M8000 3DQ LCMSn

1
M-8000 3DQ LC/MSn

• Capillary LC/MS/MS

• Ion Trap Mass Spectrometry MS/MS and MSn

• High Performance Full Scan MSn Sensitivity and
  Selectivity

2
Highly Flexible and Unique LC/MS Sources

• Four Different Atmospheric Pressure Ionization
  Sources (API)
• Sonic Spray Ionization (SSI)
• ElectroSpray Ionization (ESI)
• Atmospheric Pressure Chemical Ionization (APCI)
• Semi-Atmospheric Pressure Chemical Ionization
  (semi-APCI)

This graph illustrates the more universal
nature of the SSI source. Many compounds are
unstable at the conditions typical of an ESI or
APCI. Thermally sensitive compounds will
decompose causing reduced sensitivity and making
structural analysis by MSn difficult or
impossible. SSI will be necessary in these
cases. Also, the SSI has been shown to be much
more robust for a wider range of LC solvent
conditions.
3
Sonic Spray Ionization (SSI)
Soft Ionization without heat or high voltage -
Ionization by gas flow Suitable for unstable
components
The mechanism is based on a coaxial flow with
Nitrogen. At the exit of the capillary (diameter
of the orifice is key) sonic velocities are
achieved. It has been observed by Hitachi
researchers at Hitachi Central Research (Tokyo
Japan) that when measuring the signal as a
function of velocity, it was found that maximum
signal was obtained at sonic velocities.
Relative Ionization Efficiency
Gas velocity (l/min)
4
Sonic Spray Ionization (SSI)Example Applications
Steroids Pesticides (Carbamates) Sugars/
Oligosaccharides Herbicides Antibiotics
Peptides Aminoglycoside antibiotics Catechines
(thermally unstable polyphenols) Paclitaxel
(anticancer drug) Gangliosides
List of SSI Publications
1. Y. Hirabayashi, A. Hirabayashi, and H.
Koizumi A Sonic Spray Interface for Capillary
Electrophoresis/Mass Spectrometry, Rapid Commun.
Mass Spectrom. 13 (1999) 712-715.  2.
A.Hirabayashi Evaporation of Charged Fine
Droplets, Int. J. Mass Spectrom. Ion Processes
175 (1998) 241-245.  3. A.Hirabayashi and J.
Fernandez de la Mora Charged Droplet Formation
in Sonic Spray, Int. J. Mass Spectrom. Ion
Processes 175 (1998) 277-282.  4. Y. Hirabayashi,
A. Hirabayashi, Y. Takada, M. Sakairi and H.
Koizumi A Sonic Spray Interface for the Mass
Analysis of Highly Charged Ions from Protein
Solutions at High Flow Rates, Anal. Chem. 70
(1998) 1882-1884.  5. A. Hirabayashi, M.
Sakairi, Y. Takada and H. Koizumi Recent
Progress in Atmospheric Pressure Ionization Mass
Spectrometry, Trends Anal. Chem. 16 (1997)
45-52.  6. Y. Hirabayashi, Y. Takada, A.
Hirabayashi, M. Sakairi and H. Koizumi Direct
Coupling of Semi-micro Liquid Chromatography and
Sonic Spray Ionization Mass Spectrometry of
Pesticide Analysis, Rapid Commun. Mass Spectrom.
10 (1996) 1891-1893.  7. A.Hirabayashi, Y.
Hirabayashi, M. Sakairi and H. Koizumi
Multiply-charged Ion Formation by Sonic Spray,
Rapid Commun. Mass Spectrom. 10 (1996)
1703-1705.  8. A. Hirabayashi, M. Sakairi and H.
Koizumi Sonic Spray Mass Spectrometry, Anal.
Chem. 67 (1995) 2878-2882.  9. A. Hirabayashi, M.
Sakairi and H. Koizumi Sonic Spray Ionization
Method for Atmospheric Pressure Ionization Mass
Spectrometry, Anal. Chem. 66 (1994) 4557-4559.
5
Atmospheric Pressure Chemical Ionization (APCI)
APCI Interface Semi or Standard APCI Available
Semi-APCI (0.05 0.3 ml/min) is used for lower
flow rates than the standard APCI source (0.1
1.5 ml/min)
Nebulizer
Desolvator
Nitrogen gas not required
Mobile phase solvents that include sample
molecules are heated in the Nebulizer. Next,
they enter the Desolvator. Solvent molecules
are evaporated by heat and pressure in this
region. A Needle Electrode with high voltage is
located at the exit of the desolvator region.
Mobile Phase solvents (water, methanol, etc.) are
ionized by corona discharge around this Needle
Electrode and subsequently sample molecules are
ionized by reactions with these ions. APCI is a
relatively "soft" ionization method, which makes
it possible to obtain quasi-molecular ions of the
form MH and M-H - . With APCI,
moderately polar compounds and relatively small
molecules can be ionized efficiently.
6
The Hitachi LC/3DQ A Three-Dimensional
Quadrupole Ion Trap Mass Spectrometer
The end caps have small perforation(s) in the
center to allow the ions to enter and exit the
ion trap. The end of the ion transport optics
extend directly to the outside of the inlet end
cap. A high voltage RF potential is applied to
the ring, while the endcaps are held at ground.
The oscillating potential difference established
between the ring and endcap electrodes forms a
substantially quadrupolar field. Depending on
the level of the RF voltage, the field can trap
ions of a particular mass range. The quadrupolar
field can be thought of as a three dimensional
trough or a pseudo-potential well. Among other
things, the depth of this well is related to the
mass of the ion and the level of the RF voltage.
In practice, the range of masses that experience
a trapping force from this quadrupolar field is
wide enough that the ion trap can very
effectively produce full scan spectra while still
offering high sensitivity.
7
Benefits of Tandem Mass SpectrometryWhy use
MS/MS and MSn ?
Unparalleled Performance and Flexibility for
MS/MS and MSn

• Structural Characterization Applications
• MS/MS - especially in the context of API soft
  ionization techniques - provides structural
  information of unknown compounds, e.g., drug
  metabolites and impurities
• MSn allows multiple stages of MS molecular
  dissection, and thus provides unique evidence
  about fragments in the MS/MS spectrum and thus
  further information to an unknowns identity
• Increased Selectivity and Throughput
  (Qualitative/Quantification)
• Trace level analyses and/or enhanced
  confirmation...Especially in Complex Matrices
• Level of analyte too low or chemical interference
  too high Improved S/N
• Definitive qualitative results More confidence
  in assignment of target compounds (Confirmation
  of Identification)
• Faster HPLC analyses for screening or target
  compound applications
• Less HPLC separation required - less sensitive to
  optimal LC conditions
• MS/MS reduces the effects of chemical
  interference's or coeluting peaks

8
Unparalleled Performance and Flexibility for MS,
MS/MS and MSn

• The 3DQ LC/MSn system is based on Ion Trap Mass
  Spectrometry. In addition to the flexibility
  with the type of sources available, the 3DQ Ion
  Trap has some unique characteristics for MS/MS
  and MSn applications that make the 3DQ a very
  simple instrument to use, but very powerful.
• AutoCalculate features for easy setup of
  experiments and method development
• Unique MSn Experimental Definition Graphical User
  Interface
• Patented Filtered Noise Field (FNF) technology
  for enhanced MS/MS and MSn performance
• AutoMS/MS with AutoCID and CID Voltage Scanning
  for on-line HPLC analyses
• Complete control of all MS/MS and MSn parameters
  for the widest range of applications and problem
  solving capability

3DQ Unique Graphical User Interface
Multiple MSn Experiment Capability
Advanced MSn mode allows for quick parameter
entry or access to EDIT which allows full
control for all MSn parameters
9
MS/MS and MSn (How Filtered Noise Fields Work)
Precursor Ion Isolation and Collision Induced
Dissociation (CID)
What is FNF? Trapped ions are oscillating
along the z-axis at frequencies that are
dependent on the m/z values of the ions. If an
external rf field is added to the two end caps,
the amplitude of this oscillation is then a
function of the frequency spectrum of the rf
field. Because the initial phases of these
frequencies are randomly scrambled to make it
feasible to create such a field electronically ,
this rf filed is called Filtered Noise Field
(FNF). It is useful to think of FNFs as highly
selective programmable mass filters that can
accept or reject a complex pattern of mass to
charge (m/z) ratios . FNF is Used for Enhanced
Sensitivity and MSn Applications The trapping
capacity of any ion trap is limited. By using
FNF, dominant background ions can be excluded,
while ions of interest can be accumulated to a
much higher concentration. As a result,
detection limit and sensitivity can be
dramatically enhanced. MS/MS and MSn If the
resonant frequencies of certain ions are present
in the field with moderate field amplitudes,
those ions will be energized and undergo frequent
and energetic collisions with the background
helium buffer gas. The multiple energetic
collisions (collisional activation) cause the
ions to fragment at specific critical energy
levels (Collision Induced Dissociation, CID).
10
MS/MS and MSnExperimental Definition Graphical
User Interface
Ion Accumulation
Precursor Ion Isolation
Collision Induced Dissociation (CID)

• 3DQ MSn Unique Characteristics
• AutoCalculate feature
• Automatically calculates all MSn parameters for
  (1) Ion Accumulation, (2) Precursor Ion
  Isolation, 3) Collision Induced Dissociation
  (CID)
• Real Time Display of all three windows for MS/MS
  and MSn experiments
• Change any MS/MS parameter while monitoring the
  results
• Complete control of all parameters for the MS/MS
  process
• Unparalleled flexibility. Access to all
  parameters allows for a wider range of
  applications and better performance for existing
  applications
• Complete control of the FNFs allows for true
  Multiple Ion Monitoring, and multiple precursor
  ion isolation and excitation techniques

11
3DQ Automatic MS/MS

• Auto-MS/MS for on-line HPLC applications
• Includes Auto-CID and CID Voltage Scanning for
  more Efficient on-line MS/MS Data Acquisitions

AutoCID
CID Voltage Scan
CID Efficiencies are typically high in an ion
trap mass spectrometer. However, it is common to
use different excitation voltages to obtain
optimal CID, which depends on the mass of the
precursor ions. To minimize ion dependency for
automatic MS/MS experiments, the 3DQ utilizes an
Automatic CID function, where optimum CID
voltages are derived based on an automated FNF
calibration routine. Optimal excitation
voltages, depending on mass and CID low mass
cutoff settings, can then be calculated and
applied on-the-fly during a chromatographic
run. To some degree these optimum values may be
dependent upon the calibration compounds that are
employed. Additionally, slight drifts in ion
level calibration and other factors can shift
these optimized CID voltages. Such shifts can
result in less efficient fragmentation in the
Auto-MS/MS mode. To make this process more
robust, Hitachi affords the operator the ability
to scan the CID voltage through the optimum value
during the CID event, e.g., from 80 below
optimum to 80 above optimum. Therefore, the
ions that fragment easily will do so at lower CID
voltages (early in the CID event), and more
stable ions that require higher voltages above
those of the calibration compounds will fragment
later in the CID event.
12
High Performance Full Scan MSn Sensitivity and
Selectivity
5 pg Sensitivity for Reserpine with 3DQ ESI-MS/MS

• Conditions
• ESI
• FIA - 200 ul/min flow rate
• 5pg reserpine
• (8 femtomoles)
• S/N at 801