Food and BeverageEAAADirect nalysis of mino cids by HILIC– SI-MSLUAlexander Schriewer1, Katharina Johanna Heilen1, Heiko Hayen1, and Wen Jiang2, 1Institute ofInorganic and Analytical Chemistry, niversity of Münster, Münster, Germany, 2HI ICON AB‑Hydrophilic interaction liquid chromatography coupled toelectrospray ionization mass spectrometry (HILIC–ESI-MS) has
been established as a method to separate and quantify polar andionic analytes in a direct way for two decades. HILIC separation isbased on the polarity of analytes, so the more polar analytes havestronger retention on a HILIC column.
Highly polar amino acids carry great biological weight because they
are the basic structural units of proteins or enzymes. Accordingly, they
are an essential part of plants, animals, and humans. Quantitative
analysis of amino acids is crucial in many fields including clinicaldiagnostic, biomedical research, and food science (1). Aminoacids are classically analyzed by gas chromatography (GC), cationexchange (CEX) or reversed-phase liquid chromatography (LC) withUV absorbance, fluorescence detection (FD), or mass spectrometry(MS) (1). However, pre-column or post-column derivatization is often
needed to address either the retention problem or the detection issue.
HILIC and ESI-MS is a perfect match for analyzing amino acids in a
direct and fast manner (2).
In this application, we separated 14 amino acids with aniHILIC Fusion(+) column packed with charge modulated hydroxyethyl
amide silica. A mixed interaction—for example, hydrophilic partitioning,
weak electrostatic interactions, and hydrogen bonding—may beinvolved in the HILIC separations.

Experimental
LC–MS System: Agilent 1100er LC system and Thermo Fisher LTQ™
equipped with a HESI source, operated in positive ionization mode
for analysis of standards. For the dietary supplement, an Orbitrap™
Exactive classic equipped with a HESI source and operated inpositive ionization mode.
Column: 150 × 2.1 mm, 3.5-μm iHILIC-Fusion(+) (P/N
100.152.0310, HILICON AB)
Gradient Elution: A) acetonitrile–water–1 M ammonium acetate,
pH 5.75 (90:5:5); B) water–acetonitrile–1 M ammonium acetate,
pH 5.75 (90:5:5); 0–0.5 min (90:10) A–B; 0.5 to 25 min, gradientelution from (90:10) A–B to (60:40) A–B.
Flow Rate: 0.3 mL/min
Column Temperature: 40 °C
Injection Volume: 5 μL
Amino Acids: Arginine, asparagine, aspartic acid, glutamicacid, glutamine, hydroxyl-proline, isoleucine, leucine, lysine,phenylalanine, proline, tryptophan, tyrosine, and valine. 50 µM
of each amino acid was dissolved in water–acetonitrile (25:75)
solution.
Dietary Supplement: Whey Prime (Prozis). A 20 mg/mL measure
was dissolved in water and then filtered. The sample was further
diluted to 2 mg/mL for injection.

2LL

Figure 1: Extracted ion chromatograms of amino acid standards in HI IC separation with iHI IC-Fusion(+).

THE APPLICATIONS BOOK – July 2017

Food and Beverage

Figure 2: Extracted ion chromatograms of amino acids in dietary supplement.

Table 1: Retention times, classification of amino acids by
their side-chain, and pI values
Amino acidAbbr.

Classificationt R (min)pIa

TryptophanTrpnonpolar

4.2

6.0

PhenylalaninePhenonpolar

4.3

6.0

LeucineLeu

nonpolar

4.9

6.2

Isoleucine

Ile

nonpolar

5.3

6.2

Tyrosine

Tyrpolar

6.1

5.5

ValineValnonpolar

6.7

6.2

ProlinePropolar

8.5

7.1

Hydroxy-proline

retention and were mainly retained by hydrophilic partitioning.
The acidic and basic amino acids are not neutral and had extra
electrostatic interactions with the stationary phase. Interestingly, it
was found that the constitutional isomers leucine and isoleucine
were almost baseline separated in a generic linear gradient elution.
In the second step, we verified the applicability of the newly
developed method using a dietary supplement containing various
numbers of amino acids. As can be seen in Figure 2, leucine andisoleucine are well separated. Valine, glutamine, and hydroxyproline
present intense signals. In addition, proline, glutamic acid, arginine,and lysine were also detectable at lower intensities.
This work demonstrates that amino acids can be easily identified
through a HILIC–ESI-MS method. It offers the possibilities to quantify
this group of polar analytes in many application areas such asmedicine, biology, or nutritional science.

OH-Propolar

9.1

6.7

Glutamine

Glnpolar

10.5

5.7

Asparagine

Asnpolar

10.6

5.2

Aspartic acid

Aspacidic

14.2

3.4

Glutamic acid

Gluacidic14.4

2.8

References(1)

Arginine

Lysbasic

17.0

10.8

LysineArgbasic

18.7

9.8

apI values are based on www.chemicalize.com

H. Kaspar, K. Dettmer, W. Gronwald, and P.J. Oefner, Anal. Bioanal. Chem.
393, 445–452 (2009).

(2) M. Dell’mour, L. Jaitz, E.
Oburger, M. Puschenreiter,G. Koellensperger, and S.

Results and Conclusion

Hann, J. Sep. Sci. 33, 911–

As shown in Figure 1, 14 standards of proteinogenic amino acids
can be simultaneously determined using a iHILIC-Fusion(+)
column coupled with ESI-MS detection. The retention times,
classification of amino acids, and pI values are summarized inTable 1. As expected, the amino acids with a nonpolar side chain
like tryptophan, phenylalanine, leucine, and isoleucine eluted firstwith the lowest interactions with the HILIC stationary phase. The
polar amino acids like proline, glutamine, or asparagine had more

922 (2010).

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