Overview and Intro to LC-MS

and LC-MS/MS

Ed Malone
The State Laboratory
Types of LC-MS in use in The State Lab

6 LC-MS/MS Triple Quadrupole Instruments

1 Exactive and 1 Q-Exactive

1 LC-Quadrupole-Time of Flight, 1 LC-Ion Trap

5 QTRAP instruments

Systems from Agilent, Sciex, Thermo and Waters

Technique is used in nearly every Section in The
State Lab
Mass Spectrometry

Mass spectrometry is a very sensitive analytical technique and is widely
regarded as having good selectivity.

A Mass Spectrometer produces charged particles (ions) from chemical
substances then uses electric and magnetic fields to measure the mass
of the charged particles.

Separates and measures ions based on their mass-to-charge (m/z)
ratio.

Operates under high vacuum; key specifications are resolution, mass
measurement accuracy, and sensitivity.

Several kinds of Mass Spectrometer exist: quadrupole, time-of-flight
(TOF) and ion traps (orbital and linear) are most used.
Liquid Chromatography with Mass
Spectrometry

In many applications it is necessary to isolate the target analyte from
what could be a sample containing thousands of other different
molecules.

For example, more than 1,500 compounds may have the same
molecular mass at around 250 Da.

An additional separation technique is needed before presenting the
sample to the mass spectrometer.

Liquid chromatography-mass spectrometry (LC-MS) is the
combination of two selective techniques that allows the analyte(s) of
interest in highly complex mixtures to be isolated and measured.

LC differentiates compounds by their physico-chemical properties
and MS differentiates compounds by mass (specifically their mass-
to-charge ratio).
LC-MS Selectivity

It is this dual selectivity that makes LC-MS such a powerful
analytical tool.

The mass spectrometer acts not only as the “LC detector”
but, at least in principle, it provides the capability to identify
the species corresponding to each chromatographic peak
through its unique mass spectrum.
Why LC-MS

Directly coupling LC with MS offers the
following advantages:
 Structural information
 Speed of analysis
 Convenience
 Analysis of multicomponent mixtures
 Accurate quantitation
 Evaluation of chromatographic peak purity
Why LC-MS/MS?

Why Liquid Chromatography?
 Analysis of labile analytes
 Analysis of more polar compounds without
derivatization
 Analysis of significantly higher masses
 Reduction of lengthy clean-up

Why MS/MS?
 Additional structural elucidation
 Further reduction of clean-up (?)
 Specificity
 Useful MS modes
Sample introduction

Ion Souce
 Transforms sample molecules to ions
 Soft ionization

Places positive or negative charge on the analyte without significantly
fragmenting the analyte

M+1 ion (or M-1 ion)

No need to volatilize

Down to fmol detection limits
 Atmospheric Pressure Ionization (API)

Electrospray

MALDI

APCI

APPI
Which is Best?

It depends on the exact application.

Increasing polarity and molecular weight and
thermal instability favors electrospray.

Most drugs of abuse are highly polar and are easily analyzed
using electrospray.

High molecular weight proteins also require electrospray

Lower polarity and molecular weight favors APCI or

APPI.

Lower background, but compounds must be more thermally
stable (some steroids).
Atmospheric Pressure Ionization (API)

API techniques now provide highly sensitive detection using

conventional and capillary flow rates on benchtop MS detector
systems.

These interfaces work with typical solvent and eluent
compositions, whether the separation is achieved by isocratic or
gradient elution.

API using Electrospray Ionization (ESI) or Atmospheric Pressure
Chemical Ionization (APCI) interfaces have proven invaluable in
meeting sensitivity requirements for quantitative methods.
What is API?

(API) is a technique for interfacing the eluent flow from an LC

column to a mass spectrometer.

The API interface must:
1) separate the analytes from the solvent
2) ionize the analyte molecules
3) maintain vacuum in the mass detector.
Incompatibilities exist when combining LC and
MS

First, the eluent stream being liquid (and often aqueous) can flow
at >1 mL/min, while the MS operates at a pressure of about 10–6
torr (1.3 × 10-4 Pa).

Therefore, it is not possible to pump the eluent directly into the
source of the mass spectrometer while maintaining the
necessary vacuum.

Second, the majority of analytes likely to be separated are
relatively involatile and/or thermally labile and are not amenable
to ionization techniques used in gas chromatography/mass
spectrometry (GC/MS).
Alternative ionization and interface techniques

To overcome the incompatibilities of LC and MS alternative

ionization and interface techniques have been developed.

Two types of API interfaces are commonly encountered. These are
Electrospray Ionization (ESI) and Atmospheric Pressure Chemical
Ionization (APCI).
What is ESI?

• ESI is an API technique that provides a simple, real time means of

analyzing a wide range of polar molecules.
• For small molecules (up to 1-2 K Daltons in molecular mass), either an
[M+H]+ or [M-H]– ion is detected depending on whether positive or
negative ion detection has been selected.
• This is similar to the traditional Chemical Ionization (CI) used in GC/MS,
but ESI is a much softer ionization technique, producing significantly
less fragmentation.
• In addition, the analyte does not have to be exposed to high
temperatures (this is good for thermally labile compounds) or be
volatile.
• The mass spectrometer does require the analyte to be in the gas phase
and to be in a charged state (two basic requirements of all mass
spectral analytes) this is accomplished without the conventional GC/MS
techniques employed for vaporization and ionization.
How does ESI work?

The mechanism of electrospray ionization involves the emission of ions

from a droplet into the gas phase at atmospheric pressure, a process
known as ion evaporation.

In ESI, the eluent passes through a stainless steel capillary that carries
a high potential, typically 2 to 4 kV. The strong electric field generated
by this potential and a concentric nebulizing nitrogen gas flow cause the
formation of a fine spray of highly charged droplets at the tip of the
capillary (hence ESI).

The ion evaporation process is assisted by a flow of heated gas. This
heating process, which is close to the source entrance cone, enables
the routine use of a wide range of liquid flow rates in ESI mode (50 µL
to >1 mL min–1).
 As a sample droplet moves through the heated sheath gas, the solvent
evaporates, decreasing the size of the droplet, which increases charge-
to-volume ratio.
 When this ratio reaches the Rayleigh instability limit, the droplet
undergoes a coulombic explosion, producing a smaller droplet with a
lower charge-to-volume ratio.
 As more solvent is eliminated, the process is repeated. The end result
yields ions in the gas phase. The newly formed ions flow into the
entrance cone, pulled by the strong electric field and pressure
differential at the entrance orifice.
ESI: Droplet size reduction & fission

Droplet size reduction occurs by the
continual repetition of two processes:
1. Desolvation (evaporation of neutral solvent and
volatile buffers)
2. Droplet fission caused by electric repulsion
between like charges. ++
+
+++
+
+
+
++ +++
+
+ + +
+ + ++++ +
+
+ + + + ++
+++
+
+ + + +
+
++ +++
+ +
+
++ +++
+
+ + +
+
+ + +
++ +
+++
++ +
+ +
+
++ +++
+
The Electrospray Phenomenon
Electrospray Ionisation

19
ESI: Ionization Efficiency

Enhanced by the production of smaller droplets.

 Lower mobile phase flow rate yields smaller droplets.
 Nebulizing gas promotes droplet formation.
 Use of volatile mobile phases promotes desolvation and
droplet fission.

Enhanced by increasing the concentration of analyte

ions at the end of the capillary tip.
 Matrix modifiers to promote solution ion formation.
 Chromatography to produce narrow highly concentrated
bands of analyte.
ESI: Pros and Cons

Pros

Soft ionization technique, resulting in little
decomposition of labile analytes.

Generally produces only molecular ions.

Multi charged analytes easily produced,
allowing proteins to be analyzed.

Wide range of analytes

Efficient ion production
Restrictions of ESI

Non-volatile salts should be avoided as signal
suppression will result.

Strong ion-pairing agents such as trifluoroacetate
should similarly be avoided.

Need polar solvents for ESI.
 Normal phase not compatible with ESI (Use APCI)

Additives must be compatible with ionization mode.
(i.e. pH of separation must match desired ions)
ESI: Pros and Cons

Cons

Lower flow rates

 concentration dependent
 nL/min (nanospray)

Analyte must form solution phase ion
 HCl or Na salt good indicator of suitability

Ion Suppression
ESI: Ion Suppression

The disadvantage of electrospray.

Often results from inefficient droplet formation.

Causes:
 Nonvolatile buffers or salts (phosphates)
 Nonvolatile materials in mobile phase Ion pairing
 Reported that higher molecular weight analyte ions can
suppress smaller analytes

Generally more prominent early in an RP-LC run,
but can occur at anytime.

Underscores the need for good chromatography.
ESI: Ion Supression

Ion Suppression Study

Oxycodone Infusion with solvent
flow.
Negative control injected at ~0.1min

~90%reduction
 AfB1 without Internal std

3.50E+05

barley, slope 4072

3.00E+05
CFS, slope 3003
maize, slope 2675
2.50E+05
soln, slope 5964

2.00E+05
Area

1.50E+05

1.00E+05

5.00E+04

0.00E+00
0 10 20 30 40 50 60

-5.00E+04
ng/g

For Aflatoxin B1 without IS; Area of 50,000 counts would give

concentration of
(i) 8 ng/g using solvent curve
(ii) 11 ng/g using barley matrix curve
(iii) 14 ng/g using compound feed matrix curve
(iv) 19 ng/g using maize matrix curve
 Zon, without Internal std

8.00E+05

7.00E+05

6.00E+05

5.00E+05

barley, slope 4887

4.00E+05
CFS, slope 3983
Area

maize, slope 3985

3.00E+05 soln, slope 13271

2.00E+05

1.00E+05

0.00E+00
0 10 20 30 40 50 60

-1.00E+05
ng/g

For Zearalenone; Area of 200,000 counts would give concentration of

(i) 15 ng/g using solvent curve
(ii) 36 ng/g using barley matrix curve
(iii) 44 ng/g using maize matrix curve
Ion Suppression;
Less analyte=bigger response?
Chlormadinone Acetate in Kidney Fat;
Effect of different wash solvents on
SPE Purification; Detector Response
0% Ethyl Acetate in Hexane 70%
2.5E+06
CLA in
2.0E+06 514702.298 Wash

0
1.5E+06
CLA in
Eluate
1.0E+06 1829504
1551039.75
585682.732
5.0E+05 899148.991 887453.84
582642.368 635444.889
70091.9274
0.0E+00 49326.8744 71876.5761
0% 0% 10% 25% 40% 55% 70%
Atmospheric Pressure Chemical Ionisation
(APCI)

APCI uses analyte desolvation and charge transfer reactions in
vapour phase analyte ions.

Eluent introduced into interface using capillary similar to ESI, no
potential applied to capillary.

Liquid emerges from the capillary surrounded by a flow of inert
gas into a heated region.

Combination of gas and heat forms an aerosol that begins to
rapidly evaporate.

A pin is placed within the heated region that has a high potential
applied to it and produces an electrical discharge that ionises
eluent molecules via charge transfer reactions.
APCI Schematic
Electrospray Ionization (ESI) Atmospheric Pressure Chemical
Ionization (APCI)
Summary: The sample solution is sprayed from a Summary: A corona discharge is used to ionize
hollow capillary needle across a high-potential the analyte in the atmospheric pressure
difference (a few kilovolts) into an orifice in the region. The gas-phase ionization in APCI is
interface. Heat and gas flows are used to desolvate more effective than ESI for analyzing less-
the ions existing in the sample solution. polar species. ESI and APCI are
Electrospray ionization can produce multiply complementary methods.
charged ions with the number of charges tending to
increase as the molecular weight increases.
Sample Introduction Sample Introduction
• Flow injection • Flow injection
• LC/MS • LC/MS
• IC/MS • Typical flow rates are from 200 µL/min up to over
• Typical flow rates are from 50 µL/min up to >1 mL/min 1 mL/min

Benefits Benefits
• Good for charged, polar, or basic compounds • Good for less polar compounds
• Excellent for IC or reversed-phase LC • Excellent liquid chromatographic interface for
• Permits detection of high-mass compounds at low normal phase separations
mass-to-charge ratios(m/z less than 2000) • Complementary to ESI
• Very low chemical background yields excellent • Can control presence or absence of
detection limits fragmentation by varying the interface
• Can control presence or absence of fragmentation by potentials
varying interface potentials
Mass Range Mass Range
• Low-moderate. Typically less than 1000 Da (singly • Low-moderate. Typically less than 1000 Da
charged m/z) (singly charged m/z only in APCI)
• Med-high. Typically less than 100,000 Da (multiply
charged m/z)
 Congratulations!

You have made an ion.

Now what do you do with it?
 MS vs. MS/MS

Mass
LC Inlet Ionize Detect
Analyze

Separation
MS Identification

Mass Mass
Inlet Ionize Fragment Detect
Analyze Analyze

MS1 Collision MS2

Cell

MS/MS
 What is Tandem MS?
• Uses 2 (or more) mass analyzers in a
single instrument
– One purifies the analyte ion from a mixture
using a magnetic field.
– The other analyzes fragments of the analyte
ion for identification and quantification.
Mixture of Single Fragments
ions ion

Ion
MS-1 MS-2
source
 Analytical Assays used in
Pharmaceutical Industry Labs for New
Chemical Entities
Method 1990 1998 2000 2006
HPLC
75% 50-60% 20% 2%
(UV &Fluorescence)

GC/MS
12% 3% 2% 0

LC/MS/MS
3% 40-50% 60-75% 98%

Immunoassay
10% 10% 10% 0
(ELISA/FPIA etc.)
 Applications of Tandem MS
• Biotechnology & Pharmaceutical
– To determine chemical structure of drugs and drug metabolites.
– Detection/quantification of impurities, drugs and their metabolites
in biological fluids and tissues.
– High through-put drug screening
– Analysis of liquid mixtures
– Fingerprinting
• Nutraceuticals/herbal drugs/tracing source of natural products or
drugs
• Clinical testing & Toxicology
– inborn errors of metabolism, cancer, diabetes, various poisons,
drugs of abuse, etc.
Quadrupole Theory
Pre-filter Quadrupole Mass Filter Stable Trajectory

Unstable Trajectories

 Only ions with the correct m/z values have stable

trajectories within an RF/DC quadrupole field.
 Ions with unstable trajectories collide with the rods, or
the walls of the vacuum chamber, and are neutralised.
 Triple Quadrupole Mass Analyzer

Sample Inlet

Q2
Ion Guide (Collision cell)
EM
Q1 Detector
Q3
 Collision
cell MS2
MS1
Full Scan Acquisition Mode

Collision
MS1 Cell MS2

Scanning Rf only, pass all masses

SCANNING MODE: The first quadrupole mass
analyzer is Scanning over a mass range. The
collision cell and the second quadrupole mass
analyzer allow all ions to pass to the detector.
Full Scan Acquisition Mode Mass Spectrum: Progesterone

[M+H]+ O
CH3
315.1 CH3
100
CH3

O
%

316.1

0 m/z
200 220 240 260 280 300 320 340 360 380 400
 Collision
cell MS2
MS1
Product ion scanning

Argon gas

Products
Precursor

Static (m/z 315.1) Scanning

The first quadrupole mass analyzer is fixed at the mass-to-charge
ratio (m/z) of the precursor ion to be interrogated while the second
quadrupole is Scanning over a user-defined mass range.
 Collision induced dissociation

Argon gas
O CH3
O CH2
CH3
CH3
CH2
O
CH3

H3C

CH3
O

Precursor ion Product ions

• In the collision cell, the TRANSLATIONAL ENERGY of

the ions is converted to INTERNAL ENERGY.

Collision conditions (FRAGMENTATION) are controlled by altering:
 The collision energy (speed of the ions as they enter the cell)
 Number of collisions undertaken (collision gas pressure)
 Product Ion Scan Mode in a
Triple Quadrupole
Q1 Mass selection Q2 collision cell Q3 Full Scan

A
C
B Ion C C+Ar Products Products
D

Ions Q1 only Fragment Q3 Scans for

in transmits Ion C products
Source Ion C
Product ion scanning ↑ collision energy > ↑ fragmentation
100
%
5eV
0

100
% 10 eV
0

100
20 eV
%
0

100
%
30 eV
0

100
%
40 eV
0 m/z
20 40 60 80 100 120 140 160 180 200 220
 Product Ion Spectrum:
Progesterone
Product ion scanning
O
CH3 315.1
100 CH3

CH3 Mass Spectrum from

%
MS1
O
316.1
Precursor ion
0 m/z
300 305 310 315 320 325 330

97.0 CH2
O CH3
109.0
100 O
CH2

H3C
Product ion spectrum from MS2
% CH3

Product ions
0 m/z
100 125 150 175 200 225 250 275 300 325
 Collision
cell MS2
MS1
Multiple Reaction Monitoring

Argon gas

Product(s)
Precursor(s)

Static (m/z 315.1) Static (m/z 109.0)

Both the first and second quadrupole mass analyzers are held Static at
the mass-to-charge ratios (m/z) of the precursor ion and the most
intense product ion, respectively.
 Progesterone Analysis The State Lab

- Bovine Plasma sample fortified at 1 ppb

XIC of +MRM (21 pairs): Exp 1, 315.000/108.800 Da ID: ... Max. 4.5e6 cps. XIC of +MRM (21 pairs): Exp 1, 315.000/96.800 Da ID: ... Max. 2.9e6 cps.
13.01 13.01
4.4e6 2.8e6
4.2e6
2.6e6
4.0e6

3.8e6 2.4e6
3.6e6

3.4e6 2.2e6

3.2e6
2.0e6
3.0e6

2.8e6 1.8e6

2.6e6
1.6e6

In te n s ity , cp s
In te n sity, cp s

2.4e6

2.2e6 1.4e6
2.0e6
1.2e6
1.8e6

1.6e6 1.0e6
1.4e6
8.0e5
1.2e6

1.0e6
6.0e5
8.0e5
6.0e5 4.0e5

4.0e5
2.0e5
2.0e5

0.0 0.0
10.6 10.8 11.0 11.2 11.4 11.6 11.8 12.0 12.2 12.4 12.6 12.8 13.0 13.2 13.4 13.6 13.8 14.0 14.2 14.4 14.6 10.6 10.8 11.0 11.2 11.4 11.6 11.8 12.0 12.2 12.4 12.6 12.8 13.0 13.2 13.4 13.6 13.8 14.0 14.2 14.4 14.6
Time, min Time, min
 Multiple Reaction Monitoring (MRM)
in a Triple Quadrupole
Q1 Mass selection Q2 collision cell Q3 Mass Selection

A
C
B Ion C C+Ar C1+C2+C3 Ion C2

Ions Q1 only Fragment Q3 only transmits

in transmits Ion C Ion C2
Source Ion C
Analyte Internal Standard Precursor ion Product ions Ion ratio monitored
MS-MS (m/z)

96.4 (1)
DMZ DMZ-d3 142.2 96.4/81.4
81.4 (2)

140.1 (1)
RNZ RNZ-d3 201.2 110.3/140.1
110.3 (2)

82.5 (1)
MNZ DMZ-d3 172.0 82.5/128.2
128.2 (2)

124.3 (1)
IPZ IPZ-d3 170.0 109.4/124.3
109.4 (2)

156.1(1)
SDZ SPZ 251.0 110.3/156.1
110.3(2)

156.1(1)
SMZ SPZ 279.1 186.0/156.1
186.0(2)

130.0(1)
CAR DMZ-d3 263.1 175.0/130.0
175.0(2)

152.0(1)
CAP CAP-d5 321.0 257.0/152.0
257.0(2) 51
Specificity of Detection for LC

UV – chromophore
 all compounds with a chromophore responding at the
selected wavelength will interfere

MS – molecular mass
 interference from isobaric compounds
 chemical noise

MS/MS – molecular mass and structural information

 interference from structural isomers only

52
 HPLC-UV Analysis of Sirolimus in
Whole Blood
1. Wash all glassware in methanol x2 and tert-butyl methyl ether (TBME) x2.
2. Place 50µµL of internal standard (in methanol) into each screw-cap glass
tube.
3. Add 200µ µL Sirolimus calibrator (5x concentrated in methanol) or 200µµL
methanol for patient samples.
4. Add 1.0mL blank whole blood to calibrators or 1.0mL patient whole blood.
5. Add 2.0mL 0.1M ammonium carbonate buffer.
6. Mix thoroughly.
7. Add 7.0mL TBME and extract for 15min.
8. Transfer upper layer to clean tube and re-extract lower layer with 7.0mL
TBME.
9. Combine TBME extracts and evaporate to dryness.
10. Redissolve residue in 5.0mL ethanol and evaporate to dryness.
11. Redissolve residue in 1.0mL ethanol, transfer to Eppendorf tube and
evaporate to dryness.
12. Redissolve residue in 100µ µL 85% methanol.
13. µL (equivalent to 800µ
Inject 80µ µL whole blood) and analyse using two
4.6mm x 250mm C18 columns connected in series (30min run time).

53
Sirolimus: HPLC - UV Example

54
Immunosuppressant Sample Preparation
LC-MS/MS Analysis
Whole Blood
(10µL - 40µL)

Add ZnSO4 Soln.

Add 2 volumes MeCN

with IS, Seal & Vortex Mix

Centrifuge,
Inject 5 - 20µL
 Sirolimus:
Single ion monitoring (MS) LC-MS (SIM) vs LC-UV
100

30µg / L

HPLC-MS SIM m/z 821 %

0
1.5 min

HPLC-UV

56
Product ion scanning
Ar (2.5 – 3.0e-3mBar)
Collision
MS1 Cell MS2

Products
Precursor

Static (m/z 821.5) Scanning

The first quadrupole mass analyzer is fixed, or

Static, at the mass-to-charge ratio (m/z) of the
precursor ion to be interrogated while the second
quadrupole is Scanning over a user-defined mass
range.
 NH4+
Product ion scanning
821.5
100
Mass spectrum from MS1
822.5
%
826.5
827.5
810.5
0 m/z
790 795 800 805 810 815 820 825 830 835 840 845 850

768
100

Product ion spectrum from MS2

%

576 786
718 821
548 558 750
0 m/z
200 250 300 350 400 450 500 550 600 650 700 750 800 850 900
Multiple Reaction Monitoring
Ar (2.5 – 3.0e-3mBar)
Collision
MS1 Cell MS2

Product(s)
Precursor(s)

Static (m/z 821.5) Static (m/z 768.5)

MS/MS : Compound-Specific Monitoring

 Sirolimus
Multiple Reaction Monitoring LC-MS(SIM) vs LC-MS/MS (MRM)
100 100

3µg / L 30µg / L

% %
SIR m/z 821

0 0

100 100

% %
MRM m/z 821>768

0 Time 0 Time
0.50 1.00 1.50 0.50 1.00 1.50
Benefit: Excellent Sensitivity; Steroids in Bovine Muscle, 1 ppb
Still need for good Sample Preparation?
Methyltestosterone in Milk

Traditional method
using NaCl as only
purification step

Use of additional
purification by
dispersive SPE using
C18 sorbent material
Confirmatory Information

In the field of Vet Residues and other fields

(sports doping.)

LC-MS/MS is seen as providing unequivocal
confirmation of compound identity if certain
criteria are met.

These criteria are:
 Relative retention time
 Ion ratio
Relative retention Time

65
66
Ion Ratio
68
Benefits of LC-MS/MS

Provides confirmatory data.

Highly sensitive technique even in complex
matrices.

Requires less stringent sample preparation
methodologies.

Allows for multi analyte analysis.

Highly selective technique.
Benefits of LC-MS/MS QqQ

Very good quantitative perfomance.

Possible to detect very low levels of analyte even in
complex mixtures.

Less calibration and maintenance required than higher
resolution systems.

Allows accurate quantitation over a wide range of
concentrations.

In general cheaper to purchase than alternative high
resolution systems.
Drawbacks of LC-MS/MS QqQ

Targeted Analysis only; you won’t detect anything you have

not asked the method to look for.

Each individual transition needs to be tuned for a variety of
parameters (Collision Energy, Capillary Voltage, Ion Spray
Voltage). Gets quite detailed when trying to analyse for lots of
compounds.

Limit to the number of analytes it is possible to detect due to
limitations of the duty cycle of the instrument.

No possibility of retrospective analysis of data to check for
presence of substances which you may have new-found
suspicions about.
High Resolution and Accurate Mass LC-MS
Systems

Time of Flight and (TOF) Orbital Trapping (Orbitrap)

systems offer high resolution and accurate mass
capability.

Resolution values for TOF up to 50,000 and for Orbitrap
values greater than 250,000 are possible.

Mass Accuracy is independent of resolution and values
of < 2ppm are readily achievable.
Mass Resolution, Mass Accuracy and Precision

•Resolution: Mass resolution is typically

a large number that describes the ability
to distinguish between ions differing in
the mass/charge (m/z) value by a small
increment.
•Mass accuracy is the closeness of the
agreement between the result of a
measurement and a true value (exact
mass).
•Mass precision is the closeness of
agreement between independent mass
measurement results.
 Calculate Resolution and Mass Accuracy

Mass resolution is defined as the observed m/z value divided

by the smallest difference (Δ) m/z for two ions that can be
separated: (m/z) / Δ (m/z).

For Orbitrap technology an example of a peak at m/z
200.0000, with a peak width of 0.002 FWHM, the mass
resolution is R = m/Δm = 100,000.

For Mass Accuracy, typically reported in ppm and this is
calculated as (Theoretical Mass-Measured Mass/Theoretical
Mass)*(1,000,000)

Example: Measured Mass from LC-MS: 314.22421
Theoretical Mass of Progesterone: 314.22458
Mass Error of 1.2 ppm
Resolving Power vs Cycle Time
785.8419
R=5901 786.3435
100
R=5900
80 Resolution 7500
60
786.8447
R=5900
40 787.3463
785.5934 R=6000 787.8453
20
R=6200 R=5800
0
785.8421 786.3434
100
R=23801 R=23900
Resolution 30000
Relative Abundance

80

60 786.8446
R=24000
40 787.3457
785.5992 R=24100 787.8471
20
R=24300 R=15600
0
785.8419
100
R=48101 786.3435
80 R=47700
Resolution 60000
60 786.8446
40 R=48200 787.3458
785.5994 787.8477
20 R=47500 R=42000
R=47100
0
785.8413
100 786.3428
R=94801
R=95200
80

60 786.8442
Resolution 100000
R=93600
40 787.3458
785.5989 787.8477
20 R=98000
R=95800 R=89200
0
785.0 785.2 785.4 785.6 785.8 786.0 786.2 786.4 786.6 786.8 787.0 787.2 787.4 787.6 787.8 788.0 788.2
m/z
Benefit of Resolution
 Resolves analytes of interest from each other
 Two Pesticides:
Dimethoate (C5H12NO3PS2), m/z 230.00690 as (M+H)+
Dicryl (C10H9Cl2NO), m/z 230.01340 as (M+H)+
Benefit of Resolution
 Resolves analytes of interest from matrix components
 Isotopes

+Most elements have more than one stable isotope.

For example, most carbon atoms have a mass of 12 Da, but in
nature, 1.1% of C atoms have an extra neutron, making their mass
13 Da.

+Why do we care?
Mass spectrometers can “see” isotope peaks if their resolution is
high enough.
If an MS instrument has resolution high enough to resolve these
isotopes, better mass accuracy is achieved.
Accurate Mass and Elemental Composition

Ability to directly determine the identity of the elemental

composition of an analyte.

The accurate determination of the monoisotopic mass peak
restricts the pool of possible elemental composition
combinations significantly.

High mass resolution in combination with accurate-mass
measurements enable the determination of isotopic patterns.

From this information it is possible to produce a list of possible
chemical formula.

A database search (such as on ChemSpider), easily reveals
the identity of the compound.
Benefit of Accurate Mass

How much Mass Accuracy is needed for identification of
molecular formula.

For example Reserpine (C33H40N2O9) has a protonated ion
at m/z 609.28066.

Single quadrupole MS reports mass to +/- 0.1 = 165 ppm

Number of possible formulas using only C, H, O & N, at
various mass errors:

• 165 ppm 209, • 10 ppm 13, • 5ppm 7, • 3ppm 4, • 2ppm 2

209 (single quad resolution); 2 possibilities accurate mass
systems.

Accurate mass limits the number of possible formulae for a
given m/z measurement in this case to two possible formulas.
Benefits of High Resolution LC-MS

Ability to detect and determine unknown substances.

Not necessary to tune for each analyte singly but can use
generic conditions specified over a mass range of interest
(100-600 m/z).

Technically no limit to the number of substances you can
detect within a given mass range.

Possible to carry out retrospective analysis of data to look for
substances that you may now suspect could be present.

Can be a very specific technique.
Drawbacks of High Resolution LC-MS

Traditionally not as good at quantitation as triple quadrupole

systems.

More time required to calibrate systems and calibration needs
to be carried out more regularly.

Very large data files produced which can prove difficult to
store.

Requires higher user knowledge to trawl through the huge
amount of data produced and requires a fast PC.

Typically not as sensitive a technique as triple quadrupole
analysis.
 Thanks
and
Any Questions