molecules Article Quantification of Nineteen Bioactive Components in the Ancient Classical Chinese Medicine Formula of Wen-Dan Decoction and Its Commercial Preparations by UHPLC-QQQ-MS/MS 1,2,3, 1,2,3, , 1,2,3 1,2,3 1,2,3 Bing Zhang y, Dongli Qi * y , Xiuping Deng , Zhe Ma , Yumei Wu , Zhifeng Xue 1,2,3, Dereje Kebebe 1,2,3,4, Peng Lu 1,2,3, Jiaxin Pi 1,2,3, Pan Guo 1,2,3, Nan Li 1,2,3 and Zhidong Liu 1,2,3,* 1 Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 301600, China; [email protected] (B.Z.); [email protected] (X.D.); [email protected] (Z.M.); [email protected] (Y.W.); [email protected] (Z.X.); [email protected] (D.K.); [email protected] (P.L.); [email protected] (J.P.); [email protected] (P.G.); [email protected] (N.L.) 2 The Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 301600, China 3 Engineering Research Center of Modern Chinese Medicine Discovery and Preparation Technique, Ministry of Education, Tianjin University of Traditional Chinese Medicine, Tianjin 301600, China 4 School of Pharmacy, Institute of Health Sciences, Jimma University, P.O. Box 378, Jimma, Ethiopia * Correspondence: [email protected] (D.Q.); [email protected] (Z.L.); Tel.: +86-151-2291-5636 (D.Q.); +86-137-5258-1592 (Z.L.) These authors contribute equally to the manuscript. y Received: 29 April 2019; Accepted: 24 May 2019; Published: 28 May 2019 Abstract: A UHPLC-QQQ-MS/MS method was developed to quantify the significant constituents in Wen-Dan Decoction (WDD), a traditional Chinese medicine. Analysis of 19 compounds was conducted on an ACQUITY UPLC® BEH C18 Column (2.1 50 mm, 1.7 µm) using elution with a gradient elution × of acetonitrile and 0.05% (v/v) formic acid in water. A triple quadrupole mass spectrometer was operated in negative ionization mode and positive ionization mode by multiple reaction monitoring (MRM), respectively. All calibration curves showed acceptable linearity (r 0.9950). The RSDs of ≥ intra- and inter-day precisions of low, mid and high concentrations were 8.88%. The repeatabilities ≤ (RSDs 7.17%) and stabilities (RSD 4.79%) of the samples were qualified. The recoveries were ≤ ≤ found in the range of 93.07 3.86 to 103.98 2.98% with the RSD varying between 1.30 and 7.86%. ± ± The final rapid, sensitive, precise, accurate and reliable UHPLC-QQQ-MS/MS method was used for the simultaneous quantification of 19 constituents in WDD and its commercial preparations. The strategy of combining the contents of the 19 chemicals in a daily dose of the WDD preparations with the hierarchical cluster analysis and the 3D principal component analysis was employed to effectively distinguish the WDD preparations provided by the different suppliers, which represents a contribution to the evaluation and control of the quality of WDD (or other decoctions consisting of the same herbs) and the preparations of WDD in other dosage forms such as tablets and granules. Keywords: quantitative analysis; UHPLC-QQQ-MS/MS; Wen-Dan Decoction; commercial preparations 1. Introduction Wen-Dan Decoction (WDD), a Chinese medicine prescription, is formulated from Pinelliae Rhizoma, Bambusae Caulis in Taenias, Aurantii Fructus Immaturus, Citri Reticulatae Pericarpium, Molecules 2019, 24, 2031; doi:10.3390/molecules24112031 www.mdpi.com/journal/molecules Molecules 2019, 24, 2031 2 of 19 Glycyrrhizae Radix Et Rhizoma, Poria, Zingiberis Rhizoma and Jujubae Fructus, which are all included in the Chinese Pharmacopoeia (Version 2015) [1]. In April 2018, National Administration of Traditional Chinese Medicine of People’s Republic of China launched the “Ancient Classical Chinese Medicine Formula Catalogue (First edition)” notice containing WDD providing the basis for the second-development of WDD (http://kjs.satcm.gov.cn/zhengcewenjian/2018-04-16/7107.html). In China, WDD is clinically used for the treatment of insomnia, asthenia, dizziness, gastritis, ischemic stroke, Meniere’s disease, metabolic syndrome, depression and schizophrenia [2]. Moreover, in the past decades, it had been reported that WDD had pharmacological effects in the treatment of Alzheimer’s disease, negative emotions of insomnia, cardiac fibrosis, disorders of lipid metabolism, etc [3,4]. Although the clinical applications and pharmacological effects of WDD have been well explored, chemical composition analysis of WDD is still scant. For example, only a small number of the chemical constituents in WDD such as liquiritin, naringin, hesperidin and glycyrrhizic acid were quantified by Xu et al., 6-gingerol in WDD was determined by Li et al. and the fingerprint of WDD was reported by Wang et al. [5–7]. The development and application of mass spectrometry provides a quick and convenient method for the identification and quantification of contents in complex natural medicine extracts. For instance, Li et al. utilized UHPLC-MS for the identification and quantification of chemical constituents in the traditional Chinese medicinal formula Qi-Fu-Yin [8]. In this study, an ultra-performance liquid chromatography method combined with triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) was employed to identify and quantify some significant constituents in WDD. A total of 19 compounds, namely one organic acid (succinate), one alkaloid (synephrine), two triterpenoids (pachymic acid and dehydropachymic acid), three phenols (6-gingerol, 8-gingerol and 10-gingerol) and 12 flavonoids (rutin, eriocitrin, liquiritin, isoliquiritin, iosnaringin, naringin, hesperidin, neohesperidin, liquiritigenin, didymin, poncirin and tangeretin) were rapidly identified by the retention time and the MS/MS spectra data in both negative and positive ion modes. The structures of the 19 chemicals are shown in Figure1. The contents of the ingredients were determined by comparison with reference substances of known purities. Finally, a reliable and validated UHPLC-QQQ-MS/MS method was developed for the content determination of 19 compounds in WDD and its marketed preparations. Fick’s first law of diffusion, differences between the extraction equipment and the dosage forms were utilized to account for the content variation among batches of traditional Chinese herb preparations and its marketed preparations. Furthermore, hierarchical cluster analysis and 3D principal component analysis were used to effectively distinguish the source of the preparations with combining the contents of the 19 components in WDD in a daily dose. The radar charts of the “total daily dosage of measured ingredients” of the seven pieces of traditional Chinese medicine and the radar charts of the 19 chemicals contents in a daily dose could help the researchers and doctors to make a better choice of the source of the WDD when they were going to use WDD. This study will contribute to the quality control, pharmacological research and clinical application of WDD and its commercial preparations. Molecules 2019, 24, 2031 3 of 19 Molecules 2019, 24, x FOR PEER REVIEW 3 of 20 FigureFigure 1. 1.Structures Structures ofof 1919 chemicals analyzed analyzed in inWDD. WDD. 2. Results2. Results 2.1. Optimization2.1. Optimization of LC of LC Conditions Conditions A gradientA gradient elution elution chromatographic chromatographic procedure procedure wa wass used used to to achieve achieve chromatographic chromatographic separation separation of theof 19 the components 19 components in in a a shortshort period period of of time. time. An ACQUITY An ACQUITY UPLC® UPLC BEH C18® BEH column C18 (1.7 column μm, 2.1×50 (1.7 µm, 2.1 mm)50 mm was) wasemployed employed for better for peak better symmetries peak symmetries than others, than and others, acetonitrile and acetonitrilewas applied for was its applied better for ×chromatographic separation than that of methanol. The optimized column temperature of 35 °C and its better chromatographic separation than that of methanol. The optimized column temperature of 35 flow rate of 0.3 mL·min−1 were also contributed to the efficient separation. The 0.05% formic acid- C and flow rate of 0.3 mL min 1 were also contributed to the efficient separation. The 0.05% formic ◦ water solution provided· better− peak shapes. After these optimization procedures, all reference acid-watercompounds solution could provided be generally better and peak chromatogr shapes.aphically After theseseparated optimization within 30 minutes. procedures, all reference compounds could be generally and chromatographically separated within 30 min. 2.2. Optimization of MS Conditions 2.2. Optimization of MS Conditions Studies on the individual mass spectra of the 19 compounds were performed by injecting the Studiescorresponding on the reference individual solutions mass spectrainto the ofmass the sp 19ectrometer compounds in both were positive performed and negative by injecting ion the correspondingmodes. The referenceresults of solutionsthe pilot study into showed the mass that spectrometer the product ion in bothpeaks positiveof succinate, and liquiritin, negative ion modes.eriocitrin, The results rutin, nariruti of then, pilot naringin, study hesperidin, showed neohesperidin, that the product liquiritigenin, ion peaks isoliquiritin, of succinate, didymin, liquiritin, eriocitrin,poncirin rutin, and narirutin,dehydropachymic naringin, acid hesperidin, performed neohesperidin,well in negative ion liquiritigenin, modes. Meanwhile, isoliquiritin, synephrine, didymin, 6-gingerol, tangeretin, 8-gingerol, 10-gingerol and pachymic acid gave better