Possibilities and Limitations of Recent Developments in ...sbseinternationalmeeting.com/2011/SBSE2011_RIC.pdf · Dynamic MRM of 300 Pesticides 2 Transitions Each LC/MS Pesticide Analysis

Possibilities and Limitations of

Recent Developments

in

Micro Sample Preparation

Techniques

State-of-the-Art

Chromatographic &

Mass Spectrometric

Instrumentation

T

SUPER-CRITICAL

FLUID

P

SOLID LIQUID

GAS

Chromatographic Techniques

- Viscosity- Density- Diffusivity

Throughput

Productivity Resolution

Robustness, Flexibility, Versatility, Sensitivity, …

“21 st Century LC Triangle”

Pesticides LC/MS/MS method

Jet stream conditions

Drying gas temperature 275 ºCDrying gas flow (nitrogen) 6 L/minNebulizer gas pressure (nitrogen) 35 psigCapillary voltage 4000 VSheath gas temperature 325 ºCSheath gas flow 12 L/minNozzle voltage Off

6460A QQQ settings

MS1 and MS2 resolution Unit Time Filtering peak width = 0.03 minDynamic MRM transitions up to 4000Constant cycle time 373 msDelta EMV 400 V

Column Eclipse Plus-C18, 2.1 x 100 mm, 1.8 µmColumn temperature 35 ºCInjection volume 5.0 µL Autosampler temp 6 ºCNeedle wash flushport (MeOH:H2O 75:25), 5 secsMobile phase A = 0.01% formic acid in water

B =5 mM ammonium formate 0.01% formic acid in 95:5 acetonitrile:waterFlow rate 0.3 mL/minGradient 0 min 6% B

15 min 95% BStop time 20 min 6% BPost time 1 min

Page 5September, 2009LC/MS Pesticide Analysis

Dynamic MRM of 300 Pesticides

2 Transitions Each

September, 2009 Page 6LC/MS Pesticide Analysis

Run with 1200-SL LC 2.1 x 100 C18 Eclipse PLUS 1.8 µm

100 Pesticides in 3 min

With FULL SPECTRA because of FAST RUN TIMES


GCxGC – FID


A

B

1

2

4

5

6

3

3

1

4

5

6

2

A

B

1

2

4

5

6

3

3

1

4

5

6

2

GCxGC – ToF-MS


A

B S

S

A

B SS

SS

DAD 1V1 V2

DAD 2

Dim 1 Pump

Dim 1 Column

Dim 2 Pump 2

Dim 2 Pump 1

Dim 2 Column 1

Dim 2 Column 2

Loop 2

Loop 1

Waste

V1 V2

Comprehensive LCxLC

BSA Tryptic Digest – Contour plot 150 mm x

0 50

0.4

0.5

0.6

0.7

0.8

Dim

ensi

on 2

100

725

150 200 250 300

Dimension 1

min

min

1st dimension: 4 x HALO C18, 150 x 2.1 mm , 2.7 µm at 4 5°Cmobile phase at pH 1.8 and 100 µL/min

2nd dimension 2 parallel Zorbax 300 Extend C18, 50 x 4. 6 mm, 3.5 µmmobile phase at pH 10 and 4 mL/min Loop volume: 50 µL

725 x 35 = 25.375

Metabolomics Discovery Pipeline

Biological question

Experimental design

Sampling Samplepreparation

Dataacquisition

Dataanalysis

BiologicalInterpretation

Feature extraction

Mass & ret time

alignment

Normaliz-ation

FilteringStatistical analysis

Data recursion Identification

Metabolomics Discovery Pipeline

Biological question

Experimental design

Sampling Samplepreparation

Dataacquisition

Dataanalysis

BiologicalInterpretation

Feature extraction

Mass & ret time

alignment

Normaliz-ation

FilteringStatistical analysis

Data recursion Identification

?

Blood/plasmaBlood/plasma

350

Mas

s (D

a)

550

750

950

Retention time (min)

0 9010 30 50 70

A

C,D

E,F

G

H

B

103

105

107

In-source f ragments of cholesterol esters

Blood plasma lipidome complexity revealed

• Feature 2D retention time/mass map (+ mode) Time/mass window

Lipid Class

A Lysophospholipids

B Monoacylglycerols

C Phospholipids

D Sphingomyelins

E Ceramides

F Diacylglycerols

G Cholesterol esters

H Triacylglycerols

10 10 µµL blood plasmaL blood plasmaµµLLELLE

Insight into the skin ceramides

• 16 common ceramide classes varying in chain length and fatty acid saturation

Non-hydroxy fatty acid (N)

αααα-hydroxy fatty acid (A)

ωωωω-hydroxy fatty acid (O)

Ester linked ωωωω-hydroxy fatty acid (EO)

Dehydrosphingosine (DS)

Sphingosine (S)

Phytosphingosine (P)

Hydroxy-sphingosine (H)

The analytical variability must be smaller thanthe biological variability …

the key: sampling and sample preparation


• Sampling on a patch coated with an adhesive layer

500

Mas

s (D

a)

700

900

1300

Retention time (min)

30 40 50 60

Ester-linked

103

105

107

1100

Non-ester linked

70


• Feature 2D retention time/mass map (- mode)

~1000 ceramide related features detected in 3 out o f 3 samples

Sampling and

Sample Preparation:

“ the Key “

to Meaningful Analytical Data

Can we ... produce precise and accurate data ... ?

- PAHs in the air

- Phthalates in water, sediment, etc.

- Bisphenol A in baby bottles

- Organotin compounds in environmental

and foods samples

- Pesticides in baby food

- ...

Gaseous Samples

- Static Headspace (SHS)

- In-tube Extraction (ITEX)

- Dynamic Headspace (DHS)

- Purge & Trap (P&T)

- Solid-Phase Microextraction (SPME)

- Headspace Sorptive Extraction (HSSE)

- ...

Solid Samples

- Ultrasonic Extraction (UE)

- Supercritical Fluid Extraction (SFE)

- Accelerated Solvent Extraction (ASE)

- Pressured Liquid Extraction (PLE)

- Microwave Assisted Solvent Extraction (MASE)

- Gas Phase Stripping (GPS)

- Matrix Solid Phase Dispersion (MSPD)

- ...

Comparison …

12

4

15

13

12

11

5

6

78

910

1614

3

10 14 18 22 26 30 34

20

40

60

80

100

Abundance (x 104)

Mixed bed tube

10 14 18 22 26 30 34

20

40

60

80

100

Time, min

Direct liquid injection1

2

4

15

13

12

11

5

67 8

910

1614

3

Spike level 1 ng per PAH – 200 L air through the tube before analysis

Sorptive sampling High Volume sampling

120 mg PDMS – Glass fibre filtre + PUF60 mg Tenax

Spike d-standards

24 h at 100 mL/min 24 h at 0.9 m 3/min

Total: 144 L Total: 1.296 m 3

Thermal desorption Soxhlet Extraction

Spike d-standards

Hvol (+PUF)

Mixed Bed

Ratio

TEF (*) TEQ (*) TEQ (*)

Component ng/m³ ng/m³

Hvol (+PUF)

PDMS

Naphthalene 3.33 116.49 35.0 0.001 0.003 0.116

Acenaphtylene 3.08 6.11 2.0 0.001 0.003 0.006

Acenaphtene 0.31 7.14 23.0 0.001 0.000 0.007

Fluorene 5.38 7.92 1.5 0.001 0.005 0.008

Phenanthrene 11.74 13.85 1.2 0.001 0.012 0.014

Anthracene 0.72 0.92 1.3 0.01 0.007 0.009

Fluoranthene 3.43 4.50 1.3 0.001 0.003 0.005

Pyrene 1.98 2.90 1.5 0.001 0.002 0.003

Benzo(a)anthracene 0.36 1.01 2.8 0.1 0.036 0.101

Chrysene 0.81 1.96 2.4 0.01 0.008 0.020

Benzo(b)fluoranthene 0.57 1.78 3.1 0.1 0.057 0.178

Benzo(k)fluoranthene 0.29 0.52 1.8 0.1 0.029 0.052

Benzo(e)pyrene NM 1.49

Benzo(a)pyrene 0.48 1.11 2.3 1 0.480 1.110

Indeno(1,2,3-c,d)pyrene 0.30 0.43 1.4 0.1 0.030 0.043

Dibenzo(a,h)anthracene 0.65 1.07 1.6 1 0.650 1.070

Benzo(g,h,i)perylene 0.67 1.24 1.9 0.01 0.007 0.012

sum 34.10 170.44 1.333 2.754

Mean values 52 analyses - April 2005/March 2006

!

Liquid Samples

- Micro Liquid-Liquid Extraction (µLLE)

- Solid-Phase Microextraction (SPME)

- Stir Bar Sorptive Extraction (SBSE)

- Single Drop Microextraction (SDME)

- Liquid-Phase microextraction (LPME)

- Membrane Assisted Solvent Extraction (MemASE)

- Solid Phase Extraction (SPE)

- Dispersive Liquid-Liquid Microextraction (DLLME)

- ...

PAHs in water10 ng/L

LLE SPE DLLME SBSE

Sample size (mL) 500 100 5 5

Solvent A3 x 25 mL

DCM 5 mL 8 µL DCM none

Solvent B 1 mL hexane500 µL

acetone noneconcentration (evaporation) yes yes no no

Final volume 1 mL hexane 0,2 mL 8 µL TCE

Final concentration (*) 5 ng/mL 1 ng/200 µL 50 pg/8µL 5 0 pg/stir barInjection volume 2 µL 2 µL 2 µL TD

Amount injected (pg) 10 pg 10 pg 12,5 pg 50 pg

PAHs in Water at the 10 ppt Level (ng/L)

On-line SPE - LC-MS/MS

Load Pump

(SPE)

SPE 1-6WASTE

In-line filter

External

V

Analysis Pump 1

(Elute)

Analytical column

and QQQ

Injection valve

Loop sizes > 1 mL

Inject

Column switching valve

6 port / 2 positions

Load or Elute

Column selector valve

Multi-position

Select SPE cartridge

Inject and Load

On-line SPE - LC-MS/MS

Load Pump

(SPE)

SPE 1-6WASTE

In-line filter

External

V

Analysis Pump 1

(Elute)

Analysis Pump 2

(Add)

Analytical column

and QQQ

Injection valve

Loop sizes > 1 mL

Inject

Column switching valve

6 port / 2 positions

Load or Elute

Column selector valve

Multi-position

Select SPE cartridge

Elute and Detect

Endocrine Disrupting Chemicals

– Problem = low sensitivity even in MS

– 20 µL direct analysis vs 10 mL SPE > x50044x10x10

0.50.5

11

1.51.5

22

2.52.5

33

3.53.5

44

4.54.5

Acquisition Time (min)Acquisition Time (min)Acquisition Time (min)Acquisition Time (min)Acquisition Time (min)Acquisition Time (min)Acquisition Time (min)Acquisition Time (min)1.51.5 22 2.52.5 33 3.53.5 44 4.54.5 55 5.55.5 66 6.56.5 77 7.57.5 88 8.58.5 99 9.59.5 1010 10.510.5 1111

Direct analysisDirect analysis

100 ppb, 20 100 ppb, 20 µµLL

11 22

OnOn--line SPE analysisline SPE analysis

0.2 ppb, 10 mL0.2 ppb, 10 mL

∆∆∆∆∆∆∆∆tR= 4.5 min tR= 4.5 min

= SPE load time= SPE load time

PPCPs

– 23 compounds, 46 transitions (Only Q is shown)

– 1.5 mL SPE

3x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

Acquisition Time (min)Acquisition Time (min)Acquisition Time (min)Acquisition Time (min)3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8

PPCP LOD (ppt)

Diclofenac (Na) 0.25

Promethazin (HCl) <0.20

Propranolol <0.20

Clemastin (fumarate) 0.25

Metoclopramide (HCl) <0.20

Lidocaine (HCl.H2O) <0.20

Bupivacaine (HCl) 0.25

Fluticasone propionate 0.50

Acetominophen 0.50

Carbamazepin <0.20

Penicillin V (K) 0.50

Caffein 0.25

Ranitidine (HCl) 1.00

Sulfamethoxazole 0.25

Sulfachloropyridazine 0.25

Sulfadiazine 0.50

Sulfadimethoxine <0.20

Sulfamerazine <0.20

Sulfamethazine <0.20

Sulfamethizole 0.50

Sulfanilamide 0.25

Sulfathiazole 0.25

Standard 2.5 ppt (ng/L)Standard 2.5 ppt (ng/L)

Multipurpose Samplers

for Automated Sample Preparation/Injection

Solutions … endless

• Static (SHS) and dynamic headspace (DHS)

• Thermal desorption (ALEX and ATEX)

• Solid phase microextraction (SPME)

• Solid phase extraction (SPE)

• Twister thermal and liquid desorption

• Membrane solvent extraction

• Agitate/heat/ultrasonic extraction

• Matrix solid phase dispersion (DPX)

• Derivatization

• Fractionation

• Large volumne injection with evaporation

• LC-GC transfer

• ….

Micro-LLE-LVI-GC-MS/MS10 ng/L Organotins

upper arm

10 µL syringe

lower arm

1 mL syringe

heated tray

(Peltier)

agitator

DHSTrays

(2 mL and 20 mL vials)

SFSTDU

5 x 10 mL

wash station

3 x 100 mL

wash station

Stir Bar Sorptive Extraction (SBSE)

SBSE procedure for vegetables and fruit

Ultra-Turrax + ultrason (15 min)

SBSE: 60 min

1 mL extract

+ 10 mL water

TD-RTL-CGC-MS

15 g sample + 15 mL ACN

P. Sandra, B. Tienpont and F. DavidJ. Chromatogr., 1000 (2003) 299-309

Modified ratio ACN/waterMulti-Twister extractionN. Ochiai, K. Sasamoto, H. Kanda, T. Yamagami, F. David, B. Tienpont and P. SandraJ. Sep. Sci 28 (2005) 1083-1092

(=QuEChERS)

Analysis of Baby Food

(mixed vegetables, rice, chicken).

Detection of piperonylbutoxide

Time (min)

Abundance (*10-1)

5.00 7.00 9.00 11.00 13.00 15.00 17.00 19.00 21.00 23.00 25.00 27.00 29.00

0

400

200

600

800

1000

1200

31.00 33.00 35.00

1

1(00

A O

O

OO

O

Non-spiked

Spiked - 2 ppb

QuEChERS:

d-SPE method for pesticides

Quick Easy Cheap Effective Rugged and Safe

QuEChERS (Catchers)

M. Anastassiades et al, J. AOAC Int., 86 (2003) 412

Cryo-milling + water

Shake with Acetonitrile

Add MgSO4 + NaCl

GC - MS or LC - MS

Centrifugation

(Dispersive SPE)

Centrifugation

Automated Liner Exchange (ALEX) on MPS2

Detailed view of ALEX system1. PTV Injector, 2. ALEX support head,3. Transport adaptor, 4. Electrical gripper,5. Inlet liner tray, 6. Auto-sampler tower

QuEChERS sample prep5-10 µL LVI

PTV solvent vent mode

Automated LinerExchange System (ALEX)

QuEChERS extraction is not very selective –extracts are not clean !!

New: dispersive SPE by DPX

Disposable Pipette Extraction

Classical Luke QuEChERS SBSE

Sample size 15 g 15 g 15 g

Solvent A 30 mL acetone 15 mL acetonitrile 15 mL aceto nitrile

Solvent B 30 mL DCM dilute 1/10 in water

Solvent C 30 mL petroleum ether

extraction LLE LLE PDMS extraction

Clean-up (GPC + SPE optional) d-SPE none

Concentration yes (25 mL to 5 mL) yes (1 mL to 100 µL ) none

Final concentration 62,5 ng/5mL 50 pg/µL 5 ng on stir bar

Injection volume 1,5 µL 1 µL TD

Injected 18,75 pg 50 pg 5 ng

Pesticides in Food (non-fatty matrices) at 10 µg/kg

SBSE procedures for biological samples

Urine 5 (1) mL Blood/bile 1mL, 1g stomach content

Acetylation:

0.5 g K2CO3 + 0.5 mL AA

1mL NH4OAc + 10 µL β-glucuronidase, 90 min @ 37°C

Ethylchloroformate:

2.5 mL EtOH/PYR (2:1) + 0.1 mL ECF

+ 10 mL water

SBSE: 60 min

TD-CGC-MS

+ 1 mL MeOH+ 10 mL water

Insert vialInsert vial

PyroVial

1717

1010Pt filamentPt filament

BodyBody

Bottom part with ring Bottom part with ring electrodeselectrodes

Possibilities for Automated Analysis

BEFORE PYROLYSIS AFTER PYROLYSIS

Exchange of headspace gas

Addition of reagents/catalysts

Addition of IS or marker

Fraction collection in PyroVial

Gas phase recovery * HS injection

Liquid desorption * of pyrolysate

Addition of reagents

Addition of IS

PyroVial – (HT) LC / SFC Set-up

Pyrolysate Carcio-mutagens

500°C

700°C

900°C

Trp-P-13-amino-1,4-dimethyl-5H-pyrido[4,3-b)indole

NH

N

NH2

Multipurpose Samplers

for Automated Sample Preparation/Injection

Possibilities and Limitations of Recent Developments in ...sbseinternationalmeeting.com/2011/SBSE2011_RIC.pdf · Dynamic MRM of 300 Pesticides 2 Transitions Each LC/MS Pesticide Analysis

Documents