Download UPLC Amino Acid Analysis Solution

UPLC Amino Acid Analysis Solution
AAAFCO Meeting
Washington, DC
August 2, 2009
©2009 Waters Corporation | COMPANY CONFIDENTIAL
0.34
Lys
0.36
AMQ
UPLC® Amino Acid Analysis Solution
0.32
0.30
0.28
0.26
0.24
0.08
0.06
Pro
Ala
Glu
Deriv Peak
NH3
0.10
His
0.12
Arg
Gly
Ser
0.14
Asp
0.16
Thr
0.18
Ile
Leu
Phe
Cys
0.20
Tyr
Met
Val
0.22
0.04
0.02
0.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Minutes
5.00
5.50
6.00
6.50
7.00
7.50
8.00
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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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
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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Total System Approach
Assured 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
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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Chemistry of AQC Derivatization
O
O
O
H
N
O
N
NH2
OH 1o Amino Acid;
t1/2 <<1 s
O
N
OH
NH
+
CO2
Derivatized Amino Acids
2o Amino Acid;
t1/2 <<1 s
H
N
AMQ
H2O
t1/2 ~ 15 s
N-Hydroxy
Succinimide
NH
O
N
O
O
AQC Reagent
OH
H
N
O
N
+
N
6-Aminoquinolone (AMQ)
N
O
HO
H
N
H
N
NH2
N
O
N
bis-aminoquinoline urea (derivatization peak)
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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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
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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Chromatography Principles
Mass Transfer / Diffusion
Mobile Phase
A
Analyte Molecules
B
Porous
Particle
C
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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UPLC® Amino Acid Analysis
Application Solution
©2009 Waters Corporation | COMPANY CONFIDENTIAL
8
Lys
34
AMQ
36
UPLC® Amino Acid Analysis
Application Solution
32
30
28
26
24
08
06
Pro
Ala
Glu
Deriv Peak
NH3
10
His
12
Asp
Ser
14
Arg
Gly
16
Thr
18
Ile
Leu
Phe
Cys
20
Tyr
Met
Val
22
04
02
00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Minutes
5.00
5.50
6.00
6.50
7.00
7.50
8.00
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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Amino Acid Analysis
Application Types
Amino Acid Analysis
Hydrolysates
Cell Culture
Food Analysis
Physiological
Clinical
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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0.068
0.066
AMQ
Analysis of Acid Hydrolyzed BSA
9ng on column
Deriv Peak
0.064
0.062
0.060
0.058
0.056
0.054
0.052
0.050
0.048
0.046
Lys
0.044
0.042
0.040
0.038
0.034
0.032
Leu
0.030
0.028
Glu
0.026
0.024
0.022
Val
0.020
Ser
0.008
His
0.006
0.004
Phe
Ile
Tyr
Met
0.010
Arg
Gly
0.012
Pro
Thr
0.014
Cys
0.016
Ala
Asp
0.018
NH3
AU
0.036
0.002
0.000
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Minutes
5.00
5.50
6.00
6.50
7.00
7.50
8.00
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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Accuracy of Compositional Analysis
Bovine Serum Albumin
Amino Acid
Expected # Residues
*Observed # Residues
His
17
15.36 ± 0.19
Ser
28
26.00 ± 0.08
Arg
23
22.37 ± 0.08
Gly
16
17.68 ± 0.20
Asp
54
55.47 ± 0.21
Glu
79
80.68 ± 0.20
Thr
33
31.92 ± 0.06
Ala
47
47.51 ± 0.15
Pro
28
28.35 ± 0.14
Lys
59
57.78 ± 0.38
Tyr
20
20.19 ± 0.08
Met
4
4.16 ± 0.15
Val
36
35.67 ± 0.16
Ile
14
13.15 ± 0.15
Leu
61
63.13 ± 0.19
Phe
27
26.57 ± 0.13
* Mean ± Standard
Deviation for 75
independent
analyses
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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Measuring Protein Concentration
Amino Acid Analysis
Sample ID
Total µg AA / mL
Hydrolysate
BSA 1-1
699.64
BSA 1-2
694.09
BSA 1-3
697.88
BSA 1-4
698.21
BSA 1-5
695.43
BSA 2-1
716.66
BSA 2-2
717.87
BSA 2-3
715.65
BSA 2-4
709.75
BSA 2-5
707.93
BSA 3-1
714.93
BSA 3-2
711.50
BSA 3-3
708.46
BSA 3-4
708.14
BSA 3-5
708.10
BSA 4-1
674.16
BSA 4-2
678.43
BSA 4-3
683.07
BSA 4-4
678.62
BSA 4-5
678.94
BSA 5-1
591.4
BSA 5-2
604.3
BSA 5-3
571.78
BSA 5-4
571.57
BSA 5-5
599.18
Mean
677.43
Standard Deviation
47.98
% RSD
7.08
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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NVa
Ile
Leu
Phe
Tyr
Met
Val
AABA
Pro
Glu
Asp
MetSO2
Arg
Gly
Tau
Cya
His
NH3
Ser
Thr
Protein Hydrolysate
20X Dilution of
Concentrate A
55
CM Cys
Ala
PE Cys
Cys
Deriv Peak
Lys
Method Options
Bound vs. Free Amino Acids
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
3.00
Ile
Leu
Phe
Trp
Val
NVa
Met
Tyr
Cys
AABA
HyLys1
HyLys2
Pro
GABA
Ala
Asp
Ser
2.50
Thr
Deriv Peak
2.00
Gln
Arg
Gly
Tau
Glu
1.50
Asn
His
NH3
HyPro
Cell Culture Media
10X Dilution of
Concentrate A
60
Lys
Orn
AMQ
Minutes
3.50
4.00
4.50
Minutes
5.00
5.50
6.00
6.50
7.00
7.50
8.00
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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Amino Acid Analysis
Application Types
Amino Acid Analysis
Hydrolysates
Cell Culture
Food Analysis
Physiological
Clinical
•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
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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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
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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Foods and Feeds Analysis
1.00
1.50
2.00
ILe
Leu
Phe
AABA
Derivitization Peak
Cys
Lys
Tyr
Met
Val
NVa
Pro
Ala
Thr
Glu
Asp
MetSO2
Ser
Arg
Gly
His
NH3
Cya
Tau
AMQ
10 pmol Standard on Column
2.50
3.00
3.50
4.00
4.50
Minutes
5.00
5.50
6.00
6.50
7.00
7.50
8.00
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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0.052
AMQ
Analysis of Soybean Meal
Hydrolysate
Deriv Peak
0.050
0.048
0.046
0.044
0.042
0.040
0.038
0.036
0.034
0.032
0.030
Glu
0.026
0.020
Leu
Lys
0.022
Ala
Cys
0.006
His
0.004
Ile
Tyr
0.008
Arg
Ser
0.010
Thr
Gly
0.012
Met
0.014
Pro
0.016
Val
Asp
0.018
Phe
0.024
NH3
AU
0.028
0.002
0.000
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Minutes
5.00
5.50
6.00
6.50
7.00
7.50
8.00
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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Analysis of Soybean Meal
Hydrolysate
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
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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Leu
Phe
Ile
Val
Met
Cys
Tyr
Pro
Ala
Lys
Deriv Peak
Glu
Asp
His
0.005
Thr
0.010
Arg
NH3
AU
0.015
Gly
Poultry Diet
0.020
Ser
0.025
AMQ
Analysis of Hydrolyzed Grains and
Complex Feeds
Phe
Ile
Met
Tyr
Val
Lys
Leu
Deriv Peak
Pro
Cys
His
0.005
Thr
Ser
0.010
Ala
Glu
NH3
AU
0.015
Asp
Swine Diet
0.020
Arg
Gly
0.025
AMQ
0.000
Phe
Ile
Met
Cys
Tyr
Val
Leu
Lys
Deriv Peak
Ala
His
0.005
Thr
Ser
0.010
Pro
Asp
NH3
AU
0.015
Glu
Whole Soybean
0.020
Arg
Gly
0.025
AMQ
0.000
Phe
Ile
Met
Cys
Tyr
Val
Leu
Lys
Deriv Peak
Glu
His
0.005
Pro
0.010
Ala
Ser
0.015
Thr
NH3
AU
Asp
Soybean Meal
0.020
Arg
Gly
0.025
AMQ
0.000
0.000
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
Minutes
5.50
6.00
6.50
7.00
7.50
8.00
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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Asn
Free Amino Acids in Starting Malts
for Brewing Process
0.0105
0.0100
0.0095
0.0090
0.0085
Gln
0.0080
0.0075
0.0070
0.0065
Asp
0.0060
Ser
AU
0.0055
0.0050
0.0040
0.0035
Gly
Arg
0.0045
0.0030
0.0025
0.0020
0.0015
0.0010
0.0005
0.0000
1.80
1.90
2.00
2.10
2.20
2.30
2.40
2.50
2.60
Minutes
2.70
2.80
2.90
3.00
3.10
3.20
3.30
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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Free Amino Acids Levels
Starting Malt
35.00
30.00
2-Row Malt
25.00
Mole %
6-Row Malt
20.00
Pilsener
Malt
15.00
10.00
5.00
0.00
Pro
Asn
Gln
Amino Acids
Leu
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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Amino Acid Analysis
Application Types
Amino Acid Analysis
Hydrolysates
Cell Culture
Food Analysis
Physiological
Clinical
•Turnkey Analyzer
•Regulated and
Validated
•Diagnosis of
Metabolic Defects
•Nutritional
Monitoring
•Separate solution
for Research use
only
©2009 Waters Corporation | COMPANY CONFIDENTIAL
23
MassTrak™
Amino Acid Analysis Solution
FOR RESEARCH USE ONLY.
NOT FOR USE IN DIAGNOSTIC PROCEDURES
©2009 Waters Corporation | COMPANY CONFIDENTIAL
24
0.120
AMQ
Physiological Amino Acid Standard
Orn
0.110
0.100
Lys
0.090
HCys
Trp
Nva
Val
Cys
0.020
Phe
Cyst
GABA
AADA
Pro
BAIB
Hyl1
Hyl2
AABA
Ala
Ile
aIle
Leu
Sar
0.030
B-Ala
Thr
0.040
Glu
Cit
0.050
HyPro
NH3
His
PEA
Asn
3MH
Tau
1MH
Ser
Gln
Carn
Arg
Gly
Ans
EA
Asp
0.060
PSer
AU
0.070
Tyr
Peak
Derivatization
Met
0.080
0.010
0.000
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
Minutes
20.00
22.00
24.00
26.00
28.00
30.00
32.00
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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0.120
AMQ
MassTrak™ Amino Acid Analysis
Plasma Sample
0.110
Lys
Derivatization Peak
0.100
0.090
0.060
4.00
6.00
8.00
10.00
12.00
16.00
Val
18.00
Minutes
Nva
20.00
22.00
24.00
26.00
28.00
Trp
Phe
Leu
Ile
aIle
Met
Cys
BAIB
AADA
14.00
Hyl2
AABA
Tyr
Thr
0.000
B-Ala
Cit
EA
Asp
Glu
Arg
Carn
His
Asn
3MH
Tau
1MH
0.020
0.010
Ser
0.030
Orn
Gly
0.040
Pro
NH3
0.050
HyPro
AU
0.070
Ala
Gln
0.080
30.00
32.00
©2009 Waters Corporation | COMPANY CONFIDENTIAL
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UPLC® Amino Acid Analysis
Application Solution
 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
©2009 Waters Corporation | COMPANY CONFIDENTIAL
27