Electronic Supplementary Material
Optimisation of the Piperidine Alkaloid Extraction Method. The solid-phase partitioning method (SPP)
was optimized for the extraction of Picea abies alkaloids prior to the analyses. The SPP procedure was
adapted from that used for Pinus ponderosa (Gerson and Kelsey, 1999; Wink et al. 1995). Plant
material was collected from adult P. abies individuals from the Joensuu area (N62°, E29°), eastern
Finland. Needles were used as material for refining the method, but the suitability of other plant parts
(bark and wood) was also tested. Current and previous years’ needles were combined, and fresh or
frozen (– 23ºC) material was used. The needles were homogenised in liquid N2. Samples of 1 g were
incubated in 15 ml of 0.5 M aq. HCl for 60 min in an orbital shaker (100 rpm, RT). The filtrate was
collected and then set to basic (pH ≥11) with 6 M NaOH before loading into an Extrelut® NT 20 PE
column (refillable, 20 ml capacity, Merck, Germany). The column was then eluted with 2x 18 ml
CH2Cl2 (SupraSolv®, Merck). The eluate was collected, evaporated to ≤ 2ml with a rotary evaporator
and adjusted to 2 ml in a volumetric flask.
Several adjustments were tested for the main procedure. 1) The initial extraction solution was
changed to 15 ml of MeOH (HPLC grade) (as in Gerson and Kelsey 1999) and incubated for 60 min in
the orbital shaker (100 rpm, RT). MeOH was evaporated with a rotary evaporator and 15 ml of 0.5 M
aq. HCl were added. 2) The 60 min incubation was replaced by a 90 min incubation, or 3) the
incubation procedure was divided into two parts (incubation for 60 min with 10 ml 0.5 M aq. HCl,
collecting the filtrate, and the needle residue was incubated again for 30 min in 5 ml of 0.5 M aq. HCl.
After the incubations, the filtrates were combined). 4) CHCl3 (AnalaR Normapur®, VWR, USA), and
CH2Cl2 (SupraSolv, Merck) were tested as elution solvents (as in Gerson and Kelsey 1999), and 5)
additional rinsing with 18 ml CH2Cl2 of columns (as in Gerson and Kelsey 1999) or 6) 4x elution (24
ml, 10 ml, 10 ml, and 10 ml) was also tested. To determine the residue left in the needle mass after HCl
treatment, a separate sample was collected with an additional incubation for 60 min in 15 ml of 0.5 M
aq. HCl. Similarly, to determine the residue left in the columns after elution with CH 2Cl2, a separate
sample was collected with additional rinsing of the Extrelut column with 18 ml of CH2Cl2.
Statistical comparisons of the optimization of the extraction method for piperidine alkaloid
analyses were carried out by means of independent samples t-tests (SPSS, v. 19.0). The data were
square root-transformed when necessary.
GERSON, E. A. and KELSEY, R. G. 1999. Foliar storage and extraction methods for quantitative
analysis of piperidine alkaloids from Ponderosa pine (Pinus ponderosa). Phytochem. Anal. 10:322-327.
WINK, M., MEISSNER, C., and WITTE, L. 1995. Patterns of quinolizidine alkaloids in 56 species of
the genus Lupinus. Phytochemistry 38:139-153.
% of total epidihydropinidine detected
100
SPP
Modified SPP
90
80
70
30
20
10
0
Recovery
Elution residue
Incubation residue
Supplementary Fig. 1 Mean recovery percentage (+s.e.) of epidihydropinidine detected and residues
lost during the elution process and initial incubation from P. abies needles calculated from the total
epidihydropinidine extracted by the original (filled bars, N=4) and optimised (open bars, N=12) solidphase partitioning methods (SPP)
Supplementary Table 1 Soluble non-tannin phenolic compounds identified based on ms-ions
Compound
M
Picein
4-hydroxyacetophenone
(+)-Catechin
Cedrusin-4-O-Glc der. 1
Cedrusin-4-O-Glc der. 2
Cedrusin-Me-4-O-Glc
Cedrusin-4-O-Rha der.
Seco-isolariciresinol-9-O--Glc der.
Seco-isolariciresinol-9-O--Glc hydroxyl der.
298
136
290
508
508
522
492
524
540
Monomethylated Seco-isolariciresinol-9-O--Glc der.
Quercitrin
Astragalin
Dicoumaroylastragalin (tentatively 3’’, 6’’)
Piceatannol
Piceatannol glucoside (tentatively astringin)
Isorhapontin
a
M+Na+, bM+H+
510
448
448
740
244
406
420
Fragment ions, m/z (Relative
abundance, %)
137 (100); 321a (52)
137.1b (100)
291.1b
317.2 (100); 531.2a (80)
531.2a (100); 317.2 (70)
545.2a (100); 331.1 (55)
515.2a (80); 219.1 (40)
547.3a (100); 327.1 (50)
563.2a (60); 549.3 (50); 355 (10);
371 (5)
533.2a (100); 331.2 (20)
471.3a (7)
471.1a (70); 449.0b (18); 287.0 (10)
763a
245b
407b
443.2a (100); 259 (80); 158 (60)
Supplementary Table 2 Independent samples t-tests results from A. initial extraction phase and B.
elution from the Extrelut columns in optimisation of solid-phase partitioning method for
epidihydropinidine
A. Initial extraction
Extraction solvent (peak area)
Prolonged initial extraction (%)
Divided initial extraction (%)
0.5 M HCl
MeOH
60 min
90 min
60 min
60 + 30 min
Mean
14893320
21417668
3.39
3.28
3.39
0.51
s.e.
98800
2357194
0.03
0.37
0.03
0.04
t
-2.14
df
3
P
0.12
0.07
11
0.95
4.22
19
<0.001
6
0.75
11
0.02
19
0.70
B. Elution from the Extrelut columns
Elution solvent (peak area)
CH2Cl2
1469356 94993
0.33
CHCl3
1436059 29141
First additional elution (%)
2x elution
17.64
0.05
2.69
3x elution
4.61
0.24
Second additional elution (%)
3x elution
4.61
0.24
-0.40
4x elution
5.52
0.06
% refers to proportional size of the residue lost in initial extraction/elution process.
Supplementary Table 3 GC-MS-ions of identified piperidine alkaloid compounds
Compound
Rt
M
2-Methyl-6-propyl-1,6-piperideine
3.48
139
epidihydropinidine (preferable (+)-6- epidihydropinidine)
pinidine isomer (tentatively 2,6-trans-pinidine)
3.52
3.59
141
139
cis-pinidinol
trans-pinidinol
4.67
4.95
157
157
Fragment ions, m/z (Relative
intensity, %)
139 (18); 124 (43); 111 (58); 96
(100); 84 (10); 83 (12); 70 (32); 55
(10); 42 (18); 41 (29)
126 (8); 98 (100); 81 (4)
139 (40); 124 (100); 111 (25); 96
(79); 94 (11); 82 (46); 70 (24); 55
(24); 42 (21); 41 (25)
142 (11); 98 (100); 82 (10); 70 (17)
98 (100)