MASS SPECTROMETRY

§ 1. Definition [1-4]
Mass spectrometry is the branch of science dealing with qualitative and quantitative interpretation of
ions produced under controlled conditions by a sample submitted to analysis, by means of a mass
spectrometer.

§ 2. History
A short history of mass spectrometry is summarized in the following table.

Table 1. Milestones in development of mass spectrometry.

Period Scientist Achievement Ref.

E. Goldstein Discovery of “cathode rays” and “positively charged
1886 -
rays (canal rays)”
1897 J.J. Thomson Discovery of the electron -
Studies of electric and magnetic properties of
1898 W. Wien -
“positively charged rays”
Construction of a “parabola spectrograph” considered
1899-1911 J.J. Thomson 5, 6
as the first mass spectrometer
1918 A. J. Dempster Electron ionization and magnetic focusing 7
1919 F. W. Aston Isotope mass identification 8
Double focusing instruments for separation of 235 /
1940-1944 A.O. Nier 9
238 isotopes of Uranium
1946 W.E. Stephens Introduction of the “Time of Flight” concept 10
J.A. Hipple,
1949 H. Sommer, Ion cyclotron resonance -
H.A. Thomas
A.O. Nier
1953 Double focusing mass spectrometer 11
E.G. Johnson
W. Paul
1953 Quadrupole ion analyzers -
H.Z. Steinwedel

1
 Period Scientist Achievement Ref.
Identification of organic compounds by mass
1956 J.H. Beynon 12
spectrometry
B. Munson
1966 Chemical ionization 13
F.H. Field
1968 M. Dole Electrospray ionization 14
1969 H.D. Beckey Field desorption of organic molecules 15
A.G. Marshall
1974 Fourier transform ion cyclotron resonance 16
M.B. Comisarow
R.D. Macfarlane 252
1976 Cf plasma desorption in mass spectrometry 17
D.F. Torgerson
1978 R. Yost, C. Enke Triple quadrupole mass spectrometry -
1981 M. Barber Fast atom bombardment 18
K. Tanaka
Matrix assisted laser desorption / ionization
1983 F. Hillenkamp -
(MALDI)
M. Karas
1989 J.B. Fenn Electrospray of biomolecules 19
M. Mann
1991 Micro electrospray -
M.S. Wilm
1995 K. Hiraoka Orthogonal electrospray 20
1996 G. Siuzdak Mass spectrometry of a virus 21
1999 J. Wei Desoption / ionization without matrix 22

§3. Principles
Mass spectrometry deals with both quantitative and structural information. Basically, the
sample is stroked to generate fragments. Against the fragments generated upon impact, only positive
(+) or negative (-) ions are further considered. Selected ions are then characterized according to their
mass / charge ratio (m/z). Quantitative information is produced by counting the total number of ions
(positive or negative) which are produced or by counting the number of ions having a precise m/z being
produced during ionization stage. Structural information derives from a hypothetical reformation of the
integer from the resulting ions of known masses. Whole process is illustrated in Figure 1.

2
Figure 1. An illustration of the mass spectrometric principle.

-
+ e-
e- . +
-
e
e-
+ +
- -
e e -
e-
+

-
e- e .
-

- +
+
- +
e
+ +

+
+ + +
+

+
Total of 5 +
or ?
+
Two of + +
Quantitative Structural

3
 Mass spectrometry is thus achieved by means of the following consecutive operations: (1)
sample introduction; (2) sample ionization; (3) ion analysis / processing (mainly separation according
to their m/z); (4) ion counting / detection; (5) records of the results and interpretation. The basic
schema of a mass spectrometer is given in Figure 2.
Figure 2. Basic schema of a mass spectrometer.

Computer

Hardware
control

Data
acquisition,
computing &
Sample inlet Ion source Mass analyzer Detector interpretation

Stage (1) Stage (2) Stage (3) Stage (4) Stage (5)

Vacuum
Ion isolation
Ion isolation Product ions System
(collisionally
(precursor ion) analysis
induced)
n

Some applications require that stage (3) is expanded in order to improve the information
amount to be taken from the ionization process or to increase detection selectivity. Sequence between
brackets in Figure 2 illustrates this alternative.
Hence mass spectrometry deals with ions produced by the sample under controlled conditions
and these ions are positively or negatively charged, it clearly results that mass spectrometry should be
addressed as (+) MS or (-) MS, according to the charge sign of the ions being analyzed. In modern
mass spectrometers, analysis of positive ions can be alternatively switched to analysis of negative ions,
but in no cases, simultaneous (+) and (-) ion analysis is affordable.
It is also worthwhile to note that ion formation, separation and characterization require an
environment free of interferences, meaning that a mass spectrometer is working in deep vacuum
conditions (according to the constructive characteristics of the mass analyzer, vacuum levels ranges
between 10 -5 and 10-12 torr).
Principles and practice of mass spectrometry are detailed in general text books such as
references 23 – 30.
4
§4. The Mass Spectrum [31-33]
The mass spectrum is the plot of the individual ion abundance as function of their respective
m/z. Ions are produced during the ionization stage of a sample representing a pure compound or a
mixture.
The ion mass can be expressed in the following manners: (1) the ion average mass representing
the sum of the mean masses of the forming atoms; the mean atomic mass is calculated as a balanced
average of exact masses of the existing isotopes and their natural occurrence; (2) the ion monoisotopic
mass represents the sum of the exact masses corresponding to the most abundant natural isotope
existing for each of the forming atoms; (3) the ion nominal mass represents the sum of the integer
masses of the most abundant natural isotope corresponding to each of the forming atoms. Ion masses
are expressed in Daltons (Da). z represents the total number of charges existing on the ion, expressed in
units of elementary charge (e).
In Figure 3 a detail from the atrazine mass spectrum is presented (m/z interval ranging from
197 to 220 Da). The Profile view assumes a definite resolution of the mass analyzer, while the line
view gives the intensities only for integer values of m/z. The ion with the highest abundance in the
mass spectrum is referred as the major ion. Normalization of the abundances of the other ions with
respect to the major one leads to the relative abundance measurements (R.A.%) placed on the Oy axis.
The molecular ion is the ion produced by the molecules of the sample by means of the removal or
addition of one/more electrons.

§ 5. Sample Introduction
Samples can be brought in the ionization area either in a gas phase as well as in a condensed
phase (liquid or solid). Samples subjected to MS analysis have either organic or inorganic nature. More
often, organic samples are submitted to MS analysis in order to obtain structural information or
confirmation, generally from extremely low amounts. If such structural information is required,
simultaneous ionization of different molecular species should be avoided. This means that multi
component samples should be first separated in individual constituents and then subjected to MS
analysis. Nowadays, all separation techniques (thin layer chromatography, gas chromatography, liquid
chromatography, supercritical fluid chromatography, capillary electro chromatography, micellar
electrokinetic chromatography – micelle staking technique, capillary zone electrophoresis) have been
successfully coupled to mass spectrometers, directly or by means of especially designed interfaces. For
inorganic samples, information deals with identification and assay of atoms and related isotopic
occurrence. In such cases, no special features are imposed for sample introduction , but sample
5
ionization methods are more complex (ion bombardment, inductively coupled plasma).
Figure 3. Detail from the atrazine mass spectrum.

100
ATRAZINE
90
Molecular formula: C8H14ClN5
Average Mw = 215.69
80 Monoisotopic Mw = 215.09377
Nominal Mw = 215
70 Line
Spectra
60

50 HN

40 N N

30 HN N Cl
Ion Relative Abundance

20

10
( R.A. %)

0 212
213
214
215
216
217
218
219
220
198
199
200
201
202
203
204
205
206
207
208
209
210
211

100

90

80
Profile Spectra
70

60

50

40

30

20

10

0
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220

Mass to charge ratio (m/z)

6
§ 6. Ionization modes in mass spectrometry
§ 6.1. Gas phase ionization
§ 6.1.1. Electron impact (EI) [34-37]
Achieving electron impact ionization means that focused and accelerated electrons collide the
molecules of the analyte introduced in the ionization area in a gaseous state. Ionization arises according
to the following patterns:
(+) mode (-) mode

M + e −p → M +. + 2 es− (1) M + e −p → M -. (5)

M + e −p → M +.* + 2 es− (2) M + e −p → M -.* (6)
M +.* → Ai+. + B j (3) M −.* → Ai−. + B j (7)
M +.* → Ci+ + D .j (4) M −.* → Ci− + D .j (8)
M = target molecule; A, B, C, D = molecular fragments; p = primary; s = secondary: * = activated
energy state; . = impair electron
The basic condition for ionization is that the primary electron posses an energy at least equal to
the ionization potential of the target analyte ( Ee− ≥ ( I .P .)M ). An excess of energy is required to
p
generate molecular fragmentation (reactions 3, 4, 7, 8). As the ionization potential of organic molecules
falls in the 8 – 12 eV interval, it results that energies of the primary electron higher than 15 eV should
provide proper ionization and fragmentation. However, the plot of the ionization yield according to the
energy of the primary electrons has a profile as shown in Figure 4.
Figure 4. Ionization yield as function of the energy of the primary electrons.
100
Arbitrary units as long as only about 1% from the total

90

80
number of target molecules are ionized

70
Ion formation yield (%)

60

50

40

30

20

10

0
0 10 20 30 40 50 60 70 80 90 100
Energy of the primary electron (eV)

7
 The choice of a standardized value for E
e −p
= 70 eV is evident as long as lower energy values
lead to a significant variance of the ionization yield.
Within a mass spectrometer, three types of ionic species exist: the molecular ions, the fragment
ions produced in the ionization area and metastable ions (produced by decomposition of molecular ions
after leaving the ion source).
The residence of the ions within the source ranges in the 10-6 ÷ 5 x 10-6 seconds interval, while
the time taken for ions to run through the mass analyzer region is higher (about 10-5 seconds).
Figure 5 illustrate the relation between the lifetime of the molecular ion and the resulting mass
spectrum. It is obvious that consistent structural information is obtained if the molecular ion and
fragment ions are present with significant abundances within the mass spectrum. For chemical species
generating molecular ions with extremely short lifetimes, finding a way of reducing ionization energy
is mandatory if their observation is required. The reduction of the primary electron energy is not
recommended on that purpose as long as ion formation yield and ionization reproducibility are strongly
affected.
Figure 5. Correlation between the molecular ion lifetime and
characteristics of the mass spectrum.

Ee− > ( I .P .)M

p
Very high Fragmentation Less Practically no
fragmentation. is important. fragmentation. fragmentation.
Loss of the However, Occurrence of Only the
signal of the observation of metastable ions molecular ion
molecular ion the molecular is probable. is observable
in the mass ion is possible. Intense signal in the mass
spectrum. of the spectrum.
molecular ion
in the mass
spectrum.

0,1 1 10 100 1000

µs)
Molecular ion lifetime (µ

8
 Major advantages of Electron Impact ionization mode are its stability, ease of operation and
control of the beam intensity, lack of contamination problems, relative high sensitivity and generation
of library searchable spectra.

§ 6.1.2. Chemical ionization (CI) [38-52]

In CI, ionization of the target molecule is made by interactions with ions produced in the source
by means of the electron impact on reagent gas molecules. Existence of reagent gas molecules in large
excess with respect of the target molecules (10000:1) imposes an increase of the energy of primary
electrons (500 eV). CI is known as a “soft ionization” technique because energy transfer between
reagent gas ions and sample molecules do not exceed 5 eV. Ionization products in CI exhibit an
enhanced stability due to their even electron state (compared to impair electron state for ions generated
through EI). Interactions in CI are summarized below:
(+) mode (-) mode

RH + e −p → RH + . + 2 e s− (9)
+. +
RH + RH → [RH 2 ] + R .
(10) H 2O + e −p → OH - + H (15)
+ +
[RH 2 ] + M → [ MH] + RH (11) proton transfer
+ +
M + OH - → H 2O + [M - H] + (16)
[RH 2 ] + M → [ MRH2] (12) electrophilic addition
+. +. M + OH - → [ MOH] - (17)
RH + M → M + RH (13) charge exchange
[RH 2 ] + + MH → M + + RH 3 (14) anion abstractio n
RH = reagent gas; RH2+ = reagent ion; M or MH = target molecule; p = primary; s = secondary:
.
= impair electron

Specific interactions in the ion source are exemplified below on using methane as a reagent gas:

CH 4 + e -p → CH 4+ . + 2 es- ; CH 5+ + M → [MH] + + CH 4
CH 4+ . → CH 3+ + H . C 2 H 5+ + M → [MH] + + C 2 H 4
CH 4+ . → CH 2+ . + H 2 C 3 H 5+ + M → [MH] + + C3 H 4
CH 2+ . + CH 4 → C 2 H 3+ + H 2 + H . CH 5+ + M → [MCH 5 ] +
C 2 H 5+ + M → [MC 2 H 5 ] +
CH 4+ . + CH 4 → CH 5+ + CH 3. C 3 H 5+ + M → [MC 3 H 5 ] +
CH 3+ + CH 4 → C 2 H 5+ + H 2 C 2 H 5+ + M → [M - H] + + C 2 H 6
C 2 H 3+ + CH 4 → C 3 H 5+ + H 2

9
 The following reagents gases are commonly used in CI (reagent ions are placed between
brackets): H2 (H3+); CH4 (CH5+); C2H6 (C2H7+); H2O (H3O+); CH3OH (CH3OH2+); CH3CN
(CH3CNH+); NH3 (NH4+); CH3NH2 (CH3NH3+); H2NCH2CH2NH2 (H2NCH2CH2NH3+). For negative
CI, the following reagent gases are commonly used (reagent ions are placed between brackets): NH3
(NH2-); N2O mixed with CH4; CH4 + He; H2 + He (OH-); N2O or mixed with N2 (O-.); NF3 (F-); CHF3
(F -); O2 (O2-.); CH2Br2 (Br-); CH2Cl2 (Cl-); CHCl3 (Cl-); CF2Cl2 (Cl-).
The advantages of CI mode are: soft ionization, multiple checks of the molecular weight
possible due to a large variety of ionization processes, universal or selective action depending on the
choice of the reagent gas, easy formation of negative ions, allowing structural and thermochemical
measurements.
Both EI and CI ionization modes are realized in ion sources constructively similar to the
schema depicted in Figure 6.
Figure 6. Basic construction of an ion source designed for EI or CI ionization modes.

Reagent gas
(CI)
Accelerating and
focusing plate
N Magnet Valve
Pressure
Sample inlet Renium gauge
(vapours) Filament

Ionization
Repeller area To Mass
Analyzer
Electrode

Primary electrons
Vacuum (70 eV)
Screen system for
Anode acceleration and
focusing product ions to
mass analyzer
Magnet
Ion Source
S
Thermal electrons produced by the Renium filament are focused and accelerated by means of
the plate, the anode and/or the magnet poles. The electron beam collides the molecules of the sample
10
(or the molecules of the reagent gas) in the ionization area. Reagent gas line feeding the system
includes also a control valve and a pressure gauge for controlling the reagent ions formed within the
source. Repeller electrode is used for elimination of unwanted product ions (the electrode is positively
charged for elimination of negative product ions and negatively charged for elimination of positive
product ions). The screen system serves to focus and acceleration of product ions towards the mass
analyzer (screens are charged to increased voltages of contrary sign with respect of the product ions to
be analyzed). The whole ion source is adequately vacuumated by means of turbomolecular or diffusion
pumps.

§ 6.1.2. Field ionization (FI) [53-55]

In FI mode, molecules of the analyte in gaseous state are brought close to a surface with a high
curvature shape (generally tips, whiskers or blades) subjected to intense electric fields (10 7 ÷ 108 V x
cm-1). These molecules are readily ionized by means of quantum tunneling of valence electrons from
the molecule to the metal surface. The techniques should be considered as a “soft” ionization one,
having as a major drawback the lack in sensitivity.

§ 6.2. Ionization from condensed phases

§ 6.2.1. Liquid phases
§ 6.2.1.1. Fast Atom Bombardment (FAB) [56-62]
Target molecules are dissolved in viscous, relatively involatile liquid matrix on a porous surface
and are subjected to a bombardment of atoms or ions having keV translation energies. The use of Xe
atoms or Cs ions generates similar spectra. In the region above the surface of the frit, target molecules
are interacting in a similar way as in CI, [M+H]+ and [M-H]- ions occur. The most common viscous
liquid is glycerol. The method is suitable for polar molecules with Mw ≤ 20,000 Da. If high energy
ions are used instead of atoms, the ionization technique is also known as LSIMS (liquid secondary ions
mass spectrometry). The FAB ion source is schematically given in Figure 7.

§ 6.2.1.2. Atmospheric Pressure Chemical Ionization (APCI) [63-68]

A liquid flow containing the target molecules is pumped through a heated vaporizer. The jet of
vapors containing also liquid droplets (continuously evaporating under heat) is oriented toward a
discharge zone (typically of corona form, realized between a tip and a discoidal counter electrode
maintained at 1 – 4kV potential). The electrical discharge ionizes solvent molecules existing in the gas
state. A combination of collisions and charge transfer reactions between solvent ions and target
11
molecules lead to their ionization. Generation of protonated or deprotonated molecular ions [M+H]+ or
[M-H]- is thus possible. The ions are extracted toward the mass analyzer via a capillary tube. The set-
up is presented in Figure 8.
Figure 7. Schema of a FAB ion source.

Porous solid Atom / ion gun

surface (frit)

Liquid sample
inlet

Viscous support Ion extraction

liquid inlet optics

Vacuum To mass
analyzer

Figure 8. The APCI source (orthogonal design).

corona Liquid flow

+
discharge
N2 flow
+ + + +
Heated sheath
(up to 600 oC)

Gas phase
Counter electrode

Needle electrode Extracting capillary

To mass
analyzer

Heated N2 curtain

Corona discharge Vacuum

12
 APCI is used for a wide range polarity of target molecules and is relatively tolerant to unvolatile
salts or buffers existing in the liquid effluent. It is however especially used for apolar compounds.
Ionization pattern is not influenced to a greater extent by the solution chemistry of analytes in the
carrying phase. Unsuitable for thermally labile compounds and rarely generates multiply charged ions.
The forerunner of APCI was thermo spray (TSP). The technique was never particularly routine and has
been superseded by APCI.

§ 6.2.1.3. Electrospray ionization (ESI or AP-ESI) [69, 70]

A liquid carrier containing target molecules is mixed with a nitrogen flow and forced through a
stainless steel capillary maintained at 3-4 kV. A plume of charged liquid droplets is formed (Taylor
cone). An oriented counter current heated nitrogen curtain continuously determines the reduction of the
volume of droplets with a corresponding increase of the electric field density on the surface until a
critical volume is reached (Reyleigh limit). Resulting ions are extracted from droplets and sampled
through a capillary tube to the mass analyzer. Figure 9 is illustrating the operating principle of ESI.

Figure 9. Schematic set-up of an AP-ESI ion source.

Liquid flow
N2 flow

∼ 4 kV Counter electrode

Heated N2 curtain
Charged droplets
dispersion Extracting capillary
(Taylor cone) to mass
analyzer

Original droplet Vacuum

(+ ions predominate)
Reyleigh limit Solvent-
+ -
-+ + +- ion cluster
+ - -+ +- -+ -+- -- +
- +
+++-+ + -++-+
-++ - +
-+++ ++ - -+ +-+-+ + + +-
- -
Volume Ion extraction
Explosion
reduction

13
 ESI is the softest ionization technique available. It is especially suited for polar compounds,
even with a non-volatile character. Ions are ejected from the charged liquid droplets as [M+H]+ or
[M-H]- ions. Ionization yield is highly influenced by the solution chemistry of the analyte in the carrier
flow [71-73]. ESI is intolerant to unvolatile salts or buffers. It easily generates multiply charged ions,
allowing determination of compounds characterized by a high molecular weight.
Expanded analytical capabilities of ESI can be obtained via ion / ion reactions in dueling ion
sources. This leads to ES spectra simplification and in-depth studies of reaction mechanisms [74, 75].
Electrospray ionization is readily subjected to miniaturization. Nanospray and Electrospray
Emitter Arrays are increasing sample throughput, realizing at on-chip scale, sample preparation,
concentration and separation [76-78].
Dual APCI / AP-ESI ionization modes have been also experienced, leading to an extended
range for detecting compounds and analysis throughput [79, 80].

§ 6.2.1.4 Multiphoton ionization (MPI) [81]

The target molecules contained in a liquid carrier are nebulized by mixing with a nitrogen flow.
Solvent and analyte are then vaporized together in a quartz tube and are brought in the irradiation zone
as a homogenous gaseous phase. Direct photo ionization of the target molecules is statistically
unfavorable, resulting in lower ionization yields. Introduction in the nitrogen flow through nebulization
of a dopant can indirectly enhance on ionization. The dopant is first ionized and the resulting ions are
reacting with the analyte, by means of proton or electron transfer interactions.
The use of pulsed tunable dye lasers are strongly enhancing on ionization yields. Two stages
should be considered: 1. n photons coherently excite sample molecules to a real, intermediate electronic
state (a resonance enhanced process); 2. sequential irradiation of this intermediate with m photons
leading to final ionization.
By increasing the power density of the laser source it is possible to change from soft molecular
ion spectrum to ones dominated by fragmentation.
The schema of a MPI source is presented in Figure 10.

§ 6.2.2. Solid phases

§ 6.2.2.1 Matrix Assisted Laser Desorption Ionization (MALDI) [82-87]
The sample initially mixed with a liquid matrix (eg. sinapinic acid), chosen for its ability to
absorb and dissipate energy transferred from a laser beam. The molar ratio analyte / matrix is an
important operational parameter, readily varied by the analyst. Once prepared, the mixture is
14
transferred to a sample probe. Probe surface and geometry should also be considered as important for
the results of the analysis. After application, sample and matrix are converted to a condensed (solid)
phase by gas evaporation or vacuum drying, resulting in the co-crystallization of the sample with the
matrix. Local cluster and size variations occur across the sample deposition site. New instruments
incorporate a microscopic movement sample handler, allowing selection of the promising crystals for
excitation with the laser beam. The selection of “sweet spots” is sometimes assisted by a CCD (charge
coupled device) camera.

Figure 10. Construction of a MPI source.

Photon source
Irradiation zone
Dopant Extracting
Heated zone plates
flow
N2 flow
Gas phase To mass
analyzer
Liquid
flow Quartz tube

Heated N2 curtain

Pulsed laser fascicules generate a plume of ejected particles, including ions and neutral,
belonging to matrix and analyte, expanding at supersonic speed from the surface of the probe, generally
under right angles. UV lasers are usual (N2 laser at 337 nm or Nd-YAG). IR lasers (CO2 or Er-YAG)
are used only for specialized applications. A laser pulse takes from 1 to 10 ns and impacts an area of
about 0.01 mm2. The laser pulse is repeated over a precised interval (commonly 1 Hz frequency). This
parameter should be chosen in accordance with the type of mass analyzer which is used. Time of Flight
(TOF) and Fourier Transform – Ion Cyclotron Resonance (FT-ICR) mass analyzers are more often
coupled to the MALDI source.
MALDI is a soft ionization, tending to produce singly charged ions (resulting in very simple
spectra). Both (+) and (-) modes are known, although (+) MALDI is the most common. The (-) mode is
frequently used for nucleic acid analysis. In the default operational mode, MALDI induces no
fragmentation. Biochemical structural studies using MALDI will involve breakdown of the precursor
molecule prior to ionization, enzymically or chemically. Applications of MALDI are covering
identification of additives in food profiling and characterization of environmental macromolecules
15
(correlation of the analytical data with structure and environmental impact). The MALDI source is
presented in Figure 11.

Figure 11. The MALDI source.

Laser source
Sample
Matrix

Probe

Desolvation
+ To mass
analyzers
Desorption

Proton
y transfer
x
Probe H+
handler
Matrix / Sample CCD
co crystallization mass camera

Further improvements related to MALDI ionization mode are:

1. mixing the features of the atmospheric pressure interfaces with MALDI characteristics. The
resulting techniques are called AP – MALDI [88-90] or Laser Desorption (LD – APCI) [91-93].
Such techniques decouple desorption and ionization, eliminate the requirement that matrix
should facilitate desorption and ionization, simplify sample preparation and improve ionization
efficiencies.
2. matrix – free MALDI [94-96] requires deposition of the sample (without any matrix) on double
etched porous silicon wafers prepared from low resistivity n + Si material (0.001 ÷ 0.005 Ω x
cm). Such a technical solution simplifies sample preparation, minimizes low mass chemical
noise, and enhances analysis of low mass analytes. However, optical absorption, thermal
conductivity, pore size, and overall porosity are critical parameters for the silicon wafer surface.
16
 3. coupling liquid phase separations with MALDI – MS was achieved by electrically mediated
liquid deposition on a continuously moving plate. Application of negative voltage pulses (-2
kV) of 20 ms limitates the distribution of the column effluent to a wider area. Dihydroxy
benzoic acid and a-cyano-4-hydroxycinnamic acid were used as matrices [97]. Continuous
vacuum deposition interfaces are also available [98-100]. An excellent review of such interfaces
is made in [101].
4. imaging with MALDI of tissue sections, allowing map distribution of targeted compounds
[102-104].
5. development of affinity surfaces for MALDI probes in order to achieve selective or specific
concentration and clean-up of the analytes from complex matrices [105-106]. Such surfaces are
either chemically modified (hydrophobic, ionic, amphionic or metal ion bonded) or
biochemically modified (antibody, DNA, enzyme or receptor bonded).

§ 6.2.2.2 Fission Fragment Ionization or Plasma Desorption Ionization (PDI)

Sample molecules are deposited on a Nickel foil. Pulses of 252Cf fission fragments pass through
the probe. The thermal shock vaporizes mobile impurity ions (H+, Na+, and H-). These ions interact
with unvolatile sample molecules just after the thermal shock, converting them to ions. PDI can be
applied for large biomolecules (up to 50,000 Da). Deposition on nitrocellulose yield simple and
multiply charged ions as well, with practically no fragmentation. PDI is nowadays superseded by
MALDI.

§ 6.2.2.3 Field Deposition (FD)

Similar to FI (see § 6.1.2.), requires deposition of sample molecules in a condensed phase on
the high curvature surface subjected to intense electric fields.

§ 6.2.2.4 Secondary Ion Mass Spectrometry (SIMS)

A beam of ions (either monoatomic or polyatomic, positive or negative, obtained from inert
gases or from electropositive / electronegative elements) collides the sample deposited on a metal
surface. Secondary ions ejected from the surface are trapped and analyzed. The technique is suitable
also for inorganic samples as well as for organic molecules. In the latter case, protonated molecular
ions and sodium adducts are readily formed. Surface imaging (static SIMS) as well as 3D mapping
(Dynamic SIMS) is possible. The choice of the primary ions will determine the nature of secondary

17
ones, which are transferred to the mass analyzer. Kinetic and chemisorption theories are explaining
ejection of the different types of secondary ions.
§ 6.2.2.5 Inductively Coupled Plasma (ICP) [107-112]
Liquid or solid samples of organic or inorganic nature are dispersed in an Argon flow and
introduced in an inductively coupled plasma torch. Temperatures around 8000 oK are atomizing sample
and ionizing the resulting atoms. Ionization efficiency approaches 100 % for most elements of interest.
Ions are sampled through a skimmer directly to the mass analyzer after formation of a supersonic jet in
the differentially pumped region behind the plasma extraction cone.

§ 7. Mass analyzers
The mass analyzer is the component of mass spectrometer acting on the specific ions generated
within the source, to ordinate them according to the m/z values. The basic functioning principle of a
mass analyzer is the interaction of the target ions with external electrostatic and magnetic fields of
precised geometry.

§ 7.1 Sector mass analyzers [113-116]

Two types of sectors are used for construction of the mass analyzers: magnetic (B) and
electrostatic (E) sectors.
The electrostatic sector performs only an isokinetic arrangement of ions. When the ions are
introduced in a radial electric field, their pathways became stable only if the centrifugal force equalizes
the electric force:
m × v2 z × e × E
= (1)
rE d
where E is the potential applied between the curved electrodes, d is the distance between curved
electrodes, rE is the radius of the path followed by the ion in the electric field, v is the velocity of the
ion, m its mass, z its charge and e is the elementary unit of charge.
An ion introduced in a magnetic sector moves on a circular path with the radius rB, for which,
again, the centrifugal force should equalize the magnetic force.
m × v2
= z ×e× B×v (2)
rB
It is worthwhile to note that the magnetic field acts as a momentum (m x v) separator. As the
velocity of the ion is the same in both electrostatic and magnetic sectors, relations (1) and (2) should be
combined as following:
18
 z × e × E × rE
v= (3) from relation (1)
m×d
z × e × B × rB z × e × E × rE
= (4) introducing rel.(3) in rel.(2)
m m×d
It results that:
m e×d
= × B 2 × rB2 (5)
z E × rE
The tandem between electrostatic and magnetic sectors acts as a mass analyzer either by
scanning the magnetic field B or the electrostatic field E.
From relation (5) it seems that m/z is independent with respect to the initial acceleration
potential V, given to the ion during the transfer from the source to the mass analyzer. However, if the
velocity during the transfer is equal to the velocity in the electrostatic sector, an instrument defined
correlation is found between V and E:
E × rE
V= (6)
2×d
The former relationships are illustrated in Figure 12.

Figure 2. Pathways of a positive ion in electrostatic and magnetic sectors.

S B N
d
+ +
rB
E -
rE

+
V

Characteristics: resolution of 10,000 over a mass range up to 15,000 Da; increased resolution of
100,000 over a mass range up to 100,000 Da can be obtained with a cost in terms of sensitivity; scan

19
speeds are limited by the hysteresis and magnet heating; complex construction; needs deep vacuum
conditions (10 -10 ÷ 10-12 torr). The double focusing mass analyzer is used for high resolution
measurements (as example, the EPA method for dioxins imposes such a requirement) and fundamental
MS studies.

§ 7.2 Time of flight mass analyzers (ToF) [117-122]

ToF mass analyzers are based on the measurement of the individual flight times of ions having
identical kinetic energy through a specific path (field free drift tube) separating the ion source and the
detection area (see Figure 13A).

Figure 13. Basic functioning of a ToF mass analyzer.

V
L
Field free drift tube
Ion Detection
A Source + + + + +
Area

Acceleration
plate
V
Deflection
Electrode
Field free drift tube
Ion
B Source +
+
+

+
Acceleration L
plate +
+
Detection
Area

The kinetic energy of the ions produced within the source is established by means of the
acceleration potential V.

20
 m × v2
= z × e ×V (7)
2
The flight time is thus calculated according to the simple relationship:

L L × m1 / 2
t= = (8)
v 2 × z × e ×V
It’s clearly resulting that:
m 2 2 × e ×V
=t × (9)
z L2
For given settings of V and L, the time measurement means m/z discrimination. The specific
ToF resolution is enhanced by using the reflector design (see Figure 13B), allowing a better control
over the initial kinetic energy spread and the spatial distribution. Ion introduction in the flight zone
should be pulsed, through generation of discrete packets of ions. The flight time of the ions typically
falls in the 1 to 100 ns interval.
Characteristics: no instrumental parameters limit the upper margin of the m/z interval; high
resolution (up to 20,000) when using the reflector design; high sensitivity, especially at high ion
sampling frequencies; needs for high performance electronic controlling detection area; suitable for
applications requiring both high resolution and sensitivity for high Mw compounds; Hadamard
multiplexing technique is usable together with the ToF principle [123-125].

§ 7.3 The quadrupole mass analyzers (QMA) [126-130]

The functioning of the QMA is based on the interaction between ions and a hyperbolic field
generated within 4 rod shape electrodes arranged in a square array, interconnected two by two at
positive and negative potentials consisting in both constant and radio frequency modulated components
(see Figure 14). The potential applied to the pairs of electrodes is:
Φ 0 = ( − or + )(VDC + VRF cos(2 πνt )) (10)
The hyperbolic field generated within the electrodes should be written as following:
x2 − y 2
Φ = (VDC + VRF cos(2 πνt )) × (11)
r02
If VRF > VDC, low mass ions are lost on the Ox direction (collision with left / right electrodes
due to the fluctuation of the RF potential) and heavy ions are lost on the Oy direction (collision with
upper / lower electrodes due to their inertia toward the fast altering variable field). This combination of
both high and low pass filters yields to a stability window defined by ν, VDC, VRF and VDC/VRF ratio.
The stability window is find by solving the following Mathieu motion (on Ox, Oy and Oz directions)
21
differential equation system:
d 2x e
+ (VDC + VRF cos(2πνt ))x = 0
dt 2
m × r0
d2y e
− (VDC + VRF cos(2πνt )) y = 0 (12)
dt 2
m × r0
d 2z
=0
dt 2
The system is solved by changing to variables:
e × VDC
α= y (13)
m × π 2 × ν 2 × r02
e × VRF
q= x (14)
2 × m × π 2 × ν 2 × r02
The stability diagram is given in Figure 15.
Figure 14. The set-up of a quadrupole mass analyzer.
y
z

o x

-
+
+r o + o
+
z

+
- x
y

- z
o
+

-
-Φo +Φo

22
 Figure 15. Stability diagram for a quadrupole mass analyzer.

α (VDC, y)
unstability on the y unstability on
direction both x and y
directions

VDC/VRF=ct.
0.237 m
unstability on the x
m2 m1 direction
O Stability area
0.706
q (VRF, x)

ro

The mass interval for ions having motions on the Ox and Oy directions smaller than ro (the ions
stay within the rods) depends on the VDC/VRF ratio. Generally, VDC/VRF ratio is chosen such that 1 Da
window is selected over the entire mass range. To functioning modes are possible: a. ν is variable, VDC ,
VRF and VDC/VRF are constant; b. ν and VDC/VRF are constant, VDC and VRF are variable.
Characteristics: upper mass limit falls in the 3,000 – 4,000 Da interval; accepts relatively high
pressure regime (10 -4 – 10 -5 torr), resulting in simple vacuum generating systems; low costs; low
resolution; wide applications when coupled to GC and LC separation systems; suitable for structural
confirmation of low Mw molecules; needs serially coupling of three devices for allowing MS/MS
experiments.

§ 7.4 Ion trap mass analyzers or quistors (QIT) [131-139]

The fundamental working principles of an ion trap are the same as those described for the linear
quadrupole for the single reason that a quistor results from the imaginary bending of a linear
quadrupole to a closed loop. That imaginary operation transforms the left / right rod electrodes in
hyperbolic calottes, the upper electrode in a ring while the lower one is reduced to a mathematical point

23
(see Figure 16). The hyperbolic surface calottes (named also end-caps) are perforated to allow ion
extraction from the source and ion ejection to detection area. Constant potential is applied to end-caps.
Ions can be readily formed in the trap or captured from an external source as well.
Figure 16. The quadrupole ion trap (quistor).

Ring electrode
VRF x cos(ϖt)

Trapping ions Ion ejection

VRF low VRF high

Ion + Detection
Source Area

r0
Lissajou shaped
ion orbits

End-cap End-cap
electrode z electrode
VDC VDC
Damping gas
atoms (He)
A radio frequency modulated potential (VRF x cos(ϖt)) is applied to the inner ring electrode.
Within this three dimensional arrangement ions travel on Lissajou shaped paths. Motion equations
characterizing the linear quadrupole remain valid, except that x and y directions are replaced by r0 and
z. For low VRF amplitude, all ions covering a large mass interval are stored together within the ring
electrode, on stable orbits, characterized by precised radii and z expansions. Forcing an ion population
on stable orbits within the ring produces the risk of an uncontrolled increase of the paths radii, due to
electrostatic repulsions (collisions of ions with electrodes become possible). To avoid this, a damping
gas (usually He or H2) is feed at 10-3 torr pressure in the trap. Collisions between ions and damping gas
atoms reduce ion energies and force them to move closely around the center of the trap. This is also
improving resolution, because of the limitation of the spatial distribution (field imperfection is minimal
24
in the center of the trap). When all ions are stabilized within the ring, the amplitude VRF of the radio
frequency modulated potential starts to be scanned (see Figure 17).

Figure 17. Stability diagram for a quistor.

Measurement cycle

Potential
Ejection interval
(scaning VRF)
α (VDC, r0)

Trapping
interval
VRF

unstability on the r0 VDC

direction
Stability area
Time

+ + + +
+ + unstability on the z
O direction

q (VRF, z)

VRF1 VRF2 > VRF1

Increasing the VRF value forces the ions to be ejected from the trap to the detection area
(through the holes of the end cap electrode), starting with low masses. An ion trap holds a fixed
maximum number of ions and depending on the VRF scanning ramp, it can be filled and emptied
sequentially. Filling / emptying frequency determines the sensitivity of the instrument.
Characteristics: energy and spatial distributions of ions produced within the source are non
critical for the quistor; the use of low potentials allows a relatively high pressure and therefore,
inexpensive vacuum system is required; low costs; small dimensions; low resolution (typically 1 Da);
improved resolution is obtained for specific scanning profiles of VRF, with the proportional reduction of
the mass interval or sensitivity; inherent time controlled tandem MS capabilities; suitable for structural
confirmation; suitable for easy GC and LC interfacing.

25
§ 7.5 Ion Cyclotron Resonance mass analyzer (ICR) [140-148]
The construction of an ion cyclotron resonance mass analyzer is presented in Figure 18.

Figure 18. The Ion Cyclotron Resonance mass analyzer.

Receiver plate (up) Trapping plate (back)

Trapping plate (front)

Amplifier
Emitter plate (left) +
+ VDC
B

-V +
+
Ion beam
+ VDC Counter plate (right)
(from source)
Receiver plate (bottom)

B B B
+ +
+
+ + ++ +
+ + +
+

A strong magnetic field is used for curving the paths of the ions inside the ICR cavity. The
intensity of the magnetic field (B) is so high that ions will finally move on circular orbits within the
cavity. A small potential is applied to the trapping (front and back) plates (same sign as the ions) in
order to prevent lateral ionic loss. As for the magnetic sector, the following relation remains valid:
m×v
= z×e× B (15)
r
The angular velocity of the ion (ϖ) is given by the relation:
v
ϖ = = 2 × π× ν (16)
r
Transferring relation (16) in relation (15), it clearly results that:
26
 m e× B
= (17)
z ϖ

For an applied magnetic field of 5 T (Tesla) and a mass interval ranging from 15 to 1,500 Da,
radio frequencies of kHz to MHz result for the trapped ions.
When a radio frequency νi is applied to the emitter plate, ions having the angular velocity ϖi = 2
x π x ν i start to absorb the incoming energy (resonance phenomenon) and continuously increase their
orbit radius (from circular, the paths became spiral shaped). Once the trajectory of the resonant packet
of ions comes closer to the receiver plates, an “image current” is induced and consequently detected (if
the ions approach the top plate, electrons are attracted to this plate from ground; when the packet of
ions are situated in proximity of the bottom plate, electrons travel from the upper plate to the opposite
one, producing a detectable current). Scanning frequencies νi over a given interval results in the
observation of the signals for the corresponding m/z ions.
A faster method (FT-ICR) involves the excitation of ions by means of a fast sweep of
frequencies over a broad range. The receiver plates pick-up signals at different moments of time. The
signal at time ti corresponds to all ions reaching the resonant state by absorption of energy from the
frequency sweep applied at this specific moment. A Fourier transformation should be applied to the
“image current” variation in time, as the mathematical function is periodic. Functions resulting for each
individual cyclotron frequency are then converted to m/z values to produce the conventional mass
spectrum.
In FT-ICR, the longer the “image current” can be sampled, the higher is the resulting spectral
resolution. The most important reason for the “image current” decay rate is the collisional damping
affecting the coherent motion of the ions within the cell. Thus, the pressure inside the cell will strongly
affect spectral resolution (pressure around 10-5 torr induces milli seconds decay while pressure around
10-9 torr induces a tens of seconds decay). The unique feature of FT-ICR is that resolution and signal to
noise ratio increase as pressure reduces.
At high frequencies, low m/z detection is limited by the speed of the digitizers; at low
frequencies, high m/z detection is limited by the amplifiers and the low frequency noise. The dynamic
range is limited by the maximum number of ions held in the ICR cavity (a maximum of 106 ions is
allowed within the cell). Note that ion detection is made without loss of the respective ions.
Characteristics: unsurpassed resolution (around 50,000 for an m/z ∼ 10,000); high detection
efficiency (1 molecule detection); detection process is not destructive; m/z scale higher than 100 Da

27
(usually higher than 15,000); inherent tandem MS capabilities; high costs due to cooling systems of
superconducting magnets, high vacuum systems and demanding computer facilities; difficult to couple
to atmospheric pressure ion sources; suitable for extremely high resolution measurements, fundamental
ion chemistry and stability.

§ 8. Ion detection systems [149 - 150]

Ion current intensities resulting from the mass analyzer are ranging in an extremely wide
interval (10-9 ÷ 10 -18 A). Ion detection is achieved mainly by means of electron (photon) multipliers
(see Figure 19). The primary ions hit an electrode to generate secondary electrons. Secondary electrons
are thus multiplied through successive acceleration / impact processes between electrodes (dynodes
made from high emissive Beryllium – Copper alloys) having an increased positive potential (12 to 20
dynodes are usually coupled – see Figure 19 A). A different approach is the continuous channel
multiplier or channeltron (see Figure 19 C). The body of the channeltron is built up from a Lead doped
glass with high secondary emissive properties and electrical resistivity. A voltage applied between the
ends of the conical shaped curved cavity generates a field gradient on the inner walls. If the speed of
the incoming ions is less than 1,8 x 104 m/s [151], a lower detection sensitivity may be obtained due to
a poorer extraction capacity of the secondary electrons. A solution to this problem is to increase the
velocity of the ions ejected from the mass analyzer just before detection (see Figure 19 C).
In such a post acceleration set-up (PAD) [152,153] an electrode operated at high potential (up to 30
kV) is placed before the electron multiplier and strongly increase electrostatically the velocity of the
incident ions, with a corresponding gain in terms of secondary electron yield. Because photon
multipliers are much robust compared to electron multipliers, some manufacturers prefer to introduce a
phosphorescent screen between the post acceleration electrode and the multiplier it-self (see Figure
19B).
For magnetic sector instruments allowing spatial discrimination of ions according to their m/z,
simultaneous detection can be made using array detectors [154] (see Figure 19D). Micromachined
multipliers are used as primary receptors and are placed in the focal plane of the magnetic sector. The
amplified motion of secondary electrons is then focused on a phosphorescent screen. Emitted photons
are transmitted by means of optic fibers to a photodiode array producing the final response. The gain of
electron multipliers is usually higher than 106 and is produced with virtually no noise and very small
time constants.
Figure 19. Ion detection systems in mass spectrometry

28
 V2 > V1 V4 > V3 Vi-1 > Vi-2

electron (e-s)
Secondary
+
A
ion

Amplifier
+ V1 V3 > V2 Vi > Vi-1

Phosphorescent
Micromachined
screen
multipliers
+

Photodiode Array
ion
photon

+ Secondary
ion electron (e-s)
+

photon
ion

B + V1 optic
fibers

D
Increased Phosphorescent
V (+) potentials screen

+ V gradient
ion
Secondary
+ electron (e-s)

acceleration

(-) High
potential

Good secondary emission

properties and electrically Amplifier
C resistive material

29
§ 9. Multiple Sequential MS (Tandem MS) [155]
As described already in Figure 2, for some specific purposes, it may result a real need in
isolation of a specific ion (precursor or parent) and its further fragmentation followed by the analysis
of the resulting ionic fragments (product or daughter ions). Theoretically, this loop may be repeated
few times consecutively, resulting in (MS)n experiments.
The main objective of MS/MS hyphenation is to enhance on the selectivity characterizing
methods focused on quantitative aims or structural / stability studies for the target analytes.
According to IUPAC Compendium of Chemical Terminology, tandem mass spectrometry
represents an arrangement in which ions are subjected to two or more sequential stages of analysis
(which may be separated spatially or temporally) according to the quotient mass/charge.
MS hyphenation in space requires the use of two ore more mass analyzers coupled serially (one
for each ion discrimination process). MS hyphenation in time means the use of a single mass analyzer,
while selection of precursor / product ions is made successively, at different moments with respect to
the beginning of the process.
Once the precursor ion has been isolated, it is necessary to initiate its dissociation to produce
corresponding fragment ions (products). The following modes for achieving dissociation of precursor
ions are used in practice: 1. Collision Induced Dissociation (CID); 2. Surface Induced Dissociation
(SID); 3. Infrared Multiphoton Dissociation (IRMPD); 4. Blackbody Infrared Dissociation (BIRD); 5.
Sustained Off-Resonance Irradiation (SORI); 6. Electron Capture Dissociation (ECD); 7. Post Source
Decay (PSD). Modes 1-2 have wide spread applications, modes 3-6 are characteristic for trapping mass
analyzers while the last mode is dedicated to MALDI sources. The most common mode of dissociation
of precursor ions is undoubtedly CID. The process requires Helium atoms to collide precursor ions
resulting in their further fragmentation. As the energy control upon neutral species (He atoms) is
difficult, reproducible CID processes are obtained by means of the controlled acceleration of the
precursor ions.
As example for a tandem MS instrumentation (spatially developed) the triple quadrupole
(QQQ) set-up is further discussed (see Figure 20). The second quadrupole is used only for acceleration
of the precursor (parent) ion to achieve CID. Other arrangements designed for spatial tandem MS
experiments are: 1. Multiple magnetic sectors; 2. Quadrupole / Time of Flight (Q/TOF); 3. Time of
Flight / Time of Flight (TOF/TOF).
The typical illustration for “in time” MS hyphenation is the ion trap (quistor). As ions obtained
from the source are all trapped within the ring electrode, ejection of all ions excepting the parent ones
is realized without activating detection. Once the precursor ions are trapped, an increased constant
30
 Figure 20. Triple quadrupole arrangement for tandem MS.

Collision Induced Dissociation

(CID) He atoms
Ion Source
+ +
+ + + +
+ + +
+
+ + +

+ +

Precursor ions
+

Product ions

Multiplier
Electron
Acceleration Quadrupole 1 Quadrupole 2 Quadrupole 3
electrode

potential is applied to calotte electrodes to produce their acceleration. Collision induced dissociation
arises, while VRF is set again at lower values to trap all product (daughter) ions. Scanning VRF
amplitude results in ejection of the product (daughter) ions in the increased order of their m/z value,
this time with an activated detection system. This procedure can be repeated many times (commercially
available systems are allowing up to 13 repetitions), although more than MS4 experiments are not
commonly required. The ICR based instrumentation is also suitable to work under time delayed MS
hyphenation. It seems clear that MS hyphenation in time is less expensive with respect to spatially
developed tandem instruments, as the use of a single mass analyzer is required.
An overview on the advantages/disadvantages of hyphenated MS instruments is given in Table 2.
Table 2. Comparative overview on hyphenated MS instrumentation.
Type Advantages Disadvantages
QQQ Classical mass spectra; Low electric Low energy collisions; Incomplete
[156-159] fields required; Useful for mixture fragmentation; Limited to MS3; Not suitable
screening; Tolerant to atmospheric for pulsed ionization; Limited m/z range (up
ionization sources; Relatively small to 4000); Instrument dependent spectra;
size. Limited resolution (up to 2,000).
Q/TOF Simple system; High data acquisition High voltages required for acceleration; High
[160-162] rates; No m/z range limits; More vacuum (higher than 10-9 torr); Low energy
sensitive compared to QQQ; High collisions; Incomplete fragmentation.
resolution; High ion transmission;
Suited for pulsed ionization.

31
 Type Advantages Disadvantages
TOF/TOF Same as Q/TOF Extremely large size; Not suited for
[163] continuous ionization sources; High costs.
n n
MS QITs Compact; MS capabilities; Relatively Ion limited storage capacity within the trap;
[164,165] inexpensive; Data depending scanning; Low energy collisions; 1/3 of the low mass
Less sensitive compared to QQQ. range lost in the MS2 mode; Poor quantitation
reproducibility; Low resolution.
FTICR Highest resolution; MSn capabilities; Limited dynamic range; Low energy
MSn Ion chemistry experiments capability. collisions; High vacuum requirements;
[166-168] Difficulties to couple to LC; Large size;
Relatively low throughput.

§ 10. Mass spectrometry – working modes [169]

The possible ways of achieving MS experiments are illustrated in the following figure:

Figure 21. Mass spectrometric modes (for single and multiple MS stages).

SINGLE MS STAGE

Ion Source Mass Analyzer Detector Technique

Full Scan Mode

m1/z, m2/z,…, mn/z
Qualitative and
mi/z, i = 1,…,n transmitted and R.A. = f(m/z)
quantitative purposes
arranged
(less sensitive)
Selected Ion
Monitoring (SIM)
mj/z: transmitted
mi/z, i = 1,…,n R.A. = f(mj/z) Quantitative analysis
mi/z, i ≠ j: blocked (sensitivity gain
∼1,000)
Multiple Ion Detection
R.A. = f(mj/z, mq/z,
mj/z; mq/z; mk/z: (MID)
mk/z);
Quantitative analysis
mi/z, i = 1,…,n transmitted R.A.(mj)/RA(mq)
(with increased
mi/z, i ≠ j, q, k: blocked R.A.(mj)/RA(mk)
sensitivity) + structural
R.A.(mq)/RA(mk)
confirmation

32
 MULTIPLE MS STAGES

Mass Mass
Ion
Analyzer CID Analyzer Detector Technique
Source
1 2

mj/z: mj/z m’1/z,

mi/z, transmitted fragmented to m’2/z,…, m’n/z Product Ion
R.A. = f(m’i/z)
i = 1,…,n mi/z, i ≠ j: m’i/z, transmitted and Scan
blocked i = 1,…,n arranged
m1/z, m2/z,…,
m1/z, m2/z,…, mn/z m’j/z:
mi/z, mn/z fragmented to transmitted Precursor Ion
R.A. = f(m’j/z)
i = 1,…,n transmitted and m’i/z, m’i/z, i ≠ j: Scan
arranged i = 1,…,n blocked

m1/z, m2/z,…,
m1/z, m2/z,…, mn/z m’1/z,
Constant
mi/z, mn/z fragmented to m’2/z,…, m’n/z
∆ m = ct. neutral
i = 1,…,n transmitted and m’i/z, transmitted and
loss/gain scan
arranged i = 1,…,n arranged

mj/z: mj/z m’j/z: Single

mi/z, transmitted fragmented to transmitted Reaction
R.A. = f(m’j/z)
i = 1,…,n mi/z, i ≠ j: m’i/z, m’i/z, i ≠ j: Monitoring
blocked i = 1,…,n blocked (SRM)
R.A. = f(m’j/z,
m’q/z, m’k/z);
m’j/z; m’q/z;
mj/z: mj/z R.A.(m’j) / Multiple
m’k/z:
mi/z, transmitted fragmented to RA(m’q); Reaction
transmitted
i = 1,…,n mi/z, i ≠ j: m’i/z, R.A.(m’j) / Monitoring
mi/z, i ≠ j, q, k:
blocked i = 1,…,n RA(m’k); (MRM)
blocked
R.A.(m’q) /
RA(m’k);

§ 11. References.
1. P. Price, J. Am. Soc. Mass Spectrom., 2(4), 336 (1991).
2. K. L. Busch, Spectroscopy, 16(11), 28 (2001).
3. K.L. Busch, Mass Spectrometry, 17(6S), 26 (2002).
4. J.F.J. Todd, Pure and Applied Chemistry, 63, 1541 (1991).
5. J.J. Thompson, Phil. Mag., 547 (1899).
6. J.J. Thompson, Phil. Mag., 6(20), 752 (1911).
7. A.J. Dempster, Phys. Rev., 11, 316 (1918).

33
8. F.W. Aston, Phyl. Mag., 39, 611 (1920).
9. A.O. Nier, Nat. Bur. Stand. Circ. (US), 522, 29(1953).
10. W. Stephens, Phys. Rev., 69, 694 (1946).
11. E.G. Johnson, A.O. Nier, Phys. Rev., 91, 10 (1953).
12. J.H. Beynon, Mikrochim. Acta, 437 (1956).
13. M.S.B. Munson, F.H. Field, J. Am. Chem. Soc., 88(12), 2621 (1966).
14. M. Dole, L.L. Mack, R.L. Hines, R.C. Mobley, L.D. Fergusson, M.B. Alice, J. Chem. Phys.,
49(5), 2240 (1968).
15. H.D. Beckey, Res. / Develpoment, 20(11), 26 (1969).
16. M.B. Comisarow, A.G. Marshall, Chem. Phys. Lett., 25(2), 282 (1974).
17. R.D. Macfarlane, D.F. Torgerson, Science, 191(4230), 920 (1976).
18. M. Barber, R.S. Bordoli, R.D. Sedgwick, A.N. Tyler, Nature, 293, 270 (1981).
19. J.B. Fenn, M. Mann, C.K. Meng, S.F. Wong, C.M. Whitehouse, Science, 246, 64 (1989).
20. K. Hiraoka, H. Fukasawa, F. Matsushita, K. Aizawa, Rapid Commun. Mass Spectrom., 9,
1349 (1995).
21. G. Siuzdak, B. Bothner, M. Yager, G. Brugidon, C.M. Fauquet, K. Hoey, C.M. Chang,
Chemistry & Biology, 3, 45 (1996).
22. J. Wei, J. Buriak, G. Siuzdak, Nature, 399(6733), 243 (1999).
23. J.T. Watson, Introduction to Mass Spectrometry, 3 rd Edition, Lippincott Raven Press, New
York (1997).
24. R.A.W. Johnstone, M.E. Rose, Mass Spectrometry for Chemists and Biochemists, 2nd Edition,
Cambridge University Press, New York (1996).
25. E. de Hoffman, V. Stroobant, Mass Spectrometry: Principles and Applications, 2nd Edition, J.
Wiley & Sons, Chichester (2002).
26. Analytical Mass Spectrometry Strategies for Environmental and Related Applications, W.L.
Budde Editor, American Chemical Society, Oxford University Press Inc., New York (2001).
27. Advances in Mass Spectrometry, E. Gelpi Editor, J. Wiley & Sons, Chichester (2001).
28. Mass Spectrometry: Analytical Chemistry by Open Learning, 2nd Edition, J. Barker, J.A.
David Editors, J. Wiley & Sons, Chichester (1999).
29. J.R. Chapman, Practical Organic Mass Spectrometry: A Guide for Chemical and Biochemical
Analysis, 2nd Edition, J. Wiley & Sons, Chichester (1995).
30. C. Herbert, R. Johnstone, Mass Spectrometry Basics, CRC Press, Boca Raton, Florida (2002).
31. R.M. Smith, Understanding Mass Spectra: A Basic Approach, 2nd Edition, Wiley -
34
 Interscience (1998).
32. R.A. Hites, Handbook of Mass Spectra of Environmental Contaminants, Lewis Publisher,
Boca Raton, Florida (1992).
33. F.W. McLafferty, F. Turecek, Interpretation of Mass Spectra, 4th Edition, University Science
Books, Sansalito, California (1996).
34. A.J. Dempster, Phys. Rev., 18, 415 (1921).
35. A.O. Nier, Rev. Sci. Instrum., 18, 398 (1947).
36. W.H. McFadden, Techniques of Combined Gas Chromatography / Mass Spectrometry:
Applications in Organic Analysis, Wiley – Interscience, New York (1973).
37. A. Capiello, G. Famiglini, F. Mangani, P. Palma, Mass Spectrometry Review, 20, 88 (2001).
38. T.O. Gronneberg, Chem. Scr., 13, 56 (1979).
39. E. Busker, H. Budzikiewicz, Org. Mass Spectrom., 14, 222 (1979).
40. S. Sass, T.L. Fischer, Org. Mass Spectrom., 14, 257 (1979).
41. E.J. Cone, C.W. Gorodesky, S.Y. Yeh, W.D. Darwin, W.F. Buchwald, J. Chromatogr., 230,
571 (1982).
42. A.C. Schoots, F.E.P Mikkers, C.A. Cramers, S. Ringoir, J. Chromatogr., 164, 1 (1979).
43. F.J. Evans, M.G. Lee, D.E. Games, Biomed. Mass Spectrom., 6, 374 (1979).
44. J. Vine, L. Brown, J. Boutagy, R. Thomas, D. Nelson, Biomed. Mass Spectrom., 6, 415 (1979).
45. S. Suzuki, Y. Hori, R.C. Das, O. Koga, Bull. Chem. Soc. Jpn., 53, 1451 (1980).
46. H. Budzikiewicz, E. Busker, Tetrahedron, 36, 225 (1980).
47. W.J.A. Vandenheuvel, J. Organomet. Chem., 190, 73 (1980).
48. R. Chai, A.G. Harrison, Anal. Chem., 53, 34 (1981).
49. M. Suzuki, T. Ariga, M. Sekine, E. Araki, T. Miyatake, Anal. Chem., 53, 985 (1981).
50. M. Suzuki, K. Harada, N. Takeda, A. Tatematsu, Heterocycles, 15, 1123 (1981).
51. A.G. Harrison, F.I. Onuska, C.W. Tsang, Anal. Chem., 53, 1183 (1981).
52. R.W. Giese, J. Chromatogr. A, 892, 329-346 (2000).
53. T.E. Felter, J. Vac. Sci. Technol. B, 17(5), 9/10 (1999).
54. S.E. Huq, B.J. Kent, R. Stevens, R.A. Lawes, N.S. Xu, J.C. She, J. Vac. Sci. Technol. B, 19(3),
5/6 (2001).
55. O. Kornienko, P.T.A. Reilly, W.B. Whitten, J.M. Ramsey, Anal. Chem., 72, 559-562 (2000).
56. Y. Ito, T. Takeuchi, D. Ishi, M. Goto, J. Chromatogr., 346, 161 (1985).
57. Continuous Flow Fast Atom Bombardment Mass Spectrometry, R.M. Caprioli Ed., John Wiley
& Sons, Chichester (1990).
35
58. T. Mizuno, K. Marsuura, T. Kobayoshi, K. Otsuka, D. Ishi, Anal. Sci., 4, 569 (1988).
59. J.S.M. de Witt, L.J. Deterding , M.A. Moseley, K.B. Tomer, J.W. Jorgenson, Rapid Commun.
Mass Spectrom., 2, 100 (1988).
60. M. Barber, R.S. Bordoli, G.J. Elliott, R.D. Sedgwick, A.N. Tyler, Anal. Chem., 54, 645A
(1982).
61. S.A. Martin, C. Costello, K. Biemann, Anal. Chem., 54, 2362 (1982).
62. H. Kambara, Org. Mass Spectrom., 17, 29 (1982).
63. D.A. Lane, B.A. Thomson, A.M. Lovett, N. M. Reid, Advances in Mass Spectrometry, Vol.
8B, A. Quayle Ed., Heyden & Sons, London (1980).
64. M. Sakairi, H. Kambara, Anal. Chem., 60, 774 (1988).
65. M. Sakairi, H. Kambara, Anal. Chem., 61, 1159 (1989).
66. M. Sakairi, A.L. Yergey, Rapid Commun. Mass Spectrom., 5, 354 (1991).
67. T.R. Corey, A.P. Bruins, J.D. Henion, Org. Mass Spectrom., 23, 178 (1988).
68. J.D. Henion, E. Lee, Pract. Spectrosc. (Mass Spectrom. Biol. Mater.), 8, 469 (1990).
69. J.B. Fenn, M. Mann, C.K. Meng, S.F. Wong, C.M. Whitehouse, Mass Spectrom. Rev., 9, 37
(1990).
70. A.P. Bruins, T.R. Covey, J.D. Henion, Anal. Chem., 59, 2642 (1987).
71. V. Kertesz, G.J. Van Berkel, J. Mass Spectrom., 36, 204 (2001).
72. G,J, Van Berkel, V. Kertesz, J. Mass Spectrom., 36, 1125 (2001).
73. G,J. Van Berkel, K.G. Asano, V. Kertesz, Anal. Chem., 74, 5047 (2002).
74. D.D. Ebeling, M.S. Westphall, M. Scalf, L.M. Smith, Anal. Chem., 72, 5158 (2000).
75. J.M. Wells, P.A. Chrisman, S.A. McLuckey, J. Am. Soc. Mass Spectrom., 13, 614 (2002).
76. K. Tang, Y. Lin, D.W. Matson, T. Kim, R.D. Smith, Anal. Chem., 73, 1658 (2001).
77. H. Liu, C. Felten, Q. Xue, B, Zhang, P. Jedrzejewski, B.L. Karger, F. Foret, Anal. Chem., 72,
3303 (2000).
78. G.A. Schultz, T.N. Corso, S.J. Prosser, S. Zhang, Anal. Chem., 72, 4058 (2000).
79. W.C. Byrdwell, W.E. Neff, Rapid. Commun. Mass Spectrom., 16, 300 (2002).
80. M.M. Siegel, K. Tabei, F. Lambert, L. Candela, B. Zoltan, J. Am. Soc. Mass Spectrom., 9,
1196 (1998).
81. D.B. Robb, T.R. Covey, A.P. Bruins, Anal. Chem., 72, 3653 (2000).
82. M. Karas, F. Hillenkamp, Anal. Chem., 60, 2299 (1988).
83. F. Hillenkamp, M. Karas. R.C. Beavis, B.T. Chait, Anal. Chem., 69, 4615 (1997).
84. C. Fenselau, Anal. Chem., 69, 661A (1997).
36
85. E. Nordhoff, Trends Anal. Chem., 15, 240 (1990).
86. A.I. Gusev, W.R. Wilkinson, A. Proctor, D.M. Hercules, Anal. Chem., 67, 1034 (1995).
87. W. Ens, Y. Mao, F. Mayer, K.G. Standing, Rapid Commun. Mass Spectrom., 5, 117 (1991).
88. V.V. Laiko, M.A. Baldwin, A.L. Builingame, Anal. Chem., 72, 652 (2000).
89. M.C. Galicia, A. Vertes, J.H. Callahan, Anal. Chem., 74, 1891 (2002).
90. V.V. Laiko, N.I. Taranenko, V.D. Berkout, B.D. Musselman, V.M. Doroshenko, Rapid
Commun. Mass Spectrom., 16, 1737 (2002).
91. J.J. Coon, K.J. McHale, W.W. Harrison, Rapid Commun. Mass Spectrom., 16, 681 (2002).
92. J.J. Coon, H.A. Steele, P.J. Laipis, W.W. Harrison, J. Mass Spectrom., 37, 1163 (2002).
93. J.J. Coon, W.W. Harrison, Anal. Chem., 74, 5600 (2002).
94. Z. Shen, J.J. Thomas, C. Averbuj, K.M. Broo, M. Engelhard, J.E. Crowell, M.G. Finn, G.
Siuzdak, Anal. Chem., 73, 612 (2001).
95. J.D. Cuiffi, D.J. Hayes, S.J. Fonash, K.N. Brown, A.D. Jones, Anal. Chem., 73, 1292 (2001).
96. R.A. Kruse, X. Li, P.W. Bohn, J,V. Sweedler, Anal. Chem., 73, 3693 (2001).
97. C. Ericson, Q.T. Phung, D.M. Horn, E.C. Peters, J.R. Fitchett, S.B. Ficarro, A.R. Solomon,
L.M. Brill, A. Brock, Anal. Chem., 75, 2309 (2003).
98. J. Preisler, F. Foret, B.L. Karger, Anal. Chem., 70, 5278 (1998).
99. J. Preisler, P. Hu, T. Rejtar, B.L. Karger, E. Moskovets, B.L. Karger, Anal. Chem., 74, 17
(2002).
100. T. Rejtar, P. Hu, P. Juhasz, J.M. Cambell, M.L. Vestal, J. Preisler, B.L. Karger, J. Proteome.
Res., 1, 171 (2002).
101. T. Wehr, LC-GC North America, 21(10), 974 (2003).
102. P.J. Todd, T.G. Schaaff, P. Chaurand, R.M. Caprioli, J. Mass Spectrom., 36, 355 (2001).
103. P. Chaurand, S.A. Schwartz, R.M. Caprioli, Curr. Opinion in Chem. Biol., 6, 676 (2002).
104. M. Stoeckli, P. Chaurand, D.E. Kallahan, R.M. Caprioli, Nat. Med., 7, 493 (2001).
105. R.W. Nelson, J.R Krone, J. Molec. Recognit., 12, 77 (1999).
106. M. Merchant, S.R. Weinberger, Electrophoresis, 21, 1164 (2000).
107. J.M. Carey, F.A. Byrdy, J.A. Caruso, J. Chromatogr. Sci., 31, 330 (1993).
108. N. Bradshow, E.F.H. Hall, N.E. Sanderson, J. Anal. At. Spectrom., 4, 801 (1989).
109. A.J. Walder, P.A. Freedman, J. Anal. At. Spectrom., 7, 571 (1992).
110. U. Gießmann, U. Greb, Fresenius’ J. Anal. Chem., 350, 186 (1994).
111. G.C. Eiden, C.J. Barinaga, D.W. Koppennaal, Rapid Commun. Mass Spectrom., 11, 37 (1997).
112. S.D. Tanner, V.I. Baramov, J. Am. Soc. Mass Spectrom., 10, 1083 (1999).
37
113. M.E. Hail, R.A. Yost, Anal. Chem., 61, 2402 (1989).
114. M.L. Trehy, R.A. Yost, J.G. Dorsey, Anal. Chem., 58, 14 (1986).
115. M.E. Hail, R.A. Yost, Anal. Chem., 61, 2410 (1989).
116. D.V. Bowen, F.S. Pullen, Rapid Commun. Mass Spectrom., 3, 67 (1989).
117. W. Stephens, Phys. Rev., 69, 691 (1946).
118. W. Stephens, Bull. Am. Phys. Soc., 21(2), 22 (1946).
119. W.C. Wiley, I.H. McLaren, Rev. Sci. Instrum., 16(12), 1150 (1955).
120. M. Guilhaus, J. Mass Spectrom., 30, 1519 (1995)
121. R.J. Cotter, Anal. Chem., 64, 1027A (1992).
122. R,J. Cotter, Biomed. Environ. Mass Spectrom., 4, 293 (1989).
123. A.G. Marshall, Fourier, Hadamard and Hilbert transforms in chemistry, Plenum Press, New
York (1982).
124. A. Brock, N. Rodriguez, R.N. Zare, Anal. Chem., 70, 3735 (1998).
125. A. Brock, N. Rodriguez, R.N. Zare, Rev. Sci. Instrum., 71, 1306 (2000).
126. W. Paul, H. Steinwedel, Naturforsch, 8A, 448 (1953).
127. W. Paul, M. Reather, Z. Physik., 140, 262 (1955).
128. J.E. Campana, Int. J. Mass Spec. Ion Phys., 33, 101 (1980).
129. P.H. Dawson, Mass Spectrom. Rev., 5(1), 1 (1986).
130. P.H. Dawson, Quadrupole Mass Spectrometry and its Applications, Elsevier, New York
(1976).
131. J.F Todd, Mass Spectrom. Rev., 10, 3 (1991).
132. R.E. Marsh, Quadrupole storage Mass Spectrometry, vol. 102, John Wiley & Sons, New York
(1989).
133. R.G. Cooks, Chem. Eng. News, 69(12), 26 (1991).
134. K.R. Jonscher, Anal. Biochem., 224(1), 1 (1997).
135. J.N. Louris, Anal. Chem., 59(13), 1677 (1987).
136. J.N. Louris, Int. Mass Spectrom. Ion Processes, 96(2), 117 (1990).
137. R.G. Cooks, Int. Mass Spectrom. Ion Processes, 118-119, 1 (1992).
138. S.A. McLuckey, Int. J. Mass Spectrom. Ion Processes, 106, 213 (1993).
139. L.C.M. Ngoka, M.L. Gross, Int. J. Mass Spectrom., 194, 247 (2000).
140. H. Sommer, H.A. Thomas, J.A. Hippl, Phys. Rev., 78, 332 (1950).
141. A.T. Lehman, M.M. Bursey, Ion Cyclotron Resonance Spectrometry, John Wiley & Sons,
New York (1976).
38
142. FT-ICR MS: Analytical Applications of Fourier Transform Ion Cyclotron Resonance Mass
Spectrometry, B. Asamoto Ed., VCH, Weiheim (1991).
143. E.R. Williams, K.D. Henry, F.W. McLafferty, J. Am. Chem. Soc., 112, 6162 (1990).
144. R.L. White, E.B. Ledford, S. Ghaderi, C.L. Wilkins, ML. Gross, Anal. Chem., 52, 1525
(1980).
145. A.G. Marshall, C.L. Hendrickson, G.S. Jackson, Mass Spectrom. Rev., 17(1), 1 (1998).
146. R.D. Smith, Nature, 369, 137 (1994).
147. L. Tang, Rapid Commun. Mass Spectrom., 9, 731 (1995).
148. K.D. Henry, J.P. Quin, F.W. McLafferty, J. Am. Chem. Soc., 113, 5447 (1991).
149. S. Evans, Methods in Enzymology, Vol. 193: Mass Spectrometry, J.A. McCloskey, Chapter 3,
Academic Press, San Diego (1990).
150. J.S. Allen, Rev. Sci. Instrum., 18, 739 (1947).
151. E.A. Kurz, American Laboratory, March (1979).
152. G.H. Wang, W. Aberth, A.M. Falick, Int. J. Mass Spectrom. Ion Processes, 69, 233 (1986).
153. H. Hedin, P. Hakansson, Int. J. Mass Spectrom. Ion Processes, 69, 233 (1986).
154. J.S. Cottrell, S. Evans, Anal. Chem., 59, 1990 (1987).
155. Tandem Mass Spectrometry, F.W. McLafferty Ed., John Wiley & Sons, New York (1983).
156. R.A. Yost, C.G. Enke, J.Am.Chem. Soc., 100, 2274 (1978).
157. D. Arnott, Proteome Research: Mass Spectrometry, P. James Ed., Springer-Verlag, Germany
(2001).
158. P.E. Miller, M.B. Denton, J. Chem. Educ., 7, 617 (1986).
159. I. Yang, Rapid Commun. Mass Spectrom., 16(21), 2060 (2002).
160. G.L. Glish, D.E. Goeringer, Anal. Chem., 56, 2291 (1984).
161. K.F. Medzihradszky, Anal. Chem., 72, 552 (2000).
162. A. Wattenberg, J. Am. Soc. Mass Spectrom., 13(7), 772 (2002)
163. E.P. Go, Anal. Chem., 75, 2504 (2003).
164. R.E. Marsh, Quadrupole Storage Mass Spectrometry, vol. 102, John Wiley & Sons, New York
(1989).
165. L.C.M. Ngoka, M.L. Gross, Int. J. Mass Spectrom., 194, 247 (2000).
166. Fourier Transform MS, M.V. Buchanan Ed., ACS, Washington DC (1987).
167. F.W. McLafferty, Acc. Chem. Res., 27, 379 (1994).
168. E.R. Williams, Anal. Chem., 70, 179A (1998).
169. F. Lemiere, Guide to LC-MS, Dec. 2001, LC-GC, 4 (2001).
39