Basics of LC/MS

Primer
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 MSn 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 D3 in poultry feed supplements using MS3 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
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
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.
R

metabolites to peptides
and proteins.

Traditional detectors for

CH2CH2CH2NHCOCH3
liquid chromatography CH2CH2CH2OH
include refractive index, CH2CH2CH(NH2)COOH
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
% Rel. Abundance

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.
% Rel. Abundance

m/z

Figure 1. Two-dimensional abundance data and three-dimensional

mass spectral data from a mass spectrometer

4
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.
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.

1200000

800000 MS TIC
400000

4000
m/z
2000
648

12000

8000
m/z
4000
376

300

200 m/z
100 1325
5 10 15 20 25 30 35 40 min.

Figure 2. Identification of three components in a chromatographically unresolved peak

5
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
100,000
Electrospray Ionization
10,000
Molecular Weight
1000 APPI
APCI
100
10
Nonpolar
Polarity
6
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)
Figure 3. Applications of various
LC/MS ionization techniques
Very Polar
Electrospray ionization Some gas-phase reactions, mostly proton
transfer and charge exchange, can also
Electrospray relies in part on chemistry to gen-
occur between the time ions are ejected
erate analyte ions in solution before the analyte
from the droplets and the time they reach
reaches the mass spectrometer. The LC eluent
the mass analyzer.
is sprayed (nebulized) into a chamber at atmos-
pheric pressure in the presence of a strong
Electrospray is especially useful for analyzing
electrostatic field and heated drying gas.
large biomolecules such as proteins, peptides,
and oligonucleotides,
Ions but can also analyze
Nebulizer gas smaller molecules
like benzodiaze-
pines and sulfated
Heated nitrogen drying gas conjugates.
Solvent spray

Large molecules
+ +
+
+ +
often acquire more
+ + + + + + + + + + + than one charge.
+
Thanks to this
multiple charging,
Dielectric capillary entrance electrospray can
be used to analyze
Figure 4. Electrospray ion source
molecules as large
as 150,000 u even
though the mass range (or more accurately
The electrostatic field causes further mass-to-charge range) for a typical LC/MS
dissociation of the analyte molecules. instruments is around 3000 m/z. For example:
The heated drying gas causes the solvent
100,000 u / 10 z = 1,000 m/z
in the droplets to evaporate. As the droplets
shrink, the charge concentration in the
When a large molecule acquires many charges,
droplets increases. Eventually, the repulsive
a mathematical process called deconvolution
force between ions with like charges exceeds
is often used to determine the actual molecular
the cohesive forces and ions are ejected
weight of the analyte.
(desorbed) into the gas phase. These ions
are attracted to and pass through a capillary
sampling orifice into the mass analyzer.

Evaporation Analyte ion ejected Figure 5. Desorption of ions

from solution

+++ +
+

+ -- - + ++- ++ +
++- ++
+ - -+ ++-- --- + + -- --- +
++++
+ - + + ++ + -+-+
++ +-+- +

7
Atmospheric pressure APCI is applicable to a wide range of polar
chemical ionization and nonpolar molecules. It rarely results in
multiple charging so it is typically used for
In APCI, the LC eluent is sprayed through
molecules less than 1,500 u. Due to this,
a heated (typically 250°C – 400°C) vaporizer
and because it involves high temperatures,
at atmospheric pressure. The heat vaporizes
APCI is less well-suited than electrospray
the liquid. The resulting gas-phase solvent
for analysis of large biomolecules that may
molecules are ionized by electrons discharged
be thermally unstable. APCI is used with
from a corona needle. The solvent ions then
normal-phase chromatography more often
transfer charge to the analyte molecules
than electrospray is because the analytes are
through chemical reactions (chemical ioniza-
usually nonpolar.
tion). The analyte ions pass through a capillary
sampling orifice into the mass analyzer.

Figure 6. APCI ion source

HPLC inlet

Nebulizer (sprayer)
Nebulizing gas

Vaporizer (heater)

Drying gas

+ +
+ +
+ + + + + + + + + + +

Corona
discharge
needle Capillary

8
Atmospheric pressure photoionization APPI is applicable to many of the same
compounds that are typically analyzed by
Atmospheric pressure photoionization (APPI)
APCI. It shows particular promise in two
for LC/MS is a relatively new technique. As
applications, highly nonpolar compounds
in APCI, a vaporizer converts the LC eluent to
and low flow rates (<100 µl/min), where APCI
the gas phase. A discharge lamp generates
sensitivity is sometimes reduced.
photons in a narrow range of ionization
energies. The range of energies is carefully
In all cases, the nature of the analyte(s)
chosen to ionize as many analyte molecules
and the separation conditions have a strong
as possible while minimizing the ionization
influence on which ionization technique:
of solvent molecules. The resulting ions pass
electrospray, APCI, or APPI, will generate
through a capillary sampling orifice into the
the best results. The most effective technique
mass analyzer.
is not always easy to predict.

Figure 7. APPI ion source

HPLC inlet

Nebulizer (sprayer)
Nebulizing gas

Vaporizer (heater)

UV lamp Drying gas

hv +
+
+ +
+ + + + + + + + + + +

Capillary

9
Mass Analyzers through the filter at a given time. Quadrupoles
tend to be the simplest and least expensive
Although in theory any type of mass analyzer
mass analyzers.
could be used for LC/MS, four types:
• Quadrupole Quadrupole mass analyzers can operate in
• Time-of-flight two modes:
• Ion trap • Scanning (scan) mode
• Fourier transform-ion cyclotron resonance • Selected ion monitoring (SIM) mode
(FT-ICR or FT-MS)
In scan mode, the mass analyzer monitors a
are used most often. Each has advantages and range of mass-to-charge ratios. In SIM mode,
disadvantages depending on the requirements the mass analyzer monitors only a few mass-
of a particular analysis. to-charge ratios.

Quadrupole SIM mode is significantly more sensitive than

scan mode but provides information about
A quadrupole mass analyzer consists of four
fewer ions. Scan mode is typically used for
parallel rods arranged in a square. The analyte
qualitative analyses or for quantitation when
ions are directed down the center of the
all analyte masses are not known in advance.
square. Voltages applied to the rods generate
SIM mode is used for quantitation and monitor-
electromagnetic fields. These fields determine
ing of target compounds.
which mass-to-charge ratio of ions can pass

Figure 8. Quadrupole mass analyzer

To detector

From ion
source
1 Scan Mass Range
Scan
Abundance
m/z

time m/z

SIM 1 Scan Discrete Masses

Abundance
m/z

Figure 9. The quadrupole mass

analyzer can scan over a range
of mass-to-charge ratios or
time m/z
alternate between just a few

10
Time-of-flight travel faster and arrive at the detector first,
so the mass-to-charge ratios of the ions are
In a time-of-flight (TOF) mass analyzer, a
determined by their arrival times. Time-of-
uniform electromagnetic force is applied to
flight mass analyzers have a wide mass
all ions at the same time, causing them to
range and can be very accurate in their
accelerate down a flight tube. Lighter ions
mass measurements.

Ion trap
Flight tube (field-free region)
An ion trap mass analyzer consists of
+ a circular ring electrode plus two end
Repeller + Detector 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
Accumulation
from the trap.
From ion source
Ion traps have the advantage of being able
Flight tube (field-free region) to perform multiple stages of mass spec-
trometry without additional mass analyzers.
+
Repeller Detector

Ejection

+
+ +
+ +
+
+ +
Abundance

Accumulation Ejection

m/z
Abundance

Figure 10. Time-of-flight mass analyzer

m/z

Figure 11. Ion trap mass analyzer

11
Fourier transform-ion Like ion traps, FT-ICR mass analyzers can
cyclotron resonance (FT-ICR) perform multiple stages of mass spectrometry
An FT-ICR mass analyzer (also called FT-MS) without additional mass analyzers. They also
is another type of trapping analyzer. Ions have a wide mass range and excellent mass
entering a chamber are trapped in circular resolution. They are, however, the most expen-
orbits by powerful electrical and magnetic sive of the mass analyzers.
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.

Figure 12. FT-ICR mass analyzer

+

12
Collision-Induced Dissociation

279.1
and Multiple-Stage MS
CH3
The atmospheric pressure ionization 350000 [M + H]
+

O
techniques discussed are all N
O S
relatively “soft” techniques. They 300000 NH N CH3
generate primarily:
250000

• Molecular ions M+ or M – H2N

200000
• Protonated molecules [M + H]+
• Simple adduct ions [M + Na]+ 150000

• Ions representing simple losses 100000

280.0
such as the loss of a water
+
[M + H – H2O]+

301.0
[M + Na]

281.0
50000

0
The resulting molecular weight 100 200 300 m/z
information is very valuable, but
complementary structural infor-
mation is often needed. To obtain Figure 13. Mass spectrum of
structural information, analyte ions are sulfamethazine acquired
without collision-induced
fragmented by colliding them with neutral dissociation exhibits
molecules in a process known as collision- little fragmentation
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.

Figure 14. Mass spectrum

m/z 156
124.1

of sulfamethazine acquired
CH3
m/z 186 with collision-induced
O N dissociation exhibits more
80000
O S fragmentation and thus
NH N
186.0

CH3
more structural information
m/z 108
279.1

m/z 213
156.1

+ H2N
60000 [M + H]
m/z 124
108.2

40000
+
301.0

[M + Na]
323.0
213.2
107.1

20000
280.1
125.1

187.0
157.1

0
100 200 300 m/z

13
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
LC/MS
Abundance
Capillary CID Q1
LC/MS/MS
Capillary Q1 Q2 (CID)
Figure 16. MS/MS in a triple-quadrupole mass spectrometer
14
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 MSn) 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.
Figure 15. In-source
CID with a single-
quadrupole mass
analyzer
m/z
Abundance
Q3
m/z
The third stage (quadrupole or TOF) then A major advantage of multiple-stage MS
generates a spectrum of the resulting is its ability to use the first stage of MS to
product ions. It can also perform selected discard nonanalyte ions. Sample cleanup
ion monitoring of only a few product ions and chromatographic separation become
when quantitating target compounds. much less critical. With relatively pure
samples, it is quite common to do away
In ion trap and FT-ICR mass spectrometers, with chromatographic separation altogether
all ions except the desired precursor ion and infuse samples directly into the mass
are ejected from the trap. The precursor spectrometer to obtain product mass spectra
ion is then energized and collided to generate for characterization or confirmation.
product ions. The product ions can be
ejected to generate a mass
spectrum, or a particular product A. B.
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
Accumulation Nonprecursor ions ejected
molecules. It is also a powerful
technique for obtaining peptide C. D.
mass information that relates to
the sequence of amino acids in
a peptide. + +
++
+
+
+

Collision Product ions ejected

and detected
Abundance

Figure 17. MS/MS in an ion m/z

trap mass spectrometer

15
Adapting LC Methods Changes to LC methods required for modern
LC/MS systems generally involve changes in
Early LC/MS systems were limited by funda-
sample preparation and solution chemistry to:
mental issues like the amount of LC eluent the
mass spectrometer could accept. Significant • Ensure adequate analyte concentration
changes to LC methods were often required to • Maximize ionization through careful
adapt them to MS detectors. selection of solvents and buffers
• Minimize the presence of compounds that
Modern LC/MS systems are more versatile.
compete for ionization or suppress signal
Many mass spectrometers can accept flow
through gas-phase reactions
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.

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

16
Sample preparation • Use solvents that have low heats of vapor-
ization and low surface tensions to enhance
Sample preparation generally consists of
ion desorption
concentrating the analyte and removing
compounds that can cause background ions • Make sure that gas-phase reactions do
or suppress ionization. Examples of sample not neutralize ions through proton transfer
preparation include: or ion pair reactions
• On-column concentration to increase
If pH adjustments interfere with proper
analyte concentration
chromatography, postcolumn modification
• Desalting to reduce the sodium and of the solvent may be a good solution. This
potassium adduct formation that can improve MS response without compro-
commonly occurs in electrospray mising chromatography.
• Filtration to separate a low-
molecular-weight drug from proteins
in plasma, milk, or tissue +13
pH 2.5
+12
Ionization chemistry
+14
Because formation of analyte ions
in solution is essential to achieving +11
good electrospray results, careful +15 +10
attention must be paid to proper +9
solution chemistry. For electrospray:
• Select more volatile buffers to
+12 +11
reduce the buildup of salts in the
ion source +10 pH 6
• Adjust solvent pH according to the +13
polarity of ions desired and the pH +9
of the sample
+14 +8
+15

pH 12

Figure 19. Effect of solvent pH on the abundance of multiply charged

ions of the protein Lysozyme in electrospray mode

17
Solution chemistry is less critical for APCI Vaporizer temperature also affects APCI
operation because ionization occurs in the ionization results. The temperature must be
gas phase, not the liquid phase, but solvent hot enough to vaporize the solvent but not
selection can still have a significant effect on so hot as to cause thermal degradation of the
APCI analyte signal response. analyte molecules.
• 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

200˚C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

400˚C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Figure 20. APCI analysis with an inadequate vaporizer temperature (200°C) yields poor
results for some compounds compared to a more typical vaporizer temperature (400°C)

18
Capillary LC/MS and CE/MS Capillary electrophoresis (CE) is a technique
with a high separation efficiency and the
Capillary LC and capillary electrophoresis
added benefit of being able to handle very
(CE) are commonly used alongside HPLC.
complex matrices. It has proven useful for
Capillary LC at microliter to nanoliter flow
such diverse samples as: peptides, drugs of
rates often provides better sensitivity than
abuse, drugs in natural products, flavanoids,
conventional flow rates for extremely small
and aromatic amines.
sample quantities. It is commonly used for
protein and peptide analysis but has also
With specialized nebulizers and method
proven very useful for the analysis of small
adjustments, electrospray ionization can be
quantities of drugs.
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.

2.0 Base Peak

50 mM NaPO3
Salbutamol
Terbutaline
5 µg/mL
% Relative Abundance

1.5 5 µg/mL
HO
HO
OH
NH
1.0
HO
NH
OH
HO
0.5

0.0
2 4 6 8 10 12
Time (min)

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

19
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.

Figure 22. Mass spectra

differentiating two very
similar octapeptides

858.5
100
Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr
80

60

40
739.3

880.3
444.2

576.3
498.2

841.3
343.1

397.2

20

0
857.5

100 Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr amide

80

60
879.5

40
739.3

840.3
576.3
444.2
379.1

20

400 600 800 m/z

20
Determining the molecular weight The upper part of the display in Figure 23
of green fluorescent protein shows the full scan mass spectrum of GFP.
The pattern of mass spectral peaks is charac-
Green fluorescent protein (GFP) is a 27,000-
teristic of a multiply charged analyte. Each
Dalton protein with 238 amino acids. It emits
peak represents the molecule with a different
a green light when excited by ultraviolet light.
number of charges. The lower display is a
deconvoluted mass spectrum generated by
During electrospray ionization, GFP acquires
the data system for the singly charged analyte.
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.

Figure 23. Molecular

weight determination
789.95
839.45
895.45 866.45

of green fluorescent
protein by electro-
spray LC/MS 120000
767.55
746.15

100000
926.15
959.15

80000
726.25

1032.95
994.85

1074.15

60000
1118.85
1167.55
1220.45
1278.75

40000
1342.65

1413.25

1579.65
20000

1000 1500

1000000

800000
Deconvoluted spectrum
600000 MW = 26828.84
400000

200000

26700 26800 26900 27000

21
Structural Determination MSn analysis in an ion trap mass spectrometer
permits multiple stages of precursor ion
Another fundamental application of LC/MS
isolation and fragmentation. This stepwise
is the determination of information about
fragmentation permits individual fragmentation
molecular structure. This can be in addition
pathways to be followed and provides a great
to molecular weight information or instead
deal of structural information.
of molecular weight information if the identity
of the analyte is already known.
Figure 24 shows the full scan mass spectrum
from a direct infusion of the ginsenoside Rb1.
Structural determination of
The most prominent feature is the sodium
ginsenosides using MSn analysis
adduct ion [M + Na]+ at m/z 1131.7. MS/MS
Ginseng root, a traditional Chinese herbal of m/z 1131.7 yields a product ion at m/z 789.7
remedy, contains more than a dozen corresponding to cleavage of a single glyco-
biologically active saponins called ginseno- sidic bond (Figure 25). Subsequent isolation
sides. Since most ginsenosides contain and fragmentation of m/z 789.7 (Figure 26)
multiple oligosaccharide chains at different yields two products: a more abundant ion at
positions in the molecule, structural elucidation m/z 365.1 corresponding to loss of the oligo-
of these compounds can be quite complicated. saccharide chain (–Glc 2 –Glc), and a less
abundant ion at m/z 627.5 representing the
loss of a deoxyhexose sugar.

Figure 24. Full scan mass

spectrum of ginsenoside
Rb1 showing primarily
100 HO 1131.7
OO
sodium adduct ions
OH O
OH O
OH
OH OH
80 OH
% Relative Abundance

OH

[M + Na]+
m/z 1131.7
60 HO
OO
OH
HO
HO
40 OO
OH

HO
OH

20

0
200 400 600 800 1000 1200
m/z

22
 Figure 25. Full scan
product ion (MS/MS)
100
HO 789.7 spectrum from the sodium
OO MS/MS of 1131.7
OH O adduct at m/z 1131.7
OH O
OH
OH OH
OH
m/z 789.7
80
OH
% Relative Abundance

+
[M + Na]
m/z 1131.7
60 HO
O
O
OH
HO
HO O m/z 365.2
O
40 OH

HO
OH

20 365.2

0 1131.7

200 400 600 800 1000 1200

m/z

Figure 26. Subsequent

full scan product ion
365.1 1131.7 789.7
spectrum (MS3) from
the ion at m/z 789.7 100
OH
Na+
m/z 627.5
80 m/z 365.1
% Relative Abundance

HO
O
O
60 OH

HO OH
O
O
OH

40 OH
OH

20 627.5

0
200 300 400 500 600 700 800
m/z

23
Pharmaceutical Applications Identification of bile acid
metabolites
Rapid chromatography
of benzodiazepines The MSn capabilities of the ion trap mass
spectrometer make it a powerful tool for
The information available in a mass spectrum the structural analysis of complex mixtures.
allows some compounds to be separated Intelligent, data-dependent acquisition tech-
even though they are chromatographically niques can increase ion trap effectiveness
unresolved. In this example, a series of benzo- and productivity. They permit the identification
diazepines was analyzed using both UV and of minor metabolites at very low abundances
MS detectors. The UV trace could not be from a single analysis. One application is
used for quantitation, but the extracted ion the identification of metabolic products of
chromatograms from the MS could be used. drug candidates.
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.

Figure 27. MS identification

and quantification of
Rapid chromatography and isotopic information individual benzodiazepines
mAU from an incompletely
2 UV resolved mixture
1.5
1 343.1
0.5
Triazolam
0 345.1
2 Cl
1 2 3 4 min

350
MS
300
Triazolam
250 285.2
Clobazam
200 Diazepam
Diazepam
150 Alprazolam
287.2
1 Cl
100 Estrazolam

Lorazepam
50 250 300 350
oxazepam Diazepam
0
1 2 3 4 min

24
This example uses the in vitro incubation of corresponding to a predicted minor metabolite
the bile acid deoxycholic acid with rat liver (cholic acid) that eluted at 9.41 minutes. The
microsomes to simulate metabolism of a full scan MS/MS product spectrum (Figure 28C)
drug candidate. Intelligent, data-dependent from the ion at m/z 407 confirms the identity.
acquisition was used to select the two most
abundant, relevant ions in each MS scan. Significant time was saved because the
These precursor ions were automatically confirming MS/MS product ion spectra were
fragmented and full scan product ion acquired automatically in the same run as the
spectra collected. full scan MS data.

Figure 28A shows the base peak chromato-

gram. Figure 28B shows the extracted ion
chromatogram of the [M-H] – ion at m/z 407

Figure 28. Identification of a minor

metabolite of deoxycholic acid
11.64 A through MS/MS
5 Base Peak:
m/z 150–500
4 12.01
Minor
Metabolite
3 12.28
10.24
2 0.77 13.49

0
2 4 6 8 10 12 14

4 9.41 B

3 m/z 407

0
2 4 6 8 10 12 14

289.5 343.4 C
100
H O

80 OH
OH
MS/MS of 407
H at 9.41 min
H
60
325.3 389.5
40 H H
OH OH
H
20
Cholic Acid
0
100 200 300 400 500 m/z

25
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/µl 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.

Intens. 18.39
x107
17.55

0.8
31.37
0.6
13.43
30.37 Base Peak:
14.43
0.4 4.43 20.59 m/z 150 –2000
11.75 21.09
0.2 22.35

0.0
5 10 15 20 25 30 35 Time [min]

Figure 29. Base peak chromatogram generated from 1 pmol total material injected on column

26
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 LLDNWDSVTSTFSK
human apolipoprotein, one of the proteins in Theoretical 971.5

Y(9)
the sample mixture. 670.3 856.4 1386.6

Y(12)
Y(8)
Y(6)
381.2 569.3

Y(5)
Y(3)
1157.5
769.4 1271.6

Y(10)

Y(11)
Y(7)
971.6
100 400 600 800 1000 1200 1400
670.4
80
% Relative Abundance

856.5

1386.7
60

569.3
Observed
40 381.3 MS/MS of m/z 807.2
769.5
1271.5
20 1157.5

0
200 600 1000 1400 1800 m/z

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

27
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).

Figure 31. Trimipramine and

thioridazine in a urine extract
mAU
UV
200
280 nm
0

80000 Trimipramine EIC

m/z 295
0

40000 Trimipramine EIC

m/z 100
0

20000 EIC Thioridazine

m/z 371
0
8000
EIC Thioridazine
m/z 126
0
1 2 3 4 5 6 min

28
 Figure 32. Total ion
chromatogram of a
Scan of 2 ng mixture of aflatoxins
4
TIC
200000

160000
1) Aflatoxin G2 3
2
120000 2) Aflatoxin G1
1

3) Aflatoxin B2
80000
4) Aflatoxin B1

40000

6.00 8.00 10.00 12.00 14.00 16.00 18.00

Figure 33. Unique

mass spectra
315
allow positive 331 [M+H]
+ +
[M+H]
identification Aflatoxin G2
90 90 Aflatoxin B2
of structurally O O
313 O O
similar aflatoxins O O [M-OH]
+
O
50 50
353
O O
OCH 3
+ 337
[M+Na] O O OCH3 287 +
[M+Na]

120 160 200 240 280 320 360 120 160 200 240 280 320

329 313
[M+H] + [M+H] +
90 Aflatoxin G1 311 90 Aflatoxin B1
O O +
[M-OH] O O
O O
50 50 O
351 285 335
O O OCH3 243
283 [M+Na]+ O O OCH3 [M+Na]
+

120 160 200 240 280 320 360 120 160 200 240 280 320

29
Determination of vitamin D3 in preparation and derivatization. Further, the
poultry feed supplements using MS3 multiple-stage MS capability of the ion trap
eliminates the need for chromatographic
Vitamin D is an essential constituent in human
separation, greatly speeding analyses.
and animal nutrition. Livestock diets deficient
in vitamin D can cause growth abnormalities.
Flow injection analysis of a poultry feed
extract yields a peak at m/z 385 suggesting
Traditional GC/MS analysis methods for
the presence of vitamin D3. Isolation and
vitamin D3 in feed extracts require extensive
fragmentation of the precursor ion at m/z 385
and time-consuming sample preparation and
is inconclusive. The full scan product ion
derivatization prior to analysis. Atmospheric
spectrum shows a prominent peak at m/z 367
pressure chemical ionization with ion trap
representing the loss of a single water mole-
detection provides a sensitive analytical
cule but little other fragmentation (Figure 34).
method without the need for extensive sample

m/z 367
367.4
100
[M+H]+ – H2O

80 Poultry Feed
Extract
% Relative Abundance

H MS2
60 m/z 385 →

40 HO

20

255.2 287.3
199.3 219.0
325.4
0
150 200 250 300 350 400 450 500
m/z

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

30
Isolation and fragmentation of the ion at match with a similar analysis of a pure
m/z 367 yields a full scan MS3 spectrum standard (Figure 35B) and conclusively
(Figure 35A) rich in structurally specific confirms the presence of vitamin D3.
product ions. This spectrum is an excellent

100 255.1
A
Poultry Feed Extract
% Relative Abundance

80
MS3
m/z 385 → 367 →
60
241.2
40
213.1 227.2
285.4
185.0 271.1 311.2
20 325.5 353.2
158.5

0
150 200 250 300 350 400 500
m/z

100 255.2
B
Pure Standard
% Relative Abundance

80
Vitamin D3
271.1 MS3
60
213.0
227.2
241.3 m/z 385 → 367 →
40 185.0 285.3
325.5
20 158.6
349.2 367.6
0
150 200 250 300 350 400 500
m/z

Figure 35. Full scan MS3 product ion spectra show much more structural information

31
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

Figure 36. Chromato-

gram of phenylurea
UV shows class; MS identifies species herbicide with UV
and MS spectra
233.1
Norm. Diuron
500
400
199.2
300 Monuron
200
100 291.2
0 Chloroxuron

200 250 300 350 nm 100 200 300 400 500

mAU *
100
80
* *
60
40
20
0

5 10 15 20 25 min

32
at m/z 145 (Figure 37) generated by collision- Ion trap analysis was more sensitive than pre-
induced dissociation provided confirmation of vious analysis using a single-quadrupole mass
carbaryl and was used for subsequent quanti- spectrometer operating in scanning mode and
tative analysis. more sensitive than fluorescence detection.

Ion trap LC/MS/MS also con-

firmed false positives in the
145 m/z 145
+H
HPLC fluorescence analysis
100 O

O C N CH3 H+ = 202 caused by a coeluting compound.

H M
Based on the MS/MS spectrum
80 of the coeluting compound, a
Carbaryl possible structure was assigned
as shown in Figure 38.
Abundance

H + H
60 O

O C2H4N
40 214
100
N O C NH C2H5
H
+ O
86 H
20 145
80
[M + H] + O
mw 213

202
+
Abundance

0 N
60 H H
100 125 150 175 200 225 250 275
86
m/z
O C2H4N
40

Figure 37. Full scan product ion spectrum N O+

H
generated by collision-induced dissociation
of the [M + H]+ carbaryl ion at m/z 202 20
145

0
60 80 100 120 140 160 180 200 220
m/z

Figure 38. Full scan product ion spectrum of coeluting

compound that produced false positives in HPLC
fluorescence analysis

33
CE/MS Applications synthetic peptides. When analyzing ppm-levels
of analytes in complex matrices, minimal
Analysis of peptides using CE/MS/MS
sample preparation and short analysis times
Capillary electrophoresis (CE) is a powerful enable high throughput.
complement to liquid chromatography.
Different selectivity and higher chromato- CE/MS with an ion trap mass spectrometer is
graphic resolution are its biggest advantages demonstrated using a standard peptide mix.
when analyzing clean samples such as The data-dependent acquisition capability of
the ion trap triggers
MS/MS on the most
Intensity intense ion in each
[x107] TIE, All ±
MS scan. Figure 39
1.0
MS shows total ion electro-
0.5 MS/MS pherogram (TIE) and the
base peak electrophero-
0.0 gram (BPE). Drops in
6
[x10 ] BPE 200-1350, All MS ±
the TIE indicate where
2 3
4.0 4 MS/MS spectra were
1
acquired automatically.
2.0
The averaged mass
0.0 spectrum of peak 4
8.50 9.00 9.50 10.00 10.50 Time [min] (Figure 40) shows a
singly charged mole-
cule with m/z of 574.3.
Figure 39. Total ion electropherogram (top) and base peak electropherogram
(bottom) of a standard peptide mixture This is confirmed
as Met-enkephalin
(molecular weight 573.7)
Intensity by the examination
[x105] Intensity
5 574.3 of the MS/MS mass
[x10 ]
[M+H]+ spectrum. The MS/MS
4
4 574.3 575.2
576.2
spectrum shows all
239.1 0 of the b- and y-series
2 570 574 578
fragments within the
493.1 812.1 1147.2 scan range as well
0
200 400 600 800 1000 [m/z] as fragments from
other series.
Intensity
[x104]
a4 b4
6 425.2
397.2
4
b3 y2
b2 278.1 297.1
2 221.0 x2 y3 x3 y4
232.9 262.1 323.1 354.2 380.1 411.1 Figure 40. Full scan mass spec-
0 trum (top) and MS/MS spectrum
225 275 325 375 [m/z] (bottom) of Met-enkephalin from
peak 4 of the electropherogram

34
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