Solvent-assisted SBSE by using swollen PDMS for enhanced recovery of polar solutes in aqueous samples

Nobuo Ochiai, Kikuo Sasamoto, GERSTEL K.K.

Frank David, Pat Sandra, RIC

Introduction

Solvent assisted stir bar sorptive extraction (SA-SBSE)

· Comparison of extraction efficiency betweenconventional SBSE and SA-SBSE

Application

· Analysis of aroma compounds in roasted green tea and beer

· Ultra trace analysis of polyfunctional thiols in wine

Conclusion

Outline

E. Baltussen et al, J. Microcol. Sep., 11 (1999) 737-747.

PDMS

Glass

JacketMagnet

The extraction mechanism of SBSE is based

on Sorptive Extraction, whereby the solutes

are extracted into a polymer coating (PDMS)

on a magnetic stirring rod.

Stir Bar Sorptive Extraction (SBSE)

Kow1

Kow

m

m

0

SBSE

SBSE recovery can be estimated if the octanol

– water partitioning coefficient (Kow) of

the analyte is known, and the recovery

can be expressed using this equation.

Theoretical

Recovery (%)

0

20

40

120

logKow

Rec

ove

ry (

%)

1.00 2.00 3.00 4.000

60

80

100

PDMS: 63 µL

Sample: 5 mL

β: 79

log Kow > 3.0

Kow1

Kow

m

m

0

SBSE

E. Baltussen et al, J. Microcol. Sep., 11 (1999) 737-747.F. David, P. Sandra, J. Chromatogr. A, 1152 (2007) 54-69.

Theoretical recovery as a function of log Kow

Theoretical

Recovery (%)

0

20

40

120

logKow

Rec

ove

ry (

%)

1.00 2.00 3.00 4.000

60

80

100

PDMS: 63 µL

Sample: 5 mL

β: 79

log Kow > 3.0

Kow1

Kow

m

m

0

SBSE

log Kow < 2.0

E. Baltussen et al, J. Microcol. Sep., 11 (1999) 737-747.F. David, P. Sandra, J. Chromatogr. A, 1152 (2007) 54-69.

Theoretical recovery as a function of log Kow

DifferentPhase?

Although the commercial stir bars coated with polyethylene-modified silicone (EG Silicone) and polyacrylate are available, most applications have been still performed with PDMS because of its excellent reproducibility as well as robustness.

Commercial stir bar:

Several types of extraction phase with different polarities have been proposed. However, those phases are mostly only compatible with liquid desorption (LD) and/or have inferior performance characteristics related to robustness, bleedings, stability, etc. compared to PDMS.

Attempt:

J. Manuel and F. Nogueira, Trends Anal. Chem. 71 (2015) 214-223.

These research works highlight the challenges in developing new phases for SBSE in combination with thermal desorption (TD).

New Phases for coating

An alternative LPME approach using a silica monolith as extraction phase holder instead of hollow fiber was developed. This LPME method was termed solvent-bar microextraction using a silica monolith (SBME/SM).

Li Xu, Hian Kee Lee, J. Chromatogr. A, 1216 (2009) 5483-5488.

A similar approach using a monolithic stir bar with immobilized solvent was also demonstrated.

Bernd Spangenberg et al, J. Planar Chromatogr. 28 (2015) 472-425.

LPME using alternative phase holders

An interesting headspace sorptive extraction (HSSE) technique was introduced by Prof. Bicchi’s group. The authors developed solvent-enhanced (SE) HSSE using a PDMS tubing device and solvent inside.

B. Sgorbini et al, J. Sep. Sci. 2010, 33, 2191-2199.

“Solvent-enhanced (SE)” HSSE

SBME/SM

SE-HSSE

Conventional PDMSStir bar (conditioned)

Solvent swollen PDMS Stir bar

SBSE using a solvent swollen PDMS stir bar

SA-SBSE

Solvent-assisted SBSE (SA-SBSE) using a solvent swollen PDMS stir bar

Solvent-assisted SBSE (SA-SBSE) using a solvent swollen PDMS stir bar

Introduction

Solvent assisted stir bar sorptive extraction (SA-SBSE)

· Comparison of extraction efficiency betweenconventional SBSE and SA-SBSE

Application

· Analysis of aroma compounds in roasted green tea and beer

· Ultra trace analysis of polyfunctional thiols in wine

Conclusion

Outline

SBSE Rinsing in water

Remove water

TD-GC-MS

LD-LVI-GC-MS

Soaking in solventRe-conditioning

(TE, TC, TDU, TDS)

Solvent swelling

GERSTEL TE

SA-SBSE Workflow

▪ The most popular Twister with 24 μL PDMS (1 cm x 0.5 mm) gives very poor results in SA-SBSE because of less solvent volume in the swollen PDMS.

▪ The dedicated Twister with 63 μL PDMS (1 cm x 1 mm) namely FLEX-Twister is highly recommended for better sensitivity and reproducibility of polar solutes.

FLEX-Twister has a narrower tolerance width of PDMS volume for more uniform solvent volume in swollen PDMS.

FLEX-Twister for SA-SBSE

FLEX-Twister(63 μL PDMS)

1. Using a syringe, known amount of solvent (typically 100-150 μL) is added into the 2 mL-vial containing the FLEX-Twister.

2. The sealed vial is laid down and left for > 30 min.

3. The solvent swollen FLEX-Twister can be left at room temperature (typically for a week)

① ② ③

Solvent swelling of FLEX-Twister in a 2 mL-vial

Solvent1.Ethyl acetate (log Kow: 0.86)2.Diethyl ether (log Kow: 1.05)3.MIBK (log Kow: 1.15)4.Dichloromethane (log Kow: 1.34)5.Diisopropyl ether (log Kow: 1.86)6.Toluene (log Kow: 2.54)7.Cyclohexane (log Kow: 3.18)8.Hexane (log Kow: 3.90)

Stir bar: 63 μL PDMSSample: 5 mL water + 30 % NaClStirring time: 60 minStirring rate: 800 rpm

Spiked water Test solutes (5.0 ng/mL each)

1. 2-Acetyl pyrrole (log Kow: 0.56)2. Benzyl alcohol (log Kow: 1.08)3. Guaiacol (log Kow: 1.34)4. Indole (log Kow: 2.05)5. Phenethyl acetate (log Kow: 2.57)

SA-SBSE recovery for 5 test solutes in water

21 %SBSE

SA-SBSE

Recovery (%)

Comparison of recovery between SA-SBSE and SBSE

21 %SBSE

SA-SBSE

Phase volume: PDMS (63 μL) + Solvent volume (12-83 μL)

Phase polarity: PDMS (≈ octanol) + Solvent (log Kow: 0.86-3.90)

Recovery (%)

62 %55 %

52 %50 %

21 %

25 %21 %

22 %“log Kow”

of solvent1.16-2.54

Comparison of recovery between SA-SBSE and SBSE

Theoretical

Recovery (%)

0

20

40

120

logKow

Rec

ove

ry (

%)

1.00 2.000

60

80

100

Benzyl alcohollog Kow: 1.08

Rec: 11 %

Guaiacollog Kow: 1.34

Rec: 21 %

Indolelog Kow: 2.05

Rec: 51 %

Phenethyl acetatelog Kow: 2.57

Rec: 87 %

2-Acetyl pyrrolelog Kow: 0.56

Rec: 6.8 %

Actual recoveries for test solutes in SBSE

Theoretical

Recovery (%)

0

20

40

120

logKow

Rec

ove

ry (

%)

1.00 2.000

60

80

100

Comparison of recovery between SA-SBSE and SBSE

Theoretical

Recovery (%)

0

20

40

120

logKow

Rec

ove

ry (

%)

1.00 2.000

60

80

100

Comparison of recovery between SA-SBSE and SBSE

Theoretical

Recovery (%)

0

20

40

120

logKow

Rec

ove

ry (

%)

1.00 2.000

60

80

100

Comparison of recovery between SA-SBSE and SBSE

Theoretical

Recovery (%)

0

20

40

120

logKow

Rec

ove

ry (

%)

1.00 2.000

60

80

100

Comparison of recovery between SA-SBSE and SBSE

These data basically demonstrate that the increased phase volume (decreased phase ratio) cannot fully explain the enhanced recovery obtained from SA-SBSE using DIPE and DCM. Also, polarity modification of PDMS with these solvents is very important.

Important concepts of SA-SBSE for polar solutes

Addition of solvent increases the phase volume.The type of solvent changes the

polarity and appearance of the phase (log Kow: 1.0-2.0).

Making the phase much more attractive to the analytes

Introduction

Solvent assisted stir bar sorptive extraction (SA-SBSE)

· Comparison of extraction efficiency betweenconventional SBSE and SA-SBSE

Application

· Analysis of aroma compounds in roasted green tea and beer

· Ultra trace analysis of polyfunctional thiols in wine

Conclusion

Outline

GERSTEL TDU/CIS4 w/CCD2

TDU: 30 ºC (0.3 min) - 10 ºC/min - 60 ºC (5 min)

- 35 ºC/min - 250 ºC (5 min)CIS: -40 ºC (0.5 min) - 720 ºC/min - 240 ºC (hold)

(Tenax TA packed CIS liner)

Injection mode: Low split 1/3

Roasted green tea: 5 mL (30 % NaCl)

FLEX-Twister: 63 μL PDMS

SBSE & SA-SBSE (DCM, DEE, DCM/DEE)

for 60 min@800 rpm

Agilent 7890A GCDB-WAX UI (20 m x 0.18 mm i.d. x 0.30 μm thickness)

GC Temp: 40 ºC (3 min) - 5 ºC/min - 240 ºC (7 min)

Sample prep.

Sample Introduction

Separation

DetectionMS (Agilent 5975C)

Mass range: m/z 29 – 350

Acquisition speed: 2.8 Hz

CCD2

Analytical conditions of SA-SBSE-TD-GC-MS for roasted green tea

DEE-SA-SBSE

DCM-SA-SBSE

SBSE

1. 1-Ethyl pyrrole, 2. 2-Methyl pyrazine, 3. 2,3-Dimethl pyrazine, 4. Furfural, 5. 2-Acetyl furan, 6. 5-Methyl furfural,

7. Furfuryl alcohol, 8. Guaiacol, 9. Maltol, 10. 2-Acetyl pyrrole, 11. Furaneol, 12. 4-Vinyl guaiacol, 13. Indole,

14. Methoxy eugenol, 15. Raspberry ketone.

12 3

4

5

6

7

9

10

11

12

13 14 158

Comparison of TICs between SA-SBSE and SBSE of roasted green tea

0,00

0,20

0,40

0,60

0,80

1,00

1,20

DCM-DEE-SA-SBSE DCM-SA-SBSE DEE-SA-SBSE SBSE

Relative responses of the selected aroma compounds

log Kow < 1.57

0,00

0,20

0,40

0,60

0,80

1,00

1,20

DCM-DEE-SA-SBSE DCM-SA-SBSE DEE-SA-SBSE SBSE

Relative responses of the selected aroma compounds

0,00

0,20

0,40

0,60

0,80

1,00

1,20

DCM-DEE-SA-SBSE DCM-SA-SBSE DEE-SA-SBSE SBSE

Relative responses of the selected aroma compounds

0,00

0,20

0,40

0,60

0,80

1,00

1,20

DCM-DEE-SA-SBSE DCM-SA-SBSE DEE-SA-SBSE SBSE

Relative responses of the selected aroma compounds

R² = 0,9979

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

0 20 40 60 80 100

Guaiacol

R² = 0,9978

0

500000

1000000

1500000

2000000

0 20 40 60 80 100

Vanillin

* Quantification was performed with 5-fold diluted samples. Then, the determined values were multiplied by 5.

log Kow: 1.34 log Kow: 1.05

log Kow: 0.82

290 ng/mL*(RSD 5.0 %, n=7)

log Kow: -0.19

860 ng/mL*(RSD 5.3 %, n=7)

15 ng/mL*(RSD 2.8 %, n=7)

21 ng/mL*(RSD 7.0 %, n=7)

Quantitation of polar aroma compounds in Roasted green tea by DCM/DEE-SA-SBSE-TD-GC-MS using standard addition calibration

GERSTEL TDU/ATEX/CIS4LVI: 100 μL LVI (ATEX w/micro-vial)

TDU: 30 ºC (0.3 min) - 140 ºC/min - 80 ºC (7 min)

CIS: 20 ºC (0.5 min) - 720 ºC/min - 240 ºC (hold)

Injection mode: Pulsed split 1:3

MS (Agilent 5975C)

Mass range: m/z 29 – 350

Acquisition speed: 2.8 Hz

Japanese beer 5 mL (30 % NaCl)

FLEX-Twister: 63 μL PDMSSBSE & SA-SBSE (w/DCM, and DIPE) for 60 min@800 rpm

LD with 500 μL acetone for 30 min@800 rpm

Agilent 7890A GCDB-WAX UI (20 m x 0.18 mm i.d. x 0.30 μm thickness)

GC Temp: 40 ºC (3 min) - 5 ºC/min - 240 ºC (7 min)

Sample prep.

Sample Introduction

Separation

Detection

Micro syringe

ATEX

Micro vial

CCD2

Analytical conditions of SA-SBSE-LD-LVI-GC-MS for beer

SBSE

DCM-SA-SBSE

DIPE-SA-SBSE

Butanoic acidlog Kow: 1.07

Methionollog Kow: 0.44

Maltollog Kow: - 0.19

4-Vinyl phenollog Kow: 2.41

Comparison between SBSE and SA-SBSE of beer

0,00

0,20

0,40

0,60

0,80

1,00

1,20 SBSE DCM-SA-SBSE DIPE-SA-SBSE

Relative responses of the selected aroma compounds

log Kow < 2.5

0,00

0,20

0,40

0,60

0,80

1,00

1,20 SBSE DCM-SA-SBSE DIPE-SA-SBSE

Relative responses of the selected aroma compounds

0,00

0,20

0,40

0,60

0,80

1,00

1,20 SBSE DCM-SA-SBSE DIPE-SA-SBSE

Relative responses of the selected aroma compounds

C4-C5 fatty acids

Butanoic acidlog Kow: 1.07

RSD: 5.3 %

3-methyl butanoic acidlog Kow: 1.56

RSD: 3.8 %

Hexanoic acidlog Kow: 2.05

RSD: 3.6 %

Octanoic acidlog Kow: 3.03

RSD: 3.2 %

Decanoic acidlog Kow: 4.02

RSD: 3.7 %

Repeatability (n=6) of C4-C10 fatty acids in beer (DIPE-SA-SBSE)

Butanoic acid (log Kow: 1.07)

1.1 μg/mLRSD: 5.3 %

Furaneol (log Kow: 0.82)

150 ng/mLRSD: 6.2 %

Guaiacol (log Kow: 1.34)

1.6 ng/mLRSD: 8.0 %

Indole (log Kow: 2.05)

1.2 ng/mLRSD: 5.4 %

Quantitation of polar aroma compounds in Beer by DIPE-SA-SBSE-LD-LVI-GC-MS using standard addition calibration

Introduction

Solvent assisted stir bar sorptive extraction (SA-SBSE)

· Comparison of extraction efficiency betweenconventional SBSE and SA-SBSE

Application

· Analysis of aroma compounds in roasted green tea and beer

· Ultra trace analysis of polyfunctional thiols in wine

Conclusion

Outline

* T. Kishimoto, A. Wanikawa, K. Kono, K. Shibata,J. Agric. Food Chem. 56 (2008) 1051-1057.

4MMP 3MH 3MHA

Odor threshold: 0.0015 μg/L*

Blackcurrant, Fruity, and catty.

Odor threshold: 0.055 μg/L*

Passion fruit, Grapefruit, and catty.

Odor threshold: 0.005 μg/L*

Grapefruit, Passion fruit, and sweaty.

Polyfunctional thiols in beverages

Polyfunctional thiols, e.g. 4-mercapto-4-methyl pentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH), and 3-mercaptohexyl acetate (3MHA), have received special attention due to their extremely low odor threshold levels at low ppt and high sensory impact with tropical aroma.

Established method for these thiols in beer and wine uses p-Hydroxy mercurybenzoate (pHMB)….

CH3

O

S

CH3

CH3 CH3

O

O+

CH3

O

O SH

CH3

CH3 CH3

O

SPE-GC-MS(SIM)

Ethyl propiolate (ETP) 4MMPMW: 132logKow: 1.07

cis-/trans-4MMP ETP derivative

MW: 230logKow: 1.83

Ethyl propiolate as derivatization agent

der-SBSE-GC-MS/MS

Jan 2015

Dr. Toru KishimotoAsahi Breweries, Limited

4MMP 3MH 3MHA

Beer

Hop

Wort

H3C

O

O

HC

H3C

O

OSR

R-SH

PDMS

up to 20 samples

TD-GC-MS/MS

ng/L

PDMS

PDMS

PDMSder-SBSE

R-S-ETP

PDMSR-S-ETP

R-S-ETPETP

log Kow

4MMP: 1.07

3MH: 1.69

3MHA: 2.70

log Kow

4MMP-ETP: 1.83

3MH-ETP: 2.45

3MHA-ETP: 3.45

ETP Conc.

EtOH Conc.

Salt add.

Ext. time

Buffer Conc.

IdentificationGC-SCD/EI-TOF-MS

GC-SCD/PCI-TOF-MS

GC-SCD/PCI-Q-TOF-MS

QuantificationGC-EI-QQQ-MS(SRM)

GC-PCI-QQQ-MS(SRM)

SBSE with in-situ derivatization (der-SBSE) followed by TD-GC-MS/MS

H3C

O

O

HC

H3C

O

OSR

R-SH

PDMS

up to 20 samples

TD-GC-MS/MS

ng/L

PDMS

PDMS

PDMSder-SBSE

R-S-ETP

PDMSR-S-ETP

R-S-ETPETP

log Kow

4MMP: 1.07

3MH: 1.69

3MHA: 2.70

log Kow

4MMP-ETP: 1.83

3MH-ETP: 2.45

3MHA-ETP: 3.45

ETP Conc.

EtOH Conc.

Salt add.

Ext. time

Buffer Conc.

IdentificationGC-SCD/EI-TOF-MS

GC-SCD/PCI-TOF-MS

GC-SCD/PCI-Q-TOF-MS

QuantificationGC-EI-QQQ-MS(SRM)

GC-PCI-QQQ-MS(SRM)

SBSE with in-situ derivatization (der-SBSE) followed by TD-GC-MS/MS

CH3

O

S

CH3

CH3 CH3

O

O

log Kow: 1.83

4MMP-ETP

Wine

H3C

O

O

HC

H3C

O

OSR

R-SH

PDMS

TD-GC-MS/MS

ng/L

PDMS

PDMS

PDMSder-SA-SBSE

R-S-ETP

PDMSR-S-ETP

R-S-ETPETP

DCM DCM

DCM DCMDCM

DCM

up to 20 samples

log Kow

4MMP: 1.07

3MH: 1.69

3MHA: 2.70

log Kow

4MMP-ETP: 1.83

3MH-ETP: 2.45

3MHA-ETP: 3.45

ETP Conc.

Solvent type

Salt add.

Ext. time

Buffer Conc.

QuantificationGC-EI-QQQ-MS(SRM)

SA-SBSE with in-situ derivatization (der-SA-SBSE) followed by TD-GC-MS/MS

CH3

O

S

CH3

CH3 CH3

O

O

log Kow: 1.83

4MMP-ETP

GERSTEL TDU/CIS4 w/CCD2

TDU: 30 ºC (0.3 min) - 10 ºC/min - 60 ºC (5 min)

- 35 ºC/min - 180 ºC (3 min)CIS: 10 ºC (0.5 min) - 720 ºC/min - 280 ºC (hold)

(Quartz wool packed CIS liner)

Wine: Sauvignon Blanc 5 mL (2-fold diluted)

w/ETP + Tris-HCl (30 % NaCl)

FLEX-Twister®: 63 μL PDMS

SBSE & SA-SBSE (DCM) for 60 min@800 rpm

Sample prep.

Sample Introduction

Separation

Detection

QQQ (Agilent 7000B)

Acquisition mode: SRM

Collision energy: 2.5 V

4MMP-ETP: m/z 230 → m/z 99, m/z 132

3MH-ETP: m/z 232 → m/z 152, m/z 234 → m/z 141, 199

Acquisition speed: 5 Hz

Agilent 7890B GCColumn: DB-Waxetr (15 m x 0.25 mm i.d. x 0.25 μm thickness)

GC Temp.: 100 ºC (3 min) - 10 ºC/min - 250 ºC (1 min) + post run (Back-flush)

Analytical conditions

Retention time (min)

Comparison between der-SA-SBSE and der-SBSE for 4MMP-ETP in non-spiked wine

cis-4MMP-ETP

cis-4MMP-ETP

trans-4MMP-ETP

der-DCM-SA-SBSE

der-SBSE (JCA 2015)

S/N: 51

S/N:13

Improved sensitivity by a factor of 3.9

CH3

O

S

CH3

CH3 CH3

O

O

log Kow: 1.83

Retention time (min)

cis-3MH-ETP

trans-3MH-ETP

cis-3MH-ETP

der-DCM-SA-SBSE

der-SBSE (JCA 2015)

Comparison between der-SA-SBSE and der-SBSE for 3MH-ETP in non-spiked wine

S/N: 62

S/N: 5.5

Improved sensitivity by a factor of 11

log Kow: 2.45

CH3

O

S

O

CH3 OH

cis-3MH-ETPcis-4MMP-ETP

1.0 ng/mL spiked

Non-spike

2.0 ng/mL spiked

3.0 ng/mL spiked

5.0 ng/mL spiked

10 ng/mL spiked

20 pg/mL spiked

Non-spike

40 pg/mL spiked

60 pg/mL spiked

100 pg/mL spiked

150 pg/mL spiked

r2 = 0.9984 r2 = 0.9976

Inte

nsi

ty (x

10

5a.

u.)

Rel

ativ

e In

ten

sity

Relative spiked concentration(ISTD: d2-3MH-ETP 3.0 ng/mL)

Spiked concentration (pg/mL)

11 pg/mL(RSD 3.5 %, n=7)

2.2 ng/mL(RSD 6.7 %, n=7)

Quantification with standard addition calibration

CH3

O

S

CH3

CH3 CH3

O

O CH3

O

S

O

CH3 OH

Introduction

Solvent assisted stir bar sorptive extraction (SA-SBSE)

· Comparison of extraction efficiency betweenconventional SBSE and SA-SBSE

Application

· Analysis of aroma compounds in roasted green tea and beer

· Ultra trace analysis of polyfunctional thiols in wine

Conclusion

Outline

Conclusion

♦A new SBSE method referred to as solvent-assisted (SA)-SBSE has been developed. SA-SBSE using a solvent swollen PDMS stir bar with relatively polar solvents can enhance the extraction efficiency of polar solutes with log Kow < 2.5 in aqueous sample.

♦ SA-SBSE significantly improved extraction efficiencies for aroma compounds with log Kow < 1.5 (e.g. Maltol, Furaneol, Guaiacol, Raspberry ketone, and C4-C5 fatty acids) in roasted green tea and beer by factors of 5-20, compared to those obtained from conventional SBSE.

der-SA-SBSE significantly improved extraction efficiencies for 4MMP-ETP and 3MH-ETP in white wine by factors of 3.9-11, compared to those obtained from conventional der-SBSE.

♦

Acknowledgements

Merci pour votre attention!

Dr. Toru Kishimoto, Asahi Brewery Ltd, JapanDr. Frank David, RIC, BelgiumDr. Pat Sandra, RIC, BelgiumMr. Andreas Hoffmann, GERSTEL GmbH & Co. KG, Germany

Kikuo SasamotoGERSTEL K.K.