September 2013 Regular Article Chem. Pharm. Bull. 61(9) 941–951 (2013) 941 Simultaneous Determination of Isoflavones, Saponins and Flavones in Flos Puerariae by Ultra Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry Jing Lu,a Yuanyuan Xie,a Yao Tan, a Jialin Qu,a Hisashi Matsuda,b Masayuki Yoshikawa,b and Dan Yuan*,a a School of Traditional Chinese Medicine, Shenyang Pharmaceutical University; 103 Wenhua Rd., Shenyang 110016, P.R. China: and b Department of Pharmacognosy, Kyoto Pharmaceutical University; Shichono-cho, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan. Received April 7, 2013; accepted June 6, 2013; advance publication released online June 12, 2013 An ultra performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (QTOF/MS) method is established for the rapid analysis of isoflavones, saponins and flavones in 16 samples originated from the flowers of Pueraria lobata and P. thomsonii. A total of 25 isoflavones, 13 saponins and 3 flavones were identified by co-chromatography of sample extract with authentic standards and comparing the retention time, UV spectra, characteristic molecular ions and fragment ions with those of authentic standards, or tentatively identified by MS/MS determination along with Mass Fragment software. Moreover, the method was validated for the simultaneous quantification of 29 components. The samples from two Pueraria flowers significantly differed in the quality and quantity of isoflavones, saponins and flavones, which allows the possibility of showing their chemical distinctness, and may be useful in their standardiza- tion and quality control. Dataset obtained from UPLC-MS was processed with principal component analysis (PCA) and orthogonal partial least squared discriminant analysis (OPLS-DA) to holistically compare the dif- ference between both Pueraria flowers. Key words Pueraria flower; isoflavone; saponin; flavone; ultra performance liquid chromatography; quadru- pole time-of-flight mass spectrometry Pueraria lobata (WILLD.) OHWI and P. thomsonii BENTH. possess some different biological activities. For example, tec- (Leguminosae) are two commonly used medicinal plants. toridin shows potent effects on the inhibition of prostaglandin Radix Puerariae, the roots on both plants, is a well known E2 (PGE2) production, while kakkalide has no significant herbal medicine mentioned in Chinese and Japanese Pharma- effect.17) Actually, Flos Puerariae Lobatae and Flos Puerariae copoeias,1,2) and has been used to treat common cold, influen- Thomsonii have been indiscriminate in clinical use and in the za, and wrist and shoulder stiffness, and as an antidipsotropic preparation of phytopharmaceuticals, which is a serious issue agent.3,4) On the other hand, Flos Puerariae, the flowers on in relation to the efficacy, quality control and safety. both plants, is a medicinal and edible cognate that has been Several holistic chemical profiling methods of the flowers used to ameliorate liver injury5) and relieve some symptoms of P. lobata and P. thomsonii have been reported, such as caused by excessive drinking of alcohol, such as drunkenness, quantification of 3–12 isoflavonoid glycosides and 3 saponins headache, and red face, in China, Japan and Korea.6) Many using HPLC-UV or HPLC-evaporative light scattering detec- pharmacological studies reported the extensive biological ac- tor (ELSD).9,18–20) Liu et al.21) and Shi et al.22) reported the tivities of the flower, including hepatoprotective, antioxidant, screening and identification of 13 or 18 active isoflavones hypoglycemic, and hypolipidemic activities.5,7,8) The phytoes- from ethanolic extract of P. lobata flower by using bovine trogenic isoflavonoids and saponins are the major components serum albumin (BSA) functionalized iron oxide magnetic 9–11) in the flowers as well as roots, and should be responsible nanoparticles (Fe3O4 MNPs) coupled with HPLC-MS/MS and for these aforementioned activities.12–14) 2,2′-diphenyl-1-picrylhydrazyl (DPPH) spiking HPLC-MS/ The current study is focusing on both Pueraria flowers MS. These studies demonstrate the differences in the isofla- because the phytopharmaceuticals containing the Pueraria vonoids that are present in rich amount in both flowers, i.e., flower, such as the Pueraria flower tea and drink,15) have be- kakkalide is the major component of P. lobata flower, and come popular for the treatment of alcohol intoxication and tectoridin is that of P. thomsonii flower. Concerning saponins, liver injury in China and Japan. Radix Puerariae has been only kaikasaponin III, kakkasaponin I and soyasaponin I were separately listed and recorded as Radix Puerariae Lobatae and analyzed,9,18) which possess hypoglycemic, hypolipidemic, Radix Puerariae Thomsonii in Chinese Pharmacopoeia 2005 and hepatoprotective activities.12,23) It is known that saponins and 2010 editions due to a great difference in puerarin amount are one of the important components in Pueraria sp.,24) thus, between both roots.1,16) The differences in isoflavone composi- chemical profiling and quantification of saponins are neces- tion between both Pueraria flowers consist in that kakkalide is sary for the quality control of the raw materials of Flos Puer- the major isoflavone of Flos Puerariae Lobatae, and that tec- ariae as well. toridin is that of Flos Puerariae Thomsonii. Although as phy- Generally, using conventional HPLC methods is difficult toestrogens, kakkalide and tectoridin show similar estrogenic and time-consuming for simultaneous analysis of isoflavones, effects, several studies demonstrate that both isoflavones also saponins and flavones due to the relatively low efficient sta- tionary phases. It is also one of the challenges for the accurate The authors declare no conflict of interest. quantification of the saponins possessing weak UV absorption * To whom correspondence should be addressed. e-mail: [email protected] © 2013 The Pharmaceutical Society of Japan 942 Vol. 61, No. 9 using a UV detector. Compared to HPLC, ultra performance Shenyang Pharmaceutical University. liquid chromatography (UPLC) can provide a high peak ca- Instrumentation and Chromatographic Conditions pacity, greater resolution, increased sensitivity, and higher UPLC analysis was performed on a Waters ACQUITY speed of analysis. For example, Wu et al. simultaneously iden- HSSC18 column (2.1×100 mm, 1.8 µm, Waters, Milford, MA, tified 92 compounds, including steroidal saponins, homoisofla- U.S.A.) at 40°C. The mobile phase consisted of (A) water con- vanones and other components in Sheng-Mai San, a traditional taining 0.2% formic acid and (B) acetonitrile containing 0.2% Chinese medical formula, within 14 min by UPLC-high defi- formic acid with gradient elution (linear gradient from 5% B nition mass spectrometry (HDMS).25) TOF/MS can provide to 15% B in 1 min, followed by linear gradient from 15% B accurate molecular formula for unknown compounds, which to 45% B between 1 and 7 min, and linear gradient from 45% is very helpful for structural identification. Chu et al. charac- B to 50% B between 7 and 9 min, finally a 9–10 min linear terized 22 astragalosides from the methanolic extract of Radix gradient from 50% B to 65% B). Re-equilibration duration Astragali using quadrupole time-of-flight mass spectrometry was 2 min between individual runs. The flow rate was kept (QTOF/MS) method.26) Since many unknown saponins may at 0.6 mL/min and 2 µL of standard and sample solution were be present in the Pueraria flower and most are new or without injected in each run. reference standards, using TOF/MS to identify them is very Identification of marker compounds by UPLC-MS was per- important based on the molecular formula and fragmentation formed on Waters QTOF Xevo G2 (Waters Corp., Manchester, of the known saponins. U.K.) equipped with an electrospray ionization (ESI) source, In the present study, we developed a UPLC-QTOF/MS which gives a resolution of 10000 (FWHM) and mass accu- method for the simultaneous analysis of isoflavones, saponins racy error less than 5 ppm. Leucine-enkephalin was used as and flavones for the evaluation of the chemical consistency the lock mass to generate an [M−H]− ion (m/z 554.2615) in between in the flowers of P. lobata and P. thomsonii. the LockSpray mode at a concentration of 50 pg/µL at an infu- sion flow rate of 10µ L/min. The ESI source was operated in Experimental negative ionization mode with the capillary voltage at 2.5 kV, Chemicals and Reagents Thirty-one reference com- and the cone voltage was set to 40 V. Source and desolvation pounds were used in the present study, and the chemical temperatures were set at 130 and 450°C, respectively. The structures were shown in Fig. 1. Puerarin (1), daidzin (5), cone and nebulization gas flows were 50 and 800 L/h, respec- daidzein (16) and luteolin (18) were purchased from Funa- tively. All data collected in centroid mode were acquired using koshi Co., Ltd. (Tokyo, Japan), and glycitin (6) was from Masslynx™ NT 4.1 software (Waters Corp., Milford, MA, Shanghai Forever Biotech Co., Ltd. (Shanghai, China). Other U.S.A.). used reference compounds were isolated from the extracts Two different MS scanning experiments were used. (1) MSE of the flowers of P. lobata and P. thomsonii in our previous experiment (E represents collision energy) uses an intelligent studies.9,10,13,27) They included 6-hydroxygenistein-6, 7-di-O- approach where parallel alternating scans are acquired both at glucoside (2), 6-hydroxygenistein-7-O-glucoside (7), rutin (8), low-collision