Primer

Basics of LC/MS
Contents
Why Liquid Chromatography/Mass Spectrometry? 4
Instrumentation 6
Ion Sources 6
Electrospray ionization 7
Atmospheric pressure chemical ionization 8
Atmospheric pressure photoionization 9
Mass Analyzers 10
Quadrupole 10
Time-of-flight 11
Ion trap 11
Fourier transform-ion cyclotron resonance (FT-ICR) 12
Collision-Induced Dissociation and Multiple-Stage MS 13
CID in single-stage MS 14
CID and multiple-stage MS 14
Adapting LC Methods 16
Sample preparation 17
Ionization chemistry 17
Capillary LC/MS and CE/MS 19
Applications 20
Molecular Weight Determination 20
Differentiation of similar octapeptides 20
Determining the molecular weight of green fluorescent protein 21
Structural Determination 22
Structural determination of ginsenosides using MS
n
analysis 22
Pharmaceutical Applications 24
Rapid chromatography of benzodiazepines 24
Detection of degradation products for salbutamol 24
Identification of bile acid metabolites 25
Biochemical Applications 26
Rapid protein identification using capillary LC/MS/MS and database searching 26
Clinical Applications 28
High-sensitivity detection of trimipramine and thioridazine 28
Food Applications 28
Identification of aflatoxins in food 28
Determination of vitamin D
3
in poultry feed supplements using MS
3
30
Environmental Applications 32
Detection of phenylurea herbicides 32
Detection of low levels of carbaryl in food 32
CE/MS Applications 34
Analysis of peptides using CE/MS/MS 34
4
Why Liquid Chromatography/
Mass Spectrometry?
Liquid chromatography is a fundamental
separation technique in the life sciences
and related fields of chemistry. Unlike gas
chromatography, which is unsuitable for
nonvolatile and thermally fragile molecules,
liquid chromatography can safely
separate a very wide range
of organic compounds,
from small-molecule drug
metabolites to peptides
and proteins.
Traditional detectors for
liquid chromatography
include refractive index,
electrochemical, fluores-
cence, and ultraviolet-visible
(UV-Vis) detectors. Some
of these generate two-
dimensional data; that is,
data representing signal
strength as a function of
time. Others, including
fluorescence and diode-
array UV-Vis detectors,
generate three-dimensional
data. Three-dimensional
data include not only signal
strength but spectral data
for each point in time.
Mass spectrometers also generate three-
dimensional data. In addition to signal
strength, they generate mass spectral
data that can provide valuable information
about the molecular weight, structure,
identity, quantity, and purity of a sample.
Mass spectral data add specificity that
increases confidence in the results of both
qualitative and quantitative analyses.
CH
2
CH
2
CH
2
NHCOCH
3
CH
2
CH
2
CH
2
OH
CH
2
CH
2
CH(NH
2
)COOH
R
%

R
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.

A
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%

R
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A
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m/z
Figure 1. Two-dimensional abundance data and three-dimensional
mass spectral data from a mass spectrometer
5
Some mass spectrometers have the ability to
perform multiple steps of mass spectrometry
on a single sample. They can generate a
mass spectrum, select a specific ion from
that spectrum, fragment the ion, and generate
another mass spectrum; repeating the entire
cycle many times. Such mass spectrometers
can literally deconstruct a complex molecule
piece by piece until its structure is determined.
For most compounds, a mass spectrometer is
more sensitive and far more specific than all
other LC detectors. It can analyze compounds
that lack a suitable chromophore. It can also
identify components in unresolved chromato-
graphic peaks, reducing the need for perfect
chromatography.
Mass spectral data complements data from
other LC detectors. While two compounds
may have similar UV spectra or similar mass
spectra, it is uncommon for them to have both.
The two orthogonal sets of data can be used
to confidently identify, confirm, and quantify
compounds.
400000
800000
1200000
MS TIC
2000
4000
m/z
648
min.
5 10 15 20 25 30 35 40
100
200
300
m/z
1325
4000
8000
12000
m/z
376
Figure 2. Identification of three components in a chromatographically unresolved peak
6
Instrumentation
Mass spectrometers work by ionizing mole-
cules and then sorting and identifying the ions
according to their mass-to-charge (m/z) ratios.
Two key components in this process are the
ion source, which generates the ions, and the
mass analyzer, which sorts the ions. Several
different types of ion sources are commonly
used for LC/MS. Each is suitable for different
classes of compounds. Several different types
of mass analyzers are also used. Each has
advantages and disadvantages depending on
the type of information needed.
Ion Sources
Much of the advancement in LC/MS over the
last ten years has been in the development
of ion sources and techniques that ionize the
analyte molecules and separate the resulting
ions from the mobile phase.
Earlier LC/MS systems used interfaces that
either did not separate the mobile phase
molecules from the analyte molecules (direct
liquid inlet, thermospray) or did so before ion-
ization (particle beam). The analyte molecules
were then ionized in the mass spectrometer
under vacuum, often by traditional electron
ionization. These approaches were successful
only for a very limited number of compounds.
The introduction of atmospheric pressure
ionization (API) techniques greatly expanded
the number of compounds that can be suc-
cessfully analyzed by LC/MS. In atmospheric
pressure ionization, the analyte molecules
are ionized first, at atmospheric pressure.
The analyte ions are then mechanically
and electrostatically separated from neutral
molecules. Common atmospheric pressure
ionization techniques are:
Electrospray ionization (ESI)
Atmospheric pressure chemical ionization
(APCI)
Atmospheric pressure photoionization (APPI)
M
o
l
e
c
u
l
a
r

W
e
i
g
h
t
Polarity
1000
100,000
10,000
Nonpolar Very Polar
100
10
Electrospray Ionization
APCI
APPI
Figure 3. Applications of various
LC/MS ionization techniques
7
Electrospray ionization
Electrospray relies in part on chemistry to gen-
erate analyte ions in solution before the analyte
reaches the mass spectrometer. The LC eluent
is sprayed (nebulized) into a chamber at atmos-
pheric pressure in the presence of a strong
electrostatic field and heated drying gas.
The electrostatic field causes further
dissociation of the analyte molecules.
The heated drying gas causes the solvent
in the droplets to evaporate. As the droplets
shrink, the charge concentration in the
droplets increases. Eventually, the repulsive
force between ions with like charges exceeds
the cohesive forces and ions are ejected
(desorbed) into the gas phase. These ions
are attracted to and pass through a capillary
sampling orifice into the mass analyzer.
Some gas-phase reactions, mostly proton
transfer and charge exchange, can also
occur between the time ions are ejected
from the droplets and the time they reach
the mass analyzer.
Electrospray is especially useful for analyzing
large biomolecules such as proteins, peptides,
and oligonucleotides,
but can also analyze
smaller molecules
like benzodiaze-
pines and sulfated
conjugates.
Large molecules
often acquire more
than one charge.
Thanks to this
multiple charging,
electrospray can
be used to analyze
molecules as large
as 150,000 u even
though the mass range (or more accurately
mass-to-charge range) for a typical LC/MS
instruments is around 3000 m/z. For example:
100,000 u / 10 z = 1,000 m/z
When a large molecule acquires many charges,
a mathematical process called deconvolution
is often used to determine the actual molecular
weight of the analyte.
Analyte ion ejected Evaporation
+
+
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+ + +
- -
-
- -
-
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+
Dielectric capillary entrance
Nebulizer gas
Solvent spray
Ions
+ + + + + + + +
+
+
+
+
+
+
+
Heated nitrogen drying gas
+ +
Figure 4. Electrospray ion source
Figure 5. Desorption of ions
from solution
8
Atmospheric pressure
chemical ionization
In APCI, the LC eluent is sprayed through
a heated (typically 250C 400C) vaporizer
at atmospheric pressure. The heat vaporizes
the liquid. The resulting gas-phase solvent
molecules are ionized by electrons discharged
from a corona needle. The solvent ions then
transfer charge to the analyte molecules
through chemical reactions (chemical ioniza-
tion). The analyte ions pass through a capillary
sampling orifice into the mass analyzer.
APCI is applicable to a wide range of polar
and nonpolar molecules. It rarely results in
multiple charging so it is typically used for
molecules less than 1,500 u. Due to this,
and because it involves high temperatures,
APCI is less well-suited than electrospray
for analysis of large biomolecules that may
be thermally unstable. APCI is used with
normal-phase chromatography more often
than electrospray is because the analytes are
usually nonpolar.
+ + + + + + + + +
Drying gas
Capillary
Corona
discharge
needle
Vaporizer (heater)
Nebulizer (sprayer)
HPLC inlet
Nebulizing gas
+
+
+
+
+
+
Figure 6. APCI ion source
9
APPI is applicable to many of the same
compounds that are typically analyzed by
APCI. It shows particular promise in two
applications, highly nonpolar compounds
and low flow rates (<100 l/min), where APCI
sensitivity is sometimes reduced.
In all cases, the nature of the analyte(s)
and the separation conditions have a strong
influence on which ionization technique:
electrospray, APCI, or APPI, will generate
the best results. The most effective technique
is not always easy to predict.
Atmospheric pressure photoionization
Atmospheric pressure photoionization (APPI)
for LC/MS is a relatively new technique. As
in APCI, a vaporizer converts the LC eluent to
the gas phase. A discharge lamp generates
photons in a narrow range of ionization
energies. The range of energies is carefully
chosen to ionize as many analyte molecules
as possible while minimizing the ionization
of solvent molecules. The resulting ions pass
through a capillary sampling orifice into the
mass analyzer.
+ + + + + + + + +
+
+
+
Drying gas
Capillary
UV lamp
Vaporizer (heater)
Nebulizer (sprayer)
HPLC inlet
Nebulizing gas
+
hv
+
+
Figure 7. APPI ion source
10
Mass Analyzers
Although in theory any type of mass analyzer
could be used for LC/MS, four types:
Quadrupole
Time-of-flight
Ion trap
Fourier transform-ion cyclotron resonance
(FT-ICR or FT-MS)
are used most often. Each has advantages and
disadvantages depending on the requirements
of a particular analysis.
Quadrupole
A quadrupole mass analyzer consists of four
parallel rods arranged in a square. The analyte
ions are directed down the center of the
square. Voltages applied to the rods generate
electromagnetic fields. These fields determine
which mass-to-charge ratio of ions can pass
through the filter at a given time. Quadrupoles
tend to be the simplest and least expensive
mass analyzers.
Quadrupole mass analyzers can operate in
two modes:
Scanning (scan) mode
Selected ion monitoring (SIM) mode
In scan mode, the mass analyzer monitors a
range of mass-to-charge ratios. In SIM mode,
the mass analyzer monitors only a few mass-
to-charge ratios.
SIM mode is significantly more sensitive than
scan mode but provides information about
fewer ions. Scan mode is typically used for
qualitative analyses or for quantitation when
all analyte masses are not known in advance.
SIM mode is used for quantitation and monitor-
ing of target compounds.
From ion
source
To detector
m
/
z
time m/z
A
b
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d
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c
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m/z
A
b
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d
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c
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m
/
z
time
1 Scan
1 Scan
Mass Range
Discrete Masses
Scan
SIM
Figure 8. Quadrupole mass analyzer
Figure 9. The quadrupole mass
analyzer can scan over a range
of mass-to-charge ratios or
alternate between just a few
11
Time-of-flight
In a time-of-flight (TOF) mass analyzer, a
uniform electromagnetic force is applied to
all ions at the same time, causing them to
accelerate down a flight tube. Lighter ions
Repeller Detector
From ion source
Accumulation
Ejection
Repeller Detector
Flight tube (field-free region)
Flight tube (field-free region)
m/z
A
b
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n
d
a
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c
e
+
+
+
Figure 10. Time-of-flight mass analyzer
Accumulation
+
+
+
+
+
Ejection
+
+
+
m/z
A
b
u
n
d
a
n
c
e
Figure 11. Ion trap mass analyzer
travel faster and arrive at the detector first,
so the mass-to-charge ratios of the ions are
determined by their arrival times. Time-of-
flight mass analyzers have a wide mass
range and can be very accurate in their
mass measurements.
Ion trap
An ion trap mass analyzer consists of
a circular ring electrode plus two end
caps that together form a chamber. Ions
entering the chamber are trapped there
by electromagnetic fields. Another field
can be applied to selectively eject ions
from the trap.
Ion traps have the advantage of being able
to perform multiple stages of mass spec-
trometry without additional mass analyzers.
12
Fourier transform-ion
cyclotron resonance (FT-ICR)
An FT-ICR mass analyzer (also called FT-MS)
is another type of trapping analyzer. Ions
entering a chamber are trapped in circular
orbits by powerful electrical and magnetic
fields. When excited by a radio-frequency
(RF) electrical field, the ions generate a time-
dependent current. This current is converted
by Fourier transform into orbital frequencies
of the ions which correspond to their mass-to-
charge ratios.
Like ion traps, FT-ICR mass analyzers can
perform multiple stages of mass spectrometry
without additional mass analyzers. They also
have a wide mass range and excellent mass
resolution. They are, however, the most expen-
sive of the mass analyzers.
Figure 12. FT-ICR mass analyzer
+
13
m/z 100 200 300
0
20000
40000
60000 [M + H]
+
[M + Na]
+
80000

1
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1
NH
H
2
N
O
O
N
N
CH
3
CH
3
S
m/z 156
m/z 186
m/z 124
m/z 213
m/z 108
Figure 14. Mass spectrum
of sulfamethazine acquired
with collision-induced
dissociation exhibits more
fragmentation and thus
more structural information
Collision-Induced Dissociation
and Multiple-Stage MS
The atmospheric pressure ionization
techniques discussed are all
relatively soft techniques. They
generate primarily:
Molecular ions M
+
or M

Protonated molecules [M + H]
+
Simple adduct ions [M + Na]
+
Ions representing simple losses
such as the loss of a water
[M + H H
2
O]
+
The resulting molecular weight
information is very valuable, but
complementary structural infor-
mation is often needed. To obtain
structural information, analyte ions are
fragmented by colliding them with neutral
molecules in a process known as collision-
induced dissociation (CID) or collisionally
activated dissociation (CAD). Voltages are
applied to the analyte ions to add energy to
the collisions and create more fragmentation.
m/z 100 200 300
0
50000
100000
150000
200000
250000
300000
350000

2
7
9
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1

3
0
1
.
0

2
8
0
.
0

2
8
1
.
0[M + Na]
+
[M + H]
+
S
NH
H
2
N
O
O
N
N
CH
3
CH
3
Figure 13. Mass spectrum of
sulfamethazine acquired
without collision-induced
dissociation exhibits
little fragmentation
14
CID in single-stage MS
CID is most often associated with multistage
mass spectrometers where it takes place
between each stage of MS filtering, but CID
can also be accomplished in single-stage
quadrupole or time-of-flight mass spectrome-
ters. In single-stage mass spectrometers,
CID takes place in the ion source and is thus
sometimes called source CID or in-source CID.
Analyte (precursor) ions are accelerated and
collide with residual neutral molecules to yield
fragments called product ions.
The advantage of performing CID in single-
stage instruments is their simplicity and
relatively low cost. The disadvantage is that
ALL ions present are fragmented. There is
no way to select a specific precursor ion
so there is no sure way to determine which
product ions came from which precursor ion.
The resulting spectra may include mass peaks
from background ions or coeluting compounds
as well as those from the analyte of interest.
This tradeoff may be acceptable when analyz-
ing relatively pure samples, but does not give
good results if chromatographic peaks are not
well resolved or background levels are high.
CID and multiple-stage MS
Multiple-stage MS (also called tandem MS or
MS/MS or MS
n
) is a powerful way to obtain
structural information. In triple-quadrupole or
quadrupole/quadrupole/time-of-flight instru-
ments (see Figure 16), the first quadrupole is
used to select the precursor ion. CID takes
place in the second stage (quadrupole or
octopole), which is called the collision cell.
LC/MS
Q1 CID Capillary
m/z
A
b
u
n
d
a
n
c
e
Figure 15. In-source
CID with a single-
quadrupole mass
analyzer
LC/MS/MS
Q1 Capillary Q2 (CID) Q3
m/z
A
b
u
n
d
a
n
c
e
Figure 16. MS/MS in a triple-quadrupole mass spectrometer
15
A major advantage of multiple-stage MS
is its ability to use the first stage of MS to
discard nonanalyte ions. Sample cleanup
and chromatographic separation become
much less critical. With relatively pure
samples, it is quite common to do away
with chromatographic separation altogether
and infuse samples directly into the mass
spectrometer to obtain product mass spectra
for characterization or confirmation.
The third stage (quadrupole or TOF) then
generates a spectrum of the resulting
product ions. It can also perform selected
ion monitoring of only a few product ions
when quantitating target compounds.
In ion trap and FT-ICR mass spectrometers,
all ions except the desired precursor ion
are ejected from the trap. The precursor
ion is then energized and collided to generate
product ions. The product ions can be
ejected to generate a mass
spectrum, or a particular product
ion can be retained and collided to
obtain another set of product ions.
This process can be sequentially
automated so that the most
abundant ion(s) from each stage
of MS are retained and collided.
This is a very powerful technique
for determining the structure of
molecules. It is also a powerful
technique for obtaining peptide
mass information that relates to
the sequence of amino acids in
a peptide.
Accumulation
A. B.
Nonprecursor ions ejected
+
+
+
+
m/z
A
b
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d
a
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c
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D. C.
Collision Product ions ejected
and detected
+
+
+
+
+
+
+
Figure 17. MS/MS in an ion
trap mass spectrometer
16
Adapting LC Methods
Early LC/MS systems were limited by funda-
mental issues like the amount of LC eluent the
mass spectrometer could accept. Significant
changes to LC methods were often required to
adapt them to MS detectors.
Modern LC/MS systems are more versatile.
Many mass spectrometers can accept flow
rates of up to 2 ml/min. With minor modifica-
tions, the same instruments can also generate
good results at microliter and nanoliter flow
rates. Ion sources with orthogonal (off-axis)
nebulizers are more tolerant of nonvolatile
buffers and require little or no adjustment,
even with differing solvent compositions and
flow rates.
Changes to LC methods required for modern
LC/MS systems generally involve changes in
sample preparation and solution chemistry to:
Ensure adequate analyte concentration
Maximize ionization through careful
selection of solvents and buffers
Minimize the presence of compounds that
compete for ionization or suppress signal
through gas-phase reactions
Figure 18. Salt deposits in this Agilent
APCI ion source had little effect on
performance thanks to orthogonal
spray orientation and robust ion
source design
Use solvents that have low heats of vapor-
ization and low surface tensions to enhance
ion desorption
Make sure that gas-phase reactions do
not neutralize ions through proton transfer
or ion pair reactions
If pH adjustments interfere with proper
chromatography, postcolumn modification
of the solvent may be a good solution. This
can improve MS response without compro-
mising chromatography.
17
Sample preparation
Sample preparation generally consists of
concentrating the analyte and removing
compounds that can cause background ions
or suppress ionization. Examples of sample
preparation include:
On-column concentration to increase
analyte concentration
Desalting to reduce the sodium and
potassium adduct formation that
commonly occurs in electrospray
Filtration to separate a low-
molecular-weight drug from proteins
in plasma, milk, or tissue
Ionization chemistry
Because formation of analyte ions
in solution is essential to achieving
good electrospray results, careful
attention must be paid to proper
solution chemistry. For electrospray:
Select more volatile buffers to
reduce the buildup of salts in the
ion source
Adjust solvent pH according to the
polarity of ions desired and the pH
of the sample
pH 2.5 pH 2.5
pH 6
+15
+14
+13
+12
+11
+10
+9
+15
+14
+13
+12
+11
+10
+9
+8
pH 12
Figure 19. Effect of solvent pH on the abundance of multiply charged
ions of the protein Lysozyme in electrospray mode
18
Solution chemistry is less critical for APCI
operation because ionization occurs in the
gas phase, not the liquid phase, but solvent
selection can still have a significant effect on
APCI analyte signal response.
Select more volatile solvents
Select solvents with a lower charge affinity
than the analyte
Protic solvents generally work better than
nonprotic solvents for positive ion mode
For negative ionization, solvents that readily
capture an electron must be used
Ammonium salts in the mobile phase can
cause ammonium adduct formation
Vaporizer temperature also affects APCI
ionization results. The temperature must be
hot enough to vaporize the solvent but not
so hot as to cause thermal degradation of the
analyte molecules.
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1
200C
400C
Figure 20. APCI analysis with an inadequate vaporizer temperature (200C) yields poor
results for some compounds compared to a more typical vaporizer temperature (400C)
19
Capillary electrophoresis (CE) is a technique
with a high separation efficiency and the
added benefit of being able to handle very
complex matrices. It has proven useful for
such diverse samples as: peptides, drugs of
abuse, drugs in natural products, flavanoids,
and aromatic amines.
With specialized nebulizers and method
adjustments, electrospray ionization can be
used for both capillary LC/MS and CE/MS.
The mass spectrometry benefits of selectivity
and sensitivity are available to both of these
separation techniques.
Capillary LC/MS and CE/MS
Capillary LC and capillary electrophoresis
(CE) are commonly used alongside HPLC.
Capillary LC at microliter to nanoliter flow
rates often provides better sensitivity than
conventional flow rates for extremely small
sample quantities. It is commonly used for
protein and peptide analysis but has also
proven very useful for the analysis of small
quantities of drugs.
2.0
1.5
1.0
0.5
0.0
2 4 6 8 10 12
HO
HO
OH
NH
HO
HO
OH
NH
Time (min)
%

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t
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v
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Terbutaline
5 g/mL
Salbutamol
5 g/mL
Base Peak
50 mM NaPO
3
Figure 21. CE/MS/MS analysis of terbutaline and salbutamol shows good chromatographic separation and good
signal even in the presence of a high concentration of sodium phosphate salt
20
Applications
LC/MS is suitable for many applications, from
pharmaceutical development to environmental
analysis. Its ability to detect a wide range
of compounds with great sensitivity and speci-
ficity has made it popular in a variety of fields.
Molecular Weight Determination
One fundamental application of LC/MS is
the determination of molecular weights. This
information is key to determining identity.
Differentiation of
similar octapeptides
Figure 22 shows the spectra of two peptides
whose mass-to-charge ratios differ by only
1 m/z. The only difference in the sequence
is at the C-terminus where one peptide has
threonine and the other has threonine amide.
The smaller fragments are identical in the two
spectra, indicating that large portions of the
two peptides are very similar. The larger frag-
ments contain the differentiating peptides.
0
20
40
60
80
100

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2
m/z 400 600 800
0
20
40
60
80
100

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5

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5

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1
Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr
Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr amide
Figure 22. Mass spectra
differentiating two very
similar octapeptides
21
The upper part of the display in Figure 23
shows the full scan mass spectrum of GFP.
The pattern of mass spectral peaks is charac-
teristic of a multiply charged analyte. Each
peak represents the molecule with a different
number of charges. The lower display is a
deconvoluted mass spectrum generated by
the data system for the singly charged analyte.
Determining the molecular weight
of green fluorescent protein
Green fluorescent protein (GFP) is a 27,000-
Dalton protein with 238 amino acids. It emits
a green light when excited by ultraviolet light.
During electrospray ionization, GFP acquires
multiple charges. This allows it to be analyzed
by a mass spectrometer with a relatively
limited mass (mass-to-charge) range. Mass
deconvolution is then used to determine
the molecular weight of the protein.
1000 1500
100000
120000
20000
40000
60000
80000

7
2
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5

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1
5

7
6
7
.
5
5

7
8
9
.
9
5

8
3
9
.
4
5

8
6
6
.
4
5

8
9
5
.
4
5

9
2
6
.
1
5

9
5
9
.
1
5

9
9
4
.
8
5

1
0
3
2
.
9
5

1
0
7
4
.
1
5

1
1
1
8
.
8
5

1
1
6
7
.
5
5

1
2
2
0
.
4
5

1
2
7
8
.
7
5

1
3
4
2
.
6
5

1
4
1
3
.
2
5

1
5
7
9
.
6
5
26700 26800 26900 27000
200000
400000
600000
800000
1000000
Deconvoluted spectrum
MW = 26828.84
Figure 23. Molecular
weight determination
of green fluorescent
protein by electro-
spray LC/MS
22
Structural Determination
Another fundamental application of LC/MS
is the determination of information about
molecular structure. This can be in addition
to molecular weight information or instead
of molecular weight information if the identity
of the analyte is already known.
Structural determination of
ginsenosides using MS
n
analysis
Ginseng root, a traditional Chinese herbal
remedy, contains more than a dozen
biologically active saponins called ginseno-
sides. Since most ginsenosides contain
multiple oligosaccharide chains at different
positions in the molecule, structural elucidation
of these compounds can be quite complicated.
MS
n
analysis in an ion trap mass spectrometer
permits multiple stages of precursor ion
isolation and fragmentation. This stepwise
fragmentation permits individual fragmentation
pathways to be followed and provides a great
deal of structural information.
Figure 24 shows the full scan mass spectrum
from a direct infusion of the ginsenoside Rb1.
The most prominent feature is the sodium
adduct ion [M + Na]
+
at m/z 1131.7. MS/MS
of m/z 1131.7 yields a product ion at m/z 789.7
corresponding to cleavage of a single glyco-
sidic bond (Figure 25). Subsequent isolation
and fragmentation of m/z 789.7 (Figure 26)
yields two products: a more abundant ion at
m/z 365.1 corresponding to loss of the oligo-
saccharide chain (Glc 2 Glc), and a less
abundant ion at m/z 627.5 representing the
loss of a deoxyhexose sugar.
m/z
200 400 600 800 1000 1200
%

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b
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a
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0
20
40
60
80
100
1131.7
[M + Na]
+
m/z 1131.7
HO
OH
OH
OH
OH
HO
HO
HO
HO
O
O
O
O
OH
OH
OH
OH
OH
OH
O
O
O
O
Figure 24. Full scan mass
spectrum of ginsenoside
Rb1 showing primarily
sodium adduct ions
23
Figure 26. Subsequent
full scan product ion
spectrum (MS
3
) from
the ion at m/z 789.7
789.7
1131.7
MS/MS of 1131.7
365.2
m/z
200 400 600 800 1000 1200
%

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A
b
u
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d
a
n
c
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0
20
40
60
80
100
HO
HO
OH
HO
OH
O
O
O
O
OH
HO
OH
HO
OH
OH
O
O
O
O
OH
OH OH
OH
[M + Na]
+
m/z 1131.7
m/z 365.2
m/z 789.7
m/z
200 300 400 500 600 700 800
%

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A
b
u
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a
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c
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0
20
40
60
80
100
HO
HO
O
O
O
O
OH
OH
OH
OH
OH
OH
Na
+
m/z 627.5
m/z 365.1
365.1
627.5
1131.7 789.7
Figure 25. Full scan
product ion (MS/MS)
spectrum from the sodium
adduct at m/z 1131.7
24
Pharmaceutical Applications
Rapid chromatography
of benzodiazepines
The information available in a mass spectrum
allows some compounds to be separated
even though they are chromatographically
unresolved. In this example, a series of benzo-
diazepines was analyzed using both UV and
MS detectors. The UV trace could not be
used for quantitation, but the extracted ion
chromatograms from the MS could be used.
The mass spectral information provides addi-
tional confirmation of identity. Chlorine has a
characteristic pattern because of the relative
abundance of the two most abundant isotopes.
In Figure 27, the triazolam spectrum shows
that triazolam has two chlorines and the
diazepam spectrum shows that diazepam
has only one.
Identification of bile acid
metabolites
The MS
n
capabilities of the ion trap mass
spectrometer make it a powerful tool for
the structural analysis of complex mixtures.
Intelligent, data-dependent acquisition tech-
niques can increase ion trap effectiveness
and productivity. They permit the identification
of minor metabolites at very low abundances
from a single analysis. One application is
the identification of metabolic products of
drug candidates.
Rapid chromatography and isotopic information
min 1 2 3 4
0
50
100
150
200
250
300
350
oxazepam Diazepam
Lorazepam
Estrazolam
Alprazolam
Diazepam
Clobazam
Triazolam
343.1
345.1
250 300 350
285.2
287.2
Triazolam
Diazepam
2 Cl
1 Cl
min 1 2 3 4
mAU
0
0.5
1
1.5
2
MS
UV
Figure 27. MS identification
and quantification of
individual benzodiazepines
from an incompletely
resolved mixture
25
corresponding to a predicted minor metabolite
(cholic acid) that eluted at 9.41 minutes. The
full scan MS/MS product spectrum (Figure 28C)
from the ion at m/z 407 confirms the identity.
Significant time was saved because the
confirming MS/MS product ion spectra were
acquired automatically in the same run as the
full scan MS data.
This example uses the in vitro incubation of
the bile acid deoxycholic acid with rat liver
microsomes to simulate metabolism of a
drug candidate. Intelligent, data-dependent
acquisition was used to select the two most
abundant, relevant ions in each MS scan.
These precursor ions were automatically
fragmented and full scan product ion
spectra collected.
Figure 28A shows the base peak chromato-
gram. Figure 28B shows the extracted ion
chromatogram of the [M-H]

ion at m/z 407

m/z 407
9.41
0
1
2
3
4
2 4 6 8 10 12 14
0
1
2
3
5
4
2 4 6 8 10 12 14
10.24
11.64
12.01
12.28
13.49
0.77
Minor
Metabolite
Base Peak:
m/z 150500
MS/MS of 407
at 9.41 min
289.5
343.4
389.5
325.3
100 200 300 400 500 m/z
0
20
40
60
80
100
Cholic Acid
H H
H
OH OH
OH
OH
H
H
O
H
A
B
C
Figure 28. Identification of a minor
metabolite of deoxycholic acid
through MS/MS
26
Biochemical Applications
Rapid protein identification
using capillary LC/MS/MS
and database searching
Traditional methods of protein identification
generally require the isolation of individual
proteins by two-dimensional gel electro-
phoresis. The combination of capillary
LC/MS/MS with intelligent, data-dependent
acquisition and probability-based database
searching makes it possible to rapidly identify
as many as 100 proteins in a single analysis.
In this example, a capillary LC and ion trap
mass spectrometer were used to acquire
data from a mixture of five tryptically digested
proteins at a concentration of 1 pmol/l each
(Figure 29). Using intelligent, automated data-
dependent acquisition, a full scan product
ion (MS/MS) spectrum was acquire from the
most abundant relevant ion in each mass scan
throughout the entire run. All MS and MS/MS
data were acquire from a single analysis.
Base Peak:
m/z 1502000
0.0
0.2
0.4
0.6
0.8
Intens.
x10
7
4.43
11.75
13.43
14.43
17.55
18.39
21.09
20.59
22.35
30.37
31.37
5 10 15 20 25 30 35 Time [min]
Figure 29. Base peak chromatogram generated from 1 pmol total material injected on column
381.3
569.3
670.4
856.5
769.5
971.6
1157.5
1271.5
1386.7
Observed
MS/MS of m/z 807.2
0
20
40
60
80
100
200 600 1000 1400 1800 m/z
%

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A
b
u
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d
a
n
c
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27
Protein identification was accomplished
using MASCOT software that correlated the
uninterpreted MS/MS data with sequences
in a database. Figure 30 demonstrates the
excellent match between the observed
MS/MS spectrum from the most abundant
ion (m/z 807.2) in the chromatographic peak
at 17.55 minutes and the theoretical y-ion
series predicted for a tryptic peptide from
human apolipoprotein, one of the proteins in
the sample mixture.
Y
(
1
2
)
Y
(
1
0
)
Y
(
1
1
)
Y
(
9
)
Y
(
8
)
Y
(
7
)
Y
(
6
)
Y
(
5
)
Y
(
3
)
Theoretical
LLDNWDSVTSTFSK
400 600 800 1000 1200 1400
1386.6
971.5
856.4
1157.5
1271.6
769.4
670.3
569.3 381.2
Figure 30. Full scan MS/MS spectra from the doubly charged parent ion m/z 807.2 and the matching theoretical
sequence identified by database searching
28
Clinical Applications
High-sensitivity detection of
trimipramine and thioridazine
For most compounds, MS is more sensitive
than other LC detectors. Trimipramine is
a tricyclic antidepressant with sedative
properties. Thioridazine is a tranquilizer.
Figure 31 shows these compounds in a urine
extract at a level that could not be detected
by UV. To get the maximum sensitivity from
a single-quadrupole mass spectrometer, the
analysis was done by selected ion monitoring.
Food Applications
Identification of aflatoxins in food
Aflatoxins are toxic metabolites produced in
foods by certain fungi. Figure 32 shows the
total ion chromatogram from a mixture of four
aflatoxins. Even though they are structurally
very similar, each aflatoxin can be uniquely
identified by its mass spectrum (Figure 33).
mAU
0
200
0
80000
0
40000
0
20000
min 1 2 3 4 5 6
0
8000
Trimipramine
Thioridazine
UV
280 nm
EIC
m/z 295
EIC
m/z 100
EIC
m/z 126
EIC
m/z 371
Trimipramine
Thioridazine
Figure 31. Trimipramine and
thioridazine in a urine extract
29
Scan of 2 ng
6.00 8.00 10.00 12.00 14.00 16.00 18.00
40000
80000
120000
160000
200000
1
2
3
4
1) Aflatoxin G2
2) Aflatoxin G1
3) Aflatoxin B2
4) Aflatoxin B1
TIC
Figure 32. Total ion
chromatogram of a
mixture of aflatoxins
120 160 200 240 280 320
50
90
285
Aflatoxin B1
335
[M+Na]
+
313
[M+H]
+
O
OCH3
O O
O
O
120 160 200 240 280 320 360
50
90
Aflatoxin G2
353
[M+Na]
+
331 [M+H]
+
313
[M-OH]
+
O O
O O
O O
OCH3
120 160 200 240 280 320 360
50
90
329
[M+H]
243
283
Aflatoxin G1
351
[M+Na]
+
311
[M-OH]
+
+
O O
O O
O O
OCH3
120 160 200 240 280 320
50
90
287
Aflatoxin B2
O
OCH3
O O
O
O
315
[M+H]
+
337
[M+Na]
+
Figure 33. Unique
mass spectra
allow positive
identification
of structurally
similar aflatoxins
30
Determination of vitamin D
3
in
poultry feed supplements using MS
3
Vitamin D is an essential constituent in human
and animal nutrition. Livestock diets deficient
in vitamin D can cause growth abnormalities.
Traditional GC/MS analysis methods for
vitamin D
3
in feed extracts require extensive
and time-consuming sample preparation and
derivatization prior to analysis. Atmospheric
pressure chemical ionization with ion trap
detection provides a sensitive analytical
method without the need for extensive sample
preparation and derivatization. Further, the
multiple-stage MS capability of the ion trap
eliminates the need for chromatographic
separation, greatly speeding analyses.
Flow injection analysis of a poultry feed
extract yields a peak at m/z 385 suggesting
the presence of vitamin D
3
. Isolation and
fragmentation of the precursor ion at m/z 385
is inconclusive. The full scan product ion
spectrum shows a prominent peak at m/z 367
representing the loss of a single water mole-
cule but little other fragmentation (Figure 34).
199.3 219.0
255.2 287.3
m/z 367
325.4
367.4
m/z
%

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b
u
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a
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c
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0
20
40
60
80
100
150 200 250 300 350 400 450 500
H
[M+H]
+
H
2
O
HO
Poultry Feed
Extract
MS
2
m/z 385
Figure 34. Full scan MS/MS product ion spectrum from the precursor ion at m/z 385 showing primarily
the nonspecific loss of a water molecule
31
match with a similar analysis of a pure
standard (Figure 35B) and conclusively
confirms the presence of vitamin D
3
.
Isolation and fragmentation of the ion at
m/z 367 yields a full scan MS
3
spectrum
(Figure 35A) rich in structurally specific
product ions. This spectrum is an excellent
158.6
185.0
213.0
255.2
271.1
325.5
Pure Standard
Vitamin D
3
MS
3
m/z 385 367
349.2
367.6
241.3
285.3
227.2
0
20
40
60
80
100
150 200 250 300 350 400 500
m/z
%

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A
b
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a
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c
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B
Poultry Feed Extract
MS
3
m/z 385 367
185.0
213.1
255.1
285.4
311.2
353.2
241.2
271.1
325.5
227.2
158.5
0
20
40
60
80
100
150 200 250 300 350 400 500
m/z
%

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A
b
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c
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A
Figure 35. Full scan MS
3
product ion spectra show much more structural information
32
Environmental Applications
Detection of phenylurea herbicides
Many of the phenylurea herbicides are very
similar and difficult to distinguish with a UV
detector (Figure 36). Monuron and diuron have
one benzene ring and differ by a single chlorine.
Chloroxuron has two chlorines and a second
benzene ring attached to the first by an oxygen.
The UV-Vis spectra are similar for diuron and
monuron, but different for chloroxuron. When
analyzed using electrospray ionization on an
LC/MS system, each compound has a uniquely
identifiable mass spectrum.
Detection of low levels
of carbaryl in food
Pesticides in foods and beverages can be a
significant route to human exposure. Analysis
of the carbamate pesticide carbaryl in extracts
of whole food by ion trap LC/MS/MS proved
more specific than previous analyses by HPLC
fluorescence and single-quadrupole mass
spectrometry. The protonated carbaryl mole-
cule (m/z 202) was detected in full scan mode
using positive ion electrospray. A product ion
UV shows class; MS identifies species
nm
Norm.
200 250 300 350
0
100
200
300
400
500
min 5 10 15 20 25
mAU
0
20
40
60
80
100
*
*
*
233.1
199.2
100 200 300 400 500
291.2
Chloroxuron
Diuron
Monuron
Figure 36. Chromato-
gram of phenylurea
herbicide with UV
and MS spectra
33
Ion trap analysis was more sensitive than pre-
vious analysis using a single-quadrupole mass
spectrometer operating in scanning mode and
more sensitive than fluorescence detection.
Ion trap LC/MS/MS also con-
firmed false positives in the
HPLC fluorescence analysis
caused by a coeluting compound.
Based on the MS/MS spectrum
of the coeluting compound, a
possible structure was assigned
as shown in Figure 38.
at m/z 145 (Figure 37) generated by collision-
induced dissociation provided confirmation of
carbaryl and was used for subsequent quanti-
tative analysis.
A
b
u
n
d
a
n
c
e
100
80
60
40
20
0
100 125 150 175 200 225 250 275
m/z
145 m/z
[M + H]
+
202
O C
= 202
O
N
H
CH
3
H
+
M
145
+H
Carbaryl
O
+ H H
Figure 37. Full scan product ion spectrum
generated by collision-induced dissociation
of the [M + H]
+
carbaryl ion at m/z 202
A
b
u
n
d
a
n
c
e
100
80
60
40
20
0
60 80 100 120 140 160 200 180 220
m/z
O
N
H
+
H
O
C
2
H
4
N
N
H
O
+
86
145
214
O
C
2
H
4
N
N
H
O
+
H
C
O
NH C
2
H
5
145
mw 213
86
Figure 38. Full scan product ion spectrum of coeluting
compound that produced false positives in HPLC
fluorescence analysis
CE/MS Applications
Analysis of peptides using CE/MS/MS
Capillary electrophoresis (CE) is a powerful
complement to liquid chromatography.
Different selectivity and higher chromato-
graphic resolution are its biggest advantages
when analyzing clean samples such as
synthetic peptides. When analyzing ppm-levels
of analytes in complex matrices, minimal
sample preparation and short analysis times
enable high throughput.
CE/MS with an ion trap mass spectrometer is
demonstrated using a standard peptide mix.
The data-dependent acquisition capability of
the ion trap triggers
MS/MS on the most
intense ion in each
MS scan. Figure 39
shows total ion electro-
pherogram (TIE) and the
base peak electrophero-
gram (BPE). Drops in
the TIE indicate where
MS/MS spectra were
acquired automatically.
The averaged mass
spectrum of peak 4
(Figure 40) shows a
singly charged mole-
cule with m/z of 574.3.
This is confirmed
as Met-enkephalin
(molecular weight 573.7)
by the examination
of the MS/MS mass
spectrum. The MS/MS
spectrum shows all
of the b- and y-series
fragments within the
scan range as well
as fragments from
other series.
TIE, All
BPE 200-1350, All MS
8.50 9.00 9.50 10.00 10.50
Time [min]
0.0
0.5
[x10
7
]
0.0
2.0
1.0
Intensity
[x10
6
]
4.0
1
4
3 2
MS
MS/MS
Figure 39. Total ion electropherogram (top) and base peak electropherogram
(bottom) of a standard peptide mixture
239.1
493.1
[M+H]
+
574.3
812.1 1147.2
0
2
4
200 400 600 800 1000
[m/z]
574.3
575.2
576.2
0
4
Intensity
[x10
5
]
570 574 578
232.9 262.1
0
2
4
6
Intensity
[x10
5
]
Intensity
[x10
4
]
225 275 325 375
b2
221.0
b3
278.1
y2
297.1
x2
323.1
y3
354.2
x3
380.1
a4
397.2
b4
425.2
y4
411.1
[m/z]
Figure 40. Full scan mass spec-
trum (top) and MS/MS spectrum
(bottom) of Met-enkephalin from
peak 4 of the electropherogram
34
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Agilent Technologies
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in this publication are subject to change
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