INTRODUCTION

SFC-MIP SPE-MIP

Poster presented at the 2nd International Symposium on HTSP , Brugge, Belgium, 31st January - 3rd February 2012

GC-MS : purity assessment

Material

Application on urinary steroids for IRMS analysis Conclusion

LABoratoire d’Étude des Résidus et Contaminants dans les Aliments (LABERCA) USC INRA 1329, Oniris, LUNAM Université, BP 50707, 44307 Nantes Cedex 3, France

Fax : +33 (0)2 40 68 78 78 - Tél : +33 (0)2 40 68 78 80 laberca@oniris-nantes.fr - www.laberca.org

Mickaël DOUE, Emmanuelle BICHON, Fabrice MONTEAU, Bruno LE BIZEC

High efficiency of semi-preparative Supercritical Fluid Chromatography

with Molecularly Imprinted Polymer as stationary phase (SFC-MIP).

Application on urinary steroids purification for IRMS analysis.

In spite of numerous and significant advances in terms of analytical instrumentation during the past two decades, sample preparation still remain a key and a critical

step that determines the final performances of the developed analytical process. Recently, the use of sorbents like Molecularly Imprinted Polymers (MIPs) has been

considered as a relevant technique of sample purification since such material can present an adjusted selectivity for a specific class of compounds. Nevertheless, as

shown later, MIPs sometimes behave like a reverse-phase sorbent and specific interactions are limited particularly when biological samples are loaded on it. In this context,

the aim of the present study was to assess the capability of SFC-MIP to reach a better selectivity. The optimized sample preparation strategy was then implemented on

urine samples in the scope of confirmation of anabolic steroid abuse in cattle by GC-C-IRMS. IRMS analysis was chosen because of the high degree of purification

needed due to its poor specificity.

SAMPLE PREPARATION

SPE-MIP Sampling Washing Eluting

3 mL H2O/MeCN (65:35, v:v)

5 mL H2O/MeCN (80:20, v:v)

5 mL H2O/MeCN (90:10, v:v)

Washing

Derivatisation Acetylation

Injection in GC-MS for purity assessment

All purified extracts were analyzed by GC-MS and their chromatograms, as well the associated mass spectra and the absence of co-elution with targeted steroids, allowed

us to assess their purity. Regarding the mass spectra obtained, the noise was divided by a factor of ten between SPE-MIP and SFC-MIP methods. Moreover, targeted

steroids present less co-elution with SFC-MIP methods. To conclude, SFC-MIP technique was more efficient than the SPE MIP. Indeed, the presence of water

prevents specific recognition to take place. Because of the higher purification of the fraction F(E) obtained with the gradient MeCN/MeOH, this method was applied to the

selective isolation of urinary steroids in the further scope of IRMS analysis.

5 mL of urine from untreated pregnant cow was spiked with 12 natural steroids (11 androgens (A) and 17α-estradiol (E))

and then deconjugated by β-Glucuronidase E. Coli

Instrument : GC-MS-EI 5973 Agilent

Column: Optima17-MS, 30 m×0.25 mm×0.25 µm

Acquisition : Full Scan (50 – 500 m/z)

Injection : 2 µL in pulsed splitless mode at 250°C

Oven : 60°C (1.5 min), 20°C.min-1 to 220°C, 5°C.min-1 to 270°C

(1 min), 1°C.min-1 to 290 °C, 20°C.min-1 to 320°C (3 min).

Instrument : Investigator SFC system,

Waters

Column : AffiniMIP ® Estrogens, Polyintell

(450 × 2.6 mm i.d., 12-25 µm particle size)

Detector : PDA

Semi-preparative SFC-MIP

SPE C18 / Liquid Liquid Extraction

Gradient MeOH (5 to 30 % in CO2)

F(A): 15 – 24 min F(E): 26 – 32 min

MIP-SPE: AffiniMIP ®

Estrogens, Polyintell,

Particles diameter

range: 25–80 µm.

SPE-MIP GC-MS SFC-MIP

Sample preparation with SFC-MIP MeCN/MeOH method was applied on urine

samples collected from treated cows with 17β-boldenone (B), 17β-testosterone

(T), 17β-estradiol (E2) and androstenedione (AED) (sampling between 1 and 3

days after injection)

17α-estradiol

Experiences / Direct metabolites M

δ13C values of ERC :

DHEA (‰)

δ13C values of M (‰)

Δ(M – ERC) (‰)

Results

T / 17α-testosterone - 26,72 - 31,48 - 4,76 / NC

Es / 17α-estradiol - 23,96 -29,80 - 5,84 / NC

AED / androstanediol -24,97 -33,13 -8,16 / NC

B / 17α-boldenone -22,75 -29,16 -6,41 / NC

MIP-SFC showed a better selectivity compared to MIP-SPE method. Indeed,

removal protic solvent allowed to increase specific recognition to estradiol by

electrostatic interactions. Furthermore, chromatographic separation of estradiol,

mono- and dihydroxylated steroids has been achieved with aprotic solvent. The

purification obtained by the MIP-SFC method on the urine samples from treated cows

was sufficient for IRMS analysis. Thus, with this strategy, differences of δ13C

values between Endogenic Reference Compounds (ERC), boldenone and AED

metabolites from urine samples of treated cows were demonstrated for the first

time. In this study, we proved the high potential of MIP when used as a stationary

phase in SFC as purification technique. To conclude, this sample preparation

technique can be used for small organic compounds purification.

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A

SPE-MIP

Total Ion Chromatogram acquired in scan mode : 2 µL injected of the

eluted fraction from SPE-MIP

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B

SFC-MIP MeOH

Total Ion Chromatogram acquired in scan mode : 2 µL injected of the

collected fraction F(A) from SFC-MIP MeOH method

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SFC-MIP MeCN/MeOH C

Total Ion Chromatogram acquired in scan mode : 2 µL injected of the

collected fraction F(A) from SFC-MIP MeCN/MeOH method

Criteria : |Δ (M – ERC)| > 3 ‰ : Non compliant (NC) |Δ (M – ERC)| < 3 ‰ : Compliant (C)

Chromatogram obtained with 2 µL of the

purified extract F(E) injected in GC-C-IRMS

Gradient MeCN (5 to 60 % in CO2)

F(A): 30 – 37 min F(E): retain on the column

Gradient MeCN/MeOH (95:5) (5 to 40 % in CO2)

F(A): 15 – 23 min F(E): 31 – 40 min

A

E

The use of MeCN in SFC-MIP increased specific recognition. Chromatographic

separation of mono-(AM) and dihydroxylated (AD) androgens has been achieved.

Nevertheless, estradiol was still retained on the column using these conditions.

Addition of 5 % of MeOH in MeCN allowed to elute estradiol while preserving specificity.

AM AD

A

E

Mix steroids Mix steroids Mix steroids