Long-term detection of clenbuterol in human scalp hair by gas chromatography–high-resolution mass spectrometry

Abstract

A method for the detection of clenbuterol in human scalp hair by gas chromatography–high-resolution mass spectrometry (GC–HRMS) is described. The sample preparation involved chemical digestion of the protein structure, which was achieved by incubating the hair with 1 M KOH at 70°C. A single extraction step with tert.-butyl methyl ether provided approximately 90% of the analyte, which was dried and derivatized with N-methyl- N-trimethylsilyltrifluoroacetamide (MSTFA) to yield clenbuterol N, O-bis-trimethylsilyl (TMS). Hair was collected from four pregnant women who were therapeutically treated with Spiropent ® (clenbuterol–HCl) and from the infant of one female patient. Hair samples were taken during the application time and two to six months after completion of clenbuterol administration. The detection limit of the method was approximately 4 ng clenbuterol/g hair when 25 mg hair material were processed and 2 ng/g for 50 mg hair samples (corresponds to 4 pg per injection). The method allows clenbuterol to be measured retrospectively for up to at least six months. The levels of clenbuterol determined in hair ranged from 2 to 236 ng/g. No clenbuterol was found in the hair of the infant, which was taken five and a half months after delivery. To improve sample preparation, an additional purification step via immuno affinity chromatography (IAC) was integrated. The IAC purified extracts showed reduced biological background interference and an improved limit of detection (0.8 ng/g).