ThP 409
High Throughput simultaneous analysis of 39 amino acids in various foods without derivatization using LC-MS/MS
Manabu Yukiyama1, Keiko Matsumoto1, Jun Watanabe1, Itaru Yazawa2
1 Shimadzu Corporation, Kyoto, JAPAN; 2 Imtakt Corporation , Kyoto, JAPAN1. Introduction
As amino acids in foods are essential components of nutrition and taste, it is expected todevelop an easy and efficient analytical method. Amino acids are highly polar compounds,
so it needs their derivartization or addition of ion-pair reagent in mobile phase in order toseparate them by reversed phase mode. In the case of hydrophilic interaction
chromatography, it may be difficult to separate isomers or to analyze comprehensively.
In our previous study2), we developed a simultaneous analysis method of 20 amino acids by
LC-MS/MS with mix-mode column (ion exchange and normal phase), without derivartization.
In this study, we tried to increase the number of targeted amino acids (39 amino acids), anddetected them in various foods with high sensitivity.

Mix-mode column “Intrada Amino Acid” provides normal phase separation and ionexchange. In the condition that amino acids were not derivartized and ion-pairing reagent
wasn’t used for this analysis, 39 amino acids were retained and separated excellently bycontrolling pH, salt concentration and acetonitrile ratio. Although alanine and sarcosine aresame molecule weight, the mix-mode column enabled these 2 amino acids to be separated
chromatographically.
Arg

Aminoadipic acid
Aminoethanol
Threonine

2. Methods and Materials

Sarcosine

Amino acid standard regents and food samples were purchased from the market. Standardsof 39 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 abinary gradient system and LC-MS/MS with electrospray ionization was operated in multiplereaction-monitoring (MRM) mode.

Carnosine
Hydroxylysine(Cys)2

Beta‐aminoisobutylic acid
Hydroxyproline

Ornithine
Beta‐Ala

Methylhistidine

Citrulline

Cysteine

4‐Aminobutylic acid

Tyr

High Speed Mass Spectrometer

AsnProUF-MRM
High-Speed MRM at 555ch/sec

Leu

2.5

3.0

3.5

4.0

4.5GABA
AsnAraTrp

Citrulline

5.05.5Aminoethanol

6.0

6.5

7.0minGlnProOrnithine

HisPhe2.0

3.0

Figure 5

4.0

5.0

6.0

7.0

8.0

Arg

Lys9.0min

Mass Chromatograms of Wine (100 fold dilution with 0.1N HCl)

Recovery rate was evaluated using analytical data of Katsuobushi and beer spiked withstandard amino acids (50nmol/mL). Both Katsuobushi and beer showed good recoveries
with almost 70~120% (Figure 6,7).
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0

Asp

MetIｌeSer

Trp

Gly

PheGluGluValTyrMetTrp

Figure 6 Recovery data (extract of Katsuobushi spiked with standard amino acids)

Ser
Asp
Asn
Citrulline

Hydroxyproline

GlnTaurine

UFswitching
High-Speed Polarity Switching 5msec

GluHydroxyproline

Phe%RSD*Amino acid%RSD*
17.29
Sarcosine
3.86
9.54
β-Ala
5.36
8.52
Citrulline
4.92
5.49 4-Aminobutyric acid(GABA) 2.68
3.06
Cystathionine
3.48
1.95
Aminoethanol
1.73
8.70
Anserine
0.88
6.11
Carnosine
1.19
7.63
Hydroxylysine
5.64
8.23
Methylhistidine
3.91
7.53
β-Aminoisobutyric acid
7.36
4.76
2-Aminobutyric acid
3.30
5.29
Methionine sulfone
15.88

Dried Bonito fish (Katsuobushi), pork and wine were analyzed by using this method.
Katsuobushi (extracted by hot water) and wine were diluted with 0.1N HCl. Pork wasdelipidated with n-Hexane after a deproteinizing preparation with 5% sulfosalicylic acid.
These were filtered through a 0.2um filter and then analyzed. MRM chromatograms of eachfood sample are shown in Figure 3,4,5. In the case of Katsuobushi, Histidine carnosine andanserine which are typical substances in a migratory fish were detected with high intensity.
It is known that carnosine is contained in muscle and improves muscle function. Carnosine
also was plentifully detected in pork. On the other hand, for wine, abundant amino acids
detected were proline, glutamic acid, glutamine, arginine and GABA.

His

Cystathionine

Theanine

Amino acidGln
GlyAsn(Cys)2
His
Lys
Arg
Theanine
Aminoadipic acid
TaurineOrnithine
Cysteine
Hydroxyproline

3-2. The analysis of 39 amino acids in food samples

Anserine

2‐Aminobutylic acid%RSD*
0.90
0.73
4.97
0.78
0.84
0.90
1.90
5.54
1.17
9.19
3.47
2.01
10.67

*@ 5nmol/mL : except for Gly 10nmol/mL Taurine 50nmol/mL

LysAlaMethionine Sulfone

ValTable 2 Repeatability of 39 amino acids
Amino acidTrp
PheTyrMet
Lue
IIeValGlu
Pro
AspThrAla
SerAlaTau

Ornithine
120.00

Met-His100.00Anserine

80.00
60.00

Figure 1

2.0

3.0

4.0

5.0

6.0

7.0

8.0

LCMS-8050 triple quadrupole mass spectrometer
0.0

1.0

2.0

Figure 2

3. Result3-1. Method development
First, MRM method of 19 amino acids was optimized in order to increase the number ofanalytical amino acids. As a result, all compounds were able to be detected high sensitively
with ESI. All amino acids except for methionine sulfone were detected in positive mode.
Methionine sulfone was detected in negative mode. Ultra Fast Polarity Switching of 5msec
enabled simultaneous analysis of the compounds in both positive and negative modes.

HPLC conditions (Nexera UHPLC system)
Column: Intrada Amino Acid (3.0mmI.D. x 50mm, 3um, Imtakt Corporation, Kyoto, Japan)
Mobile phase:
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)
Flow rate: 0.6 mL/min Injection volume: 2 uL Column temperature: 40 Celsius

MS conditions (LCMS-8050)
Ionization: ESI, Positive / Negative MRM mode
MRM transition are shown in Table 1.

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

minTable 1
MRMTransition
Trp
Phe
Tyr
Met
Lue,LleVal
GluPro
Asp
Thr
Ala
Ser
Gln
GlyAsn(Cys)2
His
Lys
Arg

205.10>188.10
166.10>120.10
182.10>136.00
150.10>56.10
132.10>86.15
118.10>72.05
148.10>84.10
116.10>70.10
134.20>74.10
120.10>74.00
90.10>44.10
106.10>60.20
147.10>84.10
76.20>29.90
133.10>74.05
241.00>151.95
156.10>110.10
147.10>84.10
175.10>70.10

20.00
0.00

CarnosineMet

Mass Chromatograms of 39 Amino acids(concentration of each compound : 10nmol/mL)

The dilution series of these compounds were analyzed. Most of amino acids were detectedwith good linearity (Table1) and repeatability (Table 2).

40.00

His

Phe
0
0.0

1.0

2.0

Leu

Pro
Ile

3.0

Lys

Hydroxyproline
ValGlu

4.0

5.0

6.0

7.0

Arg

8.0

9.0

min

4. Conclusions

Linearity of 39 amino acids
Linearity
Linearity
MRM
Range
Coefficient
RangeCoefficient
Transition(nmol/mL)(r2)(nmol/mL)(r2)
0.01‐1000.995
Theanine
174.50>84.10
1‐500
0.988
0.01‐1000.997
Aminoadipic acid161.90>98.20
0.1‐500.979
0.05‐1000.99
Taurine126.20>44.05
10‐5000.998
0.05‐2000.996
Ornithine133.10>70.10 0.05‐1000.997
0.01‐1000.995
Cysteine
122.00>76.05
5‐500
0.996
0.05‐1000.999
Hydroxyproline
132.10>86.05
0.1‐500
0.991
0.05‐100.996
Sarcosine
89.90>43.85
0.05‐1000.999
0.01‐500.993
β‐Ala89.70>72.10
0.5‐500
0.999
0.5‐5000.995Citrulline
176.10>70.05 0.05‐100
0.993
0.1‐500.992
2‐Aminobutyric acid104.10>58.05
0.5‐500
0.996
0.5‐500
0.998
Cystathionine
223.00>88.05
0.1‐100.995
0.5‐500
0.998
Aminoethanol
62.00>44.10
0.1‐500
0.999
0.05‐1
0.995
Anserine
240.70>109.10 0.1‐100
0.997
5‐200
0.997
Carnosine
227.10>110.05 0.05‐100
0.995
0.05‐20
0.993
Hydroxylysine
162.90>82.15
0.1‐500
0.996
0.05‐20
0.99
Methylhistidine
169.90>124.15 0.01‐10
0.999
0.05‐200
0.998
3‐Aminoisobutyric acid103.80>86.10 0.05‐500
0.991
0.05‐50.99
4‐Aminobutyric acid (GABA） 104.10>87.05 0.05‐500
0.995
0.01‐1000.996
Methionine sulfone
180.00>79.20
0.5‐1000.973

Figure 7 Recovery data (beer spiked with standard amino acids)

Figure 3 Mass Chromatograms of Extract of Katsuobushi (500 fold dilution with 0.1N HCl)
2-aminobutyric acid
TrpPheMet

Citrulline

GluProOrnithine
LysGlnMethylhistidine

Lue Ile
3.0

4.0

5.0

6.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

8.0

9.0

 The mix-mode column enabled isomers-separation completely.
AnserineCitrulline
4.04.5

 Ultra Fast Polarity Switching of 5msec enabled simultaneous analysis of the compounds
in both positive and negative modes

Carnosine

GlnPro0.0

 39 amino acids could be separated without derivatization by using a typical volatile mobile
phase suitable for LC/MS analysis and detected with high sensitivity.

His

7.0

2-aminobutyric acid

Arg

5.0

5.5

His
6.0

6.5

7.0

7.5

Figure 4 Mass Chromatograms of pork (10 fold dilution with 0.1N HCl)

8.08.5

 This method was able to be applied to the analysis of amino acids in various foodsamples.

Arg
9.0min5. Reference
1. Imtakt Technical Report T1734E (Imtakt Co., Kyoto)
2. Keiko Matsumoto et al., Poster No.TP510, ASMS2014 in Baltimore, June 15-19, 2014