A rapid and sensitive LC-MS/MS method for determination of the active component K6 in serum of patients with...

Depression is a mental health disorder characterized by chronic negative mood, and depression has become a major threat to human health and quality of life. Anyupeibo capsule, a fifth-class new Chinese medicine, was prepared with extracts of Piper laetispicum C.DC. (Piperaceae), and the alkaloid K6 (5'-methoxy-3',4'-methyl-enedioxycinnamic-acidisob-utylamide-isobutylamide) was found to be the main active component. Using high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (LC-MS/MS), we developed a method to quantify the concentration of K6 in serum samples from patients with depression. Pretreatment of samples was completed based on solid-phase extraction, and the mobile phase for subsequent LC analysis consisted of aqueous ammonium acetate (0.1 mmol/L, phase A) and acetonitrile (phase B) with isocratic elution at 60% B. Chromatographic separation of K6 was achieved within 3 min with an Agilent ZORBAX SB-C₁₈ column (2.1 × 150 mm, 3.5 µm) at a flow rate of 0.3 mL/min. A linear regression equation for K6 yielded correlation coefficients of r² > 0.99 within a linear range 0.0503-100.5000 ng/mL. Extraction recovery ranged from 85.33% to 101.18%, and the matrix effect ranged from 87.15% to 100.28%. The inter-day and intra-day precision values-expressed as relative standard deviation-were less than 15%, and the corresponding accuracy values were within ±15%. All validation results for stability, specificity, and carry-over met the requirements of Pharmacopoeia. The LC-MS/MS method was applied to determine the K6 concentration in serum samples from patients with depression in a phase III clinical trial of Anyupeibo capsule.