Development and Validation of an LC-MS/MS Assay to Quantitate 2',4',6'-Trihydroxyacetophenone in Rat and Dog Plasma and its Application to a Pharmacokinetic Study.

In the present study, a simple, rapid, and reliable bioanalytical method was developed using liquid chromatography with tandem-mass spectrometry (LC-MS/MS) to quantify 2',4',6'-trihydroxyacetophenone (THAP) in rat and dog plasma with 2',4',6'-trihydroxybenzaldehyde as an internal standard (IS). The LC-MS/MS instrument was operated in the multiple reaction monitoring (MRM) mode to detect THAP at m/z transition 166.89 > 82.8 and IS at 152.89 > 82.8, respectively. A simple, one-step protein precipitation (PP) method was employed with acetonitrile for sample preparation. Utilizing a Gemini C18 column, THAP and IS were separated with an isocratic mobile phase consisting of 10 mM ammonium acetate and methanol (10:90, v/v) at a flow rate of 0.2 mL/min. Total chromatographic run time was 2.5 min per sample injection. The standard calibration curve for THAP was linear (r2 ≥ 0.9987) over the concentration range of 0.1 to 100 µg/mL with the lower limit of quantitation (LLOQ) of 0.1 µg/mL (S/N ratio > 10). According to the regulatory guidelines from the U.S. Food and Drug Administration (FDA) and the Korea Ministry of Food and Drug Safety (MFDS), our newly developed biomedical analytical method was fully and adequately validated in terms of selectivity, sensitivity, linearity, intra- and inter-day precision and accuracy, recovery, matrix effect, stability, and dilution integrity. Our validated assay was successfully utilized in a nonclinical pharmacokinetic study of THAP in rats and dogs.

Development and Validation of Simple LC-MS-MS Assay for the Quantitative Determination of Ticagrelor in Human Plasma: its Application to a Bioequivalence Study.

A sensitive, rapid, reproducible and reliable high-performance liquid chromatography-electrospray ionization tandem mass spectrometric method has been developed and fully validated for the determination of ticagrelor in human plasma using ticagrelor-d7 as an internal standard (IS) after one-step liquid-liquid extraction with ethyl acetate. Ticagrelor and IS were detected in the multiple reaction monitoring mode at m/z transition 523.4 > 127.0 and 530.4 > 127.0, respectively. Chromatographic separation was performed on a Phenomenex Luna® C18 column using a mobile phase consisting of 0.1% formic acid in water-acetonitrile (20:80, v/v) at a flow rate of 0.2 mL/min. The retention times of ticagrelor and IS were 1.03 min and 1.02 min, respectively. The calibration curve was linear [correlation coefficient (r) ≥ 0.9991] over the concentration range of 2-1,500 ng/mL. Intra- and inter-day precisions ranged from 1.0% to 4.9% and from 1.8% to 8.7%, and the accuracy ranged from 97.0% to 105.9% and from 97.5% to 102.9%, respectively. The developed method was fully validated with respect to selectivity, linearity, lower limit of quantitation, recovery, matrix effect and stability. This validated method was successfully applied to the bioequivalence study of ticagrelor in 44 healthy Korean male volunteers.