Liquid Extraction Surface Analysis Mass Spectrometry (LESA MS)

-Drug Distribution and Metabolism of Diclofenac

in the Mouse

Jack Henion (1), Daniel Eikel (1), Stefan L. Linehan (2), Dennis Hel ler (2), Keeley Murphy (3), Patrick J. Rudewicz (4) and Simo n J. Prosser (1)

(1) Advion Biosystems Inc., NY, USA(2) XenoBiotic Laboratories Inc., NJ, USA(3) Thermo Fisher Scientific Inc., CA, USA

(4) Elan Pharmaceuticals Inc., CA, USA

1

in the Mouse

• Introduction to liquid extraction surface analysis mass spectrometry (LESA-MS)

• Example analysis of Diclofenac from thin whole body mouse sections

Overview

body mouse sections

• Diclofenac distribution analysis and metabolite identification

• Summary and Conclusions

2

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 dispense/dispense

cycles for analyte extraction

Step 3 – Analyte Ionization

Robot aspirates extracted analytes from

target and initiates electrospray at a

400 nozzle nano ESI chip

3

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

Introduction to LESA– Schematic of Tip-to-nESI-Chip –

Mass SpectrometerIon Orifice

Robotic Sampling Probe~1300 V

nESI Chip

NozzleInlet

4

Conductive Pipette TipSample

Micro Fabricated nano ESI chip– 400 nozzles/samples per Chip –

5

Nozzle dimensions: 5.5 µm ID x 28 µm OD x 55 µm height

Universal sample support holder platform

Chemical Structures– Diclofenac and selected Metabolites –

HN

Cl

O

OH

6

Cl

Experimental

• Male balb-C mouse (18-26 g)– P.O. dosed with 10 mg/kg Diclofenac (or vehicle only for control mouse),– Sacrificed after 15 min in CO2, blocked and sectioned in cryomicrotome

• LESA parameters :– Extraction solvent 80/20 Methanol/Water

• 0.1 vol % formic acid for positive ion mode

• 0.1% Ammonium hydroxide for negative ion mode.• 0.1% Ammonium hydroxide for negative ion mode.

– 1.5 µL extraction solvent/pick up– 1.1 µL dispense followed by aspiration of 1.3 µL from the tissue, repeat dis/asp 2 more times.– nESI generated using 0.9 psi and -1.25 kV or 0.3 psi and 1.35 kV respectively.

• Thermo Exactive Benchtop Orbitrap– calibrated daily and tuned for m/z 195.0877 and m/z 265.1479 in positive and negative ion mode

respectively– operated at 100,000 nominal resolution with AGC set to high sensitivity (5e6), 250 ms fill time limit.

• AB SCIEX 5500 QTRAP– Tuned and calibrated per manufacturer’s recommendations– Operated in Enhanced Product Ion (EPI) Mode using optimal collision energy for the respective compound

as determined by analytical standard7

LESA Points SoftwareExample for sequence list generation –

BrainStomach Content

Intestinal Content

8

Liver

LESA Spatial ResolutionExample of the Liquid Junction and Tissue Hydration

0.8 mm

0.8 mm

9/19

Spatial resolution depends on:Solvent CompositionVolume dispensed on the surfaceSurface Tension (e.g. hydration state of the surface)

1.1 mm

0.9 mm

LESA MS Determination of Diclofenac: ExactiveBrain: Negative Ions

Neg_Diclofenac_mouse1_Brain_03 #1-58 RT: 0.11-0.89 AV: 58 NL: 6.03E4T: FTMS {1,1} - p NSI Full lock ms [250.00-550.00]

60

65

70

75

80

85

90

95

100294.0097

294.0694

Diclofenac_negOH_mouse3_Brain_5 #1-51 RT: 0.11-0.79 AV: 51 NL: 4.63E4T: FTMS {1,1} - p NSI Full lock ms [250.00-550.00]

60

65

70

75

80

85

90

95

100294.0694

293.9998

Dosed Mouse Control Mouse

Diclofenac(1 ppm deviation)

HN

Cl

Cl

O

O

(M-H)-

10

293.96 293.98 294.00 294.02 294.04 294.06 294.08m/z

0

5

10

15

20

25

30

35

40

45

50

55

Rel

ativ

e A

bund

ance

293.9998

294.0461

294.0999

294.0213293.9797 294.0362 294.0821

294.0541

293.96 293.98 294.00 294.02 294.04 294.06 294.08m/z

0

5

10

15

20

25

30

35

40

45

50

55

Rel

ativ

e A

bund

ance

294.0460

294.0822

Parent Diclofenac readily observed in brain tissue with Exactive at high resolution

POS_Mouse1_Stomach_03 #62-103 RT: 0.95-1.53 AV: 42 NL: 1.22E7T: FTMS {2,1} + p NSI Full ms2 1000.00@hcd20.00 [50.00-550.00]

65

70

75

80

85

90

95

100245.0775

Diclofenac_pos_std #63-97 RT: 0.85-1.35 AV: 35 NL: 1.56E6T: FTMS {2,1} + p NSI Full lock ms2 1000.00@hcd20.00 [50.00-550.00]

65

70

75

80

85

90

95

100215.0499

LESA All-Ion MS/MS Diclofenac: ExactiveStomach: Positive Ions

Diclofenac analytical standard Stomach, 15 min post-dose

215.0499 (M-H20-CO-Cl)+

HN

Cl

HN

Cl

200 205 210 215 220 225 230 235 240 245m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rel

ativ

e A

bund

ance

203.0520

229.1039

215.0490

219.0259

223.0958204.0554 232.1324

247.0830

213.1592

239.0160

242.1490227.1747

200 205 210 215 220 225 230 235 240 245m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rel

ativ

e A

bund

ance

217.0469

225.0432

239.0952203.0530 223.0639

226.9517 234.1127221.0846208.9550 240.2324 248.0030

204.1022

212.2376

m/z 215.0490

C13H10ClNExact Mass: 215.0502

C13H10ClNExact Mass: 215.0502

LESA MS/MS Confirmation of Diclofenac: 5500Heart/Blood: Positive Ions

Analytical standard

+EPI (296.00) CE (40): 0.057 to 1.080 min from Sample 1 (Diclofenac pos std 500 ng/mL MSMS) of Diclofenac_MSMS_pos_std500ngm... Max. 1.3e4 cps.

2500.00

3000.00

3500.00

4000.00

4500.00

5000.00

5500.00

6000.00

6500.00

7000.00

7500.00

8000.00

8500.00

9000.00

9500.00

1.00e4

1.05e4

1.10e4

1.15e4

1.20e4

1.25e4

Intens

ity, c

ps

214.1

278.0

214

(M+H)+

296

Diclofenac Standard

250 278

H2N

Cl

Cl

O

OH

(M+H)+ = 296

12

+EPI (296.00) CE (40) CES (25): Exp 4, 0.047 to 3.635 min from Sample 1 (2ndanimal_2_BloodHeart_pos_3) of 2ndanimal_2_BloodHe... Max. 4.3e4 cps.

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340m/z, Da

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

Intensity, cps

296.0

236.1

104.1

214.1

204.1170.083.0 278.0149.1 250.174.1 86.1

264.0177.1179.171.1 115.1 206.1 228.1136.1 146.2 245.1

214

250 278

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340m/z, Da

0.00

500.00

1000.00

1500.00

2000.00296.0

250.0179.1

178.1151.1

296

Heart/Blood(M+H)+296

(M+H)+ = 296

LESA MS Confirmation of Diclofenac-beta-D-glucuroni de: ExactiveLiver Tissue: Negative Ions

Dosed Mouse Control Mouse

Glucuronide(0.6 ppm deviation)

Neg_Diclofenac_mouse1_Liver_03 #1-61 RT: 0.11-0.93 AV: 61 NL: 1.75E4T: FTMS {1,1} - p NSI Full lock ms [250.00-550.00]

70

75

80

85

90

95

100470.0418

Diclofenac_negOH_mouse3_Liver_2 #1-42 RT: 0.11-0.67 AV: 42 NL: 1.79E4T: FTMS {1,1} - p NSI Full lock ms [250.00-550.00]

70

75

80

85

90

95

100470.3203

H

Cl

O

OO

OHOH

COO

470.0418

(M-H)-

13

469.6 469.8 470.0 470.2 470.4m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rel

ativ

e A

bund

ance

470.3206

470.3568

470.2401

469.7390469.6 469.8 470.0 470.2 470.4

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rel

ativ

e A

bund

ance

470.3570

470.2842

470.2418

469.7383 470.0259

Closest signal is 33 ppm deviation from theoretical value

HN

Cl

HO

C20H19Cl2NO8m/z 470.0415

Facile Detection of Glucuronide Metabolite in liver tissue with high resolution MS

LESA MS/MS Confirmation of Diclofenac-beta-D-glucuronide: 5500Liver Tissue: Positive Ions

Mouse Liver Tissue (M+H)+ = m/z 472.05

14

Analytical standard

H2N

Cl

Cl

O

OO

HO

OHOH

COOH

(M+H)+ = m/z 472.05

LESA MS Detection of Diclofenac Hydroxy Metabolites : ExactiveKidney Tissue: Negative Ions

Dosed MouseControl Mouse

Neg_Diclofenac_mouse1_Kidney_03 #1-53 RT: 0.11-0.83 AV: 53 NL: 1.57E4T: FTMS {1,1} - p NSI Full lock ms [250.00-550.00]

60

65

70

75

80

85

90

95

100310.1506

310.1147

309.9531 310.0052

Hydroxy-Diclofenac(2.9 ppm deviation)

Diclofenac_negOH_mouse3_Kidney_6 #1-60 RT: 0.11-0.92 AV: 60 NL: 5.50E4T: FTMS {1,1} - p NSI Full lock ms [250.00-550.00]

60

65

70

75

80

85

90

95

100310.1775

(M-H)-

HN

Cl

Cl

O

O

HO

15

309.90 309.95 310.00 310.05 310.10 310.15 310.20m/z

0

5

10

15

20

25

30

35

40

45

50

55

Rel

ativ

e A

bund

ance

309.9640

309.9065

310.0195

310.0681

310.1772310.1019

310.0598

309.90 309.95 310.00 310.05 310.10 310.15 310.20m/z

0

5

10

15

20

25

30

35

40

45

50

55

Rel

ativ

e A

bund

ance

310.1559

(M-H)- =310.0043

Facile Detection of Hydroxy Metabolite in kidney tissue with high resolution MS

LESA MS/MS Detection of Diclofenac Hydroxy Metaboli tes : 5500Kidney Tissue: Negative Ions

- E P I ( 3 1 0 . 0 0 ) C E ( - 2 5 ) C E S ( 1 0 ) : E x p 3 , 0 . 1 0 4 t o 3 . 8 5 9 m i n f r o m S a m p l e 1 ( 2 n d _ a n i m a l _ n e g N H 4 O H _ K i d n e y _ 2 ) o f 2 n d _ a n i m a l _ n e g N . . . M a x . 1 . 9 e 4 c p s .

2 0 0 0 . 0

3 0 0 0 . 0

4 0 0 0 . 0

5 0 0 0 . 0

6 0 0 0 . 0

7 0 0 0 . 0

8 0 0 0 . 0

9 0 0 0 . 0

1 . 0 e 4

1 . 1 e 4

1 . 2 e 4

1 . 3 e 4

1 . 4 e 4

1 . 5 e 4

1 . 6 e 4

1 . 7 e 4

1 . 8 e 4

1 . 9 e 4

Inte

ns

ity, c

ps

3 1 0 . 1

9 6 . 9

7 8 . 9 1 2 4 . 01 3 0 . 1

1 9 3 . 12 6 6 . 0

1 6 8 . 1

9 4 . 91 4 7 . 18 9 . 0 1 2 8 . 1

9 2 . 9 2 1 4 . 11 8 2 . 0 2 5 0 . 1

(M-H)-

266.1

Kidney Tissue310.0

-44

-CO2

HN

Cl

Cl CH2

HOC13H10Cl2NOm/z 266.1

16

6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0m / z , D a

0 . 0

1 0 0 0 . 0

2 5 0 . 12 3 6 . 11 3 4 . 1 1 7 1 . 1 2 7 4 . 11 9 7 . 9 2 1 2 . 1 2 9 2 . 01 5 3 . 0 2 5 6 . 31 9 4 . 11 1 7 . 11 1 0 . 9 2 2 2 . 17 4 . 0 8 0 . 97 1 . 0 2 2 6 . 0

- E P I ( 3 1 0 . 0 0 ) C E ( - 3 0 ) C E S ( 1 0 ) : 0 . 0 1 0 t o 0 . 6 2 3 m i n f r o m S a m p l e 1 ( 5 H y d r o x y D i c l o f e n a c s t d n e g M S M S 5 0 0 n g / m L ) o f 5 H y d r o x y D i c l o f . . . M a x . 2 . 6 e 6 c p s .

6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0m / z , D a

0 . 0

1 . 0 e 5

2 . 0 e 5

3 . 0 e 5

4 . 0 e 5

5 . 0 e 5

6 . 0 e 5

7 . 0 e 5

8 . 0 e 5

9 . 0 e 5

1 . 0 e 6

1 . 1 e 6

1 . 2 e 6

1 . 3 e 6

1 . 4 e 6

1 . 5 e 6

1 . 6 e 6

1 . 7 e 6

1 . 8 e 6

1 . 9 e 6

2 . 0 e 6

2 . 1 e 6

2 . 2 e 6

2 . 3 e 6

2 . 4 e 6

2 . 5 e 6

2 . 6 e 6

Inte

ns

ity, c

ps

2 6 6 . 0

2 3 0 . 1

1 9 4 . 12 2 9 . 12 1 5 . 1 3 1 0 . 21 6 7 . 1

Analytical standard

(M-H)-

266.1

HN

Cl

Cl

O

O

HO

310.0(M-H)- =310.0

Negative ion MS/MS provides limited characterizatio n information

LESA MS Detection of Diclofenac Hydroxy Metabolites : 5500Kidney Tissue: Positive Ions

Pos Ions

(M+H)+

Kidney

312.0

17

4-Hydroxy-Diclofenac

(M+H)+

H2N

Cl

Cl

O

OH

HO

4'-Hydroxy-Diclofenac, C14H11Cl2NO3(M+H)+=312.0189

Positive ion MS/MS provides more structural charact erization

312.0

1000000

10000000

100000000

Confirmed by all ion HCD-MS/MS

Confirmed by QQQ-MS/MS

Summary Drug Tissue Distribution Analysis– Diclofenac 15 min after P.O. 10 mg/kg Diclofenac –

Sig

nal I

nten

sity

18

100

1000

10000

100000

Stomach Intestine Liver Kidney Brain Heart/Blood Lung Skeletal Muscle Control

XIC of 294.0094 (± 5 ppm), the baseline signal from control is 0 for all tissues (!)

Average of n=4 locations on tissue and SD

Sig

nal I

nten

sity

Detection of Isobaric Metabolites in Tissue

Hydroxy-Diclofenac in Kidney 15 min P.O 10 mg/kg Di clofenac in mouse

• LESA followed by direct infusion could not differentiate between the two hydroxylated metabolites.

– Positive ion mode MS3 (QTRAP) produced – Positive ion mode MS3 (QTRAP) produced indistinguishable spectra (data not shown)

• Differentiation could be facilitated by:– LC separation of LESA extract.– Differential ion mobility employing solvent gas

additives

Set Up for LESA Directly Coupled with nanoLC/MS

3

4 6

1

2

Rheodyne

Pos 1 Pos 2

3

4 6

1

2

Rheodyne

Column

Syringe

SyringeLoop

Transfer capillary

Transfer capillary

LC Pump

HV RefHV Ref

Loop

LC Pump

Column

5

4 6

FC Mandrel

5

FC Mandrel

Solvents reservoir

Solvents reservoir

LC Pump

Courtesy R. Almeida, Advion BioSystems, Inc.

Comparison LESA vs. LESA-nanoLC

LESA LESA-nanoLC1 uL � 5 min at 200nL/min 1 uL injected on column 35 min Gradient

XIC 457.3

BPI

1mm-2mm 0.2mm-1mm

FS 170 um OD/100um ID

Courtesy R. Almeida, Advion BioSystems, Inc.

Summary• Diclofenac can be determined by electrospray-MS and MS/MS in thin whole body sections using

LESA-MS– These techniques are often complementary

• LESA with positive and negative ion analysis modes provides optimal capabilities

• LESA-MS for Diclofenac is most sensitive in negative ion mode and MS analysis – High resolution/accurate mass accuracy provides high selectivity

• Parent drug confirmed by MS/MS experiments– Positive ion MS/MS is most useful for confirmation purposes; – Confirmation may be provided by:

• HCD-all ion MS/MS• QQQ-MS/MS

– this mode is more sensitive

• Two Different types of Diclofenac Metabolites were detected– beta-D-Glucuronides in Liver – Hydroxy-Diclofenac in Kidney

22

Conclusions/Future Work

• LESA-MS can provide helpful drug and metabolite distribution information in tissue sections– LESA-nanoLC/MS could provide additional analytical utility

• Implementation of differential mobility • Implementation of differential mobility spectrometry(DMS) between ionization and MS stages could facilitate isobaric metabolite differentiation

• Comparable MALDI imaging data for diclofenac and its metabolites has not been found in the literatur e

AcknowledgementsMore LESA @ ASMS 2011:

MP 037 – Cox et al.

MP 258 – Himmelsbach et al.

MP 269 – Almeida et al.

TOF 2:30 – Wagner et al.

TOF 4:10 – Eikel et al.

WOF 9:50 – Parson et al.

WP 413 – Blatherwick et al.

ThP 285 – Taguchi et al.

ThP 379 – Porta et al.

ThP 380 – Prosser et al.

We would like to thank Thermo Fisher Scientific and AB/Sciex and for the generous loan of anExactive Benchtop Orbitrap and QTrap 5500 mass spectrometer, respectively.

... or at www.advion.com

24/19

Advion BioSciences Headquarters in Ithaca, NY

Presented at the 59th Conference of the American Society for Mass Spectrometry, ASMS in Denver, CO, 2011