Analysis of Triglycerides by Liquid

Chromatography/Mass Spectrometry
Application

Food

Author Table 1. Chemical Structure of Triglycerides

Hiroki Kumagai CH2 OCO R1

CH OCO R2
CH2 OCO R3
Abstract
Triglyceride R1=R2=R3
Triglycerides were readily analyzed using liquid Trilaurin C11
chromatography/mass spectrometry with atmospheric Trimyristin C13
pressure chemical ionization in positive ion mode. Tripalmitin C15
Tristearin C18
Triolein C18:1
Background Trilinolein C18:2

Oil is a primary component of many foods, such as cook-

ing oils and dairy products. Consequently, it is desirable
to conduct compositional analyses of oil-based fatty acid Method
components in food. Such analyses may use either gas • Instrument: Agilent 1100 LC/MS with APCI in posi-
chromatography (GC) or high-performance liquid chro- tive mode
matography (HPLC), but the GC methods have problems
with the complexity of sample preprocessing, which Mass range: 100 to 1000 m/z
requires saponification.
Drying gas: N2 4 L/min, 350 °C
With HPLC a direct analysis is possible; however, these
Nebulizer: N2 50 psi
methods are low in sensitivity because the compounds
of interest do not absorb ultraviolet and separation is Fragmentor: 160 V
barely adequate.
Vaporizer: 400 °C
In this study, six triglycerides (Table 1) with identical
• LC Conditions:
fatty acid compositions were analyzed using liquid chro-
matography/mass spectrometry (LC/MS) and atmos- Mobile phase: (CH3)2CO/H2O (98/2)
pheric pressure chemical ionization (APCI) as the
Flow rate: 1.0 mL/min
ionization mode.
Oven temperature: 40 °C
Injection volume: 15 µL
• Column: Develosil ODS OG-3, 4.6 mm id × 75 mm long
Results
The following figures show total ion chromatogram (TIC)
and selected ion mode (SIM) chromatograms, and mass
spectra for the selected triglyceride standards.

Abundance 2
1. Trilaurin
2. Trilinolein
40000
3. Trimyristin
4. Triolein
5. Tripalmitin
35000
6 6. Tristearin
5
4
30000
3

25000
1

20000

15000

10000

5000

1.00 2.00 3.00 4.00 5.00 6.00 min

Figure 1. TIC of triglyceride standards, each at 0.2 ppm.

439.5 T il i
Trilaurin Trilinolein 599.5 879.8 (MH)+ 495.5 Trimyristin
90 90
90

50 50
183 50
305 211
150 250 350 450 550 650 750 850 150 250 350 450 550 650 750 850 150 250 350 450 550 650 750 850

Tripalmitin 551.5 Tristearin 607.5

Triolein 603.5 90
90
90

50 50 50

881
150 250 350 450 550 650 750 850 150 250 350 450 550 650 750 850 150 250 350 450 550 650 750 850

Figure 2. Mass spectra of individual triglyceride standards at 0.2 ppm.

The tallest peak in each case represents the

[MH-HOOCR]+ ion created after the loss of a fatty acid
group from the protonated psuedomolecular ion. With
the exception of Trilinolein, the protonated psuedomol-
ecular ion was not observed.

2
 Trilaurin Trilinolein 7000
Trimyristin
5000
439.5 m/z 5000 599.5 m/z 6000
495.5 m/z
4000 4000 5000
3000 4000
3000
3000
2000 2000
2000
1000 1000 1000
0 0
0.40 0.80 1.20 1.60 2.00 2.40 2.80 0.40 0.80 1.20 1.60 2.00 2.40 2.80 0.40 0.80 1.20 1.60 2.00 2.40 2.80

14000
Tripalmitin
Triolein 551.5 m/z Tristearin
8000
603.5 m/z 20000 607.5 m/z
10000
6000 15000
6000 10000
4000
5000
2000
2000
3.00 4.00 5.00 6.00 3.00 4.00 5.00 6.00 3.00 4.00 5.00 6.00

Figure 3. SIM chromatograms of individual triglyceride standards at 1 ppb.

The protonated molecular ions were rarely

observed—trilinolein in the scan mode is the
exception—and the base peaks consisted of fragmented
ions from which fatty acid had been detached.
Sensitivity was favorable, extending down to 0.2 ppm in
TIC mode. In the SIM mode, measurements at 1 ppb
were possible by selecting the base peak in the mass
spectrum of each composition of monitored ions. With
this technique, it is possible to measure fat-soluble sub-
stances such as triglycerides with a high degree of
sensitivity.

Conclusions
LC/MS, with APCI in positive ion mode, readily detected
selected triglyceride standards with high sensitivity.
The analytical peaks were the positvely charged frag-
mented ions from which fatty acid had been detached.
SIM mode yielded sensitivity to 1 ppb.

Hiroki Kumagai is an application chemist at

Agilent/Yokogawa Analytical Systems, Tokyo, Japan.

3
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October 9, 2001
5988-4235EN