Download Amino Acid Presentation

Comparison of Different Methods for
Determination of Amino Acids Contents
in Food and Feed
Y. Zhang and J.M. Sido
(National Corn-to-Ethanol Research Center,, Edwardsville, IL)
S.N. Daniels
(Applied Biosystems, Framingham, MA)
R. Dickerson and L. Reimann
(Eurofins US in Des Moines,Des Moines, Iowa)
H. Hewitson and T.E. Wheat
(Waters Corporation, Milford, MA)
L. Novotny
(Olson Biochemistry Labs, South Dakota State University, Brookings SD)
J. Reuther, S. Ruiz and F. Sudradjat
(Eurofins Central Analytical Laboratories,Metairie, Louisiana)
K. Sjogren
(Minnesota Valley Testing Laboratories, New Ulm, MN)
Analytical Methods Used
•
HPLC – ion exchange with ninhydrin post-column derivatization (3
labs)
•
HPLC – ion exchange with o-phthaldialdehyde post-column
derivatization (1 lab)
•
HPLC – pre-column derivatization (ACCQ-Fluor Reagent Kit) with
FLD (1 lab)
•
UPLC – pre-column derivatization (6-aminoquinolyl-Nhydroxysuccinimidyl carbamate) (2 labs)
•
LC/MS/MS – pre-column derivatization (iTRAQ™ reagent, an amine
reactive compound) (2 labs).
Amino Acid Round Robin Design
• 15 amino acids:
Alanine, arginine, aspartic acid, glycine, glutamic acid, histidine, isoleucine, leucine, lysine,
phenylalanine, proline, serine, threonine, tyrosine and valine
• Seven samples:
1 amino acid hydrolysate standard (NIST)
1 peanut butter (NIST)
5 DDGS hydrolysates (triplicate and 2 NIST-STD spiked hydrolysates)
• Performance Criteria:
Precision (DDGS hydrolysate triplicate)
Accuracy (amino acid hydrolysate standard from NIST, food hydrolysate
from NIST, DDGS hydrolysate spiked with NIST standard)
Amino Acid Round Robin Design
(AOCS)
15 amino acids:
Alanine, arginine, aspartic acid, glycine, glutamic acid, histidine, isoleucine,
leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine and valine
Five samples:
soybean, soybean meal, swine diet, poultry diet and BSA
Test Methods:
GC/MS/MS (1 lab)
UPLC – pre-column derivatization (6-aminoquinolyl-N-hydroxysuccinimidyl
carbamate) (2 labs)
Test Scheme:
Test each sample three times a week in 5 consecutive weeks
Data Evaluation
•
•
•
•
Variation by individual methods
Variation by individual amino acids
Variation by individual labs
Variation by sample matrix
Amino Acid Round Robin Studies
Precision
Precision (RSD, %)
(Based on DDGS hydrolysate triplicate)
14
HPLC-PRE
UPLC-PRE
LCMSMS-PRE HPLC-POS
Asp
12
Precision (RSD, %)
Thr
Ser
10
Glu
Pro
Gly
8
Ala
Val
6
Isl
Leu
Tyr
4
Phe
Lys
His
2
Arg
0
Lab
B
Lab
C
Lab
G
Lab
E
Lab
J
Lab
H
Lab I
Lab
D
Precision (RSD, %)
(Based on DDGS hydrolysate triplicate)
Methods
Lab 1
Lab 2
Lab 3
Mean
(of all labs)
HPLC
Post–Column
Deriv.
UPLC
Pre-Column
Deriv.
LC/MS/MS
Pre-Column
Deriv.
HPLC
Pre-Column
Deriv.
*
1.9*
2.4
1.1
1.8
( 0.3)
( 0.7)
( 0.5)
( 0.7)
3.5
0.6
2.1
( 1.3)
( 0.1)
( 2.1)
2.1
4.9
3.5
( 1.4)
( 1.2)
( 2.0)
8
Mean of 15 amino acids
8
Precision (RSD, %)
(Soy bean sample, tested in 5-week period, mean of 15 data points)
4.0
GC-MS-MS
UPLC-PRE
3.5
Asp
Thr
Ser
3.0
Precision (RSD, %)
Glu
Pro
2.5
Gly
Ala
Val
2.0
Isl
Leu
Tyr
1.5
Phe
Lys
His
1.0
Arg
0.5
0.0
Lab 1
Lab 2
Lab 3
Precision (RSD, %)
(Soy bean sample, tested in 5-week period, mean of 15 data points)
Methods
Lab 1
Lab 2
Mean
(of all labs)
GC-MS-MS
UPLC
Pre-Column
Deriv.
*
1.6*
1.6
( 0.5)
( 0.5)
1.6
0.8
1.2
( 0.9)
( 0.7)
( 0.5)
Mean of 15 amino acids
Summary on Precision
•
•
•
Precision is method dependent: HPLC post–
column derivatization has the best
performance (RSD < 2%) and HPLC precolumn derivatization has the highest RSD.
Most methods investigated show satisfying
precision for within batch and between batch
runs.
More data points needed to produce
statistically sound conclusion.
Amino Acid Round Robin Study
Accuracy
Accuracy (Recovery, %)
(Based on NIST amino acid hydrolysate standard)
Accuracy (Recovery, %)
140
A sp
HPLC-POS
UPLC-PRE
LCMSMS-PRE
HPLC-PRE
T hr
Ser
130
Gl u
Pr o
120
Gl y
Al a
110
V al
I sl
100
Leu
T yr
90
P he
Lys
Hi s
80
La b B La b C La b G
La b E La b J
La b H La b I
La b D
Ar g
Accuracy (Recovery, %)
(Based on NIST amino acid hydrolysate standard)
Methods
Lab 1
Lab 2
Lab 3
Mean
(of all labs)
HPLC
Post–Column Deriv.
UPLC
Pre-Column Deriv.
LC/MS/MS
Pre-Column Deriv.
HPLC
Pre-Column Deriv.
98*
104
105
102
( 8)
( 8)
( 8)
( 3)
96
105
100
( 9)
( 7)
( 6)
93
107
100
( 5)
( 8)
( 10)
105
* Mean of 15 amino acids
105
Amino Acid Contents in DDGS Hydrolysate
70.0
Amino Acid Content in DDGS Hydrolysate (ug / ml)
HPLC-POS
UPLC-PRE
LCMSMS-PRE
HPLC-PRE
60.0
Asp
Thr
Ser
Glu
50.0
Pro
Gly
40.0
Ala
Val
30.0
Isl
Leu
Tyr
20.0
Phe
Lys
10.0
His
Arg
0.0
La b
B
La b
C
La b
G
La b E La b J
La b
H
La b I
La b
D
Total Amino Acid Contents in DDGS Hydrolysate
(ug / ml)
Methods
Lab 1
Lab 2
Lab 3
Mean
(of all labs)
HPLC
Post–Column
Deriv.
UPLC
Pre-Column
Deriv.
LC/MS/MS
Pre-Column
Deriv.
HPLC
Pre-Column
Deriv.
247*
282
256
261
( 5)
( 7)
( 3)
( 18)
216
289
253
( 8)
( 2)
( 52)
253
250
252
( 5)
( 12)
( 2)
266
* Mean of triplicate
266
Accuracy (Recovery, %)
(DDGS hydrolysate spiked I)
145
HPLC-POS
UPLC-PRE
LCMSMS-PRE HPLC-PRE
Asp
135
Thr
Accuracy (Recovery, %)
Ser
125
Glu
Pro
Gly
115
Ala
Val
105
Isl
Leu
Tyr
95
Phe
Lys
85
His
Arg
75
65
Lab B Lab C Lab G
Lab E Lab J
Lab H
Lab I
Lab D
Accuracy (Recovery, %)
(DDGS hydrolysate spiked I)
Methods
Lab 1
Lab 2
Lab 3
Mean
(of all labs)
HPLC
Post–Column Deriv.
UPLC
Pre-Column Deriv.
LC/MS/MS
Pre-Column Deriv.
HPLC
Pre-Column Deriv.
105*
94
102
101
( 5)
( 8)
( 7)
( 6)
115
99
107
( 4)
( 6)
( 11)
102
98
100
( 3)
( 13)
( 2)
114
* Mean of 15 amino acids
114
Accuracy (Recovery, %)
(DDGS hydrolysate spiked II)
155
HPLC-POS
UPLC-PRE
LCMSMS-PRE
HPLC-PRE
Asp
145
Thr
Ser
Accuracy (Recovery, %)
135
Glu
Pro
125
Gly
Ala
Val
115
Isl
Leu
105
Tyr
Phe
95
Lys
His
85
Arg
75
65
Lab B Lab C Lab G
Lab E Lab J
Lab H Lab I
Lab D
Accuracy (Recovery, %)
(DDGS hydrolysate spiked II)
Methods
Lab 1
Lab 2
Lab 3
Mean
(of all labs)
HPLC
Post–Column Deriv.
UPLC
Pre-Column Deriv.
LC/MS/MS
Pre-Column Deriv.
HPLC
Pre-Column Deriv.
103*
100
100
101
( 5)
( 8)
( 8)
( 2)
106
97
101
( 5)
( 8)
( 6)
102
98
100
( 6)
( 17)
( 3)
93
* Mean of 15 amino acids
93
Summary on Accuracy
(in testing hydrolysate)
•
•
•
The three methods: HPLC post–column, UPLC
and LC/MS/MS give similar performance,
which have 100% recovery for most amino
acids, with the HPLC post–column method
being the most consistent.
Except for LC/MS/MS, all the other methods
have higher than 120% recovery for histidine.
More data points needed to produce
statistically sound conclusion.
Amino Acid Round Robin Studies
(Based on food and feed samples)
Accuracy (Recovery, %)
(Peanut Butter Standard, NIST)
175
HPLC-POS
UPLC-PRE
LCMSMS-PRE HPLC-PRE
165
Asp
Thr
Ser
155
Accuracy (RSD, %)
Glu
Pro
145
Gly
135
Ala
Val
125
Isl
Leu
115
Tyr
Phe
105
Lys
95
His
Arg
85
75
Lab
B
Lab
C
Lab
G
Lab
E
Lab
J
Lab
H
Lab
I
Lab
D
Record of Accuracy
(Recovery, %, based on NIST Peanut Butter)
Methods
Lab 1
Lab 2
Lab 3
Mean
(of all labs)
HPLC
Post–Column Deriv.
UPLC
Pre-Column Deriv.
LC/MS/MS
Pre-Column Deriv.
HPLC
Pre-Column Deriv.
105 *
114
106
108
( 8)
( 17)
( 12)
( 5)
98
120
98
( 8)
( 7)
( 16)
101
99
100
( 9)
( 6)
( 1)
102
* Mean of 15 amino acids
102
Amino Acid Contents in Soybean
4.00
Amino Acid Content (%, wt/wt, as rec.)
GC-MS-MS
UPLC-PRE
Asp
3.50
Thr
Ser
Glu
3.00
Pro
Gly
Ala
Val
2.50
Isl
Leu
Tyr
2.00
Phe
Lys
His
Arg
1.50
1.00
Lab 1
Lab 2
Lab 3
Total Amino Acid Contents in Soy Bean
(wt/wt, %, as-rec.)
Methods
Lab 1
Lab 2
Mean
(of all labs)
GC-MS-MS
45.6*
45.6
( 0.5)
UPLC
Pre-Column
Deriv.
41.7
45.6
( 0.5)
( 0.2)
* Mean of 15 runs
43.6
( 2.7)
Comparison on other Factors
Methods
Run Time
(minutes)
Price
($)
60
10
180
100
LC/MS/MS
Pre-Column Deriv.
24
200
HPLC
Pre-Column Deriv.
40
250
HPLC
Post–Column Deriv.
UPLC
Pre-Column Deriv.
Summary on Accuracy
in testing food and feed
•
•
The three methods: HPLC post–column,
UPLC and LC/MS/MS give similar
performance, which have 100% recovery
for most amino acids, with the HPLC
post–column method being the most
consistent.
More data points needed to produce
statistically sound conclusion.