Supplementary Information for Young Jin Lee et al.,
“High-throughput Analysis of Algal Crude Oils using
High-resolution Mass Spectrometry”
4.20x10
C16 TAG + K
845.7002
7
ESI
C16 TAG + Na
829.7297
0.00
1.40x10
C16 DAG Fragment
551.5025
9
APCI
Signal intensity
E
379.3328
S
411.3976
0.00
3.0x10
9
E
379.3328
E
396.3367
S
410.3878
C16 DAG Fragment
551.5025
APPI
0.0
400
3.0x10
600
800
5
E
396.3383
C16 TAG + Na
829.7252
MALDI
0.0
400
600
800
m/z
Supplementary Figure 1. Orbitrap MS spectra of simulated algal solution ionized with ESI,
APCI, APPI, and MALDI. Most ions are detected as protonated (APCI) or free radicals (APPI),
except potassium or sodium ion adducts for TAG in ESI and MALDI. Ergosterol (E) is detected
as a water-loss or free radical, [M-H2O+H]+ or [M]. Squalene (S) is detected as protonated or
free radical, [M+H]+ or [M].
9.4E+08
1.2E+09
Cholesterol
0.0E+00
0.0000
R² = 0.975
0.0050
0.0100
0.0150
0.0200
DAGs
R² = 0.9854
0.0E+00
0.0250
0
0.005
% of solix Algae Oil
4.9E+07
2.0E+09
R² = 0.9558
0.005
0.010
0.015
0.020
0
0.005
1.0E+09
R² = 0.982
0.01
0.015
% of solix Algae Oil
0.01
0.015
0.02
0.025
% of solix Algae Oil
0.0E+00
0.005
0.025
R² = 0.986
0.0E+00
0.025
Squalene
0
0.02
TAGs
% of solix Algae Oil
1.8E+07
0.015
% of solix Algae Oil
Ergosterol
0.0E+00
0.000
0.01
0.02
0.025
Chlorophyll A
R² = 0.9968
0.0E+00
0
0.005
0.01
0.015
0.02
% of solix Algae Oil
Supplementary Figure 2. Ion signals for each lipid class at various algal oil concentrations.
0.025
Supplementary Table 1. MS/MS and MS3 of some TAG.
Precursor
Assignment
MS/MS and MS3 peak assignment
Mass
775.68
C16:1/C16:1/C14:0
757.6 (-H2O), 547.5 (-R(C14:0)COOH), 521.5 (-R(C16:1)COOH)
777.70
C16:0/C16:1/C14:0
759.8 (-H2O), 549.6 (-R(C14:0)COOH), 523.6 (-R(C16:1)COOH)
799.68
C14:0/C16:0/C18:4
781.5 (-H2O), 571.6 (-R(C14:0)COOH), 543.4 (-R(C16:0)COOH)
801.70
C14:0/C16:1/C18:2
783.8 (-H2O), 573.6 (-R(C14:0)COOH), 547.6 (-R(C16:1)COOH)
803.71
C14:0/C18:2/C16:0
575.5 (-R(C14:0)COOH), 547.4 (-R(C16:0)COOH)
805.73
C16:0/C16:0/C16:1
787.8 (-H2O), 551.5 (-R(C16:1)COOH), 549.6 (-R(C16:0)COOH)
823.68
C20:5/C16:1/C14:0
805.6 (-H2O), 595.4 (-R(C14:0)COOH: 267.3(RCO-H2O; C20:5), 285.3(RCO+; C20:5), 311.3(RCOO+58; C16:1), 569.4 (-R(C16:1)COOH),
827.71
C18:3/C16:0/C16:1
809.6 (-H2O), 571.4 (-R(C16:0)COOH), 549.3 (-R(C18:3)COOH)
829.73
C18:3/C16:0/C16:0
811.8 (-H2O), 573.6 (-R(C16:0)COOH), 551.4 (-R(C18:3)COOH)
831.75
C16:0/C16:1/C18:1
813.6 (-H2O), 575.5 (-R(C16:0)COOH), 549.5 [-R(C18:1)COOH: 219.2(RCO-H2O; C16:1), 237.2 (RCO; C16:1),
551.4 (-R(C16:1)COOH+H2O), 521.4 [-R(C20:5)COOH: 211.1(RCO; C14:0), 219.2(RCO-H2O; C16:1), 237.3(RCO; C16:1)]
293.2(RCOO+58-H2O; C16:1), 313.3 (RCOO+58; C16:0)]
849.70
C20:5/C16:1/C16:1
831.7 (-H2O), 595.5 [-R(C16:1)COOH: 267.2(RCO-H2O; C20:5), 285.3(RCO+; C20:5), 311.3(RCOO+58; C16:1)],
547.5 [-R(C20:5)COOH: 219.3(RCO-H2O; C16:1), 237.2(RCO+; C16:1), 293.3(RCOO+58-H2O; C16:1), 311.3(RCOO+58; C16:1)]
851.71
C20:5/C16:1/C16:0
833.7 (-H2O), 595.5 [-R(C16:0)COOH: 267.2(RCO-H2O; C20:5), 285.3(RCO+; C20:5), 311.3(RCOO+58; C16:1)]
549.5 [-R(C20:5)COOH: 219.3(RCO-H2O; C16:1), 237.2 (RCO; C16:1), 293.3(RCOO+58-H2O; C16:1), 313.3 (RCOO+58; C16:0)]
853.73
C20:4/C16:1/C16:1
835.8 (-H2O), 599.6 (-R(C16:1)COOH), 597.5 (-R(C16:0)COOH), 549.6 (-R(C20:4)COOH)
875.73
C20:4/C16:1/C18:3
857.8 (-H2O), 621.6 (-R(C16:1)COOH), 597.3 (-R(C18:3)COOH)
877.73
C20:5/C18:1/C16:1
859.6 (-H2O), 623.4 [-R(C16:1)COOH: 267.2(RCO-H2O; C20:5), 285.3 (RCO; C20:5), 339.3 (RCOO+58; C18:1)]
595.4 [-R(C18:1)COOH: 267.3(RCO-H2O; C20:5), 285.3(RCO; C20:5), 311.3 (RCOO+58; C16:1)], 575.50 [-R(C20:5)COOH:
237.3(RCO; C16:1), 247.3(RCO-H2O; C18:1), 265.3 (RCO; C18:1), 311.3 (RCOO+58; C16:1), 339.3 (RCOO+58; C18:1)]
879.75
C20:5/C18:1/C16:0
861.9 (-H2O), 623.6(-R(C16:0)COOH), 597.6 (-R(C18:1)COOH), 577.6 (-R(C20:5)COOH)
897.70
C20:5/C20:5/C16:1
879.6 (-H2O), 643.4 (-R(C16:1)COOH), 595.4 (-R(C20:5)COOH)
899.71
C20:5/C20:5/C16:0
881.6 (-H2O), 643.4 (-R(C16:0)COOH), 597.4 [-R(C20:5)COOH: 267.3(RCO-H2O; C20:5), 285.3(RCO; C20:5),
945.71
C20:5/C20:5/C20:5
927.7 (-H2O), 643.5 (-R(C20:5)COOH)
313.3 (RCOO+58; C16:0), 341.3 (RCOO+58-H2O; C20:5)]
* When MS3 is acquired, its information is given in [].
Supplementary information for cholesterol quantification using GC-MS.
To verify cholesterol amount in algal crude oils using an alternative method, a simple
one-point quantification was made using GC-MS. Namely, as described in the experimental
section, algal crude is silyated and diluted with toluene. One microliter of the final solution
(0.04% v/v of the original oil) is subjected to GC-MS analysis along with cholesterol standard
(final concentration of 20μM after silyation and toluene dilution).
Below is GC-MS
chromatogram of algal oil and cholesterol standard. Cholesterol peak appears at the retention
time of 18.4min and NIST database search gave an excellent score of 936 and 938 forward and
reverse match score as shown in Supplementary Figure 4. Four replicate of algal oil and three
replicate of cholesterol standard were run, of which the result is summarized in Supplementary
Table 2. The ratio of cholesterol peak area between algal oil and standard is 2.56 (rsd=4.3%).
Accordingly, the cholesterol amount in the original sample is calculated as 128mM (=2.56 x
20μM / 0.04%).
Supplementary Figure 3. GC-MS chromatograms of algal crude oil (Top) and cholesterol
standard (bottom).
Supplementary Figure 4. NIST MS database search of GC-MS spectrum of algal crude oil at
Rt=18.4min.
Supplementary Table 2. Summary of GC-MS analysis of cholesterol peak at Rt=18.4 min.
Std #1
Std #2
Std #3
Algal crude #1
Algal crude #2
Algal crude #3
Algal crude #4
Peak Area
7.27E+08
7.22E+08
7.00E+08
1.73E+09
1.86E+09
1.88E+09
1.85E+09
Average
Std dev
7.16E+08
1.45E+07
Average
Std dev
1.83E+09
6.90E+07