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

• 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 50 μL 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 (150.0 mm × 2.1 mm i.d., particle size 3.5 μm) under isoscratic elution. The mobile phase was composed of a 7% 10 mM ammonium formate water solution in acetonitrile and pumped at a flow rate of 0.20 mL min−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 2.5–250 ng mL−1 for alprazolam and 2.5–50 ng mL−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 (50 μL human plasma) and a run time less than 10.0 min for each sample the method can be used to support a wide range of clinical studies concerning alprazolam quantification.