Presentation Outline
• Why analyze?• Older Techniques and Limitations• EZ:faast advantages• EZ:faast product overview• EZ:faast applications • Conclusion
Why Analyze?
• Why analyze amino acids?– Determination of metabolic disorders– Fermentation/cell culture analysis– Nutritional labeling/ feed analysis– Quality control of foods/beverage– Protein identification/characterization– Peptide quality control
Older Techniques
• Past advancements– IEX with post-column derivatization introduced in 1950s
– Achievements made in • Derivatization & detection• Separation science (chromatography media)
Older Techniques
• Derivatization advancements– Pre-Column derivitization increase sensitivity– Attachment of chromaphore allows for UV or
florescence detection
Older Techniques
• Separation science advancements– Chromatography media improvements increased
resolution• Ion exchange medias (post-column derivatization)• Reversed phase medias (pre-column derivatization)
Older Techniques
• Ion exchange with post-column derivatization– Derivatizing agents
• Ninhydrin or OPA
– Products• Beckman® 6300, Pickering® Labs, Hitachi® L880,
Biochrom® 30 series
– Strengths• Established methods• Chemistry well characterized
Older Techniques
• Reversed phase with pre-column derivatization– Derivatizing agents
• ACQ, OPA, PITC, FMOC
– Products• Waters® Pico-Tag® & AccQ-Tag™
– Strengths• Faster analysis times / improved throughput• More sensitive
Older Techniques
• Ion chromatography with electrochemical detection– Products
• Dionex® AAA Direct®
– Strengths• No derivatization necessary• Compatible with gel hydrolysis
Older Techniques• Despite advancements
– Method limitations• Sample preparation• Derivatization • Analysis time/ throughput• Cost
– Performance limitations• Resolution/ dynamic range• Accuracy/ reproducibility• Limits of detection
Older Techniques
• Method limitations
Problem with buffer
preparation
Poor derivative stability
Dedicated instrument
OtherOther
High costs associated with labor and materialsCosts Costs
75 minutes40-70 minutes60-120 minutesAnalysis TimeAnalysis Time
N/ASalt & proteins affect reaction
Ninhydrin- limited sensitivity
OPA- no proline
DerivatizationDerivatization
Remove salts & detergents
Desalting required prior derivatization
Complex protein & detergent removal
Sample PreparationSample Preparation
IC with IC with ECD detectionECD detection
RP with RP with prepre--column column
derivatizationderivatization
IEX with IEX with postpost--column column derivatizationderivatization
Older Techniques
• Performance limitations
Injection volumes limited (25µL)
Limits high for poorly resolved amino acids
Proline and hydroxyproline can’t be
detected using OPA derivatization
Quantitation Quantitation LimitsLimits
Salts cause retention
time shifts
Biological contaminants co-elute with certain
amino acids
N/A
Accuracy/ Accuracy/ ReproducibilityReproducibility
Poor peak capacity
Poor resolution of critical amino acids
Poor resolution of critical amino acids
Resolution/ Resolution/ Dynamic RangeDynamic Range
IC with IC with ECD ECD
detectiondetection
RP with RP with prepre--column column
derivatizationderivatization
IEX with IEX with postpost--column column derivatizationderivatization
Older Techniques
• Shared limitations of all1. Labor intensive2. Complicated sample preparation3. Sensitive to salts and other contaminants4. Incapable of resolving certain amino acids5. Lack of sample throughput/ speed
• How do you overcome these?
EZ:faast Advantages
• Limitation #1: – Labor intensive
• Sample prep 3 hours + analysis ~1 hour
• EZ:faast advantage: – Very simple protocol
• 3 short phases 15 minutes total
EZ:faast Advantages
• Limitation #2: – Complicated sample preparation
• De-proteinization >3 hours
• EZ:faast advantage: – 7 minute sample preparation and derivatization
• No protein or urea removal required
EZ:faast Advantages
• 7 minute sample preparation and derivatization
DerivatizationDerivatizationadvantagesadvantages
– At room temperature
– In aqueous solution
– Very rapid
– Stable
EZ:faast Advantages
• Limitation #3: – Sensitive to salt and other contaminants
• Columns deteriorate rapidly• Effects derivitization• Poor Quantitation • Analysis results not reproducible
• EZ:faast advantage: – Extremely rugged system
• Salts and proteins don’t affect derivatization/analysis• Works for all complex matrices
EZ:faast Advantages
• Extremely rugged systemAnalyze amino acids in Analyze amino acids in these matricesthese matrices
– Serum
– Urine
– Wine/Beer
– Food Stuffs
– Plasma
– Fermentation Broths
– Cell Cultures
– Protein Hydrolysates
EZ:faast Advantages
• Limitation #4: – Incapable of resolving certain amino acids
• EZ:faast advantage: – Excellent baseline resolution of all amino acids
EZ:faast Advantages
• Limitation #5: – Lack of sample throughput/ speed
• 30 min-2 hours (60min on average)
• EZ:faast advantage: – 7 minute analysis time for GC– 16 minute analysis time for LC
EZ:faast Advantages
• 7 minute analysis time (GC)
pA
60
40
20
0
5 6 min42 3
HY
PG
LU
PR
OS
ERT
HR
aILE
ILE
LEU
VA
LA
BA
GLYALA S
AR
ME
T
TP
RA
SP
AS
N
C-C
HIS
LYS
OR
N GP
R
AA
A
GLN
AP
A
CT
H
PH
P
HLY
ß-A
LAIS
PH
E
TY
R
TR
P
EZ:faast Advantages• Method advantage
$US 4.00/sample
7 min (GC) 16 min (LC)
Salts & proteins don’t
affect
Easy protocol (8 min)
EZ:faastEZ:faast
High costs associated with labor and materialsCosts Costs
75 minutes40-70 minutes60-120 minutesAnalysis Analysis TimeTime
N/ASalt & proteins affect reaction
Ninhydrin-limited
sensitivityOPA- no proline
DerivatizationDerivatization
Remove salts & detergents
Desalting required prior derivatization
Complex protein & detergent removal
Sample Sample PreparationPreparation
IC with IC with ECD detectionECD detection
RP with RP with prepre--column column
derivatizationderivatization
IEX with IEX with postpost--column column derivatizationderivatization
EZ:faast Advantages• Performance advantage
Down to 1 nmol/mL (low picomole level
possible with modification)
Internal standard corrects for sample
preparation variability
All amino acids separated well
EZ:faastEZ:faast
Injection volumes limited (25µL)
N/A
Proline/hydroxyprolinearen’t detected
using OPA derivatization
Quantitation Quantitation LimitsLimits
Salts cause retention
time shifts
Biological contaminants co-elute with certain amino
acids
N/A
Accuracy/ Accuracy/ ReproducibilityReproducibility
Poor peak capacity
Poor resolution of critical amino
acids
Poor resolution of critical
amino acids
Resolution/ Resolution/ Dynamic RangeDynamic Range
IC with IC with ECD ECD
detectiondetection
RP with RP with prepre--column column
derivatizationderivatization
IEX with IEX with postpost--column column derivatizationderivatization
EZ:faast product overview
• Simple 3 phase protocol (7 simple steps)1. Sample preparation
– Using solid phase extraction (SPE)– 4 steps
2. Derivatization– 2 steps
3. Analysis– 1 step
EZ:faast product overview
• Phase 1: sample preparation Step 1
• Dispense sample and add internal standard• pH adjustment if necessary
EZ:faast product overview
• Phase 1: sample preparationStep 2
• Sample is pipetted through SPE tip • Amino acids bind to sorbent
EZ:faast product overview
• Phase 1: sample preparationStep 3
• Add reagent 2 and draw up through sorbent tip to wash away matrix interferences
EZ:faast product overview
• Phase 1: sample preparationStep 4
• Reagent 3A/3B are added to sample and amino acids along with sorbent are expelled from tip
EZ:faast product overview
• Phase 2: derivatizationStep 5
• Add derivatizing reagents 4 and 5 to sample
EZ:faast product overview
• Phase 2: derivatizationStep 6
• Add reagent 6 to sample• Extraction from aqueous to organic layer occurs
EZ:faast product overview
• Phase 3: analysisStep 7
• Inject sample onto GC/FID, GC/MS or LC/MS• Chromatogram is generated in 7 minutes & peak areas
are used to quantitate amino acid levels
pA
60
40
20
0
5 6 min42 3
HYP
GLU
PRO
SERTH
R
aILE ILE
LEU
VAL
ABA
GLYAL
A SAR
MET
TPR
ASP
ASN
C-C
HIS
LYS
OR
N GPR
AAA
GLN
APA
CTH
PHP
HLY
ß-AL
AIS
PHE
TYR
TRP
EZ:faast product overview
• Kit options– Based on sample
• Free (physiological) amino acids• Protein hydrolysates
– Based on analysis technique• GC/FID• GC/MS• LC/MS
EZ:faast product overview
• Free (physiological) amino acids kits– Determine concentrations of individual amino acids in
solids and liquids
EZ:faast product overview
• Free (physiological) amino acids kits– Typical application areas
• Biotechnology– Measure amino acid depletion in fermentation broths
• Clinical/ toxicology– Detect metabolic disorders
• Food and beverage– QC testing amino acid content in finished product
EZ:faast product overview
• Protein hydrolysate kits– Determine concentrations and compositions of amino
acids from hydrolyzed proteins
EZ:faast product overview
• Protein hydrolysate kits– Typical application areas
• Biotechnology– Identify modifications/mis-incorporations of amino acids
into proteins
• Academic– Protein characterization/ identification
• Food and beverage– Amino acid composition analysis of protein supplements
EZ:faast product overview
• GC kits– Utilizes GC/FID or GC/MS to analyze sample– Excellent baseline separation– 7 minute analysis time
– Note: not suitable for Arginine (Arg), Citrulline (Cit), or Taurine (Tau) analysis
EZ:faast product overview
• LC/MS kits– Utilizes an LC/MS system to analyze sample– 16 minute analysis time
– Note: Suitable for Arginine (Arg) & Citrulline (Cit)
EZ:faast product overview
• Kit components– Amino acid standards– Sample Rack– Sample vials– Internal Standards & Solutions– SPE Sorbent Tips and Syringes– Derivatizing Reagents– GC or HPLC Column– Focus Liners (for GC Kit)
• The EZ:faast kits contain the reagents and accessories for, the sample preparation, derivatization and analysis of 384 samples.
• ** Kit does not contain reagents and accessories for protein hydrolysis
EZ:faast Applications
–– BiotechnologyBiotechnology
–– ClinicalClinical
–– Food / BeverageFood / Beverage
–– AgriculturalAgricultural
2.0 3.0 4.0 5.0Tim e (m in)
100
200
300
pA
3 4
56 7
8
910
11
1213
14 15
16 17 18
19 20
21
22
EZ:faast Applications
• Biotechnology: mammalian cell culture
Amino Acids in Fermentation BrothKit: EZ:faast GC/FID Kit
Free (Physiological) Amino Acid KitOrder No.: KG0-7165Injection: Split 1:15 @ 250°C, 2.5 µLCarrier Gas: Helium 1.5 mL/minute, constant flow @ 110°COven Program: 30°C/min from 110° to 320°C, hold at 320° for 1 min.Detector: FID @ 320°CSample: Derivatized amino acids in fermentation
broth (0.1mL). Norvaline is the internalstandard added at a concentration of 200 µmol/L1. Alanine 9. Glutamic Acid2. Glycine 10. Phenylalanine3. α-Aminobutyric acid 11. α-Aminoadipic Acid4. Norvaline (IS) 12. Ornithine5. Serine 13. Glycine-proline (dipeptide)6. Proline 14. Lysine7. Asparagine 15. Histidine8. Methionine
pA
60
40
20
0
80
11
5 min42 3
15
14
13
12109
87
6
5
4
3
2
1180
160
140
120
100
200
EZ:faast Applications
• Biotechnology: fermentation broth
5 7 min642 3
1716
15
14
13
1211
10
9
87
6
5
4
32
1
Amino Acids in Corn Meal Hydrolysate by GC/FIDKit: EZ:faast GC/FID Kit
Protein Hydrolysate KitOrder No.: KG0-7167Injection: Split 1:15 @ 250°C, 2.5 µLCarrier Gas: Helium 1.5 mL/minute, (60 kPa) @ 110°COven Program: 30°C/min from 110° to 320°C, hold at 320° for 1 min.Detector: FID @ 320°CSample: Derivatized amino acids from a corn meal
hydrolysate sample. Norvaline is the internalstandard added at 200 µmol/L1. Alanine 10. Aspartic Acid2. Glycine 11. Methionine3. Valine 12. Hydroxyproline4. Norvaline (IS) 13. Glutamic Acid5. Leucine 14. Phenylalanine6. Isoleucine 15. Lysine7. Threonine 16. Histidine8. Serine 17. Tyrosine9. Proline
EZ:faast Applications
• Biotechnology: corn meal hydrolysate
Amino Acids Composition of a Standard ProteinKit: EZ:faast GC/FID Kit
Protein Hydrolysate KitOrder No.: KG0-7167Injection: Split 1:15 @ 250°C, 2.0 µLCarrier Gas: Helium 1.5 mL/minute, constant flow @ 110°COven Program: 30°C/min from 110° to 320°C, hold at 320° for 1 min.Detector: FID @ 320°CSample: Acid Hydrolyzed Carbonic Anhydrase: Sample was
hydrolyzed using vapor phase acid hydrolysis; 6N HCl with 1% phenol for 24 hours at 110ºC. Sample is neutralized and analyzed directly by EZ:faast.1. Alanine 9. Proline2. Glycine 10. Aspartic Acid3. Valine 11. Methionine4. Norvaline (IS) 12. Glutamic Acid5. Leucine 13. Phenylalanine6. Isoleucine 14. Lysine7. Threonine 15. Histidine8. Serine 16. Tyrosine
1.50
20
40
60
80
10 0
pA
2
1
2
3
5
4 6 7
8
9
10
13
12
11
14
15
16
2.5 3 3.5 4 4.5 5 5.5 6 min
EZ:faast Applications
• Biotechnology: protein composition
5 6 7 min42 31
150
100
50
200
0
[mV
]
23
2221
20
19
18
17
16
15
1413
1211
10
98
7
6
5
4
3
2
1
Free Amino Acids in Human Serum by GC/FIDKit: EZ:faast GC/FID Kit
Free (Physiological) Amino Acid KitOrder No.: KG0-7165Injection: Split 1:15 @ 250°C, 2.5 µLCarrier Gas: Helium 1.5 mL/minute, (60 kPa) @ 110°COven Program: 30°C/min from 110° to 320°C, hold at 320° for 1 min.Detector: FID @ 320°CSample: Derivatized amino acids in human serum (0.1mL).
Norvaline is the internal standard added at a concentration of 200 µmol/L
1. Alanine 13. Methionine2. Glycine 14. 4-Hydroxyproline3. α-Aminobutyric acid 15. Glutamic Acid4. Valine 16. Phenylalanine5. Norvaline (IS) 17. Glutamine6. Leucine 18. Ornithine7. Isoleucine 19. Lysine8. Threonine 20. Histidine9. Serine 21. Tyrosine
10. Proline 22. Tryptophan11. Asparagine 23. Cystine12. Aspartic Acid
EZ:faast Applications
• Clinical: human serum
5 6 7 min42 31
150
100
50
200
0
[mV]
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
76
5
43
2
1
Free Amino Acids in Human Urine by GC/FIDKit: EZ:faast GC/FID Kit
Free (Physiological) Amino Acid KitOrder No.: KG0-7165Injection: Split 1:15 @ 250°C, 2.5 µLCarrier Gas: Helium 1.5 mL/minute, (60 kPa) @ 110°COven Program: 30°C/min from 110° to 320°C, hold at 320° for 1 min.Detector: FID @ 320°CSample: Derivatized amino acids in human urine (0.1mL).
Norvaline is the internal standard added at a concentration of 200 µmol/L
1. Alanine 13. Glutamic Acid2. Glycine 14. Phenylalanine3. Valine 15. α-Aminoadipic Acid4. β-Aminoisobutyric acid 16. Glutamine5. Norvaline (IS) 17. Lysine6. Leucine 18. Histidine7. Isoleucine 19. Tyrosine8. Threonine 20. Proline hydroxyproline (dipeptide)9. Serine 21. Tryptophan
10. Proline 22. Cystine11. Asparagine12. Aspartic Acid
EZ:faast Applications
• Clinical: human urine (GC/FID)
Amino Acids in Human UrineKit: EZ:faast LC/MS for Free (Physiological) Amino AcidsOrder No.: KH0-7338HPLC Column: EZ:faast 4µ AAA-MS 250 x 3.0mmMobile Phase: A: 10mM ammonium formate in water
B: 10mM ammonium formate in methanolGradient: 68 to 92%B in 12min, re-equilibration at
68%B for 4minFlow Rate: 0.5 mL/minInjection Volume: 5µLDetection: Bruker Esquire 2000 IT ESIScan Range: 100-600 m/zSample: Derivatized amino acids in human urine (0.1mL).
Internal standards (HARG, d3MET and HPHE) were added at a concentration of 200 µmol/L each.
1. Arginine 12. Alanine 23. Glutamic acid2. Homoarginine IS 13. β-Aminoisobutyric acid 24. Tryptophan3. Citrulline 14. α-Aminobutyric acid 25. a-Aminoadipic acid4. Glutamine 15. Ornithine 26. Leucine5. Serine 16. Methionine 27. Isoleucine6. Asparagine 17. d3-Methionine 28. Phenylalanine7. Proline-hydroxyproline 18. Proline 29. Cystathionine8. 4-Hydroxyproline 19. Lysine 30. Homophenylalanine9. Glycine 20. Aspartic acid 31. Cystine
10. Glycine-proline 21. Histidine 32. Tyrosine11. Threonine 22. Valine
ARGm/z=303
Intens.
0
THRm/z=248
x106
0
VALm/z=246
Intens.
0
m/z=304CITx10 5
0
BAIBABA m/z=232
x107
0m/z=333
TRPx10 5
0
SERm/z=234
x10 6
0
METx105
0m/z=260
LEUILE
x105
0
HYPx10 6
0m/z=260
LYSm/z=361
x106
0m/z=308
IS=HPHEx10 7
0
IS=HARGm/z=3 1 7
x10 7
0
ALAm/z=218
x106
0m/z=318
GLUx10 5
0
m/z=275GLNx10 6
0
ORNm/z=347
x105
0m/z=332
AAAx105
0
ASNm/z=243
x10 6
0
IS=d3METm/z=281
x107
0m/z=294
PHEx105
0
PHPm/z=375
x10 6
0
PRO m/z=244x105
0m/z=479
CTHx105
0
GLYm/z=204
x1 07
0
ASPm/z=304
x105
0m/z=497
C-Cx10 5
0
GPR
2 3 4 5 6 Time(min)
x10 5
0m/z=301
HIS
4 5 6 7 8 9 Time (min)
m/z=370
x107
0m/z=396
EZ:faast Applications
• Clinical: human urine (LC/MS)
0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4Tim e (m in)
2040
pA
23
4
5
6
7
8
EZ:faast Applications
• Clinical: Phenylketonuria (PKU)
5 7 min642 3
17
16
151413
1211
10
98
765
4
321
Free Amino Acids in Corn Meal by GC/FIDKit: EZ:faast GC/FID Kit
Free (Physiological) Amino Acid KitOrder No.: KG0-7165Injection: Split 1:15 @ 250°C, 2.5 µLCarrier Gas: Helium 8psi (60 kPa)Oven Program: 32°C/min from 110° to 320°C, hold at 320°C for 1 min.Detector: FID @ 320°CSample: Derivatized amino acids in corn meal (0.1mL).
Norvaline is the internal standard added at 200 µmol/L1. Alanine 11. Aspartic Acid2. Glycine 12. Glutamic Acid3. Valine 13. Phenylalanine4. Norvaline (IS) 14. Lysine5. Leucine 15. Histidine6. Isoleucine 16. Tyrosine7. Threonine 17. Tryptophan8. Serine9. Proline
10. Asparagine
EZ:faast Applications
• Food / Beverage: corn meal
Amino Acids in Fermentation BrothKit: EZ:faast GC/FID Kit
Free (Physiological) Amino Acid KitOrder No.: KG0-7165Injection: Split 1:15 @ 250°C, 2.5 µLCarrier Gas: Helium 1.5 mL/minute, constant flow @ 110°COven Program: 30°C/min from 110° to 320°C, hold at 320° for 1 min.Detector: FID @ 320°CSample: Derivatized amino acids in fermentation
broth (0.1mL). Norvaline is the internalstandard added at a concentration of 200 µmol/L1. Alanine 9. Glutamic Acid2. Glycine 10. Phenylalanine3. α-Aminobutyric acid 11. α-Aminoadipic Acid4. Norvaline (IS) 12. Ornithine5. Serine 13. Glycine-proline (dipeptide)6. Proline 14. Lysine7. Asparagine 15. Histidine8. Methionine
pA
60
40
20
0
80
11
5 min42 3
15
14
13
12109
87
6
5
4
3
2
1180
160
140
120
100
200
EZ:faast Applications
• Food / Beverage: fermentation broth
5 6 min42 3
1918
17
16
1514
13
12
1110
9
87
654
3
2
1
Free Amino Acids in Wine by GC/MSKit: EZ:faast GC/MS Kit
Free (Physiological) Amino Acid KitOrder No.: KG0-7166Injection: Split 1:15 @ 250°C, 2.5 µLCarrier Gas: Helium 1.5 mL/minute @ 110°COven Program: 30°C/min from 110° to 320°CDetector: MS @ 300°CSample: Derivatized amino acids in wine (0.1mL).
Norvaline is the internal standard added at a concentration of 200 µmol/L
1. Alanine 11. Aspartic Acid2. Glycine 12. Methionine3. Valine 13. 4-Hydroxyproline4. β-Aminoisobutyric acid 14. Glutamic Acid5. Norvaline (IS) 15. Phenylalanine6. Leucine 16. Ornithine7. Isoleucine 17. Lysine8. Threonine 18. Tyrosine9. Proline 19. Tryptophan
10. Asparagine
EZ:faast Applications
• Food / Beverage: wine
16
11
5 6 min421 3
15
141312
109
8
7
65
4
32
1
Free Amino Acids in Beer by GC/FIDKit: EZ:faast GC/FID Kit
Free (Physiological) Amino Acid KitOrder No.: KG0-7165Injection: Split 1:15 @ 250°C, 2.5 µLCarrier Gas: Helium 8psi (60 kPa)Oven Program: 32°C/min from 110° to 320°C, hold at 320°C for 1 min.Detector: FID @ 320°CSample: Derivatized amino acids in beer.
Norvaline is the internal standard added at 200 µmol/L1. Alanine 9. Asparagine2. Glycine 10. Aspartic Acid3. Valine 11. Glutamic Acid4. Norvaline (IS) 12. Phenylalanine5. Leucine 13. Ornithine6. Isoleucine 14. Histidine7. Serine 15. Tyrosine8. Proline 16. Tryptophan
EZ:faast Applications
• Food / Beverage: beer
pA
60
40
20
0
801611
5 6 min42 3
21
20
19
18
17
15
14
13
12
10
9
8
7
6
5
4
321
Free Amino Acids in Potato Tissue by GC/FIDKit: EZ:faast GC/FID Kit
Free (Physiological) Amino Acid KitOrder No.: KG0-7165Injection: Split 1:15 @ 250°C, 2.5 µLCarrier Gas: Helium 1.5 mL/minute, (60 kPa) @ 110°COven Program: 30°C/min from 110° to 320°C, hold at 320° for 1 min.Detector: FID @ 320°CSample: Derivatized amino acids in potato tissue (0.1mL).
Norvaline is the internal standard added at a concentration of 200 µmol/L
1. Alanine 12. Aspartic Acid2. Glycine 13. Methionine3. α-Aminobutyric Acid 14. Glutamic Acid4. Valine 15. Phenylalanine5. Norvaline (IS) 16. Glutamine6. Leucine 17. Ornithine7. Isoleucine 18. Lysine8. Threonine 19. Histidine9. Serine 20. Tyrosine
10. Proline 21. Tryptophan11. Asparagine
EZ:faast Applications
• Food / Beverage: potato tissue
5 min42 3
10
9
87
65
432
1
11
201918 21
17
16
15
14
13
12
EZ:faast Applications
• Food / Beverage: tomato tissue
5 7 min642 3
1716
15
14
13
1211
10
9
87
6
5
4
32
1
Amino Acids in Corn Meal Hydrolysate by GC/FIDKit: EZ:faast GC/FID Kit
Protein Hydrolysate KitOrder No.: KG0-7167Injection: Split 1:15 @ 250°C, 2.5 µLCarrier Gas: Helium 1.5 mL/minute, (60 kPa) @ 110°COven Program: 30°C/min from 110° to 320°C, hold at 320° for 1 min.Detector: FID @ 320°CSample: Derivatized amino acids from a corn meal
hydrolysate sample. Norvaline is the internalstandard added at 200 µmol/L1. Alanine 10. Aspartic Acid2. Glycine 11. Methionine3. Valine 12. Hydroxyproline4. Norvaline (IS) 13. Glutamic Acid5. Leucine 14. Phenylalanine6. Isoleucine 15. Lysine7. Threonine 16. Histidine8. Serine 17. Tyrosine9. Proline
EZ:faast Applications
• Food / Beverage: corn meal hydrolysate
5 min42 3
15
14
13
12
10
9
87
6
5
4
321
11
16
17
EZ:faast Applications
• Food / Beverage: meat hydrolysate
5 7 min642 3
17
16
151413
1211
10
98
765
4
321
Free Amino Acids in Corn Meal by GC/FIDKit: EZ:faast GC/FID Kit
Free (Physiological) Amino Acid KitOrder No.: KG0-7165Injection: Split 1:15 @ 250°C, 2.5 µLCarrier Gas: Helium 8psi (60 kPa)Oven Program: 32°C/min from 110° to 320°C, hold at 320°C for 1 min.Detector: FID @ 320°CSample: Derivatized amino acids in corn meal (0.1mL).
Norvaline is the internal standard added at 200 µmol/L1. Alanine 11. Aspartic Acid2. Glycine 12. Glutamic Acid3. Valine 13. Phenylalanine4. Norvaline (IS) 14. Lysine5. Leucine 15. Histidine6. Isoleucine 16. Tyrosine7. Threonine 17. Tryptophan8. Serine9. Proline
10. Asparagine
EZ:faast Applications
• Agricultural: corn meal
5 7 min642 3
1716
15
14
13
1211
10
9
87
6
5
4
32
1
Amino Acids in Corn Meal Hydrolysate by GC/FIDKit: EZ:faast GC/FID Kit
Protein Hydrolysate KitOrder No.: KG0-7167Injection: Split 1:15 @ 250°C, 2.5 µLCarrier Gas: Helium 1.5 mL/minute, (60 kPa) @ 110°COven Program: 30°C/min from 110° to 320°C, hold at 320° for 1 min.Detector: FID @ 320°CSample: Derivatized amino acids from a corn meal
hydrolysate sample. Norvaline is the internalstandard added at 200 µmol/L1. Alanine 10. Aspartic Acid2. Glycine 11. Methionine3. Valine 12. Hydroxyproline4. Norvaline (IS) 13. Glutamic Acid5. Leucine 14. Phenylalanine6. Isoleucine 15. Lysine7. Threonine 16. Histidine8. Serine 17. Tyrosine9. Proline
EZ:faast Applications
• Agricultural: corn meal hydrolysate
pA
60
40
20
0
801611
5 6 min42 3
21
20
19
18
17
15
14
13
12
10
9
8
7
6
5
4
321
Free Amino Acids in Potato Tissue by GC/FIDKit: EZ:faast GC/FID Kit
Free (Physiological) Amino Acid KitOrder No.: KG0-7165Injection: Split 1:15 @ 250°C, 2.5 µLCarrier Gas: Helium 1.5 mL/minute, (60 kPa) @ 110°COven Program: 30°C/min from 110° to 320°C, hold at 320° for 1 min.Detector: FID @ 320°CSample: Derivatized amino acids in potato tissue (0.1mL).
Norvaline is the internal standard added at a concentration of 200 µmol/L
1. Alanine 12. Aspartic Acid2. Glycine 13. Methionine3. α-Aminobutyric Acid 14. Glutamic Acid4. Valine 15. Phenylalanine5. Norvaline (IS) 16. Glutamine6. Leucine 17. Ornithine7. Isoleucine 18. Lysine8. Threonine 19. Histidine9. Serine 20. Tyrosine
10. Proline 21. Tryptophan11. Asparagine
EZ:faast Applications
• Agricultural: potato tissue
Conclusion
• EZ:faast overcomes limitations of current amino acid analysis methods
• EZ:faast procedure is very easy with 3 simple phases & very fast with a 15 minute (GC) / 25 minute (LC) protocol
• EZ:faast contains all of the reagents and accessories for the sample preparation, derivatization, and analysis of 384 samples
Q: For how long are the derivatized amino acids stable?
A: Amino acids derivatized by the EZ:faast method are stable for up to a day at room temperature and for several days if refrigerated.
Q: How many samples can be analyzed with the kit?
A: 384 samples can be analyzed with the reagents and supplies of one kit
Frequently Asked Questions
Q: How long will the EZ:faast GC and LC column last?
A: We have found that the GC and LC columns will last for the duration of the kit. For best results columns should be replace with each new kit.
Q: What is the shelf life of the reagents?
A: EZ:faast reagents have a guaranteed shelf life of 12 months if properly stored as indicated on the bottle in a refrigerator or freezer
Frequently Asked QuestionsFrequently Asked Questions
Q: What are the derivatization reagents and reaction?
A: The derivatization reagents is a unique chloroformate reagent that derivatizes both the amine and carboxyl groups of the amino acids forming a highly stable derivative.
Frequently Asked QuestionsFrequently Asked Questions
NH2O
OH
ROCl
OR'
R
NHOR'
O
O
OR'+ + 2HCl + COcat.2 2
Trademarks
Phenomenex, Inc.© copyright 2005, all rights reserved.
EZ:faast is a registered trademark of Phenomenex Inc. Beckman 6300 is a trademark of Beckman Coulter. Phenomenex is in no way affiliated with Beckman Coulter. Hitachi is a registeredtrademark. Phenomenex is in no way affiliated with Hitachi. Pico-tag is a trademark and AccQ-tag is a registered trademark of Waters Corporation. Phenomenex is in no way affiliated with Waters Corporation. Pickering Labs is a registered trademark. Phenomenex is in no way affiliated with Pickering Labs. Dionex AAA Direct is a registered trademark of Dionex. Phenomenex is in no way affiliated with Dionex. Comparisons shown in this presentation may not be representative of every application.
Extraction Methods
• Cheese:– Suspend 50 mg of cheese powder in 1ml of 25% Acetonitrile in
water for 1 hour at 40ºC. Filter the top layer using a 0.45µm syringe filter.
• Honey:– Dilute Honey sample in deionized water in the ratio of 1:1.
• Plant leafs:– Add 0.5 ml of 25% Acetonitrile in 0.01N HCl to 0.5g of dried leaf
samples. Vortex the vial for 5-10 min, and place on the bench top to settle for one hour. Filter the top layer using a 0.45µm syringe filter..
Extraction Methods• Corn Meal:
– Mix 50mg of dry powder into 3 ml of 25% Acetonitrile; vortex this mixture until the entire sample is homogenized. Centrifuge the vials for 3 minutes, then filter the top layer using a 0.45µm syringe filter.
• Soil sample:– Mix 0.5g of soil sample by vortexing for a few minutes into 25%
Acetonitrile in 0.01 M HCl. Centrifuge the vials for 3 minutes, then filter the top layer using 0.4 µm syringe filter.
• Coffee and wheat:– Suspend 10 g of well ground solid in150 ml water, and mix for 15
min. Place the suspended samples on a bench top for the solids to settle. Filter the top layer using a 0.45 µm pore size syringe filter.
Extraction Methods
• Tea sample:– Mix well ground tea in boiling water in a ratio of 0.2g: 15ml solvent,
and keep at boiling temperature for 4 min. Shake the vials repeatedly and then place on a bench top for the solids to settle. Filter the top layer using a 0.45 µm pore size syringe filter.
• Tomato and Potato samples:– Suspend 50mg of dry powder in 1ml of 25% Acetonitrile in 0.01N
HCl. Centrifuge the vials for 3 minutes, then filter the top layer using 0.4 µm syringe filter.
– An alternative method of extraction suggested in the literature: use 2 ml of Ethanol:0.25M HCl, in the ratio of 80:20) as extracting solvent. In our evaluation extraction with acetonitrile producedhigher values for amino acid levels.
Amino acids AnalyzedAmino acids AnalyzedPlasma & Physiological FluidsPlasma & Physiological Fluids
• Alanine• Aminoethylcysteine• Arginine• Carnosine• Cysteine• Glucosamine• Glutamine• Homocystine• Isoleucine• Methionine• Ornithine • Phosphoserine• Serine• Trytophan
• Aminoadipic acid• Aminoisobutyric
acid• Asparagine• Citrulline• Cystine• Glucosaminic acid• Glycine• Hydroxylysine• Leucine• 1-Methylhistidine• Phenylalanine• Proline • Tyrosine
• Aminobutyric acid• Anserine• Aspartic Acid• Cystathionine• Ethanolamine• Glutamic Acid• Histidine• Hydroxyproline• Lysine• 3-Methylhistidine• Phosphoethanlolamine• Sarcosine• Threonine• Valine
* Amino acids in red only analyzed by LC/MS kit
Amino acids AnalyzedAmino acids AnalyzedCell Culture MediaCell Culture Media
• Alanine• Aminoethylcysteine• Arginine• Carnosine• Cysteine• Glucosamine• Glutamine• Homocystine• Isoleucine• Methionine• Ornithine • Phosphoserine• Serine• Trytophan
• Aminoadipic acid• Aminoisobutyric acid• Asparagine• Citrulline• Cystine• Glucosaminic acid• Glycine• Hydroxylysine• Leucine• 1-Methylhistidine• Phenylalanine• Proline • Tyrosine
• Aminobutyric acid• Anserine• Aspartic Acid• Cystathionine• Ethanolamine• Glutamic Acid• Histidine• Hydroxyproline• Lysine• 3-Methylhistidine• Phosphoethanlolamine• Sarcosine• Threonine• Valine
* Amino acids in red only analyzed by LC/MS kit
Amino acids AnalyzedAmino acids AnalyzedDairy ProductsDairy Products
• Alanine• Aminoethylcysteine• Arginine• Carnosine• Cysteine• Glucosamine• Glutamine• Homocystine• Isoleucine• Methionine• Ornithine • Phosphoserine• Serine• Trytophan
• Aminoadipic acid• Aminoisobutyric acid• Asparagine• Citrulline• Cystine• Glucosaminic acid• Glycine• Hydroxylysine• Leucine• 1-Methylhistidine• Phenylalanine• Proline • Tyrosine
• Aminobutyric acid• Anserine• Aspartic Acid• Cystathionine• Ethanolamine• Glutamic Acid• Histidine• Hydroxyproline• Lysine• 3-Methylhistidine• Phosphoethanlolamine• Sarcosine• Threonine• Valine
* Amino acids in red only analyzed by LC/MS kit
Amino acids AnalyzedAmino acids AnalyzedFermentation MediaFermentation Media
• Alanine• Aspartic Acid• Glutamine• Hydroxyproline• Lysine• Ornithine • Serine• Trytophan
• Arginine• Cystine• Glycine• Isoleucine• Methionine• Phenylalanine• Tyrosine
• Asparagine• Glutamic Acid• Histidine• Leucine• 3-Methylhistidine• Proline • Threonine• Valine
* Amino acids in red only analyzed by LC/MS kit
Amino acids AnalyzedAmino acids AnalyzedFruit ProductsFruit Products
• Alanine• Aspartic Acid• Glutamine• Hydroxyproline• Lysine• Ornithine • Serine• Trytophan
• Arginine• Cystine• Glycine• Isoleucine• Methionine• Phenylalanine• Tyrosine• GABA
• Asparagine• Glutamic Acid• Histidine• Leucine• 3-Methylhistidine• Proline • Threonine• Valine
* Amino acids in red only analyzed by LC/MS kit
Amino acids AnalyzedAmino acids AnalyzedFeeds & GrainsFeeds & Grains
• Alanine• Aspartic Acid• Glutamine• Hydroxyproline• Lysine• Ornithine • Serine• Trytophan
• Arginine• Cystine• Glycine• Isoleucine• Methionine• Phenylalanine• Tyrosine• GABA
• Asparagine• Glutamic Acid• Histidine• Leucine• 3-Methylhistidine• Proline • Threonine• Valine
* Amino acids in red only analyzed by LC/MS kit
PerformancePerformanceSpeedSpeed
Chromatograms of 33 amino acids in < 7 minutes
EZ:faast® free (physiological) amino acid kit
PerformancePerformanceRobustnessRobustness
Fermentation broth
EZ:faast® free (physiological) amino acid kit
PerformancePerformanceRobustnessRobustness
Human blood sample
EZ:faast® free (physiological) amino acid kit
PerformancePerformanceAccuracyAccuracy
Linear dynamic range (3 log+)
Linear dynamic range for ASN from 88ng to > 3000ng
PerformancePerformanceReproducibilityReproducibility
Derivative stability = reproducible results
Derivative stable > 8 hrs at room temp., Multiple days at 4C
PerformancePerformanceSensitivitySensitivity
Quantitation limit 1nmol/mL (100 picomoles)
Minor modifications can easily lower detection limit 20 fold
Kit comparisonsKit comparisonsDifference between GC Hydrolysis & Physiological Kits
Hydrolysis PhysiologicalReagent 1 Internal Hyd. Std. SD Internal Std. SD 1,2,3Reagent 2 Sodium Carbonate Wash solutionReagent 3A Eluting medium I sameReagent 3B Eluting medium II sameReagent 4 Organic solution I sameReagent 5 Organic solution II sameReagent 6 Acid solution same*One standard for Hydrolysis kit - three for the Physiological kit*Sodium carbonate is used to neutralize the sample after hydrolysis.Difference between GC/FID & GC/MS Kits All reagents as above except for:
FID MSReagent 6 Acid solution Reconstitution solution*Vials are also included in the MS kit, but not in the FID kit.Difference between LC/MS & GC Kits
All reagents as above except for:Phys. Hydr.
Reagent 1 LC STD LC STDReagent 6 none none*Sample is blown down in the same way as for GC/MS, butfor reconstitution we recommend the mobile phase used for the LC/MS.*Internal standards for LC/MS kits are the same (methionine d3, homophenyl alanine, homoarginine) for both the hydrolysis kit & physiological kit, however differ from the GC kit standards.*Focus Liners are also included in the GC kits, but excluded in the LC kits.