comprehensive solutions and technical workflows … · gc/ms and lc/ms data analysis differential...

December 11, 20121

COMPREHENSIVE SOLUTIONS AND TECHNICAL WORKFLOWS FOR THE ANALYSIS ON ENVIRONMENTAL POLLUTANTS

Jaume C. MoralesMS Product Specialist Agilent Technologies

• Introduction

• Traditional approach • New improvements for new problems

Backflush for GCMSGCQTOFt-MRMQual/Quant approachIon funnel Q-TOF technology overview

–Software for compound identification and quantitationAll Ions MS/MSMSC, MetIDMass Profiler Professional

• Example of QUAL/QUANT. Pharmaceuticals in surface water

2

Actual situation on the Enviro and Food analysis

Main concern: reduce impact of contaminants to population

Warranty regulatory MRLs controlContract LabsContract Labs

Manuf/Distribution LabsManuf/Distribution Labs

Research LabsResearch Labs

Off.Control LabsOff.Control Labs Regulatory MRLs control

Surveillance and risk evaluation, emerging risks studies, (treatment, transport, new contaminants)

Main concern: reduce impact of contaminants to population

Warranty regulatory MRLs controlContract LabsContract Labs

Manuf/Distribution LabsManuf/Distribution Labs

Research LabsResearch Labs

Off.Control LabsOff.Control Labs Regulatory MRLs control

Surveillance and risk evaluation, emerging risks studies, (treatment, transport, new contaminants)

Target compoundsTarget compoundsUnknowns & Untarget compounds

Unknowns & Untarget compounds

Target compoundsTarget compounds

Target compoundsTarget compounds

Sampling

Extraction

Workflow: Screening Confirmation & Quantification

Clean-up

S C Q

C16 H19 N3 P ClC15 H25 O P S ClC18 H21 O P Cl

LC/TOF or QTOF – for Unknowns

S

Identification by querying Exact Mass

Data Base

Molecular Formula Generation

Q C2nd injection on MS/MS (QQQ or QTOF)

3 1

LC/QQQ MRM – for Targets

S C Q

3 1

GC/QQQ MRM – for Targets

S C Q3 1

GC/MS (MMI) SIM/Scan– for Targets & Unknowns S

C

Q

Final Report from

Deconvolution

If required

If required

Screening Confirmation Quantification

Optimal Workflow for Target analysis• Quantification of all regulated compounds in one shot can compromise robustness and

sensitivity of the system. Multiresidue methods can show limits depending on chromatography.• A previous screening analysis, using identification techniques, allows to have a list of

compounds which are present on the sample.• Quantification of ONLY those compounds which were positive on the screening analysis is

more robust, sensitive and reliable than trying to afford all regulated compounds at the same time.

Screening Confirmation Cuantification

GC/QQQ

Optimal Workflow for Target analysis

LC/TOF/QTOF

LC/QQQGC/SQ

Sampling

Extraction


Clean-up

S C Q



S


Data Base



3 1


S C Q

3 1


S C Q3 1


C

Q

Final Report from

Deconvolution

If required

If required

Unknowns Analysis w TOF systems

TOF

QTOF

TOFFull Scan HR spectra acquisition

QTOFFull Scan HR spectra acquisitionHR MS/MS spectra acquisition :

S

O

O

O

NH

CH3

N

O

CH3H3C

H3C

Tiapride 0.8 ppm

1x10

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

+ESI Scan (3.893 min) Frag=150.0V 962_D_direkt…

C15 H25 N2 O4 S329.1529

Counts (%) vs. Mass-to-Charge (m/z)328 329 330 331 332

1x10

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

+ESI Scan (3.893 min) Frag=150.0V 962_D_direkt…

C15 H25 N2 O4 S329.1529

Counts (%) vs. Mass-to-Charge (m/z)328 329 330 331 332

Tiapride 0.8 ppm

Unknowns Analysis w TOF systems

11

Analysis with LC/MS TOF/QTOF of Unknown compounds wants to iedentify these compounds through :

• Exact mass• Isotopic distribution • MS/MS spectrum (QTOF)

On top of that, Full Scan acquisition allows for retrospective analysis.

Monoisotopic mass(Diff in ppm)

Isotope spacing(Diff in ppm)

Isotopic Distribution(Diff in %)

Scoring based on

Sampling

Extraction


Clean-up

S C Q



S


Data Base



3 1


S C Q

3 1


S C Q3 1


C

Q

Final Report from

Deconvolution

If required

If required

This has been traditional WORKFLOW.

AGILENT

Is introducing new IMPROVEMENTS

Hardware, Software & Tools

to make it more reliable, easy, robust and sensitive

Sampling

Extraction


Clean-up

S C Q



S


Data Base



3 1


S C Q

3 1


S C Q3 1


C

Q

Final Report from

Deconvolution

(+backflush)

If required

If required

(+backflush)

Microflow Technology Backflush Configurations

Inlet MS/MS

Purged Ultimate Union

EPC

Constant Pressure with Post-column Backflush

Flexibility to add GC detectors and scalable for shorter runtime!

Inlet MS/MS

EPC

Constant Flow with Mid-column Backflush

Provides optimal performance and shortest cycle time!.

Purged Ultimate Union

30 m HP-5ms UI

15 m HP-5ms UI

15 m HP-5ms UI

Extract Analysis

5 10 15 20 25 30 35 40 45 50 55 60 65 70

Run stopped at 42 min and back flushed at 280oC for 7 min

It took additional 33 minand column to 320oC to remove these high boilers.

Blank run after back flushing

min

Column is clean.

Once set, backflush timing will work for ALL high boilers.- heaviest compounds elute later

Sampling

Extraction


Clean-up

S C Q



S


Data Base



3 1


S C Q

3 1


S C Q3 1


C

Q

Final Report from

Deconvolution

(+backflush)

If required

If required

(+backflush)

GCMS?

Sampling

Extraction


Clean-up

S C Q


LC/QTOF or GC/QTOF– for Unknowns

S


Data Base



3 1


S C Q

3 1


S C Q3 1


C

Q

Final Report from

Deconvolution

(+backflush)

If required

If required

(+backflush)

GC/QTOF

GC/Q-TOF for Target, Non-target and Unknowns: High Resolution, Accurate Mass and Fast Acquisition

Rates MS and MS/MS

7200 GC-QTOF A new analytical tool to solve complex analytical problems

=+

What is it?7200 GC/Q-TOF = 7890 + 7000 + 6500

Quadrupole Time of Flight MS

Time of Flight MS

Triple Quadrupole MS

The merging of two platforms

Turbo 2

Ion Pulser

Turbo 3

Ion Source

Turbo 1b Turbo 1a

Quad Mass Filter (Q1)

Collision Cell

Transferoptics

6500 LC/MSQ-TOF based

7000 GC/MSQQQ based

Ion Mirror

Ion Detector

New . . . Yet Totally ProvenDual-stage ion mirror improves second-order time focusing for high mass resolution.

Hexapole collision cell accelerates ion through the cell to enable faster generation of high-quality MS/MS spectra without cross-talk

Split-flow turbo differentially pumps the ion source and quadrupole analyzer compartments

4GHz ADC electronics enable a high sampling rate (32 Gbit/s) which improves the resolution, mass accuracy, and sensitivity for low-abundance samples. Dual gain amplifiers simultaneously process detector signals through both low-gain and high gain channels, extending the dynamic range to 105.

Analog-to-digital (ADC) Detector:Unlike time-to-digital (TDC) detectors which record single ion events, ADC detection records multiple ion events, allowing very accurate mass assignments over a wide mass range and dynamic range of concentrations.

New Removable Ion Source includes repeller, ion volume, extraction lens and dual filaments

Proprietary INVAR flight tube sealed in a vacuum-insulated shell eliminates thermal mass drift due to temperature changes to maintain excellent mass accuracy, 24/7. Added length improves mass resolution.

Hot, quartz monolithic quadrupole analyzer and collision cell identical to the 7000 Quadrupole MS/MS

New Internal Reference Mass can be delivered to the source at a low and high concentration

Two 300L/s t urbos pump the focusing optics and flight tube

1. Internal Reference Mass for routine 2ppm mass accuracy even in heavy matrix

2. Removable Ion Source for quick source cleaning, filament replacement and EI/CI swapping without breaking vacuum

3. Q-TOF MS/MS:• Chemical noise reduction.• Selectivity. • Structural information• Method development

4. Software tools – formula calculator

Key Features of the 7200

GC/MS and LC/MS data analysis Differential analysis with Mass Profiler Professional

23

CE-MS, LC/MS

GC/MS

Sample prep

Data AnalysisChtromatogramMass Spectrum

MassHunter, MSD ChemStation

Detection

Sample prep

Detection

6x10

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

+ TIC スキャン ICA01.D

カウント対測定時間 (min)2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15

Advanced workflow by MPP

Component ExtractionCreate the component list

You can utilize the result of MPP in further data analysis in MassHunter and MSD ChemStation.

Differential analysis using statistic method

Mass Profiler Professional

Feature extraction by MassHunter or AMDIS

Agilent Multi-omics WorkflowSeparate &

Detect

GC-QTOFGC/MSDGC-QQQ

LC-TOF/QTOFLC-QQQ

Feature Finding

MassHunter QualFind by

Chromatogram Deconvolution

MassHunter QualMFE,

Find by Formula,Find by Ion

Alignment & Statistics

Mass ProfilerProfessional

(MPP)

Identify

ID Browser

Mass Profiler(MP)

Pathways

Pathway Architect

24

Pathway Directed Experiment Creation

Propose new experiments based on pathway analysis•Re-examine acquired untargeted metabolomics data based on pathway analysis•Design new experiments (metabolite, protein or genes) based on pathway results interpretation

PCDL

eArray

Spectrum Mill

Build custom metabolite database

Targeted MS/MS

Custom microarray or NGS design

25

Coffee extract has a much more complex matrix although LLE

EIC of 2-Formyl thiophene in Coffee extract at different extraction window

MS/MS Chemical Noise Reduction (EI) When R and Accurate Are Not Enough (1pg OFN in PFTBA Background)

MSm/z 27254:1 S/N

MS/MS272:222216:1 S/N

Analyte ions

Matrix ions Analyte ions with minimal matrix ions

Summary of MS/MS ExperimentsAccurate mass measurement of molecular ion and fragments

C4H5

C10H14N

C9H14NC7H8N

C9H11N

C6H7NO

C6H8NC6H6NO

C5H6N

C5H7N

C4H5

C4H6N

ON

C10H15NO

109122

C7H8NO 136C9H14N

148C10H14N

133C9H11N

80C5H6N

94C6H8N

108C6H6NO

81C5H7N

55C3H5N 53 41

C3H5

66C4H4N78

C5H4N

C6H7NOMS

MS/MS

Summary of MS/MS ExperimentsCalculate possible empirical formulas

C4H5

C10H14N

C9H14NC7H8N

C9H11N

C6H7NO

C6H8NC6H6NO

C5H6N

C5H7N

C4H5

C4H6N

ON

C10H15NO

109C6H7NO

122C7H8NO 136

C9H14N

148C10H14N

133C9H11N

80C5H6N

94C6H8N

108C6H6NO

81C5H7N

55C3H5N 53 41

C3H5

66C4H4N78

C5H4N

MS

MS/MS

Summary of MS/MS ExperimentsMS/MS on fragments + accurate mass to find empirical formulas

C4H5

C10H14N

C9H14NC7H8N

C9H11N

C6H7NO

C6H8NC6H6NO

C5H6N

C5H7N

C4H5

C4H6N

ON

C10H15NO

109C6H7NO

122C7H8NO 136

C9H14N

148C10H14N

133C9H11N

80C5H6N

5378

C5H4N

MS

MS/MS

‐OH

‐CH3‐C5H8

‐CHN‐H2

Summary of MS/MS ExperimentsMS/MS on other fragments

C4H5

C10H14N

C9H14NC7H8N

C9H11N

C6H7NO

C6H8NC6H6NO

C5H6N

C5H7N

C4H5

C4H6N

ON

C10H15NO

109C6H7NO

122C7H8NO 136

C9H14N

148C10H14N

133C9H11N

80C5H6N

94C6H8N

108C6H6NO

81C5H7N

55C3H5N 53 41

C3H5

66C4H4N78

C5H4N

MS

MS/MS

Formula Calculator: formulas consistent with accurate mass and formula of parent molecule

C5H12O2PS3 m/z = 230.9732

MassHunter Tools for GC/Q-TOF

Overlaid chromatograms for each deconvoluted compound found

Compound mass spectrum with mass caliper tool displaying m/z

difference between two ion fragments

Molecular and fragment formula calculation results based on

accurate mass of the ion

Sampling

Extraction


Clean-up

S C Q



S


Data Base



3 1


S C Q

3 1


S C Q3 1


C

Q

Final Report from

Deconvolution

(+backflush)

If required

If required

(+backflush)

SCREENING, QUANTIFICATION & CONFIRMATIONTriggered MRM

36

“Triggered MRM (tMRM) Data Dependent”: ~“Scan”Specificity with MRM Speed and Sensitivity

Quantification & Confirmation with MRM speed and sensitivity

1 monitoring MRM per compound triggers up to 8 confirmation MRM of each of the detected compounds

Very useful for massive quantification and confirmationwith Triple Quadrupole

Página 37

Threshold

Secondary MRM Transitions are “Triggered”

tMRM Product Ion Spectrum

x103

876543210

80 100 120 140 160 180 200 220 240 260 280 300 320 340

334.2171.1

145.0

132.1

117.0

105.191.176.0

tMRM Product Ion Spectrum

x103

876543210

80

76.0

x103

876543210 80 100

91.1

x103

876543210 80 100 120

105.1

x103

876543210 80 100 120

117.0

x103

876543210 100 140

132.1

x103

876543210 80 100 120

119.0

x103

876543210 100 140 180

145.0 x103

876543210 100 140 180

147.0

x103

876543210 80 100 120 140 160 180

171.1

334 > 76 334 > 91 334 > 105 334 > 117 334 > 119

334 > 132 334 > 145 334 > 147 334 > 171

tMRM Library SearchingTebufenpyrad Standard 50 ppb

2x10

0

0.2

0.4

0.6

0.8

1

Cpd 1: Tebufenpyrad: +ESI MRM:361 (9.611-9.752 min, 15 Scans) Frag=165.0V [email protected] (334.2…

147.1000

117.0000

132.100091.1000 105.1000

2x10

-1

-0.5

0

0.5

1

Cpd 1: Tebufenpyrad: +ESI MRM:361 (9.611-9.752 min, 15 Scans) Frag=165.0V [email protected] (334.2…

147.1000

117.0000132.100091.1000 105.1000

2x10

0

0.2

0.4

0.6

0.8

1

Tebufenpyrad C18H24ClN3O + MRM CAS_Library.mslibrary.xml

117.0000 145.0000

132.100091.1000 105.1000

Counts vs. Mass-to-Charge (m/z)75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170

Library match score: 96.75

40

Pesticide AnalysisLC/MS QQQ tMRM Pesticide Application Kit

1200 Series LC and 6400 Series QQQ LC/MS

http://www.chem.agilent.com/Library/flyers/Public/5990-5309EN.pdf

Sampling

Extraction


Clean-up

S C Q



S


Data Base



3 1


S C Q

3 1


S C Q3 1


C

Q

Final Report from

Deconvolution

(+backflush)

If required

If required

(+backflush)

QUAL/QUAN SolutionsNew strategy for High Sensitive QTOF Instruments

QuantAnalysis

QualitativeAnalysis

QUAL/QUAN Solutions :Agilent 6550 Q-TOF with iFunnel Technologies

New- Ion Beam Compression and Shaping (resolution + sensitivity)

New- iFunnel (10X sensitivity gain)

Hexapole axial focusing collision cell (faster MS/MS spectra)

New- Dual Agilent JetStream orthogonal spray (robust and stable mass calibration)

4 GHz digitizer + ADC detector (resolution, mass accuracy, dynamic range)

Longer Invar flight tube(resolution + stable mass accuracy)

New embedded processor(50 spectra/sec)

43

iFunnel Technology Revolutionizes Ion Sampling

More efficient ionization• Thermal confinement of ESI

ion plume• Efficient desolvation to

create gas phase ions• Creates an ion rich zone

44

Increased ion sampling• 6 capillary inlets• Samples 12x more ion rich

gas from the source• Captures the majority of

the gas from the source region

Greater ion transfer

• Removes the gas but captures the ions

• Helps to remove source generated noise

• Extends turbo pump life

HighPressureFunnel

Low PressureFunnel

MS Inlet

Nebulizer

Heated Sheath

GasThermal Gradient Focusing Region

Heat Sink with Forced Air

Cooling

44

But having just a sensitive QTOF is not enough

Data process with a SMART Software is key for

Qual and Quant workflow

45

MassHunter 64 – Integrated Software Tools

Confidence

MassHunter 64 – Harness the Power of 64-bit

Improved performance for the most complex data sets:• Windows 7 64-bit and native 64-bit applications can utilize up to 192 GB of RAM• When a native 64-bit program such as MassHunter Qual runs on Windows 7,

it can use all the remaining memory!

2 GB 6 GB (shipping Workstation)Up to 24 GB optional Windows 7 Pro

--MassHunter Qual--MassHunter Quant

=

What MassHunter enables for Users?Identify MORE Compounds with Greater Confidence

Unique

Find by Formula

Compound information from formula or Database

Ion Information

5x10

1

2

3

4

5

6

7

Cpd 4: Tramadol: +ESI EIC(264.1958; 281.2224; 28…

6.92

Counts vs. Acquisition Time (min)5.5 6 6.5 7 7.5 8 8.5

Ion chromatogram drawn from information

Spectra extracted from integrated peak

Spectral performance calculated

All Ions MS/MS NEW!AIM for confidence in identification

50

Retrospective Analysis

MassHunter 64 – Identify with ConfidenceAccurate Mass LC/MS Application KitsUntargeted Screening with TOF or QTOF

51

All Ions MS/MS is enabled on all high-res instruments• 6200 Series TOF Instruments

• 6500 Series QTOF Instruments

• 6550 Series iFunnel Q-TOF

52

Ion Source Region

Collision Cell

ALL Ions MSMS Workflow

53

NEW Find by Formula Algorithm for All ions

Use PCDL Lib as source of fragments

Export modified CEF file for Quant method building

Quant requirement : Ability to convert new CEF file in Quant method and Quantitate on All Ions data file.

PCDL Requirement : PCDL library content for pesticide available.

Acquisition PCDLPCDLFindbyFormula on MS low channel

EICs for fragments on MS high channel

Correlate qualified fragments with target

Qual

.d data file Targets

Find

m/z, ion species

Extract

Qualify and confirm fragments

Export

Align EICs of fragments with

parent EIC

Targets fragment ions

All Ions MSMS Results Overview

54

1

3

2

4 5

Compound results .cef file Quant method- Accurate mass

EICs for quantifier and qualifier

- Relative responses- Retention times

+ Scan (6.299-6.547 min, 15 scans) Tomato 10ppb-r003.d

Mass-to-Charge (m/z)741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756

4x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6 746.4838(M+H)+

747.4871(M+H)+

748.4901(M+H)+

749.4929(M+H)+ 751.4984

(M+H)+

Adduct patternReference pattern library

Import and build Qual/Quant Method from Qual

55

MH Qual

MH Quant

Quant Method after import of All Ions MS/MS results from Qual

56

Collision energies

Product ion information Relative response ratios

Uncertainty values updated following point system rules

Quant Batch at a Glance

57

Pharmaceuticals in surface water

Direct injection of water samples using high resolution mass spectrometry utilizing the new 6550 Series Agilent QTOF

58 Vancouver , May 2012ASMS 2012

Content

• Direct Analysis of pharmaceutical in water

• Method Setup

• Quantitative Performance

• Qualitative Performance

• Summary


Instrumental Setup

• Agilent 1290 UHPLC System including high performance auto sampler and column compartment

• Up to 1200 bar pressure• Low dead volume for maximum turn around time

• Agilent 6550 iFunnel- QTOF system• New iFunnel sample introduction for maximum sensitivity• 40000+ resolution for best matrix discrimination and compound

identification• 4.5 orders in spectral linearity for best quantitative performance


QTOF Parameters – Mass Spectrometer

61

MS Screening Method:

MS 1 Mode: 1.5 scans/sec, Mass Range: 100 –1000 m/z

Auto-MS/MS method (data-dependent):

MS/MS: 5 scans/sec, Mass Range: 100 – 1000 m/z for MS1

5 scans/sec, Mass Range: 50 – 1000 m/z for MS2Precursors per cycle: 10, Threshold 3500 countsActive Exclusion after 1 spectra for 0.08 minutes

The collision energy (CE) varied proportionally to the precursor m/z valueat a slope of 6 eV/100 m/z and an offset of 4 eV.

Method 1: Long Gradient 0.4 mL/min

Nebulizer: 27 psigDrying gas: 8 L N2/min @320°CSheath gas: 11 L N2/min @380°CNozzle: 500 VVCap: 3500 VFragmentor: 120 V

Method 2: Short Gradient 0.8 mL/min

Nebulizer: 35 psigDrying gas: 9 L N2/min @325°CSheath gas: 11 L N2/min @400°CNozzle: 500 VVCap: 3500 VFragmentor: 120 V

Vancouver , May 2012ASMS 2012

Compound results .cef file Quant method- Accurate mass

EICs for quantifier and qualifier

- Relative responses- Retention times

+ Scan (6.299-6.547 min, 15 scans) Tomato 10ppb-r003.d

Mass-to-Charge (m/z)741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756

4x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6 746.4838(M+H)+

747.4871(M+H)+

748.4901(M+H)+

749.4929(M+H)+ 751.4984

(M+H)+

Adduct patternReference pattern library

Import and build Qual/Quant Method from Qual

62 Vancouver, May 2012ASMS 2012

MH Qual

MH Quant

Find by Formula to evaluate compounds, adducts and isotopes, here Diazepam as an example

63

+-10ppm extraction window

0.27 ppm mass errorResolution 30000


Quantitative Performance MS ScanPropranolol

64

3x10

0

1

2

3

Cpd 17: Propranolol: +ESI EIC(260.1645, 277.1911) Scan Frag=225.0…Noise (RMS) = 653.06; SNR (3.510min) = 5.0

3.510

4.365

Counts vs. Acquisition Time (min)2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4

4x10

0

1

2

3

4


3.511


5x10

0

0.5

1

1.5

2


3.515

4.370


MRL 100 ppt20 ppt

LOD 2 ppt

Propranolol - 15 Levels, 15 Levels Used, 30 Points, 30 Points Used, 0 QCs

Concentration (ng/ml)-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210

6x10

-0.4

-0.20

0.20.4

0.60.8

1

1.21.4

1.61.8

22.2

2.42.6

2.83

3.2

3.43.6

3.84

y = 20410.370029 * x - 8277.405922R^2 = 0.99773342Type:Linear, Origin:Include, Weight:1/x


Quantitative Performance MS ScanKetoprofen

65

3x10

0

0.5

1

1.5

2

Cpd 16: Ketoprofen: +ESI EIC(255.1016, 272.1281) Scan Frag=225.0V …Noise (RMS) = 495.84; SNR (4.225min) = 3.5

4.225

Counts vs. Acquisition Time (min)3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5

3x10

0

0.5

1

1.5

2

2.5

3

3.5

Cpd 16: <Ketoprofen>: +ESI EIC(255.1016, 272.1281) Scan Frag=225.…Noise (RMS) = 475.95; SNR (4.231min) = 6.4

4.231


4x10

0

0.2

0.4

0.6

0.8

1

1.2

Cpd 16: Ketoprofen: +ESI EIC(255.1016, 272.1281) Scan Frag=225.0V …Noise (RMS) = 514.75; SNR (4.224min) = 24.1

4.224


MRL 100 ppt20 ppt

LOD 10 ppt

Res

pons

es


Direct injection 100µL surface water spikeResults Table

Compound Name LOD LOQAtenolol 0.84 3.59Sotalol 0.44 2.01Metronidazole 0.73 3.19Ronidazole 2.65 9.73Sulfadiazine 2.69 9.87Trimethoprim 3.02 11.02Sulfamerazine 2.55 9.39Sulfadimidine 2.59 9.53Phenazone 0.47 2.14Pentoxifylline 1.07 4.47Metoprolol 0.56 2.51Furazolidone 2.27 8.47Dapson 3.85 14.70Sulfamethoxazole 2.97 10.84Carbamazepine 10,11 epoxide 0.82 3.52Phenacetin 0.81 3.50Bisoprolol 1.38 5.52Propranolol 1.13 4.67Carbamazepine 0.53 2.39Betaxolol 1.06 4.40n,n‐Diethyl‐3‐methylbenzamide (DEET) 0.95 4.02Bezafibrate 1.12 4.62Crotamitron 0.49 2.22Diazepam 0.43 1.97Fenofibrate 3.35 12.31

All Values in ng/L (ppt)

Based on DIN 32645 calculation usingSQS 2010, a visual basic add on for ExcelCourtesy of Dr. Joachim Kleiner(www.kleiner-j.de)

All values not more than 15% of the100 ng/L MRL typical for water analysis


Qual/Quan Results MS-Scan

67

50 ng/L

100 ng/L


Qual/Quan Results data-dependent MSMS

68

50 ng/L


Quantitative Performance MS-Scan vs. data dependent MSMS mode

69

Res

pons

es

Res

pons

es

MS-Scan


Compound at a glance – Outlier


Correlate observed AM MS/MS fragments into proposed structure• Using “novel systematic bond breaking approach”, based on work from

Alastair Hill, RCM 2005, 19, 3111-3118)• The fewer bonds need to be broken, the higher the probability that a proposed

fragment will show in the MS/MS spectrum. Breaking double bonds gets assigned a higher penalty than breaking a single bond

• Same scientific approach as WATERS Mass Fragment• Much faster than MassFragment• Unique: Can do parallel processing of hundreds of structures !

Availability of MassHunter MSC• Will ship with MassHunter B.05.00 as a Tool on Supplemental Disk for FREE!

TOF Generate Formula

Correlate Structure

MSQ-TOF

MS/MS MS/MS

Harness the full power of AM MS/MSMS/MS Structural Correlation (MSC)

Confirmstructures

Unknown analysis

MassHunter MS/MS Structural Correlation (MSC)USE CASE 1: Confirm proposed structures

• Provides highest confidence in confirmation of proposed structures in minutes instead of hours• USE CASE 1: Assigns fragment ions to substructures of a proposed

parent structure• Metabolite ID• Metabolomics• Applied Markets Screening

MassHunter MSC – USE CASE 1Confirm proposed structure via accurate mass MS/MS

All fragment ions exhibit very high scores (few bond breakages), but multiple candidate isomeric structures are possible, e.g. 14 for 197.0752 .

All fragment ions exhibit very high scores (few bond breakages), but multiple candidate isomeric structures are possible, e.g. 14 for 197.0752 .

Showing individual substructures with penalties for 197.0752

Showing individual substructures with penalties for 197.0752

Total score very high (97.1) due to high individual scores and explanation of all high intensity fragment ions.

Total score very high (97.1) due to high individual scores and explanation of all high intensity fragment ions.

MSC calculates all possible subformulas for each fragment ion with mass deviation in mDa

MSC calculates all possible subformulas for each fragment ion with mass deviation in mDa

MFG score based on accurate mass of each MS/MS fragment ion and its neutral loss from precursor

MFG score based on accurate mass of each MS/MS fragment ion and its neutral loss from precursor

MSC calculates molecular formulas (MFG) based on precursor and fragment ions (unique!). The correct formula has a lower precursor score, because only two isotopes were put in and the abundance could not be accurately determined by measuring the printout!

MSC calculates molecular formulas (MFG) based on precursor and fragment ions (unique!). The correct formula has a lower precursor score, because only two isotopes were put in and the abundance could not be accurately determined by measuring the printout!

Beta Jan 2011Release Mid 2011

MassHunter MS/MS Structural Correlation (MSC)USE CASE 2: Aid true unknown analysis

• Help identify true unknown compounds • STEP 1: Generate as few as possible molecular formulas from unknown

compounds using MS and MS/MS

• STEP 2: Retrieve all possible structures for the high scoring formulas

• STEP 3: Run MS/MS Structural Correlation in batch mode on all formulas=> unique to MassHunter MSC

• STEP 4: Review structures from high scoring structures !

Retrieved 588 structures and ran MS/MS structural correlation on those.RESULTS in 40 SECONDS !

Retrieved 588 structures and ran MS/MS structural correlation on those.RESULTS in 40 SECONDS !

94% of Fragment ions explained94% of Fragment ions explained

Correct structure in 3rd positionCorrect structure in 3rd position

Generate Molecular Formula GenerationGenerate Molecular Formula Generation

MassHunter MSC – USE CASE 2Correlate total unknown versus large compound set

O

NO

FF

F

N S

O

NOO

NO

O

NO

FF

F

N S

O

NOO

NO

O

NO

FF

F

N S

O

NOO

m/z 568, major

O

NO

FF

F

N S

O

NOO

m/z 554, major

O

NO

FF

F

N S

O

NOO

NO

O

m/z 625, major

m/z 655, 2 in rat species

Parent, [M+H]+ m/z 639

O

NO

FF

F

N

m/z 341, major

O

NO

FF

F

N S

O

NOO

NO

O

NO

FF

F

N S

O

NOO

NO

m/z 611

m/z 597

O

NO

FF

F

N S

O

NOO

O

m/z 584

O

NO

FF

F

N S

O

NOO

O

m/z 570

O

NO

FF

F

N S

O

NOO

NO

Om/z 641

O

NO

FF

F

N S

O

NOO

m/z 540

- CH2

- CH2

- CH2

+ O2

- CH2

+ O2

+ O2

Structure Pool, created by either:• 3rd party Metabolite prediction Software

• Data base search / look-up

• Self-drawn (e.g. with ACD/Labs ChemSketch)

MetID software Looking for metabolites of my compounds

m/z

MS/MS

10

5

1

4

2

3

98

67

14

12

15

13

16

1711

23

2022

2425

NH

28 26

CH319H

H

HCH321 H

CH327H

CH318

Measured MS/MS spectrum of metabolite

Assigned structure to metabolite

Metabolite Structure Elucidation – Fragment Assignment

m/z

MS

MS/MS

Fragmentation

Metabolite Sample

m/z

10

5

1

4

2

3

98

67

14

12

15

13

16

1711

23

2022

2425

NH

28 26

CH319H

H

HCH321 H

CH327H

CH318

Manual or software guided solution for fragment assignment with identification of metabolism reaction by detection of chemical shifts

HO

10

5

1

4

2

3

98

67

14

12

15

13

16

1711

23

2022

2425

NH

28 26

CH319H

H

HCH321 H

CH327H

CH318

m/z

MS

m/z

MS/MS

Fragmentation

Control Sample

QTOF MS/MS spectrum of Buspirone. Measured accurate masses of the fragments are annotated in red and calculated relative mass accuracies are annotated in blue.

A

4x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3+ Product Ion (399.3 sec) (386.2536 -> **)

122.0717-3.49 ppm

222.1499-4.70 ppm

265.1921-3.94 ppm 386.2539

2.98 ppm180.1021-1.08 ppm

150.1030-2.84 ppm

G

A FD

C

E

Abundance vs. Mass-to-Charge (m/z)60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

N

O

O N NN

N

A) C9H14NO2168.1019

H) C17H27N2O2291.2067

E) C8H12N3150.1026

B) C12H19N4219.1604

C) C10H14NO2180.1019

D) C13H20NO2222.1489

F) C15H25N2O2265.1911

G) C6H8N3122.0713

Pyrimidine (P)Butyl piperazine

(BP)

Azaspirone decane dione

(ADD)

C-chain

420

168.1026-4.13 ppm

MS/MS spectrum of the monohydroxy metabolite of buspirone. The mass shifts of the fragments (green) in comparison to the MS/MSspectrum of buspirone and the comparable fragments (red) indicate the hydroxylation at the ADD Group (calculated relative mass accuracies are annotated in blue).

4x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Abundance vs. Mass-to-Charge (m/z)110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410

+ Product Ion (5.5 min) (402.2484 -> **)122.07111.42 ppm

139.0759

148.0867

238.14303.23 ppm 281.1870

-3.67ppm

402.2501-0.33 ppm

192.1381

G

F196.09604.18 ppm

150.1028-1.51 ppm

E

D

C

N

O

O N NN

N

E) C8H12N3150.1026C) C10H14NO3

196.0968 D) C13H20NO3238.1438

F) C15H25N2O3281.1860

G) C6H8N3122.0713

Pyrimidine (P)Butyl piperazine

(BP)

Azaspirone decane dione

(ADD)

C-chain

80

An Application Kit contains the methods, consumables, databases, and standards needed to quickly implement a select GC, GC/MS or LC/MS application at the customer’s laboratory.

Analyzer is a factory-configured GC or GC/MS system configured, tuned and tested for the application prior to delivery to the customer’s laboratory.

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