Purdue-UAB Botanicals Center for Age-Related Disease

GettingGetting readyready toto dodo massmass spectrometryspectrometry

Stephen Barnes, Ph.D.

Purdue-UAB Botanical Center Workshop 2002 Mass Spectrometry Methods in Botanicals Research Purdue-UAB Botanicals Center for Age-Related Disease

ObjectivesObjectives

• What is electrospray ionization (ESI)?

• What is heated nebulizer-atmospheric pressure chemical ionization (HN-APCI)?

• Conditions for extraction and analysis by mass spectrometry Purdue-UAB Botanicals Center for Age-Related Disease

WhatWhat isis massmass spectrometry?spectrometry?

• Carried out on charged ions in the gas phase

• These ions move in an electric or magnetic field according to their mass-to-charge ratio (m/z)

• To ensure that the motion of the ions is unaffected by collisions with molecules in air, the analysis is carried out in a hard vacuum (better than 10-6 Torr) Purdue-UAB Botanicals Center for Age-Related Disease

TheThe challengechallenge

How to transfer biological molecules from the liquid or solid phase to the gas phase without damaging them? Purdue-UAB Botanicals Center for Age-Related Disease

EvaporatingEvaporating moleculesmolecules

• Forces that control volatility are molecular weight and polarity (difficult to break up a charged or hydrogen bond network)

• For gas chromatography, the solution was to derivatize the polar groups

• Not suitable for delicate or large molecules Purdue-UAB Botanicals Center for Age-Related Disease

TypicalTypical derivativesderivatives forfor GCGC

R.COOH R.COOCH3

R.CH2OH R.CH2OOCH3

R.CH2OSi(CH3)3

R.CH2NH2 R.CH2NHCOCH3 Purdue-UAB Botanicals Center for Age-Related Disease

SolutionsSolutions forfor liquidliquid--toto--gasgas phasephase transfertransfer ofof complex,complex, underivatizedunderivatized biomoleculesbiomolecules

• Thermospray (not in use any more)

• Electrospray ionization (Fenn)

• Heated nebulizer atmospheric pressure chemical ionization Purdue-UAB Botanicals Center for Age-Related Disease

ElectrosprayElectrospray ionizationionization

• Creation of a spray of finely divided, charged droplets

• Rapid evaporation at atmospheric pressure, resulting in the droplets remaining cold

• Coulombic repulsion of droplets in a jet and then formation of a Taylor cone where solutes are ejected into the gas phase NanoElectrosprayNanoElectrospray

5 µm ElectrosprayElectrospray IonizationIonization (ESI)(ESI)

N2 curtain gas nebulizing gas sample Mass solution Analyzer +HV

Atmospheric pressure Vacuum + + + + + + + + n+ ++ + + [M + nH] ++ + ++ + 1. Solvent evaporation 2. Coulombic repulsion Purdue-UAB Botanicals Center for Age-Related Disease HNHN--APCIAPCI interfaceinterface

Corona discharge

N2 curtain gas

From HPLC column Ionized air (1 ml/min) and solvent Mass + + + Analyzer

Vacuum Quartz tube at Atmospheric o 500 C pressure UseUse ofof HNHN--APCIAPCI--MSMS

• This method can accommodate mobile phase flow rates up to 1 ml/min, even when it is mostly water • Useful for compounds that are not naturally ionizable • Some problems with compounds that are thermally labile • Possible advantages with electron-capturing derivatives TheThe KudzuKudzu asas aa supplementsupplement

Buried in kudzu The purple flower of Pueraria lobata Purdue-UAB Botanicals Center for Age-Related Disease

LinkingLinking ofof LCLC analysisanalysis toto ESIESI--MSMS

m/z 415 puerarin 100 m/z 341 m/z 253 m/z 415 DZN DZ’N 75 m/z 547 ) %

( m/z 283 y m/z 445 50 m/z 267 Formononetin

m/z 445 25 m/z 431 m/z 431 G’N Relative Intensit

0 0 4 8 12 16 Time (min) IonizationIonization ofof polyphenolspolyphenols Unconjugated or C-glycosides of polyphenols form simple molecular ions, [M+H]+ or [M-H]-, by gaining or losing a proton

100 431 [M-H]- of C-glycoside ) % ( 75

50 Relative Intensity 25 227

0 200 250 300 350 400 450 500 m/z IonizationIonization ofof polyphenolspolyphenols

Conjugated polyphenols may also form molecular ions - however, depending on the energy given to the ion prior to analysis, the molecular ion may largely decompose to give the parent polyphenol ion, as well as other fragment ions

[M-H-162]- 100 269 ) % (

75 [M-H]- 431 [M-H+AcH]- 491 50

[M-H]- Relative Intensity 25 [M-H-162]- 447 285

0 200 250 300 350 400 450 500 m/z Purdue-UAB Botanicals Center for Age-Related Disease

ExtractingExtracting compoundscompounds forfor MSMS analysisanalysis -- whatwhat cancan itit tolerate?tolerate? In ESI-MS, only the solutes being studied should be ionized and preferably be in one charge state – Na+, K+ and other metal ions are a problem – Cannot use polyionic buffers for LC mobile phase, i.e., no phosphate Purdue-UAB Botanicals Center for Age-Related Disease

SampleSample extractionextraction-- polyphenolicspolyphenolics

MeOH or EtOH (50-100%) in water (for glycoside, glucuronide conjugates) - extraction

Ethyl acetate or diethyl ether (for aglucones) - solvent partition

Solid-phase cartridges - can require organic counter ion for hydrophilics Purdue-UAB Botanicals Center for Age-Related Disease

HPLCHPLC mobilemobile phasesphases forfor ESIESI--MSMS

• Solvents for reverse-phase LC – MeCN, MeOH, EtOH, n-Propanol, iso- propanol

• Aqueous phase – 0.1- 0.5% acetic or formic acid – 2 - 25 mM ammonium acetate or formate