liquid-liquid extraction (lle) - lc/ms on-line · 2016-01-19 · traditional liquid-liquid...
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Liquid-Liquid Extraction
(LLE)
• Analyte(s) polarity
- non polar
- polar
- ionic
• Extraction solvent polarity/selectivity
• Extraction/sample volume size
• Recovery of analyte(s)
• Blow-down step
• Reconstitution solvent (mobile phase)
Copyright by Jack Henion, 2015 Lecture 1, Page 11
Liquid-Liquid Extraction via
96-Well Format
Generic Schematic of Liquid/Liquid Extraction in the 96-Well Format
1. Reconstitution (via Tomtec) 2. Rotate/Vortex
3. Centrifuge 4. Injection
100 mL plasma Tomtec addition of internal
standard and buffer
-Cap
-Rotate/Vortex
-Centrifuge
1. Tomtec addition of liquid extraction solvent
2. Cap, rotate/vortex, centrifuge
Tomtec transfer of
organic layer to new
wells
N2 (gas) blow-down
1.4 mL 96-well plate matrix
S. Steinborner and Jack Henion, “Liquid-Liquid Extraction in the 96-
well Plate Format with SRM LC/MS Quantitative Determination of
Methotrexate and its Major Metabolite in Human Plasma”, Anal.
Chem., 1999, 71, 2340-2345. Copyright by Jack Henion, 2015 Lecture 1, Page 12
ISOLUTE® SLE+: Supported Liquid Extraction Plates
Extraction of Corticosteroids from Plasma
Supported-liquid extraction (SLE) is a 96-well sample preparation technique that is analogous to
traditional liquid-liquid extraction (LLE). The extraction interface occurs between the buffered sample
absorbed onto an inert solid support and a water immiscible solvent (see Figure 1). This provides excellent
extraction efficiency while alleviating many of the liquid handling and emulsion formation issues associated
with traditional LLE.
Copyright by Jack Henion, 2015 Lecture 1, Page 13
Supported Liquid Extraction
(SLE)
Supported-liquid extraction (SLE) is a 96-well sample preparation technique that is analogous to
traditional liquid-liquid extraction (LLE). The extraction interface occurs between the buffered sample
absorbed onto an inert solid support and a water immiscible solvent (see Figure 1). This provides excellent
extraction efficiency while alleviating many of the liquid handling and emulsion formation issues associated
with traditional LLE. Copyright by Jack Henion, 2015 Lecture 1, Page 14
Supported Liquid Extraction (SLE)
Procedure:
• Plate: ISOLUTE SLE+ 200 mg supported-liquid extraction plate (part
number 820-0200-P01)
• Sample: Blank human plasma (100 μL) was spiked with the corticosteroids at 200 ng/mL. The plasma was then diluted 1:1 v/v with water prior to loading. This sample dilution results in approximate loading pH of 8.0.
• Sample Application: The pretreated plasma was loaded onto the plate, a pulse of vacuum applied to initiate flow and the samples left to absorb for 5 minutes.
• Analyte Extraction: Ethyl acetate (1 x 1 mL). Allow solvent to flow for 5 minutes under gravity. Apply vacuum
• (-15 "Hg / -0.5 bar) for 2 minutes to complete elution.
• Post Extraction: The extracts were evaporated to dryness and the analytes reconstituted in 500 μL of 80:20 (v/v) H2O/MeOH prior to analysis.
Copyright by Jack Henion, 2015 Lecture 1, Page 15
Extraction of Corticosteroids from Plasma using
ISOLUTE® SLE+ Supported Liquid Extraction Plates
Copyright by Jack Henion, 2015 Lecture 1, Page 16
Solid-Phase Extraction
(SPE)
• Analyte(s) polarity
- non polar
- polar
- ionic
• SPE stationary phase polarity/selectivity
• Recovery of analyte(s)
• Conventional cartridge vs. 96-well format
• SPE bed size
• Silica vs. polymer based support
• Off-line vs. On-line SPE (PROSPEKT)
• Blow-down and reconstitution of residue
- Recon solvent/mobile phase
Copyright by Jack Henion, 2015 Lecture 1, Page 17
SPE Steps
1. Sample pre-treatment
2. Column solvation (wetting)
3. Column equilibration
4. Sample application
5. Interference elution
6. Analyte elution
Copyright by Jack Henion, 2015 Lecture 1, Page 18
The Solid-Phase Extraction (SPE)
Process
Copyright by Jack Henion, 2015 Lecture 1, Page 19
SPE Issues
• Selective absorption of analyte
• ‘Wash’ sequence
• Adsorptive losses
• Recovery from cartridge material
• Cleanliness of eluate
• Analyte elution
- min of two bed volumes
- elution solvent: weak relative to HPLC mobile phase
- elution rate
• Eluate ‘blow-down/reconstitution
- adsorptive losses
- integrity of analyte
- redissolve in weak HPLC eluent
• Recovery
Copyright by Jack Henion, 2015 Lecture 1, Page 20
Summary:
SPE Positive Features
1. Can be automated
2. Amenable to 96 and 384-well plates
3. Minimize solvent waste
4. Amenable to minimum sample
quantities
Copyright by Jack Henion, 2015 Lecture 1, Page 21
Summary:
SPE Negative Features
1. Extracts not necessarily as clean
as LLE
2. Selectivity may be limited
3. Fines and particulates may be
present in the ‘extract’. This can
cause clogging, column pressure
buildup, etc.
Copyright by Jack Henion, 2015 Lecture 1, Page 22
SPE via 96-Well Format:
Issues
• Loading the plate with sample
• Bed volume
• Stationary phase capacity
• Sample capacity
• Cross-contamination
• Reproducibility
• Accuracy/precision
• Blow-down/reconstitution with solvent
• Recovery
Copyright by Jack Henion, 2015 Lecture 1, Page 23
Advantages of 96-Well
SPE Plates
• Parallel multi-sample preparation
• Readily amenable to automation.
• Reduces required sample and elution
volumes.
• 96-well collection block can be directly
inserted into autoinjector reducing sample
handling.
Copyright by Jack Henion, 2015 Lecture 1, Page 24
Commercial Liquid Handlers
for Processing SPE Plates
Tecan Freedom Evo
Beckman Biomek
Hamilton MicroLab Star Tomtec Formatter
Copyright by Jack Henion, 2015 Lecture 1, Page 25
‘Quechers’
Courtesey of Ron Majors, Agilent Copyright by Jack Henion, 2015 Lecture 1, Page 26
Work Flow for Quechers
Courtesey of Ron Majors, Agilent Copyright by Jack Henion, 2015 Lecture 1, Page 27
Courtesey of Ron Majors, Agilent Copyright by Jack Henion, 2015 Lecture 1, Page 28
Courtesey of Ron Majors, Agilent Copyright by Jack Henion, 2015 Lecture 1, Page 29