中药白芷中欧前胡素、异欧前胡素体外经皮渗透特性的串联质谱研究
In Vitro Percutaneous Permeability of Imperatorin and Isoimperatorin in Radix Angelicae Dahuricae by Tandem Mass Spectrometry
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摘要: 欧前胡素和异欧前胡素属于6,7-呋喃香豆素类化合物，是中药白芷的主要药效成分。为了考察二者的经皮渗透特性，采用改进型的Franz扩散池，以大鼠离体皮肤作为渗透屏障，以高效液相色谱-串联质谱（HPLC-MS/MS）法测定白芷提取液的经皮渗透液中欧前胡素和异欧前胡素的含量，据此来考察白芷提取液中欧前胡素和异欧前胡素的经皮渗透特性。实验结果表明，欧前胡素和异欧前胡素在48 h内，单位面积的累积渗透量分别为116.11 μg/cm2和140.84 μg/cm2，累积渗透百分率分别为37.75%和71.86%。欧前胡素的稳态渗透速率为26.5 μg/h•cm2，其体外经皮渗透符合Higuchi方程。本研究阐明了欧前胡素的体外经皮渗透机制，可为白芷作为外用经皮给药制剂的发展提供实验依据和理论基础。Abstract: Imperatorin and isoimperatorin are 6,7-furocoumarin compounds, which are the major medicinal effective ingredients of Radix Angelicae Dahuricae. Coumarin compounds are widely used in topical formulations because it has many properties, such as anti-bacterial, anti-inflammatory, antipyretic and analgesic effects. At present, the chemical components of traditional Chinese medicine are mainly analyzed by thin layer chromatography (TLC), gas chromatography (GC) and high performance liquid chromatography (HPLC). However, because there are some difficulties in Chinese medicine analysis, such as complex composition, difficulty in separation and low-content, traditional methods (TLC, HPLC, etc.) were difficult to determine and analyze the composition sometimes. HPLC/MS is a comprehensive technique, it has become an important modern separation and analysis technique because of its high separation ability, high sensitivity, wide application range and strong specificity. Tandem mass spectrometry (MS/MS) has higher sensitivity and selectivity than unipolar mass spectrometry and can obtain more structural information. In this study, the in vitro percutaneous permeability of imperatorin and isoimperatorin in Radix Angelicae Dahuricael extracting solution was evaluated by the modified Franz diffusion cell with isolated rat skin. The content of imperatorin and isoimperatorin was determined by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The chromatographic conditions were as follows: Dimonsil C18 column (250 mm×4.6 mm, 5 μm), the temperature of column is 35 ℃, mobile phase was methanol-water (80∶20, V/V) and flow rate was 0.8 mL/min. A tandem mass spectrometer coupled with positive electro-spray ionization (ESI) source was used for detection. The quantitative and qualitative analysis were performed on selective ion chromatograms acquired by a multiple reaction monitoring (MRM) mode of following transitions: m/z 271.1→m/z 203.04, m/z 271.1→m/z 147.05. The results showed that the limits of detection (LODs) of imperatorin and isoimperatorin were 0.01 and 1 μg/L, and the limits of quantification (LOQs) of imperatorin and isoimperatorin were 0.25 and 2 μg/L, respectively. Calibration curves were linear over the range of 0.001-1 mg/L for imperatorin and isoimperatorin. Both the inter-day and intra-day reproducibility (RSDs) were lower than 4%, the precision and stability of the method were lower than 2%, which indicated that the method has good precision, and the test solution could keep steady within 24 h. The mean recoveries of imperatorin and isoimperatorin were 98.09% and 100.60%. The cumulative permeation amounts of imperatorin and isoimperatorin were 116.11 μg/cm2 and 210.06 μg/cm2 and the percentage of cumulative permeation were 37.75% and 71.86%, respectively. The steady state permeation rate of imperatorin was 26.5 μg/h•cm2 and its in vitro percutaneous permeabilities was conformed to Higuchi equation. This study clarified the percutaneous permeation mechanism of imperatorin in vitro and provided a theoretical basis for the development of Radix Angelicae Dahuricae as a topical transdermal preparation.
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