A validated HPLC-ESI-MS/MS method for the determination of lycopsamine in aqueous samples

In the last three decades more than 350 different hepatotoxic, mutagenic and teratogenic pyrrolizidine alkaloids (PAs) have been identified in approximately 6000 plant species [1]. The PA content of Symphytum officinale (common comfrey) may vary between 0.04 – 0.6% [2]. For safety reasons, comfrey can be used only externally and the alkaloid quantity in products is limited. Due to the confirmed good absorption of PAs from the gastrointestinal tract the prohibition of oral use is rational, however the limitation of external application is not supported by relevant scientific data. The aim of the present work was to develop and validate a HPLC-MS/MS method for the determination of a major PA of comfrey (lycopsamine) in aqueous samples as a basis for the development of a method for the determination of absorption of lycopsamine on human skin. Chromatographic separation of aqueous extracts was performed using a Shimadzu LC system equipped with a Phenomenex Gemini C18 (100 × 4.6 mm, 5 µm) column. Isocratic elution was applied with using MeCN and water with 0.1% acetic acid (50:50) mixture. The HPLC was coupled to an API 2000 MS/MS with an ESI interface. Measurements were carried out in positive ionization mode and the quantification was accomplished by using MRM. Linear calibration curve was established in the range of 1.32 – 440 ng/injection. The intraday precision resulted during the 3-day validation was ranged between 0.57 and 2.48% while the interday precision was 2.20% and 2.40% for quality control samples. LOD was 0.014 ng/injection and recovery was above 97%. The lycopsamine content of the samples stored for 9 and 25 days at 22 °C, 10 °C and -25 °C were invariable. These results underline the good repeatability and accuracy of our method.

Acknowledgements: This research was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP 4.2.4. A/1 – 11 – 1-2012 – 0001 'National Excellence Program'.

Keywords: Symphytum officinale, comfrey, pyrrolizidine alkaloid, lycopsamine, HPLC-MS, validation

References:

[1] Betz JM, Eppley RM, Taylor WC, Andrzejewski D. Determination of pyrrolizidine alkaloids in commercial comfrey products (Symphytum sp.). J Pharm Sci 1994; 83: 649 – 653

[2] Mattocks AR. Toxic pyrrolizidine alkaloids in comfrey. Lancet 1980; 316: 1136 – 1137