2864 Wen Xu ∗ Jing Zhang Dayuan Zhu Juan Huang Zhihai Huang Junqi Bai Xiaohui Qiu Lab of Chinese Materia Medica Preparation, the Second Clinical College, Guangzhou University of Chinese Medicine, Guangzhou, China Received April 1, 2014 Revised July 22, 2014 Accepted July 23, 2014
J. Sep. Sci. 2014, 37, 2864–2873
Research Article
Rapid separation and characterization of diterpenoid alkaloids in processed roots of Aconitum carmichaeli using ultra high performance liquid chromatography coupled with hybrid linear ion trap-Orbitrap tandem mass spectrometry† The lateral root of Aconitum carmichaeli, a popular traditional Chinese medicine, has been widely used to treat rheumatic diseases. For decades, diterpenoid alkaloids have dominated the phytochemical and biomedical research on this plant. In this study, a rapid and sensitive method based on ultra high performance liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry was developed to characterize the diterpenoid alkaloids in Aconitum carmichaeli. Based on an optimized chromatographic condition, more than 120 diterpenoid alkaloids were separated with good resolution. Using a systematic strategy that combines high resolution separation, highly accurate mass measurements and a good understanding of the diagnostic fragment-based fragmentation patterns, these diterpenoid alkaloids were identified or tentatively identified. The identification of these chemicals provided essential data for further phytochemical studies and toxicity research of Aconitum carmichaeli. Moreover, the ultra high performance liquid chromatography with linear ion trap-Orbitrap mass spectrometry platform was an effective and accurate tool for rapid qualitative analysis of secondary metabolite productions from natural resources. Keywords: Aconitum carmichaeli / Diterpenoid alkaloids / Liquid chromatography with mass spectrometry DOI 10.1002/jssc.201400365
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction The processed daughter root of Aconitum carmichaeli debx. (Fuzi) is commonly used in China as an analgesic, a cardiotonic, and as an antirheumatism treatment. Its traditional clinical practice is to be prepared as decoctions together with other crude drugs (such as Sini Tang, Fuzi Lizhong wan, Correspondence: Professor Xiaohui Qiu, Lab of Chinese Materia Medica Preparation, the Second Clinical College, Guangzhou University of Chinese Medicine, The 55th Neihuan Xi road, HEMC Panyu district, Guangzhou 510006, China E-mail: [email protected] Fax: +86-20-39318571
Abbreviations: AC, aconitine; ADA, amine-diterpenoid alkaloids; BA, 14-benzoylaconine; BH, 14-Benzoyhypaconine; BM, 14-benzoylmesaconine; DDA, diester-diterpenoid alkaloids; EIC, extracted ion chromatogram; Fuzi, the processed daughter root of Aconitum carmichaeli debx.; HA, hypaconitine; IT, ion trap; LDA, lipo-diterpenoid alkaloids; LTQ, linear ion trap quadrupole; MA, mesaconitine; MDA, monoesterditerpenoid alkaloids; TIC, total ion chromatogram C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
and Fuzi Tang) for oral administration [1]. Pharmacological studies indicate that Fuzi possesses extensive biological activities, including analgesic, anti-inflammatory, cardiovascular [2], and anti-epileptiform actions [3]. However, the wellknown toxicity and the narrow therapeutic window restricts its clinical use. Diterpenoid alkaloids, notably aconitine, mesaconitine, and hypaconitine, have been proved as the major components to contribute to the bioactive and toxic effects of Fuzi. A variety of analytical methods has been applied to isolate and analyze the diterpenoid alkaloids in Fuzi [4–7]. Liu’s group [8–10] has performed studies on the structural elucidation of diesterditerpenoid alkaloids (DDA) and lipo-diterpenoid alkaloids (LDA) by means of ESI ion trap (IT) multistage MS/MS and initially proposed the summarization of general fragmentation rules for DDA and LDA. Rong [11] paid more attention
∗ In
commemoration of Professor Xinsheng Yao’s 80th birthday.
† This
paper is included in the virtual special issue on traditional chinese medicine available at the Journal of Separation Science website.
www.jss-journal.com
Liquid Chromatography
J. Sep. Sci. 2014, 37, 2864–2873
to the fragmentation behaviors of monoester-diterpenoid alkaloids (MDA) and amine-diterpenoid alkaloids (ADA) and characterized 38 diterpenoid alkaloids in methanolic extract of Fuzi by HPLC–ESI-TOF. Hao’s group [12] separated and detected 81 LDAs using an HPLC–ESI-IT-MS/MS method, and the accurate mass measurements of these compounds were independently performed in an Fourier transform ion cyclotron resonance MS. MALDI-MS and direct-infusion electrospray ionization (DI-ESI-MS) method [13] was developed for rapidly qualitative profiling of alkaloids in Fuzi and 15 of them were tentatively identified. Recently, UHPLC coupled with linear ion trap Orbitrap hybrid (LTQ-Orbitrap) MS has spread in the field of medicinal herbs analysis [14–16]. Benefiting from the advantages of the sub-2 m particles columns, UHPLC [17, 18] produces highly efficient chromatographic separations, and consequently shortens run times. Besides, narrower peaks are obtained, resulting in increased sensitivity and improved peak resolution. The Orbitrap instrument, on the other hand, was proved to show unique advantages in terms of mass resolution. The hybrid LTQ-Orbitrap analytical platform, consisting of a 2D ion trap coupled with an Orbitrap, allows two different scan types to be acquired simultaneously and independently [19,20]. The ion trap provides multistage MSn mass spectra using data-dependent analysis and mass accuracies of
J. Sep. Sci. 2014, 37, 2864–2873
Research Article
Rapid separation and characterization of diterpenoid alkaloids in processed roots of Aconitum carmichaeli using ultra high performance liquid chromatography coupled with hybrid linear ion trap-Orbitrap tandem mass spectrometry† The lateral root of Aconitum carmichaeli, a popular traditional Chinese medicine, has been widely used to treat rheumatic diseases. For decades, diterpenoid alkaloids have dominated the phytochemical and biomedical research on this plant. In this study, a rapid and sensitive method based on ultra high performance liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry was developed to characterize the diterpenoid alkaloids in Aconitum carmichaeli. Based on an optimized chromatographic condition, more than 120 diterpenoid alkaloids were separated with good resolution. Using a systematic strategy that combines high resolution separation, highly accurate mass measurements and a good understanding of the diagnostic fragment-based fragmentation patterns, these diterpenoid alkaloids were identified or tentatively identified. The identification of these chemicals provided essential data for further phytochemical studies and toxicity research of Aconitum carmichaeli. Moreover, the ultra high performance liquid chromatography with linear ion trap-Orbitrap mass spectrometry platform was an effective and accurate tool for rapid qualitative analysis of secondary metabolite productions from natural resources. Keywords: Aconitum carmichaeli / Diterpenoid alkaloids / Liquid chromatography with mass spectrometry DOI 10.1002/jssc.201400365
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction The processed daughter root of Aconitum carmichaeli debx. (Fuzi) is commonly used in China as an analgesic, a cardiotonic, and as an antirheumatism treatment. Its traditional clinical practice is to be prepared as decoctions together with other crude drugs (such as Sini Tang, Fuzi Lizhong wan, Correspondence: Professor Xiaohui Qiu, Lab of Chinese Materia Medica Preparation, the Second Clinical College, Guangzhou University of Chinese Medicine, The 55th Neihuan Xi road, HEMC Panyu district, Guangzhou 510006, China E-mail: [email protected] Fax: +86-20-39318571
Abbreviations: AC, aconitine; ADA, amine-diterpenoid alkaloids; BA, 14-benzoylaconine; BH, 14-Benzoyhypaconine; BM, 14-benzoylmesaconine; DDA, diester-diterpenoid alkaloids; EIC, extracted ion chromatogram; Fuzi, the processed daughter root of Aconitum carmichaeli debx.; HA, hypaconitine; IT, ion trap; LDA, lipo-diterpenoid alkaloids; LTQ, linear ion trap quadrupole; MA, mesaconitine; MDA, monoesterditerpenoid alkaloids; TIC, total ion chromatogram C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
and Fuzi Tang) for oral administration [1]. Pharmacological studies indicate that Fuzi possesses extensive biological activities, including analgesic, anti-inflammatory, cardiovascular [2], and anti-epileptiform actions [3]. However, the wellknown toxicity and the narrow therapeutic window restricts its clinical use. Diterpenoid alkaloids, notably aconitine, mesaconitine, and hypaconitine, have been proved as the major components to contribute to the bioactive and toxic effects of Fuzi. A variety of analytical methods has been applied to isolate and analyze the diterpenoid alkaloids in Fuzi [4–7]. Liu’s group [8–10] has performed studies on the structural elucidation of diesterditerpenoid alkaloids (DDA) and lipo-diterpenoid alkaloids (LDA) by means of ESI ion trap (IT) multistage MS/MS and initially proposed the summarization of general fragmentation rules for DDA and LDA. Rong [11] paid more attention
∗ In
commemoration of Professor Xinsheng Yao’s 80th birthday.
† This
paper is included in the virtual special issue on traditional chinese medicine available at the Journal of Separation Science website.
www.jss-journal.com
Liquid Chromatography
J. Sep. Sci. 2014, 37, 2864–2873
to the fragmentation behaviors of monoester-diterpenoid alkaloids (MDA) and amine-diterpenoid alkaloids (ADA) and characterized 38 diterpenoid alkaloids in methanolic extract of Fuzi by HPLC–ESI-TOF. Hao’s group [12] separated and detected 81 LDAs using an HPLC–ESI-IT-MS/MS method, and the accurate mass measurements of these compounds were independently performed in an Fourier transform ion cyclotron resonance MS. MALDI-MS and direct-infusion electrospray ionization (DI-ESI-MS) method [13] was developed for rapidly qualitative profiling of alkaloids in Fuzi and 15 of them were tentatively identified. Recently, UHPLC coupled with linear ion trap Orbitrap hybrid (LTQ-Orbitrap) MS has spread in the field of medicinal herbs analysis [14–16]. Benefiting from the advantages of the sub-2 m particles columns, UHPLC [17, 18] produces highly efficient chromatographic separations, and consequently shortens run times. Besides, narrower peaks are obtained, resulting in increased sensitivity and improved peak resolution. The Orbitrap instrument, on the other hand, was proved to show unique advantages in terms of mass resolution. The hybrid LTQ-Orbitrap analytical platform, consisting of a 2D ion trap coupled with an Orbitrap, allows two different scan types to be acquired simultaneously and independently [19,20]. The ion trap provides multistage MSn mass spectra using data-dependent analysis and mass accuracies of
