**1. Introduction**

Myocardial ischemia-induced infarction is one of the leading causes of human death worldwide. The benefits of either *Carthamus tinctorius* extract (CTE) or notoginseng total saponins (NGTS) towards myocardial ischemia injury on rats have been well defined, and more interestingly, previous studies have demonstrated that better cardio-protective effects were observed when using their combination

preparation (CNP) [1–3]. However, the underlying synergetic mechanisms of CTE and NGTS combination, their pharmacokinetic (PK) interactions in particular, still remain unclear.

It is widely accepted that the drug–drug interactions (DDIs) and herb–herb interactions (HHIs) can cause changes of pharmacokinetic profiles, which result in the possible improvement of drug efficacy and in the decrease of side effects, or vice versa [4,5]. However, most of the literature has merely focused on the pharmacokinetic profile variations of these primary components between individual dosing and combined use, but has overlooked the reasons responsible for the changed pharmacokinetic behaviors, which may be caused, at least in part, by cytochrome p450 (CYP450) and/or transporter-mediated HHIs [6]. Therefore, the objective of this study was to gain insight into the synergistic actions between CTE and NGTS by determination of the pharmacokinetic profiles of six major active components from CTE and NGTS, as well as their CYP450-based synergetic mechanisms. An in vitro cocktail assay, which is an efficient and widely favored approach for CYP450-mediated HHIs, was employed to pursue the factors accounting for the different pharmacokinetic patterns before and after compatibility.

Our previous pharmacological evaluations optimized a relatively low dosage CNP for the anti-myocardial ischemia effect [2]. Furthermore, the cocktail method usually suffers from extensive CYP450 crossover within the probe substrates [7]. Therefore, the emerging demand is to develop a sensitive and efficient method for reliable detection and determination of the trace ingredients for the PK and cocktail studies. Attempts were made herein to propose and apply a large volume direct injection ultra-high performance liquid chromatography–tandem mass spectrometry (LVDI-UHPLC-MS/MS) method for direct and sensitive multiple-component PK and cocktail studies.
