This is the third lecture in series of lectures on mass spectrometry for pesticides residue analysis. This lecture (3) include: Electrospray ionization and Atmospheric pressure chemical ionization

1. MASS SPECTROMETRY
for
pesticides residue analysis
“Electrospray Ionization and Atmospheric pressure chemical ionization”

2. Contents
Lecture 1: Pesticides classification with an introduction to mass spectrometry&
vacuum system for GC MS/MS and LC MS/MS
Lecture 2 : Electron ionization and Chemical ionization
Lecture 3 : Electrospray Ionization and Atmospheric pressure chemical ionization
Lecture 4 : The commonly used mass to charge analyzer for pesticides residue analysis

3. Types of Ionization
There are Different ionization techniques that can be used for mass spectrometry. In
these series of lectures , the most commonly used ionization techniques for mass
spectrometry analysis of pesticides residue will be discussed, especially:
• Electron Ionization (EI) as an example for Hard ionization technique.
• Chemical ionization (CI), Atmospheric pressure chemical ionization (APCI)
and Electron spray ionization (ESI) as examples for Soft ionization techniques.

4. • Used for analysis of thermo labile and nonvolatile analytes (reverse to EI)
• produce ions by passing a solution into narrow charged electrode (protonated
/deprotonated)
• Produce multi charged ions (with lower m/z ratio) for analyte molecules of
high molecular weight (above 1000 u) with multi active atoms
• ESI is applicable for a wide range of analyte
molecules varying in mwt and polarity
Electrospray ionization (ESI)
j.gross, Mass Spectrometry, a text book, Springer-Verlag Berlin Heidelberg 2004, 2011

5. The first electrospray–mass spectrometry interface
(Fenn group,1980s)
• Fenn and his group have showed the formation
of multiply charged ions from proteins allowing
their molecular weight to be determined with
instruments of low mass range
• ESI was first used for protein analysis after that
for polymers, co-polymers and for smaller
molecules
E.Hoffmann, Mass Spectrometry Principles and Applications, John Wiley & Sons Ltd, England (2007)
https://en.wikipedia.org/wiki/Electrospray_ionization

6. Mechanism of Electrospray Ionization
• ESI works by online HPLC MS/MS, Where a liquid is converted to an charged
aerosol droplets by passing through a metal capillary which also maintained at
high voltage (around 4 kV) in a chamber held near atmospheric pressure
• ESI can work in positive and negative modes
• Positive ESI produce both positive and negative charges only positive
charged particles will be attracted to mass vacuum (the reverse in –ve ESI)
P. Kebrale, Electrospray: From ions in solution to ions in the gas phase, what we know now, Mass Spectrometry Reviews, 2009
https://www.youtube.com/watch?v=r6TGvG7RUyo

7. Shape of the charged droplets
• The shape of liquid passed through 0.1-mm-ID horizontally positioned
capillary at :
No voltage ------------------------drops fall off under (gravity)
Moderate voltage --------------drops with reduced size are formed
Higher potentials (Von)----------------reduced size droplets are formed with
sharp point at its tip, the droplet divided with spray formation (Taylor cone, the
formed droplets attracted to the counter electrode)
At further higher potentials------- liquid cone vanishes and a fine mist of
droplets is produced (avoid discharge)
W. Niessen, Liquid Chromatography– Mass Spectrometry, Taylor and Francis Group, LLC (2006)
E.Hoffmann, Mass Spectrometry Principles and Applications, John Wiley & Sons Ltd, England (2007)

8. Shape of the charged droplets
• Calculation of the onset voltages (Von)
σ is the surface tension of the liquid
rc is the inner diameter of needle
d is the distance between capillary and counter electrode
For rc = 0.1 mm and d = 40 mm,
Von = 2.2 kV (MeOH), 2.5 kV (Acetonitrile) , 4.0 kV (Water)
• For stable ESI performance, the voltage should be set a few hundred volts higher than the
onset voltage. But, too high potential cause discharge especially when applying Negative ESI
mode
W. Niessen, Liquid Chromatography– Mass Spectrometry, Taylor and Francis Group, LLC (2006)

9. Formation of charged ions
J. Throck, Introduction to Mass Spectrometry Instrumentation, John Wiley & Sons Ltd, England (2007)
E.Hoffmann, Mass Spectrometry Principles and Applications, John Wiley & Sons Ltd, England (2007)
El-Aneed A., Mass spectrometry review of the basics : Electrospray, maldi and commonly used mass analyser, APPLIED SPECTROSCOPY REVIEWS (2009)
• As the droplet size decrease the repulsive forces between the excess
charged ions increase. Which result in “coulomb explosion” of the droplet
before reaching the Rayleigh limit (the point at which repulsive forces
between like charges in an electrolytic solution overcome the cohesive forces
of the solvent).
• Finally, when the solvent molecules have evaporated, quasi molecular ions of
the analyte will be formed; protonated for positive ESI, protons attach at
sites of high Lewis basicity. While, deprotonated for -ESI

10. Desolvation of the charged droplets
J. Throck, Introduction to Mass Spectrometry Instrumentation, John Wiley & Sons Ltd, England (2007)
1.0073
Positive ESI Mass spectrum of lysozyme

11. ESI For high flow rate
• ESI introduce aerosol from liquid of low flow rates of 1–20 μl/ min,
Therefore further modification have been established
• Applying a constant flow inert gas (N2) around
the electrospray needle (Nebulizer Gas) to
reduce the influence of the surface tension of
the used solvent.
• Although, ultrasonic nebulizer handles liquid
flow rates of 50–1000 μl/min but, the
ultrasonically created droplets are comparatively
large and this hinders ion formation
Pneumatically assisted ESI
j.gross, Mass Spectrometry, a text book, Springer-Verlag Berlin Heidelberg 2004, 2011

12. Improved geometries for ESI
• In order to decrease the system contamination (orifice,…) different
geometries for ESI are produced
j.gross, Mass Spectrometry, a text book, Springer-Verlag Berlin Heidelberg 2004, 2011

13. Increase the desolvation of the charged droplets
• A fast desolvation for the charged droplets can be
obtained by introducing a heated inert gas. Which,
ionization of the sample.
• The higher the liquid flow or the higher of its aqueous
composition, the higher heater temperature and gas flow
required.
• Temperature lead to a premature vaporization of the
solvent, chemical background noise.
• Heater Gas flow can produce a noisy, or unstable
signal.
API LC/MS/MS TurboIonSpray Ion Source Manual

14. Declustering using curtain gas
API LC/MS/MS Turbo IonSpray Ion Source Manual
W. Niessen, Liquid Chromatography– Mass Spectrometry, Taylor and Francis Group, LLC (2006)
• Curtain gas (inert gas (N2) is used as an insulator between the ion source
(prevent entering of air, solvent, non polar matrices) and vacuum chamber. So,
it should be optimized at highest possible setting.
• This gas is introduced between curtain plate and orifice plate, Part of this
gas passes through the orifice nozzle into the differentially pumped interface,
while the remaining gas flows back through the aperture in the curtain plate to
make a declusting collisions.
• The resulted ionized gas molecules are in a cluster form (with some residue
from solvent molecules). A cluster may be resulted from the cooling that
happened after solvent evaporation or by incomplete evaporation.
• Introducing curtain gas in the way of these ion clusters leads to a complete
separation for the analyte ions from solvent molecules.

15. Declustering using potential and ion focusing
API 4000™ LC/MS/MS System Hardware Manual
http://sciex.com/products/mass-spectrometers/triple-quad-systems/triple-quad-6500-system-x36184
• A cluster may also be resulted from the cooling that
happened under the first vacuum stage (after passing
orifice plate) that induce adiabatic expansion that favor
cluster formation.
• Applying a potential (declustring potential, DP ) on the orifice
is needed to decluster the aggregated ions between orifice
and skimmer or between orifice and Qjet .
• DP should be high enough to reduce chemical noisy with
avoiding fragmentation especially, for MRM analysis.
• The entrance potential (EP) guides and focuses the ions
through Q0 region.

16. Turbo V™ ESI
API LC/MS/MS TurboIonSpray Ion Source Manual
Turbo V™ Ion Source Operator Guide
Turbo heater
Electrode adjustment cap
Retaining ring
X-axis
Y-axis
Turbo heater
Exhaust
Spray is directed
away from orifice
Probe
Electrode

17. Turbo IonSpray ESI (operating conditions for the at three different flow rates)
Turbo V™ Ion Source Operator Guide
API LC/MS/MS TurboIonSpray Ion Source Manual
Spray is directed
away from orifice
• The solvent composition used
for optimization was
1:1 water: acetonitrile
• ISV : Positive mode 4000 to 5500 V.
Negative mode usually lower
-3000 to - 4500 V.
(P ISV of 4500 for 6500 system)
• For too high ISV, a blue glow can
be seen at the tip of the
TurboIonSpray (a corona discharge)
decreasing the sensitivity.
• Curtain Gas flow should be as high
as possible. Start with 20 for 4000
and 4500 & 25 for 5000 and 5500
& 30 for 6500

18. Turbo V™ APCI (APCI Heated nebulizer)
API 4000™ LC/MS/MS System Hardware Manual
Turbo V™ Ion Source operator guide
T. Portol, Potential of atmosphericpressure chemical ionization source in GCQTOF MS for pesticide residue analysis, journal of mass spectrometry (2010).
• The liquid sample is nebulized in the heated tube
into finely dispersed droplets. Where, a corona
discharge needle ionize first the solvent molecules
(most abundant) which subsequently collision
ionize the analyte molecules with minimum thermal
decomposition (least fragmentation) at API. (like
Chemical ionization)
• This interface can be used for
GC or LC

19. • This ion source combines the two most
common ionization techniques : ESI probe and
APCI probe in a single source housing
• Increase the applicability of Triple TOF™
5600 system for a wider range of analytes.
Z. Rhoades, Broader Coverage and Automatic Mass Calibration Using the TripleTOF™ 5600 System with DuoSpray™ Ion Source, AB SCIEX,
Foster City, CA, USA and Concord, Ontario, Canada
The DuoSpray™ Ion Source

20. Comparing the Extracted ion chromatogram and mass spectra of dieldrin in TOF MS EI
source Vs TOF MS APCI source.
T. Portol, Potential of atmosphericpressure chemical ionization source in GCQTOF MS for pesticide residue analysis, journal of mass spectrometry (2010).

21. The influence of electrospray ion source design on matrix effects
H. Stahnke, The influence of electrospray ion source design on matrix effects, J. Mass. Spectrom. 2012, 47, 875–884
• The matrix effect by these ESI designs for
45 pesticides (in different matrices include:
grapefruit, orange, pear and sweet pepper)
doesn’t largely differ
• The Jet Stream ESI (orthogonal geometry)
With heated sheath gas Give a higher
sensitivity With a stronger signal suppression
than Without using sheath gas

22. Positive ESI Mass spectrum of Atrazine using ABSCIEX 6500
TIC of +Q3: from Sample 92 (Boscalid_0.10_SCan_Q3) of 131114_Boscalid.wiff (Turbo Spray IonDrive) Max. 1.1e10 cps.
0 2 4 6 8 10 12 14 16 18 20 22 24 26
Time, min
0.00
1.00e9
2.00e9
3.00e9
4.00e9
5.00e9
6.00e9
7.00e9
8.00e9
9.00e9
1.00e10
1.10e10
Intensity,cps
20.37
22.96
21.37
23.30
24.34 27.8626.30
20.03
19.62
18.16
17.4216.3815.510.04
0.73 14.4313.451.49 2.26 12.392.99 11.47
+Q3: 20.074 to 20.651 min from Sample 92 (Boscalid_0.10_SCan_Q3) of 131114_Boscalid.wiff (Turbo Spray IonDrive), Centroided Max. 7.0e8 cps.
335.0 336.0 337.0 338.0 339.0 340.0 341.0 342.0 343.0 344.0 345.0 346.0 347.0 348.0 349.0 350.0
m/z, Da
0.0
5.0e7
1.0e8
1.5e8
2.0e8
2.5e8
3.0e8
3.5e8
4.0e8
4.5e8
5.0e8
Intensity,cps
343.0
345.0
346.9346.1344.0
341.2 348.0339.1335.1 337.1 349.1338.2336.2 340.2
N
O
NH
Cl
Cl
Flow rate 30μ l/min
CUR: 20.00
IS: 5500.00
TEM: 400.00
GS1: 35.00
GS2: 35.00
DP 102.00
EP 10.00
CXP 26.00342
342+ 1
QCAP Egypt

23. TIC of -Q3: from Sample 5 (Boscalid_Only_Coverd15M_) of 131121_Boscalid.wiff (Turbo Spray IonDrive) Max. 4.9e7 cps.
0 2 4 6 8 10 12 14 16 18 20 22 24 26
Time, min
0.0
5.0e6
1.0e7
1.5e7
2.0e7
2.5e7
3.0e7
3.5e7
4.0e7
4.5e7
4.9e7
Intensity,cps
20.21
1.23 21.16
1.87 21.65
23.15
22.242.65 3.87 24.564.39 5.19 6.02 27.4026.906.75
12.80 14.46 14.8311.59 17.4911.17 15.768.26 10.04 18.18
-Q3: 20.224 min from Sample 5 (Boscalid_Only_Coverd15M_) of 131121_Boscalid.wiff (Turbo Spray IonDrive), Centroided Max. 1.8e7 cps.
333.0 334.0 335.0 336.0 337.0 338.0 339.0 340.0 341.0 342.0 343.0 344.0 345.0 346.0 347.0 348.0 349.0 350.0
m/z, Da
0.0
2.0e6
4.0e6
6.0e6
8.0e6
1.0e7
1.2e7
1.4e7
1.6e7
1.8e7
Intensity,cps
340.9
342.9
341.8 343.8 344.9
346.0
N
O
NH
Cl
Cl
Flow rate 30μ l/min
CUR: 20.00
TEM: 400.00
GS1: 35.00
GS2: 35.00
IS: -4500.00
DP -102.00
EP -10.00
CXP -26.00
342
342- 1
Negative ESI Mass spectrum of Atrazine using ABSCIEX 6500
QCAP Egypt

24. Negative ESI Mass spectrum of Atrazine
https://www.researchgate.net/figure/258856265_fig10_Figure-1-Mass-spectrum-of-atrazine-with-a-chlorine-adduct-(reprinted-with-permission [accessed Oct 15, 2015]
215+35
Cl adduct
215
215-1

25. Summary
In an ESI; a liquid is converted to an charged aerosol (containing both positive and negative ions) by
passing through a metal capillary which at high voltage (around 4-5.5 kV P ESI, 3- 4.5 kV N ESI)
A Nebulizer Gas reduce the influence of the surface tension of the used solvent (allow high flow rate
with fast desolvation)
ESI needle should be at suitable geometry with the orifice plate( for example : orthogonal, a way
from the orifice aperture) In order to minimize the system contamination
A Higher heater temperature and gas flow values are required for higher liquid flow of high aqueous
composition (Avoid fragmentation)
The huge presence of vapors (evaporated solvent, volatile matrices) and gases (nebulizer, heat
gas,..) are separated from the vacuum system by applying a curtain gas (between curtain plate and
orifice) should be optimized to a high value
Applying a declustering potential is needed to decluster the aggregation of the charged ions after its
passing through the first vacuum region (between orifice and skimmer)

26. Videos :
ESI : https://www.youtube.com/watch?v=r6TGvG7RUyo
GC APCI MSMS : https://www.youtube.com/watch?v=lnAwbfiMfUE
ESI with APCI : https://www.youtube.com/watch?v=9QraBDViT2Y
LC MSMS 6500 : https://www.youtube.com/watch?v=um_ELG02wpY&list=PLRe-
FFrwYkWD216bgzEUE1amMrMEem-mD
Note : The optimizing information that found in this lecture is used only for
education and may simplify your understanding to the corresponding
parameters, Not to be used for optimizing your system.
This lecture and all lectures in these series are introduced for QCAP Egypt colleagues, we have 5 LC
MS MS (Ab Sciex) and 5 GC MS MS (Agilent). So, I have mad a more detailed information about
optimizing parameter on AB SCIEX LC MS MS and that for GC MS MS from the available
information online .
Thanks for AB SCIEX and Agilent for presenting many manuals, Guides,.. available online

27. sherif2taha@gmail.com
Sherif.taha@qcap-Egypt.com