Liquid Extraction Surface Analysis

(LESA)

Analyzing whole body animal sections

Daniel Eikel Advion BioSciences

Lecture 5, Page 60

Outline LESA is a new surface sampling technology based on liquid extraction (Liquid Extraction Surface Analysis) and nano-electrospray mass spectrometry (nESI-MS)

Example 1: LESA drug distribution profiling from whole body mouse sections: Terfenadine and its metabolite Fexofenadine

Lecture 5, Page 61

Introduction to LESA Liquid Junction Surface Sampling

Mass

spectrometer

Sample

Solvent

Sampling tip

Aspirate solvent Dispense solvent on sample Aspirate sample solution Transfer sample Spray sample

Nozzle

96- or 384-well plate

Side view Lecture 5, Page 62

Introduction to LESA – Schematic Workflow –

Step 1 – Solvent Delivery

Disposable Tip picks up extraction solvent at reservoir

Step 2 – Analyte Extraction

Robot places extraction solvent on target and initiates aspirate/dispense

cycles for analyte extraction

Step 3 – Analyte Ionization

Robot aspirates extracted analytes from target and initiates electrospray at a

400 nozzle nESI chip

Chip-based nano ESI

Kertesz V. and Van Berkel GJ:

Fully automated liquid extraction-based surface sampling and ionization using a chip-based

robotic nano electrospray platform. Journal of Mass Spectrometry 2010 45(3) p252-260 Lecture 5, Page 63

Introduction to LESA – Schematic Workflow –

LESA enabled Advion TriVersa NanoMate

Nozzle dimensions A chip:

5.5 m ID x 28 m OD x 55 m height

Lecture 5, Page 64

Example 1: LESA drug distribution profiling from whole body mouse sections: Terfenadine and its metabolite, Fexofenadine

Eikel D, Vavrek M, Korfmacher WA and Henion JD: Liquid Extraction Surface Analysis Mass Spectrometry (LESA-MS) as a Novel Profiling Tool for Drug Distribution and Metabolism Analysis: The Terfenadine Example

Lecture 5, Page 65

Example 1 - Background Information -

Terfenadine and its active metabolite, Fexofenadine, are both antihistamines

Animal experiment:

50 mg/kg PO dosed mice put down in CO2, frozen and sectioned 1 and 4 h post dose

MS experiment:

2 min Infusion

Q3 survey MS/MS and SRM analysis on AB/Sciex 5500 Qtrap

or

Exact mass experiment on Thermo Exactive Benchtop Orbitrap

Lecture 5, Page 66

Whole Body Distribution Analysis

Liver Kidney

Cecum of Intestine

Stomach

Spleen

Brain Skeletal muscle

Small Intestine

Lecture 5, Page 67

Lecture 5, Page 68

LESA spatial resolution

0.8 mm

0.8 mm

1.1 mm

0.9 mm

Lecture 5, Page 69

Example of Single Organ Profiling

Brain

Lecture 5, Page 70

Terfenadine MALDI-MS

1 h post dose

LESA comparison to other ‘imaging’ technologies (MALDI-MS comparison data, Chen et al. 2008)

Fexofenadine MADLI-MS

Optical microscope pictures

from mouse tissue

Lecture 5, Page 71

LESA Comparison to other ‘imaging’ technologies (Whole Body Autoradiography comparison data, Leeson et al. 1982)

Lecture 5, Page 72

Comparing different Analysis Approaches regarding Sensitivity

14C-terfenadine* 10 mg/kg PO rat

[Ref. 22]

MALDI-IMS 50 mg/kg PO mouse

[Ref. 23]

LESA-MS-Profiling 50 mg/kg PO mouse

(Orbitrap-Exact Mass)

LESA-MS-Profiling 50 mg/kg PO mouse

(QQQ-SRM)

Terfenadine Fexofenadine Terfenadine Fexofenadine Terfenadine Fexofenadine

Blood

Brain

Liver

Lung n.a. n.a. n.a. n.a.

Kidney

Spleen

Skeletal Muscle n.a.

Stomach

Cecum of Intestine

Small Intestine

Large Intestine

Lecture 5, Page 73

Summary Example 1

LESA …

… a profiling technique with a maximum spatial resolution of 1 mm on target

… can analyze drug distribution within a single organ or within a whole body

… is at least 20-fold more sensitive than MALDI-imaging MS and only ca. 5 fold less sensitive than QWBA in the given Terfenadine/ Fexofenadine example

Lecture 5, Page 74

References

• Sheerin Khatib-Shahidi, Malin Andersson, Jennifer L. Herman, Todd A. Gillespie, and Richard M. Caprioli, Anal. Chem., 2006, 78 (18), pp 6448–6456

• Hsieh et al. RCMS, 20, 965-72 (2006).

• Angus C. Grey, Pierre Chaurand, Richard M. Caprioli and Kevin L. Schey, J. Proteome Res., 2009, 8 (7), pp 3278–3283

• Nemes and Vertes, Anal. Chem., 2007, 79, 8098.

• D. Eikel and J. D. Henion, Liquid Extraction Surface Analysis (LESA) of Food Surfaces Employing Chip-Based Nano Electrospray Mass Spectrometry, RCMS 2011. Rapid Commun. Mass Spectrom. 2011, 25, 1–10.

• Girod, M.; Shi, Y.; Cheng, J.-X.; Cooks, R. G., Desorption Electrospray Ionization Imaging Mass Spectrometry of Lipids in Rat Spinal Cord. Journal of the American Society for Mass Spectrometry 2010, 21 (7), 1177-1189.

• Li, H.; Hummon, A. B., Imaging Mass Spectrometry of Three-Dimensional Cell Culture Systems. Analytical Chemistry 2011, 83 (22), 8794-8801.

• Luo, G.; Marginean, I.; Vertes, A., Internal Energy of Ions Generated by Matrix-Assisted Laser Desorption/Ionization. Analytical Chemistry 2002, 74 (24), 6185-6190.

. Lecture 5, Page 75