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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

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.

“The best way to have a good idea is to have lots of ideas.”

‐ Linus Pauling

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

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

High Throughput QuantitativeHigh Throughput Quantitative Binding Determinations gusing FIA‐ESI‐MS

F t

“Static Titration”

From… to…

Flow Injection AnalysisFlow Injection Analysis

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.

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

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)

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.)

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

Fryčák, P.; Schug, K.A. Anal. Chem. 2008, 80, 1385‐1393.

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

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

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)

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.

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.

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.

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.

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

Photo Hunt:h ’ iff ?What’s Different?

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.

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

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.

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

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

“If you do not know where you are going, any road will get you there.”

‐Lewis Carroll

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?

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.

Continuous Flow – Extractive Desorption pElectrospray Ionization (CF‐EDESI‐MS)

Orthogonal Continuous Liquid Flow Containing

Analytes (CF)

Hypodermic Needle

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MS Inlet•

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MS Inlet

d = 0.8 cm

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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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

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

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

CF‐EDESI‐MS:  Non‐ESI‐Friendly Solvents

80

100

danc

e

421.4 NL: 2.21E5

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OO

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40

60

Rel

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865 4

OS OOO Na

[M-C16H34O2Na]-[2M-Na]-

Dioctyl sulfosuccinate sodium salt (M)

40

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Rel

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e Ab

und

865 4

OS OOO Na

[M-C16H34O2Na]-[2M-Na]-

Dioctyl sulfosuccinate sodium salt (M)

200 400 600 800 1000 1200 1400 1600 1800 2000/

0

20

R 865.4162.9

843.2 1309.41753.5

[M C16H34O2Na]

[2M-2Na+H]- [3M-2Na]-

[4M-3Na]-

200 400 600 800 1000 1200 1400 1600 1800 2000/

0

20

R 865.4162.9

843.2 1309.41753.5

[M C16H34O2Na]

[2M-2Na+H]- [3M-2Na]-

[4M-3Na]-

Continuous Flow (CF)Sulfonic acid surfactant in chloroform

m/zm/z

Liquid‐liquid extraction with 

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MS Inlet

charged ESI droplets?

Partition coefficients between ESI droplets and immiscible solvents?

•• •• •• • •••• •• • ••

•••

HV

••••• ••

90/10 Water/IPA (‐) ion mode

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

CF‐EDESI‐MS:  Non‐ESI‐Friendly Solvents

Extraction or Mixing?

Analyte: 10 µM progesterone

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

CF‐EDESI‐MS:  VariablesCF EDESI MS:  Variables

CF‐EDESI‐MS:  VariablesCF EDESI MS:  Variables

Relative Flow Rates

Analyte: 10 µM progesterone in hexane

CF‐EDESI‐MS:  VariablesCF EDESI MS:  Variables

Temperature Pneumatic assistance

Analyte: 10 µM progesterone in hexane

To understand To understand something new, you have to look at it from many different angles…y g

A quick aside…  CHROMmunityhttp://chrommunity.ning.com/

SPE‐NP‐HPLC‐CF‐nanoEDESI‐IT‐TOF‐MS/MS ??

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

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Ft. Worth, TX June 10th – 13th, 2012

(at the Hilton in downtown)

www.chirality2012.comy201

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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:

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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

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Pharmaceutical Industry Job FairSpecial Session: “The Practice and Consequences of Chiral Drug Development” (3 featured talks and panel discussion)

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Submit Abstracts by March 15

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“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

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.