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Chapter 5

Amino Acid Analysis via LCMS Method After


Derivatization with Quaternary Phosphonium
Shinsuke Inagaki and Toshimasa Toyooka

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.

Key words: (5-N-Succinimidoxy-5-oxopentyl)triphenylphosphonium bromide, Liquid chromatography


electrospray ionization-tandem mass spectrometry, Charged derivatization, Quaternary phosphonium
regent, Amino acid analysis, 3-Nitrotyrosine, GABA, Amino acid analysis

1. Introduction

High-performance liquid chromatographyelectrospray ionization-


tandem mass spectrometry (LCESI-MS/MS) is rapidly emerging
as a powerful method for the highly sensitive and selective analysis
of biological compounds, including amino acids. However, as a
detection method, mass spectrometry (MS) is not as versatile as
differential refractive index detection (RI) or charged aerosol
detection (CAD) (1), and there are compounds for which this
technique is not appropriate. The sensitivity of the technique is
insufficient for the microanalysis of certain samples because the
ionization efficiencies of the compounds in the samples are
sometimes low; such compounds cannot be detected with high

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

sensitivity. Furthermore, in LCESI-MS/MS, a reversed-phase


column such as an octadecylsilane (ODS) is commonly used.
Highly polar analytes, including some amino acids, cannot be
retained on such column. Therefore, it is difficult to separate and
detect such compounds. To address these issues, chemical derivati-
zation is performed to introduce charged or proton-affinitive
species in a target functional group prior to analysis by LCESI-MS/
MS to facilitate retention of analytes in the reversed-phase column
and to promote ionization during ESI-MS. Chemical derivatization
also improves the detection sensitivity. In particular, the introduc-
tion of permanently charged or polar substituents can improve the
ionization of nonpolar compounds in the ESI mode (24).
The following conditions should be satisfied when derivatization
reagents are used in LCESI-MS/MS: (1) the derivatized analytes
should have high ionization efficiency, and they should be detected
with high sensitivity; (2) reagents should react with the target
functional group under mild conditions; (3) reagents should have
a hydrophobicity that is appropriate for the separation of the
derivatized analytes by a reversed-phase system; (4) reagents should
have low susceptibility to ion suppression; (5) fragmentation of the
target analytes should be accomplished easily by collision-induced
dissociation (CID) to efficiently generate a particular production
for sensitive MS/MS detection; (6) reagents should be inexpensive
and easily obtainable, as compared to the numerous conventional
derivatization reagents that are commercially available. Deriva-
tization reagents for LCESI-MS/MS that meet these require-
ments have been highly anticipated.
A variety of techniques for converting amino acids into sensitive,
analyzable fluorescent derivatives have been developed in the past
three decades. The reagents used to produce the derivatives include
o-phthalaldehyde (OPA), 5-(dimethylamino)-naphthalene-1-
sulfonyl chloride (Dansyl-Cl), 9-fluorenylmethylchloroformate
(Fmoc-Cl), 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F),
and 6-aminoquinolyl-N-hydroxysuccinimidyl carbonate (AQC).
Very recently, Shimbo et al. (57) developed derivatization reagents
for the high-speed analysis of amino acids using LCESI-MS/MS,
including p-N,N,N-trimethylammonioanilyl N -hydroxysuccimidyl
carbamate iodide (TAHS) and 3-aminopyridyl-N-hydroxysuccimidyl
carbamate (APDS). These reagents have a reaction group for ana-
lytes, a chargeable group, a nonpolar region, and a readily cleavable
group for the high-sensitivity detection using LCESI-MS/MS.
In this chapter, we describe the synthesis and application of a
new derivatization reagent, (5-N-succinimidoxy-5-oxopentyl)
triphenylphosphonium bromide (SPTPP), which has a permanently
positively charged quaternary phosphonium functional group; it is
a highly sensitive precolumn derivatization reagent for amino acids
in LCESI-MS/MS (8). Amino acids are detected via selected
reaction monitoring (SRM), whereby precursor ions are isolated in
5 Amino Acid Analysis via LCMS Method After Derivatization 49

the first stage of the mass spectrometer followed by CID to generate


fragment ions, which are detected after an additional stage of mass
spectrometer isolation. SPTPP has been used for the analysis of a
neurotransmitter, 4-aminobutanoic acid (GABA), and oxidative
stress markers such as o-tyrosine (o-Tyr), m-tyrosine (m-Tyr),
3-nitrotyrosine (3NO2-Tyr), and 3-chlorotyrosine (3Cl-Tyr), in
biological samples.
In the near future, simultaneous and sensitive detection of D,
L-amino acids can be potentially achieved by developing optically
active derivatization reagents for LCESI-MS/MS.

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.

2.2. Synthesis of 1. (4-Carboxybutyl)triphenylphosphine (120 mg, 0.25 mmol)


(5-N-Succinimidoxy- was dissolved in 20 mL of acetonitrile.
5-Oxopentyl) 2. N-Hydroxysuccinimide (29 mg, 0.25 mmol) and dicyclohexy-
Triphenylphosphonium lcarbodiimide (52 mg, 0.25 mmol) were added to the solution.
Bromide 3. After incubating for 16 h at room temperature, the precipitate
was filtered off.
4. The filtrate was evaporated to dryness and washed with diethyl
ether (15 mL 2).
5. The washed sample was evaporated to dryness again to yield
SPTPP (see Notes 1 and 2).

2.3. LCMS/MS 1. The LCESI-MS/MS apparatus comprised a 1100 series LC


Components (Agilent Technologies, Santa Clara, CA, USA) and an API
3000 triple quadrupole mass spectrometer (AB SCIEX, Foster
City, CA, USA) equipped with an ESI source.
2. Reversed-phase LC was performed using Symmetry C 18
column (2.1 mm i.d. 100 mm, 3.5 mm; Waters). The column
was maintained at 40C. The flow rate of the mobile phase was
0.2 mL/min. The injection volume was fixed at 10 mL.
50 S. Inagaki and T. Toyooka

3. Methods

1. Carry out all procedures at room temperature unless otherwise


specified.

3.1. Derivatization 1. Fifty microliters of 10 mM SPTPP in acetonitrile and 100 mL


of Amino Acids of 100 mM borate buffer (pH 9.5, see Note 3) were added to
with SPTPP 50 mL of the sample solution containing amino acids (0.010
5.0 mM, each).
2. The mixed solution was heated at 40C for 10 min (Fig. 1),
and then 800 mL of H2Oacetonitrile (75:25) containing 0.1%
formic acid (mobile phase) were added to the reaction
mixture.
3. The solution was allowed to cool to ambient temperature with
ice-water, and an aliquot (10 mL) was injected into the
LCESI-MS/MS system (see Notes 3 and 4) (Figs. 2 and 3).

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. Forty microliters of 100 mM borate buffer (pH 9.5) were


added to the tube and reacted with 40 mL of 10 mM SPTPP.
4. The reaction mixture was heated at 40C for 10 min, and
320 mL of the mobile phase were added.
5. The supernatant was filtered using a 0.20-mm filter, and an
aliquot of the filtrate was injected into the LCMS/MS system
(see Note 5) (Figs. 4 and 5).

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

1. If the washed sample is oily, add a small amount of acetonitrile


(~0.1 mL) to dissolve it, and add a large amount of diethyl
ether (~5 mL). Then decant off the ether supernatant.
2. ESI-TOF-MS: m/z 460.164 ([M]+). 1H NMR in chloroform-
d (ppm), 1.83 (m, 2 H), 2.23 (s, 2 H), 2.76 (t, 3 H), 2.81
(s, 4 H), 3.99 (m, 2 H), 7.80 (m, 15 H).
3. The borate buffer was observed to be effective at pH 9.5.
SPTPP reacted with amino acids readily and with high effi-
ciency without condensation reagents. The optimized reaction
temperature was 40C, and the reaction time was 10 min.
Regular and intense product ions (m/z 363) were observed in
the product ion mass spectra of the SPTPP-derivatized amino
acids (precursor ion: [M]+) (see Fig. 2). SPTPP enables the
highly sensitive and selective detection of its derivatives in the
SRM mode. For example, in the case of tyrosine, the signal-to-
noise ratio of the SPTPP-derivatized tyrosine improved dra-
matically compared to the underivatized tyrosine. The limits of
detection for intact tyrosine and the SPTPP-derivatized
tyrosine were 170 fmol and 0.34 fmol, respectively (the amount
of analyte per injection at a signal-to-noise ratio of 3). The
signal-to-noise ratio of the former proved to be ~500 times
greater than that of the latter (see Fig. 3).
4. Using 6-aminohexanoic acid as an internal standard, the cali-
bration curve exhibited good linearity.
5. Using SPTPP, successful analysis of 4-aminobutanoic acid
(GABA) in rat serum was achieved (see Fig. 4). GABA is a major
inhibitory neurotransmitter in the central nervous system.
Moreover, the analysis of 3-nitrotyrosine, which is an oxidative
stress marker in rat serum, was also achieved (see Fig. 5).

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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