Post on 08-Oct-2018
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