Microwave-assisted derivatization: Application to steroid profiling by gas chromatography/mass spectrometry

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2014-06, Vol.960, p.8-13
• Main Authors: Casals, Gregori, Marcos, Josep, Pozo, Oscar J., Alcaraz, José, Martínez de Osaba, María Jesús, Jiménez, Wladimiro
• Format: Article
• Language: English
• Subjects: Adult; Aged; Derivatives; Female; Gas chromatography; Gas Chromatography-Mass Spectrometry - methods; Gas chromatography/mass spectrometry; Human urine; Humans; Irradiation; Limit of Detection; Male; Metabolome; Microwave assisted derivatization; Microwaves; Middle Aged; Optimization; Reproducibility of Results; Spectrometry; Steroids; Steroids - chemistry; Steroids - urine; Urine
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2014.04.015

•A microwave-assisted derivatization procedure for steroid profiling is presented.•Urine steroid profiles for 20 healthy individuals were analyzed.•Similar derivatization yields than thermal-derivatization were obtained.•Overnight thermal-derivatization can be reduced to a 3min microwave reaction. Gas chromatography–mass spectrometry (GC–MS) remains as the gold-standard technique for the study of the steroid metabolome. A main limitation is the need of performing a derivatization step since incubation with strong silylations agents for long periods of time (usually 16h) is required for the derivatization of hindered hydroxyls present in some steroids of interest. In the present work, a rapid, simple and reproducible microwave-assisted derivatization method was developed. In the method, 36 steroids already treated with methoxyamine (2% in pyridine) were silylated with 50μl of N-trimethylsilylimidazole by using microwave irradiation, and the formed methyloxime-trimethylsilyl derivatives were analyzed by GC–MS. Microwave power and derivatization time silylation conditions were optimized being the optimum conditions 600W and 3min respectively. In order to evaluate the usefulness of this technique, the urine steroid profiles for 20 healthy individuals were analyzed. The results of a comparison of microwave irradiation with the classical heating protocol showed similar derivatization yields, thus suggesting that microwave-assisted silylation is a valid tool for the rapid steroid metabolome study.