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Fundamental and Applied ResearchFundamental and Applied Research using Liquid Chromatography, Flow Injection Analysis, and Mass Spectrometryp y
Kevin A Schug Associate ProfessorKevin A. Schug, Associate ProfessorDepartment of Chemistry & Biochemistry Th U i it f T t A li tThe University of Texas at Arlington
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AcknowledgementsIn Today’s Talk:
Dr. Petr FryčákDr. Aruna B. WijeratneDr. Hien P. NguyenSam H. YangAaron A MorganAaron A. MorganLi LiHui FanLauren Tedmon
Dan W. Armstrong, UTASandy Dasgupta, UTAJane G. Wigginton, UTSWJames W. Simpkins, UNTHSCKarel Lemr, Palacky UniversityVladimir Havlicek, Czech Acad. Sci.
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“The best way to have a good idea is to have lots of ideas.”
‐ Linus Pauling
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The University of Texas at Arlington2005 ‐ Present
ory
Chiral and Biomimetic Molecular Recognition
High throughput Mass Spec Binding Determination Methods
A f N t l P d t D Di
Labo
rat Assays for Natural Product Drug Discovery
Development of Affinity Extraction Materials
Fundamentals of Electrospray Ionization
Schu
g L
Applications of Reversed Phase HPLC
Hydrophilic Interaction Liquid Chromatography
in th
e S
Trace Quantitative Analysis from Biofluids
Complex Mixture Analysis by MALDI‐MS
Analysis of Nonpolar Compounds by MALDI‐MS
search Analysis of Nonpolar Compounds by MALDI‐MS
Forensics Analysis
Tandem Mass Spectrometry Fragmentation Mechanisms
Res
De Novo Peptide Sequencing
Atmospheric Pressure Desorption Ionization
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Soft Ionizationf
0ccc DDH0,G0,G0,22 KKFIIF
Virginia TechAdduct Ion Formation
1000
10000 exp. f it exp. f itNH
O
O
ON
N1000
10000 exp. f it exp. f itNH
O
O
ON
NAdduct Ion Formation
10
100tBuCQD + DNB‐(S)‐Leu
KD = 220 μM (ΣR = 1.46)tBuCQD
G][H
H
iiI
10
100tBuCQD + DNB‐(S)‐Leu
KD = 220 μM (ΣR = 1.46)tBuCQD
G][H
H
iiI
0.1
1
0 5 10 15 20
tBuCQD + DNB‐(R)‐Leu
KD = 24 μM (ΣR = 0.31) α′MS = KD,(S)/KD,(R) = 8.9
0.1
1
0 5 10 15 20
tBuCQD + DNB‐(R)‐Leu
KD = 24 μM (ΣR = 0.31) α′MS = KD,(S)/KD,(R) = 8.9
University of Vienna
0 5 10 15 20
c0,G (µM)
0 5 10 15 20
c0,G (µM)
“Static Titration”
Chiral Molecular Recognition
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High Throughput QuantitativeHigh Throughput Quantitative Binding Determinations gusing FIA‐ESI‐MS
F t
“Static Titration”
From… to…
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Flow Injection AnalysisFlow Injection Analysis
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FIA – ESI – MS
2ttN
Modified symmetrical Gaussian
Single host – Single guest
2
,0
2exp
2 t
p
t
GG
ttQ
Nc
0ccc DDH0,G0,G0,22 KKFIIF
Frycak & Schug, Anal. Chem. 2007, 79, 5407‐5413.
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Q(30 μL/min)
Syringe pumpThermo LCQ Deca XP
(ESI‐MS)
Q(30 μL/min)
Syringe pumpThermo LCQ Deca XP
(ESI‐MS)
c0,H constantBand‐broadening device
(200 μL PEEK)
( )
n
EICc0,H constant
Band‐broadening device (200 μL PEEK)
( )
n
EIC
n
EIC
N0,G = cG * V Injection loop (2 μL)
time
Abun
iC/iH
time
c 0,G
N0,G = cG * V Injection loop (2 μL)
time
Abun
iC/iH
time
Abun
iC/iH
time
c 0,G
time
c 0,G
Vancomycin + Ac2Kaa
RT: 0.42 - 34.30
90
95
100 NL:2.38E8m/z= 910.00-913.00+
RT: 0.42 - 34.30
90
95
100 NL:2.38E8m/z= 910.00-913.00+
y 2
50
55
60
65
70
75
80
85
ve A
bund
ance
1820.00-1823.00 F: MS 20080419_002
50
55
60
65
70
75
80
85
ve A
bund
ance
1820.00-1823.00 F: MS 20080419_002
5
10
15
20
25
30
35
40
45
Rel
ativ
5
10
15
20
25
30
35
40
45
Rel
ativ
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34Time (min)
0
5 min2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Time (min)
0
5 min Kd = 2.8 ± 1.5 μMKd, lit ~ 1 μM
Schug, et al., Anal. Chim. Acta 2012, 713, 103‐110
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FIA – ESI – MS Polynomial modified Gaussian
2
Single host – Single guest
2
)(
s
tt
m
m
ehth
2)()( ttsttsss 210 )()( mm ttsttsss
Using Excel Solver…
80
100
120
Originalnsity
g
20
40
60
80 Original
PMG
SGrelative inten
0
20
0 100 200 300 400 500
time (sec)
Fan & Schug, ASMS Conference Proceedings 2011 (Denver, CO)
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FIA – ESI – MS Integrate degree of complex formation
(1 )(1.)
(2.)
Frycak & Schug, Anal. Chem. 2008, 80, 1385‐1393.
(3.)
(4.)
Multi‐host – Multi‐guest
(5 )(5.)
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Cyclodextrins injection ofNPX + FBPin mixture att = 0 min
injection ofNPX separatelyat t = 3.2 min
injection ofFBP separatelyat t = 6.6 min
1001169.4
OHO
OH
OH
Om 100
0
50
100
-CD100% rel. int. 1.08E+8 abs. int.
ance
100
90
80
70
[ -CD+Cl] -
β-CD (m = 7), γ-CD (m = 8)CD
1000
50
100
e
-CD NPX
-CD FBP
100% 5.01E+7
Rel
ativ
e A
bund
a60
50
40
30 [ CD NPX H] -
FOH
O
FBP
0
50
50
100
ativ
e A
bund
anc CD FBP
CD
100% 5.78E+7R 30
20
10
0200 400 600 800 1000 1200 1400 1600 1800 2000
603.1 1133.4[ -CD+2Cl] 2- [ -CD-H] -
[ -CD NPX-H]1363.3
O
OH
O
NPX50
1000
50
Rel
a
-CD NPX
-CD
100% 5.76E+7
100% 1.48E+8
200 400 600 800 1000 1200 1400 1600 1800 2000m/z
NPX
O
OH
0
50
1000
-CD FBP100% 1.87E+7
Fryčák, P.; Schug, K.A. Anal. Chem. 2008, 80, 1385‐1393.
IBP 0 2 4 6 8 10Time (min)
0
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Fryčák, P.; Schug, K.A. Anal. Chem. 2008, 80, 1385‐1393.
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Ongoing work…Ongoing work…• Optimization of Practical Band Broadening Parameters
– Hui Fan, ASMS– New formats
• Increase system complexity– Protein – ligand– Natural product extracts (Anal. Chim. Acta 2012, 713, 103‐110)
• Absolute binding affinities• Absolute binding affinities– Determination of response factors for noncovalent complexes
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Trace Quantitative Analysis from Trace Quantitative Analysis from C l M t i d C l M t i d Complex Matrices and Complex Matrices and Deconvolution of Complex SamplesDeconvolution of Complex Samplesp pp p
A.Estrogens
B.Endocrine DisruptorsDisruptors
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Patient A (Mid-20s Male - Good Outcome) : Estradiol (E2) Levels in the Serum and CSF FollowingEstradiol (E2) Levels in the Serum and CSF Following
Severe Traumatic Brain Injury
50
60
atio
n
30
40
once
ntra
g/m
L) CSF-E2Serum-E2
20
30
radi
ol c
o(p
g
Normal Male E2 Values
0
10
Est
Values Values << 202056 pg/ml Serum56 pg/ml Serum
2.2 pg/ml CSF2.2 pg/ml CSF
0.0 50.0 100.0 150.0Time post injury (hrs)
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1 mL CSFAdd internal standards
CSF Sample Preparation Add internal standards
X 2
Preparation
X 2
Add 2 mL of ethyl acetate Centrifuge for Remove 1 6 mlAdd 2 mL of ethyl acetateVortex for 30 sec Tumble for 30 min 30 min at
6500g
Remove 1.6 ml of ethyl acetate
Reconstitute 40 µL of buffer pH 10.14Add 40 µL dansyl chloride 1 mg/mLIncubate 15 min at 60⁰CFinal volume: 80 µL
LC‐MS
Evaporate until dryness
Nguyen, H.P. et al. J. Pharm. Biomed. Anal. 2011, 54, 83‐837.
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Validation Data
LOD ( / L)
Accuracy and Precision (n=5)Recovery
(%)Spiked Calculated Accuracy(pg/mL) (%) Concentration (pg/mL)
Concentration±SD (pg/mL)
Accuracy (%) CV (%)
E3 6170 82 ± 6 117 7
91110 101 ± 21 92 21E3 61 91110 101 ± 21 92 21220 201 ± 30 91 15
E1 1970 60 ± 9 86 15
95110 90 14 82 16E1 19 95110 90 ± 14 82 16220 176 ± 35 80 2070 63 ± 6 90 10
17β-E2 26 93110 104 ± 9 95 9220 191 ± 18 87 970 65 ± 7 93 11
17α-E2 35 104110 99 ± 8 90 8220 196 ± 25 89 13
Nguyen, H.P. et al. J. Pharm. Biomed. Anal. 2011, 54, 83‐837.
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Application of the Method
Samples E3 E1 17β‐E2 17α‐E2
1 <LOD 61 ± 2 184 ± 9 ND
2 ND <LOD ND ND
3 ND ND 208 ± 34 ND
All values in pg/mL
Nguyen, H.P. et al. J. Pharm. Biomed. Anal. 2011, 54, 83‐837.
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Estrogens from Different MatricesEstrogens from Different Matrices
y = (0.9781±0.0330)x + (0.0455±0.0212)R² = 0.9943
0.8
1
1.2
1.4
results
2 and 17
β‐E2
‐d3)
y = (0.7367±0.0332)x + (0.082±0.0214)R² = 0.9899
0.8
1
1.2
m re
sults
2 and 17
β‐E2
‐d3)Dansylated 17β‐estradiol
PBS Serum
0
0.2
0.4
0.6
PBS‐BSA r
peak ra
tio of 1
7β‐E2
0
0.2
0.4
0.6
horse serum
peak ra
tio of 1
7β‐E2
y= (0.98 ± 0.03) x + (0.04 ± 0.02) y= (0.74 ± 0.03) x + (0.08 ± 0.02)0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
(p
No matrix results (peak ratio of 17β‐E2 and 17β‐E2‐d3)
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
(p
No matrix results (peak ratio of 17β‐E2 and 17β‐E2‐d3)
1.2) 1.2)D l t d 17 t di ly = (0.9797±0.0452)x ‐ (0.0400±0.0276)
R² = 0.9916
0.6
0.8
1
1.2
A results
α‐E2
and
17α
‐E2‐d3
)
y = (1.0211±0.0233)x ‐ (0.0768±0.01427)R² = 0.9979
0.6
0.8
1
um re
sults
α‐E2
and
17α
‐E2‐d3
)Dansylated 17α‐estradiol
PBS Serum
0
0.2
0.4PBS‐BSA
(peak ratio
of 1
7α
0
0.2
0.4
horse ser
(peak ratio
of 1
7 α
y= (0.98 ± 0.05) x + (0.04 ± 0.03) y= (1.02 ± 0.02) x + (0.08 ± 0.01)
0 0.2 0.4 0.6 0.8 1 1.2
No matrix results (peak ratio of 17α‐E2 and 17α‐E2‐d3)
0 0.2 0.4 0.6 0.8 1 1.2
No matrix results (peak ratio of 17α‐E2 and 17α‐E2‐d3)
Nguyen, H.P. et al. J. Sep. Sci. 2011, 34, 1781‐1787.
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Trace Quantitative & Qualitative Analysis
• MSn
• High and stable mass accuracy• Comprehensive biomonitoring
Q tit ti /Q lit ti fili• Quantitative/Qualitative profiling
CDL DQarray
Octopole lens QIT
Dual Stage ReflectronCDL DQarray
Octopole lens QIT
Dual Stage Reflectron
Sprayer
Skimmer
LensMCP Thermostatic FLT
Sprayer
Skimmer
LensMCP Thermostatic FLT
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Photo Hunt:h ’ iff ?What’s Different?
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On‐Line Sample PrepRestricted Access MediaRestricted‐Access Media
Shi d MAYI ODS C i l A l Bi l Ch 2006 384 1462 1469Shimadzu MAYI‐ODS– Internal Surface Reversed Phase– Methylcellulose external surface
Cassiano et al. Anal. Bioanal. Chem. 2006, 384, 1462‐1469.
y– 12 nm pore size
Yamamoto et al., Anal. Sci. 2001, 17, 1155‐1159.
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binary pump ESI‐IT‐TOF‐MS
analytical column (C18)ESI IT TOF MS
Awaste pump
MAYI trap columnautosampler
MAYI trap column
binarybinary pump
analytical column (C18)ESI‐IT‐TOF‐MS
Bwaste pump
B
MAYI trap columnautosampler
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Bisphenol‐A in Human SalivaB lk D i ti ti LOD 2 9 fCH
CH3
NBulk Derivatization LOD: 2.9 pg for derivatized BPA
[M+2H]2+ = 348 m/zCH3 N
OS
OS
OO
CH3
CH3O
1.6
2.0
2.4
2.8(x1,000,000)
07 m
/zA) Blank
CH3
CH3
N
OCH3
2.42.8
(x10,000,000)0.0
0.4
0.8
1.2
m @
348.110
B) S lHot and Sour Soup (Product of Taiwan)
0.00.40.81.21.62.0
hrom
atog
ram B) Sample
137 pg detected
0.40
0.60
0.80
1.00(x100,000,000)
acted Ion Ch
C) Spiked Sample(0.04 M BPA)
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.00.00
0.20
Extra
Yang, S.H. et al. Environ. Toxicol. Chem. 2011, 30, 1243‐1251.
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PPQ detection limits for endogenous estrogens using bulk derivatizationbulk derivatization…
…on a Shimadzu CoSense – LCMS 8030 Triple Quadrupole MS
E3*
@ 100 pg/mL (100 parts‐per‐trillion):O
N
SO O
Cl
O
Odansyl chloride
dansylated E1E3
βE2* αE2*
522 171
506 171
HO
SO O
NE1 m/z = 171
dansylated E1 (E1*)
504 171E1*
/@ 1 pg/mL (1000 parts‐per‐quadrillion):
E3 αE2 βE2 E1
S/N 6:1 10:1 14:1 5:1
MAYI ODS to Kinetex XB‐C18 (2 x 100 mm, 2.6 µ)2 µL injection (200 fg on column)MRM dwell time 300 msec S/N 6:1 10:1 14:1 5:1
250 µL injection (250 fg on column)
MRM dwell time 300 msec
12 min run time
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Next Steps…Next Steps…Next Steps…Next Steps…• Trace quantitative analysis
Focus on CoSense QQQ– Focus on CoSense – QQQ– Non‐feminizing estrogens Stroke– Premarin TBI
• Other matrices– Foods, Plasma/Serum, Saliva
• Other applications• Other applications– In‐vitro (hepatocytes) vs. in‐vivo (students) metabolism studies
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“If you do not know where you are going, any road will get you there.”
‐Lewis Carroll
Page 30
llContinuous Flow Continuous Flow –– Extractive Extractive Desorption Electrospray Desorption Electrospray p p yp p yIonization (CFIonization (CF‐‐EDESI): EDESI): An Ambient Ionization TechniqueAn Ambient Ionization TechniqueAn Ambient Ionization TechniqueAn Ambient Ionization Technique
A “me too” method?
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Ambient Ionization TechniquesDesorption Electrospray Ionization (DESI)
Takats et al. Science 2004, 306, 471‐473.
Transmission Mode ‐ Desorption Electrospray Ionization (TM‐DESI)
Chipuk & Brodbelt J. Am. Soc. Mass Spectrom. 2008, 19, 1612‐1620.
Extractive Electrospray Ionization (EESI)
Chen et al. Chem. Commun. 2006, 2042‐2044.
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Continuous Flow – Extractive Desorption pElectrospray Ionization (CF‐EDESI‐MS)
Orthogonal Continuous Liquid Flow Containing
Analytes (CF)
Hypodermic Needle
• ••••• •• ••
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••
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N2 (g)•
••
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••
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••
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• •
•
N2 (g)
Electrospray (ESI)
•
••
• ••
• •
• ••
••
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••
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••
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x = 0.15 cm
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MS Inlet•
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HV
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d = 0.8 cm
Page 34
Continuous Flow – Extractive Desorption pElectrospray Ionization (CF‐EDESI‐MS)
Orthogonal Continuous Liquid Flow Containing
Analytes (CF)
Hypodermic Needle
x = 0 15 cm
Orthogonal Continuous Liquid Flow Containing
Analytes (CF)
Hypodermic Needle
x = 0 15 cm
Independent optimization of CF and ESI solvents (e.g. manipulation of protein charge states)
• ••••
••
•••
••••
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Electrospray (ESI)
HV
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x 0.15 cm
d = 0.8 cm
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d = 0.8 cm
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MS Inlet
states).
Higher tolerance and wider compatibility with “non‐ESI‐friendly” solvents andwith non ESI friendly solvents and additives.
Mechanistically distinct from EESI and
A new source for LC‐MSNormal phase separations
DESI
Normal phase separationsProtein separations
Page 35
Manipulation of Protein Charge States200 μM Cytochrome c
+6
6080
100 2039.001751.25 2446.38
1535 38+8
+7 +5
CF: Protein in 100% H2O
ESI 50/50 H O/MeOH
0204060 1535.38
1359.90+9
1529.88100
ESI: 50/50 H2O/MeOH
1748.00
2038.632040
6080
100
+10+11+12+13+14+15
+9
+16+17+18
CF: Protein in 100% H2O
ESI: 50/50 H2O/MeOH + 10% HOAc
600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
2038.63
0
+18
1529.50 1747.754060
80100
+10
+11+12+13
+14+9
ESI only: Protein in 50/50 H2O/MeOH + 10% HOAc
m/z600 1000 1400 1800 2200 2600 3000
2038.75
0
20 +15+16
Yang et al. Anal. Chem. 2011, 83, 643‐647
Page 36
Manipulation of Protein Charge States200 μM Cytochrome c
+6
6080
100 2039.001751.25 2446.38
1535 38+8
+7 +5
CF: Protein in 100% H2O
ESI 50/50 H O/MeOH
0204060 1535.38
1359.90+9
1529.88100
ESI: 50/50 H2O/MeOH
1748.00
2038.632040
6080
100
+10+11+12+13+14+15
+9
+16+17+18
CF: Protein in 100% H2O
ESI: 50/50 H2O/MeOH + 10% HOAc
600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000
2038.63
0
+18
Yang et al. Anal. Chem. 2011, 83, 643‐647
Page 37
CF‐EDESI‐MS: Non‐ESI‐Friendly Solvents
80
100
danc
e
421.4 NL: 2.21E5
O
OO
[M-Na]-
80
100
danc
e
421.4 NL: 2.21E5
O
OO
[M-Na]-
40
60
Rel
ativ
e Ab
und
865 4
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Page 38
CF‐EDESI‐MS: Non‐ESI‐Friendly Solvents
Analyte Solvent Concentration ESI CF‐EDESI (105)
Progesterone Hexane 10M None 6.5±0.5
Progesterone Chloroform 10M None 16.9±0.9Progesterone Chloroform 0M None 6.9±0.9
Progesterone Ethyl Acetate 10M None 14±1
Vit i K H 1 M N 1 5±0 3Vitamin K3 Hexane 1mM None 1.5±0.3
Hydrocortisone Chloroform 10M None 21±2
Page 39
CF‐EDESI‐MS: Non‐ESI‐Friendly Solvents
Extraction or Mixing?
Analyte: 10 µM progesterone
Page 40
CF‐EDESI‐MS vs. EESI
CF‐EDESI
Unpublished Results
ESI source
EESI
Sample nebulizer
MS inlet
Law et al., Anal. Chem. 2010, 82, 4494‐4500
Page 41
CF‐EDESI‐MS: VariablesCF EDESI MS: Variables
Page 42
CF‐EDESI‐MS: VariablesCF EDESI MS: Variables
Relative Flow Rates
Analyte: 10 µM progesterone in hexane
Page 43
CF‐EDESI‐MS: VariablesCF EDESI MS: Variables
Temperature Pneumatic assistance
Analyte: 10 µM progesterone in hexane
Page 44
To understand To understand something new, you have to look at it from many different angles…y g
Page 45
A quick aside… CHROMmunityhttp://chrommunity.ning.com/
SPE‐NP‐HPLC‐CF‐nanoEDESI‐IT‐TOF‐MS/MS ??
Page 46
W A N T E Dom
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Y O U !At the 24th International Symposium
on Chiral Discrimination
Ft Worth TX 12.c
ow
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Ft. Worth, TX June 10th – 13th, 2012
(at the Hilton in downtown)
www.chirality2012.comy201
chira
Plenary Lecture by Prof. E.J. Corey, Harvard University1990 Nobel Laureate in Chemistry
on Enantioselective Methods for the Synthesis of Polycycles
www.chirality2012.comSPECIAL FEATURES:
alit
yality
Plenary Lecture by Prof. Ron Breslow, Columbia UniversityNational Academy of Science Member
on Origin of Chirality in Life
Chirality Medal Award Presentation and AddressProf. Eric N. Jacobsen, Harvard Universitych
ira
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2012 Chirality Medal Award Winner
Pharmaceutical Industry Job FairSpecial Session: “The Practice and Consequences of Chiral Drug Development” (3 featured talks and panel discussion)
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[email protected]
Submit Abstracts by March 15
Register Now by April 15
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Page 47
“Already for 35 years he had not stopped60 minutes
Already for 35 years he had not stopped talking and almost nothing of fundamental
l h d d ”value had emerged.”
You‐ James Watson
(Referring to F. Crick)
You
MeMe
Page 48
Thank You!Thank You! CollaboratorsThank You!Thank You! • Dan Armstrong, UTA
• Sandy Dasgupta, UTA
• Frank Foss, UTA
• Jim Simpkins UNTHSC• Jim Simpkins, UNTHSC
• Jane Wigginton, UTSW
• Mehervan Singh, UNTHSC
• Dominique Torran‐Allerand, C l bi U iColumbia Univ.
• Jungmo Ahn, UTD
• Karel Lemr & Vladamir Havliček, Prague/Olomouc, CZ
• Seoung Bum Kim, Korea Univ.