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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 [email protected] - 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