Unlocking the Benefits of MicroLC with AB SCIEX and Eksigent Technology

Anthony Romanelli, Sr. Applications Manager

Anthony.romanelli@absciex.com

2 © 2014 AB SCIEX

What is Micro Flow LC?

0.5 mm

area = 18 area = 1area = 85

0.5 mm

area = 18 area = 1area = 85

HPLC using columns with inner diameter (ID) ≤ 1 mm

Typical Flow Rates: 5-200µL/min

Column diameter (mm) 0.3 0.5 1.0 2.1

Typical flow rate (mL/min) 10 35 140 600

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• Improve throughput

• Improve sensitivity (LOQ)

• Consume less sample

• Reduce LC and MS downtime

• Reduced costs

Why Use Micro LC?

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microLC 200plus System

• 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)

• Compact, Close coupling with MS source

- Small volume hybrid electrodes for Turbo VTM

and IonDrive™ Turbo V sources

Compact, high-throughput UHPLC system for LC/MS

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Electronically

Controlled

Pressure Source

Flow Meter

Control

Processor

Microfluidic Flow Control™ (MFC)

Microfluidic Flow Control Precise, stable flow for reproducible high-speed chromatography

• Proprietary Microfluidic Flow Control (MFC)

technology for rapid flow adjustment

• Continuous flow monitoring

• Operates up to 10,000 psi

• MFC and high pressure capability combine to

ensure precise and stable flow for reproducible,

high-speed chromatography

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MicroLC for Exenatide Quantitation

Assay Development Goals:

• Achieve sensitivity levels that enable

quantitation in plasma when slow release

exenatide is administered.

• Develop a selective, robust, and

reproducible method

• Demonstrate the ability to quantitate over a

wide dynamic range

QTRAP® 6500 System Nano LC400 System

IonDrive™ ion source microflow electrodes

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MicroLC for Exenatide Quantitation

Samples prepared by spiking standards into a crashed rat plasma matrix

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Stable Isotope Labeled Universal Monoclonal Antibody Standard

XIC chromatogram of signature peptide transitions in plasma matrix; SILUMab Standard in the insight

Heavy Chain DTLMISR

Heavy Chain FNWYVDGVEVHNAK

Heavy Chain VVSVLTVLHQDWLNGK

Light Chain YAASSYLSLTPEQWK

Light Chain AGVETTTPSK

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Quantitation of Signature Peptide AGEVETTPSK (light chain: +2y6 light)

• Excellent accuracy and precision over 4 orders of linear dynamic range

• Improved sensitivity compared to previous high flow data

• Over a 2 fold increase in throughput

LOQn=1

LOQn=2

LOQn=3

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J Proteome Res. 2014 Apr 4;13(4):1905-10

Peptide Quantitation with Micro Flow Publication

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MicroLC for Lipid Profiling

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MicroLC for Lipid Profiling - Chromatography

Improvement in chromatographic resolution with the microLC 200

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MicroLC for Lipid Profiling - Sensitivity

Significant gains

in sensitivity

achieved with

only 1/10th the

amount of

analyte loaded

on column

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MicroLC for Lipid Profiling - Reproducibility

Excellent reproducibility was achieved in regards to retention time,

chromatographic resolution, and signal intensity.

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Clin/Tox Robustness - Long Term Retention Time Stability

Retention time plot of quality control sample over 1000 injections in

pooled human urine

Guard column change (every ~250 injections)

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Pooled 8 Human Urine

10 ng/mL

Injection #20

1st Guard

Pooled 8 Human Urine

10 ng/mL

Injection #1000

4th Guard

Pooled 8 Human Urine

10 ng/mL

Injection #1001

No Guard

Clin/Tox Long Term Robustness – Column Performance and Peak Shape

Note: Micro flow method increased throughput ~2x

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• Improve throughput

• Improve sensitivity (LOQ)

• Consume less sample

• Reduce LC and MS downtime

• Reduced costs

Benefits of Micro LC with AB SCIEX and Eksigent Technology

18 © 2014 AB SCIEX

Trademarks/Licensing

• For Research Use Only. Not for use in diagnostic procedures.

• SCIEX and Eksigent are part of AB SCIEX. The trademarks

mentioned herein are the property of AB Sciex Pte. Ltd. or their

respective owners. AB SCIEX™ is being used under license.

• © 2014 AB SCIEX. All rights reserved. Information subject to change

without notice.

Questions and Answers

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