Download Analysis of 25 underivatized amino acids in human plasma using

RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2007; 21: 2717–2726
Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.3124
Analysis of 25 underivatized amino acids in human
plasma using ion-pairing reversed-phase liquid
chromatography/time-of-flight mass spectrometry
Michael Armstrong1, Karen Jonscher2 and Nichole A. Reisdorph1*
1
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
2
Received 1 December 2006; Revised 25 May 2007; Accepted 26 May 2007
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 80–120% 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,1–4 and nutritional studies of neonates.5–8 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).10–15 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: [email protected]
Contract/grant sponsor: Colorado Clinical Nutrition Research
Unit; contract/grant number: NIH/NIDDK P30 DK048520-09.
etry (MS/MS) utilizing flow injection analysis,4 each of
which has certain disadvantages. For example, buffers
included in kits may be incompatible with ESI.
Recently published methods have described the use of
perfluorinated acids as ion-pairing agents to improve the
separation of amino acids on C18 columns without the
requirement for specialty columns or pre-/post-column
derivatization.16,20–23 Piraud et al.23 utilized HPLC and
tandem mass spectrometry (LC/MS/MS) with tridecafluoroheptanoic acid (TDFHA) as an ion-pairing agent with a C18
column. While TDFHA improved separation, all amino acids
of interest were not completely resolved by HPLC. Therefore,
multiple reaction monitoring (MRM) was used to improve
the specificity of the method by monitoring specific transitions of precursor to product ions (e.g. glutamine ¼147>84).
The method of Piraud et al. was used to quantitate 76 amino
acids of biological interest and the quantitation of 16 amino
acids was validated.23
In the current study, we have adapted Piraud’s method of
ion-pairing reversed-phase liquid chromatography (IPRPLC) for use with an electrospray ionization time-of-flight
(ESI-TOF) mass spectrometer equipped with an analogto-digital converter (ADC) for signal processing. Sample
Copyright # 2007 John Wiley & Sons, Ltd.
2718 M. Armstrong, K. Jonscher and N. A. Reisdorph
preparation involves precipitation of proteins from plasma
using methanol fortified with stable isotope labeled internal
standards followed, in some cases, by cation exchange.
Extremely accurate mass measurements (approximately
1–10 ppm) obtained with a TOF were used to deduce the
identity of an analyte with a much higher degree of certainty
than a standard, high-resolution single quadrupole mass
spectrometer. With the exception of isobaric molecules such
as leucine and isoleucine, the mass-to-charge (m/z) of amino
acids can be verified to within 1–5 ppm, reducing misidentification of target amino acids in the presence of
co-eluting matrix components of similar molecular weight.
We found that IPRP-LC/ESI-TOF provides a quick, simple,
reproducible alternative to MS/MS analysis and used this
technique for the analysis of 25 amino acids in human
plasma. The method described in this study uses minimal
amounts of standards, reagents, and sample, can be applied
to any amino acid that ionizes by ESI, and can easily be
adapted to high-throughput sample analysis.
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 Bonfil’s 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).
Standards preparation procedure
Amino acid calibration and spike standards were prepared at
physiological concentration ranges from pure powder or
commercially available standards. Amino acid mix #1
contained amino acids which are stable in 0.1% hydrochloric
acid solution such as the branched chain amino acids and the
hydroxyl group containing amino acids, including Asp, Hyp,
Ser, Gly, Thr, Glu, Ala, (Cys)2, Pro, Cys, Val, Met, Tyr, Ile,
Leu, Phe, His, Trp, Arg, and Lys. Amino acid mix #2
contained the amino acids which are not stable in an acid
solution, such as Tau, Gln, Asn, Cit, Ala-Gln and Orn. All
calibration stocks and working standards were stored at
208C until use.
In addition to calibration standards, two separate internal
standard (IS) mixes were used to quantitate. IS mix #1
contained glutamine-d5 and methionine-d3 in water. IS mix
Copyright # 2007 John Wiley & Sons, Ltd.
#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 1–2 days prior to thawing for extraction and analysis.
Sample preparation procedure
Calibration standards were prepared by combining 10 mL
each of amino acid mixes 1 and 2 and 100 mL of IS working
solution. Standards were vortexed briefly and then centrifuged at 10 000 g for 5 min at 48C. An aliquot (70 mL) of
supernatant was transferred to a 96-well plate or HPLC vial
containing 30 mL of 1.7 mM TDFHA in water, providing a
final concentration of 0.5 mM TDFHA.
Samples were prepared by adding 20 mL of plasma to
100 mL of IS working solution and briefly vortexing. Samples
were then centrifuged at 10 000 g for 5 min at 48C, resulting in
a protein precipitate that was subsequently discarded. An
aliquot (70 mL) of supernatant was transferred to a 96-well
plate or HPLC vial containing 30 mL of 1.7 mM TDFHA in
water.
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 manufacturer’s
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.
High-performance liquid chromatography
Liquid chromatography was carried out using an Agilent
1100 series HPLC system equipped with a binary pump and
a micro wellplate autosampler (Agilent Technologies, Palo
Alto, CA, USA). Amino acids were separated using an
XDB-C18 column (2.1 50 mm) with a 1.8 mM particle size
(Agilent Technologies) operated at ambient temperature.
Buffer A was 0.5 mM TDFHA in HPLC-grade water, and
buffer B was 100% acetonitrile. The initial flow rate was
0.2 mL/min. Separation was accomplished using a gradient
as follows: 0% B for 2 min, then 0% to 15% B from 2 to 3 min,
hold at 15% B from 3 to 8 min, then 15% to 25% B from 8 to
11 min. The column was held at 25% B from 11 to 18 min, and
Rapid Commun. Mass Spectrom. 2007; 21: 2717–2726
DOI: 10.1002/rcm
Analysis of 25 underivatized amino acids in human plasma
then returned to 0% B from 18 to 19 minutes. The flow rate
was then increased to 0.4 mL/min from 19.01 to 29 min to
recondition the column. The flow rate was then returned to
0.2 mL/min at 29.01 min, and allowed to equilibrate for
3 min. A long reconditioning time and re-equilibration time
is required to obtain consistent retention times with this
column. The column was flushed with 100% acetonitrile for
1 h after every 30 injections to wash off accumulated
non-target analytes. Failing to flush the column with
acetonitrile after 30 injections will result in a degradation
in chromatography and retention time drift.
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 50–350 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
ing purine (m/z 121.050873) and hexamethoxyphosphazine
(m/z 322.048121) (Agilent Technologies) at 20 mL/min.
Ions monitored for quantitation
Ions monitored for quantitation (see Table 1) were extracted
using Analyst QS software (Applied Biosystems, Foster City,
CA). Signals from internal standards were extracted with a
window ranging from 0.05 to 0.15 Da, while target amino
acids were provided a 0.02 Da extraction window.
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.
Method accuracy and precision
To test method accuracy and precision, pooled human
plasma was analyzed unspiked and spiked at 100 mM
(nominal) of each amino acid. Intra-day accuracy and
precision (n ¼ 5) and inter-day accuracy and precision
(n ¼ 3) were calculated.
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
[MþH]
Extracted
ion window
Low cal std
(nM/mL)
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
126.00–126.04
134.01–134.05
132.03–132.07
106.02–106.06
76.01–76.05
152.05–152.15
147.04–147.08
133.03–133.07
120.03–120.07
151.05–151.15
148.03–148.07
90.02–90.06
241.01–241.05
176.01–176.05
116.04–116.08
204.10–204.30
218.08–218.12
118.05–118.09
153.05–153.15
150.03–150.07
182.05–182.09
132.08–132.12
142.00–142.30
132.08–132.12
166.06–166.10
156.05–156.08
205.08–205.12
210.00–210.30
175.09–175.13
133.08–133.12
147.09–147.13
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.
Copyright # 2007 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2007; 21: 2717–2726
DOI: 10.1002/rcm
2720 M. Armstrong, K. Jonscher and N. A. Reisdorph
Table 2. Amino acids and internal standards used for quantitation, including the curve fit used and the correlation coefficient
obtained
Without cation exchange
With 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
Intra-day accuracy and precision (n ¼ 5) was also
measured on samples that were prepared using SPE prior
to analysis.
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
time of 32 min. While this method is an improvement in
throughput and specificity over traditional amino acid
analysis methods, the throughput could be almost doubled
by using a quaternary or additional HPLC pump, a
column-switching module, and an additional column to
alternate column regeneration and sample analysis.
Chromatographic separation of amino acids
A brief comparison was conducted using Agilent XDB-C18
2.1 50 mm columns with either 1.8 or 3.5 mm solid-phase
particle sizes. Under identical gradient conditions the 1.8 mm
column showed superior resolution of the early eluting polar
amino acids (data not shown). An attempt was made to
improve the separation efficiency of the 3.5 mm column using
different gradient profiles and flow rates; however, the
1.8 mm column still appeared to provide the best separation
(data not shown).
Although the ESI-TOF provides excellent specificity via its
high mass accuracy, there are still instances where complete
or partial chromatographic resolution must be obtained for
accurate quantitation. One example is when an isotopomer,
or m þ n (where n ¼ the number of Daltons the ion is shifted
from the m þ 0 ion), in the mass spectrum of a compound
adds to the m þ 0 area of another compound (e.g. Gln and
Glu) as a result of co-elution. Another is the differentiation of
isobaric compounds such as Leu and Ile. The chromatographic resolution obtained using the 1.8 mm column was
>90%, separating Gln from Glu, and allowing for complete
resolution of Ile and Leu isobars.
When the final gradient was optimized and established, all
of the amino acids eluted within 16.5 min (Fig. 1). A relatively
long column re-equilibration time resulted in a total cycle
Copyright # 2007 John Wiley & Sons, Ltd.
Integration reproducibility and signal-to-noise
ratio
The ability of the ESI-TOF to maintain consistent mass
accuracy and peak integration over time was assessed. The
extracted ion chromatograms for glutamine in five replicate
spiked plasma samples were integrated and compared
(Fig. 2). The mass window for each replicate sample was
m/z 147.04–147.08. The relative standard deviation (RSD)
over the five replicates was 7.29, showing good sampleto-sample integration reproducibility.
Signal-to-noise (S/N) ratios were also calculated for all
amino acids (See Table 1). Most S/N ratios were greater than
200:1, with Gly being the lowest at 22:1 and Hyp being the
highest at 1750:1.
Matrix interference in plasma samples
There were significant differences in retention times for
amino acids from extracted standards versus plasma
samples, particularly for the later eluting compounds.
Retention times for amino acids eluting after 4 min were
shifted to as much as 1.5 min earlier in plasma (e.g. Ile and
Leu). The retention time shift in plasma samples did not
typically result in a decreased chromatographic resolution
except for the peak shape of Orn, which was significantly
Rapid Commun. Mass Spectrom. 2007; 21: 2717–2726
DOI: 10.1002/rcm
Analysis of 25 underivatized amino acids in human plasma
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.
Ion suppression from TDFHA adducts
While examining data acquired in a wider mass range to
determine the major source of column overload, a very large
peak at a retention time of 8–9 min was observed that could
not be detected when using the normal acquisition
parameters. This peak was almost non-existent in calibration
standards, but appeared at an extremely high abundance in
plasma samples. Using the accurate mass obtained from the
ESI-TOF data, the empirical formula for the most abundant
ion in the spectrum was calculated to be C7O2F13Na2. This
empirical formula corresponds to a sodium adduct of
tridecafluoroheptanoate, a product of the binding of sodium
salt in plasma with the ion-pairing agent in the aqueous
buffer. The exact reference corrected mass of this ion was
m/z ¼ 408.9483, with a theoretical mass of m/z ¼ 408.9486 (see
Fig. 3(A)). The less abundant ions in the spectrum
corresponded to clusters of this compound with
additional C7O2F13Na (m/z ¼ 385.9647) subunits.
Alanyl-glutamine (Ala-Gln) dipeptide and Val co-eluted
with this peak, which significantly suppressed the signal of
Figure 2. Glutamine integration reproduciblity. Overlay of glutamine extracted ion chromatograms (m/z 147.04–147.08) of
five replicates of spiked plasma, showing sample-to-sample integration reproducibility.
Copyright # 2007 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2007; 21: 2717–2726
DOI: 10.1002/rcm
2722 M. Armstrong, K. Jonscher and N. A. Reisdorph
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: 2717–2726
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
Analyte peak name
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 ¼ 5) (day 2)
(n ¼ 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
Avg. conc. (mM)
Avg. conc. (mM)
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 ¼ 5) (day 3)
Avg. conc. (mM)
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
Copyright # 2007 John Wiley & Sons, Ltd.
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
Spike recovery (%)
Inter-day
200.00
(mM)
Spike conc.
Analysis of 25 underivatized amino acids in human plasma
2723
Rapid Commun. Mass Spectrom. 2007; 21: 2717–2726
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
Spike recovery (%)
(mM)
(%RSD)
Std Dev.
Average
(n ¼ 5) (day 3)
(n ¼ 5) (day 2)
(n ¼ 5) (day 1)
Analyte peak name
Avg. conc. (mM)
Avg. conc. (mM)
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.
Accuracy and precision of method
Sample name
Table 3. (Continued)
Avg. conc. (mM)
Inter-day
Inter-day
Precision
Spike conc.
Inter-day
2724 M. Armstrong, K. Jonscher and N. A. Reisdorph
Copyright # 2007 John Wiley & Sons, Ltd.
To measure accuracy and precision, human plasma was
prepared and analyzed without amino acids spiked, and
with amino acids spiked at levels equivalent to the level 4
calibration standard (100 mM nominal concentrations). The
plasma samples were spiked and frozen at 808C for at least
24 h before thawing, preparing and analyzing. Three
separate aliquots of the unspiked/spiked plasma were
prepared and analyzed on three different days. Results
from the first day were used to calculate the intra-day
accuracy and precision and results from all three days were
used to calculate inter-day accuracy and precision (Table 3).
The same unspiked/spiked samples were prepared using
cation-exchange cleanup, and intra-day precision was
calculated.
Intra-day precision of all amino acids without cationexchange cleanup was <20 RSD (n ¼ 5) for all amino acids
measured. Only Cit, Gly, His, and Val had RSDs >10.
Intra-day spike recoveries for most amino acids was within
80–120% with the exception of Ala-Gln (21.4%), Asn (67.4%),
His (209%), Hyp (66.6%), Orn (440%), Pro (126%) and Tau
(30.1%). Due to extremely poor chromatography, His and
Orn peaks were poorly integrated, resulting in aberrantly
high recoveries. Ion suppression from plasma co-extractives
eluting in the void volume reduced the recovery of Tau,
while Ala-Gln recovery was affected by ion suppression
from TDFHA adducts.
Inter-day precision of most amino acids was <20 RSD (see
Table 3). However, some amino acids had very high
inter-day RSD due to low endogenous concentration
(Cys2), poor chromatography (Orn), or ion suppression
(Ala-Gln, Val). Inter-day spike recoveries for most amino
acids were within 80–120% with the exception of Cys2
(154%), Ala-Gln (26.7%), Asn (63.4%), His (160%), Hyp
(66.7%), Orn (274%), Tau (32%) and Val (67.7%). Reproducibility of Val quantitiation was good on the first two days of
the study; however, due to progressively degrading
chromatography, Val eventually co-eluted with the TDFHA
adduct peak and its signal was suppressed. This degradation
in chromatography can be improved through more frequent
washes with 100% acetonitrile, as was noted by Piraud et al.23
Intra-day precision of amino acids following cationexchange cleanup was <20 RSD (n ¼ 5) for all amino acids
measured. Intra-day accuracy following cation-exchange
cleanup was vastly improved over analysis without
cation-exchange cleanup. Recoveries for all amino acids
were between 78–127%, with only Ala (127%), Asn (78.3%),
Cit (78.3%) and Glu (79.2%) outside of 80–120%. The most
dramatic improvements occurred with Tau (118%), His
(93.4%) and Orn (84.8%).
We attribute the improvement in the results to the removal
of co-extracted non-target analytes and reduction of the
TDFHA adduct that resulted in significantly diminished ion
suppression. The degradation in chromatography over time
was also much less pronounced. While similar intra-day
precision was achieved without cation-exchange cleanup,
Rapid Commun. Mass Spectrom. 2007; 21: 2717–2726
DOI: 10.1002/rcm
Analysis of 25 underivatized amino acids in human plasma
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.
the few additional steps involved with the cleanup greatly
improve the overall performance of the method.
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 1–2 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 (0.5–1.0 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.
specific and sensitive, some analytes may be subject to ion
suppression due to the complex nature of the sample
matrix,23 resulting in anomalous quantitation levels.
While there have been some promising advances in amino
acid analysis utilizing sample introduction and ionization
methods such as matrix-assisted laser desorption/ionization
(MALDI)24 and high-field asymmetric waveform ion mobility spectroscopy (FAIMS),25 as of the time of writing neither
of these techniques has been investigated for analysis of
amino acids in biological fluids such as plasma or urine.
In spite of advantages such as high mass accuracy, TOFMS
has not been used extensively for quantitative analysis due to
limitations imposed by time-to-digital converters (TDC),
which have poor dynamic range and can have considerable
dead times when measuring high concentrations of analyte.
An analog-to-digital converter (ADC) can more accurately
measure signal intensity than a TDC. ADC technology allows
TOF mass spectrometers to be used for quantitative analysis
while retaining a high degree of mass accuracy. The mass
accuracy for amino acids obtained by this method of
correction is well below 5 ppm (Fig. 4).
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: 2717–2726
DOI: 10.1002/rcm
2726 M. Armstrong, K. Jonscher and N. A. Reisdorph
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.
Copyright # 2007 John Wiley & Sons, Ltd.
10. Mahwhinney TP, Robinett RS, Atalay A, Madson MA.
J. Chromatogr. 1986; 358: 231.
11. Calder AG, Garden KE, Andersen SE, Lobley GE. Rapid
Commun. Mass Spectrom. 1999; 13: 2080.
12. Wood PL, Amin Khan M, Moskal JR. J. Chromatogr. B 2006;
831: 313.
13. Husek JP. J. Chromatogr. 1991; 552: 289.
14. Husek JP. J. Chromatogr. B 1998; 717: 57.
15. Namera A, Yashiki M, Nishida M, Kojima T. J. Chromatogr. B
2002; 776: 49.
16. Petritis K, Chaimbault P, Elfakir C, Dreux M. J. Chromatogr. A
1999; 833: 147.
17. Muller C, Schafer P, Stortzel M, Vogt S, Weinmann W.
J. Chromatogr. B 2002; 773: 47.
18. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Anal.
Chem. 1998; 70: 882.
19. Cohen SA, Michaud DP. Anal. Biochem. 1993; 211: 279.
20. Petritis K, Chaimbault P, Elfakir C, Dreux M. J. Chromatogr. A
2000; 896: 253.
21. Chaimbault P, Petritis K, Elfakir C, Dreux M. J. Chromatogr. A
1999; 855: 191.
22. Piraud M, Vianey-Saban C, Petritis K, Elfakir C, Steghens
JP, Morla A, Bouchu D. Rapid commun. Mass Spectrom. 2003;
17: 1297.
23. Piraud M, Vianey-Saban C, Petritis K, Elfakir C, Steghens
JP, Bouchu D. Rapid Commun. Mass Spectrom. 2005; 17:
1587.
24. Alterman M, Gogichayeva N, Kornilayev B. Anal. Biochem.
2004; 335: 184.
25. McCooeye M, Mester Z. Rapid Commun. Mass Spectrom.
2006; 20: 1801.
Rapid Commun. Mass Spectrom. 2007; 21: 2717–2726
DOI: 10.1002/rcm