Solid phase and immunoaffinity extraction methods in the analysis of 17a-methyl steroids
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Date
2001
Authors
Kooi Ling, Yong
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Journal ISSN
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Abstract
The analysis of anabolic steroids at low concentrations presents a major problem in many
analytcal laboratories. The need for a better detection limit requires improvements in
I
currently used analytical methods. Although sensitive instruments e.g. HRMS are available
for the trace analysis of steroids, improvement in sample preparation has not received
much attention. In the present study, two sample preparation methods i.e. direct hydrolysis
and immunoaffinity extraction (IAE) are evaluated in order to improve analytical
performance. Various SPE cartridges were studied for direct hydrolysis and mixed mode
cartridges (C8/SAX) showed comparable recoveries for clenbuterol, norandrosterone,
17a-methylandrostanediol and 3-hydroxystanozolol to that obtained from indirect
hydrolysis, i.e. 61.6 %, 75.3 %, 88.3% and 90.9% for indirect hydrolysis and 65.1 %,
74.6 %, 92.2 % and 86.1 % for direct hydrolysis respectively. However, when the urine
background becomes dirtier, indirect hydrolysis was more robust.
An immunoaffinity gel was developed with antibodies raised against 17a-MT and
immobilized on to· Sepharose gels. The immunogen was synthesized using mixed
anhydride method. The antisera having the relatively higher titre value among the antisera
obtained were used for subsequent study. The antibodies were raised in rabbits and showed
cross-reactivity with a few structurally similar steroids. Especially those steroids having a
17a-methyl group such as metendienone, mestanolone, 17a-methylandrostandiol and
stanozolol were cross-reacted with 17a-MT (33.0 %, 22.6 %, 14.1 % and 8.3 %
respectively). Those steroids that lack the 17a-methyl group had poor cross-reactivity. The
optimal solvent conditions for washing and elution of 17a.-MT were studied using the ·
immunoaffinity gel. It was found that 30 % and 70 % methanol in water was the best
\Vashing and elution solvents respectively and extraction efficiency of 98.9 % 17a.-~T
could :be achieved. The binding capacity for the gel was detennined as 800 ng 17a.-MT.
Subsequently, this IAE was evaluated using individual urinary samples spiked with I 7aMT,
17a-methylandrostandiol and 3-hydroxystanozolol. The gels showed relatively good
recoveries towards 17a-MT (86.37 %), 17a-methylandrostandiol (62.14 %) as well as 3-
hydroxystanozolol (50.25 %). Full scan mass spectra of the extracts showed relatively
cleaner backgrounds, making them suitable for confinnatory analysis at concentrations as
low as 5 ng/ml.
Description
Keywords
Immunoaffinity extraction , Methyl steroids