TP 510
Simultaneous quantitative analysis of 20 amino acids in food samples without derivatization using LC-MS/MS1
12
Keiko Matsumoto ; Jun Watanabe ; Itaru Yazawa

1 Shimadzu Corporation, Kyoto, Japan; 2 Imtakt Corporation, Kyoto, Japan

1. Introduction
In order to detect many kinds of amino acids with high selectivity in food samples, the LC/MS analysis have been used widely. Amino acids are high polar compound, so they are hard to beretained to reverse-phased column such as ODS (typical method in LC/MS analysis). It needs their derivartization or addition of ion pair reagent in mobile phase to retain them. For easier
analysis of amino acids, it is expected to develop the method without using reagents mentioned above.
This time, we tried to develop a simultaneous high sensitive analysis method of 20 amino acids by LC/MS/MS with mix-mode column (ion exchange, normal-phase) and the typical volatilemobile phase suitable for LC/MS analysis.

2.Methods and Materials

Ionization: ESI, Positive MRM mode
MRM transition are shown in Table 1.
TrpMobile Phase
A: Acetonitrile/ Formic acid =100 / 0.1
B: 100mM Ammonium formate

ThrPheUF-MRMHigh-Speed MRM at 555ch/sec

ProUFswitching
High-Speed Polarity Switching 5msec

4.4

3. Result3-1. Method development
First, MRM method of 20 amino acids was optimized. As a result, all compounds wereable to be detected high sensitively and were detected in positive MRM transitions. As
the setting temperature of ESI heating gas was found to affected on the sensitivity ofamino acids, it was also optimized. Even though amino acids were not derivartized andion-pairing reagent wasn’t used, 20 amino acids were retained by using a mixed-mode
stationary phase structure and separated excellently on the below-mentioned condition.

4.5

4.7

4.8

4.9

5.0

5.1

5.2 min

LysArgValGlu

Tyr(Cys)2Asnmin
9.0
Figure 2 Mass Chromatograms of 20 Amino acids (concentration of each compound : 10nmol/mL）
2.0

3.0

4.0

5.0

6.0

7.0

8.0

Case2 (High Resolution condition)%RSD

0.01-100

0.99501.4

Phe

166.10>120.10

0.01-1000.9971

1.2

Tyr

182.10>136.00

0.05-100

0.99001.7

Met150.10>56.10

0.05-200

0.99630.1

Lue,Lle132.10>86.15

0.01-100

0.9955

0.7Val118.10>72.05

0.05-1000.9991

1.9

Glu

148.10>84.10

0.05-100.99654.5

Pro

116.10>70.10

0.01-50

0.99331.5

Asp

134.20>74.10

0.5-500

0.9953

1.4

Thr

120.10>74.00

0.1-500.99234.5

Ala

90.10>44.10

0.5-500

0.998916.2

Ser106.10>60.20

0.5-500

0.99886.5

Gln

147.10>84.10

0.05-10.99593.9

Gly

76.20>29.90

5-200

0.997411.0

Asn

133.10>74.05

0.05-20

0.9939

6.1(Cys)2241.00>151.95

0.05-200.99092.3

His

156.10>110.10

0.05-200

0.9983

1.7Lys147.10>84.10

0.05-50.9908

0.9Arg175.10>70.10

0.01-100

0.9956

0.5

Leu
Trp IleMet
2.0SerThr
GlyIle
4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 min

Trp PheLeu
TyrArgMet ValGlu

ThrGln(Cys)2AsnHis

Trp
LeuIle

LysMet
2.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

Ara

Phe

9.0

10.0 minFigure 3 Mass Chromatograms of 20 Amino acids (concentration of each compound : 10nmol/mL）

3.0

4.3

TyrValThr
4.0

4.4

4.5

4.64.7

4.8

4.9

5.0

min

6.0

7.0

5.1

5.2minHis

Lys(Cys)2
6.0

7.0

8.0

9.0

min

Beer

4.5

3.0

Figure 6

4.6

4.7

Glu Gln
4.0

4.8

4.9

5.0

3.0

Arg
Lys

6.0

7.0

8.0

9.0

min

Mass Chromatograms of Beer (10 fold dilution with 0.1N HCl)Pro

2.0min(Cys)2

5.0

4.5

Tyr

Glu

Val

Asp

Ser
Gly

4.6

Sweet Cooking Rice Wine

Asn

AlaMet

5.1

His

Thr

Leu IleAsp

Gly Ser

Trp

4.7

Gln

4.8

4.9

5.0

5.1

min

4.0

5.0

6.0

Lys

His(Cys)2
7.08.0Arg
9.0minFigure7 Mass Chromatograms of Sweet Cooking Rice Wine (100 fold dilution with 0.1N HCl)

4. Conclusions- 20 amino acids could be separated without derivatization using a typical volatile mobile
phase suitable for LC/MS analysis and detected with high sensitivity.

His

Arg

- This methods was able to be applied to the analysis of amino acids in various foodsamples.

Glu
5.0

5.0

Ala
Thr

2.0

Asp
5.1

4.9

5.0

Tyr
Leu
IleValMet

LysSer

4.0

PheSports Beverage

Gly

3.0

TrpPro
Asp

4.8

Gln
Glu
Asn
AlaPhePro4.7Asn

The analysis of the amino acids contained in sports beverage on the market was carried out. Inthe case of sports beverage, all amino acids written in the package were detected.
Ara

4.6

Pro3-2. The analysis of 20amino acids in food samples

ThrTyrVal

4.5

Figure 5 Mass Chromatograms of Japanese Sake (100 fold dilution with 0.1N HCl)*@ 0.5nmol/mL : except for Gly, 5nmol/mL : for Gly

A: Acetonitrile / Tetrahydrofuran / 25mM Ammonium formate / formic acid=9/75/16/0.3
B: 100mM Ammonium formate / Acetonitrile = 80 / 204.4Arg

Gly SerThr

Mobile Phase

HPLC conditions (Nexera system)
Column: Intrada Amino Acid (3.0mmI.D. x 50mm, 3um, Imtakt Corporation, Kyoto, Japan)
Mobile phase
Case1
A: Acetonitrile / Formic acid =100/0.1
B: 100mM Ammonium formate
Time program: B conc.14%(0-3 min) -100%(10min) - 14%(10.01-15min)Case2（High Resolution condition)
A: Acetonitrile / Tetrahydrofuran / 25mM Ammonium formate / formic acid =9/75/16/0.3
B: 100mM Ammonium formate / Acetonitrile = 80 / 20
Time program: B conc.0%(0-2 min) -5%(3min) - 30%(6.5min) -100%(12min)- 0%(12.01-17min)
Flow rate: 0.6 mL/min Injection volume: 2 uL Column temperature: 40 C

4.6HisGlnIleLeuMet

LCMS-8050 triple quadrupole mass spectrometer

GlyAraTrpAspSer

Coefficient (r2)Japanese SakeAla

Repeatability*Range (nmol/mL)

205.10>188.10

ProPheLinearity

MRM Transition

Case1

High Speed Mass Spectrometer

Figure 1

As the mobile phase condition of case1 is more simple and the result of case1 was sufficiently
well, case1 analytical condition was used for quantitative analysis. The dilution series of thesecompounds were analyzed. All amino acids were detected with good linearity and repeatability(Table1).

Table1 Linearity and Repeatability of 20 amino acids

MS conditions (LCMS-8050)

Amino acid standard regents and food samples were purchased from the market.
Standards of 20 kinds of amino acids were optimized on each compound-dependent
parameter and MRM transition.
As an LC-MS/MS system, HPLC was coupled to triple quadrupole mass spectrometer(Nexera with LCMS-8050, Shimadzu Corporation, Kyoto, Japan). Sample was eluted
with a binary gradient system and LC-MS/MS with electrospray ionization was operated
in multiple-reaction-monitoring (MRM) mode.

In this study, two conditions of mobile phase were investigated. It was found that 20 amino acids
were separated with higher resolution in case2.

Furthermore, Japanese Sake, Beer and sweet cooking rice wine (Mirin) were analyzed using
this method. Japanese Sake and Beer were diluted with 0.1N HCl. Sweet cooking rice wine
was diluted in the same way after a deproteinizing preparation. These were filtered through a0.2um filter and then analyzed. MRM chromatograms of each food samples are shown inFigure 5,6,7. Amino acids of each sample were detected with high sensitivity.

8.0

9.0minFigure 4 Mass Chromatograms of Sports Beverage (100 fold dilution with 0.1N HCl)