Introduction to Mass Spectrometry

Introduction to Mass Spectrometry
Jenny Moshin
Staff Application Specialist

What is a Mass Spectrometer
Mass Spectrometers measure:
− atomic weight of single elements (ICP-MS)
− the molecular weight of molecules (LCMS, MALDI MS)
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Newborn Screening for metabolic conditions
by LC-MS/MS
− Newborn screening is the process of testing newborn babies
for treatable diseases:
− genetic
− endocrinologic
− metabolic
− hematologic
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Neonatal Screening
Blood Specimen Collection:
− A few drops of blood taken from the baby’s heel are put on
special filter paper. The blood is then sent to a state-approved
lab for testing
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Metabolic Diseases
− Metabolic diseases affect the body’s ability to use certain parts
of food for growth, energy, and repair.
− The parts include:
– Amino acids from proteins,
– Fatty acids from fats
– Organic acids from proteins, fats, and sugars.
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Some of the metabolic diseases screened for by the
NBS Program:
− Phenylketonuria (PKU)
− Galactosemia
− Maple Syrup Urine Disease (MSUD)
− Medium-Chain Acyl-CoA Dehydrogenase Deficiency
(MCADD)
− Biotinidase Deficiency
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25-Hydroxy Vitamin D by LC/MS/MS
− Vitamin D is clinically useful to diagnose disorders related
to intestinal malabsorption and vitamin D deficiency or
intoxication.
− The 25-Hydroxy vitamin D blood test allows
monitoring for therapeutic response in patients being
treated for vitamin D-related disorders.
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Background
− 25-Hydroxy vitamin D (250HD) is the major circulating
form of vitamin D and the precursor of the active form
(1,25-dihydroxy vitamin D). It has a long half-life, which
makes its measurements ideal for assessing a patient’s
vitamin D level
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Components of a Mass Spectrometer
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LCMSMS –What can it do?
− The two most important uses of LC/MS/MS
– Identify unknown species (structural elucidation)
– Quantification (how much of an analyte is in your sample)
− Technologies
– Spherical and Linear Ion Traps
– Time-of-Flight (TOF) MS & Hybrid Quadrupole TOF MS
– Quadrupole based MS
– Hybrid Quadrupole –Linear Ion Trap MS
− Application areas
– Small molecules
– Proteomics and Biomarker research
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Benefits of LCMSMS
− Extreme detection power for single and multi analyte analysis
− Often simplified sample preparation (typically no derivatization
needed)
− Can be used to identify unknowns (structural elucidation) and
quantitation
− Simpler LC methods and shorter LC run times >> more throughput
− Analytes from low to very high polarity can be efficiently ionized
− Rugged instrument design and high throughput
– Lower cost/sample
– Increased productivity
– Easy-to-use
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Instrument performance criteria which matter
− Ion source & interface design
− Number of analytes that can be quantified from single injections
− Minimum dwell time and pause time and consequences on
Signal/noise
− Absence of cross-talk
− Detector technology
− Sensitivity, signal/noise (S/N) & detection power
− Dynamic range
− Scan speed
− Systems ruggedness when analyzing many real samples
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System Components
Eluent
Delivery
Eluent
Reservoir
Pump
Houses the mobile phase continuously
applied to the column. Usually consists of two
bottles, one aqueous and one organic.
Accurately delivers eluent
to the column. Capable of
forming precise eluent
gradients.
Introduces the sample into
the mobile phase stream
UV/VIS
Refractive Index
ELSD
Mass
Spectrometer
Detection
Sample
Introduction
Separation
Guard
Column
Analytical
Column
Thermostatted
Column
Compartment
The heart of an HPLC
system. The column
separates the individual
analytes from the matrix.
Stable temperature ensures
constant retention times and
may reduce column
backpressure.
The detector monitors the
presence of a compound
in the mixture.
Data Acquisition
and Instrument
Control
Interprets the data
collected from the
detector.
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Reversed Phase Mechanism
Pump Delivering Higher Concentration of Organic Solvent
aqueous
1. The autosampler picks up sample from the vial.
2. The sample is injected onto the column.
3. Initially, the pump delivers a low concentration of organic solvent, and the analytes “stick”
on the column.
4. As the concentration of organic solvent increases, the analyte “unsticks” from the column,
separating one analyte from another.
%B
organic
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Reversed Phase Mechanism
H 2 O
H 2 O
Aqueous Mobile Phase
Si
O
Si
O
Si
O
O
O
Si
Si
Si
H 2 O
H 2 O
H 2 O
H 2 O
O
Si
O
Si
H 2 O
silica ( -OH)
H 2 O
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Reversed Phase Mechanism
H 2 O
H 2 O
Acetonitrile in Mobile Phase
Si
O
Si
O
Si
O
O
O
Si
Si
Si
ACN
ACN
ACN
ACN
ACN
ACN
H 2 O
ACN
H 2 O
ACN
O
Si O Si
silica ( -OH)
ACN
ACN
ACN
H 2 O
ACN
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HPLC versus UHPLC
−
Conventionally, HPLC analysis is run on columns with a particle size 3µm or larger
– Useful for samples with high matrix
– Works with standard HPLC pumps
−
−
Recently, there has been a movement to columns packed with column particles ≤2µm
– Less diffusion in the column
– Decreases the chromatographic run time
– Produces sharper peaks
– Requires pump capable of increased column backpressure
Both methods have advantages, and each is easily coupled to a mass spectrometer
0 3 6 9 12 15 18 0 3 6 9 12 15 18
Minutes
Conventional Chromatography
0 0.3
0.6 Minutes 0.9
Ultra High Pressure Chromatography
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How Mass Spectrometry Works – The Basics
Ionized
compounds are
introduced into
the instrument
Ions are
sorted by m/z
1. 2.
4.
intensity
m/z
the results are displayed
the signal is
detected and
counted
3.
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Convenient “Plug and Play” Sources
Turbo V source with ESI & APCI probes
• Efficiently ionizes compounds and eliminates cross contamination
• Accommodates large sample loads and LC flow rates up to 3ml/min
• Embedded ceramic heater technology and improved gas dynamics enable low
detection limits
• Quick change TurboIonSpray® probe and APCI probe let you switch ionization
modes in seconds
Optional DuoSpray ion source
Optional PhotoSpray® source
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Ion Spray
Voltage
(IS)
LC
Flow
Gas 1
(GS1)
Electrospray Ionization Theory
(positive mode shown)
Droplet
Gas 2 (GS2)
& Heat (TEM)
Curtain Plate
+
Solvent molecule
Ion of interest
Curtain
Gas(CUR)
+
+ - +
+ +
+ +
+
+
+ +
- ++
+
+- +
++
+
+
+
+ +
+
Orifice
(DP)
1. Formation of
Charged
Droplets
2. Evaporation 3. Ionic repulsion
w/clustering
4. Ions enter
Mass Analyzer
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Corona
discharge
needle
LC
Flow
Heated
nebulizer
APCI Theory
+
Gas 1
Curtain Plate
(GS1)
+
+ .
+
+
+ +
+
+
+
+ +
+ . + .
+ .
+
+
+
+
+
+
+
+ . + . + .
O 2 or N 2
Solvent molecule
Ion of interest
Curtain
Gas(CUR)
+ . Orifice
+
1. Molecules
in gas phase
2. Corona
discharge
needle ionizes
N2 or O2 in
source
3. N 2 or O 2
pass charge
to vaporized
solvent
4. Vaporized,
charged solvent
passes charge
to Analyte
5. Ions enter
Mass Analyzer
aided by DP
(DP)
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When to use the various sources
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Triple Quadrupole Overview
6
1
Ion Production
2 Ion Transmission
2 3 4 5
3
Mass Filter
1
TM
4
Fragmentation
5
Mass Filter
6
Detector
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Triple Quadrupole (Tandem) MS schematics
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LC/MS/MS – Multiple Reaction Monitoring (MRM)
Filter precursor ion Fragmentation Filter product ion
− Superior selectivity and sensitivity for quantitation of targeted
compounds
− MRM ratio calculation for compound identification (qualifier/quantifier)
− Multi-target screening for hundreds of compounds
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Multiple Reaction Monitoring (MRM)
N 2
Q0 Q1 Q2 Q3 Detector
LC
MRM
intensity
0 5 min
time
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Single Quadrupole vs. Triple Quadrupole
SIM
MRM
1.6e5
1.5e5
1.4e5
1.3e5
6500
6000
5500
6.9
1.2e5
5000
1.1e5
1.0e5
9.0e4
8.0e4
7.0e4
11.1
4500
4000
3500
3000
6.0e4
5.0e4
4.0e4
3.0e4
2.0e4
1.0e4
0.6
7.1
9.6
2500
2000
1500
1000
500
0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time, min
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time, min
− Higher selectivity resulting in better S/N for quantitation
− Better accuracy and reproducibility
− Wider linear range
− More reliable identification using MRM ratios
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How quantitation is performed
Run calibration curve
Integrate curve
Integrate sample
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Confirmation through MRM
− LCMSMS analysis
usually requires
monitoring 2 MRM
transitions for each
compound –
Quantitation Ion and
Qualifier Ion
− Ion ratio (ratio of the
areas of the 2
transitions) needs to
be calculated
Oxazepam
Quant 287>241
Qualifier 287>269
Lorazepam
Quant 321>275
Qualifier 321>229
Nordiazepam
271>140
271>165
Temazepam
301>177
301>193
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Scheduled MRM Algorithm
1.7e6
1.5e6
1.0e6
5.0e5
0.58
0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
Time, min
− Adjusts detection windows automatically depending on retention time
− Allows detecting many more MRM transitions
− Allows using faster LC
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QTRAP® System Overview
6
1
Ion Production
2 Ion Transmission
2 3 4 5
3
Mass Filter
1
TM
4
Fragmentation
5
Mass Filter
• Q3 can be used as a quadrupole or
as a linear ion trap
6
Detector
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Q1 MS Scan vs. Enhanced MS Scan
Q0 Trap
Trap
Q0 Q1 Q2 Q3 Detector
Q1 MS
Scan
Enhanced
MS Scan
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Product Ion Scan vs. Enhanced Product Ion Scan
Q0 Trap
N 2
Trap
Q0 Q1 Q2 Q3 Detector
Product
Ion Scan
Enhanced
Product
Ion Scan
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Movie
− ..\Help\Brent\4000QTRAP_4x3
_640x480_052808.wmv
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Information Dependent Acquisition (IDA)
− Acquisition method that automatically selects candidate ions for
MS/MS study
Wide variety of survey or
independent scans possible
Survey Scan
Selection criteria
IDA Criteria
Dynamic
Exclusion
Dependant scan
(EPI, Product Ion and MS3)
Acquire MSMS
Spectra
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IDA Workflows
− MultiTarget Screening (MTS)
– MRM or sMRM survey scan will trigger EPI scans
– High sensitivity
− General Unknown Screening (GUS)
– EMS survey scan will trigger EPI scans
– Searching for unknown compounds
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EMS Triggering EPI for Library Search
EMS
intensity threshold
Threshold
EPI
297
159
69 173
81 141
109
115
176
201
255
Library Search
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sMRM Quant Survey Triggering EPI for Library
Search
Scheduled MRM Algorithm
intensity threshold
Threshold
EPI
297
159
69 173
81 141
109
115
176
201
255
Library Search
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Cliquid ® 3.0 Software
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Cliquid ® 4-Step Workflow
1. Choose a Test 2. Build Sample List
4. Submit Samples 3. Select Report
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Automatic Report Generation
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iMethod Test: Instant Methods for
Accelerated Results
− Download a lab proven method from the web or a CD
− Add your samples
− Ready to go
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http://www.absciex.com/imethods/
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iMethod Tests
– Inborn Errors of Metabolism
– Antiretrovirals
– Vitamin D
– Immunosuppressants
– Steroids Panel
– Pain Panel
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Summary
− LC-MS/MS provides the selectivity and sensitivity required to analyze
patient samples for clinical research applications.
− LC-MS/MS allows for simultaneous quantitaion and verification of
results
– Can detect non-volatile, polar and thermally labile compounds
– More sensitive than GC, LC or GC/MS
– More information for confirmation via RT, ion ratios, library searching
− LC-MS/MS has the ability to monitor hundreds of analytes per
analysis
– Monitor hundreds of analytes per analysis
– Obtain full scan confirmatory MS/MS in the same run
– High throughput capabilities – fast chromatography and multiple analytes
can be screened for in the same run
− Cliquid Software and iMethod Tests provide ease of use along with a
number of key features
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Legal Acknowledgments
− For Research Use Only. Not for use in diagnostic
procedures.
− UltiMate is a registered trademark of Dionex Corporation
− All other trademarks mentioned herein are the property of
AB SCIEX Pte. Ltd. or their respective owners.
− AB SCIEX is being used under license.
− © 2010 AB SCIEX.
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