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Abstract
(5-N-Succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP) is a highly sensitive and
positively charged precolumn derivatization reagent for the analysis of amino acids in liquid chromatography
electrospray ionization-tandem mass spectrometry. The synthesis of this reagent and handling of the
derivatization reaction are quite simple. It reacts with amino acids rapidly and with high efficiency. MS/
MS analysis revealed that the SPTPP-derivatized amino acids formed strong product ions; thus, highly
sensitive and selective detection is possible in the selected reaction monitoring mode. The limits of detec-
tion for the SPTPP-derivatized amino acids are in the sub-fmol range. The sensitivities of the derivatized
amino acids increased about 500-fold, as compared to those of underivatized amino acids.
1. Introduction
Michail A. Alterman and Peter Hunziker (eds.), Amino Acid Analysis: Methods and Protocols, Methods in Molecular Biology,
vol. 828, DOI 10.1007/978-1-61779-445-2_5, Springer Science+Business Media, LLC 2012
47
48 S. Inagaki and T. Toyooka
2. Materials
2.1. Chemicals 1. Prepare all solutions using ultra pure water (prepared by
purifying deionized water to attain a sensitivity of 18 M cm
at 25C).
2. Acetonitrile is of LCMS grade.
3. All other reagents were of analytical-reagent grade and were
used without further purification.
4. Sodium borate buffer (pH 9.5) was prepared as follows. Boric
acid was dissolved in distilled water, and the pH was adjusted
to 9.5 by adding of 1 M NaOH and monitoring the pH using
a pH meter.
5. Store all reagents at 5C unless otherwise indicated.
3. Methods
3.2. Analysis of 1. Forty microliters of rat serum were deproteinized by the addi-
Biological Samples tion of 160 mL of acetonitrile containing an internal standard,
Using SPTPP and the sample was centrifuged at 2,000 g for 5 min.
2. The resulting supernatant (40 mL) was transferred to another
tube and evaporated to dryness under a gentle stream of
nitrogen.
Fig. 1. Scheme for the derivatization reaction of amino acids with SPTPP.
Fig. 2. Product ion mass spectrum of SPTPP-derivatized tyrosine (Precursor ion: m/z 526).
5 Amino Acid Analysis via LCMS Method After Derivatization 51
Fig. 3. SRM chromatograms of tyrosine derivatized with SPTPP (a, m/z 526 363) and
underivatized tyrosine (b, m/z 182 165); the amounts of the two tyrosine samples were
equal (1.0 pmol). The mobile phase was (a) H2Oacetonitrile (75:25) containing 0.1% (v/v)
formic acid and (b) H2Oacetonitrile (98:2) containing 0.1% (v/v) formic acid.
3.3. LCESI-MS/MS 1. The conditions for ESI-MS detection were optimized to obtain
Conditions the highest signal intensity by using an optimizing program
and were as follows: mode = positive-ion mode; ionspray
voltage = 4,000 V; nebulizer gas pressure = 11 psi; curtain gas
pressure = 10 psi; collision gas pressure = 6 psi; external turbo
gas flow rate = 6 L/min; turbo gas temperature = 500C;
declustering potential = 76 V; focusing potential = 330 V;
entrance potential = 10 V; collision cell exit potential = 15 V;
and collision energy = 55 eV.
52 S. Inagaki and T. Toyooka
Fig. 4. SRM chromatograms of SPTPP-derivatized GABA obtained from rat serum. The mobile phase was H2Oacetonitrile
(80:20) containing 0.1% (v/v) formic acid.
Fig. 5. SRM chromatograms of authentic SPTPP-derivatives of (a) tyrosine, o-tyrosine, m-tyrosine, 3-chlorotyrosine, and
3-nitrotyrosine and (b) analysis results for SPTPP-derivatized oxidative stress markers obtained from rat serum. The mobile
phase was H2Oacetonitrile (79:21) containing 0.1% (v/v) formic acid.
5 Amino Acid Analysis via LCMS Method After Derivatization 53
4. Notes
Acknowledgments
The authors thank Mr. Yuma Tano and Mr. Yusuke Yamakata for
their support with the experiments. This work was supported in
part by an academic research grant from the Shimadzu Science
Foundation and by the Global COE Program of the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
54 S. Inagaki and T. Toyooka
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