Accurate and Reliable Amino

Acid Analysis of Foods and

Feeds

Chantel Lee
Chemistry Specialist
Waters Pacific

©2020 Waters Corporation COMPANY CONFIDENTIAL 1

 Topics for this Session

INTRODUCTION AMINO ACID ANALYSIS

▪ Amino acid structure and METHODS &
classification THE CHALLENGES
▪ Types of samples

SOLUTIONS APPLICATIONS
▪ AccQ-Tag
▪ Andrew Alliance

©2020 Waters Corporation COMPANY CONFIDENTIAL 2

 Amino Acids
Essential and non-essential amino acids
Amine Carboxyl a) Essential: Amino acids that cannot be synthesized de
Group Group novo by the organism
b) Non-Essential: the rest

Essential Non-Essential
Histidine (H) Alanine (A)
Isoleucine (I) Arginine* (R)
Leucine (L) Aspartic acid (D)
Lysine (K) Cysteine* (C)
Variable Group
Methionine (M) Glutamic acid (E)
Phenylalanine (F) Glutamine* (Q)
Threonine (T) Glycine* (G)
Tryptophan (W) Proline* (P)
Valine (V) Serine* (S)
Tyrosine* (Y)
Asparagine* (N)
Selenocysteine (U)
Average MW: 100-110 Da
Pyrrolysine** (O)
©2020 Waters Corporation COMPANY CONFIDENTIAL 3
 Key Characteristic for Amino Acid Analysis (AAA) Samples

 Samples requiring AAA contain either bound or free amino acids

– Bound amino acids are those amino acids which form peptides and proteins.
o This type of sample includes proteins, peptides and/or foods and feeds
o The sample must go through hydrolysis in order to analyze the individual amino acids

– Free amino acids are the individual amino acids

C
o They are not interconnected to peptides or proteins
L P
o This type of sample can include foods, plant or animal matrices H
A
o This sample does not require hydrolysis, but may require other sample preparation (i.e. D S
K
deproteinization)

©2020 Waters Corporation COMPANY CONFIDENTIAL 4

 Bound Amino Acids:
Hydrolysis for Analyzing Amino Acid Content of Peptides and
Proteins C-terminal

 An amino acid sequence is unique to a

particular peptide or protein
 To analyse bound proteins, sample N-terminal
preparation requires reaction to break up
the amino acid bonds
 Hydrolysis is the most common method for
preparing a protein sample for amino acid
analysis
– Includes acid and alkaline hydrolysis
– Each type of hydrolysis allows for
measurement of specific amino acids
– To measure all 20 hydrolysate amino acids,
multiple reactions will need to be performed
Acid hydrolysis
©2020 Waters Corporation COMPANY CONFIDENTIAL 5
 Bound Amino Acids:
Hydrolysis for Analyzing Amino Acid Content of Peptides and
Proteins

Performic Acid Oxidation

Acid Hydrolysis + Acid Hydrolysis Alkaline Hydrolysis

▪ Most common type of ▪ Analysis of cysteine, cystine and ▪ Analysis of tryptophan

hydrolysis methionine o Tryptophan is destroyed by
▪ AOAC 994.12 ▪ Performic acid oxidation (AOAC acidic hydrolysis
▪ Variability due to complete or 994.12) ▪ AOAC 988.15
partial destruction of AA o Cysteine/ Cystine → Cysteic acid
o Serine and threonine are
partially destroyed
o Tryptophan and cysteine Cys Cya
are destroyed o Methionine → Methionine sulfone
o Methionine might undergo
oxidation

Met MetSO2
©2020 Waters Corporation COMPANY CONFIDENTIAL 6
 Bound Amino Acids:
Hydrolysis Instrumentation

 Microwave hydrolysis
– Newer hydrolysis instrumentation
– Individually programmed methods
– Uses microwaves to apply energy for hydrolysis
– Temperature control also applied to sample
– Increased throughput, sample can be hydrolyzed in < 1 hour Hydrolyzed feed sample (liquid phase)
– Load up to 48 samples
– Compatible with all hydrolysis reactions

 After hydrolysis, sample preparation prior to derivatization may

include:
– Centrifugation
– Filtration
– Neutralization
– Dilution

©2020 Waters Corporation COMPANY CONFIDENTIAL 7

 Free Amino Acids

 Amino acid analysis is required to analyze “free” or non bound amino acids for a
variety of samples, including:
– Cell culture media
C
– Foods and beverages containing free amino acids L P
A H
– Physiological amino acids in human, animal and plant matrices D S
K

 Sample preparation is less than bound amino acids

– Deproteinization
– Dilution
– Filtration
https://en.wikipedia.org/wiki/Energy_drink#/media/File
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©2020 Waters Corporation COMPANY CONFIDENTIAL 8

 Amino Acid Analysis: the Challenges
Sample preparation
 Amino acids are small compounds
(~110 Daltons each)
Sample
Preparation  Complex samples
 Varied sample preparation
Separation
 Derivatization typically required (pre or
 Wide range of properties
post column)
 Slight differences between pairs LC Analysis – Needs to be uniform
 Wide range of matrices

Detection
 Low or no chromophore Data
Analysis
 Wide concentration range
 Need consistent quantitation

©2020 Waters Corporation COMPANY CONFIDENTIAL 9

 Review of Separation Techniques of Amino Acids
Free amino acids

 Post-column Derivatization
– Derivatization is done after the column
– Typically IEX separation
– Derivatization is less sensitive to matrix components column

– Derivatives are short lived – detection has to take place

immediately
Derivatization Tag
– Realistic run times near 30-60min
Cation
Exchanger R H
C
- +H2N COO-
-
- - --
Stationary - - -
Phase
- - -

Mid 1958 ▪ Pre-column derivatization

1950’s
©2020 Waters Corporation COMPANY CONFIDENTIAL ▪ Post-column derivatization10
 Review of Separation Techniques of Amino Acids

 Pre-column Derivatization
– Derivatization is done before the column
– Typically RPLC separation
o Better separation selectivity
o Better sensitivity
Derivatization Tag
o Shorter run times (10-30 min)
– Derivatives are stable (for certain reagent) – enables retesting of sample R H
C
– Many reagents used successfully; some commercialized Reveresed N COO-
Phase R
o OPA and FMOC – two reagents for all amino acids
o AQC reagent
Stationary
Phase

Mid 1958 1971 1979 1980 1983 1985 1988 1992 ▪ Pre-column derivatization
1950’s
©2020 Waters Corporation COMPANY CONFIDENTIAL ▪ Post-column derivatization11
 Pre and Post-Column Derivatizing Reagents
Ninhydrin FMOC OPA AQC
Mode Post-column Pre-column Pre-column

Instruments Dedicated system LC (modification may be required) LC

Absorbance or
Detection mode Absorbance Absorbance or Fluorescence
Fluorescence
Detection
Multiple Multiple Single
Wavelength

Sensitivity pmol fmol fmol

Derivatize stability Good Excellent Poor Excellent

Reaction kinetics Rapid Rapid Rapid

Secondary amino
Yes Yes No Yes
acids
Single peak per
Yes No Yes Yes
amino acid

©2020 Waters Corporation COMPANY CONFIDENTIAL 12

 Kit Chemistry
AccQ-TagTM Derivatization
1 º Amino Acid
t 1/2 << 1 s

1 Fast reaction time

2 Single
AQC Reagent
Patented Derivatized Amino Acids Wavelength
2º Amino Acid Detection
t 1/2 << 1 s

AMQ
H2O
t 1/2 ~ 15 s
By-Product
4 Fully Resolved
3
No special
handling to
6-Aminoquinoline remove excess bis-aminoquinoline urea (derivatization
(AMQ) peak)
reagent
©2020 Waters Corporation COMPANY CONFIDENTIAL 13
 Key Features of AccQ-Tag Chemistry
Hydrolyze sample
 Reagent is stable at room temperature
 One step derivatization; No special sample
preparation, vacuum drying, extraction required Add 70 µL of buffer to vial
 Reaction performed in largely aqueous conditions
 Enhanced sensitivity for both UV and MS detection
Add 10 µL of standard/sample to vial
methods
 High resolution and sensitivity characteristics
 Excellent quantitative results Add 20 µL of reagent to vial

 Automated derivatization using Andrew Alliance, 55 ºC for 10 min

Hamilton or Tecan Inject 1 µL

©2020 Waters Corporation COMPANY CONFIDENTIAL

5 Ready for injection
14
 Keys to Automation

 Identify system for automation

– Requires derivatized amino acids are stable (e.g. AccQ*Fluor)
– Ensure required “blocks” are included

 Ensure sample preparation kits are compatible with system

– Adequate volumes and appropriate vials

 Develop and test scripts for reproducible, accurate results

– Volume accuracy
– Mixing efficiency

 Andrew Alliance, Tecan and Hamilton kits and scripts available

with AccQ-Tag

©2020 Waters Corporation COMPANY CONFIDENTIAL 15

 Andrew Alliance:
Example Workbench

 Example workbench

1. Tip insertion system, 10-300 µL

2. Tip insertion system, 5-120 µL
3. Tip insertion system, 5-120 µL
4. Tip Rack, 100-5000 µL
5. Microplate holder
6. Microtube holder
7. 6-channel deep-well microplate
8. 96-well PCR plate Shaker+
9. 96-well PCR plate Peltier+

©2020 Waters Corporation COMPANY CONFIDENTIAL 16

 the Solution
Amino Acid Analysis: the Challenges
Sample preparation
▪ Stable
Amino acids – important
are small
derivatives compounds
for
reproducibility
(~110 Daltons each)
Sample
Preparation ▪ Ready-to-use
Complex samplesreagents and
 standards – save
Varied sample time and minimize
preparation
Separation
Separation variability
 Derivatization typically required (pre or
 Wide range of properties ▪ Automation/
 Separation on Alliance HPLC or post column) simple-to-follow
 Slight differences
ACQUITY between pairs
UPLC H-Class LC Analysis manual procedure – minimal
– Needshuman
training/ to be uniform
error
 Systems
Wide range of matrices
 Pre-optimized separation method
Detection
– high resolution, short run time,
 good
Low orreproducibility
no chromophore Data
Analysis

 Ready-to-use mobile
Wide concentration phase –
range
minimize pH error
 Need consistent quantitation
 Reliable column chemistry
©2020 Waters Corporation COMPANY CONFIDENTIAL 17
 Amino Acid Analysis Methods

HPLC – 50 min

Phe
ILe
Leu
Val
Fluorescence Detection

Ala

Met
Tyr

Lys
Pro
His

Arg
Thr
Ser
Asp

Glu

Gly

NH3

Cys
AMQ

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 34.00 36.00 38.00
Minutes

UPLC – 10 min
UV Detection

Plug & Play; NO

Method Development
Required

©2020 Waters Corporation COMPANY CONFIDENTIAL 18

 the Solution
Amino Acid Analysis: the Challenges
Sample preparation
▪ Stable
Amino acids – important
are small
derivatives compounds
for
reproducibility
(~110 Daltons each)
Sample
Preparation ▪ Ready-to-use
Complex samplesreagents and
 standards – save
Varied sample time and minimize
preparation
Separation
LC Analysis variability
 Derivatization typically required (pre or
 Separation
Wide range of onproperties
Alliance HPLC or ▪ Automation/
post column) simple-to-follow
 ACQUITY UPLCbetween
Slight differences H-Classpairs LC Analysis manual procedure – minimal
– Needshuman
training/ to be uniform
error
 Systems
Wide range of matrices
 Pre-optimized separation
method – high resolution, short
Detection Empower 3
run time, good reproducibility Data
 Low or no chromophore  Compliant-ready
 Ready-to-use mobile phase – Analysis
 minimize
Wide concentration
pH error range  Existing or customizable report
 Reliable
Need consistent template
 columnquantitation
chemistry
 Custom fields
©2020 Waters Corporation COMPANY CONFIDENTIAL 19
 Amino Acid Analysis in Foods and Feeds

 Nutritional Content
– Absolute/ total amount of amino acids
– Proportions of essential amino acids
– Optimize blended feeds
 Identification/ Adulteration
– Unusual amino acids
– Marker amino acids
– Proprietary constituents
– Authentic composition

©2020 Waters Corporation COMPANY CONFIDENTIAL 20

 Amino Acid Analysis
- Feed

 Nutritional monitoring
– Total protein required to support
health
– Specific amino acids required
o Essential amino acids not
synthesized by the animal
o Growth-limiting nutrients

©2020 Waters Corporation COMPANY CONFIDENTIAL 21

 Sample Preparation for Food and Feeds
 Free amino acids
– For beverages, free amino acids can also be evaluated
– Liquid foods may need to be diluted

 Bound amino acids

– Hydrolysis (Performic acid, acidic, alkaline)

 Additional techniques due to sample complexity

– Defatting
– Filtration, etc.

©2020 Waters Corporation COMPANY CONFIDENTIAL 22

 Hydrolysis of Cat Food:
Workflow

Pre-column Separation with

Microwave Hydrolysis Derivatization with ACQUITY UPLC H-
AccQTag Class

 Overlay of five replicate hydrolyzed cat food samples

©2020 Waters Corporation COMPANY CONFIDENTIAL 23

 Repeatability of Sample Preparation + AccQ•Tag
Derivatization
 Sample preparation:
– Large sample was ground, in blender
– Sample was hydrolyzed
– Internal standard (norvaline) was added to sample prior to hydrolysis
– Five separate samplings were hydrolyzed
– High precision was observed for procedure (<3.5% for all amino acids)

Weight
Sample His Tau Ser Arg Gly Asp Glu Thr Ala Pro Lys Tyr Met Val Ile Leu Phe
mg

C1 Cat Food 56.030 0.565 0.090 1.540 1.594 1.564 2.989 6.106 1.062 2.138 2.178 1.862 0.980 0.564 1.238 0.961 3.008 1.273

C2 Cat Food 53.800 0.550 0.088 1.470 1.565 1.544 3.059 6.095 1.039 2.130 2.108 1.922 0.941 0.561 1.244 0.974 2.925 1.219

C3 Cat Food 51.980 0.537 0.083 1.413 1.508 1.468 2.781 5.711 0.998 1.998 2.025 1.747 0.927 0.544 1.196 0.947 2.843 1.200

C4 Cat Food 51.410 0.543 0.085 1.438 1.543 1.559 2.884 5.793 1.020 2.042 2.058 1.834 0.929 0.567 1.240 0.968 2.838 1.201

C5 Cat Food 54.370 0.538 0.087 1.485 1.552 1.522 3.084 6.109 1.031 2.116 2.101 1.893 0.942 0.561 1.217 0.952 2.903 1.222

2.1 3.2 3.3 2.0 2.6 4.3 3.3 2.3 3.0 2.8 3.6 2.3 1.6 1.7 1.2 2.4 2.4

©2020 Waters Corporation COMPANY CONFIDENTIAL 24

 Complete Amino Acid Analysis
Turkey Starter
Met
Cys MetSO2
Cya

Food and Feed Amino Acid Standard

ACQUITY H-Class

Alkaline Hydrolysis

Acid Hydrolysis

Oxidation w/ Acid Hydrolysis

©2020 Waters Corporation COMPANY CONFIDENTIAL 25

 AAA in Foods and Feeds

 Nutritional Content
– Total amount of amino acids
– Proportions of essential amino acids
– Optimize blended feeds
 Identification/ Adulteration
– Unusual amino acids
– Marker amino acids
– Proprietary constituents
– Authentic composition

©2020 Waters Corporation COMPANY CONFIDENTIAL 26

 Amino Acids as Biomarkers
Free Amino Acid Method
 Free Amino Acids analysis
– Nutrients in final formulation
– Reflect metabolic state / genetics / environment / origin

 Practical Applications
– Competitive & Investigative analysis
– Identification and Adulteration
– Process / Fermentation monitoring

©2020 Waters Corporation COMPANY CONFIDENTIAL 27

 Competitor Analysis of Beer
- Food Identification

 Objective
– Discriminate between different brands of beer
– Profile amino acids present

 Sample
– Beers from various regions
– Light and dark beers
– Sonicated & filtered

©2020 Waters Corporation COMPANY CONFIDENTIAL 28

 Competitor Analysis of Beer
- Food Identification

©2020 Waters Corporation COMPANY CONFIDENTIAL Apps Note: 72000158EN 29

 Competitor Analysis of Beer
- Food Identification
3500

3000 American
American
Dutch
2500

2000

1500

1000

500

0
Ser

Tyr
HyPro

HyLys1

HyLys2

Orn
Arg

Trp
Thr

Pro

Cys

Lys

Met
Tau

Leu
GABA

AABA

Phe
Gly

Val
Asn

Gln

Glu
Asp

Ala

Ile
His

©2020 Waters Corporation COMPANY CONFIDENTIAL 30

 Identification / Adulteration of High Value Food

 Objective
– Discriminate among natural materials of different origin
– Relate profile to valuable characteristics
– Characterize blends and adulteration

 Sample
– Fresh green coffee beans
– Grind and extract
– Analyze

©2020 Waters Corporation COMPANY CONFIDENTIAL 31

 Free Amino Acids in Kona Coffee Beans
0.095
AMQ

Deriv Peak
0.090

Caffeine
Glu
0.085

0.080

0.075

Ala
0.070

0.065

0.060

0.055

0.050
AU

0.045

0.040
EA

0.035

GABA
Asn

0.030
Asp

Phe
0.025
Ser

Pro
0.020

Trp
NH3

Lys

Val
AABA
0.015

Leu
Ile
Tyr
0.010

HyLys2
HyLys1

Met
Arg
Gly

Thr
HyPro

Cys
Orn
0.005
Gln
His

0.000

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
Minutes
©2020 Waters Corporation COMPANY CONFIDENTIAL 32
 Different Free Amino Acid Levels
Green Coffee Beans

Caffeine
0.100

0.095

0.090 Colombian
0.085

0.080
Kona Blend
0.075 100% Kona

Ala
0.070

0.065

GABA
0.060

0.055
AU

Glu

0.050

0.045

0.040

0.035

0.030

Pro
0.025

0.020

0.015

0.010
Thr

0.005

0.000
3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40
Minutes
©2020 Waters Corporation COMPANY CONFIDENTIAL 33
 Summary

 Amino acid analysis is critical for evaluating a wide range of samples from
biopharmaceutical to food and nutrition
– Quantitative analysis is typically required

 Chemical and physical nature of amino acids require some type of derivatization
for accurate, precise quantitation
– Automation can increase throughput and reduce errors

 Single vendor solution ensures robust, reliable measurements

©2020 Waters Corporation COMPANY CONFIDENTIAL 34