Analysis of 25 underivatized amino acids in human plasma using ion-pairing reversed-phase liquid chromatography/time-of-flight mass spectrometry

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2007; 21: 2717–2726

) DOI: 10.1002/rcm.3124
Published online in Wiley InterScience (www.interscience.wiley.com
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*1Department of Immunology, National Jewish Medical and Research Center, Denver, CO 80206, USA2University of Colorado at Denver and Health Science Center, Clinical Nutrition Research Unit, Department of Anesthesiology, Denver,

CO 80262, USA

Received 1 December 2006; Revised 25 May 2007; Accepted 26 May 2007

*Corresponology, NJackson SE-mail: RContract/Unit; con

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 Tradi-

tionally, 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 chromato-

graphy/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 sup-

pression 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-

ndence to: N. A. Reisdorph, Department of Immu-ational Jewish Medical and Research Center, 1400

treet K924, Denver, CO 80206, [email protected] sponsor: Colorado Clinical Nutrition Research

tract/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 tridecafluor-

oheptanoic 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 transi-

tions of precursor to product ions (e.g. glutamine¼147>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 (IPRP-

LC) for use with an electrospray ionization time-of-flight

(ESI-TOF) mass spectrometer equipped with an analog-

to-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 mis-

identification 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

ReagentsNanopure water (18.2VOhms) was used for sample pre-

paration. 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 (And-

over, 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 procedureAmino 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


#2 contained leucine-d10, glutamic acid-d3 and tryptop-

han-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 �808Cfor 1–2 days prior to thawing for extraction and analysis.

Sample preparation procedureCalibration standards were prepared by combining 10mL

each of amino acid mixes 1 and 2 and 100mL of IS working

solution. Standards were vortexed briefly and then centri-

fuged at 10 000 g for 5 min at 48C. An aliquot (70mL) of

supernatant was transferred to a 96-well plate or HPLC vial

containing 30mL of 1.7 mM TDFHA in water, providing a

final concentration of 0.5 mM TDFHA.

Samples were prepared by adding 20mL of plasma to

100mL 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 (70mL) of supernatant was transferred to a 96-well

plate or HPLC vial containing 30mL of 1.7 mM TDFHA in

water.

Solid-phase extractionFor 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, 100mL 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 chromatographyLiquid 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 1.8mM 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


DOI: 10.1002/rcm

Analysis of 25 underivatized amino acids in human plasma 2719

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-TOFMSDetection 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 Tech-

nologies). The data acquisition mass range was 50–350m/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-

Table 1. List of experimental parameters for amino acids and si

injection

CompoundMolecularformula

Exact mass[MþH]

Extractedion window

L

Taurine C2H7NO3S 126.0224 126.00–126.04Aspartic acid C4H7NO4 134.0453 134.01–134.05Hydroxyproline C5H9NO3 132.0660 132.03–132.07Serine C3H7NO3 106.0504 106.02–106.06Glycine C2H5NO2 76.0398 76.01–76.05Glutamine-d5� C5H5D5N2O3 152.1000 152.05–152.15Glutamine C5H10N2O3 146.0769 147.04–147.08Asparagine C4H8N2O3 133.0613 133.03–133.07Threonine C4H9NO3 120.0660 120.03–120.07Glutamic acid-d3� C5H6D3NO4 151.1000 151.05–151.15Glutamic acid C5H9NO4 148.0609 148.03–148.07Alanine C3H7N02 90.0555 90.02–90.06(Cysteine)2 C6H12N2O4S2 241.0316 241.01–241.05Citrulline C6H13N3O3 176.1035 176.01–176.05Proline C5H9NO2 116.0711 116.04–116.08Gly-Gln� C7H13N3O4 204.2000 204.10–204.30Ala-Gln C8H15N3O4 218.1140 218.08–218.12Valine C5H11NO2 118.0868 118.05–118.09Methionine-d3� C5H8D3NO2S 153.1000 153.05–153.15Methionine C5H11NO2S 150.0588 150.03–150.07Tyrosine C9H11NO3 182.0817 182.05–182.09Isoleucine C6H13NO2 132.1024 132.08–132.12Leucine-d10� C6H3D10NO2 142.2000 142.00–142.30Leucine C6H13NO2 132.1024 132.08–132.12Phenylalanine C9H11NO2 166.0868 166.06–166.10Histidine C6H9N3O2 156.0773 156.05–156.08Tryptophan C11H12N2O2 205.0977 205.08–205.12Tryptophan-d5� C11H7D5N2O2 210.1000 210.00–210.30Arginine C6H14N4O2 175.1195 175.09–175.13Ornithine C5H12N2O2 133.0977 133.08–133.12Lysine C6H14N2O2 147.1133 147.09–147.13

� Internal standard.


ing purine (m/z 121.050873) and hexamethoxyphosphazine

(m/z 322.048121) (Agilent Technologies) at 20mL/min.

Ions monitored for quantitationIons 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 curvesCalibration 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 400mM. The

more abundant amino acids (Gln, Glu, Gly and Ala) were

calibrated from 25 to 3200mM (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 precisionTo 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 (n¼ 5) and inter-day accuracy and precision

(n¼ 3) were calculated.

gnal-to-noise (S/N) ratios obtained from a 125pm (nominal)

ow cal std(nM/mL)

High calstd (nM/mL)

S/N ratio(pM injected)

IS usedfor quantitation

1.56 400 851 (125) Glutamine-d51.56 400 52.5 (125) Glutamic acid-d31.56 400 1750 (125) Glutamine-d51.56 400 267 (125) Glutamic acid-d325 3200 22.3 (3125) Glutamic acid-d3

NA NA 637 (1000) NA25 3200 1450 (3125) Glutamine-d5

1.56 400 94.9 (125) Glutamine-d51.56 400 315 (125) Glutamic acid-d3NA NA 15.2 (1000) NA12.5 1600 714 (1562) Glutamic acid-d312.5 1600 345 (125) Leucine-d101.56 400 1160 (125) Methionine-d31.56 400 383 (125) Glutamine-d51.56 400 355 (125) Glutamine-d5NA NA 2560 (1000) NA1.56 400 508 (125) Gly-Gln1.56 400 163 (125) Leucine-d10NA NA 1810 (1000) NA1.56 400 737 (125) Methionine-d31.56 400 722 (125) Leucine-d101.56 400 340 (125) Leucine-d10NA NA 1450 (1000) NA1.56 400 228 (125) Leucine-d101.56 400 1010 (125) Leucine-d101.56 400 1090 (125) Tryptophan-d51.56 400 383 (125) Tryptophan-d5NA NA 1110 (1000) NA1.56 400 1700 (125) Tryptophan-d51.56 400 544 (125) Tryptophan-d51.56 400 448 (125) Tryptophan-d5


DOI: 10.1002/rcm

Table 2. Amino acids and internal standards used for quantitation, including the curve fit used and the correlation coefficient

obtained

Amino acid

Without cation exchange With cation exchange

IS Curve fit R2 IS Curve fit R2

Taurine (TAU) Glutamine-d5 Quadratic 1 Glutamine-d5 Linear 0.9965Aspartic acid (ASP) Glutamic acid-d3 Quadratic 0.9997 Glutamine-d5 Quadratic 0.9996Hydroxyproline (HYP) Glutamine-d5 Quadratic 1 Glutamine-d5 Linear 0.9994Serine (SER) Glutamic acid-d3 Linear 0.9986 Glutamine-d5 Quadratic 0.9999Glycine (GLY) Glutamic acid-d3 Quadratic 0.9996 Glutamine-d5 Linear 0.9978Glutamine (GLN) Glutamine-d5 Quadratic 1 Glutamine-d5 Linear 0.9996Asparagine (ASN) Glutamine-d5 Quadratic 1 Glutamine-d5 Linear 0.9992Threonine (THR) Glutamic acid-d3 Quadratic 0.9997 Glutamine-d5 Quadratic 0.9999Glutamic acid (GLU) Glutamic acid-d3 Quadratic 0.9998 Glutamate-d3 Linear 0.9992Alanine (ALA) Leucine-d10 Quadratic 0.9997 Leucine-d10 Linear 0.9993(Cysteine)2 Methionine-d3 Quadratic 0.9999 Glutamine-d5 Linear 0.9999Citrulline (CIT) Glutamine-d5 Quadratic 1 Glutamine-d5 Linear 0.9988Proline (PRO) Glutamine-d5 Quadratic 0.9999 Glutamine-d5 Linear 0.9984Valine (VAL) Leucine-d10 Quadratic 0.9999 Leucine-d10 Linear 0.9968Methionine (MET) Methionine-d3 Linear 0.9999 Methionine-d3 Linear 0.9997Tyrosine (TYR) Leucine-d10 Linear 0.9999 Leucine-d10 Linear 0.999Isoleucine (ISO) Leucine-d10 Linear 0.9999 Leucine-d10 Linear 0.9989Leucine (LEU) Leucine-d10 Quadratic 1 Leucine-d10 Linear 0.9994Phenylalanine (PHE) Leucine-d10 Quadratic 0.9999 Leucine-d10 Linear 0.997Histidine (HIS) Leucine-d10 Linear 0.9999 Tryptophan-d5 Linear 0.9999Tryptophan (TRP) Tryptophan-d5 Quadratic 1 Tryptophan-d5 Linear 0.9986Arginine (ARG) Leucine-d10 Quadratic 0.9999 Tryptophan-d5 Linear 0.995Ornithine (ORN) Leucine-d10 Quadratic 0.9997 Tryptophan-d5 Linear 0.9997Lysine (LYS) Leucine-d10 Quadratic 0.9998 Tryptophan-d5 Linear 0.9998


Intra-day accuracy and precision (n¼ 5) was also

measured on samples that were prepared using SPE prior

to analysis.

RESULTS

Chromatographic separation of amino acidsA brief comparison was conducted using Agilent XDB-C18

2.1� 50 mm columns with either 1.8 or 3.5mm solid-phase

particle sizes. Under identical gradient conditions the 1.8mm

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 3.5mm column using

different gradient profiles and flow rates; however, the

1.8mm 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 n¼ 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 chromato-

graphic resolution obtained using the 1.8mm 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


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.

Integration reproducibility and signal-to-noiseratioThe 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 sample-

to-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 samplesThere 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


DOI: 10.1002/rcm

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 2mL 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 adductsWhile 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

Figure 2. Glutamine integration reproduciblity. Overlay of glutam

five replicates of spiked plasma, showing sample-to-sample in


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/z¼ 408.9483, with a theoretical mass of m/z¼ 408.9486 (see

Fig. 3(A)). The less abundant ions in the spectrum

corresponded to clusters of this compound with

additional C7O2F13Na (m/z¼ 385.9647) subunits.

Alanyl-glutamine (Ala-Gln) dipeptide and Val co-eluted

with this peak, which significantly suppressed the signal of

ine extracted ion chromatograms (m/z 147.04–147.08) of

tegration reproducibility.


DOI: 10.1002/rcm

Figure 3. A tridecafluoroheptanoate (TDFHA) adduct elutes between 8 and 9min 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 9min (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 cleanupTo 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 chro-

matographic 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).


Calibration linearityCalibration linearity was compared between the samples

analyzed with and without cation-exchange cleanup. Over-

all, 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 improve-

ment in performance could also be due to a 5-fold increase

in sample amount used for the preparation with cation-

exchange 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


DOI: 10.1002/rcm

Table

3.Inter-dayaccuracyandprecisionofaminoacidsin

humanplasmawithoutcation-exchangecleanup

Sam

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Av

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c.(m

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Inte

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ayIn

ter-

day

Pre

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Sp

ike

con

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ter-

day

(n¼

5)(d

ay1)

(n¼

5)(d

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(n¼

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Av

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tdD

ev.

(%R

SD

)(m

M)

Sp

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)

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(Cy

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ne)

239

.12

6.37

14.5

020

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17.0

585

.28

PL

AS

MA

100-

5(C

yst

ein

e)2

220.

4027

9.00

482.

0032

7.13

137.

2841

.97

200.

0015

3.57

PL

AS

MA

0-5

Ala

nin

e(A

LA

)38

2.40

368.

8037

7.00

376.

076.

851.

82P

LA

SM

A10

0-5

Ala

nin

e(A

LA

)77

2.80

716.

0072

9.00

739.

2729

.76

4.03

400.

0090

.80

PL

AS

MA

0-5

Ala

ny

l-G

luta

min

e(A

LA

-GL

N)

0.00

0.00

0.00

0.00

0.00

0.00

PL

AS

MA

100-

5A

lan

yl-

Glu

tam

ine

(AL

A-G

LN

)21

.42

25.3

033

.30

26.6

76.

0622

.71

100.

0026

.67

PL

AS

MA

0-5

Arg

inin

e(A

RG

)69

.40

61.6

064

.90

65.3

03.

926.

00P

LA

SM

A10

0-5

Arg

inin

e(A

RG

)16

5.60

138.

0017

2.00

158.

5318

.07

11.4

010

0.00

93.2

3P

LA

SM

A0-

5A

spar

agin

e(A

SN

)8.

7310

.20

10.0

09.

640.

808.

29P

LA

SM

A10

0-5

Asp

arag

ine

(AS

N)

76.1

277

.00

65.9

073

.01

6.17

8.45

100.

0063

.36

PL

AS

MA

0-5

Asp

arti

cac

id(A

SP

)0.

000.

000.

000.

000.

000.

00P

LA

SM

A10

0-5

Asp

arti

cac

id(A

SP

)10

7.60

98.4

010

9.00

105.

005.

765.

4810

0.00

105.

00P

LA

SM

A0-

5C

itru

llin

e(C

IT)

13.4

011

.00

13.0

012

.47

1.29

10.3

1P

LA

SM

A10

0-5

Cit

rull

ine

(CIT

)97

.82

93.8

094

.60

95.4

12.

132.

2310

0.00

82.9

4P

LA

SM

A0-

5G

luta

mic

acid

(GL

U)

227.

2025

5.00

254.

0024

5.40

15.7

76.

43P

LA

SM

A10

0-5

Glu

tam

icac

id(G

LU

)57

1.40

663.

0068

9.00

641.

1361

.77

9.64

400.

0098

.93

PL

AS

MA

0-5

Glu

tam

ine

(GL

N)

27.6

830

.10

39.1

032

.29

6.02

18.6

3P

LA

SM

A10

0-5

Glu

tam

ine

(GL

N)

773.

0081

9.00

686.

0075

9.33

67.5

58.

9080

0.00

90.8

8P

LA

SM

A0-

5G

lyci

ne

(GL

Y)

410.

2045

0.00

486.

0044

8.73

37.9

28.

45P

LA

SM

A10

0-5

Gly

cin

e(G

LY

)12

18.0

013

50.0

013

60.0

013

09.3

379

.25

6.05

800.

0010

7.58

PL

AS

MA

0-5

His

tid

ine

(HIS

)10

3.94

88.4

086

.40

92.9

19.

6010

.33

PL

AS

MA

100-

5H

isti

din

e(H

IS)

313.

2020

0.00

247.

0025

3.40

56.8

722

.44

100.

0016

0.49

PL

AS

MA

0-5

Hy

dro

xy

pro

lin

e(H

YP

)5.

475.

175.

245.

290.

162.

94P

LA

SM

A10

0-5

Hy

dro

xy

pro

lin

e(H

YP

)72

.10

78.7

065

.10

71.9

76.

809.

4510

0.00

66.6

7P

LA

SM

A0-

5Is

ole

uci

ne

(IS

O)

31.7

428

.80

22.5

027

.68

4.72

17.0

5P

LA

SM

A10

0-5

Iso

leu

cin

e(I

SO

)13

0.20

119.

0088

.10

112.

4321

.80

19.3

910

0.00

84.7

5P

LA

SM

A0-

5L

euci

ne

(LE

U)

54.9

658

.70

52.2

055

.29

3.26

5.90

PL

AS

MA

100-

5L

euci

ne

(LE

U)

153.

4015

4.00

138.

0014

8.47

9.07

6.11

100.

0093

.18

PL

AS

MA

0-5

Ly

sin

e(L

YS

)13

3.20

116.

0013

1.00

126.

739.

367.

39P

LA

SM

A10

0-5

Ly

sin

e(L

YS

)24

2.20

189.

0024

4.00

225.

0731

.25

13.8

810

0.00

98.3

3P

LA

SM

A0-

5M

eth

ion

ine

(ME

T)

12.6

011

.80

10.2

011

.53

1.22

10.6

0P

LA

SM

A10

0-5

Met

hio

nin

e(M

ET

)11

0.20

122.

0012

0.00

117.

406.

325.

3810

0.00

105.

87P

LA

SM

A0-

5O

rnit

hin

e(O

RN

)31

8.00

277.

0023

8.00

277.

6740

.00

14.4

1P

LA

SM

A10

0-5

Orn

ith

ine

(OR

N)

757.

8037

6.00

522.

0055

1.93

192.

6534

.90

100.

0027

4.27

PL

AS

MA

0-5

Ph

eny

lala

nin

e(P

HE

)47

.46

47.6

040

.70

45.2

53.

948.

72P

LA

SM

A10

0-5

Ph

eny

lala

nin

e(P

HE

)15

0.00

163.

0015

1.00

154.

677.

234.

6810

0.00

109.

41P

LA

SM

A0-

5P

roli

ne

(PR

O)

42.6

041

.80

50.8

045

.07

4.98

11.0

5P

LA

SM

A10

0-5

Pro

lin

e(P

RO

)16

8.80

145.

0015

3.00

155.

6012

.11

7.78

100.

0011

0.53

PL

AS

MA

0-5

Ser

ine

(SE

R)

71.4

876

.10

81.2

076

.26

4.86

6.38

PL

AS

MA

100-

5S

erin

e(S

ER

)16

4.60

170.

0019

6.00

176.

8716

.79

9.49

100.

0010

0.61

PL

AS

MA

0-5

Tau

rin

e(T

AU

)3.

415.

454.

454.

441.

0223

.04

PL

AS

MA

100-

5T

auri

ne

(TA

U)

33.5

242

.40

33.6

036

.51

5.10

13.9

810

0.00

32.0

7P

LA

SM

A0-

5T

hre

on

ine

(TH

R)

68.8

075

.30

74.5

072

.87

3.54

4.86

Copyright # 2007 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2007; 21: 2717–2726

DOI: 10.1002/rcm


(Continues)

Table

3.(C

ontinued)

Sam

ple

nam

eA

nal

yte

pea

kn

ame

Av

g.

con

c.(m

M)

Av

g.

con

c.(m

M)

Av

g.

con

c.(m

M)

Inte

r-d

ayIn

ter-

day

Pre

cisi

on

Sp

ike

con

c.In

ter-

day

(n¼

5)(d

ay1)

(n¼

5)(d

ay2)

(n¼

5)(d

ay3)

Av

erag

eS

tdD

ev.

(%R

SD

)(m

M)

Sp

ike

reco

ver

y(%

)

PL

AS

MA

100-

5T

hre

on

ine

(TH

R)

166.

2017

2.00

173.

0017

0.40

3.67

2.15

100.

0097

.53

PL

AS

MA

0-5

Try

pto

ph

an(T

RP

)28

.86

27.5

029

.80

28.7

21.

164.

03P

LA

SM

A10

0-5

Try

pto

ph

an(T

RP

)14

1.40

137.

0014

2.00

140.

132.

731.

9510

0.00

111.

41P

LA

SM

A0-

5T

yro

sin

e(T

YR

)37

.38

30.4

025

.50

31.0

95.

9719

.19

PL

AS

MA

100-

5T

yro

sin

e(T

YR

)13

2.60

109.

0096

.40

112.

6718

.38

16.3

110

0.00

81.5

8P

LA

SM

A0-

5V

alin

e(V

AL

)88

.34

72.8

028

.00

63.0

531

.33

49.6

9P

LA

SM

A10

0-5

Val

ine

(VA

L)

189.

6015

1.00

51.7

013

0.77

71.1

454

.40

100.

0067

.72



to do for all amino acids, but if only a few amino acids are

targeted, the benefits of improved accuracy would con-

ceivably outweigh the costs.

Accuracy and precision of methodTo 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 (100mM 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 cation-

exchange cleanup was <20 RSD (n¼ 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%). Reprodu-

cibility 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 cation-

exchange cleanup was <20 RSD (n¼ 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,


DOI: 10.1002/rcm

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 quanti-

tation. The method also generally requires a dedicated

system for online derivatization of samples in order to obtain

consistent results. Conversely, GC/MS methods tradition-

ally 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


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 mobi-

lity 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 spec-

trometry 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.


DOI: 10.1002/rcm


AcknowledgementsSupport 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.

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DOI: 10.1002/rcm

Documents

Analysis of 25 underivatized amino acids in human plasma using ion-pairing reversed-phase liquid chromatography/time-of-flight mass spectrometry