In Vitro Percutaneous Permeability of Imperatorin and Isoimperatorin in Radix Angelicae Dahuricae by Tandem Mass Spectrometry

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.