Application Note

LC/MS DETERMINATION OFAMINO ACIDS
Using Compact Mass Spectrometry

INTRODUCTION
Amino acids are the building blocks of the protein
backbone and universally contain an amine andcarboxylic acid functional group. Free amino acid blood
determination is important for the early detection ofinborn errors of metabolism in the newborn screening
program. Also, amino acid usage has increased in thefood industry and requires product control analysis.
HPLC is the most common separation technique forthe analysis of samples containing amino acids, withtraditional approaches using pre or post column
derivatization and UV detection. However, derivatizationis adding unnecessary sample preparation steps andusing mass spectrometry as the detector allows foroverlapping analyte peaks and hence faster HPLC
analysis times.
Here, we show examples for the determination ofamino acids on a combined HPLC compact mass
spectrometry (LC/CMS) system for the rapid detection
and quantification of amino acids utilizing a novel
mixed mode LC column. This sample analysis strategy
results in a cost effective analysis system to detect andquantify free amino acids in mixtures, protein amino acidcompositions and amino acid biomarkers.

Figure 1: UHPLC-CMS set up for the detection and quantification of amino acids in complex mixturesutilizing a JASCO X-LC system (Jasco, USA) , an Intrada Amino Acid HPLC column (Imtakt Corporation,Japan) and the expressionL compact mass spectrometer (Advion, Inc. USA).

METHODLC method using a 150 mm x 2mm Intrada column with 3 um particles (Imtakt Corporation, Japan)
and a flow rate of 200-400 µL/min. Solvent A: Acetonitrile/Tetrahydrofuran/25 mM Ammonium
formate/Formic Acid 9/75/16/0.3 (v:v:v:v) and Solvent B: Acetonitrile/100 mM Ammonium formate
20/80 (v:v).

RESULTS
A first separation example is shown in Figure 1 witha 60 min chromatography separation and LC-CMS
detection of a standard 20 amino acid mixture using
selected ion monitoring (SIM) acquisition for eachanalyte. Here, a complete baseline separation canbe achieved and isotopic analytes such as Leucine
and Iso-Leucine are baseline separated and readily
distinguishable by their unique retention times.
However, due to the selectivity of the CMS
detector, a baseline separation of all analytes is notnecessarily required and faster chromatographycan be used to analyze the 20 amino acid mixture
in only 15 min (Figure 2) – a significant increasein sampling speed. Now, the Leu/Ile pair is still
resolved, albeit not baseline separated, and someother analytes are co-eluting. But since the coeluting peaks have different mass-to-charge
ratios they can easily be distinguished by mass
spectrometry, something not possible in UV
detection with or without derivatization.
Figure 3 shows example calibration functions forfour amino acids (Proline, Tyrosine, Phenylalanineand Threonine) with good linearity in the range of
5 to 1000 pmol on-column (ca. 600 pg to 180 ng of
the respective amino acid). This analysis employed
the 15 min chromatography for a faster sample
throughput. Observed linearity and sensitivity iswell within the required levels for eg. amino acidblood determination.

SIM(cts)

A: ACN/THF/25 mM
Ammoniumformate
10:75:15 0.3 vol% formic
acid
B:ACN/100 mM
Ammoniumformate 20:80

A: ACN/THF/25 mM
Ammoniumformate
10:75:15 0.3 vol% formic
acid
B:ACN/100 mM
Ammoniumformate 20:80

Iso-Leucine

Gradient:0-2 min 0% B
2-5.5 min 0-20%B
5.5-9 min 20-100 %B
9-12 min 100 %B
12-12.5 min 100-0 %B
12.5-15 min 0%B

Gradient:
0-12 min 0% B
12-26 min 0-20%B
26-40 min 20-100 %B
40-46 min 100 %B
46-47 min 100-0 %B
47-60 min 0%B

0.4 mL/min
Intrada Amino Acid 2x150mm

0.2 mL/min
Intrada Amino Acid 2x150mm

5 nmol on column

5 nmol on column

Time(sec)

Time(sec)

Figure 2: Example of a 60 min LC-CMS analysis of a 20 aminoacid mixture using SIM (5 nmol on column).

Figure 3: Example of a 15 min LC-CMS analysis of a 20 amino acidmixture using SIM acquisition of the unique m/z of each aminoacid.

SRM (cts)

SRM (cts)

OHNH
COOH

H2N

COOH

Amount oncolumn (pmol)

SRM (cts)

Amount oncolumn (pmol)

SRM (cts)
OH

H2N

COOH

H2N

COOH

Amount oncolumn (pmol)

Amount oncolumn (pmol)

Figure 4: Example calibration functions for four amino acids:
Proline, Tyrosine, Phenylalanine and Threonine (15 min LC-CMS
method).

H3C

H2N
O

OHO
H2N

L-Leucine

NH2OH

3-Aminobutyric acid

H3C

CH3

H3C

OH
H2N

2-Aminobutyric acidTo further show the potential of the LC-CMS
analysis system, Figure 4 shows the separationand detection gamma-aminobutyric acid (GABA)
isomers. All three isotopic and isobaric analytes
are baseline-separated and can be readily
distinguished. The same can be shown for a
mixture of isotopes of Leucine (Figure 5). Here, themobile phase solvents had to be slightly adjusted
and gradient conditions changed as indicated toachieve a separation of all three isotopes.

SIM(cts)

Methionine
LeucineOCH3

Ƴ-Aminobutyric acid (GABA)
A: ACN/THF/25 mM Ammoniumformate
10:75:15 0.3 vol% formic acid
B:ACN/100 mM Ammoniumformate
20:80
Gradient:
0-2 min 0% B
2-5.5 min 0-20%B
5.5-9 min 20-100 %B
9-12 min 100 %B
12-12.5 min 100-0 %B
12.5-15 min 0%B
0.4 mL/min Intrada Amino Acid 2x150mm

OHO

NH2

H3CHOO

CH3
NH2

H3C
HO

allo-L-iso-LeucineO

L-iso-Leucine

A: MeOH/H2O 85:15 0.3
vol% formic acid
B: ACN/100mM
Ammoniumformate 20:80
Gradient:
0-7 min 8% B
7-9 min 8-100%B
9-12 min 100%B
12-12.5 min 100-8%B
12.5-15 min 8%B
0.4 mL/min Intrada Amino
Acid 2x150 mm
500 pmol on column
MS:
ESI (+) SIM m/z 132.1

500 pmol on column
MS:
ESI (+) SIM m/z 104.1

Figure 5: Separation of GABA isomers using thesame 15 min LC-CMS method than before

Figure 6: Separation of L-Leucine isomers using amodified15 min gradient

CONCLUSIONS
A combination of ion-ion interaction chromatography and a compact mass spectrometer presents a costeffective and powerful analysis system for the detection and quantification of amino acids in complex
mixtures without the need for extra sample processing steps such as derivatizations.

ACKNOWLEDGEMENT
We would like to thank Itaru Yazawa from Imtakt Corporation, Japan for the generous donation of an
Intrada Amino Acid HPLC column and JASCO Corporation, USA for the loan of a UHPLC system.

www.expressioncms.com
info@advion.com

Advion is a leader in mass spectrometry & synthesis solutions. The expression CMS is a high performance, compact, aordable single quadmass spectrometer. Its compact size allows it to t in space-limited labs for direct access and immediate results for chemists requiring massconrmation, reaction monitoring, quality control and purity analysis.