ity enhancement values represent lower limits and would likely
be even greater following LC purification that would remove
compounds likely to cause ion suppression (e.g., the excess
cholamine and triethylamine). The sample of unlabeled acids did
not contain any interfering compounds, but even so, it yielded
lower signal intensities than the sample of labeled fatty acids.
The improvement offered by labeling fatty acids was further
explored by estimating the limits of detection for the labeled fatty
acids and for the unlabeled ones under typical acidic LC-MS
conditions. An equimolar mixture was prepared of the three
cholamine-labeled fatty acids and the three unlabeled fatty acids,
where each of the six compounds was 15 µM in a solution of 75:
25 water/acetonitrile with 0.1% formic acid. These mixtures were
separated by reversed-phase HPLC followed by positive-mode ESI-
MS as well as negative-mode ESI-MS (in separate LC runs). The
limits of detection (LODs) for the labeled fatty acids in positive
mode were 15 ( 5, 23 ( 4, and 30 ( 8 fmol for myristic, oleic,
and archidonic acids, respectively. The LODs for the unlabeled
fatty acids in negative mode were in the picomole range and as
such were 110×, 300×, and 30× higher than for their labeled
counterparts in positive mode. The poor LOD values for the
unlabeled fatty acids were caused predominantly by the acidic
buffer, which limits deprotonation of the carboxylic acid groups.
The acidic buffer was employed because it is highly preferred
Table 1. Experimental Isotope Ratios for 10 Fatty
Acids in a Simulated Relative Quantification
Experiment where the Expected Ratio was 4.00.
fatty
experimental
precision
CV (%)
accuracy
(% error)
a
b
acid
ratio
1
1
1
1
1
1
2
2
2
2
6:0
6:1
8:0
8:1
8:2
8:3
0:3
0:4
2:5
2:6
4.21
4.04
4.30
4.22
4.29
3.98
3.50
4.15
4.04
3.94
3.1
0.8
2.3
2.6
1.4
6.3
4.8
1.8
1.7
1.3
5.4
1.1
7.5
5.5
7.4
0.6
12.5
3.7
1.0
1.4
average
4.07
2.6
4.6
a
Fatty acids are referred to by their number of carbons and degree
b
of unsaturation. A 1:1 mixture was used to normalize ratios for minor
variations in reactivity between heavy and light cholamine
(Scheme 1). Not only is the ionizability of the acid not destroyed
during the coupling reaction but the mode of ionization switches
from negative to positive mode, which facilitates the use of acidic
buffers during the LC separation. Second, the reaction conditions
are such that no sample cleanup is required. The product-
containing solution is simply diluted with LC loading buffer,
separated into its components by chromatography, and then mass
analyzed. Third, the size and mass of the label is smaller than
the targeted analytes; the added masses for the light and heavy
labels are 85 and 94 Da, respectively. Thus, the chemical
structures of the original analytes still contribute significantly to
the chromatographic separation, as observed in Figure 2. In other
words, the label does not dominate the separation and cause all
labeled compounds to elute at the same time, which could increase
detection limits because of ion suppression effects,15 and thereby
impair observation of some minor constituents. Fourth, the
placement of the deuterium on the methyl groups of the
quaternary ammonium group, rather than at some more hydro-
phobic position, ensures coelution of light- and heavy-isotope
derivatives of equivalent metabolites from two different samples,
23
for RPLC of fatty acids, which makes the above experiment a
fair comparison between cholamine-labeled and unlabeled
fatty acids when considering the entire LC-MS analysis
method.
Relative Quantification of Fatty Acids from Hydrolyzed
Egg Lipid. Dietary lipid composition can have significant effects
on the growth and composition of chickens and their eggs. For
example, animals fed CLA exhibit improved growth, efficiency of
32-36
food use, and resistance to certain diseases;
but it also leads
to dramatic decreases in egg hatchability. Aydin et al. have shown
that combining other fatty acids with CLA eliminates the hatch-
37
ability problem. These fatty acids in the diet impact the fatty
acid composition of the eggs, which affects hatchability. The
following experiment used cholamine labeling for relative quan-
tification of fatty acids in egg lipids in order to examine the
incorporation of dietary fat into egg yolks.
Results are shown in Figure 3 for the fatty acid analysis of
egg lipids after supplementing the standard table diet with OO
and SO, with and without CLA. The various supplemented diets
are all compared to the standard table diet, and a ratio of 1 in
Figure 3a indicates that the supplement had no effect on the
amount of that particular fatty acid. Many substantial changes in
the fatty acid composition are observed. The error bars in Figure
30
as observed in Figure 2 and reported in the literature. In contrast,
some deuterium isotope derivatives that are used in relative
quantification of peptides produce an undesirable chromatographic
shift, which introduces a source of error in calculation of relative
13,19
abundances.
Finally, the relatively large 9-Da shift between the
light- and heavy-labeled compounds prevents the natural isotope
peaks of the light-labeled compound from overlapping with the
monoisotopic peak of the heavy-labeled compound.
The sensitivity enhancement produced by cholamine labeling
was determined by comparing ESI-MS intensities of labeled and
unlabeled fatty acids of various lengths and degrees of unsatura-
tion. Myristic acid (14:0), oleic acid (18:1), and arachidonic acid
3
represent the standard deviations obtained from two LC-MS
(32) Cook, M. E.; DeVoney, D.; Drake, B.; Pariza, M. W.; Whigham, L.; Yang,
M. In Advances in Conjugated Linoleic Acid Research; Yurawecz, M. P.,
Mossaba, M. M., Kramer, J. K. G., Pariza, M. W., Nelson, G. J., Eds.; AOCS
Press: Champaign, IL, 1999; Vol. 1, pp 226-237.
(
7
20:4) were labeled with cholamine and then diluted to 15 µM in
5:25 water/acetonitrile with 0.1% formic acid. Direct infusion
(33) Banni, S.; Heys, S. D.; Wahle, K. W. J. In Advances in Conjugated Linoleic
Acid Research; Sebedio, J.-L., Christie, W. W., Adlof, R., Eds.; AOCS Press:
Champaign, IL, 2003; Vol. 2, pp 267-282.
positive-mode ESI-MS of this unpurified mixture, which contained
excess labeling reagents, gave signal intensities that were 1.5-3
times higher than the three unlabeled acids infused under their
optimal conditions (negative-mode ESI-MS at 15 µM in 75:25
water/acetonitrile with 1% ammonium hydroxide). These sensitiv-
(
34) Chin, S. F.; Storkson, J. M.; Albright, K. J.; Pariza, M. W. J. Nutr. 1994,
124, 694-701.
(
(
35) Pariza, M. W.; Y., P.; M. E., C. Prog. Lipid Res. 2001, 40, 283-298.
36) Ostrowska, E.; Muralitharan, M.; Cross, R. F.; Bauman, D. E.; Dunshea, F.
R. J. Nutr. 1999, 129, 2037-2042.
(37) Aydin, R.; Pariza, M. W.; Cook, M. E. J. Nutr. 2001, 131, 800-806.
Analytical Chemistry, Vol. 79, No. 14, July 15, 2007 5147