micro extraction by packed sorbent · • separation is based on the affinity of the solutes with...
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MEPSMicro Extraction by Packed Sorbent
Jade AntonioSGE Europe Ltd
What is MEPS?
MEPS is a technique which combines sample preparation by solid phase
extraction with syringe based sample injection.
Objectives of Sample Preparation
• Make sure the analytes of interest are in solution
• Remove any interferences found in the sample matrix
• Concentrate the analytes to a level which can be detected
History of Sample Preparation
1941 – First use of Liquid:Liquid extraction
1966 – First paper on HPLC
1974 – First reported use of SPE
1993 – Solid Phase Micro Extraction (SPME)
2007 – MEPS
Impact of Sample Preparation
• An estimated 75% of labor time in an analytical labs are spent preparing and processing samples
• Any reduction in this time will have a big impact on a laboratory costs and increase profit.
Liquid-Liquid Extraction (LLE)
• Liquid-liquid extraction is used to separate compounds based on how easily they dissolve in two different liquids that will not mix.
• These liquids are usually water and an organic solvent.
• The extraction moves the compound from one liquid phase to the other.
Liquid Liquid Extraction
LLE has three main draw backs
• It requires large quantities of solvent
• It is not automated (labor intensive)
• Operator Inconsistency is enhanced
Solid Phase Extraction
• Used to isolate analytes of interest from a wide variety of matrices, including urine, blood, water samples
• Separation is based on the affinity of the solutes with the stationary phase and impurities are washed away
• Analytes that are retained on the stationary phase are then eluted from the cartridge and analysed
SPE advantages over LLE
• Smaller volumes of solvent and sample are required
• Processing times are much faster
• The process allows some steps to be automated but in most cases the entire process could not be automated.
• Requires a transfer step before sample introduction into analytical equipment
How Similar are SPE and MEPS?
• MEPS and SPE function in the same manner and use the same phases
• MEPS and SPE are physically very similar
• Methods created for SPE simply require scaling down of the volumes used for use with MEPS
MEPS Advantages
• MEPS combines the sample preparation of SPE with the sample injection capability of autosamplers
• Uses 10 - 100x less solvent and sample
• Increases through put from 10 to 15 minutes per sample to 1 to 2 minutes
• Ability to concentrate the analytes onto the sorbent material
MEPS Application benefits
• MEPS allows existing SPE methodology and applications to be applied to small sample volumes.
• Provides labs a method for priority handling for critical samples.
• Samples maybe concentrated by cycling multiple aliquots through the sorbent bed
• Ability to automate SPE without requiring expensive dedicated on-line systems
MEPS Product
MEPS Products available
• Volumes– 100µL for CTC, Thermo, HTA, Varian 8400 Autosamplers– 250µL for CTC , Thermo, HTA, Varian 8400 Autosamplers
• Phases– C18 end capped, 45µm, 60Å– C8, 45µm, 60Å– C2, 45µm, 60Å– C8/SCX (cation exchange), 45µm, 60Å– Silica, 45µm, 60Å
MEPS operation
MEPS Animation
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Performance
Performance
Calibration Curves 5-2000 ng/mL
Compound 1 name: BusCoefficient of Determination: 0.996893Calibration curve: 3.55292e-8 * x 2̂ + 0.00177372 * x + 0.00148196Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )Curve type: 2nd Order, Origin: Exclude, Weighting: 1/x, Axis trans: None
0.0 200.0 400.0 600.0 800.0 1000.0 1200.0 1400.0 1600.0 1800.0 2000.0ng0
3.83
Response
Repeated 3x per/day
Assay The coefficients of determination(R2)
Day 1 0.9969Day 2 0.9997Day 3 0.9998
Assay The coefficients of determination(R2)
Day 1 0.9969Day 2 0.9997Day 3 0.9998
Assay The coefficients of determination(R2)
Assay The coefficients of determination(R2)
Day 1 0.9969Day 2 0.9997Day 3 0.9998
Day 1 0.9969Day 2 0.9997Day 3 0.9998
Carry Over
• All classes of compound influence MEPS and conventional SPE chemistry in exactly the same way.
• The small quantity of sorbent used and incorporation of MEPS into automation provides for multiple washes of the bed that are not practical for full scale SPE.
• One paper on this technique states washing 4 times with solvent followed by 4 washes with water reduced carry over to 0.02%
Carry Over and Extraction Efficiency
Comparing MEPS, SPE and SPME
Factor MEPS SPE SPME
Amount sorbent 0.5-2 mg 50-2000 mg thickness 150 mm
Sample prep. time 1-2 min 10-15 min 10-40 min
Usage 100 extractions once 50-70 extractions
Recoveries good good low
Sensitivety good good low
Correlation between MEPS & LLE
0 250 500 750 1000 1250 1500 17500
250
500
750
1000
1250
1500
1750
ng/mL (MEPS)
ng/m
L (P
P)
Good correlation between MEPS & LLE with patient sample,but 1 min vs. 45 min, plus automation with MEPS
Bisulphan concentration
ng/m
L (L
LE)
Market Sectors
• Environmental
– Pesticide – Herbicides – POP’s– Water screening
• Food (liquid), Flavors and Fragrances
– Trace Analysis– POP’s– Essential Oils
Market Sectors
• Pharmaceutical
– Bioanalysis– Drug Metabolism & Pharmacokinetics (DMPK). – High Through-put System
• Important: users in high throughput laboratories will require full support and specialist advice (hand holding) for adoption of MEPS as part of their procedure.
• Clinical
– Urine & Plasma Screening– Drugs of Abuse
Market Sectors
• Forensic– Drugs of abuse (Human and Animal)
• SPE methods have been developed for many drugs of abuse.
– Amphetamine and Barbiturates– Benzodiazepines (Valium®)– Flunitrazepam (Rohypnol®)
– Tetrahydrocannabinol (THC)– Cocaine
Who are users of MEPS?
• Anyone who is currently using any of the following techniques in their lab is a potential customer for MEPS:
– Solid Phase extraction (SPE) – Solid Phase Micro Extraction (SPME) – Liquid-Liquid Extraction (LLE) – Protein Precipitation
Which MEPS Sorbent?
C18 BIN’s are like 5% phenyl GC columns – 70% or more of all analysis are made using a C18 packed bed cartridge. This is because most
samples are water based and the analytes need to be transferred from the water matrix into a
non-polar solvent for analysis.
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Which Sorbent?
• For aqueous based fluid, i.e. water, biological fluids, use a reversed phase ‘BIN’ – C18, C8 or C2
• For non-polar organic solvent based samples, hexane, dichloromethane, chloroform, acetonitrile , use a normal phase ‘BIN’ – Silica
• For aqueous or polar organic eluent samples with basic analytes, i.e. some drugs and amines, use a cation ion-exchange ‘BIN’ – M1 (C8/SCX)
Miniaturising your method
SPE Method MEPS Method
Sorbent C18, (500mg sorbent - 6mL volume) Sorbent C18, (2mg sorbent – 7µL bed
volume)
Conditioning 5mL Methanol, 5mL HPLC grade Water Conditioning 2 x 50µL Methanol, 2 x 50µL
HPLC grade Water
Sample 20mL (4mL/minute) Sample 50uL (10-20uL/sec)
Wash 5mL of HPLC grade water Wash 2 x 50µL HPLC grade water
Elution 1mL Methanol/Water 95:5 (v/v) Elution 25µL Methanol/Water 95:5 (v/v)
Benefits of MEPS
• Extraction and injection combined in one process
• On-Line and fully automated (for LC and GC).
• Reduce the time to prepare and inject samples from hours to minutes
• Sample volumes as small as 3.6µL
• Minimum solvent required is 15µL for elution
• 'BIN' maybe reused 40 to 100 times (or greater)
SGE would like to acknowledge an agreement for the development and commercialisation of MEPS with:
• Mohamed Abdel-Rehim AstraZeneca Södertälje, Sweden
• Lars BlombergDepartment of ChemistryKarlstad UniversityKarlstad, Sweden
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