85.00 90.00 95.00 100.00 105.00 110.00 115.00 0 5 10 15 20 25 30 35 40 45 50 55 Normaliz ed δ Reeks1 Reeks2 Reeks3 Reeks4 Ethiocholanolone DHEA AED α‐Estradiol
Background
The use of hormones as growth promoters for fattening purposes in cattle has been banned in the European Union since the early eighties. Control of the illegal use of natural steroid hormones in cattle is still a challenge since no conclusive method and non-ambiguous analytical criteria are available. The ability of gas chromatography / combustion / isotope ratio mass spectrometry (GC-c-IRMS) to confirm the
administration of estradiol and testosterone to cows has been investigated. This was done by comparison of the 13C/12C isotopic ratio of the main
urinary metabolites, i.e. 17α-estradiol and 17α-testosterone, with endogenous reference compounds (ERCs) to differentiate the endogenous or
exogenous origin. For this purpose sample clean-up methods were developed based on humane urine analysis protocols1 which were used to
determine whether an athlete has been abusing an exogenous natural hormone. Preliminary results obtained with the developed clean-up method (LC-fractionation using a UPLC column resulting in small (<0.3 min) selective fractions ) and GC-c-IRMS analysis are presented.
Conclusions
To determine the stability of the delta (δ13C) value of the GC-c-IRMS
system, standards of different concentration (1-50 ng absolute)
containing different steroids were analysed. Stable delta values were obtained > 10 ng steroids absolute injection.
GC-c-IRMS for analysis of natural hormone
abuse in urines of cattle
Paul Zoontjes, Hennie van Rossum, Marco Blokland, Leen van Ginkel, Saskia Sterk
The figure below shows GC-c-IRMS chromatograms of a standard, urine of a non-treated animal, a non-treated-spiked urine, an urine of an estradiol treated animal and a suspected sample of urine
(lower trace is 13C, upper trace isotopic swing).
Samples of urine from a bovine population with and without
treatment of testosterone or estradiol were analysed and their
corresponding ∆δ13C values were compared. A difference of more
than 3 ∆δ13C value is considered proof of abuse2. See figure below
for the average ∆δ13C values of these measurements.
Concentration (ng steroid absolute) δ measur ed/ av er ag e δ 20 ‐50 ng
• Delta values start deviating below 10 ng absolute steroids
• Sensitivity can possibly be improved by use of large volume injection • High-resolution UPLC-fractionation gives clean extracts
• First experiments demonstrate that applied method gives a
pronounced discrimination between exogenous and endogenous natural steroids
Acknowledgment: This project was financially supported by the Dutch Ministry of Economic Affairs and the EC - DG SANCO
References
1) Steroids. 2012;77(11):1050-60
2) J Agric Food Chem. 2013;61(30):7242-9
Δ δ 13 C( ‰ )= δ 13 C ER C(a ‐T) ‐δ 13 C TC (a ‐E2) -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4
Reference Female Estradiol treatment
Estradiol treatment Compliant Non‐compliant -6 -5 -4 -3 -2 -1 0 1 2 3
Reference Male Testosterone treatment Testosterone treatment Δ δ 13 C( ‰ )= δ 13 C ER C(a ‐E2) ‐δ 13 C TC (a ‐T) Compliant Non‐compliant RIKILT Wageningen UR
P.O. Box 230, 6700 AE Wageningen Contact: paul.zoontjes@wur.nl
T + 31 (0)317 48 04 37
Scan for PDF
St. mixture ‐ Injection of 4 ng steroid‐acetates (absolute) (internal standards : 20 ng)
DHEA α‐Testosterone α‐estradiol
β‐testosterone‐d3 β‐estradiol‐d3 α‐ Es tr adiol Δ 35.03 B ‐Es tr adiol – d3 Δ 35.91 UPLC standard α‐E2+ β‐E2d3 B ‐Es tr adiol Δ 33.78 Urine 2007M3902 animal non‐treated α‐ Es tr adiol Δ 32.68 B ‐Es tr adiol Δ 32.97 Urine 2007M3903 animal non‐treated spiked α‐ Es tr adiol Δ 33.85 B ‐Es tr adiol Δ 34.86 Urine 2007M3875 treated animal with Estradiol Urine 2009M1486 animal Survey program α‐ Es tr adiol Δ 23.34 B ‐Es tr adiol Δ 33.99
