ionization of carboxyl groups is suppressed.21 This problem could
be circumvented by using a postcolumn pH adjustment with
Scheme 1. Fatty Acid Derivatization Reactions
18
ammonia, but would increase the complexity of the analysis and
dilute analytes. Another possibility would be to derivatize carboxyl
groups with a reagent that causes them to have a positive charge
in acidic mobile phases. This strategy has been very successful
22,23
in the HPLC/ESI-MS analysis of amino acids and peptides
along with neutral analytes.24,25
Quantification in LC/MS is also related to ionization efficiency.
Matrix suppression of ionization can greatly reduce sensitivity and
complicate quantification. The problem is that ionization efficiency
of analytes is variable, being related to the concentration of other
analytes in the mixture that suppress their ionization. One way
to deal with this problem has been to increase ionization efficiency
through the introduction of positive charge into analytes, as
discussed above. Another is to add an internal standard that
experiences the same suppression of ionization. When it is the
objective to quantify large numbers of analytes as in proteomics
and metabolomics, large numbers of isotopically coded internal
standards of structure identical to analytes are added to a mixture.
sion of ionization.19,30,31 The advantage of this approach is that
internal standards have the same chromatographic properties as
the analytes but can still be differentiated from analytes on the
basis of mass. The disadvantage of this strategy is that a different
internal standard has to be synthesized for each analyte. Also,
this can only be done with known analytes with known structure.
An alternative approach is to attach a stable isotope coded
derivatizing agent to analytes.32 The attractive feature of this
strategy is that it allows all analytes in a sample to be coded with
one isotopomer of the derivatizing agent while those from another
sample can be coded with a second isotopomer.33 Moreover, both
relative and absolute comparisons of concentration between
samples are easily achieved with this method.
2
6
Isotope coded affinity tagging and global internal standard
2
7
technology are examples of the large-scale use of internal
standards for relative quantification in proteomics. Very similar
differential coding methods have been described for metabolom-
2
8,29
ics.
Functional groups in a control sample are derivatized in
vitro with an isotopically coded reagent to target a particular
functional group or molecular feature of analytes in a mixture.
The experimental sample in contrast is derivatized with an isotopic
isoform of the reagent. After the differentially coded samples are
mixed, they are analyzed and as a last step the isotope ratio of
the isotopomers is determined by mass spectrometry. In this way,
differences in concentration between control and experimental
samples are easily determined. Although there is substantial
similarity between global internal standard coding methods in
proteomics and metabolomics, there are differences. The major
one is that almost all peptides contain an amino group or carboxyl
group that can be isotopically coded and used in relative
quantification studies. Metabolites on the other hand have no
single, common functional group that can be used for isotope
coding. This means that a number of reagents will have to be
used to achieve truly global internal standard quantification.
In the analysis of carboxylic acids, stable isotope coded fatty
acids are often used as internal standards due to matrix suppres-
This paper describes a strategy in which carboxyl-containing
analytes are derivatized with a reagent that both increases their
ionization efficiency in HPLC/ESI-MS analysis and isotopically
codes them for internal standard-based quantification.
EXPERIMENTAL SECTION
Materials and Reagents. Fatty acids (FAs), human serum,
2
-bromopyridine, 3-carbinolpyridine, triethylamine (TEA), iodo-
methane, and anhydrous acetonitrile (ACN), were purchased from
Sigma-Aldrich (St. Louis, MO). HPLC grade acetonitrile, acetone,
and formic acid were obtained from Mallinckrodt Baker (Phil-
3
lipsburg, NJ). Iodomethane-d was a product of Cambridge Isotope
Laboratories (Andover, MA). Double-deionized water was pro-
duced by a Milli-Q gradient A10 system from Millipore (Bedford,
MA).
(
(
(
19) Valianpour, F.; Selhorst, J. J. M.; van Lint, L. E. M.; van Gennip, A. H.;
Wanders, R. J. A.; Kemp, S. Mol. Genet. Metab. 2003, 79, 189-196.
20) Perret, D.; Gentili, A.; Marchese S.; Sergi M.; Caporossi, L. Rapid Commun.
Mass Spectrom. 2004, 18, 1989-1994.
Synthesis of 2-Bromo-1-methylpyridinium Iodide (BMP),
3
-Carbinol-1-methylpyridinium Iodide (CMP), and 3-Carbinol-
1-methyl-d -pyridinium Iodide (CMP-d ). Five-fold excess
iodomethane-d or -d was added to 2-bromopyridine (10 mmol,
.97 mL) or 3-carbinolpyridine (10 mmol, 0.96 mL). The solution
21) Jemal, M.; Zheng, O.; Teitz, D. Rapid Commun. Mass Spectrom. 1998, 12,
3
3
4
29-434.
0
3
(
(
22) Yang, W.-C.; Mirzaei, H.; Liu, X.; Regnier, F. E. Anal. Chem. Submitted.
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0
4
530.
24) Quirke, J. M. E.; Adams, C. L.; Van Berkel, G. J. Anal. Chem. 1994, 66,
302-1315.
was stirred at room temperature for 1 h. The crystals were washed
with cold acetone and dried in vacuum. H NMR properties were
as follows: (200 MHz, ACN-d ) for BMP, δ 9.12 (d, 1H, C(3)H),
3
(
1
1
(
(
25) Quirke, J. M. E.; van Berkel, G. J. J. Mass Spectrom. 2001, 36, 1294-1300.
26) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R.
Nat. Biotechnol. 1999, 17, 994-999.
(30) Lagerstedt, S. A.; Hinrichs, D. R.; Batt, S. M.; Magera, M. J.; Rinaldo, P.;
McConnell, J. P. Mol. Genet. Metab. 2001, 73, 38-45.
(
(
(
27) Ji, J.; Chakraborty, A.; Geng, M.; Zhang, X.; Amini, A.; Bina, M.; Regnier, F.
J. Chromatogr., B 2000, 745, 197-210.
28) Yang, W.-C.; Mirzaei, H.; Liu, X.; Regnier, F. E. Anal. Chem. 2006, 78, 4702-
(31) Yang, Y. J.; Choi, M. H.; Paik, M.-J.; Yoon, H.-R.; Chung, B. C. J. Chromatogr.,
B 2000, 742, 37-46.
4
708.
(32) Cowen, A. E.; Hofmann, A. F.; Hachey, D. L.; Thomas, P. J.; Belobaba, D.
T.; Klein, P. D.; Tokes, L. J. Lipid Res. 1976, 17, 231-238.
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Analytical Chemistry, Vol. 79, No. 14, July 15, 2007 5151