1 © 2011 AB SCIEX
Presentation title Name of Speaker and Identification, Location, Date
Micro LC/MS: Miniaturizing LC for LC/MS applications in bioanalysis Tina Settineri, Ph.D. Director, HPLC Products
2 © 2011 AB SCIEX
What is Microflow LC?
0.5 mm
area = 18 area = 1area = 85
0.5 mm
area = 18 area = 1area = 85
• MicroLC: HPLC using columns with inner diameter (ID) ≤ 1.0 mm. • Typical Flow Rates: 5-200µL/min
3 © 2011 AB SCIEX
� Improve throughput
� Use less sample
� Improve sensitivity (LOQ) of LC/MS
� Improve LC and MS uptime
� Save money spent on mobile phases
Why Miniaturize to Micro flow UHPLC for LC/MS Bioanalysis?
4 © 2011 AB SCIEX
ekspertTM microLC 200 micro flow UHPLC
• Microflow UHPLC (10,000 psi) • System delay (void) volume: 1 – 3 µL • Total system volume: ~ 4.5 µL
• Supports 1.0, 0.5, and 0.3 mm ID columns • Flow rates 5 – 200 µL/min • Programmable injection - from 15 nL (metered)
to 10 µL (without changing loop) • Close coupling with MS source
- Small volume hybrid electrodes for Turbo VTM source
Compact, high-throughput UHPLC system for LC/MS
5 © 2011 AB SCIEX
Microfluidic Flow Control™ (MFC) Unique pumping system - flow control at nano/micro low flow rates • Direct flow rate feedback to a rapidly adjustable
pressure source
• Maintains flow rate independent of backpressure
• Enables highly reproducible and accurate analytical separations
• Single-stroke-per-run design for reliability and performance
0 60 120 180 240 300395
396
397
398
399
400
401
402
Flow
Rat
e (n
L/m
in)
Time (sec)
1 nL/min step
2 nL/min step
Electronically Controlled Pressure Source
Flow Meter
Control Processor
6 © 2011 AB SCIEX
Flow lamination to decrease diffusion length by 2x and hence improve mixing by 4x
A
B B A
Pumps
Purge Valves
Filters
Pressure Transducers
Pb
A
B Pc
Pa
Flow Module (restrictors)
Pumping system - Unique 3 Layer Mixing Design Significantly Decreases mixing & delay time
7 © 2011 AB SCIEX
Decreasing diffusion length, x, decreases time required for mixing by x2
Required mixing time t (or mixer length)
Two thick streams
Three thinner streams
t = x2
x
x’
1 2 3 4
• In a two layer mixer, the longest diffusion distance needed for full mixing is the tube diameter
• In a three layer the max diffusion distance needed to travel is only the tube radius
Pumping system - Unique 3 Layer Mixing Design Significantly Decreases mixing & delay time
8 © 2011 AB SCIEX
Improved Throughput with Low Delay Times: Micro Flow UHPLC – actual gradient = programmed gradient High Flow UHPLC – actual gradient ≠ programmed gradient - especially with fast separations
- reduces inject-to-inject times - allows a wider range of compounds eluted during gradient - improves accuracy with fast gradients
Time
Programmed gradient
Actual microLC gradient
Actual analytical UHPLC gradient
Column Diameter (mm)
Delay Volume/ Void Volume
UHPLC 2.1 1.0
MicroLC 0.5 0.13
Improving Throughput with Micro Flow UHPLC:
9 © 2011 AB SCIEX
Improved Throughput with Low Delay Times:
Traditional UHPLC Microflow UHPLC
Flow Rate 1.5 mL/min 0.060 mL/min
Column Diameter 2.1 mm 0.50 mm
Mobile Phase / Solvent Waste Volume / run
1925 µL 66 µL
Solvent Used / 1200 injections 2.31 L 2 mL
10 20 30 40 50 60 70
-500
0
500
1000
1500
2000
14
13
12
11
109
8
7
6
5
4
2
mAU
Seconds
1
3
10 20 30 40 50 60 70
-200
0
200
400
600
800
14
13
1211
10
9
8
7
65
4
3
2
mAU
Seconds
1
Analytical UHPLC, 77 sec Microflow UHPLC, 48 sec
10 © 2011 AB SCIEX
RT: 0.00 - 2.50
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative
Abunda
nce
1.16
1.98
1.44
1.44
1.45
1.43
1.671.66
1.460.89 0.89 1.65
1.40 1.47
1.481.21 1.70 2.05 2.452.310.84 1.010.710.58 1.950.03 0.14 0.39
NL:2.76E6TIC MS RT9019-24
RT: 0.00 - 1.00
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
0.82
0.820.48
0.48
0.82
0.530.83
0.56
0.610.47 0.830.81
0.61
0.83
0.62 0.640.47 0.81 0.840.840.65 0.810.75 0.86 0.920.470.350.120.07 0.19 0.310.25 0.37
NL:2.40E7TIC MS Dev-10
10
Traditional LC: Multiple Analytes
Low Flow: Multiple Analytes
Exceptional Separation Equivalent Sensitivity using 10-20x less sample 1/3 the overall run time
Improved Throughput with Low Delay Times:
Data courtesy of Rob Turner, CoMentis
11 © 2011 AB SCIEX 11
� Excellent separation with 0.5 to 1 minute chromatography
� Solvent usage decreased from ~ 2-3 Liters/week to ~ 20 mls/week
� Reduced sample injection size from 10-20 µLs to 1-2 µLs without a loss in sensitivity, reduces sample required, enabling multiple sampling and Single animal PK/PD even with rodent species
� Extended linear range observed
� Decrease in matrix effect observed
Summary - MicroLC System Incorporation at CoMentis
Slide courtesy of Rob Turner, CoMentis
12 © 2011 AB SCIEX
Electrode Dispersion Comparison (10 µL/min)
• Peak dispersion as observed in a flow injection experiment at 10 µL/min for four ESI electrodes with different internal diameter sizes.
• The orange and blue lines illustrate the peak dispersion improvement utilizing the new smaller ID hybrid electrodes
13 © 2011 AB SCIEX
600µL/min Standard electrode
800µL/min Standard electrode
38µL/min 65µm ID
80µL/min 65µm ID
38µL/min 50µm ID
80µL/min 50µm ID
Example Data: 1 ng/mL, 1 µL injection, same gradient Improvement in peak width at base with smaller ID electrodes
Shimadzu system - high flow ekspert™ microLC 200 system - micro flow
1.8 sec 1.8 sec 1.5 sec
2.1 sec 2.4 sec 1.5 sec
14 © 2011 AB SCIEX
� Two new smaller ID electrodes for use in the TurboV™ source – 25 µm ID - ideal for 0.3 mm columns (5-20 µL/min) – 50 µm ID - ideal for 0.5 mm columns (20-100 µL/min)
� Hybrid PEEKSIL/stainless steel tip electrodes
� Easy to install by replacing standard electrode
25 µm ID 50 µm ID
1 cm
Column very close to source
MicroLC system uses new hybrid electrodes
15 © 2011 AB SCIEX
Improved Chromatography with hybrid electrodes & ekspert™ microLC 200 System
1A9 Peptide 1
2B7 Peptide 2
2B7 Peptide 1
1A6 Peptide 2
Endogenous
1A6 Peptide 1 1A1 Peptide 1
Endogenous
1A9 Peptide 1
2B7 Peptide 2
2B7 Peptide 1
1A6 Peptide 2
1A6 Peptide 1
1A1 Peptide 1
Endogenous
Endogenous
1A1 peptide 2
Data courtesy of Pharmacadence (in collaboration with Merck)
With hybrid electrode
With hybrid electrode
Old style larger ID electrode
Old style larger ID electrode
16 © 2011 AB SCIEX
0.25 µL sample pickup and injection with 0.6% RSD (n=3)
y = 3E+06x - 346238R2 = 0.9973
0
1
2
3
4
5
6
0 0.5 1 1.5 2
Millions
Sample Volume (µL)
Peak
are
a (c
ount
s)
Verapamil Peak Area (counts)
Linear fit (blank injection excluded)
Use less sample: AS/valve design enables very small volume injections with low RSDs
10 ng/mL Verapamil, 53 sec cycle time 5-90% B gradient in 30 seconds, 0.5 x 50 mm Halo C18 column.
Sample Vol (µL) %RSD, n=3 0.25 0.643407
0.50 1.109915
0.75 0.891552
1.0 0.630341
1.25 1.750287
1.50 1.474698
1.75 1.229087
2.0 1.691631
17 © 2011 AB SCIEX
% Sampling Efficiency, entire flow range, 0.05-500 µL/min
Flow Rate (µL/min)
0
5
10
15
20
25
0.5 100 200 300 400 500 5
20 60
Sampling Efficiency %
0.05
120 250
22.0%
% Sampling Efficiency, high flow range only, 5-500 µL/min 2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8 Sampling Efficiency %
0.20% 0.27%
0.40% 0.50%
0.80%
1.50%
7.5x
110x
Covey,T.R.; Thomson B.A.; , Schneider, B.S. “Atmospheric Pressure Ion Sources,” Mass Spec. Reviews, 2009, 00, 1-29. Covey TR, Schneider BB, Kovarik, Corr, Javahari et al. “”The Central Analytical Figures of Merit of ESI, MALDI, and APCI.” In:Cole RB, ed. Electrospray and MALDI mass spectrometry. 2010, Chapter 13. Hoboken: John Wiley & Sons, Inc.
Sampling Efficiency vs. Flow Rate in ESI - Turbo V™ Source API 5000 System
18 © 2011 AB SCIEX
High Flow Steroid Method vs. Micro Flow Method � Equal sample volume & concentration was injected on column for both methods
� Peak areas increased by about 10X on average and S/N improved by 4X
XIC of +MRM (8 pairs): 347.200/293.200 Da ID: Corticosterone_57 from Sample 40 (std 3, 11,111.11 pg_mL) of InertSustain 2.1x... Max. 1.3e4 cps.
1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8Time, min
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
1.5e4
1.6e4
1.7e4
1.8e41.8e4
Inten
sity, c
ps
XIC of +MRM (8 pairs): 347.200/293.200 Da from Sample 43 (std 3, 11,111.11 pg_mL) of 0.3x100mm Sustain calcurve.wiff (Tur... Max. 8.2e4 cps.
2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
Inten
sity, c
ps
1.4e5
1.8e4
600 µL/min 2 µL inj.
10 µL/min 2 µL inj.
Signal to Noise Comparison of MicroLC vs. High Flow Steroid Method. Data points and replicates which produced accuracies of 100% ± 10% were used to calculate average values in the table. Ratios are expressed as low flow/high flow.
Compound Conc. (pg) Area Ratio
Peak Height Ratio S/N Ratio
Corticosterone 2.47 10.5 6.0 6.0
7.41 11.0 5.6 5.6
22.22 11.6 5.3 5.2
66.67 11.5 5.8 5.6
Average 11.2 5.7 5.6
%CV 4.4 5.6 5.9
Aldosterone 7.41 9.2 7.6 3.9
22.22 8.8 6.7 3.2
66.67 8.3 6.5 2.9
Average 8.8 6.9 3.3
%CV 5.2 8.3 16.2
19 © 2011 AB SCIEX
LC-MS/MS Overlayed Chromatograms (HPLC vs. MFLC/QTRAP® 5500 System Bioanalysis of Methotrexate (400 ng/mL) from Human Plasma
Similar linear velocities Same stationary phase Prontosil, C18, 3 µm
0.2 0.6 1.0 1.4 1.8 2.2 2.6 3.0 3.4 3.8 4.2 4.6 Time, min
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 7.5e5
Intensity, cps
HPLC – 700 µL/min, 2.0 mm ID x 50 mm Injection volume = 2 µL
MFLC – 35 µL/min, 0.5 mm ID x 50 mm, Injection volume = 2 µL
9.6x Signal Increase!
Data courtesy of Alturas Analytics, Inc.
20 © 2011 AB SCIEX
Overall LLOQ Sensitivity Compared to Traditional Analytical LC Method
Column ID (mm)
Flow rate (µl/min)
Max Injection
Volume (µl) Avg Area % Diff.
2.0 400 40 1.27E06 N/A
0.3 10 10 9.37E05 -26
Data produced from injection of propranolol on QTRAP® 5500
Data courtesy of Alturas Analytics, Inc.
21 © 2011 AB SCIEX
� Improve throughput Very low delay volume of system enable ultrafast gradients for LC/MS withough compromising chromatography
� Use less sample – inject smaller volumes, minimize waste Injection system enables sub 1 µL injections and minimal sample wasted
� Improve sensitivity (LOQ) for LC/MS Improved ionization efficiency translates & improved chromatographic peak capacity translates into improved sensitivity & S/N
� Improve LC and MS Uptime 1-stroke per run minimizes LC maintanence, lower flow rates & injection volumes provide reduced solvent load on MS
� Save Money spent on mobile phases Up to 95% lower mobile phase costs, including storage and disposal
Miniaturization to Micro flow UHPLC
22 © 2011 AB SCIEX
Trademarks/Licensing
For Research Use Only. Not for use in diagnostic procedures.
The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.
© 2012 AB SCIEX.
23 © 2011 AB SCIEX
Robustness for DMPK DMPK - Robustness Test – 10 µL/min, 5+ days, 750+ injections
Peak Area Response Ratio of Imipramine vs. Clomipramine spiked into rat plasma
0.00.20.40.60.81.01.21.41.6
10/31 11/1 11/2 11/3 11/4 11/5 11/6 11/7
Are
a R
espo
nse
Rat
io
Date of acquisitionXIC of +... Max. 2.1e5 cps.
5Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e5
1.8e5
1.9e5
2.0e5
2.1e5
Intensity, cp
s
1.06
XIC of +... Max. 2.1e5 cps.
5Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e5
1.8e5
1.9e5
2.0e5
2.1e5
Intensity, cp
s
1.18
XIC of +... Max. 2.0e5 cps.
5Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e5
1.8e5
1.9e5
2.0e5
Intensity, cp
s
1.27
XIC of +... Max. 2.2e5 cps.
5Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e5
1.8e5
1.9e5
2.0e5
2.1e5
2.2e5
Intensity, cp
s
1.31
Day 1 Day 3 Day 4 Day 5
2.1e5 2.1e5 2.0e5 2.2e5
No loss in signal intensity or chromatographic performance
observed across the 750+ replicate injections.
0.3 mm ID × 50 mm ChromXP™ HALO C18 column
Avg. response ratio = 1.3, Std Dev. = 0.05 3.8% SD over the entire analysis
24 © 2011 AB SCIEX
Inject time = Required volume
Measured flow rate
• Technique for delivering nanovolume injections
• Programmable / variable injection volumes
• Excellent precision / linearity
• Multiple injections from single A/S sample load allows method development with a small volume of sample
• Faster method development by more efficient system utilization
y = 1428.7 x - 238256R2 = 0.9979
0.E+00
2.E+06
4.E+06
6.E+06
8.E+06
1.E+07
1.E+07
1.E+07
0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
Injected Volume (uL)
Pea
k A
rea
Metered, ‘Time-slice’ Injections for nanovolume injections to save sample for microLC
0 2 4 6 8 10
R2=0.9998
Injected Volume (nL) 0 500 1000
Pea
k A
rea
25 © 2011 AB SCIEX
Eksigent UHPLC Product Portfolio
� From Analytical Flow to Micro Flow to Nano Flow LC
ekspert™ ultraLC 100 & 100-XL systems The only analytical flow UHPLC systems designed for use with AB SCIEX mass spectrometers
ekspert™ microLC 200 system The world’s only dedicated micro-UHPLC designed to deliver faster separations and improved sensitivity for LC/MS
NanoLC-Ultra® & cHiPLC system High Performance splitless flow nano-UHPLC coupled with the plug and play, high reproducibility cHiPLC system
ExpressLC® – UV System High Performance microflow UHPLC system
26 © 2011 AB SCIEX
Delay volume effect – high flow vs. microflow 25 µm electrode, 2 µL inj. (Triazine Pesticide Mixture – 10 µL/min flowrate) Chromatogram for 0.5 X 50 mm Halo™ C18 column run with the conventional LC system optimized for low flow Chromatogram for 0.5 X 50 mm Halo™ C18 column run with the ekspert™ microLC 200 MS: AB SCIEX 3200 QTRAP®
27 © 2011 AB SCIEX
Tubing, flow rates & electrodes for various column diameters
Column ID (mm)
Flow rate range (µL/min)
Connection tubing ID (µm)
Recommended ESI electrode
2.1 200-1000 ~125 (0.005”) Standard TurboV electrode
1.0 50-200 75 pre-column 50 post-column
65µm ID stainless steel or 50µm ID PEEKsil/stainless steel
0.5 10-50 50 pre-column 50 post-column
50µm ID PEEKsil/stainless steel
0.3 4-20 50 pre- column ≤50 post-column
25µm or 50µm ID hybrid PEEKsil/stainless steel
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