高通量筛选

– DNA damage
• Oxidants • Carcinogenic compounds
• Future directions
– GC-AMS: dual isotope measurements – Tritium AMS
BEAMS Lab
BEAMS Lab
BEAMS Lab
Sample Preparation and Analysis:
BEAMS Lab
Outline of Presentation
• History and technology development • Scientific studies
– ADME
• Pharmacokinetics • Metabolite profiling • Drug delivery to selected targets
Raw Data using Multi-well Combustion Interface
BEAMS Lab
1584.250 (40361.629) 1600 1689.250 (12148.523) Sec
Acetaminophen PK Studies. 2.
Objective: Animals: Dose:
He in CuO bed Alumina cup
He + CO2 out
BEAMS Lab
Combustion Interface (v. 2.0) Sample Holder
BEAMS Lab
1000
1250
1500
1750
2000
2250
Counts
250
500
750
0 45.250 (9020.408) 167.250 (163.396) 200 386.750 (139.953) 400 480.000 (215.865) 580.500 (239.450) 600 688.000 (461.236) 800 793.250 (553.789) 1000 1200 1236.250 (9728.563) 1344.750 (10386.208) 1400 1464.750 (42402.176) 1021.607 (3263.763) 1126.547 (2691.743) 900.500 (15643.945) *C14 File (C:\DOCUME~1\ROSALI~1\MYDOCU~1\AMS\DATA\2005\ANALYSIS\PFIZER\CALIBR~1\FEB23\0223R010.CSV)
1999 1994 2000 2002
2004 2005
Laser-induced combustion proof-of-principle Patent apps. filed
Gas ionization detector 1st Patent issues
BEAMS Lab
A Brief History of AMS at MIT
Sample collection: Blood: Urine: Feces: 1, 2, 5, 8, 24 and 48 hours post dose 0-8, 8-24 and 24-48 hours post dose 0-8, 8-24 and 24-48 hours post dose
BEAMS Lab
He in
(f)
(f)
He + CO2 out
(g)
(a) (b) (c) (d)
CuO bed (e)
BEAMS Lab
Multi-well Sample Combustion Interface (v. 2.0): Cross-sectional View
Infrared laser
IR window
Calibration Curves for CH3CN Plasma Extracts
3.0
2.5
Peak area ratio
2.0
1.5
1.0
0.5
2-23 2-25 3-2 0 2 4 6 8 10
0.0
Actual 14C concentration
BEAMS Lab
AMS Detection of Oxidative DNA Damage
Combustion interface v. 2.0 1st Detection of 14C 1st SBIR grant to NSI 2nd Patent issues Combustion interface v. 1.0 Development of GC interface
1999 1994 2000 2002
BEAMS Lab
APAP Urinary metabolites
100
Percent of total metabolites
10 µCi dose by LSC 10 nCi dose by AMS 80
60
40
20
0
Glucuronide Sulfate
APAP Mercapturate
Βιβλιοθήκη Baidu
BEAMS Lab
Laser-induced heating of CuO causes sample combustion Resulting CO2 transported to AMS ion source
BEAMS Lab
Detection of [14C]-Acetaminophen with Laser-induced Sample Combustion
3500
1000
3000
acetaminophen
UV C14 800
2500
benzoic acid
600
mAU
2000
tyrosine
1500 400 1000
500
200
0 0 100 200 300 400
Time [sec]
BEAMS Lab
14C counts
Cross-sectional View of Interface (v. 1.0)
sample 14C sample 12C
MIT
Peak area ratio:
sample 14C ext.std. 14C
10-3 – 10-2 M 100 zmol To statistical precision: 1 sec – 30 min Limited by sample prep or
1400
0.21 dpm
Carbon-14 Count Rate (counts/sec)
1200
1000
0.14 dpm
800
0.084 dpm 0.042 dpm
600
400
0.014 dpm
200
0
0 1 2 3 4 5 6 7 8 9 10
Time (minute)
BEAMS Lab
HPLC-AMS with Laser-induced Combustion Interface
10 – 1 M 100 zmol Duration of CO2 pulse: 1 – 30 sec > 1 per min
BEAMS Lab
A Brief History of AMS at MIT
Combustion interface v. 2.0 1st Detection of 14C 1st SBIR grant to NSI 2nd Patent issues Combustion interface v. 1.0 Development of GC interface
Reduction to Graphite or Titanium Hydride
Continuous-flow, Single Target, Multi-sample Ion Source
Solid Cathode, Multiple Target Ion Source
Higher Throughput
BEAMS Lab
Calibration Curves for Whole Plasma
60
50
Peak area ratio
40
30
20
10
3-1 3-2 (1) 3-2 (2) 0 20 40 60 80 100 120
0
Actual 14C concentration (dpm/mL)
BEAMS Lab
Combustion interface v. 2.0 1st Detection of 14C 1st SBIR grant to NSI 2nd Patent issues Combustion interface v. 1.0 Development of GC interface
1999 1994 2000 2002
High Sample Throughput Approaches to Accelerator Mass Spectrometry
P. L. Skipper, R. G. Liberman, J. Flarakos, and S. R. Tannenbaum BEAMS Lab Biological Engineering Division Massachusetts Institute of Technology
2004 2005
Laser-induced combustion proof-of-principle Patent apps. filed
Gas ionization detector 1st Patent issues
BEAMS Lab
A Brief History of AMS at MIT
Plasma Clearance: Unbound APAP + Metabolites
200
APAP equivalents (µg/mL)
100 50
10 µCi dose by LSC 10 nCi dose by AMS
10 5
1
0
5
10
15
20
25
Time after dose (hours)
2004 2005
Laser-induced combustion proof-of-principle Patent apps. filed
Gas ionization detector 1st Patent issues
BEAMS Lab
Laser-induced Combustion Interface (v. 1.0)
Effect of plasma proteins on AMS sensitivity
Plasma carbon concentration is ~3 M
14C:12C
ratio is ~1.4 x 10-12 in living organisms
Plasma [14C] is ~4 pM
= 0.5 dpm/mL
BEAMS Lab
Quantitative Analysis: Comparison of Instrument Types
Graphite
Method of quantitation Limits of quantitation Measurement period Sample throughput Isotope ratio:
Comparison of Methods Conventional AMS
Collect Fractions Chromatographed sample (LC, GC, CE effluent)
BEAMS Lab
Bulk sample (tissue, blood, urine, …) Oxidation to CO2 or H2O Online generation of CO2 or H2
Sample deposition* CuO
Transfer to interface chamber
*Dropwise at discrete points (pipette) or by continuous flow (HPLC)
BEAMS Lab
Interface Operation
IR laser He in He and CO2 out
Validate analytical methods for low-level isotope detection at therapeutic doses Rat/Sprague-Dawley/males/250-300 g/ 2 Groups of N=4 All: Group 1: Group 2: 150 mg/kg + 10 µCi [14C]-Acetaminophen + 10 nCi [14C]-Acetaminophen