Development and Validation of LC/MS/MS Method with Ultra Small-Volume Injection for Quantitative Determination of Alprazolam in Human Plasma

A reversed phase gradient LC method using Shim-Pack FC-ODS C18 column (150mmL x 3mmID, 3 um) was employed. The MRM parameters of alprazolam was optimized on LCMS-8080 and two transitions were used, with 309.1>281.1 for quantification and 309.1>274.1 for confirmation. The alprazolam peak appeared at 3.2 minutes and the total run time was 4.5 minutes including column washing and equilibrium. A linear calibration curve was established. The concentrations of the plasma standards used were from 0.33 pg/uL to 10.7 pg/uL, corresponding to 33.3 to 1066.7 femtogram (fg) on column. The LOD and LOQ of the method were 14.2 fg (S/N =3) and 46.9 fg (S/N = 10), respectively. The repeatability was evaluated at three levels: 33.3, 66.7 and 266.7 fg on column with an injection volume of 0.1uL. The repeatability (RSD %, n=6) of peak areas obtained were 24.6%, 8.7% and 2.9%, respectively. The concentration accuracy for the three levels was 100~191%, 19~101% and 108~117%, respectively. The above performance data were compared with that of different injection volume from 0.5uL to 10uL and the results showed that no significant difference was found. In addition, matrix effect and robustness of the method were also investigated. The results indicates that this extra small injection volume LC/MS/MS method did not change matrix effect behaviour but contamination of the dirty plasma matrix to the interface and ion optics was reduced greatly. As such, the method is more robust and suitable for bioanalysis in heavily loaded research and service laboratory. The method also makes it possible to handle very small amount of samples available due to limited sample sources.

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Liquid Chromatography, Liquid Chromatograph-Mass Spectrometry, Mass Spectrometry
Alprazolam, Human Plasma, MRM technique, Pharmaceutical, Life Science, Drug discovery, LCMS-8080, Nexera LC-30A
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