principle of nanoelectrospray ionization and high
TRANSCRIPT
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Purdue-UAB Botanicals Center for Age-Related Diseases
Nanoelectrospray Nanoelectrospray IonizationIonizationandand
High Sensitivity Analysis High Sensitivity Analysis
Chao-Cheng (Sam) Wang, Ph.D.
Purdue-UAB Botanicals Center Workshop 2002Mass Spectrometry Methods in Botanicals Research
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Purdue-UAB Botanicals Center for Age-Related Disease
Outline
• Concentration rules in ESI
• Micro-nano flow ESI
• Development of microESI-MS method
• Application to polyphenols
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Electrospray Electrospray Ionization (ESI)Ionization (ESI)
VacuumAtmospheric pressure
+HV
nebulizinggas
samplesolution
MassAnalyzer
N2 curtain gas
+++++
++
++
[M + nH]n++++
+ ++ +
+ +
1. Solvent evaporation2. Coulombic repulsion
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Properties of Properties of ElectrosprayElectrospray
Purdue-UAB Botanicals Center for Age-Related Disease
• 10 ~ 1000 µl/min flow ratewith pneumatically assisted interface
• Concentration-sensitive type detection- flow rate affects peak width and area,but intensity
• 10-5 ~ 10-7M• Picomole (10-12mol) detection limit
- 10 ~ 20 µl for LC-MS analysis
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What about small volume and low amount samples ??
Scale Down !!
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MicroMicro-- & & NanoNano-- ElectrosprayElectrospray
• Spraying tips (2~50 µm) • Flow Rate: 1000 ~ 10 nl/min• Electrical contact (1~2 kV)
Au-coated
HV
Gold coated glass needle
SS Union
HV
Liquid-junction interface
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NanoElectrosprayNanoElectrospray
5 µm
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Comparison of ESI and Comparison of ESI and NanoNano--ESIESI-- Ionization Efficiency Ionization Efficiency --
1 µm
MS
N2
5 kV
nebulizergas
samplesolution
samplesolution
1~2 KV 0.1 µm
MS
N2
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Comparison of ESI and Comparison of ESI and NanoNano--ESIESI-- Ion Transfer Efficiency Ion Transfer Efficiency --
ElectrosprayFlow rate: 25 µl/minIon efficiency: 0.01 %
Nano-ElectrosprayFlow rate: < 500 nl/minIon efficiency: 50~60%
--- -
-1~2 KV1~2 mm
-+
+ ---
--- -- --
-
Gas
-5 KV
-
20 mm
MS
N2
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Purdue-UAB Botanicals Center for Age-Related Disease
Advantages of Advantages of NanoElectrosprayNanoElectrospray
• sensitivity (femto - attomole level)
• tolerance toward buffer composition-- aq. and salt concentration (10 mM NaCl)
• longer analysis time (for MS-MS)
• more compatible with high efficiencyseparation techniques (nano LC & CE)
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• Many, many of them ??
Microscale method• on-line sample cleanup• microESI-MS !!
• How about biologicalsamples with 150 mM salt ?
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Microscale Microscale SystemSystem
Micro Preconcentrator
FS Capillary50 µm id
Teflon tubing
FS Capillary50 µm id
RP polymer
MicroES Interface
-++ ---
- --- -- -
N 2
HVMS
< 1000 nl/min
RPMPC
MS
Elutingbuffer
Loading and washingbuffer
Waste
1 µlInjection
Waste
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Operation of Operation of MicroscaleMicroscale SystemSystem
RPMPC
MS
Waste
1 µlInjection
Waste70% ACN
300-1000 nl/min
5% ACN3 µl/min x 3 min
Sample Elution
RPMPC
MS
Waste
1 µlInjection
Waste
70% ACN300-1000 nl/min
5% ACN3 µl/min x 3 min
SampleLoading & Washing
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Purdue-UAB Botanicals Center for Age-Related Disease
MicroESI MicroESI -- Nebulizer Nebulizer InterfaceInterface
MS
N2
Nebulizer gasAir 20 psi
FSC50 / 360 µm
SS tubing180 / 330 µmSS Tee
150 µm through holefor ESI voltage
PEEK Tee 250 µm throughhole
FSC25 / 150 µm
2 mm
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MicroESI MicroESI -- Nebulizer Nebulizer InterfaceInterface
Purdue-UAB Botanicals Center for Age-Related Disease
1 mm
MS orifice
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Purdue-UAB Botanicals Center for Age-Related Disease
Applications to Applications to PolyphenolsPolyphenols
• Qualitative analysis : MS and MS-MS
• Quantitative analysis : MRM-MS
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Characterization of Characterization of MicroESI MicroESI InterfaceInterface
Solvent Composition
Methods: flow injectionCarrier: 70% ACN, 10 mM NH4OAc
1000 nl/min Sample: isoflavone standards
in various solvent composition 50%70% 20% 5%60%% ACN in sample solvent
5001000 300 200Flow Rate (nl/min)
Flow Rate
Methods: flow injectionSample: isoflavone standards in
70% ACN, 10mM NH4OAc Carrier: 70% ACN, 10 mM NH4OAc
at various flow rate
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Microscale Microscale System for Qualitative MS System for Qualitative MS Analysis of Analysis of Isoflavones Isoflavones in PBSin PBS
Sample: 1 µl, 100 nM isoflavones in PBS (100 fmol)
0.0 1.2 2.4 3.6Time (min)
100
50
0
Rel
ativ
e In
tens
ity (%
)
220 260 300 340m/z
90
45
0
Rel
ativ
e In
tens
ity (%
) 223
247261
283
305
329
m/z220 260 300 340
100
50
0
Rel
ativ
e In
tens
ity (%
)217
223
241253
269281
299
305
255257
m/z = 241m/z = 253m/z = 255m/z = 257m/z = 269
EquolDaidzeinDHDO-DMAGenistein
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m/z50 100 150 200 250
24,500
34 5980
92
108
136
100
50
0Rel
ativ
e In
tens
ity (%
) 25721,000
91
120134
148
100
50
0Rel
ativ
e In
tens
ity (%
)
50 100 150 200 250m/z
2550.4 1.2 2.1 3.0 3.8
Time (min)
100
50
0
Rel
ativ
e In
tens
ity (%
)
Sample: 1 µl, 100 nM isoflavone in PBS (100 fmol)
MicroscaleMicroscale System for Qualitative MSSystem for Qualitative MS--MS Analysis of MS Analysis of IsoflavonesIsoflavones in PBSin PBS
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LCLC--MRMMRM--MS Analysis of MS Analysis of IsoflavonesIsoflavonesEquol (241/119)
Daidzein (253/223)
Apigenin (269/149)
O-DMA (257/108)
Genistein (269/133)
DHD (255/149)
199
76
6348(5 µM)
246
194
210
0.00 1.00 2.00 3.00 4.00 5.00 6.00Time (min)
Column: RP C8 column4.6 mm id x 10 cm
Flow rate : 1 ml/min25 µl/min to MS
Sample : isoflavones50 nM in 80% MeOH
Injection volume : 20 µl
Sample consumed : 1 pmol
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Application of Application of MicroscaleMicroscale System System for MRM Analysis of for MRM Analysis of IsoflavonesIsoflavones in PBSin PBS
Equol (241/119)
Daidzein (253/223)
Genistein (269/133)
DHD (255/149)
O-DMA (257/108)
Apigenin (269/149)
594
719
515
3408
2142
2169(500 nM)
1.00 1.50 2.00Time (min)
Column: 3M SDB-XC polymerpacked bed: 85 nl
Flow rate : 1 µl/min
Sample : isoflavones12.5 nM in PBS
Injection volume : 1 µl
Sample consumed : 12.5 fmol
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Application of Application of Microscale Microscale System System for MRM Analysis of for MRM Analysis of Isoflavones Isoflavones in PBSin PBS
Load : 1 µl , Wash: 5% ACN 3 µl x 3 minElute :10 mM NH4OAc in 70% ACN, 1000 nl/min
Equol (241/119)
Daidzein (253/223)
Genistein (269/133)
DHD (255/149)
O-DMA (257/108)
Apigenin (269/149)
151
107
245
598
311
1733
1.00 1.50 2.00Time (min)
2.5 nM isoflavones in PBS155
43
130
138
68
1454
1.00 1.50 2.00Time (min)
PBS blank w\ internal std.
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Application of Application of Microscale Microscale System System for MRM Analysis of for MRM Analysis of Isoflavones Isoflavones in PBSin PBS
-2
0
2
4
6
8
10
12
14
0 20 40 60 80 100 120 140Conc. (nM)
Peak
Are
aR
atio
(vs
Api
geni
n)
R2 = 0.999 DHD
R2 = 0.999 O-DMA
R2 = 0.998 Genistein
R2 = 0.999 DaidzeinR2 = 0.994 Equol
Conc. Range: 2.5 ~ 125 nM
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Summary
Purdue-UAB Botanicals Center for Age-Related Disease
• Micro- to nano- flow ESI provides many advantages, especially sensitivity, over conventional ESI, due to better ion formation and transfer efficiency.
• The microscale system, combining on-line sample cleanup (desalt) and concentration with MicroESI-MRM-MS, provides detection limits at low femtomole levels in 1 µl of physiological samples.
• The microscale system will be ideal for analysis ofpolyphenols from minimal physiological samples, such as blood extracts of small animals and microdialysates.
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DemonstrationDemonstration
• Making of micropreconcentrator(hand-on)
• Analysis of isoflavones in saline(mimic microdialysis samples)- desalt or not desalt !!