principle of nanoelectrospray ionization and high

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

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

Electrospray Electrospray Ionization (ESI)Ionization (ESI)

VacuumAtmospheric pressure

+HV

nebulizinggas

samplesolution

MassAnalyzer

N2 curtain gas

+++++

++

++

[M + nH]n++++

+ ++ +

+ +

1. Solvent evaporation2. Coulombic repulsion

Properties of Properties of ElectrosprayElectrospray


• 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

What about small volume and low amount samples ??

Scale Down !!

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

NanoElectrosprayNanoElectrospray

5 µm

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

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


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)

• Many, many of them ??

Microscale method• on-line sample cleanup• microESI-MS !!

• How about biologicalsamples with 150 mM salt ?

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

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


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

MicroESI MicroESI -- Nebulizer Nebulizer InterfaceInterface


1 mm

MS orifice


Applications to Applications to PolyphenolsPolyphenols

• Qualitative analysis : MS and MS-MS

• Quantitative analysis : MRM-MS

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

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

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

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

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

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.

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

Summary


• 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.

DemonstrationDemonstration

• Making of micropreconcentrator(hand-on)

• Analysis of isoflavones in saline(mimic microdialysis samples)- desalt or not desalt !!

principle of nanoelectrospray ionization and high

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