Supplementary Materials:A New Chemical Pathway
Yielding A-Type Vitisins in Red Wines
Paula Araújo, Ana Fernandes, Victor de Freitas and Joana Oliveira
S1. Identification of the new compound formed during the reaction of mv-3-glc with OAA
560.93
55000000
50000000
45000000
40000000
Intensity
35000000
30000000
25000000
20000000
A
15000000
1054.53
10000000
5000000
399.13
333.00
0
300
495.07
400
1614.20
1547.93
1681.73
1120.20
672.93 724.07
450.87
500
600
700
827.60
800
909.33
900
1006.60
1000
1200.87
1100
m/z
1200
1335.53 1409.73
1300
1400
1500
1600
1779.00
1700
1862.27 1913.73
1800
1900
2000
399.07
36000000
34000000
32000000
30000000
28000000
26000000
Intensity
24000000
22000000
20000000
18000000
16000000
14000000
12000000
10000000
8000000
B
6000000
4000000
2000000
0
160
180
200
220
240
260
280
300
320
340
360
380
400
420
440
460
480
500
520
540
560
m/z
Figure S1. MS-ESI analysis performed in the positive-ion mode of the new compound formed during
the reaction of mv-3-glc with OAA: (A) full mass spectrum; (B) MS2 spectrum of the fragment
obtained in full mass spectrum (m/z 561).
S2. Decomposition of oxaloacetic acid into pyruvic acid in model solution at pH 3.5
3000
OAA
Absorbance at 214 nm (mAU)
2500
2000
1500
PA
1000
t=0
t = 1.5 h
t = 2.5 h
500
0
5
10
15
20
25
30
Retention time (min)
Figure S2. HPLC chromatograms recorded at 214 nm of a model solution (pH 3.5) containing OAA
(7.6 mM) at pH 3.5 at time 0, 1.5, and 2.5 h at room temperature.
S3. Detection and quantification of OAA and PA in grape musts
3.1. Derivatization of Keto-Acids (OAA and PA)
The derivatization (oxime-derivatives) of the keto-acids OAA and PA was performed according
to the procedure described in the literature by Noguchi and co-workers, 2014, with some
changes [15].
Twenty microliters of the sample were mixed with 20 µL of PFBHA (O-(2,3,4,5,6pentafluorobenzyl)hydroxylamine hydrochloride) 10 mg/mL in a microtube and allowed to react at
0 °C during 30 min (conversion of the keto-acids to the corresponding O-PFBO derivatives (O(2,3,4,5,6-pentafluorobenzyl)oxime)). The derivatization reaction was quenched by the addition of 10
µL of a solution of acetone and acetonitrile (1:1, v/v), and then the resulting mixture was diluted with
25 µL of 0.1% NaOH and acetonitrile (1:1, v/v). This final mixture was then stored at 4 °C until LCESI-MS analysis. Standards of OAA and PA were prepared similarly as described for grape must
samples. The samples and standards were analysed in triplicates.
3.2. LC/ESI-MS Analysis
Keto-acids O-(2,3,4,5,6-pentafluorobenzyl)oxime derivatives were analyzed by LC/ESI-MS on a
Finnigan Surveyor series liquid chromatograph and their detection was done by mass spectrometry.
The auto-sampler temperature and injection volume were set at 4 °C and 25 µL, respectively. Liquid
chromatography was performed at 40 °C using a BDS Hypersil C18 column of (150 × 4.6 mm, 3 µm)
and a gradient elution system with the mobile phase consisting of solvent A (50 µM formic acid in
water) and solvent B (acetonitrile). The flow rate was maintained at 0.5 mL/min through the analysis.
The gradient elution conditions consisted of 10–100% B for 30 min. The column was washed with
100% B for 6 min and then stabilised at the initial conditions for 5 min. The mass detector was a
Finnigan LCQ DECA XP MAX (Finnigan Corp., San Jose, CA, USA) quadrupole ion trap equipped
with atmospheric pressure ionization (API) source, using electrospray ionization (ESI) interface. The
MS was operated in a Select Ion Monitoring (SIM) mode with negative detection. The vaporizer and
the capillary voltages were 5 kV and 4 V, respectively. The capillary temperature was set at 325 °C.
Nitrogen was used as both sheath and auxiliary gas at flow rates of 80 and 30, respectively (in
arbitrary units).