Amino Acid Analysis of Spinach and Apple using a %RSD QuEChERS
1
SA Introduction Conclusions y = 0.0879x - 0.2168 R² = 0.9999 0 20 40 60 80 100 0 200 400 600 800 1000 1200 Peak Area Concentration, pmol/μL Glutamic Acid Eclipse Plus C18, 2.1 mm x 150 mm, 3.5 μm y = 0.1029x - 0.5381 R² = 0.9998 0 20 40 60 80 100 120 0 200 400 600 800 1000 1200 Peak Area Concentration, pmol/μL Alanine Eclipse Plus C18, 2.1 mm x 150 mm, 3.5 μm y = 0.069x + 0.8281 R² = 0.9995 0 10 20 30 40 50 60 70 80 0 200 400 600 800 1000 1200 Peak Area Concentration, pmol/μL Proline Eclipse Plus C18, 2.1 mm x 150 mm, 3.5 μm y = 0.1193x - 3.0495 R² = 0.9972 0 20 40 60 80 100 120 140 0 200 400 600 800 1000 1200 Peak Area Concentration, pmol/μL Lysine Eclipse Plus C18, 2.1 mm x 150 mm, 3.5 μm Linearity Calibration 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 Reproducibility Overlay 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. min 2 4 6 8 10 12 14 16 18 mAU 0 10 20 30 40 50 60 70 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 Derivatizations The 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 acids will be used to analyze QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extracts of apple and spinach produce. Amino acid analysis of the food extracts will be compared. Scalability, batch-to-batch reproducibility, linearity, and longevity data of the amino acid method will be presented. Several column options will be shown, ranging from rapid nine minute analyses of 23 amino acids including re-equilibration using 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 °C Mobile Phase A: 10 mM Na 2 HPO 4 : 10 mM Na 2 B 4 O 7 , pH 8.2: 5 mM NaN 3 Mobile Phase B: Acetonitrile: Methanol: Water (45:45:10, v: v: v) Injection Diluent: (0.25 mL H 3 PO 4 + 100 mL H 2 O) 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 vial 3) Mix 3.5 μL in washport 5X 4) Wait 0.2 min 5) Draw 0.5 μL from OPA vial (Agilent PN 5061-3335) 6) Mix 4.0 μL in washport 10X max speed 7) Draw 0.4 μL from FMOC vial (Agilent PN 5061-3337) 8) Mix 4.4 μL in washport 10X max speed 9) Draw 32 μL from Injection Diluent vial 10) Mix 20 μL in washport 8X 11) Inject 12) Wait 0.1 min 13) Valve bypass The Automated Derivatization The QuEChERS Technique Method Ruggedness Amino Acid Identification and Detection References Amino Acid Analysis of Spinach and Apple using a QuEChERS Sample Preparation Technique and Automated OPA/FMOC Derivatization LC Method John W Henderson Jr, Thierry Faye, Ulrik Wittek, Joan Stevens Agilent 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 min 2.5 5 7.5 10 12.5 15 17.5 20 22.5 mAU 0 1 2 3 4 min 2.5 5 7.5 10 12.5 15 17.5 20 22.5 mAU 0 1 2 3 4 min 2.5 5 7.5 10 12.5 15 17.5 20 22.5 mAU 0 1 2 3 4 B8022 B7107 B7044 α 4,3 =1.21 α 5,4 =1.06 α 6,5 =1.04 α 4,3 =1.20 α 5,4 =1.06 α 6,5 =1.04 α 4,3 =1.20 α 5,4 =1.06 α 6,5 =1.04 α 14,13 =1.02 α 14,13 =1.02 α 14,13 =1.02 3 4 6 5 Columns 2.1x 150 mm, 3.5 μm . Lifetime Overlay 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 min 5 10 15 20 25 30 mAU 0 5 10 15 20 25 Rs= 2.4 4.6 x 250 mm, 5 μm PN 959990-902 1 2 34 5 6 7 8 9 12 10 11 13 14 15 16 17 19 18 20 21 22 23 RRHT 1.8 μm Options min 2 4 6 8 10 12 14 mAU 0 10 20 30 40 50 Rs= 2.6 4.6 x 100 mm, 1.8 μm PN 959964-902 min 1 2 3 4 5 6 7 mAU 0 20 40 60 80 Rs= 1.9 Rs= 1.9 4.6 x 50 mm, 1.8 μ m PN 959941 -902 1 2 3 4 6 78 9 12 10 11 15 16 19 20 21 22 23 5 20 min 2.5 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 μm PN 959963-902 1 2 4 6 7 8 9 12 10 11 13 14 15 16 19 20 21 22 23 3 5 The Linear Gradients The 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 resolution method 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 from QuEChERS vial Results Apple Fruit Amino Acids from the QuEChERS Vial Acetonitrile Fraction Aqueous Fraction SER LEU GABA ASP GLU ASN GLN ALA ILE GABA” min 0 2 4 6 8 10 12 14 16 18 LU 0 10 20 30 40 50 60 70 80 min 0 2 4 6 8 10 12 14 16 18 LU 0 10 20 30 40 50 60 70 80 min 0 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 min 0 2 4 6 8 10 12 14 16 18 LU 0 100 200 300 400 500 600 700 min 0 2 4 6 8 10 12 14 16 18 LU 0 100 200 300 400 500 600 700 min 0 2 4 6 8 10 12 14 16 18 LU 0 100 200 300 400 500 600 700 Acetonitrile Fraction Aqueous Fraction AA Standard Pulp Layer Salt solids Aq. Layer Organic Layer ASP GLU ASN SER ALA ILE 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 MgSO 4 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
Transcript of Amino Acid Analysis of Spinach and Apple using a %RSD QuEChERS
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
20
30
40
50
60
70
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
0
5
10
15
20
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
0
10
20
30
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
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10
20
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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
0
10
20
30
40
50
60
70
80
min0 2 4 6 8 10 12 14 16 18
LU
0
10
20
30
40
50
60
70
80
min0 2 4 6 8 10 12 14 16 18
LU
0
100
200
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400
500
600
700
Pulp
Salt
solids
Aq. layer
Organic
Layer
AA Standard
min0 2 4 6 8 10 12 14 16 18
LU
0
100
200
300
400
500
600
700
min0 2 4 6 8 10 12 14 16 18
LU
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500
600
700
min0 2 4 6 8 10 12 14 16 18
LU
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100
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400
500
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700
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
