Basics of LC/MS
Primer
Contents
Why Liquid Chromatography/Mass Spectrometry?
4
Instrumentation
6
Ion Sources
Electrospray ionization
Atmospheric pressure chemical ionization
Atmospheric pressure photoionization
6
7
8
9
Mass Analyzers
Quadrupole
Time-of-flight
Ion trap
Fourier transform-ion cyclotron resonance (FT-ICR)
10
10
11
11
12
Collision-Induced Dissociation and Multiple-Stage MS
CID in single-stage MS
CID and multiple-stage MS
13
14
14
Adapting LC Methods
Sample preparation
Ionization chemistry
16
17
17
Capillary LC/MS and CE/MS
19
Applications
20
Molecular Weight Determination
Differentiation of similar octapeptides
Determining the molecular weight of green fluorescent protein
20
20
21
Structural Determination
Structural determination of ginsenosides using MSn analysis
22
22
Pharmaceutical Applications
Rapid chromatography of benzodiazepines
Detection of degradation products for salbutamol
Identification of bile acid metabolites
24
24
24
25
Biochemical Applications
Rapid protein identification using capillary LC/MS/MS and database searching
26
26
Clinical Applications
High-sensitivity detection of trimipramine and thioridazine
28
28
Food Applications
Identification of aflatoxins in food
Determination of vitamin D3 in poultry feed supplements using MS3
28
28
30
Environmental Applications
Detection of phenylurea herbicides
Detection of low levels of carbaryl in food
32
32
32
CE/MS Applications
Analysis of peptides using CE/MS/MS
34
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
metabolites to peptides
and proteins.
R
% Rel. Abundance
CH2CH2CH2NHCOCH3
CH2CH2CH2OH
CH2CH2CH(NH2)COOH
% Rel. Abundance
Traditional detectors for
liquid chromatography
include refractive index,
electrochemical, fluorescence, and ultraviolet-visible
(UV-Vis) detectors. Some
of these generate twodimensional data; that is,
data representing signal
strength as a function of
time. Others, including
fluorescence and diodearray 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 threedimensional 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.
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 chromatographic peaks, reducing the need for perfect
chromatography.
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.
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.
1200000
MS TIC
800000
400000
4000
m/z
648
2000
12000
8000
m/z
376
4000
300
m/z
1325
200
100
5
10
15
20
25
30
35
40
min.
Figure 2. Identification of three components in a chromatographically unresolved peak
5
molecules from the analyte molecules (direct
liquid inlet, thermospray) or did so before ionization (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.
Instrumentation
Mass spectrometers work by ionizing molecules 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.
The introduction of atmospheric pressure
ionization (API) techniques greatly expanded
the number of compounds that can be successfully 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:
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.
• Electrospray ionization (ESI)
• Atmospheric pressure chemical ionization
(APCI)
• Atmospheric pressure photoionization (APPI)
Earlier LC/MS systems used interfaces that
either did not separate the mobile phase
Figure 3. Applications of various
LC/MS ionization techniques
100,000
Electrospray Ionization
Molecular Weight
10,000
1000
APPI
APCI
100
10
Nonpolar
Very Polar
Polarity
6
Electrospray ionization
Electrospray relies in part on chemistry to generate analyte ions in solution before the analyte
reaches the mass spectrometer. The LC eluent
is sprayed (nebulized) into a chamber at atmospheric pressure in the presence of a strong
electrostatic field and heated drying gas.
Ions
Nebulizer gas
Solvent spray
+
+ +
+ +
+
+
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 benzodiazepines and sulfated
conjugates.
Heated nitrogen drying gas
+ + + + + + + +
Dielectric capillary entrance
Figure 4. Electrospray ion source
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.
Evaporation
+++
+ -- - +
+ - -+
+ - +
++
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
+
++- ++
++-- --- +
+ ++
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:
Figure 5. Desorption of ions
from solution
+
+
++- ++
+ -- --- +
++++
+ -+-+
+-+- +
7
Atmospheric pressure
chemical ionization
In APCI, the LC eluent is sprayed through
a heated (typically 250°C – 400°C) 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 ionization). 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.
Figure 6. APCI ion source
HPLC inlet
Nebulizer (sprayer)
Nebulizing gas
Vaporizer (heater)
Drying gas
+
+
Corona
discharge
needle
8
+
+
+
+ + + + + + + + + +
Capillary
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.
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 µl/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.
Figure 7. APPI ion source
HPLC inlet
Nebulizer (sprayer)
Nebulizing gas
Vaporizer (heater)
UV lamp
Drying gas
hv
+
+
+
+
+
+ + + + + + + + + +
Capillary
9
through the filter at a given time. Quadrupoles
tend to be the simplest and least expensive
mass analyzers.
Mass Analyzers
Although in theory any type of mass analyzer
could be used for LC/MS, four types:
•
•
•
•
Quadrupole mass analyzers can operate in
two modes:
Quadrupole
Time-of-flight
Ion trap
Fourier transform-ion cyclotron resonance
(FT-ICR or FT-MS)
• 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 massto-charge ratios.
are used most often. Each has advantages and
disadvantages depending on the requirements
of a particular analysis.
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 monitoring of target compounds.
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
Figure 8. Quadrupole mass analyzer
To detector
From ion
source
Mass Range
1 Scan
m/z
Abundance
Scan
m/z
time
1 Scan
Abundance
Discrete Masses
m/z
SIM
Figure 9. The quadrupole mass
analyzer can scan over a range
of mass-to-charge ratios or
alternate between just a few
10
time
m/z
Time-of-flight
travel faster and arrive at the detector first,
so the mass-to-charge ratios of the ions are
determined by their arrival times. Time-offlight mass analyzers have a wide mass
range and can be very accurate in their
mass measurements.
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
Ion trap
Flight tube (field-free region)
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.
+
Repeller
Detector
+
Accumulation
From ion source
Ion traps have the advantage of being able
to perform multiple stages of mass spectrometry without additional mass analyzers.
Flight tube (field-free region)
Repeller
+
Detector
Ejection
+
+
+
+
Abundance
+
+
+ +
Ejection
Accumulation
Figure 10. Time-of-flight mass analyzer
Abundance
m/z
m/z
Figure 11. Ion trap mass analyzer
11
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 timedependent current. This current is converted
by Fourier transform into orbital frequencies
of the ions which correspond to their mass-tocharge 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 expensive of the mass analyzers.
Figure 12. FT-ICR mass analyzer
+
12
279.1
Collision-Induced Dissociation
and Multiple-Stage MS
CH3
The atmospheric pressure ionization
techniques discussed are all
relatively “soft” techniques. They
generate primarily:
O
O
300000
S
NH
N
CH3
250000
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 – H2O]+
H2N
200000
150000
50000
100
+
200
300
m/z
Figure 13. Mass spectrum of
sulfamethazine acquired
without collision-induced
dissociation exhibits
little fragmentation
CH3
m/z 186
O
O S
186.0
[M + Na]
0
m/z 156
124.1
301.0
280.0
100000
The resulting molecular weight
information is very valuable, but
complementary structural information is often needed. To obtain
structural information, analyte ions are
fragmented by colliding them with neutral
molecules in a process known as collisioninduced dissociation (CID) or collisionally
activated dissociation (CAD). Voltages are
applied to the analyte ions to add energy to
the collisions and create more fragmentation.
80000
+
N
281.0
•
•
•
•
[M + H]
350000
N
NH
N
CH3
Figure 14. Mass spectrum
of sulfamethazine acquired
with collision-induced
dissociation exhibits more
fragmentation and thus
more structural information
279.1
m/z 108
156.1
m/z 213
+
H2N
m/z 124
+
323.0
[M + Na]
280.1
213.2
187.0
157.1
125.1
20000
107.1
301.0
40000
[M + H]
108.2
60000
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 spectrometers. 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 singlestage 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 analyzing 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 instruments (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 singlequadrupole mass
analyzer
Abundance
LC/MS
Capillary
CID
Q1
m/z
Capillary
Abundance
LC/MS/MS
Q1
Q2 (CID)
Q3
m/z
Figure 16. MS/MS in a triple-quadrupole mass spectrometer
14
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
A.
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
Accumulation
molecules. It is also a powerful
technique for obtaining peptide
C.
mass information that relates to
the sequence of amino acids in
a peptide.
+ +
+
B.
+
Nonprecursor ions ejected
D.
++
+
+
Product ions ejected
and detected
Abundance
Collision
Figure 17. MS/MS in an ion
trap mass spectrometer
m/z
15
Adapting LC Methods
Early LC/MS systems were limited by fundamental 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 modifications, 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
16
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 lowmolecular-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
• Use solvents that have low heats of vaporization 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 compromising chromatography.
+13
pH 2.5
+12
+14
+11
+15
+10
+9
+12 +11
pH 6
+10
+13
+9
+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
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.
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
200˚C
1
2
3
4
5
6
7 8
5
6
9 10
11
12 13 14 15
16
400˚C
1
2
3
4
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
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.
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.
% Relative Abundance
2.0
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.
Base Peak
50 mM NaPO3
Salbutamol
5 µg/mL
Terbutaline
5 µg/mL
1.5
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
Differentiation of
similar octapeptides
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 specificity has made it popular in a variety of fields.
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 fragments contain the differentiating peptides.
One fundamental application of LC/MS is
the determination of molecular weights. This
information is key to determining identity.
100
Figure 22. Mass spectra
differentiating two very
similar octapeptides
858.5
Molecular Weight Determination
Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr
80
880.3
841.3
576.3
498.2
444.2
397.2
20
343.1
40
739.3
60
857.5
0
100
Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr amide
80
879.5
739.3
576.3
444.2
20
379.1
40
840.3
60
0
400
20
600
800
m/z
Determining the molecular weight
of green fluorescent protein
The upper part of the display in Figure 23
shows the full scan mass spectrum of GFP.
The pattern of mass spectral peaks is characteristic 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.
Green fluorescent protein (GFP) is a 27,000Dalton protein with 238 amino acids. It emits
a green light when excited by ultraviolet light.
1000
1579.65
1413.25
20000
1342.65
40000
1220.45
60000
994.85
1032.95
1074.15
1118.85
1167.55
80000
1278.75
789.95
100000
746.15
767.55
120000
726.25
Figure 23. Molecular
weight determination
of green fluorescent
protein by electrospray LC/MS
839.45
895.45 866.45
926.15
959.15
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.
1500
1000000
800000
Deconvoluted spectrum
MW = 26828.84
600000
400000
200000
26700
26800
26900
27000
21
MSn 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.
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.
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 glycosidic 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 oligosaccharide chain (–Glc 2 –Glc), and a less
abundant ion at m/z 627.5 representing the
loss of a deoxyhexose sugar.
Structural determination of
ginsenosides using MSn analysis
Ginseng root, a traditional Chinese herbal
remedy, contains more than a dozen
biologically active saponins called ginsenosides. Since most ginsenosides contain
multiple oligosaccharide chains at different
positions in the molecule, structural elucidation
of these compounds can be quite complicated.
100
1131.7
HO
OH
OO
O
OH
OH
OH
% Relative Abundance
80
OH
O
OH
OH
[M + Na]+
m/z 1131.7
60
HO
OH
40
HO
OO
HO
OH
OO
HO
OH
20
0
200
400
600
800
m/z
22
1000
1200
Figure 24. Full scan mass
spectrum of ginsenoside
Rb1 showing primarily
sodium adduct ions
100
HO
789.7
OO
OH
OH
OH
O
OH
OH
% Relative Abundance
80
Figure 25. Full scan
product ion (MS/MS)
spectrum from the sodium
adduct at m/z 1131.7
MS/MS of 1131.7
O
OH
m/z 789.7
OH
+
[M + Na]
m/z 1131.7
60
HO
O
OH
HO
40
O
HO
O
O
m/z 365.2
OH
HO
OH
20
365.2
1131.7
0
200
400
600
1000
800
1200
m/z
Figure 26. Subsequent
full scan product ion
spectrum (MS3) from
the ion at m/z 789.7
365.1
1131.7
789.7
100
OH
% Relative Abundance
80
Na+
m/z 627.5
m/z 365.1
HO
O
O
OH
60
HO
OH
O
O
OH
40
OH
OH
627.5
20
0
200
300
400
500
600
700
800
m/z
23
Identification of bile acid
metabolites
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 benzodiazepines 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 MSn capabilities of the ion trap mass
spectrometer make it a powerful tool for
the structural analysis of complex mixtures.
Intelligent, data-dependent acquisition techniques 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.
The mass spectral information provides additional 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
individual benzodiazepines
from an incompletely
resolved mixture
Rapid chromatography and isotopic information
mAU
2
UV
1.5
1
343.1
0.5
Triazolam
0
345.1
2 Cl
1
2
3
4
min
350
300
MS
Triazolam
250
285.2
Clobazam
200
Diazepam
Diazepam
150
Alprazolam
100
Estrazolam
287.2
1 Cl
Lorazepam
50
oxazepam
250
Diazepam
0
1
24
2
3
4
min
300
350
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.
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.
Figure 28A shows the base peak chromatogram. Figure 28B shows the extracted ion
chromatogram of the [M-H] – ion at m/z 407
11.64
5
Base Peak:
m/z 150–500
4
12.01
Minor
Metabolite
3
A
Figure 28. Identification of a minor
metabolite of deoxycholic acid
through MS/MS
12.28
10.24
2
13.49
0.77
1
0
2
4
6
8
4
10
12
14
9.41
B
m/z 407
3
2
1
0
2
4
6
8
289.5
100
OH
MS/MS of 407
at 9.41 min
OH
325.3
H
40
OH
H
20
C
343.4
H
H
60
14
12
O
H
80
10
389.5
H
OH
Cholic Acid
0
100
200
300
400
500
m/z
25
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 datadependent 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.
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 electrophoresis. 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.
Intens.
18.39
x107
17.55
0.8
31.37
0.6
0.4
13.43
30.37
14.43
4.43
11.75
20.59
21.09
0.2
Base Peak:
m/z 150 –2000
22.35
0.0
5
10
15
20
25
30
Figure 29. Base peak chromatogram generated from 1 pmol total material injected on column
26
35
Time [min]
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.
LLDNWDSVTSTFSK
Y(8)
1157.5
Y(7)
Y(10)
769.4
1271.6
Y(11)
Y(6)
Y(5)
1386.6
Y(12)
856.4
670.3
569.3
Y(3)
381.2
Y(9)
971.5
Theoretical
971.6
100
400
600
800
1000
1200
1400
% Relative Abundance
670.4
80
856.5
1386.7
60
381.3
40
Observed
MS/MS of m/z 807.2
569.3
769.5
1157.5
20
1271.5
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
Food Applications
High-sensitivity detection of
trimipramine and thioridazine
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).
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.
Figure 31. Trimipramine and
thioridazine in a urine extract
mAU
UV
280 nm
200
0
80000
Trimipramine
EIC
m/z 295
Trimipramine
EIC
m/z 100
0
40000
0
20000
EIC
m/z 371
Thioridazine
EIC
m/z 126
Thioridazine
0
8000
0
1
28
2
3
4
5
6
min
Figure 32. Total ion
chromatogram of a
mixture of aflatoxins
Scan of 2 ng
4
200000
TIC
160000
1) Aflatoxin G2
120000
1
2) Aflatoxin G1
2
3
3) Aflatoxin B2
80000
4) Aflatoxin B1
40000
6.00
Figure 33. Unique
mass spectra
allow positive
identification
of structurally
similar aflatoxins
8.00
10.00
12.00
14.00
16.00
18.00
315
+
[M+H]
+
331 [M+H]
90
Aflatoxin G2
90 Aflatoxin B2
O
O
O
313
+
[M-OH]
O
O
50
O
O
120
353
+
[M+Na]
OCH 3
160
200
240
280
320
50
O
360
O
O
O
120
287
OCH3
200
160
240
280
O
O
50
O
120
O
200
Aflatoxin B1
O
O
OCH3
160
90
311
+
[M-OH]
O
50
243
240
351
[M+Na]+
283
280
320
360
320
313
[M+H] +
329
[M+H] +
90 Aflatoxin G1
337
+
[M+Na]
O
O
285
O
120
O
OCH3
160
200
240
280
335
+
[M+Na]
320
29
preparation and derivatization. Further, the
multiple-stage MS capability of the ion trap
eliminates the need for chromatographic
separation, greatly speeding analyses.
Determination of vitamin D3 in
poultry feed supplements using MS3
Vitamin D is an essential constituent in human
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
the presence of vitamin D3. 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 molecule but little other fragmentation (Figure 34).
Traditional GC/MS analysis methods for
vitamin D3 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
m/z 367
367.4
100
[M+H]+ – H2O
Poultry Feed
Extract
MS2
m/z 385 →
% Relative Abundance
80
H
60
HO
40
20
199.3
219.0
255.2
287.3
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
m/z 367 yields a full scan MS3 spectrum
(Figure 35A) rich in structurally specific
product ions. This spectrum is an excellent
255.1
100
% Relative Abundance
match with a similar analysis of a pure
standard (Figure 35B) and conclusively
confirms the presence of vitamin D3.
A
Poultry Feed Extract
MS3
m/z 385 → 367 →
80
60
241.2
40
213.1 227.2
285.4
271.1
185.0
20
311.2
325.5
353.2
158.5
0
150
200
250
300
350
400
500
m/z
255.2
% Relative Abundance
100
B
Pure Standard
Vitamin D3
MS3
m/z 385 → 367 →
80
271.1
60
241.3
213.0
227.2
185.0
40
285.3
325.5
20
158.6
349.2
0
150
200
250
300
350
367.6
400
500
m/z
Figure 35. Full scan MS3 product ion spectra show much more structural information
31
Detection of low levels
of carbaryl in food
Environmental Applications
Detection of phenylurea herbicides
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 molecule (m/z 202) was detected in full scan mode
using positive ion electrospray. A product ion
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.
Figure 36. Chromatogram of phenylurea
herbicide with UV
and MS spectra
UV shows class; MS identifies species
233.1
Norm.
Diuron
500
400
199.2
300
Monuron
200
100
291.2
Chloroxuron
0
200
250
300
350
nm
100
200
300
400
500
*
mAU
100
80
*
60
*
40
20
0
5
32
10
15
20
25
min
at m/z 145 (Figure 37) generated by collisioninduced dissociation provided confirmation of
carbaryl and was used for subsequent quantitative analysis.
145 m/z
Ion trap LC/MS/MS also confirmed 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.
+H
145
100
Ion trap analysis was more sensitive than previous analysis using a single-quadrupole mass
spectrometer operating in scanning mode and
more sensitive than fluorescence detection.
O
O
C
N
H
CH3
H+ = 202
M
80
Abundance
Carbaryl
H
60
+
O
H
O
40
C2H4N
214
100
20
150
175
200
225
m/z
250
275
Abundance
125
C2H5
mw 213
+
N
60
H
H
86
40
Figure 37. Full scan product ion spectrum
generated by collision-induced dissociation
of the [M + H]+ carbaryl ion at m/z 202
NH
O
H
O
0
100
+
145
80
[M + H] +
202
C
O
N
H
86
O
C2H4N
N
H
O+
145
20
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
sample preparation and short analysis times
enable high throughput.
Analysis of peptides using CE/MS/MS
Capillary electrophoresis (CE) is a powerful
complement to liquid chromatography.
Different selectivity and higher chromatographic resolution are its biggest advantages
when analyzing clean samples such as
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
Intensity
intense ion in each
[x107]
TIE, All ±
MS scan. Figure 39
1.0
MS
shows total ion electropherogram (TIE) and the
0.5
MS/MS
base peak electrophero0.0
gram (BPE). Drops in
6
the TIE indicate where
[x10 ]
BPE 200-1350, All MS ±
3
2
MS/MS spectra were
4.0
4
acquired automatically.
1
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 molecule with m/z of 574.3.
Figure 39. Total ion electropherogram (top) and base peak electropherogram
This is confirmed
(bottom) of a standard peptide mixture
as Met-enkephalin
(molecular weight 573.7)
by the examination
Intensity
Intensity
[x105]
of the MS/MS mass
574.3
5
[x10 ]
[M+H]+
spectrum. The MS/MS
4
4
574.3
575.2
spectrum shows all
576.2
239.1
0
of the b- and y-series
2
570
574
578
fragments within the
812.1
1147.2
493.1
scan range as well
0
as fragments from
200
400
600
800
1000
[m/z]
other series.
Intensity
[x104]
a4
397.2
6
4
2
0
b3
y2
278.1 297.1
b2
221.0
232.9
225
34
262.1
275
x2
323.1
325
y3
354.2
x3
380.1
375
b4
425.2
y4
411.1
[m/z]
Figure 40. Full scan mass spectrum (top) and MS/MS spectrum
(bottom) of Met-enkephalin from
peak 4 of the electropherogram
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Agilent Technologies
Information, descriptions and specifications
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