Escolar Documentos
Profissional Documentos
Cultura Documentos
Department of Immunology, National Jewish Medical and Research Center, Denver, CO 80206, USA
University of Colorado at Denver and Health Science Center, Clinical Nutrition Research Unit, Department of Anesthesiology, Denver,
CO 80262, USA
Amino acids in biological fluids have previously been shown to be detectable using liquid
chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with perfluorinated acids
as ion-pairing agents. To date, these studies have used precursor mass, retention time and tandem
mass spectrometry (MS/MS) to identify and quantify amino acids. While this is a potentially
powerful technique, we sought to adapt the method to time-of-flight (TOF)MS. A new application
of a recently described liquid chromatographic separation method was coupled with TOFMS to
employ accurate mass for qualitative identification; resulting in additional qualitative data not
available with standard single quadrupole data. In the current study, we evaluated 25 physiological
amino acids and one dipeptide that are routinely quantified in human plasma. Accuracy and
precision of the method was evaluated by spiking human plasma with a mix of the 25 amino acids;
in addition, the inclusion of a cation-exchange cleanup step was evaluated. The calibration curves
were linear over a range from 1.56 to 400 mM. The dynamic range was found to be within
physiological levels for all amino acids analyzed. Accuracy and precision for most of the amino
acids was between 80120% spike recovery and <10% relative standard deviation (RSD). The LC/MS
technique described in this study relies on mass accuracy and is suitable for the quantitation of
free amino acids in plasma. Copyright # 2007 John Wiley & Sons, Ltd.
Free amino acid analysis has applications in a variety
of areas, including the diagnosis of inherited metabolic
disorders,14 and nutritional studies of neonates.58 Traditionally, free amino acids in plasma have been analyzed by
ion chromatography (IC) using ninhydrin post-column
derivatization,9 or by cation-exchange solid-phase extraction
followed by derivatization and analysis by gas chromatography/mass spectrometry (GC/MS).1015 Both of these
methods have disadvantages, including long run times and
extensive sample preparation, respectively.
Although useful for a broad range of compounds, neither
high-performance liquid chromatography (HPLC) nor MS
techniques were generally employed for amino acid analysis
due to the inability to separate more polar amino acids using
reversed-phase (RP)-HPLC,16 and amino acid signal suppression in electrospray ionization (ESI)-MS caused by
co-elution of components in complex biological matrices.17,18
Some approaches to enable the application of LC/MS to
amino acid analysis include dedicated amino acid analysis
kits such as Waters AccQtag,19 and tandem mass spectrom*Correspondence to: N. A. Reisdorph, Department of Immunology, National Jewish Medical and Research Center, 1400
Jackson Street K924, Denver, CO 80206, USA.
E-mail: ReisdorphN@njc.org
Contract/grant sponsor: Colorado Clinical Nutrition Research
Unit; contract/grant number: NIH/NIDDK P30 DK048520-09.
EXPERIMENTAL
Reagents
Nanopure water (18.2 VOhms) was used for sample preparation. Water (HPLC grade) and acetonitrile (UV) used for
HPLC mobile phases was obtained from Burdick and
Jackson (Morristown, NJ, USA). HPLC-grade methanol
was obtained from Fisher Scientific (Hampton, NH, USA).
Tridecafluoroheptanoic acid (TDFHA) was obtained from
Aldrich Chemicals (St. Louis, MO, USA). Hydrochloric acid
was obtained from Sigma (St. Louis, MO, USA). The primary
amino acid calibration standard at 2.5 mM (standard H) was
obtained from Pierce (Rockford, IL, USA). Hyp, Gly, Glu,
Ala, Trp, Tau, Asn, Gln, Cit, Ala-Glu, Nor and Orn were
obtained from Sigma. Stable isotope labeled analogs of amino
acids used as internal standards (glutamine-d5, glutamic
acid-d3, methionine-d3, leucine-d10 and tryptophan-d5)
were obtained from Cambridge Isotope Laboratories (Andover, MA, USA). Outdated human blood plasma was
provided by Bonfils Blood Center (Denver, CO, USA).
The use of outdated plasma samples for method validation
and quality control purposes was considered exempt by the
Colorado Multiple Institutional Review Board (COMIRB).
#2 contained leucine-d10, glutamic acid-d3 and tryptophan-d5 in 0.1% hydrochloric acid. The internal standard
working solution was prepared immediately prior to sample
preparation, by adding equal parts of IS #1 and IS #2 to eight
parts methanol (1:1:8). All internal standard stocks and IS
mix #1 and #2 were stored at 208C until use.
Pooled human plasma samples, used for accuracy and
precision measurements, were spiked with amino acid
calibration mixes 1 and 2 (100 mM) and frozen at 808C
for 12 days prior to thawing for extraction and analysis.
Solid-phase extraction
For some samples, cleanup was performed via solid-phase
extraction (SPE) using a cation-exchange cartridge. Strata
X-C cartridges with a capacity of 30 mg (Phenomenex,
Torrance, CA, USA) were placed on a vacuum SPE manifold,
conditioned with 1 mL of methanol, then equilibrated with
1 mL of 0.1 N HCl in water, as per the manufacturers
protocol. Subsequently, 100 mL of plasma was mixed by
vortexing with 100 mL of the IS working solution prepared in
0.2 M HCl. The entire sample was then loaded onto the SPE
cartridge and drawn through by vacuum. Afterwards, the
cartridge was washed with 1 mL of methanol, and sample
was eluted into a new test tube using 5% ammonium
hydroxide in methanol. The eluate pH was neutralized by
vacuum evaporation of the ammonium hydroxide. Samples
were then lyophilized to dryness and reconstituted with
100 mL of 50 mM TDFHA in 1:1 methanol/water prior to
analysis. The final volume results in a 5-fold increase in
sample over the samples not extracted by SPE.
ESI-TOFMS
Detection of amino acids was accomplished using an
Agilent 1969 orthogonal TOF mass spectrometer coupled to
a positive ESI source with dual spray needles for continuous
infusion of reference mass solution. Heated (3508C) drying
gas flowing at 9.0 L/min, with a nebulizer pressure of 40
psig, was used for droplet desolvation. Spray was induced
with a capillary voltage of 3000 V and the fragmentor
voltage was 100 V. The TOF was tuned and calibrated using
Agilent ESI-TOF calibration and tuning mix (Agilent Technologies). The data acquisition mass range was 50350 m/z at
10058 transients/scan and 0.93 scans/s. Reference mass
correction on each sample was performed with a continuous
infusion of Agilent TOF biopolymer analysis mix contain-
2719
Calibration curves
Calibration curves for each amino acid were constructed
using Analyst QS software and prepared so all amino acids
would be within expected physiological concentrations.
Most amino acids were calibrated from 1.56 to 400 mM. The
more abundant amino acids (Gln, Glu, Gly and Ala) were
calibrated from 25 to 3200 mM (see Table 2). Analyst QS was
used to choose the best fit for the calibration curve. Either a
quadratic or linear fit was applied to quantify most amino
acids.
Table 1. List of experimental parameters for amino acids and signal-to-noise (S/N) ratios obtained from a 125pm (nominal)
injection
Compound
Molecular
formula
Taurine
Aspartic acid
Hydroxyproline
Serine
Glycine
Glutamine-d5
Glutamine
Asparagine
Threonine
Glutamic acid-d3
Glutamic acid
Alanine
(Cysteine)2
Citrulline
Proline
Gly-Gln
Ala-Gln
Valine
Methionine-d3
Methionine
Tyrosine
Isoleucine
Leucine-d10
Leucine
Phenylalanine
Histidine
Tryptophan
Tryptophan-d5
Arginine
Ornithine
Lysine
C2H7NO3S
C4H7NO4
C5H9NO3
C3H7NO3
C2H5NO2
C5H5D5N2O3
C5H10N2O3
C4H8N2O3
C4H9NO3
C5H6D3NO4
C5H9NO4
C3H7N02
C6H12N2O4S2
C6H13N3O3
C5H9NO2
C7H13N3O4
C8H15N3O4
C5H11NO2
C5H8D3NO2S
C5H11NO2S
C9H11NO3
C6H13NO2
C6H3D10NO2
C6H13NO2
C9H11NO2
C6H9N3O2
C11H12N2O2
C11H7D5N2O2
C6H14N4O2
C5H12N2O2
C6H14N2O2
Exact mass
[MH]
Extracted
ion window
High cal
std (nM/mL)
126.0224
134.0453
132.0660
106.0504
76.0398
152.1000
146.0769
133.0613
120.0660
151.1000
148.0609
90.0555
241.0316
176.1035
116.0711
204.2000
218.1140
118.0868
153.1000
150.0588
182.0817
132.1024
142.2000
132.1024
166.0868
156.0773
205.0977
210.1000
175.1195
133.0977
147.1133
126.00126.04
134.01134.05
132.03132.07
106.02106.06
76.0176.05
152.05152.15
147.04147.08
133.03133.07
120.03120.07
151.05151.15
148.03148.07
90.0290.06
241.01241.05
176.01176.05
116.04116.08
204.10204.30
218.08218.12
118.05118.09
153.05153.15
150.03150.07
182.05182.09
132.08132.12
142.00142.30
132.08132.12
166.06166.10
156.05156.08
205.08205.12
210.00210.30
175.09175.13
133.08133.12
147.09147.13
1.56
1.56
1.56
1.56
25
NA
25
1.56
1.56
NA
12.5
12.5
1.56
1.56
1.56
NA
1.56
1.56
NA
1.56
1.56
1.56
NA
1.56
1.56
1.56
1.56
NA
1.56
1.56
1.56
400
400
400
400
3200
NA
3200
400
400
NA
1600
1600
400
400
400
NA
400
400
NA
400
400
400
NA
400
400
400
400
NA
400
400
400
S/N ratio
(pM injected)
851 (125)
52.5 (125)
1750 (125)
267 (125)
22.3 (3125)
637 (1000)
1450 (3125)
94.9 (125)
315 (125)
15.2 (1000)
714 (1562)
345 (125)
1160 (125)
383 (125)
355 (125)
2560 (1000)
508 (125)
163 (125)
1810 (1000)
737 (125)
722 (125)
340 (125)
1450 (1000)
228 (125)
1010 (125)
1090 (125)
383 (125)
1110 (1000)
1700 (125)
544 (125)
448 (125)
IS used
for quantitation
Glutamine-d5
Glutamic acid-d3
Glutamine-d5
Glutamic acid-d3
Glutamic acid-d3
NA
Glutamine-d5
Glutamine-d5
Glutamic acid-d3
NA
Glutamic acid-d3
Leucine-d10
Methionine-d3
Glutamine-d5
Glutamine-d5
NA
Gly-Gln
Leucine-d10
NA
Methionine-d3
Leucine-d10
Leucine-d10
NA
Leucine-d10
Leucine-d10
Tryptophan-d5
Tryptophan-d5
NA
Tryptophan-d5
Tryptophan-d5
Tryptophan-d5
Internal standard.
Table 2. Amino acids and internal standards used for quantitation, including the curve fit used and the correlation coefficient
obtained
Without cation exchange
Amino acid
IS
Curve fit
R2
Taurine (TAU)
Aspartic acid (ASP)
Hydroxyproline (HYP)
Serine (SER)
Glycine (GLY)
Glutamine (GLN)
Asparagine (ASN)
Threonine (THR)
Glutamic acid (GLU)
Alanine (ALA)
(Cysteine)2
Citrulline (CIT)
Proline (PRO)
Valine (VAL)
Methionine (MET)
Tyrosine (TYR)
Isoleucine (ISO)
Leucine (LEU)
Phenylalanine (PHE)
Histidine (HIS)
Tryptophan (TRP)
Arginine (ARG)
Ornithine (ORN)
Lysine (LYS)
Glutamine-d5
Glutamic acid-d3
Glutamine-d5
Glutamic acid-d3
Glutamic acid-d3
Glutamine-d5
Glutamine-d5
Glutamic acid-d3
Glutamic acid-d3
Leucine-d10
Methionine-d3
Glutamine-d5
Glutamine-d5
Leucine-d10
Methionine-d3
Leucine-d10
Leucine-d10
Leucine-d10
Leucine-d10
Leucine-d10
Tryptophan-d5
Leucine-d10
Leucine-d10
Leucine-d10
Quadratic
Quadratic
Quadratic
Linear
Quadratic
Quadratic
Quadratic
Quadratic
Quadratic
Quadratic
Quadratic
Quadratic
Quadratic
Quadratic
Linear
Linear
Linear
Quadratic
Quadratic
Linear
Quadratic
Quadratic
Quadratic
Quadratic
1
0.9997
1
0.9986
0.9996
1
1
0.9997
0.9998
0.9997
0.9999
1
0.9999
0.9999
0.9999
0.9999
0.9999
1
0.9999
0.9999
1
0.9999
0.9997
0.9998
RESULTS
IS
Curve fit
R2
Glutamine-d5
Glutamine-d5
Glutamine-d5
Glutamine-d5
Glutamine-d5
Glutamine-d5
Glutamine-d5
Glutamine-d5
Glutamate-d3
Leucine-d10
Glutamine-d5
Glutamine-d5
Glutamine-d5
Leucine-d10
Methionine-d3
Leucine-d10
Leucine-d10
Leucine-d10
Leucine-d10
Tryptophan-d5
Tryptophan-d5
Tryptophan-d5
Tryptophan-d5
Tryptophan-d5
Linear
Quadratic
Linear
Quadratic
Linear
Linear
Linear
Quadratic
Linear
Linear
Linear
Linear
Linear
Linear
Linear
Linear
Linear
Linear
Linear
Linear
Linear
Linear
Linear
Linear
0.9965
0.9996
0.9994
0.9999
0.9978
0.9996
0.9992
0.9999
0.9992
0.9993
0.9999
0.9988
0.9984
0.9968
0.9997
0.999
0.9989
0.9994
0.997
0.9999
0.9986
0.995
0.9997
0.9998
2721
Figure 1. Extracted ion chromatograms for 25 amino acids. Amino acid calibration and spike standards were prepared as
described in the Experimental section and analyzed according to the parameters listed in Table 1. Overlaid extracted ion
chromatograms of all 25 amino acids in a 400 nM/mL (nominal concentration) standard are shown. Note the separation
between the isobaric amino acids leucine and isoleucine. All peaks are displayed to scale.
broadened. The peak shape for Orn was significantly worse
in plasma samples (data not shown).
In order to determine if this retention time shift was due to
column overloading, 10, 5 and 2 mL of a spiked plasma
sample were loaded onto the column, and the retention time
for leucine-d10 was compared to that of the calibration
standard. The data show that a reduction in the amount of
sample loaded decreased the shift in retention times,
suggesting that the column is indeed overloaded. However,
decreasing the sample load significantly raised the lower
limit of quantitation for several compounds.
Figure 2. Glutamine integration reproduciblity. Overlay of glutamine extracted ion chromatograms (m/z 147.04147.08) of
five replicates of spiked plasma, showing sample-to-sample integration reproducibility.
Copyright # 2007 John Wiley & Sons, Ltd.
Figure 3. A tridecafluoroheptanoate (TDFHA) adduct elutes between 8 and 9 min and interferes with the analysis of
Ala-Gln and Val. Plasma samples were spiked with internal standards and analyzed as described in the Experimental
section. The mass spectrum of a large peak eluting between 8 and 9 min (A) and extracted ion chromatograms of TDFHA
adduct, Ala-Gln and Val (B) are shown. The exact mass of m/z 408.948579 corresponds to the molecular formula of
disodium tridecafluoroheptanoate, an adduct formed between the TDHFA ion-pairing agent and sodium in plasma. The
Ala-Glu dipeptide and Val are almost completely obscured by the adduct (B).
both analytes (Fig. 3(B)). In an effort to separate Ala-Gln and
Val from this peak, we experimented with (a) increasing the
hold at 15% B from 5 to 8 min, (b) changing the hold from 15%
B to 12 % B, and (c) changing the hold from 12% B to 10% B.
None of these changes improved the separation of Ala-Gln
and Val from the TDHFA adduct peak enough to reduce ion
suppression.
SPE cleanup
To eliminate the TDFHA adduct and additional non-target
compounds from the sample extract, a cation-exchange
cleanup step was performed on a series of calibration
standards, unspiked plasma, and spiked plasma. Although
slightly more time-consuming, the improvement in chromatographic performance provided by the cation-exchange
cleanup was significant. Retention time shift between the
calibration standards and plasma samples was virtually
eliminated. The abundance of the TDFHA adduct was also
decreased significantly enough to dramatically improve both
the accuracy and the precision of Val in plasma and spiked
plasma. The accuracy and precision of Ala-Gln was not
improved significantly after cation-exchange cleanup (data
not shown).
Copyright # 2007 John Wiley & Sons, Ltd.
Calibration linearity
Calibration linearity was compared between the samples
analyzed with and without cation-exchange cleanup. Overall, much better results were obtained with cation-exchange
cleanup. In the calibration without cation-exchange cleanup,
a quadratic regression was selected as the preferred fit for
most of the amino acids whilst, with cation exchange, a linear
regression fit was determined to be optimal. This improvement in performance could also be due to a 5-fold increase
in sample amount used for the preparation with cationexchange cleanup.
In the cation-exchange cleanup, glutamine-d5 was used
with much better quantitative results as an internal standard
for several of the early eluting amino acids when compared
to the samples that had not been cleaned up. Also for the later
eluting amino acids, tryptophan-d5 produced much better
quantitative results in the cation-exchange cleanup.
Amino acids which used an isotopically labeled analog for
quantitation (e.g. glutamine/glutamine-d5, methionine/
methionine-d3) produced excellent calibration curves as
expected. Much more accurate results could be obtained with
this method if stable isotope labeled analogs were utilized for
all target amino acids. This would be prohibitively expensive
Rapid Commun. Mass Spectrom. 2007; 21: 27172726
DOI: 10.1002/rcm
(Cysteine)2
(Cysteine)2
Alanine (ALA)
Alanine (ALA)
Alanyl-Glutamine (ALA-GLN)
Alanyl-Glutamine (ALA-GLN)
Arginine (ARG)
Arginine (ARG)
Asparagine (ASN)
Asparagine (ASN)
Aspartic acid (ASP)
Aspartic acid (ASP)
Citrulline (CIT)
Citrulline (CIT)
Glutamic acid (GLU)
Glutamic acid (GLU)
Glutamine (GLN)
Glutamine (GLN)
Glycine (GLY)
Glycine (GLY)
Histidine (HIS)
Histidine (HIS)
Hydroxyproline (HYP)
Hydroxyproline (HYP)
Isoleucine (ISO)
Isoleucine (ISO)
Leucine (LEU)
Leucine (LEU)
Lysine (LYS)
Lysine (LYS)
Methionine (MET)
Methionine (MET)
Ornithine (ORN)
Ornithine (ORN)
Phenylalanine (PHE)
Phenylalanine (PHE)
Proline (PRO)
Proline (PRO)
Serine (SER)
Serine (SER)
Taurine (TAU)
Taurine (TAU)
Threonine (THR)
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
100-5
0-5
Sample name
6.37
279.00
368.80
716.00
0.00
25.30
61.60
138.00
10.20
77.00
0.00
98.40
11.00
93.80
255.00
663.00
30.10
819.00
450.00
1350.00
88.40
200.00
5.17
78.70
28.80
119.00
58.70
154.00
116.00
189.00
11.80
122.00
277.00
376.00
47.60
163.00
41.80
145.00
76.10
170.00
5.45
42.40
75.30
(n 5) (day 2)
(n 5) (day 1)
39.12
220.40
382.40
772.80
0.00
21.42
69.40
165.60
8.73
76.12
0.00
107.60
13.40
97.82
227.20
571.40
27.68
773.00
410.20
1218.00
103.94
313.20
5.47
72.10
31.74
130.20
54.96
153.40
133.20
242.20
12.60
110.20
318.00
757.80
47.46
150.00
42.60
168.80
71.48
164.60
3.41
33.52
68.80
14.50
482.00
377.00
729.00
0.00
33.30
64.90
172.00
10.00
65.90
0.00
109.00
13.00
94.60
254.00
689.00
39.10
686.00
486.00
1360.00
86.40
247.00
5.24
65.10
22.50
88.10
52.20
138.00
131.00
244.00
10.20
120.00
238.00
522.00
40.70
151.00
50.80
153.00
81.20
196.00
4.45
33.60
74.50
(n 5) (day 3)
Table 3. Inter-day accuracy and precision of amino acids in human plasma without cation-exchange cleanup
20.00
327.13
376.07
739.27
0.00
26.67
65.30
158.53
9.64
73.01
0.00
105.00
12.47
95.41
245.40
641.13
32.29
759.33
448.73
1309.33
92.91
253.40
5.29
71.97
27.68
112.43
55.29
148.47
126.73
225.07
11.53
117.40
277.67
551.93
45.25
154.67
45.07
155.60
76.26
176.87
4.44
36.51
72.87
Average
Inter-day
17.05
137.28
6.85
29.76
0.00
6.06
3.92
18.07
0.80
6.17
0.00
5.76
1.29
2.13
15.77
61.77
6.02
67.55
37.92
79.25
9.60
56.87
0.16
6.80
4.72
21.80
3.26
9.07
9.36
31.25
1.22
6.32
40.00
192.65
3.94
7.23
4.98
12.11
4.86
16.79
1.02
5.10
3.54
Std Dev.
Inter-day
85.28
41.97
1.82
4.03
0.00
22.71
6.00
11.40
8.29
8.45
0.00
5.48
10.31
2.23
6.43
9.64
18.63
8.90
8.45
6.05
10.33
22.44
2.94
9.45
17.05
19.39
5.90
6.11
7.39
13.88
10.60
5.38
14.41
34.90
8.72
4.68
11.05
7.78
6.38
9.49
23.04
13.98
4.86
(%RSD)
Precision
63.36
105.00
82.94
98.93
90.88
107.58
160.49
66.67
84.75
93.18
98.33
105.87
274.27
109.41
110.53
100.00
100.00
100.00
400.00
800.00
800.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
(Continues)
32.07
93.23
100.00
100.00
26.67
100.00
100.61
90.80
400.00
100.00
153.57
Inter-day
200.00
(mM)
Spike conc.
DOI: 10.1002/rcm
67.72
81.58
111.41
97.53
100.00
100.00
100.00
100.00
2.15
4.03
1.95
19.19
16.31
49.69
54.40
3.67
1.16
2.73
5.97
18.38
31.33
71.14
170.40
28.72
140.13
31.09
112.67
63.05
130.77
173.00
29.80
142.00
25.50
96.40
28.00
51.70
172.00
27.50
137.00
30.40
109.00
72.80
151.00
Threonine (THR)
Tryptophan (TRP)
Tryptophan (TRP)
Tyrosine (TYR)
Tyrosine (TYR)
Valine (VAL)
Valine (VAL)
100-5
0-5
100-5
0-5
100-5
0-5
100-5
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
PLASMA
166.20
28.86
141.40
37.38
132.60
88.34
189.60
to do for all amino acids, but if only a few amino acids are
targeted, the benefits of improved accuracy would conceivably outweigh the costs.
Sample name
Table 3. (Continued)
Inter-day
Inter-day
Precision
Spike conc.
Inter-day
2725
Figure 4. Mass accuracy of the ESI-TOF is under 1 ppm for His and under 3 ppm for Lys and Arg. Plasma samples
were prepared and analyzed using RP-LC/ESI-TOF as described in the Experimental section. The mass spectra for
Lys, His and Arg are shown and mass accuracy was calculated using experimental mass and actual mass values as
shown. Overall the mass accuracy is well below 5 ppm, thereby reducing the number of possible empirical formulas for
these peaks and improving the qualitative spectral data.
DISCUSSION
Analysis of amino acids by ion-pairing RP-LC/ESI-TOF is a
viable alternative to traditional amino acid analysis methods,
such as GC/MS and ninhydrin methods, both of which
require derivatization. While the IC/ninhydrin method
requires very little sample preparation, relatively long
(approximately 12 h per sample) analysis times are needed
in order to achieve baseline separation suitable for quantitation. The method also generally requires a dedicated
system for online derivatization of samples in order to obtain
consistent results. Conversely, GC/MS methods traditionally have shorter run times, excellent chromatography, and
increased specificity; however, GC/MS methods require
extensive sample preparation and derivatization. Increased
handling of the sample due to numerous steps is not only
time-consuming, but can potentially lead to increased error
and variability in the results.
Although the preparation of samples for LC/MS analysis
using an amino acid kit is relatively simple compared to
derivatization, the kits are designed to be used with the
specific derivative chemistry they were developed for and
may not be optimal for every amino acid, nor for every
detection technique. Analysis of the resultant samples
requires very high flow rates (0.51.0 mL/min) and the
use of non-volatile salt buffers which are not readily
compatible with ESI-MS.
Tandem mass spectrometry with flow injection analysis
can be used to identify amino acids without chromatographic
separation through the use of monitoring the transition of
precursor ions to product ions or multiple reaction
monitoring (MRM). While this technique is extremely
Copyright # 2007 John Wiley & Sons, Ltd.
CONCLUSIONS
Ion-pairing reversed-phase chromatography coupled with
the current generation of small particle size columns makes
separation of amino acids possible without derivatization,
allowing for quick and reproducible sample preparation.
Mass accuracy obtained through time-of-flight mass spectrometry can be used in addition to retention time to provide
qualitative data that is not available with single quadrupole
or triple quadrupole mass spectrometers. The described
method can be utilized to provide quick and accurate results
for amino acids in human plasma.
Rapid Commun. Mass Spectrom. 2007; 21: 27172726
DOI: 10.1002/rcm
Acknowledgements
Support for this work was generously provided through the
Colorado Clinical Nutrition Research Unit (Funding through
NIH/NIDDK P30 DK048520-09, PI Dr. James Hill). The
authors would like to thank Dr. Patti Thureen for her helpful
comments.
REFERENCES
1. Jellum E, Kvittingen EA, Stokke O. Biomed. Environ. Mass
Spectrom. 1988; 16: 57.
2. Deng C, Deng Y. J. Chromatogr. B Analyt. Technol. Biomed. Life
Sci. 2003; 792: 261.
3. Magni F, Arnoldi L, Galati G, Galli Kienle M. Anal. Biochem.
1994; 220: 308.
4. Pitt JJ, Eggingtin E, Kahler SG. Clin. Chem. 2002; 48: 1970.
5. Shen X, Deng C, Wang B, Dong L. Anal. Bioanal. Chem. 2006;
384: 931.
6. Deng C, Li N, Zhang X. Rapid Commun. Mass Spectrom. 2004;
18: 2558.
7. des Robert C, Le Bacquer O, Piloquet H, Roze JC, Darmaun
D. Pediatr. Res. 2002; 51: 87.
8. Miller RG, Jahoor F, Reeds PJ, Heird WC, Jaksic T. J. Pediatr.
Surg. 1995; 30: 1325.
9. Spackman DH, Stein WH, Moore S. Anal. Chem. 1958; 30:
1185.