uplc amino acid analysis solution...©2009 waters corporation | company confidential 2 uplc® amino...

©2009 Waters Corporation | COMPANY CONFIDENTIAL

UPLC Amino Acid Analysis Solution

AAAFCO Meeting

Washington, DC

August 2, 2009

©2009 Waters Corporation | COMPANY CONFIDENTIAL 2

UPLC® Amino Acid Analysis Solution

AM

Q

NH

3 His

Se

r

Arg

Gly A

sp

Glu T

hr

Ala

Pro

De

riv

Pe

ak

Cy

sL

ys

Ty

r

Me

tV

al

Ile

Le

uP

he

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.32

0.34

0.36

Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00


UPLC® Application Solution for Amino Acid Analysis

Turn-key UPLC® Amino Acid Analysis application

— Optimized for the ACQUITY UPLC System

— Dedicated QC-tested column and reagents

— Application-specific Performance Qualification

— Same result day-to-day, instrument-to-instrument, lab-to-lab,

around the world

Target Applications

— Protein and peptide ID and quantitation

— Monitoring cell culture media

— Nutritional content of food and feed


Total System ApproachAssured Performance

ACQUITY UPLC® System with TUV Detector

Empower software control

Application Start Up Kit Containing:

— Manual

— CD with method templates and example data/calculations

— Necessary instrument accessories

AccQ●Tag™ Ultra Chemistry Kit

— Reagent

— Buffers/diluents/eluents

— QC tested ACQUITY UPLC BEH 2.1 x 100mm, 1.7 µm C18

column

Add-on Kit for existing ACQUITY UPLC Users


Chemistry of AQC Derivatization

N

N

NH

O

O

O

ON

NH

O

NH

OH

O

N

NH2

NH2

O

OH

NH

O

OH

N

N

NH

OOH

O

NN

NH

NH

O

AQC Reagent

Derivatized Amino Acids

6-Aminoquinolone (AMQ)

1o Amino Acid;

t1/2 <<1 s

2o Amino Acid;

t1/2 <<1 s

bis-aminoquinoline urea (derivatization peak)

N-HydroxySuccinimide

+ CO2

+

AMQH2O

t1/2 ~ 15 s


Ultra Performance LC® Technology

A new class of separation science

— Based on chromatography columns with very small particles

— Based on instruments designed to take advantage of the small

particles

Provides improved Resolution, Speed, and Sensitivity with

no compromises

Suitable for chromatographic applications in general

— Appropriate for developing new methods

— Appropriate for improving existing methods


Chromatography PrinciplesMass Transfer / Diffusion

Porous

Particle

Analyte Molecules

Mobile Phase

A

B

C


UPLC® Amino Acid AnalysisApplication Solution



AM

Q

NH

3 His

Se

r

Arg

Gly As

p

Glu T

hr

Ala

Pro

De

riv

Pe

ak

Cy

sL

ys

Ty

r

Me

tV

al

Ile

Le

uP

he

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.32

0.34

0.36

Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00


Amino Acid AnalysisApplication Types

Amino Acid Analysis

Hydrolysates Cell Culture Food Analysis PhysiologicalClinical


Analysis of Acid Hydrolyzed BSA9ng on column

AM

Q

NH

3

His

Se

r

Arg

Gly

As

p

Glu

Th

r

Ala

Pro

De

riv

Pe

ak

Cy

sL

ys

Ty

r

Me

t

Va

l

Ile

Le

u

Ph

e

AU

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

0.030

0.032

0.034

0.036

0.038

0.040

0.042

0.044

0.046

0.048

0.050

0.052

0.054

0.056

0.058

0.060

0.062

0.064

0.066

0.068

Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00


Accuracy of Compositional AnalysisBovine Serum Albumin

26.57 ± 0.1327Phe

63.13 ± 0.1961Leu

13.15 ± 0.1514Ile

35.67 ± 0.1636Val

4.16 ± 0.154Met

20.19 ± 0.0820Tyr

57.78 ± 0.3859Lys

28.35 ± 0.1428Pro

47.51 ± 0.1547Ala

31.92 ± 0.0633Thr

80.68 ± 0.2079Glu

55.47 ± 0.2154Asp

17.68 ± 0.2016Gly

22.37 ± 0.0823Arg

26.00 ± 0.0828Ser

15.36 ± 0.1917His

*Observed # ResiduesExpected # ResiduesAmino Acid

26.57 ± 0.1327Phe

63.13 ± 0.1961Leu

13.15 ± 0.1514Ile

35.67 ± 0.1636Val

4.16 ± 0.154Met

20.19 ± 0.0820Tyr

57.78 ± 0.3859Lys

28.35 ± 0.1428Pro

47.51 ± 0.1547Ala

31.92 ± 0.0633Thr

80.68 ± 0.2079Glu

55.47 ± 0.2154Asp

17.68 ± 0.2016Gly

22.37 ± 0.0823Arg

26.00 ± 0.0828Ser

15.36 ± 0.1917His

*Observed # ResiduesExpected # ResiduesAmino Acid

* Mean ± Standard Deviation for 75

independent analyses


Measuring Protein ConcentrationAmino Acid Analysis

677.43Mean

47.98Standard Deviation

699.64BSA 1-1

694.09BSA 1-2

7.08% RSD

599.18BSA 5-5

571.57BSA 5-4

571.78BSA 5-3

604.3BSA 5-2

591.4BSA 5-1

678.94BSA 4-5

678.62BSA 4-4

683.07BSA 4-3

678.43BSA 4-2

674.16BSA 4-1

708.10BSA 3-5

708.14BSA 3-4

708.46BSA 3-3

711.50BSA 3-2

714.93BSA 3-1

707.93BSA 2-5

709.75BSA 2-4

715.65BSA 2-3

717.87BSA 2-2

716.66BSA 2-1

695.43BSA 1-5

698.21BSA 1-4

697.88BSA 1-3

Total µg AA / mL

Hydrolysate

Sample ID

677.43Mean

47.98Standard Deviation

699.64BSA 1-1

694.09BSA 1-2

7.08% RSD

599.18BSA 5-5

571.57BSA 5-4

571.78BSA 5-3

604.3BSA 5-2

591.4BSA 5-1

678.94BSA 4-5

678.62BSA 4-4

683.07BSA 4-3

678.43BSA 4-2

674.16BSA 4-1

708.10BSA 3-5

708.14BSA 3-4

708.46BSA 3-3

711.50BSA 3-2

714.93BSA 3-1

707.93BSA 2-5

709.75BSA 2-4

715.65BSA 2-3

717.87BSA 2-2

716.66BSA 2-1

695.43BSA 1-5

698.21BSA 1-4

697.88BSA 1-3

Total µg AA / mL

Hydrolysate

Sample ID


Method OptionsBound vs. Free Amino Acids

NH

3

Cya

His

Tau

Ser A

rg

Gly

Asp

MetS

O2

Glu

Thr

CM

CysA

la

PE

Cys

Pro

AA

BA

Deri

v P

eak

Cys

Lys

Tyr

Met

Val

NV

a

Ile

Leu

Phe

Minutes

1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Protein Hydrolysate20X Dilution ofConcentrate A55

AM

Q

NH

3H

yP

ro

His

Asn

Tau

Ser

Gln Arg G

ly Asp

Glu Thr

Ala

GA

BA

Pro

HyLys1

HyLys2

AA

BA

Orn

Deri

v P

eak

Cys

Lys

Tyr

Met

Val NV

a

Ile

Leu Phe T

rp

Minutes

1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Cell Culture Media10X Dilution ofConcentrate A60



Amino Acid Analysis

Hydrolysates Cell Culture PhysiologicalClinical

Food Analysis

•Nutritional Content•Absolute amount of amino acids•Complex sample handling

•Identification/Adulteration•Unusual amino acids•Marker amino acids•Proprietary

•Criteria•Unequivocal identification•Accuracy, precision, linearity•Sensitivity and speed•Rugged and robust


Amino Acid Analysis of Foods and Feeds

Nutritional monitoring

— Total protein required to support health

— Proportions of specific amino acids required

o Essential amino acids that are not synthesized by the

animal

o Growth-limiting nutrients

Identity testing

— Free amino acids related to metabolic pathways

— Free amino acids derived from the environment of the product


Foods and Feeds Analysis10 pmol Standard on Column

AM

Q

NH

3C

ya H

is

Tau

Ser Arg

Gly A

sp MetS

O2

Glu

Thr

Ala

Pro

AA

BA

Derivitiz

ation P

eak

Cys

Lys

Tyr

Met

Val

NV

a

ILe

Leu

Phe

Minutes

1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00


Analysis of Soybean MealHydrolysate

AM

Q

NH

3

His

Se

r

Arg

Gly

As

p

Glu

Th

r

Ala

Pro

De

riv

Pe

ak

Cy

sL

ys

Ty

r

Me

t

Va

l

Ile

Le

u

Ph

e

AU

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

0.030

0.032

0.034

0.036

0.038

0.040

0.042

0.044

0.046

0.048

0.050

0.052

Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00


Analysis of Soybean MealHydrolysate

Amino Acid Weight % Amino Acid Weight %

His 1.87 ± 0.03 Pro 3.86 ± 0.03

Ser 3.65 ± 0.02 Lys 4.67 ± 0.03

Arg 5.82 ± 0.03 Tyr 2.90 ± 0.02

Gly 2.98 ± 0.02 Met 1.08 ± 0.02

Asp 9.06 ± 0.05 Val 3.66 ± 0.02

Glu 14.49 ± 0.08 Ile 3.45 ± 0.02

Thr 2.92 ± 0.02 Leu 6.12 ± 0.03

Ala 3.17 ± 0.02 Phe 3.92 ± 0.02

Mean ± Standard Deviation for 75 independent replicate analyses


Analysis of Hydrolyzed Grains and Complex Feeds

AM

Q

NH

3

His

Se

r

Arg

Gly A

sp

Glu

Th

r

Ala

Pro

De

riv P

ea

k

Cy

sL

ys

Ty

r

Me

t

Va

l

Ile

Le

u

Ph

e

AU

0.000

0.005

0.010

0.015

0.020

0.025

AM

Q

NH

3

His

Se

r

Arg

Gly

As

p

Glu

Th

r

Ala

Pro

De

riv P

ea

k

Cy

sL

ys

Ty

r

Me

t

Va

l

Ile

Le

u

Ph

e

AU

0.000

0.005

0.010

0.015

0.020

0.025

AM

Q

NH

3

His

Se

r

Arg G

ly

As

p

Glu

Th

r Ala

Pro

De

riv P

ea

k

Cy

sL

ys

Ty

r

Me

t

Va

l

Ile

Le

u

Ph

e

AU

0.000

0.005

0.010

0.015

0.020

0.025

AM

Q

NH

3

His

Se

r

Arg G

ly

As

p

Glu

Th

r Ala

Pro

De

riv P

ea

k

Cy

sL

ys

Ty

r

Me

t

Va

l

Ile

Le

u

Ph

e

AU

0.000

0.005

0.010

0.015

0.020

0.025

Minutes

1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Poultry Diet

Swine Diet

Whole Soybean

Soybean Meal


Asn

Ser

Gln

Arg

Gly

Asp

AU

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

0.0045

0.0050

0.0055

0.0060

0.0065

0.0070

0.0075

0.0080

0.0085

0.0090

0.0095

0.0100

0.0105

Minutes1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30

Free Amino Acids in Starting Malts for Brewing Process


Free Amino Acids Levels Starting Malt

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

Pro Asn Gln Leu

Amino Acids

Mo

le %

2-Row Malt

6-Row Malt

PilsenerMalt



Amino Acid Analysis

Hydrolysates Cell Culture Food Analysis PhysiologicalClinical

•Turnkey Analyzer•Regulated and Validated

•Diagnosis of Metabolic Defects

•Nutritional Monitoring

•Separate solution for Research use only


MassTrak™Amino Acid Analysis Solution

FOR RESEARCH USE ONLY.NOT FOR USE IN DIAGNOSTIC PROCEDURES


Physiological Amino Acid StandardP

Ser

AM

QH

yP

roN

H3 H

isP

EA A

sn

3M

HT

au

1M

H

Ser

Gln

Carn

Arg

Gly Ans

EA

Asp

Sar

Glu C

it B-A

la Thr Ala GA

BA

AA

DA

Pro

BA

IB Hyl1 Hyl2

AA

BA

Cyst

Orn

Cys

Lys

Tyr

Deri

vatization P

eak

Met

Val

Nva

Ile

aIle

Leu

HC

ys

Phe

Trp

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

0.110

0.120

Minutes

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00


MassTrak™ Amino Acid Analysis Plasma Sample

AM

QH

yP

roN

H3

His

Asn

3M

H Tau

1M

H

Ser

Gln

Carn

Arg

Gly

EA

Asp

Glu

Cit

B-A

laT

hr

Ala

AA

DA

Pro

BA

IB

Hyl2 AA

BA

Orn

Cys

Lys

Tyr

Derivatization P

eak

Met

Val

Nva

Ile

aIle

Leu

Phe

Trp

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

0.110

0.120

Minutes

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00



Turnkey System

— Newly developed analytical method

— Based on established chemistry and instruments

— Components tested for ruggedness

Demonstrated for most common applications

— Protein structure

— Cell culture

— Nutritional analysis of foods and feeds

Right Answer Assured

uplc amino acid analysis solution...©2009 waters corporation | company confidential 2 uplc® amino...

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