SHORT COMMUNICATION Identification of Silymarin Constituents: An Improved HPLC–MS Method A ´ kos Kuki • Lajos Nagy • Gyo ¨rgy Dea ´k • Miklo ´s Nagy • Miklo ´s Zsuga • Sa ´ndor Ke ´ki Received: 16 August 2011 / Revised: 24 October 2011 / Accepted: 7 November 2011 / Published online: 20 November 2011 Ó Springer-Verlag 2011 Abstract A high-performance liquid chromatographic/ tandem mass spectrometric method was developed for the determination of the major bioactive flavonolignans in silymarin, a herbal remedy extracted from the milk thistle Silybum marianum. In this study, eight active components of silymarin with the same elemental composition, including silychristins A and B, silydianin, silybin A and B, isosilybin A and B and an unknown compound were completely separated. Furthermore, three additional components were detected and partly separated; presumably two silybin stereoisomers and one isosilybin stereoisomer. The colli- sion-induced dissociation (CID) MS/MS spectra of these silymarin constituents were studied: the spectral similarity values of the component pairs were determined, and simple criteria were found for distinguishing the components. Keywords High-performance liquid chromatography  Tandem mass spectrometry  Milk thistle  Silymarin  Silybin Introduction Silymarin, extracted from the milk thistle plant, Silybum marianum, has been successfully applied for the treatment of various liver diseases [1, 2], and recently its exceptionally high anti-tumor promoting activity and many other phar- macological activities have also been reported [3]. Silymarin is a mixture of flavonolignans including silychristins A and B, silydianin, silybins A and B and isosilybins A and B as the main bioactive constituents [4–6]. The 2,3-cis-silybin A and B have also been reported [7]. The chemical structures of the nine major silymarin components are shown in Fig. 1. Because of the extensive medical application of milk thistle, there is a strong need for the development of highly sensitive and effective analytical methods for the characterization of its major components. Liquid chromatography–mass spec- trometry combined with soft ionization methods, such as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), can satisfy these requirements. Furthermore, if tandem MS (MS/MS) is applied, structural information can also be obtained and the efficient differen- tiation between the isomeric flavonolignans can be achieved. Khan et al. [8] previously reported the fragmentation study of silymarin using electrospray ionization mass spectrometry (ESI–MS/MS) without separation. Ding et al. [9] separated six silymarin components by high-performance liquid chromatography (HPLC) with UV detection. Later, Lee et al. [10] reported the separation and fragmentation analysis of six silymarin components using HPLC ESI–MS/MS. Shib- ano et al. [11] studied the collision-induced dissociation (CID) MS/MS and MS 3 spectra of the seven flavonolignans using hybrid ion-trap and time-of-flight (IT-TOF) mass spectrometry. A detailed fragmentation behavior of the protonated silybin and its main building blocks by atmo- spheric pressure chemical ionization quadrupole time-of- flight tandem mass spectrometry in the positive ion mode (APCI(?)-QqTOF MS/MS) has been also reported [12]. In this study, we separated 11 components of silymarin, and report the detailed HPLC MS/MS analysis of the silymarin constituents. To our best knowledge, no report on Electronic supplementary material The online version of this article (doi:10.1007/s10337-011-2163-7) contains supplementary material, which is available to authorized users. A ´ . Kuki  L. Nagy  G. Dea ´k  M. Nagy  M. Zsuga  S. Ke ´ki (&) Department of Applied Chemistry, University of Debrecen, Debrecen 4032, Hungary e-mail: keki.sandor@science.unideb.hu 123 Chromatographia (2012) 75:175–180 DOI 10.1007/s10337-011-2163-7