Simultaneous LC-HRMS determination of 28 benzodiazepines and metabolites in hair

Abstract

A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for the simultaneous identification and quantification of 28 benzodiazepines, including 6 metabolites, in 50 mg of hair has been validated. Positive ion electrospray ionization and HRMS determination in full-scan mode were realized on an Orbitrap mass spectrometer at a nominal resolving power of 60,000. In-source collisional experiments were conducted to obtain additional information for a more reliable identification of the investigated drugs. HRMS in full-scan mode allowed the exact determination of molecular masses of all analytes eluting in the HPLC run, so that both the immediate and retrospective screening of results for drugs and their metabolites were available. Sample preparation consisted of an overnight incubation in phosphate buffer pH 8.4 and a subsequent liquid/liquid extraction with methylene chloride/diethyl ether (90:10). Gradient elution was performed on a Luna C18 analytical column and four deuterated analogues were used as internal standards (IS). Validation was performed using both spiked hair samples and hair samples from subjects treated with benzodiazepines. Selectivity was evaluated by analysis of 20 certified blank hair samples. Extraction efficiency and matrix effects were evaluated by analysis of true positive samples. The lowest limits of quantification (LLOQs) ranged from 1 to 10 pg/mg. Linearity was investigated in the range from LLOQ to 1,000 pg/mg, for each compound (R(2) 0.998-0.999). Mean relative errors, calculated at three concentration levels, ranged from 1 to 20% (absolute value). Precision, at concentrations higher than the LLOQs, was always less than 15% expressed as percentage relative standard deviation. After validation, the procedure was applied to real samples collected for clinical and forensic toxicology purposes from subjects who were assumed to have taken benzodiazepines.

Rapid detection and quantification of 35 benzodiazepines in urine by GC-TOF-MS

“…5 Extracted ion chromatogram of a patient sample, where three benzodiazepines and seven other drugs/therapeutic drugs could be identified (sample preparation using Oasis MCX SPE) 16,21], GC-MS is still the method of choice in drug abuse testing. The capillary GC separation technique provides excellent separation efficiency owing to low height equivalent to a theoretical plate values.…”

10years of MS instrumental developments – Impact on LC–MS/MS in clinical chemistry

“…As expected this approach not only provides quantitative results for compounds of interest, but also will afford data on other analytes present in the sample. As proof an example of the identification of Vogliardi et al also showed that LC-HRMS can be used to identify and quantify simultaneously 28 benzodiazepines, including 6 metabolites, in hair [88]. Positive ion ESI and HRMS determination in the full-scan mode were realized on an Orbitrap mass spectrometer at a nominal resolving power of 60,000.…”

Determination of Benzodiazepines in Beef by Magnetic Solid Phase Extraction and High-Performance Liquid Chromatography–Tandem Mass Spectrometry

“…Benzodiazepines, extensively prescribed and readily available psychoactive pharmaceuticals (Vogliardi et al 2013) are used as muscle relaxants, anticonvulsants, sedative-hypnotics, and anxiolytics (Ming and Heathcote 2011;Uddin, Samanidou, and Papadoyannis 2013). However, they are associated with adverse effects, such as addiction with continued use and toxicological emergencies, either alone or in combination with alcohol and other drugs (Samanidou, Uddin, and Papadoyannis 2009).…”