Derivatization procedures for the detection of beta(2)-agonists by gas chromatographic/mass spectrometric analysis.
An evaluation of derivatization procedures for the detection of beta(2)-agonists is presented. The study was performed with the beta(2)-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol and terbutaline. Different derivatizating agents were employed, aiming to obtain derivatives with high selectivity to be used in the gas chromatographic/mass spectrometric analysis of beta(2)-agonists in biological samples. Trimethylsilylation was compared with different agents and the role of some catalysts was evaluated. Acylation, combined trimethylsilylation and acylation, and the formation of cyclic methylboronates were also studied. Sterical hindrance caused by different substituents at the nitrogen atom of the beta-ethanolamine lateral chain of beta(2)-agonist molecules is mainly responsible for differences in the abundances of the derivatives obtained. The use of catalysts produces an increase in the derivatization yield, especially for compounds with low steric hindrance (substituents with primary and secondary carbon atoms). The formation of trimethylsilyl (TMS) ethers is not influenced by structural molecular differences when only hydroxy groups are involved in derivatization. Combined trimethylsilylation and acylation showed that compounds with a secondary carbon atom linked to the nitrogen atom form mainly N-TFA-O-TMS derivatives, with a small amount of N-TMS-O-TMS derivatives. Compounds with tert-butyl substituents at the amino group (bambuterol, salbutamol and terbutaline) formed O-TMS derivatives as the main products, although a limited amount of trifluoroacylation at the nitrogen atom also occurred. Cyclic methylboronates were formed with bambuterol, clenbuterol, formoterol, salbutamol and salmeterol. Owing to hydroxy substituents in unsuitable positions for ring formation, this procedure was not effective for fenoterol and terbutaline. Mass spectra of different derivatives and tentative fragmentation profiles are also shown. For screening purpose (e.g. sports drug testing), derivatization with MSTFA or BSTFA alone is recommended as a comprehensive derivatization technique for beta(2)-agonists owing to minimal by-product formation; formation of cyclic methylboronates can be useful for confirmation purposes. Detection limits were obtained for the TMS and cyclic methylboronate derivatives using the derivatizing reagents MSTFA and trimethylboroxine, respectively. For most of the compounds, lower detection limits were found for the TMS derivatives.
Damasceno
L
L
Unitat de Farmacologia, Institut Municipal d'Investigaci o M edica, E-08003 Barcelona, Spain.
Ventura
R
R
Ortuño
J
J
Segura
J
J
D000081:Acetamides; D000215:Acylation; D000318:Adrenergic beta-Agonists / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism / Q000652:urine; D001896:Boron Compounds / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D005463:Fluoroacetates; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D012680:Sensitivity and Specificity; D014269:Trifluoroacetic Acid / Q000737:chemistry / Q000378:metabolism; D014297:Trimethylsilyl Compounds / Q000737:chemistry / Q000378:metabolism
D000081:Acetamides; D000318:Adrenergic beta-Agonists; D001896:Boron Compounds; D005463:Fluoroacetates; D014297:Trimethylsilyl Compounds; C086665:N-methyl-N-(trimethylsilyl)trifluoroacetamide; C047270:N,N-bis(trimethylsilyl)-2,2,2-trifluoroacetamide; D014269:Trifluoroacetic Acid