MSQ Plus North America Sales Training Chapter 1 – Basic MS Beta Training Version B 20-Dec-11

MSQ Plus North America Sales Training

Chapter 1 – Basic MS

Beta Training

Version B

20-Dec-11

2 Proprietary & Confidential

Agenda for the NA MSQPlus training program

• Comparison of Detectors: General, MS only, MS/MS

• Functional modules common to all MS systems

• MS Features and their importance to customers

• MSQPlus Strengths, Weaknesses, Opportunity, and Threats

• Decision Tree to better understand when to pursue an MS only order or an MS/MS order


MSQPlus beta Sales Training

Attending

Sales Rep Region Manager

Alex Tagol North West Ken Larkey

Zee Nadjie West Ken Larkey

Natalie Odneal Mid West Ken Larkey

Matt Dilts Ohio Valley Ken Larkey

Tucker Lincoln North East Andy Rodriguez

Chuck Costanza South West Mike Hey

Rita Clement South East Mike Hey

Mark Netsch CoE Matt Neely

Charles Ontiveros CoE Matt Neely



Program Goals

•Transfer general knowledge of the MSQPlus, and the competitive landscape, our market position in order to better sell the product.

•Provide feedback on beta training modules to optimize them prior to launch to the NA Sales Team for Chromatography/Advanced Detection.

•Impact on MSQPlus order receipts within the 1st quarter of 2012

•Resource within your region for MSQPlus sales



By the end of this module you will know….

•The key components of Mass Spectral Analysis

•Advantages and limitations of MS in the determinations of unknowns

•Advantages of using Collision Induced Dissociation with MS and MS/MS instruments.

•Advantage of MS over general detection.

•Advantage of MS/MS over MS only detection


Today’s Agenda - Comparison of Detectors

• Detection and Mass Spectrometry Overview

• Analyte Identification by Mass & MS Patterns

• Analyte screening: Retention Time vs. Mass

• Improved MS Specificity through Fragmentation

• What is MS/MS?

• Differentiating Factors: General, MS, & MS/MS Detection

Detection and MS Overview


What does Mass Spectrometry Do?

• Confirm the identity of pesticides and herbicides – Contract Labs

• Determine toxic substances in Catfish - USDA

• Determine Low level ClO4 Analysis – EPA, Universities, Municipalities, Contract Labs

• Determine explosives residues – RCMP, ATF, Fire depts., MRI

• As III / As V Speciation - EPA

• Determine Haloacetic Acids & Bromate – EPA, Municipalities

• Determine the molecular weight of small molecules with biological activity – Pharmaceutical Labs

• Fraction collection of compounds based upon molecular ion - Pharmaceutical Labs

• Determine plating bath contaminants – Semiconductor Labs

Did you know that mass spectrometry is used to...

Mass Spectrometers have a broad range of applications: ICMS & LCMS.ThermoFisher is the ICMS market leader.


Attributes of the Ideal Detector

•High sensitivity and the same predictable response

•Response to all solutes, or has a predictable specificity

•Linear response over a wide range of solute amounts

•Unaffected by changes in temperature and mobile phase

•Does not contribute to extra-column band broadening

•Reliable and convenient to use

•Fast response

•Provides qualitative information on the detected peak

•Does not destroy the solute

A combination of different detectors can help customers achieve attributes of the Ideal Detector


The Power of Ultra High Performance Liquid Chromatography

0.0 0.5 1.0 1.5 2.0 2.5 3.0Time (min)

UltiMate 3000UHPLC+

AccelaSystems

ViperFittings

Columnsand

Consumables

World-Class Thermo Scientific Chromatography PortfolioUltra High Performance Liquid

Chromatography with UV/VIS detection is the primary workhorse in most analytical labs around the world

HPLC with UV/VIS is a very valuable analytical tool because it offers:

1. High analytical data quality for routine analysis

2. Robust and easy-to-maintain operation

3. Straightforward method building


What if You Want More Information?

Absorbance trace shows a number of components and their possible identity if tR standards are available.

Full-scan MS gives additional/alternate components, possible chemical formulae but can still leave co-eluters unresolved.

Tandem mass spectrometry, MSn, can offer differentiation of co-eluting species and information about structure.

Mass Spectrometry Offers

1.Sensitivity

2.Selectivity

3.Information

LC AbsorbanceUV-VIS

0

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100000

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LC Full Scan MSXIC

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ance

MS2 XIC 167.9

MS2 XIC 181.9

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135.96181.89 300.83107.89

167.90

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107.96 301.01

181.9

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0


+ +++

Mass Spectrometer: Basic Schematic

LC SampleInlet

DataSystem

IonizationSource

MassAnalyzer(s) Detector

++

+++ + +

++

+

+

+ +

++

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Mass Spectra

Creation of Ions

Mixture of Solvent and Compounds

Detection of Ions

Separation of Ions

“A device to measure the mass to charge ratio of individual molecules that have been converted ions”


Key Advantages of using MS Detection

•Selectivity – Co-eluting peaks can be isolated by mass selectivity and are not constrained by chromatographic resolution.

•Peak assignment – A unique mass for the compound of interest is generated, ensuring correct peak assignment in the presence of complex

matrices.

•Molecular weight information – Confirmation and identification of known and unknown compounds.

•Structural information – In-source Collision Induced Dissociation (CID) enables molecular ion fragmentation providing structural determination.

•Sample matrix adaptability – Decreases sample preparation time.

•Quantitation – Quantitative and qualitative data can be obtained easily with limited instrument optimization and simultaneous scan modes.


MSQPlus Mass Detector: Advanced LC Detection

For cost chemists who require increased

productivity through dependable simplicity,

the ThermoFisher MSQPlus is a turnkey

HPLC/MS solution providing superior

chromatographic efficiency and enhanced

mass detection with unsurpassed ease of

use, performance, and ruggedness.

MSQPlus Value Statement

Analyte ID by Mass - Specificity


Identification/Confirmation by Mass

Mass Spectrum of Methomyl - LCMS

Molecular Ion

Base Peak

Adduct

MW = 162

Formula: C5H10N2O2S

m/z 163

m/z 231

m/z 100 m/z 300

The mass spectrum and its interpretation require experience and knowledge in order to identify an analyte. Software can help but does not provide the answer.


Identification/Confirmation by Mass

Mass Spectrum

Chromatogram

Unique pattern can identify compound

m/z 79 m/z 81

Target Analyte

Isotopic patterns can aid in compound identification

Isotope ratios are “gifts of nature” that give patterns to help indentify analytes


Mass vs. Chromatography Selectivity

Analytes can be distinguished by mass even of they co-elute with other compounds or matrix


Mass Detection Enhances Chromatography

Compound B(RT: 1.43min)

Compound A(RT: 1.40min) 205 m/z285 m/z

Which is which?

Compound A or Compound B

Co-eluting peaks can be distinguished by mass – SIM or Extracted Ion


Chromatogram Overlays help Indentify Analytes

(1) Scan Masses

(2) Sum All Ions

(3) Plot

“Total” Ion Chromatogram (TIC)

“Extracted” Ion Chromatogram“Extracted” Ion Chromatogram (EIC)

Mass Extactions and the resulting Mass Chromatogram help to correlate a specific mass to a chromatographic peak

(4) Mass Spectrum

Analyte screening: Mass Spectrometric Detection complements General Detection


Screen Detection Comparison: Conductivity vs MS

Extracted ion chromatograms:1. Sodium, m/z 232. Ammonium, m/z 183. Hydrazine, m/z 334. Potassium, m/z 395. 2-(2-Aminoethoxy)ethanol,

m/z 1066. 5-Amino-1-pentanol, m/z 1047. Morpholine, m/z 888. 2-Diethylaminoethanol, m/z 1189. Cyclohexylamine, m/z 10010. Magnesium, 2411. Calcium, m/z 20

0 2 4 6 8 10 12 14 16

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1Cond.

Extracting Ions from the Full Scan and then overlaying the resulting Mass Chromatograms, Correlate Mass with Peaks from a General Detector


Screen Detection Comparison: Conductivity vs MS

Column: IonPac® CS12A-MS, 100 x 2 mm I.D.EG40 Gradient: 8-24 mM MSA in 4 min, then hold, using EG40Flow Rate: 0.25 mL/minSuppressor: CAES™, 21 mADetection: 1. Conductivity

2. Mass specMS Conditions: MSQ™, +ESI,

monovalents @ 50 V, divalents @100 V,

350 ºC, SIM channels as indicatedPeaks:

1. Lithium 500 µg/L2. Sodium 500 3. Ammonium 5004. Potassium 5005. Magnesium 2506. Calcium 250

NH4+, 18 m/z

Na+, 23 m/z

Li+.H2O, 25 m/z

K+, 39 m/z

Mg2+, 24 m/z

½ Ca2+, 20 m/z

Cond.

0 84

Selected Ion Monitoring (SIM) can help Screen for Known Compounds and also, Correlate Mass with Peaks from a General Detector


Chromatogram Overlays help Identify Analytes

Extracted Mass Chromatogram m/z 81

Mass spectrum

Conductivity & Mass Chromatogram overlays – Link peaks time

m/z 79 m/z 81

General Detector Chromatogram

Chromatograms

Compound Identification - Chromeleon makes the process of Extracting Ions from the Full Scan and then overlaying the applicable Chromatograms - Easy


Quantitation using MS

ClO4-QuanY = 0.0122265+0.0012984*X R 2̂ = 0.9996 W: 1/X

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Are

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ClO4-QuanY = 0.0122265+0.0012984*X R^2 = 0.9996 W: 1/X

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ppt

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Selected Ion Monitoring (SIM – MS) or Selected Reaction Monitoring (SRM – MS/MS) are used for Quantitation

Improved MS Specificity through Fragmentation


MS/MS on MSQ Plus? In-Source CID

A: MorphineThere is no significant fragmentation when cone voltage is set at 70 V.

B: HydromorphoneFragmentation dominates the spectrum when cone voltage is set at 125 V.

The MSQPlus via Collision Induced Dissociation can produce fragments of the molecular ion creating “MS/MS like” Spectra


CID – More Energy Increases Fragmentation

Mass Spectra - Anatoxin-a

Cone voltage 50 V

Cone voltage 30 V

Cone voltage 70 V

HN CH3

O

IncreasingEnergy

IncreasesFragmentation

Potential

Increasing Collision Energy (CID – MSQPlus Cone Voltage), will cause fragmentation of the molecular ion, adducts, etc. This can help in analyte ID.


CID can help Elucidate Structure

Functional Group has m/z 44

Mass spectral peaks separated by 44 Daltons

Mass Chromatogram of Triton-X 100 (surfactant)

By increasing Collision Energy (CID), functional groups can fragment away from the molecular ion, helping to elucidate molecular structure

MS/MS – General Overview


MS/MS – Basic Description

Q1

Product Ions

Precursor Ions

Q2Q3

● MS/MS or MSn enable determination of product ions. ● Compared to a single quads, MS/MS instruments have greater specificity due to the uniqueness

product ions. ● Compared to a single quads, MS/MS instruments have greater sensitivity due to reduced chemical

noise.

Step 1: Isolate Target Precursor Ion from the mass spectrum

Step 2: CID

Step 3a: MRM - Analyze MS/MS mass spectrum

Step 3b: SRM - Monitor Selected Ions


MS/MS – The Advantage: Low Chemical Noise

Inte

nsi

ty [

cou

nts

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[µ

S]

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35,000

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ClO4 - 99 - 11.97

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ClO4 - 101 - 11.98

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20,000

Retention Time [min]

4 ClO4 - IStd - 11.95

Conductivity

SIM_01: 99 m/z

SIM_02: 101 m/z

SIM_03: 107 m/z

RT: 0.00 - 14.02 SM: 7G

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RT: 11.60AA: 740997SN: 5299

RT: 11.58AA: 235803SN: 6226

RT: 11.59AA: 227857SN: 21147

NL: 3.76E4TIC F: - c ESI SRM ms2 99.102 [83.012-83.312] MS Genesis Sample_E_3



MS/MS SRM – No Chemical noise MS SIM – Chemical noise present

ClO4 in Infant Formula ClO4 in Urine

Differentiating Factors: General, MS & MS/MS Detection


Differentiating Factors of LC/IC Detection

Factor General Detection Single Stage MS Multi Stage MS

Sensitivity Less SensitiveDetection levels in the PPM levels.

More SensitiveSelected Ion Monitoring (SIM) Detection levels in the low to mid PPB levels.MSQPlus Optimized for Low Mass, Low Level Detection.

More Sensitive in MatrixProduct Ions are isolated from high chemical background lowering detection levels.

Specificity Less SpecificAnalyte determination by retention time only. Confirmation may be available from 3D spectrum

More SpecificMS detectors differentiate by mass/charge

More SpecificProduct Ion mass provides more specific analyte information. Requirement is sample dependent.

Selectivity Not SelectiveTarget analytes that co-elute or elute with matrix cannot be differentiated. Different wavelengths may separate coeluters.

More Selective Co-eluting analytes can be differentiated by mass. A target compound’s mass can differentiate it from matrix.

More SelectiveProduct Ion mass provides more selective analyte information. Requirement is sample dependent.

Ease of Use Easy ModerateSimple walk-up Mass Spec. The MSQPlus is one of easiest to use MS detectors available.

ComplexRequires skilled and experienced operator.














































Speed of Analysis

SlowerChromatographic separation required.

FasterDue to analyte ID by mass, complete chromatographic separation may not be necessary. Lower requirement for sample pre-treatment.

Faster in MatrixProduct Ions are isolated from high chemical background lowering requirement for sample pre-treatment.

Cost of Operation

LessFewer consumables

More Higher cost in terms of operator skill and consumables. The MSQPlus is one of the lowest cost and easiest to use MS detectors available.

MoreHigher than Single Stage MS but may be justified depending on sample requirements.

Purchase Price

Less MoreThe benefit in terms of superior Sensitivity, Specificity, Selectivity, and Speed of Analysis, justifies higher cost.

MoreThe benefit in terms of superior analysis in matrix relative Sensitivity, Specificity, Selectivity, and Speed of Analysis, justifies higher cost.




Speed of Analysis




Cost of Operation




Purchase Price


MoreThe benefit in terms of superior analysis in matrix relative Sensitivity, Specificity, Selectivity, and Speed of Analysis, justifies higher cost.




Speed of Analysis




Cost of Operation




Purchase Price


MoreThe benefit in terms of superior analysis in matrix relative Sensitivity, Specificity, Selectivity, and Speed of Analysis, may justify higher cost.

Quiz


Quiz - Comparison of Detection

1. The Goal of this beta Sales Training exercise is.a.Obtain general knowledge of the MSQPlus and the competitive

landscape

b.Better understand the MSQPlus market position and how to sell the product

c.Impact on MSQPlus order receipts within the 1st quarter of 2012

d.Resource within your region for MSQPlus sales



2. An ideal detector has the following attributes.a.Linear response over a wide range of analyte amounts

b.Utilizes mass to identify the analyte

c.Makes use of software to provide the correct answers

d.Is made in China



3. Mass Spectral Analysis requires which one of the following.a.Physical Separation

b.Ionization

c.Fragmentation

d.Nuclear Reaction to change into a stable isotope



4. Name 2 ways and LCMS can be used to identify an unknown.a.A mass spectral library purchased from NIST

b.Unique isotope ratios

c.Mass of the molecular ion



5. Mass Chromatograms from a Mass Spectrometer can be used to Correlate Mass to a Specific Peak from a General Detector.

a.True

b.False



6. Collision Induced Dissociation (CID) is an MS feature that is used to….

a.Cause fragmentation of the Molecular Ion creating “MS/MS like” mass spectra

b.Elucidate molecular structure

c.Add clusters and adducts to the molecular ion

d.Is only available on MS/MS instruments



7. Advantages of MS/MS over MS only instruments include ….a.Lower detection levels due to lower chemical background

b.Less sample preparation for high matrix samples

c.Improved specificity

d.Superior detection at sales price equivalent to MS only instruments



8. Advantages of MS over general detection include ….a.Determinations of co-eluting analytes

b.Lower detection levels via Selected Ion Monitoring

c.Simultaneous confirmation of knowns and determination of unknowns

d.Cost and Ease of Operation


Future beta training sessions

# Training Module Date

1 Comparison of Detectors 15-Dec-11

2 Functional modules common to all MS systems

5-Jan-12

3 MS Features and their importance to customers

10-Jan-12

4 MSQPlus Strengths, Weaknesses, Opportunity, and Threats

12-Jan-12

5 Decision Tree to better understand when to pursue an MS only order or an MS/MS order

17-Jan-12

Launch of Training Modules to the entire NA Sales Team will be in the form of Adobe Connect Self Paced Sessions and occur within 2 wks beta training.


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Documents

MSQ Plus North America Sales Training Chapter 1 – Basic MS Beta Training Version B 20-Dec-11