SAIntroduction

Conclusions

y = 0.0879x - 0.2168R² = 0.9999

0

20

40

60

80

100

0 200 400 600 800 1000 1200

Peak A

rea

Concentration, pmol/µL

Glutamic AcidEclipse Plus C18, 2.1 mm x 150 mm, 3.5

µm

y = 0.1029x - 0.5381R² = 0.9998

0

20

40

60

80

100

120

0 200 400 600 800 1000 1200

Peak A

rea

Concentration, pmol/µL

AlanineEclipse Plus C18, 2.1 mm x 150 mm, 3.5

µm

y = 0.069x + 0.8281R² = 0.9995

01020304050607080

0 200 400 600 800 1000 1200

Peak A

rea

Concentration, pmol/µL

ProlineEclipse Plus C18, 2.1 mm x 150 mm, 3.5

µm

y = 0.1193x - 3.0495R² = 0.9972

0

20

40

60

80

100

120

140

0 200 400 600 800 1000 1200

Peak A

rea

Concentration, pmol/µL

LysineEclipse Plus C18, 2.1 mm x 150 mm, 3.5

µm

LinearityCalibration curves of early, middle and late eluting amino acids show linearity over 1 to 1000 pmol/µL range using the Eclipse Plus C18, 2.1 x 150 mm, 3.5 µm method

Injection-to-Injection ReproducibilityOverlay of eight sequential injections showing reproducibility of the online derivatization and gradient programs. Peak area of early, middle and late eluting amino acids are statistically tabulated below. The other amino acids had similar statistics.

min2 4 6 8 10 12 14 16 18

mAU

0

10

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30

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80

DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0010.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0009.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0008.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0007.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0006.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0005.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0004.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0003.D)DAD1 C, Sig=338,10 Ref=390,20, TT (E:\AAAREPRO 2009-04-08 16-10-39\21X150REPRO0002.D)

Peak Area

amino acid Injection 1 Injection 2 Injection 3 Injection 4 Injection 5 Injection 6 Injection 7 Injection 8 Injection 9 StDEV Mean %RSD

glu 90.7 93.7 92.4 93.1 93.8 94.7 92.5 95.7 93.2 1.43 93.3 1.5

ala 109.9 113.2 111.8 112.1 113.3 114 111.6 116 113.3 1.72 112.8 1.5

cy2 153.2 157 155 157 157.8 159 154.4 162.6 158 2.79 157.1 1.8

lys 142.3 142.5 137.1 144.4 141.9 143.5 137.9 140.7 138.9 2.55 141.0 1.8

pro 60.8 62.9 61.8 65.8 60.9 63.2 61.2 70.2 61 3.11 63.1 4.9

Columns 2.1x 150 mm 3.5µm

.

• An automated online derivatization method for amino acids using ZORBAX Eclipse Plus C18 was demonstrated as robust by longevity, lot-to-lot reproducibility, and linearity data.

• The Eclipse Plus C18 column choices offer the analyst high resolution, high speed, and reduced solvent consumption, in a combination that bests suits one’s needs.

• QuEChERS extraction techniques may be a useful for analyzing fruit or vegetable for amino acids.

• Fluorescence detection can be substituted for UV detection for higher sensitivity.

The Online Pre-Column DerivatizationsThe primary amino groups react with ortho-phthalaldehyde (OPA) in the presence of 3-mercaptopropionic acid (3-MPA) at about pH 10 to form an isoindole derivative. Secondary amino groups do not react. The OPA derivatized amino acid is then detected by UV at 338 nm.

The secondary amino groups react with 9-fluorenylmethyl chloroformate (FMOC) at about pH 10 to form a secondary amide. Secondary amino groups do not react. The FMOC derivatized amino acid is then detected by UV at 262 nm.

An online automated OPA /FMOC derivatization method for amino acidswill be used to analyze QuEChERS (Quick, Easy, Cheap, Effective, Rugged,and Safe) extracts of apple and spinach produce. Amino acid analysis ofthe food extracts will be compared. Scalability, batch-to-batchreproducibility, linearity, and longevity data of the amino acid methodwill be presented. Several column options will be shown, ranging fromrapid nine minute analyses of 23 amino acids including re-equilibrationusing short (50 mm) Rapid Resolution High Throughput columns (1.8um),to 40 minute analyses using 250 mm traditional 5 um columns.

The LC Method

Stationary Phase: ZORBAX Eclipse Plus C18 Column Temperature: 40 °CMobile Phase A: 10 mM Na2HPO4: 10 mM Na2B4O7, pH 8.2: 5 mM NaN3

Mobile Phase B: Acetonitrile: Methanol: Water (45:45:10, v: v: v)Injection Diluent: (0.25 mL H3PO4 + 100 mL H2O)

The Mobile and Stationary Phase

G1376C well plate automatic liquid sampler (WPALS):

1) Draw 2.5 µL from Borate vial (Agilent PN 5061-3339)2) Draw 1.0 µL from Sample vial3) Mix 3.5 µL in washport 5X4) Wait 0.2 min5) Draw 0.5 µL from OPA vial (Agilent PN 5061-3335)6) Mix 4.0 µL in washport 10X max speed7) Draw 0.4 µL from FMOC vial (Agilent PN 5061-3337) 8) Mix 4.4 µL in washport 10X max speed9) Draw 32 µL from Injection Diluent vial 10) Mix 20 µL in washport 8X11) Inject12) Wait 0.1 min13) Valve bypass

The Automated Derivatization

The QuEChERS Technique

Method Ruggedness

Amino Acid Identification and Detection

References

Amino Acid Analysis of Spinach and Apple using aQuEChERS Sample Preparation Technique andAutomated OPA/FMOC Derivatization LC MethodJohn W Henderson Jr, Thierry Faye, Ulrik Wittek, Joan StevensAgilent Technologies 2850 Centerville Rd. Wilmington, Del. USA 19808

RAFA2009

Prague, Czech Republic

John W Henderson Jr, and Anne Mack “Improved Amino Acid Methods using Agilent ZORBAX Eclipse Plus C18 Columns for a Variety of Agilent LC Instrumentation and Separation Goals”Agilent Pub.# 5990-4547EN (2009)

Cliff Woodward, John W Henderson Jr. and Todd Wielgos, “High-Speed Amino Acid Analysis (AAA) on Sub-Two Micron Reversed-phase (RP) Columns” Agilent Pub.# 5989-6297EN (2007)

Limian Zhao and Joan Stevens, “Analysis of Pesticide Residues in Spinach Using Agilent SampliQ QuEChERS AOAC Kits by GC/MS” Agilent Pub.# 5990-4305EN (2009)

Three lots of material, manufactured at different times, exhibit similar selectivity (α). Selectivity is determined by the nature of the particle surface. The similar selectivity indicates similar packing material, and reproducibility.

Lot-to-Lot Reproducibility

min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

1

2

3

4

min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

1

2

3

4

min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

1

2

3

4 B8022

B7107

B7044

α4,3 =1.21

α5,4 =1.06

α6,5 =1.04

α4,3 =1.20

α5,4 =1.06

α6,5 =1.04

α4,3=1.20

α5,4=1.06

α6,5=1.04

α14,13 =1.02

α14,13 =1.02

α14,13=1.02

3

4

65

Columns 2.1x 150 mm, 3.5 µm

.

LifetimeOverlay of early middle and late chromatograms of a 500 injection sequence

An Eclipse Plus C18 5 µm Option

A Rapid Resolution 3.5 µm Option

min5 10 15 20 25 30

mAU

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15

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25

Rs= 2.4

4.6 x 250 mm, 5 µmPN 959990-902

1 23 4

56

78

9

12

1011 13 14

15 16

1719

1820

21 2223

RRHT 1.8 µm Options

min2 4 6 8 10 12

14

mAU

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40

50

Rs= 2.6

4.6 x 100 mm, 1.8 µmPN 959964-902

min1 2 3 4 5 6

7

mAU

0

20

40

60

80

Rs= 1.9Rs= 1.9

4.6 x 50 mm, 1.8 µm

PN 959941 -902

12

34 6

7 89 12

10 11

15

1619

20

21 22 23

5

20min2.5 5 7.5 10 12.5 15 17.5

mAU

0

10

20

30

40

Rs= 2.6

4.6 x 150 mm, 3.5 µmPN 959963-902

1 24

67

89

12

10 1113

1415

16 19

20

2122 23

35

The Linear GradientsThe gradient profile (%B) is identical for all columns. The different gradient delay times are mitigated by reducing delay volume and the isocratic hold in the beginning of the gradient program.

Rapid Resolution method gradients

4.6 x 150, 3.5 µm 2.1x 150, 3.5 µm

PN959963-902 PN959763-902

time (min.) %B %B

0 2 2

0.5 2 2

20 57 57

20.1 100 100

23.5 100 100

23.6 2 2

25 end end

flow (mL/min.) 1.5 0.42

4.6x50, 1.8 µm

PN959941-902

time (min.) %B

0 2

0.2 2

7.67 57

7.77 100

8.3 100

8.4 2

9 end

flow (mL/min.) 2.0

Rapid Resolution High Throughput method gradients

4.6 x 100, 1.8 µm

PN959964-902

time (min.) %B

0 2

0.35 2

13.4 57

13.5 100

15.7 100

15.8 2

16 end

flow (mL/min.) 1.5

Traditional high resolutionmethod gradients

4.6 x 250, 5 µm

PN 959990-902

time (min.) %B

0 2

0.84 2

33.4 57

33.5 100

39.3 100

39.4 2

40 end

flow (mL/min.) 1.5

Spinach Leaf Amino Acids fromQuEChERS vial

Results

Apple Fruit Amino Acids from the

QuEChERS VialAcetonitrile Fraction

Aqueous Fraction

SER

LEU

GA

BA

AS

P

GLU

AS

N

GLN

ALA

ILE

GA

BA

”

min0 2 4 6 8 10 12 14 16 18

LU

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80

min0 2 4 6 8 10 12 14 16 18

LU

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80

min0 2 4 6 8 10 12 14 16 18

LU

0

100

200

300

400

500

600

700

Pulp

Salt

solids

Aq. layer

Organic

Layer

AA Standard

min0 2 4 6 8 10 12 14 16 18

LU

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300

400

500

600

700

min0 2 4 6 8 10 12 14 16 18

LU

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min0 2 4 6 8 10 12 14 16 18

LU

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400

500

600

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Acetonitrile Fraction

Aqueous

Fraction

AA Standard

Pulp

Layer

Salt

solids

Aq. Layer

Organic

Layer

ASP GLUASN

SER

ALAILE

LEU

GABA

GABA

ARG

TYR PHE

TRP

VAL

MET

LYS

THR

GLN

Weigh 15 g comminuted sample ( 0.1g) in 50 mL centrifuge tube

Add 100 µL of IS (TPP) solution, and QC spike solution if necessary, vortex 1min

Centrifuge @ 4000rpm for 5min

Vortex 1min, centrifuge @ 13,000 rpm for 2

min for 2 mL tubes

Or @ 4000 rpm for 5 min for 15 mL tubes

Add 15mL of ACN containing 1% HAc

Cap and shake vigorously for 1min

Transfer 500 µL extract to autosampler vial

Analyze by GC/MS

Add SampliQ AOAC QuEChERS Extraction salt packet PN 5982-5058

Transfer 1 mL of upper ACN layer to

SampliQ AOAC Dispersive-SPE 2 mL tube, or

8mL to SampliQ AOAC Dispersive-SPE 15

mL tube

Filter 2 mL of lower aqueous layer

through a “2 in 1 regenerated

cellulose/polypropylene 0.45 um syringe

filter, PN 5042-1392” into an autosampler

vial

Analyze by LC/ fluorescence

Flow chart of the Agilent SampliQ QuEChERS

AOAC extraction procedure for pesticides is in

the gray boxes.

The aqueous layer contains amino acids and is

not used in the AOAC Method 2007.01 or EN

Method 15662, but was analyzed with the

Eclipse Plus C18 AAA LC method

The MgSO4 in the QuEChERS procedure partitions the Acetonitrile and water,

concentrating the pesticides in the organic layer. The more polar amino acids

concentrate in the aqueous layer.

Amino acid levels were under 100 pmol/uL therefore a G1321A fluorescence

detector was used in place of UV for increased sensitivity, (λEx 340, λEm450,

PMT gain 12 for OPA –AA, λEx 266, λEm305 for FMOC-AA (a programmed

wavelength switch occurs after lysine elutes and before hydroxyproline elutes).

Apples contained less amino acids compared to spinach. GABA (γ-aminobutyric

acid) was also found in apple and spinach (GABA is not in standard but peak

retention was confirmed, data not shown), and consists of two peaks. The major

peak elutes about 8 minutes, and the minor elutes at about 12.2 minutes.

Besides amino acid content, the chromatographic “fingerprint” from the

aqueous fraction QuEChERS protocol may be useful for determining ripeness,

food quality, authenticity or adulteration, and variation of cultivars or origin.

4.6 x 150 mm, 3.5 µm PN 959963 902

4.6 x 150 mm, 3.5 µm PN 959963 902