Linda Lopez,1 Leo Wang,1 Ting Zheng,1 Mark Tracy,1 Rod Corpuz,2 and Rosanne Slingsby1
Thermo Fisher Scientific, Sunnyvale, CA, USA; 2General Atomics, San Diego, CA, USA
1
Wh ite Pa p er 7 04 9 5
Analysis of Carbohydrates and Lipids in
Microalgal Biomass with HPAE-MS and
LC-MS
Executive Summary
Comprehensive biomass analysis requires simple, robust, and accurate
methods for carbohydrate and lipid profiling. High-performance anionexchange chromatography with mass spectrometry (HPAE/MS) enables
high resolution separation and trace-level determination of carbohydrates
in lysed microalgal biomass without the need for extensive sample
preparation. High-performance liquid chromatography (HPLC) with MS
detection (LC/MS) is a fast and convenient approach for biomass lipid
profiling.
Key Words
Introduction
Biofuel, Carbohydrates, Lipids, MS
Biodiesel is rapidly becoming an attractive alternative to fossil-derived diesel fuel, and algae are a
promising feedstock. Efficient production of biodiesel from microalgae requires analysis of all cell
products, including carbohydrates, lipids, and proteins. Profiling of lipids in biomass feed stocks is
critical for the production of quality biodiesel. Determination of carbon chain length and the
degree of saturation is key to ensuring high quality biodiesel that delivers optimum performance
during engine combustion. Lipid profiling is commonly done by gas chromatography (GC) with a
flame ionization detector (FID), as specified in the ASTM D6584 and EN 14105 methods; however,
this methodology has several limitations. High-boiling triglycerides often require special inlet types
to preclude discrimination which can cause thermal degradation and interfere with the summative
mass closure calculations typically done to determine the total composition of biomass. Recently,
HPLC analysis at ambient temperatures with MS detection has emerged as the preferred method
for biomass lipid profiling because it is well-suited to analysis of nonvolatile components and can
handle very complex or dirty sample matrices without clogging of the LC/MS interface.
In addition to lipid profiling, a complete characterization of the carbohydrate breakdown products
in the algae is essential for efficient nutrient recycle to determine which sugars are best absorbed by
the algae. Carbohydrate and lipid analyses of microalgal biomass are often complex and require a
high-resolution, high-sensitivity technique capable of separating and quantifying trace-level
components in the presence of multiple interfering compounds. The use of high pH anion-exchange
chromatography mass spectrometry (HPAE-MS) permits baseline separation of key saccharides in
complex lysate mixtures.
2
Experimental
Instrument Setup
Carbohydrate analyses were performed on a Thermo Scientific™ Dionex™ ICS-3000 IC system,
which includes an ICS-3000 DP gradient pump, AS autosampler, and ICS-3000 DC column compartment with electrochemical cell. Lipid analyses were performed on a Thermo Scientific™ Dionex™
UltiMate™ 3000 RS system with HPG-3400 RS pump, WPS-3000 RS sampler, TCC-3000 RS
column thermostat, DAD-3000 RS diode-array detector, and SofTA model 400 evaporative light
scattering detector (ELSD).
Carbohydrates were detected using pulsed amperometry with a gold electrode and Ag/AgCl
reference electrode using the standard quadruple waveform we developed.1 Mass spectrometric
analyses were run on a Thermo Scientific™ MSQ Plus™ single quadrupole mass spectrometer. The
effluent of the high-performance anion-exchange chromatography (HPAEC) column was passed
through a Thermo Scientific™ Dionex™ CMD™ Carbohydrate Membrane Desalter2 (P/N 059090),
then to the MSQ Plus single quadrupole mass spectrometer. Pressurized water was pumped through
the Dionex CMD Carbohydrate Membrane Desalter as regenerating reagent. Postcolumn addition
of 0.5 mM lithium chloride was mixed into the eluent at 0.1 mL/min using a mixing tee and
Thermo Scientific Dionex AXP-MS pump (P/N 060684). Detailed instrument setup is shown in
Figure 1. The addition of lithium allowed mono- and disaccharides to form lithium adducts. The
use of lithium allows detection of the sugars in the lithium form as opposed to mixed forms which
may be caused by contamination of weaker adduct-forming cations, such as ammonium or sodium.
Lipids were detected using LC-UV/MS. Postcolumn addition of ammonium acetate was applied to
facilitate the formation of lipid-ammonium or lipid-acetate adducts. The MSQ Plus single quadrupole mass spectrometer was operated in the positive full scan mode over a scan range of m/z 100 to
1500 with a cone voltage fragmentor setting of 50 V.
Chromeleon
Chromatography
Data System
Eluent
Under N2, ∆P
Signals
Water
Pump
Autosampler
CMD
Guard
Analytical Column
To Waste
MSQ
Pump
0.5 mM LiCI
25999
Figure 1. Instrument setup for on-line HPAE-MS analysis of carbohydrates.
220
Thermo Scientific™ Dionex™ CarboPac™ MA1,
4 × 250 mm with Guard
Eluent:
500 mM NaOH, isocratic
Flow Rate: 0.2 mL/min
Temperature: 30 °C
Detection:
Integrated pulsed amperometry
Column:
nC
0
0
10
20
30
40
50
60
70
80
Minutes
90
100
110
120
130
140
26000
Figure 2. Separation profile of microalgae carbohydrates on the Dionex CarboPac MA1 column.
Chromatographic separations by LC/MS analyses were performed using a Thermo Scientific™
Acclaim™ RSLC C18 2.2 µm column, in 2.1 × 150 mm format. Chromatographic separations by
HPAE-MS were performed using a Dionex CarboPac™ MA1 (3 × 250 mm) analytical column and
MA1 (4 × 50 mm) guard column at a flow rate of 200 µL/min at 30 ˚C under isocratic conditions.
Chromatography and MS analysis were controlled by Thermo Scientific™ Dionex™ Chromeleon™
6.8 Chromatography Data System software. Reference spectra were acquired using Thermo
Scientific™ Xcalibur™ software.
Sample Preparation
Samples of algal biomass and oil were obtained from General Atomics, San Diego, CA. To prepare
biomass samples for the HPAEC-MS analysis, 2 mL of lysed microalgae sample was centrifuged at
12,000 rpm for 60 min. The supernatant was collected and centrifuged for an additional 30 min.
The supernatant was collected and filtered through a 0.2 µm syringe filter to remove any remaining
particles. The sample was passed through a Thermo Scientific™ Dionex™ OnGuard™ RP cartridge to
remove hydrophobic components. The Dionex OnGuard RP cartridge was activated first with
5 mL methanol followed by 10 mL deionized (DI) water using a 10 mL syringe. Next, a 400 µL
sample of supernatant was mixed with 600 µL water and passed through the activated cartridge
using a 1 mL syringe. The cartridge was then washed with 0.5 mL water and the eluted washing
solution was combined with the initial eluent. The sample was then passed through another
activated RP cartridge. This step was repeated until the sample was free of color. All the eluted
solutions (~3 mL) were combined and concentrated down to a volume of ~250 µL.
Results
Carbohydrate Analysis
The separation profile of carbohydrates in microalgae samples on the Dionex CarboPac MA1 is
shown in Figure 2. More than a dozen peaks were observed. On-line mass spectrometry analysis
was used on the same samples to confirm the carbohydrate identity of the peaks. Note the similarities between the total ion chromatogram (TIC) and pulsed amperometric detection (PAD) profiles
(Figure 3). The full scan spectrum of each peak is shown in Figure 4. Because many mono- and
disaccharides have identical m/z, HPAE-PAD profiles of carbohydrate standards were compared
with the sample profile. Comparison of their retention times helped to identify the peaks (Figure 5).
Table 1 shows the identification results based on the peak’s m/z and comparison with PAD profiles
of the standards. These data indicated that monosaccharide alditols (fucitol, arabitol, inositol),
monosaccharides (glucose, mannose, and others), and disaccharides (sucrose, maltose, and others)
were present in the microalgal biomass, sucrose being the major sugar component.
70E6
Current for CMD:
Postcolumn addition:
Mode:
Cone voltage:
400 mA
0.5 mM LiCI, 0.1 mL/min
+ESI, Full Scan 120–600 m/z
50V
12
Separation conditions are shown in Figure 2.
counts
2
1 34 5
7
6
8
9
10
13
11
0
0
10
20
30
40
50
60
70
80
Minutes
Figure 3. MS TIC profile microalgae carbohydrates.
90
100
110
120
130
140
26001
3
4
173.1
138.1
138.1
138.1
Peak 2
367.1
423.1
Peak 3
323.2
205.1
162.1
261.2
138.1
138.1
Peak 1
196.1
205.1
187.1
228.3
162.1
Peak 4
398.2
261.2
162.1
Peak 5
398.1
159.1
138.1
Peak 6
182.1
349.1
Peak 7
138.1
120
180
138.1
240
187.1
162.1
175.1
360
420
480
600
Peak 9
228.2
349.1
205.1
Peak 10
337.4
219.1
205.1 237.2
Peak 11
349.1
Peak 12
365.2
349.1
229.1
120
540
Peak 8
289.3
228.1
205.1
187.1
139.1
300
205.1
180
240
Peak 13
367.1
289.2
300
360
m/z
420
480
540
600
26002
Figure 4. Background-subtracted ESI mass spectra of peaks shown in Figure 3.
5A
Column:
Dionex CarboPac MA1, 4 × 250 mm, with Guard
Eluent:
500 mM NaOH, isocratic
Flow Rate: 0.2 mL/min
Temperature: 30 °C
Detection: Integrated pulsed amperometry
Samples: 1. Mannitol
2. Inositol
3. Xylitol
4. D-arabitol
5. L-arabitol
6. Dulcitol
7. Algae sample
7
6
nC 5
4
3
2
1
-0.4
5.0
10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.5
5B
Column:
Eluent:
Flow Rate:
Temperature:
9
8
7
6
nC 5
4
3
2
1
14.6
20
25
30
35
40
45
50
55
60
Dionex CarboPac MA1, 4 × 250 mm, with Guard
500 mM NaOH, isocratic
0.2 mL/min
30 °C
Detection: Integrated pulsed amperometry
Samples: 1. Galactosamine
2. Arabiose
3. Xylose
4. Glucose
5. Mannose
6. Fucose
7. Galactose
8. Glucosamine
9. Algae sample
65
5C
Column:
Eluent:
Flow Rate:
Temperature:
Detection:
Dionex CarboPac MA1, 4 × 250 mm, with Guard
500 mM NaOH, isocratic
0.2 mL/min
30 °C
Integrated pulsed amperometry
4
Samples:
1. Maltose
2. Lactose
3. Sucrose
4. Algae sample
nC 3
2
1
40
50
60
70
80
90 100 110 120 130
Minutes
142
26003
Figure 5. Overlay comparisons of algae samples with carbohydrate standards with A: alditol
standards; B: monosaccharide standards; C: disaccharide standards.
5
Table 1. Identification of sample peaks based on m/z and retention times.
Peak
m/z
Adduct
Possible Identification
1
173.1
166 + 7
(Fucitol + Li)+
2
205.1/187.1
180 + 7, 180 + 7 + 18
(Inositol + Li)+, (Inositol + Li + H2O)+
3
423.1
Unknown
Unknown
4
261.2
Unknown
Unknown
5
261.2
Unknown
Unknown
6
159.1
152 + 7
(Monoalditol + Li)+ (L-Arabitol)
7
349.1
342 + 7
(Disaccharide + Li)+
8
205.1/187.1
180 + 7
(Monosaccharide + Li)+ (Mannose)
9
201.1/187.1
180 + 7
(Monosaccharide + Li)+ (Glucose)
10
349.1
342 + 7
(Disaccharide + Li)+
11
219.1
Unknown
Unknown
12
349.1
342 + 7
(Disaccharide + Li)+ (Sucrose)
13
349.1
342 + 7
(Disaccharide + Li)+ (Maltose)
Lipid Analysis
The oil samples were analyzed under conditions that give a simplified chromatogram where
triacylglycerides (TAGs) with similar Partition Numbers or Effective Carbon Numbers3 tend to
coelute in a single peak.4 Mass spectrometry allows us to assign total carbon and total unsaturation
numbers to components within each peak. The notation x:y is used where x is the total carbon
number of the acyl chains, and y is the total number of double bonds. The typical fatty acid
compositions of lipids from various sources are well known in the literature, and with few exception, they have an even number of carbon atoms.5,6 For each x:y we enumerate the m/z value of the
[M+NH4]+ adduct, and a theoretical isotope pattern. We then assign matching x:y to the components in the chromatogram. Reference 4 shows an example of this process for soybean oil. Figure 6
shows the extracted ion chromatograms and x:y assignments for the main components of algal oil.
56:10
56:11
52:4
52:3
m/z 914.9
m/z 917.0
50:9
Chromatographic Conditions
Column:
Acclaim 120 C18
2.2 µm
2.1 × 150 mm
Column Temp.: 70 °C
Mobile Phase: CH3CN/IPA = 80/20 (v/v)
Flow Rate:
500 µL/min
Inj. Volume:
3 µL
m/z 879.2
m/z 874.9
50:8
52:2
m/z 877.0
Intensity [counts]
50:7
m/z 834.9
48:2
m/z 836.9
48:1
m/z 838.9
Mass Spectrometric Conditions
Interface:
Electrospray (ESI)
Infusion:
50 µL/min of 20%
NH4OAC buffer
in CH3CN (10 mM)
Scan Mode: Positive full scan
100–1500 amu
Probe Temp.: 400 °C
Needle Voltage: 3000 V
Cone Voltage: 50 V
50:3
m/z 846.9
48:3
50:2
m/z 848.9
m/z 818.9
46:1
m/z 820.9
m/z 822.9
m/z 794.9
0
2.5
5
7.5
Retention Time [min]
10
Figure 6. Extracted MS chromatograms of lipids in algae oil.
12.5
15
26062
Table 2. TAGs in algal oil.
Retention Time
M+NH4 Adduct m/z
Assignment
Probable TAGs
6.4
914.9
56:11
16:1/20:5/20:5
8.1
917.0
56:10
18:2/18:3/20:5
3.8
872.9
52:4
16:0/18:1/18:3
16:0/18:2/18:2
4.7
874.9
52:3
16:0/18:0/18:3
16:0/18:1/18:2
18:3/18:3/20:5
11.5
877.0
52:2
16:0/18:0/18:2
16:0/18:1/18:1
3.7
834.9
50:9
16:2/16:3/18:4
16:3/16:3/18:3
3.6
836.9
50:8
14:0/16:3/20:5
16:2/16:3/18:3
4.5
838.9
50:7
14:0/16:2/20:5
16:1/16:3/18:3
7.4
846.9
50:3
14:0/18:0/18:3
14:0/18:1/18:2
9.1
848.9
50:2
14:0/18:0/18:2
14:0/18:1/18:1
5.7
818.9
48:3
14:0/16:0/18:3
14:0/16:1/18:2
7.2
820.9
48:2
14:0/16:0/18:2
14:0/16:1/18:1
9.2
822.9
48:1
14:0/16:0/18:1
14:0/16:1/18:0
7.3
794.9
46:1
12:0/16:0/18:1
12:0/16:1/18:0
Conclusion
Simple, sensitive, and robust methods were developed for the analysis of carbohydrates and lipids
in microalgal biomass. HPAE-MS allows baseline separation and detection of key saccharides in
complex lysate samples. Lipid profiling of algal oil was achieved with LC/MS. These MS-based
methods obviate the need for extensive sample preparation.
Reference
1. Rocklin, R.D.; Clarke, A.P.; Weitzhandler, M. Anal. Chem. 1998, 70, 1496–1501.
2. Thayer, J.R; Rohrer, J.S.; Avdalociv, N; Gearing, R.P. Anal. Biochem. 1998, 256, 207–216.
3. Podlaha, O.; Töregård, B. Journal of High Resolution Chromatography, 2005, 5 (10), 553–558.
4.Tracy, M. L.; Wang, L.; Liu, X.; Lopez, L.; Analysis for Triglycerides, Diglycerides, and Monoglycerides by
High Performance Liquid Chromatography (HPLC) Using 2.2 µm Rapid Separation C18 Columns with
Alternative Solvent Systems and Mass Spectrometry. 31st Biofuels Conference, San Francisco, USA, Feb
2–4, 2009. LPN 2266. Dionex (now part of Thermo Scientific) Corporation, Sunnyvale, CA.
5.Gunstone, F. D. The Chemistry of Oils and Fats, Blackwell Publishing: Oxford, UK, 2004;
Page 4, Table 1.1.
6. Bajpai, D; Tyagi, V.K. J. Oleo. Sci. 2006, 55 (10), 487–502.
7Senanayake, N.; Shahidi, F. Concentration of Docosahexaenoic Acid (DHA) from Algal Oil via Urea
Complexation. J. Food Lipids 2004, 7, 51–61.
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Wh ite Pa p er 7 04 9 5
Table 2 lists the masses, peak assignments, and a partial list of probable TAGs in algal oil. The
possible combinations of known fatty acids to make any given x:y can be large; the list of probable
TAGs was established according to reported fatty acid composition in algal oil.7 Each list is isobaric
and the constituents cannot be distinguished by a single-stage mass spectrometer. To make a
complete assignment of TAGs found in the sample would require MS/MS. Compared to typical
plant-derived oils, there are some unusual/characteristic features to this algal oil. First, no 54-carbon
TAGs were observed in this study. Second, the presence of highly unsaturated TAGs (unsaturations
from eicosapentaenoic acid, 20:5) was confirmed and this observation agrees with reported data.7