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Latest News from Agilent Liquid Phase Mass Spectrometry Solutions
Shaun Bilsborough PhD
Page 2EMEAI LSAG
Page 3EMEAI LSAG
Page 4EMEAI LSAG
Latest News from Agilent Liquid Phase Mass Spectrometry Solutions
Shaun Bilsborough PhD
Page 6EMEAI LSAG
LC/MS Portfolio
• 6100 Single Quadrupoles
• 6200 TOF
• 6400 Triple Quadrupoles
• 6500 QTOF
• MassHunter Software
Page 7EMEAI LSAG
Key Features
• A mass detector for chromatographers
• Unified LC and MSD design
• Mass detection within existing footprint
• OpenLAB CDS, including QA Compliance &
OpenLAB CDS ChemStation Edition
• Highest robustness, reliability and
performance
The New Mass Selective Detector (MSD)
RoHS Compliant
Page 8EMEAI LSAG
New Agilent LC/MSD Series2 models
Best Value Configuration - 6125
• Quick and easy move from UV-
based methods to MS data.
• Benefits of mass detection for the
chromatographer
• Great value for the pharma,
chemistry, start-up and contract
labs for small molecule analysis
• MassHunter Walkup software for non-experts
• ESI source exceeds many detection limits
Most Flexible Configuration - 6135
• Extended mass range (3000) for
Biopharma & life science
applications
• Fastest polarity switching for high-
throughput pharma
• Best choice for trace level detection (w/
optional AJS source)
Available with OpenLAB CDS and OpenLAB Chemstation Edition
G6125B/C(A) G6135B/C(A)
Page 9EMEAI LSAG
LC/MSD SpecificationsImproved S/N
*1pg reserpine on-column
**30 ms requires upgrade, without upgrade 300 ms
LC/MSD
Model
ESI+
SIM S/N
RMS*
Jet Stream
ESI
S/N RMS*
APCI+
SIM S/N
RMS*
m/z Max
scan
speed
(u/s)
Polarity
Switching
(ms)
6135 >300:1 >650:1 >100:1 10- 3000 10,400 30
6125 >100:1 NA >100:1 2-2000 10,400 30**
LC/MSD Model Multi-mode
source
APPI Mass accuracy (Da) Dynamic
range
Min SIM
dwell (ms)
6135 Yes Yes +/-0.13 >6x106 5
6125 Yes Yes +/-0.13 >6x106 5
w/ MassWorks
Software
+/- .005 Da Spectral accuracy 98.5
Page 10EMEAI LSAG
1260 Infinity II Series LC/MSD Bundle –G6125BW
1260 Infinity II Modules
G6125BA MSD
Software OpenLAB CDS ChemStation Edition
G7111B 1260 Infinity II Quaternary Pump
G7114A 1260 Infinity II Variable Wavelength Detector
G7129A 1260 Infinity II Vialsampler (#63 ICC)
Option #001 Replace quat pump with binary pump
G7112B 1260 Infinity II Binary Pump
Option #002 Replace VWD with DAD
G7117C 1260 Infinity II Diode Array Detector
Option #004 Delete VWD
Page 11EMEAI LSAG
LC/MS Portfolio
• 6100 Single Quadrupoles
• 6200 TOF
• 6400 Triple Quadrupoles
• 6500 QTOF
• MassHunter Software
Page 12EMEAI LSAG
• Support for all Agilent LCs, GCs, and mass spectrometers
• Superior compound detection for LC/MS with techniques such as tMRM, All Ions MS/MS, and Ion Mobility provides flexibility and confidence
Acquisition
• Key software program for the identification and characterization of compounds in all markets
• Innovative and patented software algorithms extract compounds from data and determine formulas and structure
Qualitative Analysis
• High-throughput quantitation of target compounds
• Easily visualize results using the Batch Table or Compounds-at-a-Glance, then quickly generate reports or upload to a LIMS
Quantitative Analysis
Agilent MassHunter Core Programs
Page 13EMEAI LSAG
MassHunter Software for your Key Applications
• Qualitative Analysis for confident identifications and to set up methods
Food
• Quantitative Analysis for fast and accurate results
Environmental
• Acquisition for innovative techniques such as tMRM to confirm compounds
Veterinary Drugs
• Personal Compound Database and Libraries (PCDLs) for rapid identification
Forensic Toxicology
• Spectrum Mill for accurate identification of proteins
Proteomics
• BioConfirm for characterization of intact proteins, peptides, and monoclonal antibodies
Biopharma
• Mass Profiler Professional (MPP) for differential profiling
Metabolomics
• Pathway Architect for bringing together genomics, proteomics, and metabolomics
Integrated Biology
Page 14EMEAI LSAG
• Ultra-Trace Analysis
• Agilent iFunnel Technology – Attogram zeptomole sensitivity!
• Ideal for biomarker, steroids, and most demanding bioanalytical/clinical assays
6495Ultra High-
End
• Trace Analysis
• Agilent Jet Stream Technology – Sub femtogram sensitivity!
• Ideal for multi-residue pesticide analysis, clinical assay. Upgradeable to 6495!
6470High-End
• Routine Trace Analysis
• Agilent Jet Stream Technology – Sub femtogram sensitivity!
• Ideal for drugs of abuse, chemical and forensics
6460 Mid-High
End
• Routine Analysis
• Lowest cost of ownership, Robust and Reliable
• Excellent sensitivity and productivity
• Ideal for clinical research & drug analysis
6420Entry-Level
Perf
orm
an
ce
6400 Triple Quad Family
Page 15EMEAI LSAG
Agilent 6400 QQQ LC/MS
6495 QQQ
6470 QQQ
6420 QQQ
Relative Price
Rela
tive
Pe
rfo
rma
nc
e
6460 QQQ
Ultra-Trace Analysis• Pesticide, Hormones
• Emerging contaminants
• Biomarker, Steroids
• Ultra-low Assay Requirements
• Complex matrices
Trace Analysis• Pesticide, Mycotoxins
• Vet drugs, Designer drugs
• Drug of abuse
• Vitamin D
• Bioanalysis
Routine Analysis• PPCPs, PFCs
• Opiates
• Pain management
• Chemical & Forensics
Page 16EMEAI LSAG
6470 QQQ TechnologiesEnhanced Performance in a Smaller Space
Agilent Jet Stream Technology
• Thermal gradient focusing
• Efficient desolvation
• Creates an ion rich zone
• Up to 10x gains in sensitivity
• An Ion Detector with High Energy Conversion Dynode and Low Noise
• Improved ion detection3
• A Curved and Tapered Hexapole Collision Cell
• Effective ion collection and transmission
2
• Enhanced Q1 Ion Optics
• Improved ion transmission1
Page 17EMEAI LSAG
High Sensitivity for Benzodiazepines in Plasma LLOQs and MDLs at Low-pg/mL level on the 6470
• Excellent peak area precision (%RSD) and accuracy are observed at the LLOQ level
-HydroxymidazolamLLOQ = 5 pg/mLS/N = 13.6
MidazolamLLOQ = 5 pg/mLS/N = 35.2
-HydroxyalprazolamLLOQ = 5 pg/mL
S/N = 5.4
AlprazolamLLOQ = 5 pg/mL
S/N = 9.2
CompoundLLOQ
(pg/mL)%Accuracy
%RSD(n = 7)
MDL(pg/mL)
-Hydroxymidazolam 5 100.7 6.9 1.1
Midazolam 5 114.6 5.3 0.84
-Hydroxyalprazolam 5 99.4 9.9 1.6
Alprazolam 5 96.1 7.5 1.2
Page 18EMEAI LSAG
Confident Quantitation over 5 Orders of Dynamic Range
• Excellent linearity (R2 > 0.995), accuracy and precision are achieved at all levels including LLOQ
Levels %RSD (n = 7) %Accuracy
5 pg/mL 5.32 114.6
25 pg/mL 2.76 96.6
50 pg/mL 2.53 102.8
250 pg/mL 1.22 98.6
500 pg/mL 0.73 98.7
2.5 ng/mL 0.51 97.6
5 ng/mL 0.34 99.1
25 ng/mL 0.56 96.5
50 ng/mL 0.79 96.4
250 ng/mL 0.75 97.3
500 ng/mL 0.47 101.9
Midazolam, 5 pg/mL – 500 ng/mL
5 Orders of Linear Dynamic Range
R2 = 0.9994
Concentration (ng/ml)0.005 0.01 0.05 0.1 0.5 1 5 10 50 100 500 1000
Rela
tive
Res p
ons e
s 2x10
5E-06
1E-05
5E-05
0.0001
0.0005
0.001
0.005
0.01
0.05
0.1
0.5
Levels %RSD (n = 7) %Accuracy
5 pg/mL 7.54 96.1
25 pg/mL 2.77 96.9
50 pg/mL 2.95 101.9
250 pg/mL 1.62 101.3
500 pg/mL 1.20 101.8
2.5 ng/mL 0.42 99.8
5 ng/mL 1.24 102.3
25 ng/mL 2.15 100.1
50 ng/mL 0.77 99.7
250 ng/mL 2.91 100.0
500 ng/mL 0.62 86.6
Alprazolam, 5 pg/mL – 500 ng/mL
5 Orders of Linear Dynamic Range
R2 = 0.9970
Concentration (ng/ml)0.005 0.01 0.05 0.1 0.5 1 5 10 50 100 500 1000
Rela
tive
Res p
ons e
s 2x10
5E-06
1E-05
5E-05
0.0001
0.0005
0.001
0.005
0.01
0.05
0.1
0.5
Page 19EMEAI LSAG
Proven System Robustness in Human Plasma
• Stable peak area response was observed over 1,300 injections of plasma extract and 1 week
of continuous operation
• Average area ratio %RSD = 1.8 was observed with use of internal standard (ISTD)
• Average peak area %RSD = 6.6 was observed for benzodiazepines and metabolites
0
0.2
0.4
0.6
0.8
1
1.2
0 200 400 600 800 1000 1200 1400
Peak A
rea R
ati
o
Injection number
-Hydroxymidazolam
0
0.2
0.4
0.6
0.8
1
1.2
0 200 400 600 800 1000 1200 1400
Peak A
rea R
ati
o
Injection number
Midazolam
0
0.2
0.4
0.6
0.8
1
1.2
0 200 400 600 800 1000 1200 1400
Peak A
rea R
ati
o
Injection number
-Hydroxyalprazolam
0
0.2
0.4
0.6
0.8
1
1.2
0 200 400 600 800 1000 1200 1400P
eak A
rea R
ati
oInjection number
Alprazolam
Area Ratio %RSD=1.3
Peak area %RSD=5.7
Area Ratio %RSD=3.3
Peak area %RSD=7.0
Area Ratio %RSD=1.4
Peak area %RSD=7.2
Area Ratio %RSD=1.3
Peak area %RSD=6.3
Page 20EMEAI LSAG
Proven iFunnel Technology: Upgrade 6470!
Agilent Jet Stream Hexabore Capillary Dual Ion Funnel
• Thermal gradient focusing
• Efficient desolvation
• Creates an ion rich zone
• Six capillary inlets
• Samples x10 times more
ion rich gas
• Removes the gas but
captures the ions
• Removes neutral noise
Page 21EMEAI LSAG
Scan Speed, dMRM and tMRM…..
Dynamic MRM, Fast MRM Speed and Polarity Switch
• Group MRMs in RT windows
instead of time segments
• 2x shorter cycle time supports
narrow UHPLC peaks
• Supported by very fast MRM
speed (1 ms dwell) and
polarity switch (30 ms)
• tMRM database accelerates
acquisition method setup
• tMRM library – Match Score
for confirmation
• tMRM is Fast and Sensitive
• UHPLC compatible
• Simultaneous Quantitation,
Screening and Confirmation
Triggered MRM, tMRM Database and Library
Match
Score
95.1Trigger
Threshold
tMRM Product
Ion Spectrum
Apex
Page 22EMEAI LSAG
6500 QTOF Family
• Agilent iFunnel Technology – Low femtogram sensitivity in complex samples
• Ultra trace analyses for proteomics, metabolomics and glycomics
6550High-End
• Agilent Jet Stream Technology – mid femtogram sensitivity
• Enhanced resolution and mass accuracy for complex samples
• Ideal for impurity analysis, metabolite ID, food safety and environmental screening
6545Mid-Range
• Ease of use and robust operation
• Excellent value for everyday routine use
• Ideal for chemical and forensic applications
6530Entry Level
Maximum sensitivity
Performance with
robustness
Advantages of QTOF
at lower cost
Page 23EMEAI LSAG
Ultra-Complex Analysis• Lipidomics
• Proteomics
• Glycomics
• Biophysics studies
• Polymers
• Biopharm structural studies
6500 QTOF Portfolio
6560 IM QTOF
6550 QTOF
6530 QTOF
Relative Price
Rela
tive
“P
erf
orm
an
ce
”
Complex, Trace Analysis• Pesticides, Mycotoxins
• Protein ID/ Biomarkers
• Metabolomics
• Qual/ Quan
• Biopharm characterization
Routine Analysis• PPCPs, PFCs
• Impurity analysis
• Chemical & Forensics
• Quality control
6545 QTOF
Performance based upon
sensitivity and resolving power
Page 24EMEAI LSAG
Why Accurate Mass?
m/z
MS
Low Mass Accuracy
m/z
MS
High Mass Accuracy
C12 H14 N9 Cl
C13 H23 N3 P S Cl
C14 H24 N2 S2 Cl
C14 H22 N O3 S Cl
C14 H16 N6 O Cl
C15 H25 O P S Cl
C16 H19 N3 P Cl
C17 H20 N2 S Cl
C18 H21 O P Cl
C13 H23 N3 P S Cl
C14 H24 N2 S2 Cl
C14 H22 N O3 S Cl
C14 H16 N6 O Cl
C15 H25 O P S Cl
C16 H19 N3 P Cl
C17 H20 N2 S Cl
C18 H21 O P Cl
Unit accuracy instrument
e.g. ion trap, QQQ, Q
High mass accuracy
instrument
e.g. QTOF, TOF
Page 25EMEAI LSAG
Why Buy an LC-Q-TOF?
To identify compounds with the strongest confidence
Up to 5 degrees of confirmation! Accurate mass, isotopic distribution, product ion spectrum, retention time, IM drift time
Spectrum with theoretical
isotope boxesMirror plot?
Is this safe to eat? Confirm with product ion
spectral matchConfirm with accurate
mass and isotopes
Page 26EMEAI LSAG
Why Buy an LC-Q-TOF?
Analysis of very complex mixtures (cellular digest, biological fluids, environmental)
samples)
Co-eluting compounds with similar masses can be resolved for identification
Picture of complex TIC Extracted complex spectra
Complex TIC Complex Spectra Resolution of
VERY similar MW
ions
Page 27EMEAI LSAG
Why Buy an LC-Q-TOF?
To screen for thousands of targets including unknowns
Screen or profile for thousands of compounds, with high confidence from accurate mass + MS/MS
Full mass range data is collected, so data can be retrospectively queried (doping control, forensics)
Page 28EMEAI LSAG
How do we use LC/MS?
United States Secretary of Defense 2001-2006 Donald Rumsfeld
Page 29EMEAI LSAG
The Rumsfeld Conundrum
… “as we know, there are known knowns; there
are things we know that we know.
There are known unknowns; that is to say, there
are things that we now know we don't know.
But there are also unknown unknowns – there
are things we do not know we don't know.”
United States Secretary of Defense 2001-2006 Donald Rumsfeld
Page 30EMEAI LSAG
Types of Screening and Definitions
“Known Knowns” a.k.a. Target Screening
All Ions
PCDL
Qual/Quant
Customised Reporting.
“Known UnKnowns” a.k.a. Suspect ScreeningAll Ions
PCDL
Find by Formula
Auto or Target MS/MS
PCDL MS/MS Libraries
Molecular Structure Correlator
“UnKnown UnKnowns” a.k.a. Discovery ProfilingMS
Profinder
Mass Profiler Professional
Mass Profiler
Auto or Target MS/MS
PCDL MS/MS Libraries
Molecular Structure Correlator
Page 31EMEAI LSAG
Screening Workflow SolutionsScreening Target Suspect Discovery
QQQ Using MRM and tMRM
databases, method can be
quickly developed for
implementation
TOF Using PCDLs and an All-Ions
workflow Quantitation and
Qualification is possible using
four points for confirmation
(Rt, AccMass, Fragments, IP).
Suspect Screening requires
the use of Find by Formula
referring to a suspect PCDL
where MS/MS spectra are
available.
QTOF Using PCDLs and an All-Ions
workflow Quantitation and
Qualification is possible using
four points for confirmation
(Rt, AccMass, Fragments, IP).
Using a Suspect PCDL via All
Ions and or Find-by-Formula
to find proposed compounds.
MS/MS of unqualified
compounds for further
identification via Molecular
Formula Generation or
Structural Correlation.
Samples analysis using Mass
Profiler or Mass Profiler
Professional.
Page 32EMEAI LSAG
Databases and Libraries for LC/MSIntegrated with Agilent’s Workflows
Separate & Screen Untargeted w/ All Ions
LC/Q-TOF
Separate & Detect
LC/QQQ
Match spectra
Build Targeted Acquisition Methods
tMRM DatabaseSam
ple
Pre
p,
Colu
mns &
Supplie
sA
pplic
atio
n C
onsultin
g
PCDL
Quantify & Report
MassHunter Quant
Confirm Hits & Report
MassHunter Qual & Quant
Page 33EMEAI LSAG
Forensic/ToxicologyLC/QTOF Forensic Toxicology PCDL
LC/QQQ Forensic Toxicology tMRM DB
Environmental
LC/QTOF Water Screening PCDL
LC/QTOF Pesticide PCDL
LC/QQQ Pesticide tMRM DB
Food/Feed Analysis
LC/QTOF Mycotoxins PCDL
LC/QTOF Pesticide PCDL
LC/QTOF Veterinary Drugs PCDL
LC/QTOF Extractables and Leachables PCDL
LC/QQQ Pesticide tMRM DB
LC/QQQ Veterinary Drugs tMRM DB
Portfolio – Databases and Libraries
Life Sciences
LC/QTOF METLIN Personal Metabolite PCD and PCDL
LC/QTOF NIST Library PCDL
LC/QQQ Metabolomics dMRM DB & Method
Pharma LC/QTOF Extractables and Leachables PCDL
Page 34EMEAI LSAG
Agilent LC/MS Databases and Libraries
Trust Agilent’s long history in database & library development - Let our mass spectrometry experts work for you!
We did the research - you can save that time!
Use the MassHunter Software suite - streamline your analysis workflow!
Keep your testing current - free upgrades for 3 years!
Rely on Agilent application consulting and our large portfolio of sample prep products and columns – Let our application experts help you!
Instant Productivity
Relevant Content
Help you focus on
what you do best
Ease of Use
Confidence
Page 35EMEAI LSAG
Sa 6545XT AdvanceBio LC/QTOF
Introducing the…
6545XT AdvanceBio LC/QTOF
Builds upon 6545A model eXTended mass range and sensitivity
for eXTra large biomolecules
Developed for large
molecules such as intact or
digested proteins
New 6545XT QTOF
Page 36EMEAI LSAG
6545XT Development Concept
A single
Q-TOF to
drill down
through
each level
of analysis
for
BioPharma
mAB
Subunit
Peptide
Map
PTM
Intact Protein
Page 37EMEAI LSAG
AssayMAP
Bravo
Liquid Chromatography
NEW 6545XT
AdvanceBio QTOF
BioConfirm
Software
Only Agilent has the Complete Solution
Advance
BioColumns
Page 38EMEAI LSAG
What is the Advantage of Using Automation?
1. Very high reproducibility / very low variation
2. Robust method progress, no user mistakes
3. Higher quality results with more information
4. More walk-away time – high efficiency
5. Higher throughput
Automation provides major advantages even with low
sample numbers!
Page 39EMEAI LSAG
Summary
• New Single Quadrupole MS
• New BioPharma/Large Molecule QTOF
• Application-specific method databases and libraries to confirm identity
of compounds in complex matrices
• Application-specific software to support various applications
• Target analysis of knowns
• Suspect analysis on known unknowns
• Discovery profiling of unknown unknowns
Page 40EMEAI LSAG
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