Development of a new gas chromatography-mass spectrometry (GC-MS) methodology for the evaluation of 5 alpha-reductase activity

• Type: Article in International Scientific Journal
• Date: 2013

Authors

Cristina Amaral (Author) Other; Sara C Cunha (Author) Other; Jose O Fernandes (Author) FFUP; Elisiario Tavares da Silva (Author) Other; Fernanda M F Roleira (Author) Other; Natercia Teixeira (Author) FFUP; Georgina Correia da Silva (Author) FFUP

Journal

Title: Talanta

Vol. 107

Pages: 154-161

ISSN: 0039-9140

Publisher: Elsevier

Indexing

ISI Web of Knowledge - 14 Citations

Scopus - 16 Citations

Scientific classification

FOS: Natural sciences > Chemical sciences

Other information

Authenticus ID: P-005-2DN

DOI: 10.1016/j.talanta.2012.12.045

Abstract (EN): The androgens testosterone (T) and dihydrotestosterone (DHT) play a key role in the function and integrity of prostate tissue, but are also implicated in prostate cancer and benign prostatic hyperplasia (BPH). The reduction of androgen levels can be achieved by the inhibition of 3-oxo-5 alpha-steroid-4-dehydrogenase (5 alpha-reductase), which is responsible for the irreversible conversion of T into its more active metabolite DHT. In fact, the use of 5 alpha-reductase inhibitors (RIs), like finasteride, can be a valuable strategy for the treatment of BPH and in chemoprevention of prostate tumors. In this work a new method based on a dispersive liquid-liquid microextraction (DLLME) procedure, followed by gas chromatography-mass spectrometry (GC-MS), to evaluate the 5 alpha-reductase activity, by measuring the conversion percentage of T into DHT was optimized and validated. Enzymatic assays were carried out in human prostate microsomes, using T as substrate. T and DHT were extracted by the developed DLLME technique and quantified, after silylation, by GC-MS. Variables affecting the extraction efficiency and derivatization of T and DHT were evaluated. The optimized method showed good linearity (with correlation coefficients over 0.9994 for T and 0.9995 for DHT), good recoveries (higher than 80%), and good intra- and inter-day precision (below 13%, 3 levels, n=6). The detection limits for T and DHT were 0.5 nM and the limits of quantification were 5 nM. The new GC-MS method is a good alternative to the already described methods, to evaluate 5 alpha-reductase activity, since it avoids the use of radioactive compounds and corresponds to a fast and sensitive methodology with a good extraction efficiency, accuracy and high recovery. As this method allows the evaluation of 5 alpha-reductase activity, also permits the study of inhibitory efficacy of new molecules as potential RIs.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 8