TR06A

Amino Acids Separation Column for LC-MS
The world's first specialty column for intact amino acid analysis via LC-MS
Compatible with conventional LC-MS, providing fast and easy amino acid analysis

LC-MS analysis of amino acids
Amino Acid analysis without sample derivatization
Ability to separate isobaric amino acids such as Leu and Ile
High-throughput (<1 minute) analysis for selected amino acids
5-10 minutes for standard amino acids analysis
Pure spherical silica / 3um particles / unique stationary phase designed for amino acids

10min analysis using LC-MS/MSStandard amino acids
100 nmol/mL
A: CH3CN / THF / 25 mM HCOONH4 /HCOOH= 9 / 75 / 16 / 0.3 (v/v/v/v)
B: CH3CN / 100 mM HCOONH4 = 20 / 80 (v/v)
0 %B (0-3 min)
0-17 %B (3-6.5 min)
100 %B (6.5-10 min)
0.6 mL/min, 40 deg.C, 1 uL (0.1N HCl)
ESI , positive

Arg (Q1/Q3 175.1/70.1)
Lys (Q1/Q3 147.0/84.1)
His (Q1/Q3 156.1/110.1)(Cys)2 (Q1/Q3 241.0/151.95)
Asn (Q1/Q3 133.1/74.05)
Gly (Q1/Q3 75.8/30.1)
Gln (Q1/Q3 147.1/84.1)Ser (Q1/Q3 106.1/60.2)
Ala (Q1/Q3 90.1/44.1)
Thr (Q1/Q3 120.1/74.0)
Asp (Q1/Q3 134.1/73.95)

Pro (Q1/Q3 116.1/70.1)
Glu (Q1/Q3 148.1/84.1)
Val (Q1/Q3 118.1/72.05)
Leu, Ile (Q1/Q3 132.1/86.15)
Met (Q1/Q3 150.1/56.1)
Tyr (Q1/Q3 182.1/136.25)
Phe (Q1/Q3 166.1/120.1)
Trp (Q1/Q3 205.1/188.1)

12345650x3 mm
LCMS-8040 (ESI, positive), Shimadzu Corp.

78910 min

Imtakt has developed a novel column for the analysis of amino acids that couples well with MS
detection and does not require use of an amino acid analysis system.
When coupled to LC-MS the Intrada Amino Acid column achieves high-throughput analysis
without requiring tedious pre- or post-labeling methods.
Use of various column dimensions can aid in optimizing analytical time and resolution for different
amino acid samples.
Separation of leucine and isoleucine isomers, GABA isomers, and dipeptides analysis is now
possible.
Separate free amino acids in mixtures!
Study protein amino acid composition!
Isolate amino acid bio-markers!
Imtakt USA (info@imtaktusa.com)
North America

Imtakt Corp./JAPAN (info@imtakt.com)
Other Countries

TR06AE-MJ22

LC-MS analysis for 55 amino acids in 10 min* Stationary phase is specially designed for amino acid and dipeptide retention and separation.
* No pre- or post-labeling methods required* High throughput analysis via LC-MS* Separation of isobaric amino acids on LC-MS systems is finally possible.

55 Amino Acids (standard samples)
Intrada Amino Acid, 50 x 3 mm
A: acetonitrile /tetrahydrofuran /25mM ammonium formate /formic acid= 9 / 75 / 16 / 0.3
B: acetonitrile / 100mM ammonium formate = 20 / 80
0 %B (0-2.5min)
0-17 %B (2.5-6.5 min)a
100 %B (6.5-10 min)
0.6 mL/min (6MPa), 35 deg.C, 5 uL (1000nmol/mL)
ESI (SIM, positive)

arginine (Arg, m/z 175.1)
ornithine (Orn, m/z 133.1)
hydroxylysine (Hylys, m/z 163.0)
lysine (Lys, m/z 147.0)
carnosine (Car, m/z 227.1)
anserine (Ans, m/z 241.1)ba) 1-methylhistidine (1-Mehis, m/z 170.1)b) 3-methylhistidine (3-Mehis)

histidine (His, m/z 156.0)2-aminoethanol (EtOHNH2, m/z 62.1)
cystathionine (Cysthi, m/z 223.1)
5-aminolevulinic acid (m/z 132.0 )b caa)b)c)d)

cystine ((Cys)2, m/z 241.0)d-aminobutyric acid ( -Abu)-aminobutyric acid ( -Abu)-aminoisobutyric acid ( -Aiba)-aminobutyric acid (GABA, m/z 104.0)

o-phosphoethanolamine (m/z 142.0)5-aminovaleric acid (m/z 118.1)
glycylproline (Gly-Pro, m/z 173.1)

A problem with LC-MS is thedifficulty of detecting isomers
which have the same m/z.
These isomers can be betterdetected if first separated byLC.
This column can separate
isomers such as Leu/Ile (131Da), as basic amino acids,and Val/Norvaline (117 Da).
It also enables the separationof Cysteic Acid, an importantcompound in Amino Acid
metabolism.
Dipeptides such as Anserine
can also be separated and theanalysis of other free andartificial amino acids will beshown in the future.

o-phosphoserine (Ser (P), m/z 186.0)
citrulline (Cit, m/z 176.1)
asparagine (Asn, m/z 133.0)
glycine (Gly, m/z 76.4)
glutamine (Gln, m/z 147.0)
serine (Ser, m/z 106.1)ca) alanine (Ala, m/z 90.0)b) sarcosine (Sar)c) -alanine ( -Ala)a b

aspartic acid (Asp, m/z 134.0)
threonine (Thr, m/z 120.1)
aba) o-methylserine (m/z 120.1)b) allo-threonine (allo-Thr)c) homoserinechydroxyproline (Hyp, m/z 116.0)
proline (Pro, m/z 116.0)
taurine (Tau, m/z 126.0)
methionine sulfone (m/z 182.0)
glutamic acid (Glu, m/z 148.0)-aminoadipic acid ( -Aaa, m/z 162.1)
theanine (m/z 175.1)
cysteine (Cys, m/z 122.0)aba) norvalineb) valine (Val, m/z 118.0)-aminopimelic acid (Apm, m/z 162.1)

aba) leucine (Leu, m/z 132.0)b) isoleucine (Ile)allo-isoleucine (m/z 132.0)
norleucine (m/z 132.0)
methionine (Met, m/z 150.0)
tyrosine (Tyr, m/z 182.0)
phenylalanine (Phe, m/z 166.0)

tryptophan (Trp, m/z 205.0)
thioproline (m/z 134.0)
cysteic acid (Cys(O3H), m/z 170.0)

0

2.5

57.5

210 minVarious column dimensions enhance scalability and flexibility of analysis methods
Intrada Amino Acid columns allow more flexibility:
Manipulate the mobile phase composition and gradient method to accommodate a variety ofamino acid samples, sensitivity requirements, and run times.
Use of a shorter column allows for one minute analysis of non-isomer amino acids.
Left: An application for protein amino acidanalysis. Typically THF/acetonitrile organic
solvent mobile phases are used, however inthis application simple elution using
acetonitrile is acceptable.
Leu/Ile isomers separate using these mildconditions. However, mid-sized amino acids
such as Asp(133Da)/Asn(132Da) andGlu(147Da)/Gln(146) elute at similar times
on the column, so high performance MS
detection is ideal.

Example of simple elution method
Intrada Amino Acid, 50 x 3 mm
A: acetonitrile /HCOOH = 100 / 0.1
B: 100mM HCOONH4
14 %B (0-3min)14-100 %B (3-10 min)
14 %B (10-12 min)
0.6 mL/min, 35 deg.C, 5 uL
ESI, positive

Arg (m/z 175.1)
Lys (m/z 147.0)
His (m/z 156.0)(Cys)2 (m/z 241.0)
Asn (m/z 133.0)
Gln (m/z 147.0)
Ser (m/z 106.1)
Gly (m/z 76.4)
Asp (m/z 134.0)

Glu (m/z 148.0)
Thr (m/z 120.1)
Ala (m/z 90.4)
Val (m/z 118.0)
Tyr (m/z 182.0)
Pro (m/z 116.0)
Met (m/z 150.0)
Leu, Ile (m/z 132.0)
Trp (m/z 205.0)
Phe (m/z 166.0)

0

2.5

5

7.5

10 minAnalytical condition protocol to handle
Intrada Amino Acid column is described inleft table.
Analysis condition for various kinds ofanalysis purpose should be optimizedchanging gradient profile or solvent
concentration etc.

Analytical conditions protocol
A: acetonitrile /HCOOH = 100 / (0.1 - 0.5), v/v
B: (50-200mM) HCOONH4Initial - Final %B (Gradient Time)
Flow Rate: depends on column I.D.
Temperature: up to 65deg.C
Injection Solution: 0.1N HCl or 0.1 - 2% HCOOH
MS detection: ESI, positive

Separation of Leucine (131Da) isomers

Separation of GABA (103Da) isomers

OIntrada Amino Acid, 100 x 3 mm
A: acetonitrile / formic acid = 100 / 0.3
B: 100mM ammonium formate
25-30 %B (0-12 min), 100 %B (12-15min)
0.4 mL/min (4MPa), 37 deg.C, 5 uL (0.1N HCl)
3-aminoisobutyric acid
ESI (positive, m/z 104.1)
O (beta-aminoisobutyric acid)
H 2N

O

H 2N

H 3C

57.5

OOH

allo-L-isoleucine

NH2OH

L-isoleucine

2-aminobutyric acid(alpha-aminobutyric acid)

2.5

H3C

OH
NH2

NH20

NH2L-leucine
H3C

4-aminobutyric acid
(GABAgamma-aminobutyric acid)

OH

OH

H3C

O

H3C

O

H 2N

O

H3C

OH
CH3

H 3C
OH
3-aminobutyric acid(beta-aminobutyric acid)

H3C

Intrada Amino Acid, 150 x 3 mm
A: methanol / water / formic acid = 85 / 15 / 0.3
B: acetonitrile / 100mM ammonium formate = 20 / 80
12-13 %B (0-12 min)
100 %B (12-15 min)
0.4 mL/min (11 MPa)
35 deg.C, 5 uL (0.1N HCl)
ESI (positive, m/z 132.0)

510 min

7.5

10

12.5 minIntrada Amino Acid column separates amino acid isomers quickly by using an optimized column
length.
Above: Figures show aminobutyric acid isomers(103Da) and leucine isomers(131Da) using 100150mm length columns.

3

High-througput analysis of standard amino acids

10 x 3 mm

One-minute analysis of related compounds

10 x 2 mm

OOHH2Nornithine NH2Arg (m/z 175.1)

CH2
H3C

Lys (m/z 147.0)

N+OH O
O-CH2

His (m/z 156.0)

carnitineO
H2NOOHN
H

NH2citrulline

Intrada Amino Acid, 10 x 2 mm
A: CH3CN /HCOOH = 100 /0.1
B: 100mM HCOONH4
15-100 %B (0-0.8 min)
100 %B (0.8-1.0 min)
0.4 ml/min (1.6 MPa)
35deg.C, 1 uL (0.1N HCl)
ESI, positive(Cys)2 (m/z 241.0)
Asn (m/z 133.0)

m/z 133.1 (240 nmol/mL)

Gly (m/z 76.0)

m/z 176.1 (170 nmol/mL)

Gln (m/z 147.0)

m/z 162.1 (300 pmol/mL)
OS OHtaurine

Ser (m/z 106.1)
Ala (m/z 90.0)

0Asp (m/z 134.0)

12H2N24O

36

m/z 126.1 (1.6 umol/mL)

48

60 secAbove: One minute ultra high-throughput analysis can
successfully be performed on a 10mm length column.

Thr (m/z 120.1)
Pro (m/z 116.0)
Glu (m/z 148.0)

Intrada Amino Acid, 10 x 3 mm
Val (m/z 118.2) A: CH3CN /HCOOH = 100 /0.1
B: 100mM HCOONH4Leu, Ile (m/z 132.0) 27 %B (0-0.2 min),
100 %B (0.2-1.5 min)Met (m/z 150.0) 0.4 mL/min (0.8MPa)
35deg.C
Tyr (m/z 182.0)
1 uL (0.1N HCl)
ESI (SIM, positive)

Intrada Amino Acid high-throughput columns offer the next
generation amino acid analysis method for clinical aminoacid biomarkers, fermented materials, botanical aminoacids and related compounds providing amazing speed,selectivity and convenience.

Phe (m/z 166.0)

Trp (m/z 205.0)

00.25

0.5

0.75

1 min

Dipeptide analysis
L-cystine

ONH2HOSOHSONH2Glu-Glu
HOO

NH2L-anserine
ONHOHNNOHOOHOO

HN

H2NO

HN

H2N

H3C

L-carnosine NHis-Leu
HNNH

H2NHOHNNOO

0Analysis of longer chain peptides that require high ionicstrength mobile phases should use the Scherzo SS-C18
multi-mode ODS column.

OO

HOIntrada Amino Acid, 75 x 2 mm
A: CH3CN / HCOOH = 100 / 0.3
B: 100mM HCOONH4
20-100 %B (0-10 min), 100 %B(10-12 min)
0.3 mL/min, 35 deg.C
2 uL (0.02-0.32 ug, 0.1N HCl)
ESI, positive

Intrada Amino Acid is applicable not only for amino acidanalysis, but also for polar dipeptides which are difficult toretain and separate in conventional HPLC.

2.557.510 min

Product Information
Column I.D.

Column Length (depends on I.D.)

3mm, 2mm, 1mm
0.5mm - 0.075mm

10mm, 20mm, 30mm
50mm, 75mm, 100mm
150mm, 250mm

Guard column system is not available for this product.

Imtakt USA (info@imtaktusa.com)
North America

Column Recommendations
The Intrada Amino Acid column should be used onlyfor LC-MS in order to achieve adequate peakidentification. This product is not recommended forapplications involving UV or ELSD instruments.
Note that detection sensitivity is highly dependent
upon MS instrument performance. LC-MS instruments
should be carefully chosen to yield adequately
sensitive data.
In order to achieve optimized analytical data, method
development (column dimensions, gradient conditions,sample preparation, standard curve, etc.) andvalidation are required.
Please refer to general methods for sample preparations
for amino-acid composition analysis.

Imtakt Corp./JAPAN (info@imtakt.com)
Other Countries