lc-ms analysis of amino acids on a novel mixed-mode hplc … · 2018-11-01 · 2 ssi-lcms-022 lcms...

1

Liquid Chromatography Mass Spectrometry

SSI-LCMS-022

LCMS-8040

BackgroundThere are four established methods for analyzing amino acids: prelabeled, post-labeled, ion-pairing reversed-phase, and normal-phase, but each of these methods has disadvantages. The pre-labeled method has problems with derivatization e�ciency and cost, while the post-labeled method is usually not compatible with LC-MS due to non-volatile mobile phases. The ion-pairing reversed-phase method has di�culty separating polar amino acids, whereas, the normal-phase mode has problems separating all the compounds, especially the Leucine and Isoleucine isomers. To alleviate these challenges, Imtakt developed the Intrada amino acid separation column for LC-MS/MS which can separate 55 amino acids in 10 minutes using a mixed-mode

stationary phase without utilizing either pre- or post- column labelling methods. Additionally, the column can separate gamma-Aminobutyric acid (GABA) isomers and dipeptides also without derivatization. Imtakt has optimized separation and detection char-acteristics utilizing the Shimadzu LCMS-8040 and LCMS-2020.

MethodThis mixed-mode stationary phase for LC-MS analysis of amino acids consists of 3µm silica particles modi-�ed with ion ligands which couples ion exchange with normal-phase interactions. All amino acid and bovine sera standards were purchased commercially. Each �gure below provides instrument parameters speci�c to each acquisition.

Results and DiscussionFigures 1 and 2 demonstrate methods utilizing the LCMS-2020 single quadrupole mass spectrometer and LCMS-8040 triple quadrupole mass spectrom-eter to characterize a separation of the 20 standard amino acids including Leucine and Isoleucine with-out need for derivatization. Figure 3 provides calibra-tion curves of amino acids representing the four categories of these molecules, aromatic, aliphatic, basic, and acidic to demonstrate the ability of the mixed-mode column and the mass spectrometer to accurately quantitate these four classes of analytes. Figure 4 provides a simple gradient procedure for separating a mixture of amino acids in 10 minutes. A 5 minute separation of the 20 standard amino acids was able to maintain resolution between Leucine and Isoleucine, (Figure 5). Figure 6 demonstrates measurement of free amino acids over 10 minutes in bovine serum utilizing the LCMS-2020. Additionally, dipeptides and beta and gamma amino acids were measured with the LCMS-2020 as charted in Figures 7 and 8. Ornithine, citrulline, carnitine, taurine, and other related compounds are also candidate analytes

LC-MS Analysis of Amino Acids on aNovel Mixed-Mode HPLC Column

for separation using this strategy. Method conditions have been optimized to separate these compounds in less than 60 seconds. Isobaric Leucine isomers are readily resolved using the separation strategy provided in Figure 10. Figure 11 demonstrates the separation of 55 amino acid analytes over 10 minutes.

ConclusionLabel free amino acid separations and detection utilizing a mixed-mode stationary phase coupled with the single and triple quadrupole mass spec-tromters, LCMS-2020 and LCMS-8040 has been demonstrated. Amino acid samples of varying complexity and from various matrices, including isomers, dipeptides, and related compounds are separated using optimized methods spanning from 10 minutes to as short as one minute.

This combination of column, UHPLC, and MS yields a powerful tool for amino acid analysis for many di�erent biochemical applications.

2

SSI-LCMS-022

LCMS 2020 Analysis of 20 Amino Acids Standard

Figure 1: 20 standard amino acids separated and detected on LCMS-2020. Leucine and Isoleucine isomers were also separated in several minutes.

%

Trp (m/z 205.0)

Phe (m/z 166.0)

Met (m/z 150.0)Tyr (m/z 182.0)

Leu, Ile (m/z 132.0)

Val (m/z 118.0)Glu (m/z 148.0)

Pro (m/z 116.0)

Thr (m/z 120.1)Asp (m/z 134.0)Ala (m/z 90.4)

Ser (m/z 106.1)Gln (m/z 147.0)

Asn (m/z 133.0)(Cys)2 (m/z 241.0)

His (m/z 156.0)Lys (m/z 147.0)

Arg (m/z 175.1)

Intrada Amino Acid, 50 x 3 mmA: acetonitrile / tetrahydrofuran / 25 mM ammonium formate / formic acid = 9 / 75 / 16 / 0.3B: acetonitrile / 100 mM ammonium formate = 20 / 800 %B (0-2.5min), 0-17 %B (2.5-6.5 min), 100 %B (6.5-10 min)0.6 mL/min (6MPa), 35 deg.C, 5 uL, ESI (SIM, positive)

Gly (m/z 76.4)

Figure 2: 20 standard amino acids were separated and detected using the LCMS-8040.

Trp (Q1/Q3 205.1/188.1)Phe (Q1/Q3 166.1/120.1)

Met (Q1/Q3 150.1/56.1)Tyr (Q1/Q3 182.1/136.25)

Leu, Ile (Q1/Q3 132.1/86.15)Val (Q1/Q3 118.1/72.05)

Glu (Q1/Q3 148.1/84.1)Pro (Q1/Q3 116.1/70.1)

Thr (Q1/Q3 120.1/74.0)

Asp (Q1/Q3 134.1/73.95)

Ala (Q1/Q3 90.1/44.1)

Ser (Q1/Q3 106.1/60.2)Gln (Q1/Q3 147.1/84.1)

Asn (Q1/Q3 133.1/74.05)(Cys) 2 (Q1/Q3 241.0/151.95)

His (Q1/Q3 156.1/110.1)Lys (Q1/Q3 147.0/84.1)

Arg (Q1/Q3 175.1/70.1)

Intrada Amino Acid, 50 x 3 mmA: ACN / THF / 25 mM HCOONH4 /HCOOH = 9 / 75 / 16 / 0.3B: ACN / 100 mM HCOONH4 = 20 / 800 %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)

LCMS-8040, ESI (MRM, positive)100 nmol/mLamino acids standard mixture

Gly (Q1/Q3 75.8/30.1)

1 2 4 63 5 7 8 9 10 min

3

SSI-LCMS-022





Calibration Standard Curve for Various Amino Acids

Figure 3: Typical standard (aromatic, aliphatic, acidic and basic) amino acids showed good results for linearity and sensitivity.

R2 = 0.9999

Trp

Aromatic Amino Acid

0 2 4 6 8 10 12 14nmol/mL

1.4x106

1.2

1.0

0.8

0.6

0.4

0.2

Aliphatic Amino Acid

Leu

0 2 4 6 8 10 12 14nmol/mL

x106

1.2

1.0

0.8

0.6

0.4

0.2

R2 = 0.9994

Arg

Glu

Acidic Amino Acid

Basic Amino Acid

x106

5

4

3

2

1

10nmol/mL

0 20 30 40 50

x105

1.6

1.2

0.8

0.4

0 2 4 6 8 10 12 14nmol/mL

R2 = 0.9970

R2 = 0.9985

Figure 4: Simple gradient elution condition was also successful for standard amino acids separation in 10min with low pressure.

min0

Trp (m/z 205.0)Phe (m/z 166.0)

Met (m/z 150.0)

Tyr (m/z 182.0)


Val (m/z 118.0)

Glu (m/z 148.0)

Pro (m/z 116.0)

Thr (m/z 120.1)

Asp (m/z 134.0)

Ala (m/z 90.4)

Ser (m/z 106.1)

Gln (m/z 147.0)

Asn (m/z 133.0)(Cys)2 (m/z 241.0)

His (m/z 156.0)Lys (m/z 147.0)

Arg (m/z 175.1)

Intrada Amino Acid, 50 x 3 mmA: acetonitrile / formic acid = 100 / 0.1B: 100 mM ammonium formate14 %B (0-3min), 14-100 %B (3-10 min), 14 %B (10-12 min)0.6 mL/min (4MPa), 35 deg.C, 5 uLESI (SIM, positive)

Gly (m/z 76.4)




4

SSI-LCMS-022

Figure 5: High throuput analysis using Nexara UHPLC LCMS-2020 system was achieved in 5min.

5min High Throughput Analysis

0 1 2 3 4 5 minTrp (m/z 205.0)

Phe (m/z 166.0)Tyr (m/z 182.0)

Met (m/z 150.0)Leu, Ile (m/z 132.0)

Val (m/z 118.0)

Glu (m/z 148.0)

Pro (m/z 116.0)Thr (m/z 119.9)

Asp (m/z 133.9)

Ala (m/z 89.9)

Ser (m/z 105.9)

Gln (m/z 147.0)

Gly (m/z 75.8)Asn (m/z 133.0)

(Cys)2 (m/z 241.0)

His (m/z 156.0)Lys (m/z 147.0)

Arg (m/z 175.1)

Intrada Amino Acid, 150 x 2 mmA: tetrahydrofuran / 20 mM ammonium formate / formic acid = 75 / 25 / 0.3B: 200 mM ammonium formate / acetonitrile = 80 / 200 %B (0-2.5 min), 100 %B (2.5-5 min)0.6 mL/min (45 MPa), 37 deg.C, 5 uLESI (SIM, positive)

Figure 6: The novel amino acid analysis column was applied for amino acids in serum and several amino acids were detected using the LCMS-2020.

Application for Amino Acids in Serum

Trp (m/z 205.0)

Phe (m/z 166.0)

Met (m/z 150.0)

Tyr (m/z 182.0)


Val (m/z 118.0)Glu (m/z 148.0)

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

Asp (m/z 134.0)

Ala (m/z 90.4)

Ser (m/z 106.1)

Gln (m/z 147.0)

Asn (m/z 133.0)

(Cys) 2 (m/z 241.0)His (m/z 156.0)

Lys (m/z 147.0)

Arg (m/z 175.1)

Gly (m/z 76.4)

Bovine Serum (0.4N HClO4 treatment)

0.0 2.5 5.0 7.5 10.0 min

Intrada Amino Acid, 50 x 3 mmA: ACN / THF / 25 mM HCOONH4 /HCOOH = 9 / 75 / 16 / 0.3B: ACN / 100 mM HCOONH4 = 20 / 800 %B (0-2.5 min)0-17 %B (2.5-6.5 min)100 %B (6.5-10 min)0.6 mL/min(6MPa), 35 deg.C, 1 uL (0.1N HCl)ESI (SIM, positive)

5

SSI-LCMS-022





Figure 7: The amino acid analysis column could be applied for dipeptides using the LCMS-2020.

Dipeptide Analysis

0.0 2.5 5.0 7.5 10.0 min

Intrada Amino Acid, 75 x 2 mmA: acetonitrile / formic acid = 100 / 0.3B: 100 mM ammonium formate20-100 %B (0-10 min), 100 %B(10-12 min)0.3 mL/min (5 MPa), 35 deg.C2 uL (0.02-0.32 ug, 0.1N-HCl aq.)ESI (SIM, positive)

H2N

O

HN

O

OH

NHN

OH

O

NH2S OH

O

NH2

S

H2N

O

HN

O

OH

N NH3C

O

OH

OH

NH

OO

OHNH2

O

HN

NO

O

HOHN

H2N

N-(L-alpha-glutamyl)-L-glutamic acid(Glu-Glu)

L-cystine ((Cys)2)

L-anserineL-carnosine

N-L-histidyl-L-leucine(His-Leu)

Figure 8: Not only alpha-amino acids but also beta- and gamma-amino acids isomers were well separated.

0 1 2 3 4 5 6 7 8 9 10 min

Intrada Amino Acid, 75 x 2 mmA: acetonitrile / formic acid = 100 / 0.3B: 100 mM ammonium formate15-100 %B (0-8 min), 15 %B (8-10 min)0.3 mL/min (4 MPa), 35 deg.C5 uL (10 ug/mL, 0.1N-HCl aq.)ESI (SIM, positive, m/z 104.1)

NH2

O

OHH3C

H2N O

OHH3C

H2NO

OH

2-aminobutyric acid (alpha-aminobutyric acid)

3-aminobutyric acid (beta-aminobutyric acid)

4-aminobutyric acid (GABA, gamma-aminobutyric acid)

m/z 104.1

m/z 104.1

m/z 104.1




6

SSI-LCMS-022

Figure 9:Amino acid related compounds were analyzed by one-munite high throuhput separation with 10mm column using the LCMS-2020.

Intrada Amino Acid, 10 x 2 mmA: acetonitrile / formic acid = 100 / 0.1B: 100mM ammonium formate15-100 %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 (SIM, positive)

0 12 6024 36 48 sec

m/z 126.1 (1.6 umol/mL)

m/z 162.1 (300 pmol/mL)

m/z 176.1 (170 nmol/mL)

m/z 133.1 (240 nmol/mL)

taurine H2NSO

OOH

carnitine

O

O-N+H3C

CH2

CH2OH

citrulline

H2N

O

OHNH

O

NH2

ornithine

H2N

O

OHNH2

Figure 10: Separation of Leucine (131Da) isomers, detected by the LCMS-2020.

5 7.5 10 12.5 min

H3C OH

O

NH2H3C

H3C OH

O

NH2

H3CH3C OH

O

NH2

H3C

allo-L-isoleucine

L-leucine

L-isoleucine

Intrada Amino Acid, 150 x 3 mmA: MeOH / Water / Formic acid = 85 / 15 / 0.3B: ACN / 100mM Ammonium formate = 20 / 80

Gradient Conditions:12-13 %B (0-12 min)100 %B (12-15 min)0.4 mL/min (11 MPa)35 °C, 5 µL (0.1N HCl)ESI (positive, m/z 132.0)

7

SSI-LCMS-022





tryptophan (m/z 205.0)phenylalanine (m/z 166.0)

methionine (m/z 150.0)tyrosine (m/z 182.0)

a) leucine (m/z 132.0)b) isoleucine

glutamic acid (m/z 148.0)

proline (m/z 116.0)

threonine (m/z 120.1)

aspartic acid (m/z 134.0)

serine (m/z 106.1)glutamine (m/z 147.0)

asparagine (m/z 133.0)

cystine (m/z 241.0)

histidine (m/z 156.0)

lysine (m/z 147.0)

arginine (m/z 175.1)

glycine (m/z 76.4)

2-aminoethanol (m/z 62.1)

hydroxylysine (m/z 163.0) ornithine (m/z 133.1)

a) 1-methylhistidine (m/z 170.1)b) 3-methylhistidine

anserine (m/z 241.1)

carnosine (m/z 227.1)

taurine (m/z 126.0)

o-phosphoserine (m/z 186.0)

o-phosphoethanolamine (m/z 142.0)

hydroxyproline (m/z 116.0)

citrulline (m/z 176.1)

cystathionine (m/z 223.1)

cysteic acid (m/z 170.0)

methionine sulfone (m/z 182.0)

a) o-methylserine (m/z 120.1)b) allo-threonine c) homoserine

theanine (m/z 175.1)

a) norvaline b) valine (m/z 118.0)

norleucine (m/z 132.0)allo-isoleucine (m/z 132.0)

cysteine (m/z 122.0)

5-aminovaleric acid (m/z 118.1)

a-aminoadipic acid (m/z 162.1)

5-aminolevulinic acid (m/z 132.0 )

a-aminopimelic acid (m/z 162.1)

thioproline (m/z 134.0)

glycylproline (m/z 173.1)

0 2.5 5 7.5 10 min

a b

a b

a bc

a) alanine (m/z 90.0)b) sarcosine c) b-alanine

a b c

a b c d

a b

a) a-aminobutyric acid ( a-Abu)b) b-aminobutyric acid ( b-Abu)c) b-aminoisobutyric acid ( b-Aiba)d) g-aminobutyric acid (GABA, m/z 104.0)

Figure 11: 55 Amino Acids (standard samples) separated and detected utilizing the LCMS-2020.

Intrada Amino Acid, 50 x 3 mmA: ACN / THF / 25mM Ammonium formate / Formic acid = 9 / 75 / 16 / 0.3B: ACN / 100mM Ammonium formate = 20 / 80Gradient Conditions:0 %B (0-2.5min)0-17 %B (2.5-6.5 min)100 %B (6.5-10 min)0.6 mL/min (6MPa), 35 °C , 5 µL (1µmol/mL)ESI (SIM, positive)




8

SSI-LCMS-022








First Edition: October, 2012

© Shimadzu Scienti�c Instruments, 2012

SHIMADZU SCIENTIFIC INSTRUMENTS, INC. Applications Laboratory

Shimadzu Corporationwww.shimadzu.com/an/

7102 Riverwood Drive, Columbia, MD 21045Phone: 800-477-1227 Fax: 410-381-1222URL http://www.ssi.shimadzu.com

Founded in 1875, Shimadzu Corporation, a leader in thedevelopment of advanced technologies, has a distinguished history of innovation built on the foundation of contributing to society through science and technology. Established in 1975, Shimadzu Scienti�c Instruments (SSI), the American subsidiary of Shimadzu Corporation, provides a comprehensive range of analytical solutions to laboratories throughout North, Central, and parts of South America. SSI maintains a network of nine regional o�ces strategically located across the United States, with experienced technical specialists, service and sales engineers situated throughout the country, as well as applications laboratories on both coasts.

For information about Shimadzu Scienti�c Instruments and to contact your local o�ce, please visit our Web site at www.ssi.shimadzu.com

LCMS-8040LCMS-8030 LCMS-8050 LCMS-2020 LCMS-IT-TOF

For Research Use Only. Not for use in diagnostic procedures. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you “as is” without warranty of any kind including without limitation warranties as to is accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publications is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.

lc-ms analysis of amino acids on a novel mixed-mode hplc … · 2018-11-01 · 2 ssi-lcms-022 lcms...

Documents