H. LUNDBECK A/S

2

LC-MS/MS Set-Up 2009:

No. Systems Inlet Mass Spectrometre

Max. Capacity(No. Plates/Day)

3 CTC - Agilent Quattro Ultima 6

2 CTC - Agilent Quattro Ultima Pt 4

2 Cohesive System API 4000 4

Total 16

3

Intended LC-MS/MS Set-Up 2011:

No. Systems Inlet Mass Spectrometre

Max. Capacity(No. Plates/Day)

4 Acquity Xevo TQ 16

2 Acquity Xevo TQ-S 8

Total 24

4

Reality:

Current LC-MS/MS Set-Up 2011:

No. Systems Inlet Mass Spectrometre

Max. Capacity(No. Plates/Day)

4 Acquity Xevo TQ 16

1 Acquity Xevo TQ-S 4

2 Acquity Quattro Ultima Pt 4

Total 24

5

Xevo TQ: S/N = 83Peak Height 7.4*103

(25 pg/On Column)0.005ng/ml std

Time0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 1.65 1.70 1.75 1.80 1.85

%

0

100

SAT_VBA_491_034 MRM of 1 Channel ES+ TIC (Irindalone)

7.40e3

S/N:PtP=83.26

0.98

0.99

1.19

1.00

1.141.13

1.02

1.16

1.311.30

1.20

1.27

1.211.25

1.25

1.601.581.541.41

1.371.33 1.37

1.381.53

1.481.47

1.43

1.68

1.671.62

1.71

0.0005ng/ml std

Time0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 1.65 1.70 1.75 1.80

%

0

100

SAT_WAA_090_091 MRM of 1 Channel ES+ TIC (Irindalone)

5.45e4

1.19S/N:PtP=38.63

0.96

0.79 0.85

1.15

1.13

1.031.04

1.30

1.271.25

1.21

1.37

1.33

1.69

1.39 1.64

1.511.401.491.45

1.44

1.621.52

1.551.57

1.66

N

N

F

N

NO

Xevo TQ-S: S/N = 38Peak Height 5.5*104

(2.5 pg/on Column)

Sensitivity Test: Irindalone

6

Xevo TQ Linearity over 4 decades

Std. Conc(ng/mL) RT Area Height ng/mL %Dev Factor

0.005 0.99 95 4.80E+03 0.00508 1.7

0.03 0.99 509 2.50E+04 0.02844 -5.2 6

0.2 0.98 3525 1.70E+05 0.19858 -0.7 40

1.2 0.99 21539 1.00E+06 1.2148 1.2 240

7.5 0.99 137691 6.70E+06 7.7674 3.6 1500

50 0.98 881360. 4.30E+07 49.72069 -0.6 10000

Xevo TQ-S ~ Linearity over 3.5 decades

0.0005 0.98 654 2.10E+04 0.00054 8.1

0.003 0.98 3531 1.30E+05 0.00325 8.3 6

0.02 0.98 20083 7.50E+05 0.01884 -5.8 40

0.15 0.98 139340 5.20E+06 0.13117 -12.6 300

1 0.98 1082708 4.00E+07 1.0197 2.0 2000

Linearity Test: Irindalone

7

Xevo TQ 1 ng/mL plasma

1 3 14014_PQ_A

Time1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 1.65 1.70 1.75 1.80 1.85 1.90 1.95 2.00

%

0

100

14014_PQ_A_003 MRM of 2 Channels ES+ 305.24 > 190.119 (AA39959)

3.21e4

S/N:PtP=584.25

CAL 1 11 3 14016_PQ_A

Time1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 1.65 1.70 1.75 1.80 1.85 1.90 1.95 2.00

%

0

100

14016_PQ_A_0003 MRM of 2 Channels ES+ 305.3 > 190.2 (Lu AA39959)

1.33e5

S/N:PtP=107.63

1.18

S/N = 584 S/N = 107

Peak Height = 3.2*104 Peak Height = 1.2*105

Xevo TQ-S 0.1 ng/mL plasma

8

Xevo TQ

Clobazam 2 ng/mL and N-Desmethyl-Clobazam 1 ng/mL plasma

50 µL plasma precipitated with 150 µL cold ACN

Diluted 1 time with mobile phase A

15 µL Injected

Kal 1

Time0.92 0.94 0.96 0.98 1.00 1.02 1.04 1.06 1.08 1.10 1.12 1.14 1.16 1.18 1.20 1.22 1.24 1.26 1.28 1.30 1.32 1.34 1.36 1.38 1.40 1.42 1.44 1.46 1.48 1.50

%

0

100

0.92 0.94 0.96 0.98 1.00 1.02 1.04 1.06 1.08 1.10 1.12 1.14 1.16 1.18 1.20 1.22 1.24 1.26 1.28 1.30 1.32 1.34 1.36 1.38 1.40 1.42 1.44 1.46 1.48 1.50

%

0

100

Pro 420_02_3 MRM of 2 Channels ES+ 301.2 > 259 (Lu **00638)

9.88e4

S/N:PtP=142.13

Pro 420_02_3 MRM of 2 Channels ES+ 287.2 > 244.95 (Lu AF36275)

1.03e4

1.42

S/N:PtP=12.18

1.19 1.20

1.401.23

1.44

1.44

1.49

1.471.47

S/N = 142

S/N = 12

N

N

O

O

Cl

N

N

O

O

Cl

9

Xevo TQ-S:

Clobazam 2 ng/mL and N-Desmethyl-Clobazam 1 ng/mL plasma

50 µL plasma precipitated with 150 µL cold ACN

Diluted 1 time with mobile phase A

7.5 µL Injected

Kal 1

Time0.92 0.94 0.96 0.98 1.00 1.02 1.04 1.06 1.08 1.10 1.12 1.14 1.16 1.18 1.20 1.22 1.24 1.26 1.28 1.30 1.32 1.34 1.36 1.38 1.40 1.42 1.44 1.46 1.48 1.50

%

0

100

0.92 0.94 0.96 0.98 1.00 1.02 1.04 1.06 1.08 1.10 1.12 1.14 1.16 1.18 1.20 1.22 1.24 1.26 1.28 1.30 1.32 1.34 1.36 1.38 1.40 1.42 1.44 1.46 1.48 1.50

%

0

100

Pro 420_03_18 MRM of 2 Channels ES+ 301.14 > 259.161 (Lu **00638)

8.18e5

S/N:PtP=931.30

Pro 420_03_18 MRM of 2 Channels ES+ 287.14 > 245.09 (Lu AF36275)

4.46e4

S/N:PtP=36.37

1.16

1.030.96 1.08

1.431.41

1.381.361.191.311.30

1.271.19 1.241.23 1.25

1.33

1.44

1.461.461.49

S/N = 931

S/N = 36

N

N

O

O

Cl

N

N

O

O

Cl

10

Flupentixol in plasma

Sample Preparation:

100 µL plasma precipitated with 500 µL ACN

400 µL transferred and evaporated to dryness

Reconstituted in 500 µL mobile phase A

Analysis

45 µL injected

Mobile phase: 0.2% TFA/ACN A: 95:5 B:5:95

Column: Acquity BEH C18 1.7 µm - 50x2.1 mm

Flow rate: 1 mL/min

Column temperature: 80°C

Dwell time: 50 ms

S

N

N

F

FF

O

11

12

IV. RECOMMENDED STUDIES FOR ASSESSING THE SAFETY OF METABOLITES A. General Toxicity Studies The toxicity of the drug metabolite should be investigated at multiples of the human exposure or at least at levels comparable to those measured in humans.......D. Carcinogenicity Studies Carcinogenicity studies should be conducted on metabolites of drugs that are administered continuously for at least 6 months

V. TIMING OF SAFETY ASSESSMENTS If toxicity studies of a drug metabolite are warranted, studies should be completed and study reports provided to the FDA before beginning large-scale clinical trials.

13

Late stage information on new possible metabolites in man

No animal data on several of these possible metabolites

Synthetical batches not available (6 months of delivery time)

Pivotal animal and man studies about to start

Rn Rn

Rm Rm

Ro

OH

OH

Rn/m Rn/m

O

Rn/m Rn/m

OH

OH

OH

?

Parent

14

Starting with finding the expected analytes of human relevance

Crash of 50 µL urine with 150 µL ACN from human dosed with Drug to steady state

Dilute supernatant x 1 with mobile phase A (0.2% FA)

Acquity & XEVO TQ-S

- HSS T3 100 x 2.1 mm 1.7 µm

- 0.6 mL/min linear gradient 0.2% FA/0.2% ACN

- Inject 7.5 µL PLUNO Default.

11 suggested product ion scans (one by one)

Manual data-analysis of mass chromatograms

15

Total Ion Chromatogram (TIC) Product Ion Scan 1.89 min

16

MRM’s

17

TIC

Parent ion

Product ion

18

Rn Rn Rn

OH

OH

Rn

Rm Rm Rm

OH

OH

Rm

Ro

OH

OH

O

OH

OH

O

19

Set-up MRM experiment for 9 found parent-product ions(1 drug and 12 metabolites)

Try different Cone Voltages and Collision Energies Optimise chromatography (optimise gradient)

Evaluate matrix effects Spike 50 µL blank human, mouse and rat plasma with 10 µL

human urine Crash with 250 µL ACN Dilute supernatant with x 1 0.2 % FA UPLC-MRM analysis 7.5 µL PLUNO-mode 9 MRM’s with 5 ms dwell time.

Conclusion: The analysis will give the identical response of each analyte in

human, mouse and rat samples. Suitable for relative comparison of peak areas across species

20

Blank/placebo samples

Steady State condition

Relevant doses (effective dose in Man, NOAEL in mice and rats)

Be available !

Samples tested:

Man (3 placebo and 15 active samples from visit 12-15)

Rats (2/4 h - tmax) samples from 3 males & 3 females in a Toxicological study)

Mice (2 h - tmax) samples from 3 males & 3 females in an Exploratory study)

Only metabolites found in man are considered

21

Man Rat Mouse

Blank

Sample

22

Analyte Retention time(min)

Mean peak area ratioRat/Man

Mean peak area ratioMouse/Man

Parent 4.09 15 16

Metabolite A 4.04 8.6 10

Metabolite B 2.99 35 4.2

Metabolite B 3.55 67 14

Metabolite C 2.08 14 8.6

Metabolite D 3.09 51 14

Metabolite D 3.24 558 4

Metabolite D 3.42 - -

Metabolite E 2.72 0.3 1.5

Metabolite F 2.90 62 6.1

Metabolite G 1.96 8.8 3.8

Metabolite H 3.00 160 19

Metabolite H 3.16 ND ND

Low abundance !

Low abundance !

23

H. Lundbeck A/S was able to decide to carry out the pivotal studies without delays

With the 9 MRM transitions we were able to find 7 NEW HUMAN metabolites and 11 NEW ANIMAL specific metabolites due to the combination of UPLC and Xevo TQ-S.

The Xevo TQ-S proved to be very sensitive and fast in Product Ion Scan

24

Noise is the key parameter to deal with.

Meaning more focus on:

Ultra Pure Solvents

Clean bottles

Clean Source

Contamination

More focus on S/N because of the larger response

Carryover: Not really an issue !

25

Mass Spectrometre Sensitivity Selectivity Speed Dynamic Range

Quattro Ultima X X X X

Quattro Ultima Pt XX X X X

API 4000 XX X XX X

Xevo TQ XXX X XXX X/

Xevo TQ-S XXXX X/ XXX /

H. LUNDBECK A/S

H. LUNDBECK A/S