Mechanism-based perspective on metal ion-induced signal instability and development of a robust LC-MS/MS method for trace quantification of hetrombopag in plasma.

Hetrombopag (Hengqu®) is an oral nonpeptide thrombopoietin receptor agonist for the treatment of thrombocytopenia and aplastic anemia. Research on the pharmacokinetic behavior is very important, but accurate quantification of metal-sensitive hetrombopag in biological specimens is a challenging analytical problem. In view of the presence of trace metals from a variety of sources, such as chromatographic system, containers, and the mass spectrometry spray needle, which can contribute to poor peak shape, tailing, and diminished recovery of compound. To ameliorate these problems, different solvent additives, mobile phase, containers and precipitant reagents were investigated to reduce the negative effects. In the present study, a robust, selective, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of hetrombopag in dog plasma was developed based on the dual strategies: (1) 6 % perchloric acid in acetonitrile eliminates metal ion adsorption in storage containers, and (2) 0.01 % trifluoroacetic acid (TFA) in the mobile phase suppresses chelation in chromatographic systems. These two solvents are essential requirements for ensuring stable analytical signals. This method employed an InfinityLab Poroshell 120 EC-C18 column (4.6 ×50 mm, 2.7 μm, Agilent) with a gradient system comprising 0.01 % trifluoroacetic acid with 10 mM ammonium formate (phase A) and a mixture of HO: methanol: isopropanol (1:10:10, v/v/v) containing 0.01 % trifluoroacetic acid and 10 mM ammonium formate (phase B). The column temperature of 50℃ and the addition of isopropanol were beneficial for the separation of interfering peaks and target analyte peaks in plasma. The method exhibits excellent linearity over 3 orders of magnitude. The method achieved a lower limit of quantification (LLOQ) of 0.10 ng/mL using only 50 μL of plasma, with precision (RSD ≤ 8.83 %) and accuracy (88.4-102.2 %) within acceptable limits. The method was applied to determine the plasma concentration of hetrombopag in eight male beagle dogs after each administration of three 2.5 mg hetrombopag tablets. The analytical methodology described in the present work should be useful in the future work aiming to establish a model relating metal-sensitive other compounds.