Determination of 13 Free Fatty Acids in Pheretima Using Ultra-Performance LC-ESI-MS

A simple and rapid ultra-performance liquid chromatography-electrospray ionization mass spectrometry method for the simultaneous determination of thirteen free fatty acids (FFAs) in Pheretima has been developed and validated. Measurements for each FFA were linear over a wide range (0.05–3.95 μg mL) with good correlation coefficients (>0.99). The limit of detection and limit of quantification for all the fatty acids were below 26 and 78 ng mL, respectively. The intraand inter-assay precision and accuracy for the thirteen FFAs fell well within the predefined limits of acceptability. Satisfactory recoveries were in the range of 96– 103%. Article: INTROCUDTION Pheretima has been well known for its wide therapeutic properties such as anti-inflammatory, anti-oxidative [1], anti-asthmatic, thrombolytic, reducing symptoms of the central nervous system decline including memory loss in traditional Chinese medicine (TCM) for over 2,000 years [2]. Four kinds of earthworms, including Pheretima aspergillum (E. Perrier), Pheretima guillelmi (Michaelsen), Pheretima vulgaris Chen, Pheretima pectinifera Michaelsen are included in the Pharmacopoeia of the People’s Republic of China (2005). Since patients with asthma and other forms of allergy have been repeatedly reported to present an abnormal plasma fatty acid pattern [3], fatty acids extensively occurring in animal origin TCM seem to play an important role and act as the bioactive components. In our previous work, the acidic fraction isolated from Pheretima containing free fatty acids (FFAs) showed obvious anti-asthmatic activities [4], and the FFAs in the active fraction probably contributed to these pharmacological effects. In order to facilitate the future researches about these activities, it is paramount to establish a qualitative and quantitative assay method for the determination of FFAs in Pheretima. Although fatty acids have measurable absorbance in the range of 190–215 nm, the interference of most solvents is a limiting factor for sensitive detection when analyzed directly by liquid chromatography-ultraviolet spectrometry (LC-UV). To increase sensitivity, gas chromatography (GC) has been widely applied in FFA analysis after the absolute necessary derivatization of FFAs by esterification to form relative low polar analytes. Due to the high selectivity provided by mass spectrometric (MS) detection [2], and the capability of analyzing non-volatile compounds provided by liquid chromatography (LC), in most cases, derivatization to form UV-absorptive and volatile compounds is dispensable using LC combined with MS. Thus, LC-MS provides a relatively rapid, reproducible method which is suitable for the determination of thermolabile, non-volatile multi-components without obvious UV absorption. As the primary evolution of LC, ultra-performance liquid chromatography (UPLC) employs particles smaller than 2 μm in diameter to achieve superior resolution, rapid speed and higher sensitivity compared to LC. Therefore, UPLC coupled with MS is suggested as a better alternative method for FFA assay in Pheretima. Zehethofer et al. [5] had developed a method for profiling of 29 FFAs in plasma using UPLCESI-MS-MS with the positive ion mode by cationization of the FFAs via addition of barium ions for sensitive multiple reaction monitoring (MRM). However, to our knowledge the application of UPLC-MS to the analysis of FFAs in Pheretima raw materials has not been reported to date. In the present work, we aim to develop a simple UPLC electrospray ionization MS (UPLC-ESIMS) method in the negative ion mode by selected ion monitoring (SIM) without adding any additives for the determination of FFAs in Pheretima raw materials and the isolated bioactive fraction to elucidate the composition variation between the two samples for further bioactive evaluation.