Basics of LC/MS

Primer

Contents
Why Liquid Chromatography/Mass Spectrometry?

Instrumentation

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.

% 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

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

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

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

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

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 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

+
+

+ + + + + + + + + +

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 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.

Figure 7. APPI ion source

HPLC inlet
Nebulizer (sprayer)

Nebulizing gas

Vaporizer (heater)

UV lamp

Drying gas

hv
+

+
+

+ + + + + + + + + +

Capillary

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

NH

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]

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

281.0

[M + H]

350000

N
NH

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

200C

7 8

9 10

11

12 13 14 15

16

400C

10

11

12 13 14 15 16

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

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 g/mL

Terbutaline
5 g/mL

1.5
HO

HO

OH

NH

1.0
HO

NH

OH
HO

0.5

0.0
2

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

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

OH

m/z 789.7
OH
+

[M + Na]
m/z 1131.7
60

HO
O
OH
HO

40

HO
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

OH

60
HO

OH
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

345.1

2 Cl
1

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

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 150500

12.01

Minor
Metabolite

Figure 28. Identification of a minor

metabolite of deoxycholic acid
through MS/MS

12.28
10.24

13.49

0.77

1
0
2

10

12

14

9.41

B
m/z 407

3
2
1
0
2

8
289.5

100
OH

MS/MS of 407
at 9.41 min

OH

325.3
H

40
OH

20

343.4

60

14

12

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/l 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

min

Figure 32. Total ion

chromatogram of a
mixture of aflatoxins

Scan of 2 ng

4
200000

TIC

160000

1) Aflatoxin G2
120000

2) Aflatoxin G1

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

313
+
[M-OH]

50
O

120

353
+
[M+Na]

OCH 3

160

200

240

280

320

50
O

360

120

287

OCH3

200

160

240

280

O
O

50
O

120

200

Aflatoxin B1
O

OCH3

160

90

311
+
[M-OH]

50
243

240

351
[M+Na]+

283

280

320

360

320

313
[M+H] +

329
[M+H] +

90 Aflatoxin G1

337
+
[M+Na]

285
O

120

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

N
H

CH3

H+ = 202
M

80

Abundance

Carbaryl
H

60

+
O

40

C2H4N

214

100

20

150

175

200

225

m/z

250

275

Abundance

125

C2H5

mw 213

+
N

60

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

0
100

145

80

[M + H] +
202

N
H
86

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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Copyright 2001
Agilent Technologies
Information, descriptions and specifications
in this publication are subject to change
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Printed in the U.S.A. February 15, 2001
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