Synthesis, structure characterization, and enzyme screening of clenbuterol glucuronides

Alonen, Anna; Gartman, Minna; Aitio, Olli; Finel, Moshe; Yli‐Kauhaluoma, Jari; Kostiainen, Risto

doi:10.1016/j.ejps.2009.05.003

Cited by 17 publications

(11 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The synthesis of the glucuronides was based on a previously described method [32,33]. Briefly, in 0.5 mL of toluene, 100 g (0.36 mol, 1 equivalent) of either 17␣-methyl-5␣-androstan-3␣,17␤-diol or 17␣-methyl-5␤-androstan-3␣,17␤-diol were added followed by 142 g (0.36 mol, 1 equivalent) of acetobromo-␣-d-glucuronic acid methyl ester and 500 g of Ag 2 CO 3 .…”

Section: Methods Validationmentioning

confidence: 99%

Screening for anabolic steroids in sports: Analytical strategy based on the detection of phase I and phase II intact urinary metabolites by liquid chromatography tandem mass spectrometry

Balcells

Pozo

Esquivel

et al. 2015

Journal of Chromatography A

View full text Add to dashboard Cite

In order to improve the detection capabilities of anabolic androgenic steroids (AAS) in sports, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method for the simultaneous detection of AAS phase I and phase II intact urinary metabolites (glucuronides and sulfates) was developed. A total of 36 metabolites (7 unconjugated; 19 glucuronides and 10 sulfates) corresponding to 15 of the most reported AAS were included. Analytes were extracted from urine using C18 cartridges. LC and MS conditions were studied in-depth to determine the most sensitive and selective conditions for each analyte. A selected reaction monitoring method was set up. The optimization of the experimental parameters for 13 metabolites not available as standards was performed using excretion study urines. Extraction recoveries were above 77% for all 23 validated analytes. Intra-day precision was lower than 21%, and LODs were in the range 0.25-4ng/mL for 18 of the 23 analytes. Matrix effect was evaluated using post column infusion and ranged from 92 to 147%. The method was successfully applied to excretion study urines of different exogenous AAS. The suitability of the strategy was demonstrated with methyltestosterone and stanozolol excretion study urines by achieving detection times of 22 and 21 days, respectively. The method is compliant with the World Antidoping Agency requirements for most of the studied compounds. It represents a cost-effective approach that improves the detection capabilities of AAS by increasing the sensitivity for some metabolites and by including recently described phase II long-term metabolites not detectable using the current screening strategy.

show abstract

Section: Methods Validationmentioning

confidence: 99%

Screening for anabolic steroids in sports: Analytical strategy based on the detection of phase I and phase II intact urinary metabolites by liquid chromatography tandem mass spectrometry

Balcells

Pozo

Esquivel

et al. 2015

Journal of Chromatography A

View full text Add to dashboard Cite

show abstract

“…Regioisomeric glucuronides of 6β‐OH‐And (6β‐glucuronide ( 1 ) and 3α‐glucuronide ( 2 )) and 6β‐OH‐Etio (6β‐glucuronide ( 3 ) and 3α‐glucuronide ( 4 )) were synthesized based on the Koenigs‐Knorr reaction described for similar compounds (Figure ). Briefly, to 1 mL of toluene, 1 mg (3.26 µmol, 1 equivalent) of 6β‐OH‐And or 6β‐OH‐Etio was added followed by 1.30 mg (3.26 µmol, 1 equivalent) of acetobromo‐α‐D‐glucuronic acid methyl ester and 1 mg of Ag 2 CO 3 .…”

Section: Methodsmentioning

confidence: 99%

Synthesis and characterization of 6β‐hydroxyandrosterone and 6β‐hydroxyetiocholanolone conjugated with glucuronic acid

Kotronoulas

Fabregat

Alfonso

et al. 2014

Drug Testing and Analysis

View full text Add to dashboard Cite

The detection of testosterone (T) misuse by doping control laboratories is mainly based on monitoring urinary T phase I metabolites released after enzymatic hydrolysis of the corresponding phase II glucuronide metabolites by gas chromatography (tandem) mass spectrometry (GC-MS(/MS)) methods. However, this strategy fails to properly determine two recently reported phase II metabolites of T conjugated with glucuronic acid that remained mostly conjugated after the hydrolysis step. These metabolites were identified as glucuronides of 6β-hydroxyandrosterone (6β-OH-And) and 6β-hydroxyetiocholanolone (6β-OH-Etio) but their exact conjugation site remained undetermined. In this study, the four possible glucuronides of 6β-OH-And and 6β-OH-Etio were synthesized and characterized by nuclear magnetic resonance (NMR) spectroscopy. Moreover, their chromatographic properties and MS spectra were compared to those obtained for the urine samples collected after administration of T. Results confirmed that the recently reported metabolites were the 3α-glucuronides of 6β-OH-And and 6β-OH-Etio. The synthesis and the elucidation of the exact structure of the metabolites presented in this study are crucial steps for the development of analytical methods in order to explore their role in T metabolism and their potential usefulness as biomarkers of T misuse.

show abstract

“…Most of the enzymes can glucuronidate several or even many different compounds, with partial overlaps between individual UGTs with respect to the aglycones they can glucuronidate are common. It may be added, however, that the substrate specificity overlaps are less significant when kinetic parameters are taken into account and when regio-and stereo-selectivity are considered in the case of substrates that can be glucuronidated at more than one position [9][10][11][12].…”

Section: Introductionmentioning

confidence: 99%

The UDP-Glucuronosyltransferases as Oligomeric Enzymes

Finel¹,

Kurkela

2008

CDM

Self Cite

View full text Add to dashboard Cite

The UDP-glucuronosyltransferases (UGTs) are integral membrane proteins of the endoplasmic reticulum that play important roles in the defense against potentially hazardous xenobiotics. The UGTs also participate in the metabolism and homeostasis of many endogenous compounds, including bilirubin and steroid hormones. Most human UGTs can glucuronidate several substrates the chemical structures of which may vary significantly. Understanding the structural basis for the complex substrate specificity of the UGTs is a major challenge that is hampered by the lack of sufficient structural information on these enzymes. Nevertheless, there is currently a broad interest in the structure and function of the UGTs and here we have focused on their oligomeric state. The question whether or not the UGTs are oligomeric enzymes, either dimeric or tetrameric, was frequently addressed in the past, as well as in recent studies. The current knowledge of protein-protein interactions among the UGTs is limited, however, primarily due to considerable difficulties in purifying individual recombinant UGTs as fully active and mono-dispersed proteins. Such hurdles in studying the oligomeric state of the UGTs prompted researchers to develop less direct approaches for examining the quaternary structure of the UGTs and its functional significance. In this article we have reviewed, sometimes critically, most of the available studies about the oligomeric state of the UGTs. Concluding that the UGTs are oligomeric enzymes, we discuss hetero-oligomerization among UGTs and its possible implications for the structure, function and substrate specificity of the enzymes.

show abstract

Synthesis, structure characterization, and enzyme screening of clenbuterol glucuronides

Cited by 17 publications

References 37 publications

Screening for anabolic steroids in sports: Analytical strategy based on the detection of phase I and phase II intact urinary metabolites by liquid chromatography tandem mass spectrometry

Screening for anabolic steroids in sports: Analytical strategy based on the detection of phase I and phase II intact urinary metabolites by liquid chromatography tandem mass spectrometry

Synthesis and characterization of 6β‐hydroxyandrosterone and 6β‐hydroxyetiocholanolone conjugated with glucuronic acid

The UDP-Glucuronosyltransferases as Oligomeric Enzymes

Contact Info

Product

Resources

About