ez:faast™ amino acid analysis - phenomenex · 2008-10-16 · – protein...

EZ:faast™ Amino Acid AnalysisTheory and practical overview

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 TechniquesHistorical Background and Method

Limitations

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




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 AdvantagesOvercoming amino acid analysis limitations

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• 3 phase, simple protocol

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• Excellent baseline resolution of all amino acids

min6.05.04.03.02.0

2.0

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

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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




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




EZ:faast Product OverviewProtocol, product options, & kit components

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


• Phase 1: sample preparation Step 1

• Dispense sample and add internal standard• pH adjustment if necessary


• Phase 1: sample preparationStep 2

• Sample is pipetted through SPE tip • Amino acids bind to sorbent



• Add reagent 2 and draw up through sorbent tip to wash away matrix interferences



• Reagent 3A/3B are added to sample and amino acids along with sorbent are expelled from tip


• Phase 2: derivatizationStep 5

• Add derivatizing reagents 4 and 5 to sample


• Phase 2: derivatizationStep 6

• Add reagent 6 to sample• Extraction from aqueous to organic layer occurs


• 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

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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


• Kit options– Based on sample

• Free (physiological) amino acids• Protein hydrolysates

– Based on analysis technique• GC/FID• GC/MS• LC/MS


• Free (physiological) amino acids kits– Determine concentrations of individual amino acids in

solids and liquids


• 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


• Protein hydrolysate kits– Determine concentrations and compositions of amino

acids from hydrolyzed proteins


• 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


• 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


• LC/MS kits– Utilizes an LC/MS system to analyze sample– 16 minute analysis time

– Note: Suitable for Arginine (Arg) & Citrulline (Cit)


• 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 ApplicationsFor various industries

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


• 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


• 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


• 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

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60

80

10 0

pA

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1

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4 6 7

8

9

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2.5 3 3.5 4 4.5 5 5.5 6 min


• Biotechnology: protein composition

5 6 7 min42 31

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]

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2221

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1413

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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


• Clinical: human serum

5 6 7 min42 31

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100

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22

21

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12

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76

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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).


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


• 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


• 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


• 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


• 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

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11

5 min42 3

15

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13

12109

87

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• Food / Beverage: fermentation broth

5 6 min42 3

1918

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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).


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


• 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


• Food / Beverage: beer

pA

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801611

5 6 min42 3

21

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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).


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


• Food / Beverage: potato tissue

5 min42 3

10

9

87

65

432

1

11

201918 21

17

16

15

14

13

12


• Food / Beverage: tomato tissue

5 7 min642 3

1716

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1211

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• Food / Beverage: corn meal hydrolysate

5 min42 3

15

14

13

12

10

9

87

6

5

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321

11

16

17


• Food / Beverage: meat hydrolysate

5 7 min642 3

17

16

151413

1211

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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


• Agricultural: corn meal

5 7 min642 3

1716

15

14

13

1211

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9

87

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• Agricultural: corn meal hydrolysate

pA

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40

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801611

5 6 min42 3

21

20

19

18

17

15

14

13

12

10

9

8

7

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5

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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).


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


• 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

Frequently Asked Questions

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.

Appendix

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


• Aminoadipic acid• Aminoisobutyric acid• Asparagine• Citrulline• Cystine• Glucosaminic acid• Glycine• Hydroxylysine• Leucine• 1-Methylhistidine• Phenylalanine• Proline • Tyrosine



Amino acids AnalyzedAmino acids AnalyzedDairy ProductsDairy Products


• Aminoadipic acid• Aminoisobutyric acid• Asparagine• Citrulline• Cystine• Glucosaminic acid• Glycine• Hydroxylysine• Leucine• 1-Methylhistidine• Phenylalanine• Proline • Tyrosine



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 AnalyzedAmino acids AnalyzedFruit ProductsFruit Products


• Arginine• Cystine• Glycine• Isoleucine• Methionine• Phenylalanine• Tyrosine• GABA



Amino acids AnalyzedAmino acids AnalyzedFeeds & GrainsFeeds & Grains


• Arginine• Cystine• Glycine• Isoleucine• Methionine• Phenylalanine• Tyrosine• GABA



PerformancePerformanceSpeedSpeed

Chromatograms of 33 amino acids in < 7 minutes

EZ:faast® free (physiological) amino acid kit

PerformancePerformanceRobustnessRobustness

Fermentation broth


PerformancePerformanceRobustnessRobustness

Human blood sample


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.

ez:faast™ amino acid analysis - phenomenex · 2008-10-16 · – protein...

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