D) previously
converted into their trimethylsilyl oximes under optimised conditions. I, II and III:
regions from Fig. 1D. (1) Pipecolic acid, (2) glycerol, (3) glyceric acid, (4)
monosilylated methyl-pyrrolidinecarboxylic acid, (5) malic acid, (6) disilylated
methyl-pyrrolidinecarboxylic acid, (7) cis 5-hydroxypipecolic acid, (8) hydroxy-
pipecolic acid with
a
methyl group, (9) trans 5-hydroxypipecolic acid, (10)
Detection limits (LOD) and quantification limits (LOQ) of
54 ng gÀ1 of product and 180 ng gÀ1 of product respectively, were
obtained. The average recovery values ranged from 91% to 94%,
whereas precision of the method ranged from 5.9% to 7.0% for all
the target compounds which can be considered adequate for quan-
titative analysis. However, as indicated before, derivatization pro-
cedure and preservation of samples should be very carefully
controlled to obtain this reproducibility values.
fagomine, (11) ribose, (12) citric acid, (13) quinic acid, (14) gluconic acid (lactone),
(15) DNJ, (16) fructose, (17) galactose, (18) glucose, (19) gluconic acid, (20) myo-
inositol, (21) phenyl-b-glucopyranoside (i.s.), (22) glyceryl-galactoside, (23) galact-
inol, (24) isomers of galactinol, (25) disaccharides.
fagomine; other alkaloids were also detected. Pipecolic acid
(IT = 1250; peak 1) was observed at trace levels. Two peaks eluting
Table 2
Content (mg gÀ1 of product) of iminosugars, sugars and inositols in mulberry samples.
M1L
tr*
M2L
tr
M3L
tr
M3F
M3B
tr
0.14 (0.06)
tr
0.08 (0.00)
M4L
tr
M4F
tr
M4B
tr
Pipecolic acid
Methylpyrrolidine carboxylic acid
cis-5-Hydroxypipecolic acid
Hydroxypipecolic acid with a methyl group
trans-5-Hydroxypipecolic acid
Fagomine
DNJ
Ribose
Fructose
Galactose
Glucose
Myo-inositol
Galactinols
Disaccharides
tr
tr
tr
0.68 (0.05)**
0.34 (0.02)
0.55 (0.06)
0.05 (0.00)
0.36 (0.03) tr
5.98 (0.27)
1.39 (0.09)
1.53 (0.35)
0.12 (0.01)
2.68 (0.52)
1.76 (0.20)
72.88 (0.32)
2.67 (0.16)
16.50 (1.07)
11.17 (0.16)
2.04 (0.33)
1.57 (0.14)
0.52 (0.10)
0.85 (0.00)
0.31 (0.11)
0.66 (0.09)
1.23 (0.03)
1.08 (0.04)
0.11 (0.02)
0.70 (0.01)
0.25 (0.03)
0.05 (0.00)
0.04 (0.00)
0.34 (0.01)
0.02 (0.00)
0.16 (0.03)
0.30 (0.04)
0.05 (0.00)
0.22 (0.03)
0.32 (0.10)
0.31 (0.04) tr
0.58 (0.02) tr
0.54 (0.06) tr
0.22 (0.10)
2.08 (0.05)
0.41 (0.02)
0.13 (0.03)
0.11 (0.02)
0.10 (0.04)
0.11 (0.01)
0.39 (0.01)
tr
tr
0.05 (0.01)
4.75 (1.48)
0.28 (0.10)
8.91 (0.54) 38.08 (1.69) 31.71 (1.30)
0.27 (0.08)
0.13 (0.06)
6.24 (0.38)
0.86 (0.03)
1.98 (0.16)
0.75 (0.09)
0.01 (0.00)
0.06 (0.03)
57.40 (0.18) 12.84 (0.19) 14.39 (0.88)
2.07 (0.06) 0.93 (0.00) 0.35 (0.08)
20.72 (0.40) 12.16 (0.14) 25.92 (0.81) 10.44 (3.53) 40.51 (4.51) 29.40 (4.11)
6.59 (0.14)
0.41 (0.08)
0.27 (0.07)
1.72 (0.66) 5.66 (0.85) 1.34 (0.43)
3.09 (0.01)
0.28 (0.01)
0.36 (0.02)
4.37 (0.77)
0.03 (0.00)
0.12 (0.00)
6.91 (2.61)
0.06 (0.00)
0.15 (0.04)
7.12 (0.37) 12.34 (2.94)
3.37 (0.37)
2.74 (0.13)
0.27 (0.01)
0.34 (0.12)
*
tr: Traces.
**
Standard deviation in brackets.

S. Rodríguez-Sánchez et al. / Food Chemistry 126 (2011) 353–359
359
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leaves of mulberries M1L and M2L and leaves, fruit and branches
of mulberries M3 and M4. The highest values of DNJ were detected
in samples M3B, M1L and M4F (4.75, 2.68, and 2.08 mg gÀ1 of
product, respectively). However, among all samples under study,
mulberry M2L showed the highest concentration of fagomine
(0.66 mg gÀ1 of product). In general, carboxylic acid derivatives
were higher for leave samples, showing the highest values for
M1L; only minor amounts of these compounds were observed in
bark samples (M3B and M4B).
a
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According to sugars, both M1L and M2L leaves samples showed
the highest fructose and ribose concentrations; however, glucose
and galactose was the most abundant in M4L. Galactinol and its
isomers also showed the highest values in mulberry M4L.
4. Conclusions
Iminosugars are frequently present in natural products together
with other low molecular weight carbohydrates. GC–MS is a power-
ful tool for their analysis, but it requires a previous derivatization
step, whichcan lead to problems in both qualitative and quantitative
determination. The proposed procedure for derivatization to TMSO
appears to be the most appropriate method to simultaneously quan-
tify sugars, inositols and iminosugars in mulberry extracts, although
for the last compounds, control of derivatization conditions is of cru-
cial importance. Derivatizationof complex samples containingother
different iminosugars will possibly require a similar study of the ef-
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4, 6-triol]. Tetrahedrom, 44(18), 5959–5964.
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Acknowledgements
This work was financed by projects PIF-SIALOBIOTIC
200870F0101 (CSIC), AGL2009-11909 (Ministerio de Ciencia e
Innovación) and PRONAOS (CDTI, CENIT-2008 1004). O. Hernán-
dez-Hernández thanks CSIC for a JAE Predoc grant.
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