title: Hydrophilic interaction liquid chromatography/positive ion electrospray ionization mass spectrometry method for the quantification of alprazolam and α-hydroxy-alprazolam in human plasma.
abstract: A hydrophilic interaction liquid chromatography/positive ion electrospray-mass spectrometry (HILIC-ESI/MS) has been developed and fully validated for the quantification of alprazolam and its main metabolite, α-hydroxy-alprazolam, in human plasma. The assay is based on 50μL plasma samples, following liquid-liquid extraction. All analytes and the internal standard (tiamulin) were separated by hydrophilic interaction liquid chromatography using an X-Bridge-HILIC analytical column (150.0mm×2.1mm i.d., particle size 3.5μm) under isoscratic elution. The mobile phase was composed of a 7% 10mM ammonium formate water solution in acetonitrile and pumped at a flow rate of 0.20mLmin(-1). Running in positive electrospray ionization and selected ion monitoring (SIM) the mass spectrometer was set to analyze the protonated molecules [M+H](+) at m/z 309, 325 and 494 for alprazolam, α-hydroxy-alprazolam and tiamulin (ISTD) respectively. The assay was linear over the concentration range of 2.5-250ngmL(-1) for alprazolam and 2.5-50ngmL(-1) for α-hydroxy alprazolam. Intermediate precision was less than 4.1% over the tested concentration ranges. The method is the first reported application of HILIC in the analysis benzodiazepines in human plasma. With a small sample size (50μL human plasma) and a run time less than 10.0min for each sample the method can be used to support a wide range of clinical studies concerning alprazolam quantification.
authors.affiliation: University of Athens, School of Pharmacy, Division of Pharmaceutical Chemistry, Panepistimopolis - Zografou, 157 71 Athens, Greece.
authors.forename: Eleni
authors.initials: E
authors.lastname: Kalogria
authors.forename: Constantinos
authors.initials: C
authors.lastname: Pistos
authors.forename: Irene
authors.initials: I
authors.lastname: Panderi
mesh_terms: D000328:Adult; D000525:Alprazolam / Q000031:analogs & derivatives* / Q000097:blood* / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D004224:Diterpenes; D005260:Female; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D057230:Limit of Detection; D016014:Linear Models; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D004224:Diterpenes; C032318:alpha-hydroxyalprazolam; C014224:tiamulin; D000525:Alprazolam
keywords: Alprazolam; Hydrophilic interaction liquid chromatography (HILIC); Liquid chromatography/mass spectrometry; Tiamulin; α-Hydroxy-alprazolam