Access to your success SHIMADZU
Access to your success SHIMADZU
Access to your success SHIMADZU
Access to your success SHIMADZU Co-Sense for BA Co-Sense for BA + LCMS
Access to your success SHIMADZU
Access to your success SHIMADZU
Access to your success SHIMADZU
Access to your success SHIMADZU
100 ul 100 ul 50 ul 10 ul 50 ul 10 ul
10 ul 100 ul Drug Protein
Sample introduction pump Auto-sampler Pretreatment column Dilution pump 90% Sample introduction pump 10% Auto-sampler Free Drug Pretreatment column
µ
ODS
µ 0 4 8 12 16 Time (min)
µ 500 L 200 L 100 L 0 4 8 12 16 Time (min)
1 300 0 8.5 17 Time (min) 0 8.5 17 Time (min)
Pretreatment Column Shim-pack MAYI-ODS (4.6mmi.d. x 10mm) Extraction mobile phase 10mM ammonium acetate/acetonitrile = 95/5 Flow rate 3mL/min Column temperature 45ºC Injection volume 5, 10 or 50µL Extraction time 1 or 2min Dilution factor 8 fold Analysis Column Phenomenex Mercury MS (4.0mmi.d. x 10mm, 5µm) Mobile phase A : 10mM ammonium acetate, B : acetonitrile Gradient program Extraction time 1min 5%B(0-0.5min) 90%B(3-4min) - 5%B(4.01min) - STOP(5min) or Extraction time 2min 5%B(0-1.5min) 90%B(4-5min) - 5%B(5.01min) - STOP(6min) Flow rate 0.8mL/min Split ratio 1 : 3 (mass spectrometer : waste) Column temperature 45ºC Ionization Electrospray ionization Probe voltage +4.5 kv or 3.5kV Nebulizing gas flow 4.5L/min
SIM chromatograms of standards 3 4 5 1µg/mL each, 5µL 2 1
Compound RSD, % (n=5) Standard Plasma spiked Metoprolol 1.2 1.0 Propranolol 1.0 1.4 Lidocaine 0.9 2.6 Dibucaine 1.6 2.5 Bupivacaine 0.7 1.5 each 1ug/mL, 5uL
15000000 10000000 4 5 Area 3 5000000 2 1 0 0 0.2 0.4 0.6 0.8 1 1.2 Conc.(µg/mL)
1st run 101st run 201st run 301st run 0.1µg/mL each, plasma spiked, Injection volume : 50µL, Extraction time : 2min
Column pressure at start time (MPa) 10.00 9.00 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 Pump A Pump B Pump C Pump C for pretreatment 0.00 1 21 41 61 81 101 121 141 161 181 201 221 241 261 281 301 Number of Injection
LCMS UV - - UV 230nm ESI(+) SIM m/z 235
CH 3 CH 3 NH C O CH 2 N C 2 H 5 C 2 H 5 M+H + 0.1% / = 8 / 2, ESI(+)
r = 0.9996 LCMS-ESI-(+) (µg/l)
0.13% 2.99% LCMS 0.18% 1.16% ml 1 2 3 4 5
1 3 1 2 3 9 2 8 7 4 4 5 6 7 8 10 6 5 9 10
UV 10uL
α
min.
min.
57 Access to your success SHIMADZU
58 Access to your success SHIMADZU
59 - - - - - - - - - Access to your success SHIMADZU
60 Access to your success SHIMADZU
61 1 Access to your success SHIMADZU
62 2 Access to your success SHIMADZU
CPMC MTMC PHC Arprocarb NAC MPMC XMC MIPC BPMC Mercaptodim ethur
( ) (O- ) ) ( )
1994 6 9
S/N=3 2ppb
4 6 8 10 20 50 / 3-30 XMC 40 BDMC
(1)
5000e3 4500e3 4000e3 3500e3 TIC 223.05 163.00 239.15 224.05 202.05 226.05 208.10 3000e3 2500e3 2000e3 1500e3 1000e3 500e3 0e3 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 : SIM
Int. 223.05 300e3 200e3 O H 3 C S O CH 3 C H C CH 3 O N O C N H CH 3 M.W. 222 100e3 255.05 239.65 147.85 166.00 254.00 0e3 100 125 150 175 200 225 250 275 m/z Int. 202.05 250e3 200e3 150e3 100e3 M.W. 162 144.95 H 3 C C H 3 CS NOCONHCH 3 50e3 234.35 195.05 219.05 0e3 100 125 150 175 200 225 250 275 m/z Int. 163.00 150e3 100e3 50e3 106.00 M.W. 238 CH 3 OCON CH 3 H 3 C N CH 3 H 3 C N N CH 3 179.20 195.15 282.75 0e3 100 125 150 175 200 225 250 275 m/z
Int. 226.05 300e3 200e3 100e3 M.W. 223 164.05 OCONHCH 3 O C O CH 3 CH 3 106.80 258.05 0e3 100 125 150 175 200 225 250 275 m/z Int. 239.15 1000e3 750e3 500e3 M.W. 201 250e3 168.30 240.30 0e3 100 125 150 175 200 225 250 275 m/z Int. 208.10 750e3 500e3 250e3 M.W. 225 240.05 152.45 0e3 100 125 150 175 200 225 250 275 m/z
Int. 226.05 500e3 400e3 300e3 200e3 M.W. 225 100e3 258.20 168.90 207.95 0e3 100 125 150 175 200 225 250 275 m/z Int. 224.05 200e3 150e3 100e3 M.W. 207 50e3 183.90 167.30 184.65 226.05 255.65 0e3 100 125 150 175 200 225 250 275 m/z Area SIM 8 ~ 1000 ppb 800e3 700e3 600e3 500e3 400e3 300e3 200e3 100e3 0e3 0.0 1.0 2.0 3.0 4.0 Conc
GPC
GPC UV detector Column Mobile Phase Flow Rate Fraction Volume : Shodex CLN-pak EV-2000 ( 20.0mmI.D. 300mmL) Guard column : Shodex CLN-pak EV-G : Acetone/Cyclohexane = 2/8 (V/V) : 4mL/min. : 10mL
Elution Map Bio Beads SX Fraction No. Recovery % Elution Profiles from GPC(2.2cm i.d. x 50cm, CH 2 Cl 2 -cyclohexane 1:1)
RID-20A 2005 4
!!
NH2 R C COOH H
: : --- UV/VIS OPA ---
: 440nm ( ) or 570nm (primary) : 130 : : :
(Ex=350nm, Em=460nm) :
Ninhydrin OPA Reaction primary, secondary amine primary amine Reaction Temp. 130 C Ambient Reaction Speed First First Detection Visible detector Fluoresces detector Reagent Stability unstable less stable Amino Acid itself Column Oven CationExchange Column Fluorescence detector Hypochrolite OPA reagent ethanethiol 2-mercaptoethanol 3-mercaptopropionic acid N-acetylcysteine (Shimadzu patent)
Postcolumn repeatability, RSD around 1% Precolumn repeatability, RSD more than 2%
Column : Revered Phase Column (ODS) Reagent : OPA (Fluorescence) FMOC (Fluorescence) Fluorescamine (Fluorescence) DANS-Cl (Fluorescence) PITC (UV)
LC-20AT LC-20AT (x2) DGU-20A3 SIL-20A CTO-20A SPD-20A Tray CBM-20A lite LC-Solution LC-2010HT
LC-20A