Application Areas of Nanospray-HPLC- Chip/MS

Application Areas of Nanospray-HPLC-
Chip/MS
Small sample quantities require extremes in
sensitivity
Page 1
Proteomics/Biomarker discovery
Drug Metabolite ID
PharmacoKinetics
Clinical Applications
Environmental & Forensic Trace-Analyses

Sensitivity by Nanospray and Reduction
of ID
Page 2
2r = 4.6mm
2r =
1.5mm
V
@ constant
linear velocity
L
V sp
sp
=
r π
2
L
Chip: Analytical column = 0.075mm ID
Intensitiy = f(
c)
=
L
x10
L
m
V sp

Page 3
Typical set-up of Nano-Spray LC/MS
• Sensitivity
75µm ID analytical column
Challenging to set-up & maintain
• Multiple parts
Possible leaks
Possible misalignments
• Robustness & ease-of-use
System
Valve
Enrichment
Frequent clogging of spray needle
After part replacement system can take hours to stabilize
Chromatographic degradation
• Extra column volume
Diffusion leads to band broadening
Tubings
Spray tip
Nano column
Fittings

Page 4
Advantages of HPLC-Chip Nano-
• Sensitivity
75µm ID analytical column
• Multiple parts integrated on Chip
Leaks
Misalignments
• Robustness & ease-of-use
Analysis reproducibility
Clogging of spray needle
Plug-&-Play device
Converts sophisticated analysis to simple operation (multi-D)
• Extra column volume
Chromatographic performance
LC/MS
! LC-Chip Suitability for Routine Environment !

Page 5
Ready-to-use in 4 steps
1 2
3
Set up method
Insert Chip
Click
1200 Nano HPLC Chip Cube on QTOF Mass Hunter
software
4
Chip automatically moves
into spray position
MS
Chip Tip
Spray
Camera visualises
nanospray

Automated HPLC-Chip Workflow
Page 6
Stator
Rotor
Sixport valve
Autosampler
Waste
TC
All components chip-integrated
extra column volume
chromatographic performance
Production by laser ablation
leaks
misalignments
Nano pump
Nano analytical column
Nanospray tip

Page 7
Online Workflow
Stator
Rotor
Enrichment
Analysis
Rotor
Switch

Nanospray
99.5% H 2 O 90% H 2 O 65% H 2 O 10% H 2 O
Page 8
99 H 2 O
Data courtesy of Paul Goodley, Agilent Technologies
(USA)
99
ACN
---EIC m/z 433 +3 Angiotensin I
---EIC m/z 450-640 Noise Measurements
Use of the
orthogonal dual
electrode
nanospray ion
source shows
constant signal-tonoise
for all types of
Taylor cone plumes
when combined
with the polymer
chip
Flow: 320nl/min V: 2100volts
Angiotensin infused at constant flow rate over the 50 min
gradient.
•HPLC-Chip delivers robust spray performance for all solvent conditions.
•Does not require voltage change or optimization during a gradient.
•Polyimide nanospray emitter lifetime measured in weeks!

Page 9
HPLC-Chip/MS: Features and
Benefits
• High sensitivity & chromatographic performance
• Improved MS/MS data quality
• Easy to use, robust, plug-&-play device suitable for
unattended routine identification and quantification
• Automated Chip-system for sample enrichment, separation &
nebulization now into all Agilent mass spectrometers
Chip – Ion Trap
• MS & MS n
Chip – TOF
• MS (MS 2 )
• Accurate mass
Chip – QTOF
• MS & MS 2
• Accurate mass
Chip – QQQ
• MS & MS 2
• Quantitation

Ultra High Capacity (UHC)-SM-Chip
Main Objective
Page 10
Enrichment of small molecules comprising a wide range of polarities
500nL enrichment
column
6
5
1
2
4
3
Separation Column 150mm x 75µm
ID

The Consequences – The “TOF/QTOF Image”
• limited dynamic range (2 decades)
• quantification is difficult if not impossible
• mass accuracy varies with signal level. „Exact mass“ requires
highly experienced operator.
• high resolution can be achieved only by extending the ion
path length, sacrificing signal abundance.
Page 11

Page 12
Traditional Signal Processing Using TDC
LOW ion current
situation
TDC
threshold
Flight time: 50.000000 µsec
1. A single ion hits the detector.
When exceeding the intensity threshold, the flight time
gets measured.
1a. During the subsequent transient a second ion hits
the detector. Within the instrument’s resolution we
register a slightly shifted flight time.
TDC = Time to Digital Converter

TDC Principle (contd.)
Page 13
HIGH ion current
situation
TDC
threshold
Flight time:
Now the sample is more concentrated, causing
two ions reaching the detector at almost the same
time (within the instrument’s resolution). The dark
blue curve would represent the resulting signal.
Unfortunately, the TDC only registers the first
arriving ion exceeding the threshold. The clock
cannot be reset fast enough to register the
second ion arriving a little later. This mistakenly
shifts the flight times to shorter values with
increasing signal levels.
In order to compensate for this weakness, TDC
based systems apply ”dead time correction“
factors and/or ion beam attenuation at high signal
levels.

TDC Principle (contd.)
Page 14
HIGH ion current
situation
TDC
threshold
Flight time:
Now the sample is more concentrated, causing
two ions reaching the detector at almost the same
time (within the instrument’s resolution). The dark
blue curve would represent the resulting signal.
Unfortunately, the TDC only registers the first
arriving ion exceeding the threshold. The clock
cannot be reset fast enough to register the
second ion arriving a little later. This mistakenly
shifts the flight times to shorter values with
increasing signal levels.
In order to compensate for this weakness, TDC
based systems apply ”dead time correction“
factors and/or ion beam attenuation at high signal
levels.

Modern Signal Processing Using ADC
Page 15
LOW and HIGH
ion current
situation
Flight time: is represented by the centroid across all ions
1. A single ion hits the detector.
The apex of the resulting signal gets registered
as the arrival time.
2. Now the sample is more concentrated, causing
two (or up to 256 due to 8 bit ADC) ions reaching
the detector within minimal time shifts (according
to the TOF’s resolution). The dark blue curve
represents the resulting signal.
As described earlier, TDC systems would have
problems to digitize the overall arrival time
correctly.
3. ADC based systems transform the abundance of
the resulting signal in time increments of 1 nsec
(1 GHz ADC), 500 psec (2 GHz ADC) or 250 psec
(4 GHz ADC). No “dead time correction” or
attenuation of the ion beam required.
ADC = Analog to Digital Converter

Enhancements in Time-of-Flight
New Acquisition Technology Increasing Resolving Power
O
FIA, 2 scans/sec (6713 transients/scan)
2 IRM averaged over five scans
Page 16
O
O
OH
Butyl paraben
[M+H] + 195.10157
Methyl 5-acetylsalicylate
[M+H] + 195.06519
HO
O
8 bit – 1 GHz
Analog Digital
Converter
O
195.088679
6,100 Resolving
Power (FWHM)
195 195.02 195.04 195.06 195.08 195.1 195.12 195.14 195.16 195.18 195.2
Counts vs. Mass-to-Charge (m/z)

Enhancements in Time-of-Flight
New Acquisition Technology Increasing Resolving Power
O
FIA, 2 scans/sec (6713 transients/scan)
2 IRM averaged over five scans
Page 17
O
O
OH
Butyl paraben
[M+H] + 195.10157
Methyl 5-acetylsalicylate
[M+H] + 195.06519
HO
O
8 bit – 4 GHz
Analog Digital
Converter
Transient Peak
Picking
O
+ 0.8 ppm
195.065349
195.101392
0.036 Da
- 0.9 ppm
14,000 Resolving
Power (FWHM)
195 195.02 195.04 195.06 195.08 195.1 195.12 195.14 195.16 195.18 195.2
Counts vs. Mass-to-Charge (m/z)

New 4GHz Acquisition System
up to 5 Decades of In-Spectrum Dynamic Range
Max Abd
3x10 7
Page 18
x10 6
9
8.8
8.6
8.4
8.2
8
7.8
7.6
7.4
7.2
7
6.8
6.6
6.4
6.2
6
5.8
5.6
5.4
5.2
5
4.8
4.6
4.4
4.2
4
3.8
3.6
3.4
3.2
3
2.8
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
+ Scan (# 31-35, 5 scans) niacinamide_erythromycin.d
* 123.055394
9x10 6 cts
Niacinamide, 10 ng/µL
[M+H] + = 123.055289 m/z
Error = +0.8 ppm
In-Scan Dynamic Range
This Example
9x10 6 / 250 = 3.6 × 10 4
Maximum
3x10 7 / 250 = 1.2x 10 5
250 cts
207.007866 329.056151 425.138434
659.287715 759.355249 922.009798
1221.991581
1521.971969
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700
Counts vs. Mass-to-Charge (m/ z)
x10 2
2.5
2.4
2.3
2.2
2.1
2
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
+ Scan (# 31-35, 5 scans) niacinamide_erythromycin.d
734.468956
Erythromycin, 500 fg/µL
[M+H] + = 734.468518 m/z
Error = +0.6 ppm
735.416113
736.387755
734 734.5 735 735.5 736 736.5 737 737.5 738 738.5 739 739.5 740 740.5
Counts vs. Mass-to-Charge (m/ z)

TIC of A Mixture
Page 19
x10 8
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
+ TIC Scan mix995.d
1
Flow Injection
Angiotensin (m/z 1046): 16 nM
Caffeine (m/z 195): 25 nM
Reserpine (m/z 609): 25 µM
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3
Counts vs. Acquisition Time (min)
Important: 1) Mixture
2) Co-Elution
3) Wide [ ] Range
4) Wide m/z Range
An artificial sample,
but representative
of real-world
complex sample
analysis with matrix

Page 20
Flight Tube Thermal Stability
Vacuum Insulated
Flight Tube
Thermostatted
HV Power Supplies
Low CTE* Inner
Flight Tube (INVAR)
* Coefficient of Thermal Expansion

Mass Accuracy – Environmental Extremes
Page 21
Injecting every 15 minutes, from 11º - 36º C and 10 - 95 %Relative Humidity
Mass Error, ppm
5
4
3
2
1
0
-1
-2
-3
-4
-5
Mass Accuracy
0 50 100 150 200 250 300
Reserpine Injection

Conclusion
Sensitivity
no loss, as ion path remains unchanged
Resolution
2x to 3x increase by signal processing
Mass accuracy
sub-2 ppm with IRM, sub-5 ppm without IRM
Dynamic range
up to 5 decades by dual-channel signal amplifier/logic
Page 22

Page 23
Merci pour votre
attention,
Questions?
Frederic_metral@agilent.com
Tel:06 78 73 55 26