Establishment of LC-MS/MS method for determination of aloperine in rat plasma and its application in preclinical pharmacokinetics

Highlights

• An efficient LC-MS/MS method of aloperine analysis was developed and fully validated for the first time.

• Aloperine in rat plasma was quantified from 5 to 2000 ng/mL by LC-MS/MS.

• This method was successfully applied to the pharmacokinetics of aloperine after p.o. and i.v. administration in rats.

Abstract

Aloperine, a novel natural active alkaloid derived from Sophora alopecuroides L., has attracted much attention for its anti-inflammatory, antiviral, anti-tumor, anti-allergy and other pharmacological activities. In this study, we first established and validated an efficient and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of aloperine in rat plasma. Cytisine was used as the internal standard (IS). The separation of aloperine and IS was conducted on a Phenomenex Luna Omega Polar C₁₈ column (2.1 × 50 mm, 1.6 μm) with 0.3% (v/v) formic acid aqueous (containing 5 mM ammonium acetate) and 0.3% (v/v) formic acid acetonitrile using isocratic elution condition at a flow rate of 0.20 mL/min. Aloperine and IS were determined under the transitions of m/z 233.2 → 98.1 and m/z 191.2 → 148.2 (positive ionization mode), respectively. The calibration curve of aloperine was established in the range of 5 (LLOQ) to 2000 ng/mL (r² = 0.994). The well validated method was full compliance with the bioanalytical method validation of FDA, and was applied to the pharmacokinetic study of aloperine in Sprague-Dawley rats after 50 mg/kg oral administration and 5 mg/kg intravenous injection. This study provides valuable references for the further study of Sophora alopecuroides L., especially for the drug development and clinical application of aloperine.

Introduction

As recorded in the texts from China, a variety of bioactive compounds have been isolated from Chinese herbal medicines and used as drugs for thousands of years. Sophora alopecuroides L., a natural herb mainly distributed in Western and central Asia, has been widely used in the treatment of sore throat, cough, enteritis and arthritis in China [1]. Alkaloids are recognized as the major components of Sophora alopecuroides L. for its medical effects. Especially, quinolizidine alkaloids, the bioactive natural products, such as aloperine, sophoridine, matrine and oxymatrine, have been extensively reported due to their anti-tumor activities [2], [3]. Among them, aloperine is an important alkaloid with many pharmacological activities. Aloperine mainly originates from the leaves, roots and beans of Sophora alopecuroides L., and the parent nucleus of aloperine is a particular bridged tetracyclic ring system [4] (Fig. 1).

Many studies have shown that aloperine has potential anti-inflammatory, antiviral, anti-cancer, anti-allergy and other pharmacological activities [4], [5], [6]. According to reports, aloperine has achieved positive effects in the treatment of non-small cell lung cancer (NSCLC), multiple myeloma, and colon cancer by changing cell cycle, inhibiting tumor cell growth and inducing apoptosis [7], [8], [9], [10], [11]. In addition, low concentration of aloperine can also inhibit HIV-1 virus infection. In order to further enhance the antiviral ability, more than 30N-12-substitutions derivatives of aloperine were synthesized, and the results indicated that aloperine can inhibit HIV-1 infection by preventing the entry of HIV-1, which may provide a novel solution for the treatment of AIDS [4], [12].

Aloperine also presents great potential in the treatment of pulmonary hypertension and pulmonary fibrosis [13], [14]. There is evidence that aloperine can hyperpolarize the E_M of oocytes expressing KCNQ5 and active KCNQ5, thus relaxing blood vessels and protecting the body from hypertension [15]. In addition, aloperine is expected to be a candidate drug for the treatment of Alzheimer’s disease. On the one hand, it has the ability to regulate the NF-κB and AKT signaling pathways and inhibit H₂O₂-mediated apoptosis. On the other hand, it can improve the oxidative stress mode through reducing the reactive oxygen species (ROS) and 4-hydroxy-2-nonenal (4-HNE) production [16], [17], [18]. Although aloperine has been widely utilized in the treatment of many diseases, recent studies have shown that aloperine has reversible hepatotoxicity and nephrotoxicity in mice [19]. Therefore, it is very important to establish an effective method to monitor the concentration of aloperine in biological samples.

The pharmacological effects of aloperine and its application in the treatment of diseases have attracted extensive attention. However, until now, no efficient method has been found for aloperine determination. In 2010, an HPLC-UV detection method was used to separate aloperine from Sophora flavescens, but the sensitivity was limited [20]. For the last few years, LC–MS/MS system has been extensively applied in natural products analysis. In this study, we aimed to develop a sensitive and efficient LC–MS/MS determination method for the quantitative analysis of aloperine in rat plasma, and further verified its pharmacokinetic characteristics in rats, which could provide a reference for the clinical research of aloperine.