spectrometre (no. plates/day) · 2011-11-22 · h. lundbeck a/s was able to decide to carry out the...
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H. LUNDBECK A/S
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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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
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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
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Total Ion Chromatogram (TIC) Product Ion Scan 1.89 min
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MRM’s
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TIC
Parent ion
Product ion
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Rn Rn Rn
OH
OH
Rn
Rm Rm Rm
OH
OH
Rm
Ro
OH
OH
O
OH
OH
O
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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
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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
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Man Rat Mouse
Blank
Sample
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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 !
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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
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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 !
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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 /
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H. LUNDBECK A/S
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H. LUNDBECK A/S