SupelMIP™ - Highly Selective SPE for Trace Analysis from Complex Matrices

Advances in Sample Preparation Technology

• Protein precipitation• Liquid-liquid extraction• Non-selective resin SPE (hydrophobic only)• Supported liquid-liquid extraction• C18-C2 silica-based SPE• Mixed-mode SPE - silica based sorbents• Mixed-mode SPE – resin-based sorbents • SupelMIPs

Non-Selective

Highly Selective

Dirty Extracts

Clean Extracts

Sample Preparation – OverviewRelative Selectivity of Various Sample Preparation Techniques

What are MIP‘s? What is SupelMIP?(Molecularly Imprinted Polymers)?

What are MIPs?What are Molecularly Imprinted Polymers?

• Polymer-based sorbents• Pre-determined selectivity for a particular

analyte or group of analytes• Selective target recognition - Mimics of

antibodies or receptors

Functional Monomers

Template

Cross-linking Monomers

Polymerisable Groups

Solvent (Porogen)Initiator

Molecular Imprinting –The Ingredients

Molecular Imprinting → MIP

MIP Binding

Features

• High selectivity• Stable at high temperatures• Stable in organic solvents and at extreme pH• Possibilities of large scale applications • Amenable to fast production

The MIP Binding Site

The Advantage of Selectivity

• Non-selective materials - widespread general use

• Selective materials are needed for extraction of trace-level compounds from complex samples

Hydrophobic ionic/polar Immunoaffinity MIP’s

Analyte

1.1. Column conditioningColumn conditioning2.2. Sample LoadingSample Loading

3.3. Elution of interfering Elution of interfering

compoundscompounds

4.4. Elution of analyteElution of analyte

SupelMIP SPE-Methodology

SupelMIP SPE- Methodology

1. SupelMIP methodology differs from conventional SPE methodology:

- Protocols for reversed phase, ionic-exchange etc can NOT be used- Only use the supplied protocols

2. Typically the loading from aqueous samples is non-selective via hydrophobic interactions

3. Analytes are then retained selectively in the ’analyte specific cavities’ of the SupelMIP

- H-bonding/ionic - Van der Waals interactions- π – π interactions

4. Final Selectivity is introduced during the interference wash step with organic solvents

!

SupelMIP SPE- MethodologyAqueous Samples

1. Sample pre-treatment- Depending on the sample matrix: dilution, centrifugation,

filtration, enzymatic hydrolysis

2. Condition / Equilibration- Organic solvent followed by an aqueous buffer

3. Sample load- Aqueous sample loaded (volume dependent on amount

of SupelMIP phase in SPE column)

- Initial retention non-selective (hydrophobic interactions)

SupelMIP SPE- MethodologyAqueous Samples, cont.

4. Wash- SupelMIPs generate stronger interactions between

sorbent and analyte than conventional SPE sorbents- Harsher washing conditions can be used- Switch to an organic solvent after drying step- Selective interaction with the analyte (H-bonding, ionic,

van der Waals)- Solvent / modifier selection must maximise removal of

interferences and support analyte binding to the SupelMIP without losses

SupelMIP SPE- Methodology Wash Steps = Interference Elution

Aqueous Samples, cont.

• Wash solutions:- Water based solvent

- Non-polar organic solvent (e.g. toluene, heptane)

- Weak polar organic solvent (e.g. DCM)

- Polar organic solvent (acetonitrile, methanol with or without addition of acid to increase the break of H-bonds)

• Selective removal of:- Salts, sugars, and hydrophilic

matrix components - Hydrophobic interferences

- Polar and ionic bonded interferences

- Hydrogen bonded interferences

Wash steps break only one interaction at a time!

SupelMIP SPE- MethodologyAqueous Samples, cont.

5. Elution- Solvent has to overcome hydrophobic and/or ion

exchange interactions- Organic solvents - break hydrophobic interactions- Modifiers e.g. acid or base, break ionic interactions

6. Eluat post-treatment- Often necessary to evaporate and reconstitute the eluate

in mobile phase prior to LC analysis- Further improvement of detection limit

3 ml or 10 ml LRC CartridgesBed sizes 25mg or 50mg

Standard SPE-Formats

3mL10mL LRC

Analytical SupelMIP Applications

• Clenbuterol• Beta-Agonists• Beta-Blockers • Beta-Receptors (combined Beta-Agonists snd Beta-Blockers)

• Tobacco metabolites / Cancerogenes / Biomarkers- NNAL ( 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol)- TSNA‘s (NNN, NNK, NAB, NAT)

• Chloramphenicol• Triazine• Riboflavin • Amphetamines (NEW!)

Summary

NNK = 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanoneNNN = N-nitrosonornicotineNAB = N’-nitrosoanabasineNAT = N’-nitrosoanatabine

Overview on features

• Cleaner extracts with better recovery• Less Ion Suppression in LC/MS• Quicker results with almost no method development.

SupelMIP ClenbuterolSuperior Clean-up Compared to Conventional SPE

Clenbuterol

HPLC-Chromatogram (UV)

ClenbuterolBeta Agonist

Cl

H2N

Cl

HN

OH

0 2 4Time (min)

020

0

0 2 4Time (min)

020

0

Blank Urine

Clenbuterol spiked urine

(0.1ng/mL)

SupelMIP SPE Extracts

0 2 4Time (min)

020

0

0 2 4Time (min)

020

0

Blank Urine

Clenbuterol spiked urine

(0.1ng/mL)

Polymer SPE Extracts

Clenbuterol SupelMIPCleaner Extracts than Conventional SPE

HPLC/MS-ChromatogramsMS/MS, MRM Transitions (277.2/203.1 and 277.3/168.2 m/z)

0 2 4Time (min)

020

0

Clenbuterol spiked urine (0.1ng/mL)

SupelMIP SPE Extracts

0 2 4Time (min)

020

0

Clenbuterol spiked urine (0.1ng/mL)

Polymer SPE Extracts

% Recovery from UrineSpike Level

(ng/mL)

SupelMIP SPE -

Clenbuterol

Hydrophilic Polymer

SPE0.1 99% 8%0.5 75% 66%1.0 75% 69%

Clenbuterol SupelMIPHPLC/MS-Chromatogram

• Clean extracts with low background

• Higher selectivity • Lower limits of quantitation

Shimelis, O., Aurand, C., and Trinh, A., Reporter 25.2, Supelco

• Cleaner extracts & Quicker Application

Hydrophilic polymer SPE methodCAP spiked milk sample

SupelMIPCAP spiked milk sample

SupelMIPBlank milk sample

No interfering responses

Improved S/N of SupelMIP

Results from Shimelis, O., Trinh, A., Brandes, H, The Reporter 2006, 25.1* Oasis HLB SPE cartridge (500 mg)

Chloramphenicol SupelMIP ⇔ Polymer-SPE (hydrophilic)

Ionchromatograms ESI(-), 320-323 m/z Range

Results from Shimelis, O., Trinh, A., Brandes, H, The Reporter 2006, 25.1* Oasis HLB SPE cartridge (500 mg)

Hydrophilic polymer SPE* methodCAP spiked milk sample

SupelMIP CAP spiked milk sample

Chloramphenicol SupelMIP ⇔ Polymer-SPE (hydrophilic)

MS Spektra (3.65 – 4.00 Minutes)

Improved S/N of SupelMIP

Significantly cleaner MS

Chloramphenicol SupelMIP ⇔ Polymer-SPE (hydrophilic)

Simpler & Quicker Procedure!<2h ~6.5h

Chloramphenicol SupelMIP ⇔ Polymer-SPE (hydrophilic)

• SupelMIP1. Centrifugation2. SPE clean-up

• < 2 h• 75 % Reduction of

• Costs• Analysis time

• Polymer SPE1. Protein precipitation2. Vortex and heating3. Centrifugation4. Filtration5. SPE clean-up6. 3 times LLE step

• 6,5 h

Introduction of Beta-Blockers Class-selective MIP – Perfect for Screening

NH2

O

O

H3C

N N

S

N

O

Betaxolol

Metoprolol

Timolol

Atenolol

Sotalol

RCompound

HNS

O O

H3C NH

OH

CH3

CH3

O NH

CH3

CH3

ROH

Common Structure:

Pindolol

Propranolol

Carazolol

RCompound

HN

NH

Not limited to these beta-blockers

Atenolol

Atenolol

* Data courtesy of Prof. Damia Barcelo, Department of Environmental Chemistry, IIQAB-CSIC, Barcelona, Spain

y = 4E+07xR2 = 0.9986

y = 4E+07xR2 = 0.9948

0500000

100000015000002000000

250000030000003500000

40000004500000

0 0.02 0.04 0.06 0.08 0.1 0.12

Conc patró (ppm)

Àre

a pa

tró

Calibrationcurve in solvent

Calibration curve in spiked influent extracts

y = 3E+07xR2 = 0.998

y = 2E+07xR2 = 0.9974

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

0 0.02 0.04 0.06 0.08 0.1 0.12

Conc patró (ppm)

Àre

a pa

tró

Calibrationcurve in solvent

Calibration curve in spiked influent extracts

Competition Hydrophilic Polymer

Ion Suppression = Need for internal standard

Calibration curve in solvent compared with spiked influent extractsOverlap = NO Ion Suppression

SupelMIP Beta Blocker

Beta Blocker SupelMIPSupelMIP™™MinimizingMinimizing Ion Ion SuppressionSuppression

N

N NON

ONNO

N

NNO N

NNO

NNK NNN NAB NAT

NornicotineNicotine AnatabineAnabasine

N

NHN

N NHN

NHN

NNK NNN NAB NAT

Introduction of TSNA’sTobacco Origin and Specific Nitrosamines

NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanoneNNN: N‘-nitrosonornicotineNAB: N‘-nitrosoanabasineNAT: N‘-nitrosoanatabine

Introduction of TSNA’s

• Formed from tobacco alkaloids during curing and processing - Found only in tobacco products- Do not occur in any other product

• Highly carcinogenic - NNN and NNK (Group 2B, IARC*)- NAT and NAB (Group 3, IARC*)

*IARC International Agency for Research on Cancer

•Health monitoring -Monitoring of exposure to tobacco smoke

-TSNA’s are analysed in urine

•Quality assurance and product development -Analysis of TSNA’s in Tobacco products

•Chewing tobacco, smoking tobacco, and snuff, etc.

•Trace level determination needed (Low picogram range)

0

200

400

600

800

1000

1200

0 1 2 3 4 5 6 7 8

Time (min)

Inte

nsity

(cps

)

NNKNNNNABNAT

25 pg/ml spiked TSNA mix spiked into urine • Conventional Method1

- Recovery of NNN 79 %- 36 mL urine needed

• SupelMIP- Low detection limits

• 4 pg/ml LOD

- Recoveries are above 90 %- 1 to 5 mL needed

1. Stepanov I and Hecht, SS, Cancer Epidemiol Biomarkers Prev 2005; 14(4), 885-891

TSNA’s SupelMIP SPE Extraction of TSNA’s from Urine

Column: Ascentis C18, 3 µm, 50 x 3.0 mmFlow: 0.5 ml/minInj. Vol: 5 µlTemperature: 25 ºCDetection: MSMobile phase A: 10mM Ammonium Formate pH 6.1Mobile Phase B: Acetonitrile

Gradient:Time(min.) A % B%0.00 90% 10%1.00 90% 10%4.00 60% 40%5.00 30% 70%6.00 30% 70%6.10 90% 10%9.00 90% 10%

TSNA’s SupelMIP SPE: Extraction from Human Urine

Fast and Cost Efficient Method

• Conventional method1- Liquid-Liquid extraction, thrice methylene chloride. - The tubes were shaken gently on a bench top shaker for 10 min, - centrifugation. - The extracts were combined into a fresh 50-mL glass centrifuge

tube and 5 g sodium sulfate were added. - The mixture was briefly shaken- Incubate 0.5 hour, - Evaporate to dryness in portions on a Speedvac concentrator- The residue was redissolved in water, and the pH was adjusted to

2 to 3 with 300 AL of 1 N HCl. - Liquid-Liquid extraction- thrice with methylene chloride; - The aqueous portion was adjusted to pH 7 with 1 mL of potassium

phosphate buffer - Load on a SPE cartridges (10-mL ChemElut cartridge).- Eluted with 10 mL methylene chloride into a clean 50-mL glass

centrifuge tube. - The combined eluants were concentrated to dryness (Speedvac

concentrator). - Residues were dissolved in 0.5 mL of methylene chloride - Column conditioning with methylene chloride.- Load on SPE cartridges (Sep-Pak Plus Silica), - The cartridges were washed with 5 mL methylene chloride/ethyl

acetate (50:50)- NNN was eluted with 10 mL of ethyl acetate. - The ethyl acetate eluants were concentrated to dryness

(Speedvac). - The dry residues were transferred into gas chromatograph

microvials.

• TSNA’s SupelMIP method- Column conditioning using 1 mL of MeOH and 1 mL of

deionized water- Apply the sample on the SupelMIP-TSNA, 1 to 3 mL urine

sample- Elute the interferences with: 1 mL 10 mM NH4Ac pH 5.5 - 10 minutes of vacuum (~ -0.7 bar / 525 mm Hg) to dry the

column. - 1 ml Heptane- 5 minutes of vacuum - Analyte elution 2x 1 ml 10% MeOH in Dichloromethane- Evaporate eluted samples- Reconstitute in mobile phase before analysis

• Current method is elaborate and timeconsuming

• Using TSNA’s SupelMIP the total samplehandling time is reduced because just a single extraction step is needed

• Total sample handling time less than2 hours

Summary SupelMIP™ SPE Highly Selective SPE for Trace Analysis from Complex Matrices

www.miptechnologies.com www.sigma-aldrich.com/supelmip

Feature Advantage Benefit

Selective extraction of analytes from complex matrices

Cleaner extracts Lower detection limits

SupelMIP’s generate stronger interaction between the sorbent and the analyte

Simplified washingprotocols with less extraction steps

Significant time and cost savings

Interfering substances can be washed away using harsher washing conditions

Minimized matrixeffects, eliminated ion suppression

Improved MS compatibility

Applications

Information on SupelMIPs

• SupelMIP Brochure (JOZ)- www.sigma-aldrich.com/supelmip- www.miptechnologies.com

Available SupelMIPs• Clenbuterol• Beta-agonists (class selective)• Beta-blocker (class selective)• Full Beta Receptor Ligands • Chloramphenicol• NNAL • TSNAs (Tobacco specific Nitrosamines)• Riboflavin (Vitamin B2)• Triazines (class selective)• Amphetamines (NEW!)