Solid phase and immunoaffinity extraction methods in the analysis of 17a-methyl steroids
| dc.contributor.author | Kooi Ling, Yong | |
| dc.date.accessioned | 2015-07-30T03:38:24Z | |
| dc.date.available | 2015-07-30T03:38:24Z | |
| dc.date.issued | 2001 | |
| dc.description.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. | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/964 | |
| dc.language.iso | en | en_US |
| dc.subject | Immunoaffinity extraction | en_US |
| dc.subject | Methyl steroids | en_US |
| dc.title | Solid phase and immunoaffinity extraction methods in the analysis of 17a-methyl steroids | en_US |
| dc.type | Thesis | en_US |
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