Hydrophilic interaction liquid chromatography/positive ion electrospray ionization mass spectrometry method for the quantification of alprazolam and α-hydroxy-alprazolam in human plasma

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2013-12, Vol.942-943, p.158-164
• Main Authors: Kalogria, Eleni, Pistos, Constantinos, Panderi, Irene
• Format: Article
• Language: English
• Subjects: acetonitrile; Adult; Alprazolam; Alprazolam - analogs & derivatives; Alprazolam - blood; Alprazolam - chemistry; benzodiazepines; Chromatography, Liquid - methods; clinical trials; Diterpenes; electrospray ionization mass spectrometry; Female; Humans; hydrophilic interaction chromatography; Hydrophilic interaction liquid chromatography (HILIC); Hydrophobic and Hydrophilic Interactions; ionization; Limit of Detection; Linear Models; Liquid chromatography/mass spectrometry; liquid-liquid extraction; Male; metabolites; Middle Aged; monitoring; particle size; Reproducibility of Results; spectrometers; Spectrometry, Mass, Electrospray Ionization - methods; Tiamulin; α-Hydroxy-alprazolam
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2013.10.040

•HILIC chromatography enables rapid quantitation of alprazolam and α-hydroxy alprazolam in human plasma.•HILIC-ESI/MS enables the sensitive and selective quantitation of alprazolam and α-hydroxy alprazolam.•HILIC-ESI/MS was used to analyze samples obtained from patients treated for anxiety disorders. 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.