1. Agilent LC/MS Overview
Nathan Miller
LC/MS Product Specialist 1

2. Agilent’s > 40 year heritage in mass spectrometry
GC/MS
1994, 4500 Series
ICP-MS
QQQ
2000, 7500 Series
1971, 5930A
2008, 7000A
2005, 5975A
2011
7200A
1976,
5992A
LC/MS
LC/QQQ
Mass spectrometry is not new to Agilent. Over 35 years ago we introduced our first GC single quadrupole mass spectrometer. Since then, we have sold over 25,000 of these instruments. In 1994, we introduce inductively coupled mass spectrometry and have sold over 2,400 of them since. In 1996 we introduced our first LCMS instrument in the form of a single quadrupole mass spectrometer. A few years later, in the year 2000, we introduced our first tandem MS/MS instrument: the ion trap. Then, a little over three years ago, we introduced our time-of-flight instrument. Overall, we have sold more than 3500 LCMS systems.
2003
ESI on 5989
MS Engine
Direct Liquid Injection
1980, 5985 SQ
Thermospray, 5987
Particle Beam, 5988
1994, 2000
2006
LC/MSD TOF
LC/MSD, LC/MSD Ion Trap
LC/QTOF
Over 37,000 GC/MS, 4,500 ICP-MS and 6,500 LC/MS systems through June 2012
Agilent (and previously, as HP) has sold more quadrupole MS systems than any other mfr.

3. High Sensitivity HPLC-Chip/MS
Agilent High Performance Infinity Series LC: Best in class “Front End” systems for MS
Preparative LC
Capillary & Nano LC
Standard LC
Ultra High Pressure LC
High Sensitivity HPLC-Chip/MS
The industry’s most comprehensive LC portfolio to configure an integrated LC/MS system

4. MassHunter Workstation One software for all Agilent Mass Specs
Minimize the learning and optimize the use of software in your lab across different mass spec instrument platforms
Control of Agilent 6000 Series QQQ, TOF and Q-TOF
Control of most 1260 and 1290 HPLC modules, 7100 CE
Control for GC QQQ and Q-TOF
Data Processing for QQQ, TOF, Q-TOF, SQ*) and GC Ion Trap*)
Control & data processing for ICP/MS
MassHunter has become the data platform for all of Agilent’s MS systems. For LC/MS, it handles both control and data processing for the QQQ, TOF, and QTOF. Using translators, it can process the data from the single quad and former Agilent LC Ion Traps.
For GC/MS, it controls and processes data from the GC QQQ and can process data prom the GC/MSD
For ICP-MS, it does both instrument control and data processing. This minimizes learning effort and makes it easier to compare data from multiple instruments.
LC/MS
GC/MS
ICP-MS
*) after conversion to MassHunter format
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5. Qualitative Quantitative Agilent 6000 Series LC/MS portfolio
High Resolution
Qualitative
These can be broadly divided into HIGH RESOLUTION instruments – namely our TOF and QTOF instruments. These provide high mass resolution and extremely high mass accuracy. And then our low resolution, high speceficity quadrupole based instruments, the single quadrupole and triple quadrupole. These can also be divided into Single Stage instruments – measuring parent or pre-cursor mass (MS) and Tandem Stage instruments that have the added benefit of a collision cell and can be used for both MS and MS/MS.
6200 series TOF
6500 series Q-TOF
Tandem Stage
Single Stage
Quantitative
6100 series Quad
Low Resolution
6400 series QQQ

6. Single Quad LC/MS – Proven Solutions
ChemStation
Easy Access
G2725AA
6120B – 2,000 m/z
6130B – 3,000 m/z
6150B – 1,400 m/z
MassHunter Data Translator
Single Quad
Control Software
Data Analysis Software
Analytical Studio Reviewer, G3772AA
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7. Single Quad Supports Wide Range of Ion Sources seven Agilent ion sources and third party sources
MultiMode
HPLC Chip
APCI
ESI
APPI
AJS
nanoESI
Plus Direct Sampling Sources from Ion Sense:
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8. Agilent Jet Stream Gas Dynamics View
Nebulizing gas
Enhanced efficiency nebulizer
Super-heated sheath gas
Nozzle voltage
Heated drying gas
The super-heated sheath gas collimates the nebulizer spray and creates a dramatically “brighter source”
Resistive sampling capillary
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9. iFunnel Technology
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10. iFunnel Technology Revolutionizes Ion Sampling
Agilent Jet Stream
Thermal confinement of ESI plume
Efficient desolvation to create gas phase ions
Creates an ion rich zone
Hexabore Capillary
6 capillary inlets
Samples ~10 times more ion rich gas from the source
Captures the majority of the gas from the source region
Dual Ion Funnel
Removes the gas but captures the ions
Removes neutral noise
Extends turbo pump life
Captures up to 10 x more ions
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11. 6230B Accurate Mass TOF LC/MS
True Hi-Def TOF Technology:
NO PERFORMANCE COMPROMISE!
Unmatched Mass Accuracy
< 1 ppm MS
>22,000 Resolution
105 Dynamic Range
Up to 30 spectra/second
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12. Formulas for TOF performance
Resolving power = (m/z)/w1/2
1000/0.025 = 40,000
922/0.022 = 41,909
W1/2 = 0.022
921.5
922.0
922.5
923.0
923.5
924.0
924.5
m/z, amu
Two fundamental aspects to the analytical capability of the TOF and QTOF instruments are defined here. The first one is resolving power which is defined as the ion mass divided by its profile peak width. Therefore, it is important that if you are told what the resolving power if the mass spectrometer instrument is, at what mass does that definition hold? Because the Agilent TOF and QTOF instruments have such high resolving power, or distance between adjacent peaks as shown in the top figure, sensitivity comparable to a single quad in SIM mode is possible when one simply generates an extracted ion chromatogram (EIC) of 50 to 100 mDa in width. The sensitivity can be very good because so much of the co-eluting interferences is removed when creating such a narrow EIC.
The other aspect is mass accuracy, which is also defined here. Mass accuracy is the relative difference between what the instrument measures and what is the exact value, based on chemical formula. Because the TOF and QTOF instruments measure ion masses to 3 and 4 decimal places of the exact masses, the relative amount is multiplied by 1 million and the units are in parts per million, or ppm. Therefore, if the instrument can measure an ion at 1000 m/z with an accuracy of 1 ppm, then the mass is measured to within Da, or 1 mDa of the exact mass. If the same mass accuracy wer applied to an ion mass of 500, then ne would know the mass to within 0.5 mDa, or the mass of an electron.
Mass accuracy ( rel. mass error)
( )/1000)x106=1 ppm
Therefore: m/z is 0.001Da (1 mDa)
Epitestosterone
Resolving power: 6890
actual meas ppm

13. Rapid Analysis of Pharmaceutical Compounds
TOF - MS with Peak Capacity of 40 Cycle in 39 seconds
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
Intensity, cps
0.34s
0.36s
0.42s
Atenolol
Metoprolol
Primidone
Verapamil
Beclomethasone-
dipropionate
6000 Overview Diploma Training 2006
Agilent Restricted
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14. A big leap forward in 2006 6100 Series SQ 6500 Series Q-TOF
6400 Series QQQ
6100 Series SQ
Over 15 Years in LC/MS atmospheric sampling and patented orthogonal geometry - Result in industry leading sensitivity and robustness
Rough Pump
Octopole 1
Turbo 1
Quad Mass Filter (Q1)
Collision Cell
Lens 1 and 2
Quad Mass Filter (Q3)
10KV Detector
Ion Pulser
Turbo 2
DC Quad
Octopole 2
Collision cell incorporates axial acceleration for high speed MS/MS analysis
These hybrid MS systems are the result of a long standing development program in R&D in Santa Clara spanning over 25 years.
With the additional of a newly designed high performance collision cell to Agilent’s exceptional single quad and TOF analyzer systems we can now offer QQQ and QTOF instruments.

15. Agilent 6400 Triple Quadrupole Product Line
A New Agilent LC Triple Quad
Launched
Every Year
For last
4 years
6460
Triple
Quad
6430 Triple Quad
6420 Triple Quad
6490
6460 Triple Quad
Performance
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16. What is MRM? (Multiple Reaction Monitoring)
Ions
This is a nice slide comprised of four transition slides to show how the operation of the QQQ translates into actual signal in the MRM process. The spectrum at far left represents everything being ionized in the ion source. In this example, we’re looking at the ESI spectrum of a phenylurea pesticide. Why do we use a QQQ for doing low level quantitation in a dirty matrix? Why is this analyzer chosen over a single quad LC/MS? Well, we are able to utilize the QQQ to reduce the chemical noise. We do this first by selecting the pesticide of interest at m/z 210 from the co-eluting interferences seen in the rest of the spectrum. This is seen in the second spectrum as a result of passing through the first quadrupole, or Q1.
After Q1, fragment ions are generated in the collision cell. The corresponding MS/MS spectrum is shown just under the collision cell. Using the Q3 quadrupole, we can select out particular fragments ions for quantitation and confirmation. For example, the product ion at m/z 158 is more intense than the product ion at m/z Therefore, the MRM transition 210 > 158 would be used for quantitation and the 210 > 191 transition would be used for confirmation, where the m/z 191 ion is considered a qualifier ion.
This second stage of selectivity, using Q3, removes even more chemical background if the 158 and 191 ions are not fragments of isobaric interferences through Q1.
Spectrum with background ions (from ESI)
Q1 lets only target ion 210 pass through
190
210
Collision cell breaks ion 210 apart
150
170
190
210
158
191
Q3 monitors only characteristic fragments 158 and 191 from ion 210 for quant and qual.
160
158
190
191
no chemical background
170
210
250
290
222
268
280
165
For Research Use Only.

17. Single quads vs. Triple quads in complex matrix
SIM m/z 306
This slide shows how using MS-MS improves the chromatography by removing those ions not producing the 261 product ion. Several 306 ions appear in SIM mode but only one in MS-MS mode which is our analyte of interest. The exception is Imazapic where there are interferants that co-elute with it and shown in previous slides.
m/z 306 → m/z 261
MS-MS
Triple quads enjoy the benefit of filtering out the noise as observed in SIM

18. Triggered MRM (tMRM) Acquisition:
Simultaneous Quantitation and Confirmation
Dynamic
MRM
Triggered
Triple Quad MRM
Acquisition
Data
Dependent
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19. for compound confirmation
Triggered MRM (tMRM) Fast, Sensitive Compound Confirmation to Prevent False Positives
Triggered cycle (above threshold)
Additional MRMs used
for compound confirmation
Threshold
Primary cycle (below threshold)
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20. tMRM Product Ion Spectrum A Re-Constructed Product Ion Spectrum that is Library Searchable
334 > 76
334 > 91
334 > 105
334 > 117
334 > 119
x103
x103
x103
x103
x103 8 7 6 5 4 3 2 1 8 7 6 5 4 3 2 1 8 7 6 5 4 3 2 1 8 7 6 5 4 3 2 1
117.0 8 7 6 5 4 3 2 1
76.0
91.1
105.1
119.0 80
x103
334 > 132
145.0
334 > 171
x103
334 > 145
x103
334 > 147
x103 8 7 6 5 4 3 2 1 8 7 6 5 4 3 2 1 8 7 6 5 4 3 2 1 8 7 6 5 4 3 2 1
147.0
132.1
171.1
This is an illustration of how tMRM acquisition generates the product ion spectrum for each triggered analyte.
Up to ten MRM transitions are acquired for each target (the primary transitions are typically the transitions with the highest product ion abundance).
The tMRM software then compiles the primary and secondary MRM spectra into a single, library searchable, product ion spectrum
With up to ten product ions present in each tMRM product ion spectrum for each target analyte, there is sufficient data to confidently identify or confirm the analyte of interest with library confirmation. The biggest advantage of using tMRM to generate these product ion spectra (rather than conventional product ion scanning instruments), is that acquiring all ten of the MRM transitions which make up this spectrum takes about 20 ms, where a typical product ion scan on a trap or TOF instrument takes about 200 ms. The faster cycle time for qualitative analysis using tMRM acquisition means that more data points are acquired across each peak, and the quantitative data acquired is more sensitive, robust, and accurate.
tMRM Product Ion Spectrum
Tebufenpyrad
x103
145.0
117.0 8 7 6 5 4 3 2 1
76.0
91.1
105.1
132.1
171.1
334.2
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21. 6500 QTOF Product Line Excellent Value and Performance 6530 QTOF LC/MS
Agilent JetStream for greatest sensitivity
Robust, easy to use
Wide dynamic range for qual/quan applications
6540 UHD QTOF LC/MS
Sub 1-ppm mass accuracy for highest confidence
compound identification
Versatile system with highest resolution
Fast scan speed to match UHPLC separations
6550 iFunnel QTOF LC/MS
Agilent iFunnel for highest sensitivity
Perfect for most demanding applications
Fastest scan speed to match UHPLC separations
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22. 6550 iFunnel Q-TOF LC/MS System
New 6550 iFunnel QTOF
10X Sensitivity Gain Enables Applications
Sensitivity
Dramatically improved quantitative capabilities
New Qual/Quan Workflows
Superior metabolite and protein detection
Non-targeted compound screening
Comprehensive Performance Enhancements
Mass Resolution >40,000
50 spectra /sec MS and 33 spectra/sec MS/MS
5 orders of linear dynamic range
<1 ppm MS mass accuracy; <2 ppm MS/MS
Unrivalled sensitivity
The 6550 iFunnel QTOF leads the industry in sensitivity performance and will be very impactful in enabling many different applications which need increased detection levels. These applications now include both simultaneous quantitative and qualitative analysis since the 6550 sensitivity enhancements approach levels previously addressed by QQQ technology. More on the target applications in the next slide.
The 6550 QTOF generates the 10 fold gain in sensitivity to low femtogram levels while doubling in acquisition speed to over 50 spectra/ sec. At the same time we see other strong performance attributes of previous 6540 QTOF maintained at sub ppm mass accuracy and 40K mass resolution performance.
6550 iFunnel Q-TOF LC/MS System
ASMS 2010
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23. Low Femtogram Detection LevelMS
EIC
Resolution
~ 31,500
Mass error
-0.1 ppm
25 fg Reserpine
S/N = 49 (RMS)
This example shows the extracted ion chromatogram results of a 25 femtogram injection of reserpine on column. We see an excellent signal to noise ratio of 49 (root mean square) at this extremely low detection level. By comparison this detection is greater than 10 fold better than our previous 6540 QTOF. We also show excellent resolution >30,000, very accurate mass and isotopic fidelity for reserpine at the 25 fg concentration level.
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24. G3212A - GC APCI Interface
Increases MS flexibility by enabling both GC and LC compatibility
Analysis of wider range of compounds
Proven QTOF LC/MS platform
Proven 7890 GC performance
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25. GC Transfer line / APCI Source Design
Corona needle assembly
Capillary MS inlet
Bayonet lock coupling
Transfer line heat shield
Heated transfer line
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26. Simplify Your Startup LC/MS Application Kits Method Standards
On Site Training
LC Column
Databases and Libraries
Setting up a new method on a new or existing LC/MS instrument can be a time-consuming process, particularly for a user who is new to LC/MS. The process of ordering hundreds of standards, developing LC and MS methods, and creating necessary databases and libraries is a process that can easily take months to complete.
Agilent’s LC/MS Application Kits are designed to save our customers time and money, making method setup for a new instrument or application simple and efficient. Our LC/MS Application kits include consumables, such as LC columns, chemical standards, LC/MS methods, Databases, Libraries, and On Site training with an application expert. With Agilent’s complete solution, method setup can be completed in a matter of days versus months. In addition, the method that is pre-developed, customized, and optimized by our expert application engineers better suits the needs of users that require a high level of sensitivity, reproducibility, and accuracy.
LCMS Overview Jan 2013
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27. RapidFire® Mass Spectrometry
Fast autosampler & SPE system
Replaces LC in LC-MS
Integrated, automated, micro-scale solid-phase extraction
Integrates with standard ESI MS instruments
Compatible with most biological matrices
cycle time: 5–10s/sample
The RapidFire System:
Fits existing workflows
Low operational costs
Improved data quality
Strong correlation with traditional methods
Pictures show system and an up-close view of microfluidics.
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28. RapidFire™ Mass Spectrometry (RF-MS)
Microfluidic Sample
Clean-up System
96 or 384 well
Plate Handler
Monitor to track
MSMS data
This graphic shows the RapidFire system working with a 6410 QqQ MS.
Connection is via Peek tubing directly into the source, the same as for any LC system (in fact the RapidFire system can switch between RF and LC in a matter of minutes).
Standard 96 or 384-well assay plates are used to run biological assays. Typically these are enzyme assays that are incubated for a certain amount of time and then stopped via quenching with organic buffer, acid or a specific inhibitor. Once the assays are quenched the plates (capacity is 17 or 18 depending on plate format) are stacked in the plate handler for walk-away analysis.
There are no laborious sample prep or extra upstream steps needed. Using the microfluidic sample clean-up system, samples are aspirated directly from the plate in which the assay was run and applied, in a serial manner, to an on-line solid phase extraction cartridge. The cartridge is washed with about five bed volumes of wash buffer and then the analytes are eluted from the cartridge with a plug of elution buffer. The eluate is applied to the mass spectrometer where the selectivity of MRM analysis, or scanning of TOF MS provides detection and quantitation of specific analytes from the mixture.
The MS analysis is followed using the Agilent monitor and the microfluidic system is tracked using a RapidFire- supplied monitor.
Monitor
to track
RapidFire System
Agilent 6410
QqQ Mass Spectrometer
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29. THANK YOU!
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