Solving Complex Chromatographic Challenges John Van …Solving Complex Chromatographic Challenges ....

©2012 Waters Corporation 1

Convergence Chromatography: Solving Complex Chromatographic Challenges

John Van Antwerp


What is Convergence Chromatography: Why The Name?

Giddings, J.C. (1965) A critical evaluation of the theory of gas chromatography. In Gas Chromatography. 1964, edited by A. Goldup, p. 3-24. Elsevier, Amsterdam

In this article Dr. Giddings stated “One of the most interesting features of ultra high pressure gas chromatography would be convergence with classical liquid chromatography.”

Prof. Calvin Giddings (1930-1996)


Evolution of Separation Technology

Gas Chromatography Liquid Chromatography Convergence Chromatography

GC

Capillary GC

HPLC

UPLC

SFC

UPC2


UPLC and UPC2

SPEED SENSITIVITY RESOLUTION

SIMPLICITY SIMILARITY

ORTHOGONALITY


UPC² Adoption

UPC² SIMPLIFIES the workflow – Combines multiple techniques into ONE

o LC and GC – Combines multiple methods into ONE

o NP and RP – Reduces sample prep and analysis times

o Direct injection of organic solvents/extracts

UPC² separates compounds with SIMILARITY – Chiral, positional isomers, structural analogs, conjugates (biomarkers)

UPC² provides ORTHOGONALITY – More confidence in identifying impurities/degradants – Full sample characterization – Separation of analytes from matrix interferences (i.e., hydrophobic drugs

in bioanalysis)


How is SSO possible?

11

+−

kk

αα

4N=Rs

Selectivity [α] and retentivity [k] impacted by:

Stationary phase (column selectivity) Organic solvent (eluotropic series) Mobile phase additives (pH and ionic strength)

System efficiency [N] impacted by:

System dispersion Reduction in particle size

Impact on Rs % Improvement

Double N 20-40% Double k 15-20%

Double α > 400%

ConvergenceChromatography Selectivity Space

Unlimited solvent and stationary

phase selectivity

SolventPentane, Hexane, Heptane

Xylene

Toluene

Diethyl ether

Dichloromethane

Chloroform

Acetone

Dioxane

THF

MTBE

Ethyl acetate

DMSO

Acetonitrile

Isopropanol

Ethanol

Methanol

Stationary Phase

Silica / BEH

2-ethylpyridine

Cyano

Aminopropyl

Diol

Amide

PFP

Phenyl

C18 < C8


UPC2 Adoption


ORTHOGONALITY

SIMPLICITY


Combining Multiple Techniques for Lipid Analysis

Gas Chromatography Liquid Chromatography Convergence Chromatography

Free fatty acids are typically derivatized to form the methyl esters (FAMEs) Analysis time 30 min

Analyzed by both HILIC and RP HILIC separates lipid classes by polar head group RP separates based on acyl chain length and number of double bonds

Single methodology to separate complex lipids by class Faster baseline separation of lipids based on chain length and number of double bonds


UPC2 Analysis of a Mouse Heart Extract

PC

SM LPC

PE

TAG

TAG: Triacylglycerides PE: Phosphotidylethanolamine PC: Phosphotidylcholine SM: Sphynogomyelin LPC: Lysophosphotidylcholine

ACQUITY UPC2 BEH column 5-50% B


Time0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10

%

0

100

0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10

%

0

100

8:0

14:0

16:0

18:0

20:0

22:0

24:0

10:0

12:0

ESI negative mode Free Fatty Acids (FFA) containing 8-24 acyl chain

ESI positive mode Triacylglycerols (TG) and Cholesterol esters (CE)

1 7

2 8

3

4 5

6 11

10

13 12

9

Peak Lipid Species

1 15:0/15:0/15:0 TG 2 18:3(∆9,12,15Cis)/18:3(∆9,12,15Cis)/18:

3(∆9,12,15Cis) TG 3 16:0/16:0/16:0 TG 4 18:2(∆9,12Cis)/18:2(∆9,12Cis)/18:2(∆9,1

2Cis) TG 5 18:1(∆9Tr)/18:1(∆9Tr)/18:1(∆9Tr) TG 6 17:0/17:0/17:0 TG 7 18:1(∆9Tr)/18:1(∆9Tr)/18:1(∆9Tr) TG 8 18:3 CE 9 18:2 CE 10 17:0 CE

18:1 CE

11 18:0/18:0/18:0 TG 12 18:0 CE

13 19:0 CE

Separation of Neutral Lipids Based on Chain Length and Number of Double Bonds

ACQUITY UPC2 HSS C18 SB column 1-10% B


Metabolomics/Lipidomics

University of Northern Texas Collaboration

Shulaev Metabolomics Lab Metabolic Signaling Pathway Research

Cottonseed Extracts

http://www.unt.edu/

http://lab.shulaev.org/


Global Profiling Workflow

ANALYZE INTERPRET

UNT Goals: Global Profile and Targeted Analysis

Lipids from Cottonseed extracts

•UPC2 BEH Stationary Phase

•Neutral and Polars Interclass

•UPC2 HSS C18 SB Stationary Phase

•Neutral Lipids Intraclass

•Synapt G2 - S Qualitative

•Xevo TQ-S Quantitative


How does UPC2 Compare to Current Lipidomic Approaches?

Separation based on adsorption of the head group to the NP material for lipid class separation.

Separation based on hydrophobic interaction of the FA chain and RP material for lipid molecular species separation

Historically UPC2 Trends

01020304050

7.2 7.25 7.3 7.35 7.4

# F

A c

hai

n

Rt (min)

BEH: Effect of FA chain length

01020304050

7 8 9 10 11 12

# F

A c

hai

n

Rt (min)

C18: Effect of FA chain length

1. PC: 14:0/14:0 2. PC: 16:0/16:0 3. PC: 17:0/17:0 4. PC: 18:0/18:0 5. PC: 23:0/23:0

ACQUITY UPC2 BEH

ACQUITY UPC2 HSS C18 SB

Trends Similar based on stationary phase


Time0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10

%

0

100

0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10

%

0

100

8:0

14:0

16:0

18:0

20:0

22:0

24:0

10:0

12:0

ESI negative mode Free Fatty Acids (FFA) containing 8-24 acyl chain

ESI positive mode Triacylglycerols (TG) and Cholesterol esters (CE)

1 7

2 8

3

4 5

6 11

10

13 12

9

Peak Lipid Species

1 15:0/15:0/15:0 TG 2 18:3(∆9,12,15Cis)/18:3(∆9,12,15Cis)/18:

3(∆9,12,15Cis) TG 3 16:0/16:0/16:0 TG 4 18:2(∆9,12Cis)/18:2(∆9,12Cis)/18:2(∆9,1

2Cis) TG 5 18:1(∆9Tr)/18:1(∆9Tr)/18:1(∆9Tr) TG 6 17:0/17:0/17:0 TG 7 18:1(∆9Tr)/18:1(∆9Tr)/18:1(∆9Tr) TG 8 18:3 CE 9 18:2 CE 10 17:0 CE

18:1 CE

11 18:0/18:0/18:0 TG 12 18:0 CE

13 19:0 CE

UPC2 Neutral Lipid Method Mix UPC2 HSS C18 SB 1.7 µm (3.0x100mm)

ACQUITY UPC2 HSS C18 SB column 1-10% B


UNT Cottonseed Extract Biological samples targeted to Neutral Lipids

ACQUITY UPC2 BEH

Low collision energy

High collision energy

PC

TAG

DAG

NAPE

50%

2%

UPC2/Synapt G2 with MSE:


Sample Analysis

Goals: Discriminate between Wild type and treated cottonseeds Search for homo- and hetero- geneity between treatments


Combining Multiple LC Methods Fat-soluble vitamins

Vitamin A Normal phase 12 minutes

Vitamin D3 Normal phase 20 minutes

Vitamin E Normal phase 30 minutes

Vitamin K1 Reversed-phase

12 minutes

β-carotene Normal phase 10 minutes

Lycopene Normal phase 10 minutes

Simultaneous Analysis of Fat-soluble Vitamins and Carotenoids in 5 minutes

A A

ceta

te

E A

ceta

te

K2

K1

E

D2

A P

alm

itate

Lyco

pene

Bet

a ca

rote

ne

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.32

0.34

0.36

0.38

0.40

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00


Official AOAC Method

Reducing Sample Prep and Analysis Time β-carotene Analysis

Dissolve/Hydrolyze

Extract

Dilute

Filter

LC Analysis

Modified AOAC Method

30 min

120 min

30 min

20 samples ~12.5 hrs

Dissolve/Extract

Filter

LC Analysis

30 min

20 samples ~10 hrs

UPC2 Method

Dissolve/Extract

Filter

UPC² Analysis

2 min

20 samples ~ 40 minutes

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50

AU

0.0

1.0e-1

2.0e-1

3.0e-1

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50

AU

0.0

1.0e-1

2.0e-1

3.0e-1

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50

AU

0.0

5.0e-2

1.0e-1

1.5e-1

2.0e-1

2.5e-1

3.0e-1

0.91

0.91

0.91

0.77 1.31

β-carotene standard

β-carotene capsule

Carotenoids Mix

6 Replicates Peak Area Retention

Time (min) Average 10326 0.91 RSD% 0.34 0

Label Claim: 15 mg/capsule Assay

#1 Assay

#2 Assay

#3 Average RSD%

15.13 15.39 15.24 15.25 0.84%


Directly injecting organic solvent extracts Eliminating Potential Sources of Error

Sample Pretreatment/

Homogenization

Extraction

Centrifugation/ Filtration

Evaporation

Reconstitution

Desired injection

point

LLE Protein Precipitation SPE

Time0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90

%0

100

0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90

%

0

100

Blank Human Urine

0.1 ng/mL Desipramine D3 in Human Urine

Samples directly injected from mixed-mode SPE

Concentration RT (min) Area

Counts % Deviation %

Accuracy 0.1 ng/mL 1.48 16161 -3.3 96.7 0.2 ng/mL 1.48 27061 2.7 102.7 0.5 ng/mL 1.48 60531 7.9 107.9 1 ng/mL 1.48 103149 -3.6 96.4 5 ng/mL 1.48 467997 -7.9 92.1 10 ng/mL 1.48 999886 -0.9 99.1

Standard Curve results for amitriptyline


Analysis of Non-Ionic Surfactants


Background

•Non-ionic surfactants are used in cosmetics, industrial materials, and

many other products.

•Their composition has to be monitored because the differences in

ethoxy chain length affect the viscosity, solubility, polarity, and other

characteristics of the mixture


Current separation methods for non-ionic surfactants

•Normal phase HPLC •Hexane:Methylene chloride:Methanol gradient •24 min to elute all components •Approx. 16 oligomers separated and detected •(Sigma-Aldrich)

•High temperature GC •19 min to elute all components •Approx. 18 oligomers separated and detected (Atas GL)


Purpose/Competitive Technology

•Typically analyzed by HPLC, SFC, GC •Analysis by GC and HPLC very time-consuming

•SFC uses high column temperatures which can limit analysis of thermally labile compounds

•HPLC might require derivatization for non-UV absorbing surfactants

•Incomplete baseline separation for oligomers in some cases


Sample Set Id: 1462 SampleName: Triton X-100 Date Acquired: 6/21/2012 8:54:56 AM EDT Injection Id: 1522

Peak1 -

0.2

71

Peak2 -

0.4

55

Peak3 -

0.5

74

Peak4 -

0.6

62

Peak5 -

0.7

33

Peak6 -

0.7

92

Peak7 -

0.8

41

Peak8 -

0.8

84

Peak9 -

0.9

21

Peak10 -

0.9

54

Peak11 -

0.9

85

Peak12 -

1.0

13

Peak13 -

1.0

41

Peak14 -

1.0

67

Peak15 -

1.0

93

Peak16 -

1.1

19

Peak17 -

1.1

58

Peak18 -

1.1

88

Peak19 -

1.2

17

Peak20 -

1.2

48

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

Minutes0.10 0.15 0.20 0.25 0.30 0.35 0.40 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

Triton X (10 mg/mL in IPA)

•UPC2

•CO2 and Methanol gradient @ 40°C •1.4 min to elute all components •Approx. 20 oligomers separated and detected


Improving Workflow with Convergence Chromatography

Samples Incompatible with Water – Tree extracts analyzing for phenolics – Resins crash out when in contact with water – Impossible to run by Reversed Phase – Needed to be Compatible with MS so NP was out


Simplifying the Workflow with UPC2

Application Example UPC2 Advantages Other application areas this

might apply

Combining multiple techniques (LC and GC into CC) - Lipids

1) Single technique for neutral and polar lipids

2) No derivatization for neutral lipids (needed for GC)

3) Faster baseline separation than LC

1) Glyceride analysis in food and fuels 2) Lipid profiling in drug discovery

studies

Combining multiple methods (NPLC and RPLC into CC) Fat-soluble Vitamins and Carotenoids

1) Directly inject organic extracts 2) One technique to replace RPLC and

NPLC

1) Pre-mixes and formulated samples 2) Raw materials testing 3) Samples prepared by LLE

Reducing sample prep and analysis times β-carotene Analysis

1) Reduced sample prep steps 2) Faster run times 3) Higher overall throughput

1) Samples prepared by SPE, LLE, or

protein precipitation that need evaporation and reconstitution

2) Bioanalysis/DMPK Directly injecting organic solvent extracts Tricyclic anti-depressants (TCAs)

1) Direct injection of SPE extract 2) No evaporation and reconstitution =

less experiment error 3) No solvent exchange needed prior to

analysis


UPC2 Adoption


ORTHOGONALITY

SIMILARITY


Structural Similarity

Isomers and structural analogs can be challenging to separate due to small differences in structure, or due to their chirality.

In this section we will look at applications that are of interest due to their structural similarity using UPC2, including: 1. Chiral Separations (Enantiomers & diastereomers) 2. Positional isomers (differ in location of functional groups) 3. Structural analogs Biomarkers (conjugated/unconjugated) Drugs (metabolites, impurities, degradants)


Chiral Separations

Key advantages of moving to UPC2

– Results that are equal to or better than NPLC – Drastic reduction in analysis time (up to 30X) – Nearly 75X reduction in solvent – Drastic reduction in cost of analysis (up to 100X)

o Waste generation and disposal

AU

0.00

0.30

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

UPC²

NPLC 11 min

0.3 min


Cyfluthrin Chiral Separation

AU

0.00

0.10

0.20

0.30

0.40

0.50

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Time0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

AU

0.0

2.5e-2

5.0e-2

7.5e-2

1.0e-1

1.25e-1

1.5e-1

1.75e-1

2.0e-1

2.25e-1

2.5e-1

2.75e-1

(A) UPC2 IC + OJ-H

(B) Traditional SFC 2 x IC + 2 x OJ-H

cyfluthrin

Cl

Cl

O

O

CN

O

**

*

F


Methamphetamine UPC2/TQD In house Control Urine sample

d_M

etha

mph

et -

4.78

4

l_Met

ham

phet

- 5.

570

Inten

sity

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

6000.0

7000.0

8000.0

9000.0

10000.0

11000.0

12000.0

Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50


Chiral Separations

Chiral screening Chiral method development – MS and UV detection

Chiral inversion studies Enantiomeric excess

Pesticides Drugs of Abuse Beta-blockers Binol Warfarin Benzyl Mandelate (enantiomeric excess) Carprofen (chiral method development) Pantoprazole and Oxfendazole (chiral

method development with MS) Clenbuterol Phenylalanine methyl esters Flurbiprofen Cyclometalated Iridium (III) Complexes

Fast Chiral Separations

www.waters.com/upc2


Positional isomers DMBA

2.13

42.

245

2.34

2

2.55

2

2.68

2

3.43

0

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80

2,5

2,3 3,5

2,4

3,4

2,6

Mixture of 6 positional isomers of DMBA Each at 0.2 mg/mL in isopropanol (IPA) 3.0 x 100 mm, 1.7 µm ACQUITY UPLC BEH125 (custom configuration) CO2 / MeOH with 0.2% formic acid


Structural Analogs Steroids

Androstenedione

17α-Hydroxyprogesterone

Testosterone Estrone

11-Deoxycortisol

Corticosterone Aldosterone

Cortisol Estradiol


Steroids by UPC2

Steroids 1. Androstenedione 2. Estrone 3. 17α-Hydroxyprogesterone 4. Testosterone 5. 11-Deoxycortisol 6. Estradiol 7. Corticosterone 8. Aldosterone 9. Cortisol

1

2

3

4

5 6 7 8 9 AU

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14

Minutes 0.00 0.50 1.00 1.50 2.00

ACQUITY UPC2 BEH

AU

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14

Minutes 0.00 0.50 1.00 1.50 2.00

1

2,5

3

4

6 7 8 9

ACQUITY UPC2 BEH 2-EP

1

2

3

4

6 7 8 9 AU

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14

Minutes 0.00 0.50 1.00 1.50 2.00

5

ACQUITY UPC2 CSH Fluoro-Phenyl


Structural Analogs Sulfated Estrogens

Estrone (1) MW = 350.43

O C H 3

O

S O O

O H

H H

H

Equilin (2) MW = 348.41

O H

O C H 3

O

S O O

H H

∆-8,9-Dehydroestrone (3) MW = 348.41

O C H 3

O

S O O

O H

H

Equilenin (4) MW = 346.41

O C H 3

O

S O O

O H

17α-Estradiol (5) MW = 352.45

O H C H 3

O

S O O

O H

H H

H

17β-Estradiol (6) MW = 352.45

O H C H 3

O

S O O

O H

17α-Dihydroequilin (7) MW = 350.43

O H C H 3

O

S O O

O H

H H

17β-Dihydroequilin (8) MW = 350.43

O H C H 3

O

S O O

O H

H H

17α-Dihydroequilenin (9) MW = 348.41

O H

O H C H 3

O

S O O

H

17β-Dihydroequilenin (10) MW = 348.41

O H C H 3

O

S O O

O H

H

Molecular weights are color coded to denote isobaric compounds


USP 35-NF 30 GC method for Conjugated Estrogens

7

6

5

10 9 8

1

2

3 4

17α-Estradiol (5) MW = 352.45

O H C H 3

O

S O O

O H

H H

H

17β-Estradiol (6) MW = 352.45

O H C H 3

O

S O O

O H


AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

Minutes 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00

Sulfated Estrogens by UPC2

1 2

3

4

5

6

7 8

9

10

Sulfated Estrogens 1. Estrone 2. Equilin 3. ∆-8,9-Dehydroestrone 4. Equilenin 5. 17α-Estradiol 6. 17β-Estradiol 7. 17α-Dihydroequilin 8. 17β-Dihydroequilin 9. 17α-Dihydroequilenin 10. 17β-Dihydroequilenin


Structural Similarity with UPC2

Application Example UPC2 Advantages Other potential

applications

Enantiomers and diastereomers

(Chiral separations)

1) Faster and cheaper than NPLC 2) 10X faster, >85% solvent savings 3) Meets “green” initiatives

1) Chiral screening 2) Chiral method development

(MS and UV detection) 3) Chiral inversion studies 4) Enantiomeric excess

Positional isomers DMBA

1) Faster separation than NPLC 2) Low solvent usage and waste production 3) Compatibility with NP diluents and

extraction solvents 4) Separation of both geometric and

enantiomeric isomers

1) Starting materials analysis 2) Reaction monitoring 3) Asymmetric catalysis (Chemical

Materials)

Structural Analogs Steroids/ Estrogens

1) No derivatization required (takes 2.5 hrs) 2) Faster and better separation than LC or GC 3) Resolution of conjugated steroids 4) Directly compatible with MS

1) Non-polar biomarkers 2) Natural product API and

formulation analysis


UPC2 Adoption


ORTHOGONALITY


Orthogonal Separations

Why do I need an orthogonal separation mode?

1. A common concern in many applications is that an impurity, degradation peak, or similar compounds may be overlooked (isobaric, co-elution)

2. Orthogonal methods that provide different relative retention of peaks are needed to ensure full characterization

3. Ability to see more information about the sample

4. Separation of desired compounds from matrix interferences


Orthogonal to RPLC Metoclopramide Related Substances

ACQUITY UPC2

Reversed-Phase

AU

0.000

0.013

0.026

Minutes0.00 1.20 2.40 3.60 4.80 6.00 7.20 8.40 9.60 10.80

1 2

3 4

5 6

8 9

AU

-0.003

0.000

0.003

0.006

0.009

Minutes0.00 1.10 2.20 3.30 4.40 5.50 6.60 7.70 8.80 9.90

2

Metoclopramide

Metoclopramide

12 minutes

12 minutes


Separation of Compounds from Matrix Interferences (Bioanalysis)

Clopidogrel (RPLC)

Clopidogrel (UPC2)

Interfering Phospholipids (RPLC)

Interfering Phospholipids (UPC2)



Clopidogrel (RPLC)

Clopidogrel (UPC2)




Orthogonal Separations with UPC2

Application Example UPC2 Advantages Other potential applications

Full characterization Metaclopramide

1) Different elution order than RPLC 2) Resolves peaks not resolved by LC 3) Compatible with MS for

identification of unknowns

1) Impurity/degradant analysis 2) Agrochemical APIs and formulations 3) Stability indicating methods (OLEDs) 4) Non-polar compounds that elute late

in RPLC

Orthogonal to LC Chamomile

1) Direct injection of organic extracts (i.e., microwave extraction)

2) MS compatibility 1) Complex mixture analysis

Ability to see more (isobaric) Chamomile

1) Separation of isobaric species

1) Isobaric separations

Separation from Matrix Interferences Bioanalysis (phospholipids)

1) Analytes of interest are eluted away from the matrix

2) Less matrix interferences = less potential suppression

3) More precise and accurate quantitation

1) Bioanalysis, DMPK (hydrophobic compounds)

2) Other matrices containing lipids (food, fuels, tissues)


University of Mississippi Collaboration

National Center for Natural Products Research

School of Pharmacy, The University of Mississippi


Global Profiling Workflow

EXTRACT ANALYZE INTERPRET

Ole Miss Goals: To explore new entities via different extraction procedures

Chamomile Extracts

•MeOH •Hexane Solvent -

Solvent

•IPA •Hexane •IPA:Hexane

Microwave

•Different Percentages •Different Modifiers SFE

UPC2 2-EP Stationary

Phase

Xevo Q-Tof G2 S

•TOIML TransOmics


Chamomile Profiling

Compare to MeOH extracts and Hexane extracts of two species of chamomile – UPC2 method development

required

Compare to previous studies

– HPTLC v. UPLC/MS v. GC-MS

Identify benefits – New discoveries? – Examination of extracts on both

systems?

Anthemis nobilis (Roman Chamomile)

Matricaria recutita (German Chamomile)


Roman Chamomile

AU

0.00

0.05

0.10

0.15

0.20

Minutes0.00 2.20 4.40 6.60 8.80 11.00 13.20 15.40 17.60 19.80 22.00

AU

0.00

0.22

0.44

0.66

0.88

Minutes0.00 3.20 6.40 9.60 12.80 16.00 19.20 22.40 25.60 28.80 32.00

A. UPLC Reversed Phase UV 350 nm

B. UPC2 UV 350 nm

2

3

1

1

2

3

1. apigenin-7-O-glucoside 2. chamaemeloside 3. apigenin


Separation of Isobaric Species XIC of m/z = 475 Da

Inte

nsity

0

80000

160000

240000

320000

Minutes11.60 11.80 12.00 12.20 12.40 12.60 12.80 13.00 13.20 13.40 13.60 13.80 14.00 14.20 14.40 14.60 14.80 15.00 15.20 15.40

Inten

sity

0.0

4.0x105

8.0x105

1.2x106

1.6x106

Minutes4.62 5.28 5.94 6.60 7.26 7.92 8.58 9.24 9.90 10.56

Interrogation of the MS data for both LC and SFC based techniques displayed some differences when comparing isobaric separations indicated by the XIC of selected m/z = 475 XIC of m/z=475. An additional peak was found in the UPC2 trace (A)…

A. UPC2/MS

B. UPLC/MS …when compared to the UPLC-RP (B) trace.


OPLS/OPLS-DA (9172 vs. 9254) (from different extraction techniques)

RC GC

Between group variation

Wit

hin

gro

up

var

iati

on German - 9172 Roman - 9254


S-Plot (9172 vs. 9254)

Compounds up regulated in 9254

Compounds up regulated in 9172

Exported to excel file for the list of up regulated features, currently the collaborator have identified new features not previously seen by other techniques (yet to be published)


Difficult Separations - Orthogonality

Column: BEH 2-EP 1.7 um, 2.1 x 50 mm Gradient: 5-35% MeOH in 1 min Solvent: CO2, MeOH

Column: BEH C18 1.7 mm, 2.1 x 50 mm Gradient: 5-95% AcCN in 1 min Solvent: 0.1% FA in H2O, 0.1% FA in AcCN

UV Chromatogram - UPLC-MS

UV Chromatogram - UPC2-MS

Time0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85

AU

2.0e-1

4.0e-1

6.0e-1

8.0e-1

1.0

1.2

1.452Range: 1.379

UV Detector: 220 100%404.20.46

Time0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85

AU

2.0e-1

4.0e-1

6.0e-1

8.0e-1

1.0

1.2

D-JSC-000007-857, MW: 404.2

Time0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00

AU

0.0

2.5e-1

5.0e-1

7.5e-1

1.0

1.25

1.5

1.997Range: 1.997

UV Detector: 220

0.12

87%404.20.58

13%404.20.63

Time0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00

AU

0.0

2.5e-1

5.0e-1

7.5e-1

1.0

1.25

1.5

http://www.google.com/url?sa=i&rct=j&q=&source=images&cd=&cad=rja&docid=vk2nzBCXQIBIlM&tbnid=GBGEMKpASyx8TM:&ved=0CAUQjRw&url=https%3A%2F%2Fwww.smartrecruiters.com%2Fdartneuroscience%2F167833&ei=I0yvUZuvNsG9OdWEgAg&bvm=bv.47380653,d.ZWU&psig=AFQjCNGpilj6zhj7zGR3rlGEXtaeryBWTA&ust=1370529151957057


Similar Structures - Regioisomers

Column: BEH C18 1.7 mm, 2.1 x 50 mm Gradient: 5-95% AcCN in 1 min Solvent: 0.1% FA in H2O, 0.1% FA in AcCN

TIC Trace- UPC2-MS

TIC Trace - UPLC-MS

Column: BEH 2-EP 1.7 um, 2.1 x 50 mm Gradient: 5-35% MeOH in 1 min Solvent: CO2, MeOH

D-JSC-000006-0010

0010A 0010B

0010A 0010B

http://www.google.com/url?sa=i&rct=j&q=&source=images&cd=&cad=rja&docid=vk2nzBCXQIBIlM&tbnid=GBGEMKpASyx8TM:&ved=0CAUQjRw&url=https%3A%2F%2Fwww.smartrecruiters.com%2Fdartneuroscience%2F167833&ei=I0yvUZuvNsG9OdWEgAg&bvm=bv.47380653,d.ZWU&psig=AFQjCNGpilj6zhj7zGR3rlGEXtaeryBWTA&ust=1370529151957057


Conclusion

ACQUITY UPC2 Technology, using compressed carbon dioxide (CO2) as the primary mobile phase, is a separation tool that solves both routine and complex chromatographic problems, especially for samples possessing a wide range of polarities

UPC2 offers scientists unique workflow, application, and environmental impact benefits compared to LC and GC platforms

Because UPC2 is built utilizing UPLC Technology, customers are assured of its optimized performance through holistic design of instrumentation, detectors, software data systems and chemistries.

UPC2 provides an exceptional increase in available selectivity, making this technology widely applicable to a diverse range of compound types – 80-85% overlap of compounds that can be analyzed by CC and RPLC – Any compound soluble in an organic solvent


Thank You for Your Attention !

Solving Complex Chromatographic Challenges John Van …Solving Complex Chromatographic Challenges ....

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