Qualitative and quantitative analysis of an alkaloid fraction from Piper longum L. using ultra-high performance liquid chromatography-diode array detector–electrospray ionization mass spectrometry

In a previous research, an alkaloid fraction and 18 alkaloid compounds were prepared from Piper longum L. by series of purification process. In this paper, a qualitative and quantitative analysis method using ultra-high performance liquid chromatography-diode array detector–mass spectrometry (UHPLC-DAD–MS) was developed to evaluate the alkaloid fraction. Qualitative analysis of the alkaloid fraction was firstly completed by UHPLC-DAD method and 18 amide alkaloid compounds were identified. A further qualitative analysis of the alkaloid fraction was accomplished by UHPLC–MS/MS method. Another 25 amide alkaloids were identified according to their characteristic ions and neutral losses. At last, a quantitative method for the alkaloid fraction was established using four marker compounds including piperine, pipernonatine, guineensine and N-isobutyl-2E,4E-octadecadienamide. After the validation of this method, the contents of above four marker compounds in the alkaloid fraction were 57.5 mg/g, 65.6 mg/g, 17.7 mg/g and 23.9 mg/g, respectively. Moreover, the relative response factors of other three compounds to piperine were calculated. A comparative study between external standard quantification and relative response factor quantification proved no remarkable difference. UHPLC-DAD–MS method was demonstrated to be a powerful tool for the characterization of the alkaloid fraction from P. longum L. and the result proved that the quality of alkaloid fraction was efficiently improved after appropriate purification.