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A new method for hydrolyzing sulfate and glucuronyl conjugates of steroids.
Abstract: A new method for hydrolyzing steroid conjugates (both sulfates and glucuronides conjugates) that is efficient, effective, and inexpensive is described. This method comprises incubation of the conjugates--after salting-out into ethyl acetate or elution from a C18 cartridge--with anhydrous methanolic hydrogen chloride (methanolysis) for 10 min. It has been successfully applied to our routine radioimmunoassay screening and GC/MS confirmation studies of steroids in prerace and postrace equine urine samples. Comparative GC/MS studies on entire (male horse) urine samples showed that methanolysis gave amounts of free steroids (estrone, estradiols, testosterone, estrenediols, nandrolone, androstanediols) at least as large as those obtained by solvolysis. Similar studies on urine samples from a gelding that had been administered nandrolone phenylpropionate showed that methanolysis gave larger amounts of free steroids (nandrolone, estranediols) than Helix pomatia enzymatic hydrolysis or solvolysis. Also, TLC studies on methanolysis of corticosteroid conjugates such as hydrocortisone 21-sulfate and hydrocortisone 21-phosphate showed that free corticosteroid was released in 5 min.
Publication Date: 1989-11-01 PubMed ID: 2610346DOI: 10.1016/0003-2697(89)90596-4Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Comparative Study
- Journal Article
Summary
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The abstract discusses a new, cost-effective method of hydrolysing sulfate and glucuronyl conjugates of steroids. The aim of this research is to improve the efficiency of testing and confirming the presence of steroids in equine urine samples.
Objective
The objective of the research was to introduce a new method for hydrolysing, which is the act of separating or breaking down, sulfate and glucuronyl conjugates of steroids. These conjugates are substances that have been altered from their original state to make them more soluble, and they are commonly found in the urine of horses.
Method
- The new method involves inducing the conjugates with anhydrous methanolic hydrogen chloride, a process known as methanolysis, for a period of 10 minutes.
- Prior to the methanolysis, the conjugates are salted-out into ethyl acetate or run through a C18 cartridge to remove their soluble elements.
Application
- The researchers have successfully applied this method to routine radioimmunoassay screening and GC/MS confirmation studies of steroids in equine urine samples.
- The methanolysis method is being compared to solvolysis and enzymatic hydrolysis in the study. Solvolysis involves breaking down the conjugates using solvents, while enzymatic hydrolysis uses enzymes for the same purpose.
Results
- Comparative studies performed on horse urine samples show that this method of methanolysis releases at least an equivalent amount of free steroids in comparison to solvolysis.
- Furthermore, methanolysis was found to release a larger amount of free steroids when compared to Helix pomatia enzymatic hydrolysis or solvolysis in the case of a gelding that had been administered nandrolone phenylpropionate.
- Additional TLC (Thin Layer Chromatography) studies showed free corticosteroids could be released from corticosteroid conjugates such as hydrocortisone 21-sulfate and hydrocortisone 21-phosphate in as little as 5 minutes using methanolysis.
Conclusion
The new method of methanolysis hold promise as an efficient and cost-effective option to analyse horse urine for the presence of steroids. More tests are needed to further its application.
Cite This Article
APA
Tang PW, Crone DL.
(1989).
A new method for hydrolyzing sulfate and glucuronyl conjugates of steroids.
Anal Biochem, 182(2), 289-294.
https://doi.org/10.1016/0003-2697(89)90596-4 Publication
Researcher Affiliations
- Racing Laboratory, Royal Hong Kong Jockey Club, Sha Tin.
MeSH Terms
- Animals
- Chromatography, Thin Layer / methods
- Gas Chromatography-Mass Spectrometry
- Helix, Snails
- Horses
- Hydrolysis
- Kinetics
- Male
- Methods
- Nandrolone / pharmacology
- Radioimmunoassay
- Solvents
- Steroids / urine
- Testosterone / analogs & derivatives
- Testosterone / metabolism
Citations
This article has been cited 3 times.- Wang Q, Mesaros C, Blair IA. Ultra-high sensitivity analysis of estrogens for special populations in serum and plasma by liquid chromatography-mass spectrometry: Assay considerations and suggested practices. J Steroid Biochem Mol Biol 2016 Sep;162:70-9.
- Wang Q, Bottalico L, Mesaros C, Blair IA. Analysis of estrogens and androgens in postmenopausal serum and plasma by liquid chromatography-mass spectrometry. Steroids 2015 Jul;99(Pt A):76-83.
- Spink DC, Wu SJ, Spink BC, Hussain MM, Vakharia DD, Pentecost BT, Kaminsky LS. Induction of CYP1A1 and CYP1B1 by benzo(k)fluoranthene and benzo(a)pyrene in T-47D human breast cancer cells: roles of PAH interactions and PAH metabolites. Toxicol Appl Pharmacol 2008 Feb 1;226(3):213-24.
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