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Simultaneous quantification of major bioactive constituents from Zhuyeqing Liquor by HPLC-PDA

Simultaneous quantification of major bioactive constituents from Zhuyeqing Liquor by HPLC-PDA Background: Zhuyeqing Liquor (ZYQL) is a famous traditional Chinese functional liquor. For quality control of ZYQL products, quantitative analysis using high-performance liquid chromatography coupled with photodiode array detector (HPLC-PDA) was undertaken. Methods: Eighteen compounds from ZYQL were simultaneously detected and used as chemical markers in the quantitative analysis, including 3-hydroxy-4,5(R)-dimethyl-2(5H)-furanone (M1), isobiflorin (M2), vanillic acid (M3), biflorin (M4), genipin 1-O-β-D-gentiobioside (M5), 1-sinapoyl-β-D-glucopyranoside (M6), geniposide (M7), epijasmnoside A(M8), ferulic acid (M9), luteolin 8-C-β-glucopyranoside (M10), isoorientin (M11), narirutin (M12), hesperidin (M13), 6′-O-sinapoylgeniposide (M14), 3,5-dihydroxy-3′,4′,7,8-tetramethoxyl flavones (M15), 3′,4′,3,5,6,8-hexamethoxyl flavone (M16), kaempferide (M17), and tangeretin (M18). Results: The separation by gradient elution was achieved on SHIMADZU VP-ODS column (4.6 × 150 mm, 5 μm) at 30°C with methanol (A)/0.1% phosphoric acid (B) as the mobile phase. The detection wavelengths were 254, 278, and 335 nm. The optimized HPLC method provided a good linear relation (r ≥ 0.9991 for all the target compounds), satisfactory precision (RSD values less than 1.47%) and good recovery (97.40% to 103.44%). The limits of detection −4 −4 ranged between 0.20 × 10 and 64.90 × 10 μg/μL for the different analytes. Furthermore, the optimum sample preparation was obtained from HPD column eluted with water and 95% ethanol, respectively. Conclusions: Quality control of ZYQL products, in total seven samples and twelve parent plants, was examined by this method, and results confirmed its feasibility and reliability in practice. Keywords: Zhuyeqing Liquor; Bioactive constituent; Quantitative analysis; HPLC-PDA Background Lysimachia capillipes Hemsl. (Paicao), Angelica sinensis Zhuyeqing Liquor (ZYQL), authorized as a functional (Oliv.) Diels (Danggui), Kaempferia galanga L. (Shannai), health liquor in 1998 by the Ministry of Public Health in Citrus reticulata Blanco (Chenpi), Chrysanthemum mori- China, is a famous traditional Chinese functional liquor. folium Ramat. (Juhua), Amomum villosum Lour. (Sharen), The history of ZYQL could be traced back to the Warring Santalum album L. (Tanxiang), Eugenia caryophyllata Thunb. States Period and became popular among people in the (Gongdingxiang), Aucklandia lappa Decne. (Guangmuxiang), South and North Dynasties. In the Tang Dyansty and Song and Lysimachia foenum-graecum Hance (Linglingxiang). Dynasty, it had reached its climax (Yang 2007). ZYQL was According to its long-term history use, ZYQL has various designed based on the principles of traditional Chinese biological properties such as anti-oxidant, anti-fatigue, and medicine (TCM) and comprises 12 herbs: Lophatherum immunoenhancement (Han 2007). gracile Brongn. (Zhuye), Gardenia jasminoides Ellis (Zhizi), Up to now, many studies show solicitude for the color, smell, and taste of the health functional liquor; few studies pay close attention to its chemical constituents and quality * Correspondence: wjh.1972@aliyun.com School of Traditional Chinese Materia Medica 49#, Shenyang Pharmaceutical control. Currently, chemical analytical methods for the University, Wenhua Road 103, Shenyang 110016, People’s Republic of China 2 quality control of ZYQL have not been established. There- School of Pharmacy, Shihezi University, Shihezi 832002, People’s Republic of fore, it is necessary to establish a rapid and effective method China Full list of author information is available at the end of the article © 2014 Gao et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 2 of 10 http://www.jast-journal.com/content/5/1/34 for the quantitative analysis of the health functional liquor. was purified by a Milli-Q water purification system In this study, the system of high-performance liquid (Millipore, Billerica, MA, USA). Phosphoric acid (analytical chromatography coupled with photodiode array detector grade) was purchased from Tianjin Guangfu Chemical (HPLC-PDA) was used for analyzing the chemical profile Reagent Co. Ltd. (Tianjin, China). Other solvents from of ZYQL. This method includes many advantages like high Tianjin Guangfu Chemical Reagent Co. Ltd. (Tianjin, speed detection, excellent peak shapes, less solvent usage, China) were all of analytical grade. well-defined chemical constituents, and simultaneous Reference compounds of M1 to M18 (Figure 1) were detection of multi-constituents, which is better than finger- isolated previously from ZYQL by author, structures of printing. Thus, simultaneous determination by RP-HPLC which were elucidated by comparison of spectral data (UV, 1 13 method is suitable for quantitative analysis and can be used MS, HNMR, and C NMR) with the literature data (Lin as an effective tool to evaluate herbal medicine products. et al. 2006; Okamurα et al. 1998; Huang et al. 2012; Zhang and Chen 1997; Ma et al. 2009; Miyake et al. 2007; Liu et al. Methods 2011; Chen et al. 2008; Rayyan et al. 2005; Kumarasamy Chemicals and materials et al. 2004; Ke et al. 1999; Yoo et al. 2002; Dinda et al. 2011; Methanol (HPLC-grade) was purchased from Fisher Esteban et al. 1986; Ballester et al. 2013; Wang et al. 2010; Scientific Co. (Franklin, MA, USA). Water for HPLC analysis Hòrie et al. 1998). The purity of each reference standard Figure 1 Structures of compounds M1 to M18. Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 3 of 10 http://www.jast-journal.com/content/5/1/34 −1 was determined to be above 98% by HPLC analysis 6′-O-sinapoylgeniposide (M14) 5.312 mg mL ,3,5-dihy- based on a peak area normalization method, detected droxy-3′,4′,7,8-tetramethoxyl flavones (M15) 5.021 mg −1 by HPLC-PDA and confirmed by HR-ESI-TOF-MS and mL ,3′,4′,3,5,6,8-hexamethoxyl flavone (M16) 15.005 mg −1 −1 NMR spectroscopy. mL ,kaempferide (M17) 6.408 mg mL , and tangeretin −1 The samples of different batch and different alcoholicity (M18) 17.155 mg mL . A mixed solution containing all of ZYQL and the 12 parent plants were provided by the 18 standards was prepared as accurately as 108 μL M1, Shanxi XinghuaCun Fen Jiu Group Co., Ltd. (Shanxi, 6.8 μL M2,2.4 μL M3,8.0 μL M4, 165 μL M5,96 μL M6, China). The 12 parent plants were identified by Professor 106 μL M7,3.4 μL M8,8.2 μL M9,9.5 μL M10,7.9 μL Jincai Lu (Shenyang Pharmaceutical University, Shenyang, M11,35 μL M12,40 μL M13,80 μL M14,8.2 μL M15, China). The voucher specimen was deposited at Shenyang 11 μL M16,12 μL M17, and 4.6 μL M18 and were placed Pharmaceutical University (Shenyang, China) and regis- in a 2-mL flask with stopper, diluted with methanol to tered under the number ZYQL 2011050101. make sure the volume reached 2 mL. All prepared solu- tions were respectively stored in a refrigerator at 4°C Instrumentation and chromatographic conditions when not in use. Chromatographic analysis was performed on Waters 2695 Alliance HPLC system (Waters Co., Milford, MA, Treatment for samples USA) with Waters 2998 PDA detector. Chromatographic For the analysis, 40 mL of ZYQL were evaporated in vacuum separation was carried on a SHIMADZU VP-ODS column at 50°C to dryness. The dry residue was processed as follows (4.6 mm × 150 mm, 5 μm; Shimadzu, Kyoto, Japan) at a in order to obtain better analytical results: The residue was column temperature of 30°C using methanol (A) and 0.1% dissolved with water (10 mL) and applied to an HPD col- phosphoric acid (B) as mobile phase with the gradient umn eluted with water (150 mL); the water eluent was dis- elution procedure show in Table 1. The flow rate was carded and then eluted with 95% ethanol (150 mL). The 95% set at 1.0 ml/min and the detection wavelengths were ethanol eluent was condensed and dissolved with methanol 254 nm (for compounds M1 to M5, M7, M8,and M17), andthenplacedina2-mL flaskwithstopper, with a 278 nm (for compounds M12 and M13), and 335 nm methanol-metered volume. Prior to HPLC analysis, the sam- (for compounds M6, M9 to M11, M14 to M16,and ple solution was passed through a 0.22-μm millipore filter. M18), which were chosen based on the maximum absorp- The 12 crude dried parent plants were pulverized and tion of all the tested compounds. The injection volume sifted through 40 mesh sieve, respectively. One gram of the was 10 μL, and the analytes were well separated in chro- powder from the parent plant was placed in a 50-mL flask matographic conditions above. with stopper, then weighed again correctly, and extracted by ultrasonic method with 20 mL methanol for 30 min. Standard solution preparation Then standing, it was cooled down to room temperature Individual stock solutions were prepared by dissolving the (22°C) and the weight was mended to the incipient weight standards in methanol to obtain 3-hydroxy-4,5(R)-di- with methanol. Prior to HPLC analysis, the sample solution −1 methyl-2(5H)-furanone (M1)19.920 mgmL , isobiflorin was passed through a 0.22-μm millipore filter. −1 −1 (M2)8.330 mg mL , vanillic acid (M3)5.802 mg mL , −1 biflorin (M4) 3.911 mg mL ,genipin 1-O-β-D-gentiobio- Validation of the method −1 side (M5) 4.405 mg mL , 1-sinapoyl-β-D-glucopyranoside Calibration curves −1 −1 (M6) 1.115 mg mL ,geniposide (M7)23.804 mgmL , Linearity was established by the injection of 1, 2, 4, 8, −1 epijasmnoside A (M8) 12.060 mg mL , ferulic acid (M9) 12, 16, and 20 μL of the mixed reference standard solu- −1 2.515 mg mL , luteolin 8-C-β-glucopyranoside (M10) tion prepared, respectively. Calibration graphs were plot- −1 −1 1.510 mg mL , isoorientin (M11) 2.203 mg mL ,nairutin ted subsequently based on linear regression analysis of −1 −1 (M12) 1.032 mg mL ,hesperidin(M13) 4.801 mg mL , the integrated peak (Y) versus content (X, μg). Table 1 Time program of the gradient elution Time (min) Flow (mL/min) Methanol (%) 0.1% Phosphatic Limits of detection and quantitation acid (%) In order to evaluate the limits of detection (LODs) and the 01 5 95 limits of quantification (LOQs) of the compounds, mixed 70 1 55 45 standard stock solution was further diluted serially to pro- 75 1 60 40 vide a series of appropriate concentrations, and an aliquot of the diluted solutions was injected into HPLC for ana- 110 1 80 20 lysis. The LOD and LOQ for each analyte was calculated 120 1 98 2 with corresponding standard solution on the basis of a 125 1 98 2 signal-to-noise ratio (S/N) of 3 and 10, respectively. Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 4 of 10 http://www.jast-journal.com/content/5/1/34 (A) 0.15 5 16 0.10 335 nm 13 14 18 278 nm 0.05 1 10 11 12 15 254 nm 2 4 8 0.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 Minute (B) 0.15 254 nm 2 4 8 0.10 0.05 0.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 0.10 278 nm 0.05 0.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 0.15 335 nm 15 16 18 0.10 9 10 0.05 0.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 Minute Figure 2 Stack views. (A) Different detector-wavelength HPLC chromatograms of mixed reference standards (from up to down: 335, 278, 254 nm). Column: SHIMADZU VP-ODS column (4.6 mm × 150 mm, 5 μm), temperature of 30°C. (B) HPLC chromatograms of M1 to M18 and mixed reference standards (from up to down: 254 nm M1, M2, M3, M4, M5, M7, M8, M17, mixed reference standards; 278 nm M12, M13, mixed reference standards; 335 nm M6, M9, M10, M11, M14, M15, M16, M18, mixed reference standards). AU AU AU AU Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 5 of 10 http://www.jast-journal.com/content/5/1/34 Table 2 Optimization of the treatment method of Zhuyeqing Liquor (μg/mL) Compound Treatment method Method 1 Method 2 Method 3 Method 4 Method 5 M1 ND 5.2494 3.1331 9.5943 16.4270 M2 0.0888 0.5371 0.5078 0.5818 0.5942 M3 0.0922 0.0906 0.0388 0.0939 0.1063 M4 0.0263 0.4980 0.5098 0.5069 0.5153 M5 ND 44.0545 101.6907 100.6372 101.7888 M6 0.1479 0.1880 0.1902 0.1927 0.1936 M7 45.2421 563.2436 570.3556 566.0022 574.2514 M8 20.8481 24.6279 25.6049 24.5513 25.7319 M9 0.1931 0.1782 0.1906 0.1918 0.1968 M10 0.0324 0.0526 0.0600 0.0705 0.0736 M11 0.2009 0.4601 0.4871 0.4915 0.4954 M12 0.7071 1.0544 1.0683 1.0699 1.0877 M13 1.1371 2.5392 2.8461 2.8029 2.9909 M14 ND 3.4939 4.0839 4.0563 4.3756 M15 ND 0.0641 0.0678 0.0688 0.0689 M16 0.5189 0.6113 0.5842 0.3282 0.6623 M17 2.9599 2.9776 2.1520 1.6446 2.9957 M18 0.4448 0.5307 0.4278 0.4673 0.5413 Sum 72.6396 650.4512 713.9987 713.3521 733.0967 Sample in optimization of the treatment method was 45° Zhuyeqing Liquor (20130207). Method 1, acetoacetate extract; method 2, n-butanol extract; method 3, 70% ethanol treatment; method 4, SPE column eluted with methanol; method 5, HPD column eluted with ethanol. ‘ND’ in the ‘Compound’ column expressed under LOQ. Total content of the 18 investigated compounds. Precision and stability Repeatability and recovery The precision of the chromatographic system was vali- The repeatability test was analyzed by injecting six inde- dated by injecting 10 μL of the mixed reference solution pendently prepared samples (45° ZYQL (20130207), the six times during 1 day. Stability study was performed with concentration, and prepared method as the ‘Treatment for sample solution in 48 h (the time points are 0, 5, 10, 15, samples’). The RSD value of concentration was adopted to 25, 35, and 48 h, respectively). Variations were expressed evaluate repeatability. The recovery tests were studied by by relative standard deviations (RSD) of peak area. adding the proper amount of mixed-reference standard Figure 3 Stack views of 45° Zhuyeqing Liquor preparation method HPLC chromatograms (254 nm, from up to down: method 1, method 2, method 3, method 4 and method 5). Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 6 of 10 http://www.jast-journal.com/content/5/1/34 Table 3 Regression equations, correlation coefficients, and linear range for 18 analytes in Zhuyeqing Liquor Analyte Time Linear regression (t ) Regression equation (n = 3) Correlation Linear range (μg) LOD LOQ coefficients −4 −4 (10 μg/μL) (10 μg/μL) M1 14.004 Y = 5.48e + 003X − 1.45e + 003 0.9998 1.08~21.63 64.90 216.34 −2 −1 M2 22.819 Y = 2.86e + 006X − 8.84e + 002 0.9999 2.80 × 10 ~5.60 × 10 1.68 5.60 −3 −2 M3 23.573 Y = 4.73e + 006X − 5.52e + 003 0.9991 1.70 × 10 ~3.40 × 10 0.20 0.67 −2 −1 M4 25.069 Y = 1.62e + 006X − 3.23e + 003 0.9997 1.55 × 10 ~3.10 × 10 3.10 10.34 −1 M5 26.671 Y = 5.41e + 005X − 3.44e + 004 0.9994 3.63 × 10 ~7.25 2.42 8.08 −2 M6 28.087 Y = 1.31e + 006X − 4.70e + 003 0.9998 5.25 × 10 ~1.05 8.40 28.0 M7 29.646 Y = 7.08e + 005X + 4.19e + 003 0.9998 1.26~25.21 25.46 84.87 −2 −1 M8 32.875 Y = 9.81e + 005 X - 5.87 e + 003 0.9998 2.04 × 10 ~4.08 × 10 4.08 13.60 −2 −1 M9 37.264 Y = 3.17e + 006X − 1.51e + 004 0.9992 1.03 × 10 ~2.06 × 10 2.06 6.87 −3 −1 M10 40.883 Y = 1.65e + 006X − 1.26e + 002 0.9993 7.10 × 10 ~1.42 × 10 2.13 7.11 −3 −1 M11 42.396 Y = 2.38e + 006X − 6.23e + 003 0.9991 8.70 × 10 ~1.74 × 10 1.74 5.83 −2 −1 M12 46.878 Y = 2.67e + 006X − 3.22e + 002 0.9998 1.75 × 10 ~3.50 × 10 5.25 17.51 −2 M13 50.008 Y = 1.69e + 006 X + 3.71e + 003 0.9998 9.56 × 10 ~1.91 5.74 19.12 −1 M14 58.096 Y = 4.55e + 005X − 2.11e + 003 0.9998 2.13 × 10 ~4.25 12.75 42.50 −3 −2 M15 74.987 Y = 2.15e + 006X − 7.62e + 002 0.9991 4.10 × 10 ~ 8.20 × 10 3.28 10.95 −2 M16 80.609 Y = 3.59e + 006X − 2.08e + 004 0.9999 8.23 × 10 ~1.65 0.55 1.84 −2 −1 M17 83.248 Y = 1.97e + 005X − 3.36e + 003 0.9991 3.85 × 10 ~7.70 × 10 15.40 51.32 −2 −1 M18 85.890 Y = 2.69e + 006X − 1.38e + 004 0.9996 3.93 × 10 ~ 7.86 × 10 0.79 2.64 Y is the peak area and X is the content of standard solutions; LOD refers to the limits of detection, S/N = 3; LOQ refers to the limits of quantity, S/N = 10. Table 4 Precision, stability, recovery, and repeatability data of 18 analytes in Zhuyeqing Liquor Analyte Precision (n = 6) Stability Recovery (n = 6) Repeatability (n =6) RSD (%) Concentrations RSD Original Spiked Detected Recovery RSD Average RSD (%) (μg) (μg) (μg) (%) (%) concentration (%) (mg/mL) (μg/mL) M1 1.08 0.92 1.70 164.16 162.26 326.57 100.17 3.10 16.0862 ± 0.2722 1.50 −2 M2 2.80 × 10 0.82 1.52 7.11 4.20 11.41 102.44 1.84 0.5580 ± 0.0045 1.39 −3 M3 1.70 × 10 1.30 1.66 0.27 0.26 0.52 97.40 2.52 0.1072 ± 0.0020 1.96 −2 M4 1.55 × 10 0.78 1.08 5.88 2.33 8.23 100.88 2.27 0.5154 ± 0.0032 0.64 −1 M5 3.63 × 10 0.87 1.62 1217.80 54.39 1273.19 101.83 3.30 101.1963 ± 0.7206 0.72 −2 M6 5.25 × 10 0.86 1.58 6.00 7.88 14.15 103.44 1.68 0.1940 ± 0.0019 1.00 M7 1.26 0.49 1.18 1270.60 1260.30 2543.54 101.00 0.78 568.4991 ± 2.7722 0.98 −2 M8 2.04 × 10 1.04 1.76 211.50 102.00 316.34 102.79 1.46 25.1942 ± 0.2548 1.45 −2 M9 1.03 × 10 0.30 1.49 2.82 1.55 4.37 100.76 2.63 0.1947 ± 0.0018 1.00 −3 M10 7.10 × 10 1.10 1.67 3.22 1.07 4.28 99.54 3.26 0.0728 ± 0.0011 1.61 −3 M11 8.70 × 10 1.15 1.62 4.65 1.31 5.96 100.66 3.09 0.5101 ± 0.0057 1.28 −2 M12 1.75 × 10 1.09 1.39 2.43 2.63 5.14 103.13 1.48 1.0800 ± 0.0155 1.44 −2 M13 9.56 × 10 1.02 1.36 37.69 14.34 52.20 101.14 1.99 2.8911 ± 0.0351 1.21 −1 M14 2.13 × 10 0.74 1.59 943.73 31.88 975.00 98.10 2.25 4.2790 ± 0.0314 1.30 −3 M15 4.10 × 10 1.47 1.75 1.99 1.23 3.22 100.30 2.66 0.0645 ± 0.0009 1.53 −2 M16 8.23 × 10 0.91 1.58 21.52 12.35 33.77 99.20 2.44 0.6444 ± 0.0052 0.81 −2 M17 3.85 × 10 0.93 1.75 133.12 115.50 249.54 100.79 2.63 2.9457 ± 0.0498 1.36 −2 M18 3.93 × 10 1.11 1.49 10.02 11.79 21.89 100.65 1.58 0.5368 ± 0.0060 1.13 RSD refers to relative standard deviation. Samples in stability, recovery, and repeatability methods were taken from 45°Zhuyeqing Liquor (20130207). Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 7 of 10 http://www.jast-journal.com/content/5/1/34 solution to the sample (45° ZYQL (20130207)), and then phase of water and methanol rather than water and aceto- processed by the method described in the ‘Treatment for nitrile. Therefore, in this work, the optimum resolution was samples’ section to yield the final concentration. The ex- achieved using methanol (A) and 0.1% phosphatic acid (B) periment was repeated six times. as mobile phase, with a column temperature of 30°C at dif- ferent detection wavelengths, which were described in ‘In- Results and discussion strumentation and chromatographic conditions’ section, Optimization of chromatographic conditions with gradient elution (Table 1). All 18 standard analytes To improve resolution and sensitivity of analysis but reduce could be eluted with baseline separation in 90 min. Repre- analytical time, the following chromatographic conditions sentative chromatograms for the mixed reference standard were optimized (Gao et al. 2013), including different mobile and 18 standard compounds were shown in Figure 2A,B. phase compositions (methanol, acetonitrile, and aqueous phosphatic acid of different concentrations), column Optimization of sample preparation temperature, and wavelength: To inhibit ionization of the In order to eliminate the water-soluble constituents and ob- acidic ingredients in the ZYQL sample, phosphatic acid was tain the liposoluble constituents, the optimization of sample added in mobile phase. Two mobile phase systems, preparation was performed using 45° ZYQL (20130207). methanol-phosphatic acid aqueous solution and acetonitrile- Forty milliliters of ZYQL was evaporated in vacuum at 50°C phosphatic acid aqueous solution, were examined, and then to dryness. And the following five methods were choosen to column temperatures at 25°C, 30°C, 40°C, and 50°C were select the best method for sample preparation. First, the dry compared. A sensitive wavelength was determined by PDA residue was suspended with water (10 mL) and extracted with reference compounds. Present researches indicated that with acetoacetate (10 mL). The acetoacetate extract was con- better separation and results were obtained using a mobile densed and then methanol was used to meter the volume 0.10 254 nm 5 7 0.08 45°ZYQL 0.06 45°FenJiu 2 4 8 0.04 Mixed standards 0.02 3 Methanol 0.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 0.10 278 nm 0.08 0.06 0.04 0.02 0.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 0.10 335 nm 0.08 0.06 9 11 10 15 0.04 0.02 0.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 Minute Figure 4 Stack views of different detector-wavelength HPLC chromatograms. (From up to down: 45° Zhuyeqing Liquor, 45° FenJiu, mixed reference standards, blank solvent: methanol, respectively). AU AU AU Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 8 of 10 http://www.jast-journal.com/content/5/1/34 (2 mL). Second, the dry residue was suspended with water Validation of the method (10 mL) and extracted with n-butanol (10 mL) The n-buta- The method was validated in terms of linearity, LOD and nol extract was condensed and then methanol was used to LOQ, precision, repeatability, stability, and recovery test. meter the volume (2 mL). Third, the dry residues was dis- All calibration curves exhibited good linearity (r ≥ 0.9991) solved with 70% ethanol (20 mL) to precipitate the polysac- in a relatively wide linear range as shown in Table 3. For charide and then condensed the supernate, use methanol to the quantified compounds, the LOD and LOQ were −4 −4 −4 metered volume (2 mL). Fourth, the dry residues was dis- 0.20 × 10 ~64.90 × 10 μg/μL and 0.67 × 10 ~216.34 × −4 solved with water (10 mL) as fraction A, then the remanent 10 μg/μL, respectively (Table 3), which were calculated residues was dissolved with methanol (10 mL) as fraction B. with corresponding standard solution on the basic of a Fraction A was applied to an SPE column eluted with water signal-to-noise ratio (S/N) of 3 and 10, respectively. Table 4 (150 mL); the water eluent was discarded; fraction B was ap- showed the results of precision, stability, recovery and re- plied to the same SPE column eluted with methanol peatability of the 18 analytes. It was indicated that the (150 mL); the methanol eluent was condensed and methanol RSD of the precision variations were less than 1.47% for was used to meter the volume (2 mL). Fifth, the dry residue all 18 analytes. The RSD of repeatability was less than was dissolved with water (10 mL) and applied to an HPD 1.96% for all the analysis, which proved that this column eluted with water (150 mL). The water eluent was assay had good reproducibility. Stability test results, discarded and then eluted with 95% ethanol (150 mL). The with RSD less than 1.76%, indicated that the sample 95% ethanol eluent was condensed and then methanol was solution was stable at room temperature for at least used to meter the volume (2 mL). Comparing the analytical 48 h. The mean recovery rates, which ranged from results of the target constituents, though the former three 97.40% to 103.44% with RSD values less than 3.30% methods proved to be more simple than the other, they for the analytes concerned, showed that the developed could not obtain all the tested constituents and some con- analytical method had good accuracy. All these values tent too lower to accurately reflect the real content. So, these fall within acceptable limits, which indicates this three methods were deserted. The fourth one although could HPLC method is reliable with significant repeatability, obtain all the tested constituents but at a lower content. recovery rate, and precision. The results proved that Therefore, the optimized condition was selected, the fifth HPLC is appropriate for analyzing and assessing the one (Table 2, Figure 3). quality of ZYQL. Table 5 Contents of 18 analytes in different batches and different alcoholicity of Zhuyeqing Liquor (μg/mL) Compound 45° FenJiu 38° 42° 45° 20130207 20130207 20130207 20130207 20120601 20110507 20100417 20090302 M1 ND 15.1395 15.1395 16.7150 16.6674 16.6558 16.6543 16.6239 M2 ND 0.3689 0.3945 0.5851 0.5854 0.5846 0.5839 0.5840 M3 ND 0.0792 0.0830 0.1063 0.1064 0.1058 0.1032 0.1060 M4 ND 0.3028 0.3742 0.5180 0.5139 0.5106 0.5081 0.5111 M5 ND 65.7206 71.8348 101.7175 101.3777 101.0265 101.1293 100.9297 M6 ND ND ND 0.1930 0.1904 0.1901 0.1893 0.1934 M7 ND 273.1958 309.9846 574.4770 574.1508 574.1103 573.6367 573.2887 M8 ND 12.6644 19.9230 25.8595 25.4397 25.2009 25.4234 25.1453 M9 ND 0.1395 0.1659 0.1956 0.1934 0.1909 0.1933 0.1938 M10 ND 0.0536 0.0612 0.0715 0.0714 0.0715 0.0715 0.0721 M11 ND 0.2612 0.2963 0.4993 0.4972 0.4983 0.4919 0.5000 M12 ND 0.5690 0.6861 1.0884 1.0835 1.0822 1.0806 1.0830 M13 ND 2.0079 2.0709 2.9740 2.9731 2.9587 2.8982 2.9883 M14 ND 2.5058 2.7162 4.3718 4.3672 4.3615 4.3586 4.3699 M15 ND 0.0549 0.0585 0.0689 0.0686 0.0684 0.0685 0.0686 M16 ND 0.4758 0.5602 0.6434 0.6435 0.6422 0.6427 0.6383 M17 ND 2.6999 2.8338 3.0047 2.9943 2.9927 2.9836 2.9735 M18 ND 0.3534 0.4045 0.5495 0.5432 0.5427 0.5389 0.5433 Sum ND 376.5922 427.5872 733.6385 732.4671 731.7937 731.5560 730.8129 a b ‘ND’ in the ‘Compound’ column expressed under LOQ. Total content of the 18 investigated compounds. Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 9 of 10 http://www.jast-journal.com/content/5/1/34 Table 6 Contents of 18 analytes in 12 parent plants (mg/g) Compound Zhuye Zhizi Paicao Danggui Shannai Chenpi Juhua Sharen Tanxiang Gongdingxiang Guangmuxiang Linglingxiang M1 ND 2.1057 ND ND ND ND ND ND ND ND ND 19.8802 M2 ND ND ND ND ND ND ND ND ND 4.7841 ND 0.0790 M3 ND ND ND ND ND ND ND ND 0.0038 ND ND ND M4 ND ND ND ND ND ND ND ND ND 5.4547 ND 0.0498 M5 ND 19.9996 ND ND ND ND ND ND ND ND ND ND M6 ND 1.6513 ND ND ND 0.2580 ND ND ND ND ND ND M7 ND 43.3886 ND 2.5174 ND ND ND ND ND ND ND ND M8 ND 5.0078 ND ND ND 1.0468 ND ND ND ND ND ND M9 ND ND ND 0.6170 ND 0.0805 ND ND ND ND ND ND M10 0.3268 ND ND ND ND ND ND ND ND ND ND ND M11 0.4161 ND ND ND ND ND ND ND ND ND ND ND M12 ND 0.0111 ND ND ND 3.6198 0.2104 ND ND ND 0.0217 ND M13 ND ND ND ND ND 4.6719 0.3139 ND ND ND ND ND M14 ND 8.6950 0.0302 ND ND ND ND 0.0292 0.0025 ND ND ND M15 ND ND ND ND ND 0.1017 0.0472 ND ND ND ND ND M16 0.0638 0.0750 ND 0.2479 17.7933 0.6813 0.4755 ND ND ND ND 0.3214 M17 ND 1.9330 ND ND ND 0.8970 1.4942 ND ND ND ND ND M18 ND 0.0648 ND ND ND 0.5263 0.1625 ND ND ND ND ND Sum 0.8067 82.9319 0.0302 3.3823 17.7933 11.8833 2.7037 0.0292 0.0063 10.2388 0.0217 20.3304 a b ‘ND’ in the ‘Compound’ column expressed under LOQ. Total content of the 18 investigated compounds. Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 10 of 10 http://www.jast-journal.com/content/5/1/34 Sample analysis Author details School of Traditional Chinese Materia Medica 49#, Shenyang Pharmaceutical The HPLC analytical method described above was subse- University, Wenhua Road 103, Shenyang 110016, People’s Republic of China. quently used to simultaneously quantify 18 compounds in School of Pharmacy, Shihezi University, Shihezi 832002, People’s Republic of seven commercial products and 12 parent plants supplied China. Shanxi Xinghuacun Fen Jiu Group Co., Ltd, Shanxi 450000, People’s Republic of China. by Shanxi XinghuaCun Fen Wine Group Co., Ltd. (Shanxi, China). Generally, the 18 compounds were authenticated Received: 19 December 2013 Accepted: 12 June 2014 by comparison of their retention times and MS spectra with those of reference standards. The representative References HPLC chromatograms of mixed standard solution and Ballester AR, Lafuente MT, De Vos RCH, Bovy AG, González-Candelas L (2013) sample solutions are shown in Figure 4. The analytical re- Citrus phenylpropanoids and defence against pathogens. Part I: metabolic profiling in elicited fruits. Food Chem 136:178–185 sults are summarized in Tables 5 and 6. According to the Chen QC, Youn UJ, Min BS, Bae KH (2008) Pyronane monoterpenoids from the chromatographic results shown in Table 5, there was no fruit of Gardenia jasminoides. J Nat Prod 71:995–999 any constituents to be detected in 45° FenJiu (solvent of Dinda B, Debnath S, Banik R (2011) Naturally occurring iridoids and secoiridoids. An updated review, part 4. Chem Pharm Bull 59:803–833 ZYQL). Moreover, the concentration of compounds M1 Esteban MD, González Collado L, Macías FA, Massanet GM, Rodríguez Luis F to M18 in 45° ZYQL were higher than those in 42° and (1986) Flavonoids from Artemisia lanata. Phytochemistry 25:1502–1504 38°, which showed that with the increase of alcoholicity, Gao HY, Huang J, Wang HY, Du XW, Cheng SM, Han Y, Wang LF, Li GY, Wang JH (2013) Protective effect of Zhuyeqing liquor, a Chinese traditional health the content of bioactive constituents increased as well. In liquor, on acute alcohol-induced liver injury in mice. J Inflamm 10:30 addition, there was no content difference between the suc- Han Y (2007) Investigation on the immunoregulation functions of Zhuyeqing cessive 5 years of 45° ZYQL. This indicated that the qual- Liquor. Liq Making Sci Technol 2:117–119 Hòrie T, Ohtsuru Y, Shibata K, Yamashita K, Tsukayama M, Kawamura Y (1998) C ity of 45° ZYQL was stable for at least 5 years. NMR spectral assignment of the a-ring of polyoxygenated flavones. Table 6 showed the content of compounds in 12 par- Phytochemistry 47:865–874 ent plants, which exhibited that the major bioactive con- Huang Y, Chang RJ, Jin HZ, Zhang WD (2012) Phenolic constituents from Tsoongiodendron odorum chun. Tianran Chanwu Yanjiu Yu Kaifa 24:176–178 stituents were mainly from Gardenia jasminoides Ellis Ke Y, Jiang Y, Luo SQ (1999) Chemical constituents from Clinopodium chinense (Zhizi), Kaempferia galanga L. (Shannai), Citrus reticu- (Benth.) O. Ktze. Chin Traditional Herbal Drugs 30:8–10 lata Blanco (Chenpi), and Lysimachia foenum-graecum Kumarasamy Y, Byres M, Cox PJ, Delazar A, Jaspars M, Nahar L, Shoeb M, Sarker SD (2004) Isolation, structure elutiondition, and biological activity of flavone Hance (Linglingxiang). And this result was greatly useful 6-C-glycosides from Alliaria petiolata. Chem Nat Compd 40:122–128 and helpful for the quality control and further formula Lin LB, Fu XW, AI CH, Shen J, Wei K, Li W (2006) Studies on chemical constituents optimization of the technical study of Zhuyeqing Liquor. in leaves of Mallotus furetianus. China J Chin Materia Medica 31:477–479 Liu XM, Jiang Y, Sun YQ, Xu XW, Tu PF (2011) Chemical constituent study of Herba Cistanches. Chin J Pharm 46:1053–1058 Conclusions Ma ZT, Yang XW, Zhong GY (2009) A new flavonoid glucoside from Huanglian jiedutang decoction. China J Chin Materia Medica 34:1097–1100 An HPLC-PDA method has been developed for the sim- Miyake Y, Mochizuki M, Okada M, Hiramitsu M, Morimitsu Y, Osawa T (2007) ultaneous determination of 18 major compounds ex- Isolation of antioxidative phenolic glucosides from lemon juice and their tracted from ZYQL for the first time. The validation suppressive effect on the expression of blood adhesion molecules. Biosci Biotech Biochem 71:1911–1919 data indicated that this method is reliable and can be ap- Okamurα N, Hine N, Tateyamα Y, Nakazawα M, Fujiokα T, Mihashi K, Yagi A plied to determine the contents of the 18 compounds in (1998) Five chromones from Aloe vera leaves. Phytochemistry 49:219–223 different ZYQL products. This valuable information con- Rayyan S, Fossen T, Solheim Nateland H, Andersen ØM (2005) Isolation and identification of flavonoids, including flavone rotamers, from the herbal drug cerning the concentration of these bioactive constituents ‘crataegi folium cum flore’ (hawthorn). Phytochem Anal 16:334–341 in ZYQL could be of great importance for the quality as- Wang QJ, Wang YS, He L, Lou ZP, Zang S (2010) Study on chemical constituents sessment and should therefore be useful for the guidance from Ipomoea Pescaprae (L.) Sweet. Chin J Marine Drugs 29:41–44 Yang HP (2007) Zhuyeqing liquor in different historical period. Liquor Making Sci of development of the new health care products. Fur- Technol 2:110–112 thermore, this HPLC-PDA assay supplies a rapidness Yoo SW, Kim JS, Kang SS, Son KH, Chang HW, Kim HP, Bae KH, Lee CO (2002) and effectiveness method for the simultaneous determin- Constituents of the fruits and leaves of Euodia daniellii. Arch Pharm Res 25:824–830 ation of multiple constituents in ZYQL. Zhang YW, Chen YW (1997) Isobiflorin, a chromone C-glucoside from cloves (Eugenia Caryophyllata). Phytochemistry 45:401–403 Competing interests The authors declare that they have no competing interests. doi:10.1186/s40543-014-0034-1 Cite this article as: Gao et al.: Simultaneous quantification of major Authors' contributions bioactive constituents from Zhuyeqing Liquor by HPLC-PDA. Journal of HYG carried out the whole experiment, SYW participated in the sample Analytical Science and Technology 2014 5:34. preparation, HYG and JHW performed the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript. Acknowledgements Grateful acknowledgement is made to the Shanxi XinghuaCun Fen Jiu Group Co., Ltd. (Shanxi Province, China) and National Key Technology R&D Program (2012BAI30B02) for financial support of this work. The authors acknowledge Waters Co. Ltd. for Cooperation Laboratory. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png "Journal of Analytical Science and Technology" Springer Journals

Simultaneous quantification of major bioactive constituents from Zhuyeqing Liquor by HPLC-PDA

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Abstract

Background: Zhuyeqing Liquor (ZYQL) is a famous traditional Chinese functional liquor. For quality control of ZYQL products, quantitative analysis using high-performance liquid chromatography coupled with photodiode array detector (HPLC-PDA) was undertaken. Methods: Eighteen compounds from ZYQL were simultaneously detected and used as chemical markers in the quantitative analysis, including 3-hydroxy-4,5(R)-dimethyl-2(5H)-furanone (M1), isobiflorin (M2), vanillic acid (M3), biflorin (M4), genipin 1-O-β-D-gentiobioside (M5), 1-sinapoyl-β-D-glucopyranoside (M6), geniposide (M7), epijasmnoside A(M8), ferulic acid (M9), luteolin 8-C-β-glucopyranoside (M10), isoorientin (M11), narirutin (M12), hesperidin (M13), 6′-O-sinapoylgeniposide (M14), 3,5-dihydroxy-3′,4′,7,8-tetramethoxyl flavones (M15), 3′,4′,3,5,6,8-hexamethoxyl flavone (M16), kaempferide (M17), and tangeretin (M18). Results: The separation by gradient elution was achieved on SHIMADZU VP-ODS column (4.6 × 150 mm, 5 μm) at 30°C with methanol (A)/0.1% phosphoric acid (B) as the mobile phase. The detection wavelengths were 254, 278, and 335 nm. The optimized HPLC method provided a good linear relation (r ≥ 0.9991 for all the target compounds), satisfactory precision (RSD values less than 1.47%) and good recovery (97.40% to 103.44%). The limits of detection −4 −4 ranged between 0.20 × 10 and 64.90 × 10 μg/μL for the different analytes. Furthermore, the optimum sample preparation was obtained from HPD column eluted with water and 95% ethanol, respectively. Conclusions: Quality control of ZYQL products, in total seven samples and twelve parent plants, was examined by this method, and results confirmed its feasibility and reliability in practice. Keywords: Zhuyeqing Liquor; Bioactive constituent; Quantitative analysis; HPLC-PDA Background Lysimachia capillipes Hemsl. (Paicao), Angelica sinensis Zhuyeqing Liquor (ZYQL), authorized as a functional (Oliv.) Diels (Danggui), Kaempferia galanga L. (Shannai), health liquor in 1998 by the Ministry of Public Health in Citrus reticulata Blanco (Chenpi), Chrysanthemum mori- China, is a famous traditional Chinese functional liquor. folium Ramat. (Juhua), Amomum villosum Lour. (Sharen), The history of ZYQL could be traced back to the Warring Santalum album L. (Tanxiang), Eugenia caryophyllata Thunb. States Period and became popular among people in the (Gongdingxiang), Aucklandia lappa Decne. (Guangmuxiang), South and North Dynasties. In the Tang Dyansty and Song and Lysimachia foenum-graecum Hance (Linglingxiang). Dynasty, it had reached its climax (Yang 2007). ZYQL was According to its long-term history use, ZYQL has various designed based on the principles of traditional Chinese biological properties such as anti-oxidant, anti-fatigue, and medicine (TCM) and comprises 12 herbs: Lophatherum immunoenhancement (Han 2007). gracile Brongn. (Zhuye), Gardenia jasminoides Ellis (Zhizi), Up to now, many studies show solicitude for the color, smell, and taste of the health functional liquor; few studies pay close attention to its chemical constituents and quality * Correspondence: wjh.1972@aliyun.com School of Traditional Chinese Materia Medica 49#, Shenyang Pharmaceutical control. Currently, chemical analytical methods for the University, Wenhua Road 103, Shenyang 110016, People’s Republic of China 2 quality control of ZYQL have not been established. There- School of Pharmacy, Shihezi University, Shihezi 832002, People’s Republic of fore, it is necessary to establish a rapid and effective method China Full list of author information is available at the end of the article © 2014 Gao et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 2 of 10 http://www.jast-journal.com/content/5/1/34 for the quantitative analysis of the health functional liquor. was purified by a Milli-Q water purification system In this study, the system of high-performance liquid (Millipore, Billerica, MA, USA). Phosphoric acid (analytical chromatography coupled with photodiode array detector grade) was purchased from Tianjin Guangfu Chemical (HPLC-PDA) was used for analyzing the chemical profile Reagent Co. Ltd. (Tianjin, China). Other solvents from of ZYQL. This method includes many advantages like high Tianjin Guangfu Chemical Reagent Co. Ltd. (Tianjin, speed detection, excellent peak shapes, less solvent usage, China) were all of analytical grade. well-defined chemical constituents, and simultaneous Reference compounds of M1 to M18 (Figure 1) were detection of multi-constituents, which is better than finger- isolated previously from ZYQL by author, structures of printing. Thus, simultaneous determination by RP-HPLC which were elucidated by comparison of spectral data (UV, 1 13 method is suitable for quantitative analysis and can be used MS, HNMR, and C NMR) with the literature data (Lin as an effective tool to evaluate herbal medicine products. et al. 2006; Okamurα et al. 1998; Huang et al. 2012; Zhang and Chen 1997; Ma et al. 2009; Miyake et al. 2007; Liu et al. Methods 2011; Chen et al. 2008; Rayyan et al. 2005; Kumarasamy Chemicals and materials et al. 2004; Ke et al. 1999; Yoo et al. 2002; Dinda et al. 2011; Methanol (HPLC-grade) was purchased from Fisher Esteban et al. 1986; Ballester et al. 2013; Wang et al. 2010; Scientific Co. (Franklin, MA, USA). Water for HPLC analysis Hòrie et al. 1998). The purity of each reference standard Figure 1 Structures of compounds M1 to M18. Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 3 of 10 http://www.jast-journal.com/content/5/1/34 −1 was determined to be above 98% by HPLC analysis 6′-O-sinapoylgeniposide (M14) 5.312 mg mL ,3,5-dihy- based on a peak area normalization method, detected droxy-3′,4′,7,8-tetramethoxyl flavones (M15) 5.021 mg −1 by HPLC-PDA and confirmed by HR-ESI-TOF-MS and mL ,3′,4′,3,5,6,8-hexamethoxyl flavone (M16) 15.005 mg −1 −1 NMR spectroscopy. mL ,kaempferide (M17) 6.408 mg mL , and tangeretin −1 The samples of different batch and different alcoholicity (M18) 17.155 mg mL . A mixed solution containing all of ZYQL and the 12 parent plants were provided by the 18 standards was prepared as accurately as 108 μL M1, Shanxi XinghuaCun Fen Jiu Group Co., Ltd. (Shanxi, 6.8 μL M2,2.4 μL M3,8.0 μL M4, 165 μL M5,96 μL M6, China). The 12 parent plants were identified by Professor 106 μL M7,3.4 μL M8,8.2 μL M9,9.5 μL M10,7.9 μL Jincai Lu (Shenyang Pharmaceutical University, Shenyang, M11,35 μL M12,40 μL M13,80 μL M14,8.2 μL M15, China). The voucher specimen was deposited at Shenyang 11 μL M16,12 μL M17, and 4.6 μL M18 and were placed Pharmaceutical University (Shenyang, China) and regis- in a 2-mL flask with stopper, diluted with methanol to tered under the number ZYQL 2011050101. make sure the volume reached 2 mL. All prepared solu- tions were respectively stored in a refrigerator at 4°C Instrumentation and chromatographic conditions when not in use. Chromatographic analysis was performed on Waters 2695 Alliance HPLC system (Waters Co., Milford, MA, Treatment for samples USA) with Waters 2998 PDA detector. Chromatographic For the analysis, 40 mL of ZYQL were evaporated in vacuum separation was carried on a SHIMADZU VP-ODS column at 50°C to dryness. The dry residue was processed as follows (4.6 mm × 150 mm, 5 μm; Shimadzu, Kyoto, Japan) at a in order to obtain better analytical results: The residue was column temperature of 30°C using methanol (A) and 0.1% dissolved with water (10 mL) and applied to an HPD col- phosphoric acid (B) as mobile phase with the gradient umn eluted with water (150 mL); the water eluent was dis- elution procedure show in Table 1. The flow rate was carded and then eluted with 95% ethanol (150 mL). The 95% set at 1.0 ml/min and the detection wavelengths were ethanol eluent was condensed and dissolved with methanol 254 nm (for compounds M1 to M5, M7, M8,and M17), andthenplacedina2-mL flaskwithstopper, with a 278 nm (for compounds M12 and M13), and 335 nm methanol-metered volume. Prior to HPLC analysis, the sam- (for compounds M6, M9 to M11, M14 to M16,and ple solution was passed through a 0.22-μm millipore filter. M18), which were chosen based on the maximum absorp- The 12 crude dried parent plants were pulverized and tion of all the tested compounds. The injection volume sifted through 40 mesh sieve, respectively. One gram of the was 10 μL, and the analytes were well separated in chro- powder from the parent plant was placed in a 50-mL flask matographic conditions above. with stopper, then weighed again correctly, and extracted by ultrasonic method with 20 mL methanol for 30 min. Standard solution preparation Then standing, it was cooled down to room temperature Individual stock solutions were prepared by dissolving the (22°C) and the weight was mended to the incipient weight standards in methanol to obtain 3-hydroxy-4,5(R)-di- with methanol. Prior to HPLC analysis, the sample solution −1 methyl-2(5H)-furanone (M1)19.920 mgmL , isobiflorin was passed through a 0.22-μm millipore filter. −1 −1 (M2)8.330 mg mL , vanillic acid (M3)5.802 mg mL , −1 biflorin (M4) 3.911 mg mL ,genipin 1-O-β-D-gentiobio- Validation of the method −1 side (M5) 4.405 mg mL , 1-sinapoyl-β-D-glucopyranoside Calibration curves −1 −1 (M6) 1.115 mg mL ,geniposide (M7)23.804 mgmL , Linearity was established by the injection of 1, 2, 4, 8, −1 epijasmnoside A (M8) 12.060 mg mL , ferulic acid (M9) 12, 16, and 20 μL of the mixed reference standard solu- −1 2.515 mg mL , luteolin 8-C-β-glucopyranoside (M10) tion prepared, respectively. Calibration graphs were plot- −1 −1 1.510 mg mL , isoorientin (M11) 2.203 mg mL ,nairutin ted subsequently based on linear regression analysis of −1 −1 (M12) 1.032 mg mL ,hesperidin(M13) 4.801 mg mL , the integrated peak (Y) versus content (X, μg). Table 1 Time program of the gradient elution Time (min) Flow (mL/min) Methanol (%) 0.1% Phosphatic Limits of detection and quantitation acid (%) In order to evaluate the limits of detection (LODs) and the 01 5 95 limits of quantification (LOQs) of the compounds, mixed 70 1 55 45 standard stock solution was further diluted serially to pro- 75 1 60 40 vide a series of appropriate concentrations, and an aliquot of the diluted solutions was injected into HPLC for ana- 110 1 80 20 lysis. The LOD and LOQ for each analyte was calculated 120 1 98 2 with corresponding standard solution on the basis of a 125 1 98 2 signal-to-noise ratio (S/N) of 3 and 10, respectively. Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 4 of 10 http://www.jast-journal.com/content/5/1/34 (A) 0.15 5 16 0.10 335 nm 13 14 18 278 nm 0.05 1 10 11 12 15 254 nm 2 4 8 0.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 Minute (B) 0.15 254 nm 2 4 8 0.10 0.05 0.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 0.10 278 nm 0.05 0.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 0.15 335 nm 15 16 18 0.10 9 10 0.05 0.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 Minute Figure 2 Stack views. (A) Different detector-wavelength HPLC chromatograms of mixed reference standards (from up to down: 335, 278, 254 nm). Column: SHIMADZU VP-ODS column (4.6 mm × 150 mm, 5 μm), temperature of 30°C. (B) HPLC chromatograms of M1 to M18 and mixed reference standards (from up to down: 254 nm M1, M2, M3, M4, M5, M7, M8, M17, mixed reference standards; 278 nm M12, M13, mixed reference standards; 335 nm M6, M9, M10, M11, M14, M15, M16, M18, mixed reference standards). AU AU AU AU Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 5 of 10 http://www.jast-journal.com/content/5/1/34 Table 2 Optimization of the treatment method of Zhuyeqing Liquor (μg/mL) Compound Treatment method Method 1 Method 2 Method 3 Method 4 Method 5 M1 ND 5.2494 3.1331 9.5943 16.4270 M2 0.0888 0.5371 0.5078 0.5818 0.5942 M3 0.0922 0.0906 0.0388 0.0939 0.1063 M4 0.0263 0.4980 0.5098 0.5069 0.5153 M5 ND 44.0545 101.6907 100.6372 101.7888 M6 0.1479 0.1880 0.1902 0.1927 0.1936 M7 45.2421 563.2436 570.3556 566.0022 574.2514 M8 20.8481 24.6279 25.6049 24.5513 25.7319 M9 0.1931 0.1782 0.1906 0.1918 0.1968 M10 0.0324 0.0526 0.0600 0.0705 0.0736 M11 0.2009 0.4601 0.4871 0.4915 0.4954 M12 0.7071 1.0544 1.0683 1.0699 1.0877 M13 1.1371 2.5392 2.8461 2.8029 2.9909 M14 ND 3.4939 4.0839 4.0563 4.3756 M15 ND 0.0641 0.0678 0.0688 0.0689 M16 0.5189 0.6113 0.5842 0.3282 0.6623 M17 2.9599 2.9776 2.1520 1.6446 2.9957 M18 0.4448 0.5307 0.4278 0.4673 0.5413 Sum 72.6396 650.4512 713.9987 713.3521 733.0967 Sample in optimization of the treatment method was 45° Zhuyeqing Liquor (20130207). Method 1, acetoacetate extract; method 2, n-butanol extract; method 3, 70% ethanol treatment; method 4, SPE column eluted with methanol; method 5, HPD column eluted with ethanol. ‘ND’ in the ‘Compound’ column expressed under LOQ. Total content of the 18 investigated compounds. Precision and stability Repeatability and recovery The precision of the chromatographic system was vali- The repeatability test was analyzed by injecting six inde- dated by injecting 10 μL of the mixed reference solution pendently prepared samples (45° ZYQL (20130207), the six times during 1 day. Stability study was performed with concentration, and prepared method as the ‘Treatment for sample solution in 48 h (the time points are 0, 5, 10, 15, samples’). The RSD value of concentration was adopted to 25, 35, and 48 h, respectively). Variations were expressed evaluate repeatability. The recovery tests were studied by by relative standard deviations (RSD) of peak area. adding the proper amount of mixed-reference standard Figure 3 Stack views of 45° Zhuyeqing Liquor preparation method HPLC chromatograms (254 nm, from up to down: method 1, method 2, method 3, method 4 and method 5). Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 6 of 10 http://www.jast-journal.com/content/5/1/34 Table 3 Regression equations, correlation coefficients, and linear range for 18 analytes in Zhuyeqing Liquor Analyte Time Linear regression (t ) Regression equation (n = 3) Correlation Linear range (μg) LOD LOQ coefficients −4 −4 (10 μg/μL) (10 μg/μL) M1 14.004 Y = 5.48e + 003X − 1.45e + 003 0.9998 1.08~21.63 64.90 216.34 −2 −1 M2 22.819 Y = 2.86e + 006X − 8.84e + 002 0.9999 2.80 × 10 ~5.60 × 10 1.68 5.60 −3 −2 M3 23.573 Y = 4.73e + 006X − 5.52e + 003 0.9991 1.70 × 10 ~3.40 × 10 0.20 0.67 −2 −1 M4 25.069 Y = 1.62e + 006X − 3.23e + 003 0.9997 1.55 × 10 ~3.10 × 10 3.10 10.34 −1 M5 26.671 Y = 5.41e + 005X − 3.44e + 004 0.9994 3.63 × 10 ~7.25 2.42 8.08 −2 M6 28.087 Y = 1.31e + 006X − 4.70e + 003 0.9998 5.25 × 10 ~1.05 8.40 28.0 M7 29.646 Y = 7.08e + 005X + 4.19e + 003 0.9998 1.26~25.21 25.46 84.87 −2 −1 M8 32.875 Y = 9.81e + 005 X - 5.87 e + 003 0.9998 2.04 × 10 ~4.08 × 10 4.08 13.60 −2 −1 M9 37.264 Y = 3.17e + 006X − 1.51e + 004 0.9992 1.03 × 10 ~2.06 × 10 2.06 6.87 −3 −1 M10 40.883 Y = 1.65e + 006X − 1.26e + 002 0.9993 7.10 × 10 ~1.42 × 10 2.13 7.11 −3 −1 M11 42.396 Y = 2.38e + 006X − 6.23e + 003 0.9991 8.70 × 10 ~1.74 × 10 1.74 5.83 −2 −1 M12 46.878 Y = 2.67e + 006X − 3.22e + 002 0.9998 1.75 × 10 ~3.50 × 10 5.25 17.51 −2 M13 50.008 Y = 1.69e + 006 X + 3.71e + 003 0.9998 9.56 × 10 ~1.91 5.74 19.12 −1 M14 58.096 Y = 4.55e + 005X − 2.11e + 003 0.9998 2.13 × 10 ~4.25 12.75 42.50 −3 −2 M15 74.987 Y = 2.15e + 006X − 7.62e + 002 0.9991 4.10 × 10 ~ 8.20 × 10 3.28 10.95 −2 M16 80.609 Y = 3.59e + 006X − 2.08e + 004 0.9999 8.23 × 10 ~1.65 0.55 1.84 −2 −1 M17 83.248 Y = 1.97e + 005X − 3.36e + 003 0.9991 3.85 × 10 ~7.70 × 10 15.40 51.32 −2 −1 M18 85.890 Y = 2.69e + 006X − 1.38e + 004 0.9996 3.93 × 10 ~ 7.86 × 10 0.79 2.64 Y is the peak area and X is the content of standard solutions; LOD refers to the limits of detection, S/N = 3; LOQ refers to the limits of quantity, S/N = 10. Table 4 Precision, stability, recovery, and repeatability data of 18 analytes in Zhuyeqing Liquor Analyte Precision (n = 6) Stability Recovery (n = 6) Repeatability (n =6) RSD (%) Concentrations RSD Original Spiked Detected Recovery RSD Average RSD (%) (μg) (μg) (μg) (%) (%) concentration (%) (mg/mL) (μg/mL) M1 1.08 0.92 1.70 164.16 162.26 326.57 100.17 3.10 16.0862 ± 0.2722 1.50 −2 M2 2.80 × 10 0.82 1.52 7.11 4.20 11.41 102.44 1.84 0.5580 ± 0.0045 1.39 −3 M3 1.70 × 10 1.30 1.66 0.27 0.26 0.52 97.40 2.52 0.1072 ± 0.0020 1.96 −2 M4 1.55 × 10 0.78 1.08 5.88 2.33 8.23 100.88 2.27 0.5154 ± 0.0032 0.64 −1 M5 3.63 × 10 0.87 1.62 1217.80 54.39 1273.19 101.83 3.30 101.1963 ± 0.7206 0.72 −2 M6 5.25 × 10 0.86 1.58 6.00 7.88 14.15 103.44 1.68 0.1940 ± 0.0019 1.00 M7 1.26 0.49 1.18 1270.60 1260.30 2543.54 101.00 0.78 568.4991 ± 2.7722 0.98 −2 M8 2.04 × 10 1.04 1.76 211.50 102.00 316.34 102.79 1.46 25.1942 ± 0.2548 1.45 −2 M9 1.03 × 10 0.30 1.49 2.82 1.55 4.37 100.76 2.63 0.1947 ± 0.0018 1.00 −3 M10 7.10 × 10 1.10 1.67 3.22 1.07 4.28 99.54 3.26 0.0728 ± 0.0011 1.61 −3 M11 8.70 × 10 1.15 1.62 4.65 1.31 5.96 100.66 3.09 0.5101 ± 0.0057 1.28 −2 M12 1.75 × 10 1.09 1.39 2.43 2.63 5.14 103.13 1.48 1.0800 ± 0.0155 1.44 −2 M13 9.56 × 10 1.02 1.36 37.69 14.34 52.20 101.14 1.99 2.8911 ± 0.0351 1.21 −1 M14 2.13 × 10 0.74 1.59 943.73 31.88 975.00 98.10 2.25 4.2790 ± 0.0314 1.30 −3 M15 4.10 × 10 1.47 1.75 1.99 1.23 3.22 100.30 2.66 0.0645 ± 0.0009 1.53 −2 M16 8.23 × 10 0.91 1.58 21.52 12.35 33.77 99.20 2.44 0.6444 ± 0.0052 0.81 −2 M17 3.85 × 10 0.93 1.75 133.12 115.50 249.54 100.79 2.63 2.9457 ± 0.0498 1.36 −2 M18 3.93 × 10 1.11 1.49 10.02 11.79 21.89 100.65 1.58 0.5368 ± 0.0060 1.13 RSD refers to relative standard deviation. Samples in stability, recovery, and repeatability methods were taken from 45°Zhuyeqing Liquor (20130207). Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 7 of 10 http://www.jast-journal.com/content/5/1/34 solution to the sample (45° ZYQL (20130207)), and then phase of water and methanol rather than water and aceto- processed by the method described in the ‘Treatment for nitrile. Therefore, in this work, the optimum resolution was samples’ section to yield the final concentration. The ex- achieved using methanol (A) and 0.1% phosphatic acid (B) periment was repeated six times. as mobile phase, with a column temperature of 30°C at dif- ferent detection wavelengths, which were described in ‘In- Results and discussion strumentation and chromatographic conditions’ section, Optimization of chromatographic conditions with gradient elution (Table 1). All 18 standard analytes To improve resolution and sensitivity of analysis but reduce could be eluted with baseline separation in 90 min. Repre- analytical time, the following chromatographic conditions sentative chromatograms for the mixed reference standard were optimized (Gao et al. 2013), including different mobile and 18 standard compounds were shown in Figure 2A,B. phase compositions (methanol, acetonitrile, and aqueous phosphatic acid of different concentrations), column Optimization of sample preparation temperature, and wavelength: To inhibit ionization of the In order to eliminate the water-soluble constituents and ob- acidic ingredients in the ZYQL sample, phosphatic acid was tain the liposoluble constituents, the optimization of sample added in mobile phase. Two mobile phase systems, preparation was performed using 45° ZYQL (20130207). methanol-phosphatic acid aqueous solution and acetonitrile- Forty milliliters of ZYQL was evaporated in vacuum at 50°C phosphatic acid aqueous solution, were examined, and then to dryness. And the following five methods were choosen to column temperatures at 25°C, 30°C, 40°C, and 50°C were select the best method for sample preparation. First, the dry compared. A sensitive wavelength was determined by PDA residue was suspended with water (10 mL) and extracted with reference compounds. Present researches indicated that with acetoacetate (10 mL). The acetoacetate extract was con- better separation and results were obtained using a mobile densed and then methanol was used to meter the volume 0.10 254 nm 5 7 0.08 45°ZYQL 0.06 45°FenJiu 2 4 8 0.04 Mixed standards 0.02 3 Methanol 0.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 0.10 278 nm 0.08 0.06 0.04 0.02 0.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 0.10 335 nm 0.08 0.06 9 11 10 15 0.04 0.02 0.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 Minute Figure 4 Stack views of different detector-wavelength HPLC chromatograms. (From up to down: 45° Zhuyeqing Liquor, 45° FenJiu, mixed reference standards, blank solvent: methanol, respectively). AU AU AU Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 8 of 10 http://www.jast-journal.com/content/5/1/34 (2 mL). Second, the dry residue was suspended with water Validation of the method (10 mL) and extracted with n-butanol (10 mL) The n-buta- The method was validated in terms of linearity, LOD and nol extract was condensed and then methanol was used to LOQ, precision, repeatability, stability, and recovery test. meter the volume (2 mL). Third, the dry residues was dis- All calibration curves exhibited good linearity (r ≥ 0.9991) solved with 70% ethanol (20 mL) to precipitate the polysac- in a relatively wide linear range as shown in Table 3. For charide and then condensed the supernate, use methanol to the quantified compounds, the LOD and LOQ were −4 −4 −4 metered volume (2 mL). Fourth, the dry residues was dis- 0.20 × 10 ~64.90 × 10 μg/μL and 0.67 × 10 ~216.34 × −4 solved with water (10 mL) as fraction A, then the remanent 10 μg/μL, respectively (Table 3), which were calculated residues was dissolved with methanol (10 mL) as fraction B. with corresponding standard solution on the basic of a Fraction A was applied to an SPE column eluted with water signal-to-noise ratio (S/N) of 3 and 10, respectively. Table 4 (150 mL); the water eluent was discarded; fraction B was ap- showed the results of precision, stability, recovery and re- plied to the same SPE column eluted with methanol peatability of the 18 analytes. It was indicated that the (150 mL); the methanol eluent was condensed and methanol RSD of the precision variations were less than 1.47% for was used to meter the volume (2 mL). Fifth, the dry residue all 18 analytes. The RSD of repeatability was less than was dissolved with water (10 mL) and applied to an HPD 1.96% for all the analysis, which proved that this column eluted with water (150 mL). The water eluent was assay had good reproducibility. Stability test results, discarded and then eluted with 95% ethanol (150 mL). The with RSD less than 1.76%, indicated that the sample 95% ethanol eluent was condensed and then methanol was solution was stable at room temperature for at least used to meter the volume (2 mL). Comparing the analytical 48 h. The mean recovery rates, which ranged from results of the target constituents, though the former three 97.40% to 103.44% with RSD values less than 3.30% methods proved to be more simple than the other, they for the analytes concerned, showed that the developed could not obtain all the tested constituents and some con- analytical method had good accuracy. All these values tent too lower to accurately reflect the real content. So, these fall within acceptable limits, which indicates this three methods were deserted. The fourth one although could HPLC method is reliable with significant repeatability, obtain all the tested constituents but at a lower content. recovery rate, and precision. The results proved that Therefore, the optimized condition was selected, the fifth HPLC is appropriate for analyzing and assessing the one (Table 2, Figure 3). quality of ZYQL. Table 5 Contents of 18 analytes in different batches and different alcoholicity of Zhuyeqing Liquor (μg/mL) Compound 45° FenJiu 38° 42° 45° 20130207 20130207 20130207 20130207 20120601 20110507 20100417 20090302 M1 ND 15.1395 15.1395 16.7150 16.6674 16.6558 16.6543 16.6239 M2 ND 0.3689 0.3945 0.5851 0.5854 0.5846 0.5839 0.5840 M3 ND 0.0792 0.0830 0.1063 0.1064 0.1058 0.1032 0.1060 M4 ND 0.3028 0.3742 0.5180 0.5139 0.5106 0.5081 0.5111 M5 ND 65.7206 71.8348 101.7175 101.3777 101.0265 101.1293 100.9297 M6 ND ND ND 0.1930 0.1904 0.1901 0.1893 0.1934 M7 ND 273.1958 309.9846 574.4770 574.1508 574.1103 573.6367 573.2887 M8 ND 12.6644 19.9230 25.8595 25.4397 25.2009 25.4234 25.1453 M9 ND 0.1395 0.1659 0.1956 0.1934 0.1909 0.1933 0.1938 M10 ND 0.0536 0.0612 0.0715 0.0714 0.0715 0.0715 0.0721 M11 ND 0.2612 0.2963 0.4993 0.4972 0.4983 0.4919 0.5000 M12 ND 0.5690 0.6861 1.0884 1.0835 1.0822 1.0806 1.0830 M13 ND 2.0079 2.0709 2.9740 2.9731 2.9587 2.8982 2.9883 M14 ND 2.5058 2.7162 4.3718 4.3672 4.3615 4.3586 4.3699 M15 ND 0.0549 0.0585 0.0689 0.0686 0.0684 0.0685 0.0686 M16 ND 0.4758 0.5602 0.6434 0.6435 0.6422 0.6427 0.6383 M17 ND 2.6999 2.8338 3.0047 2.9943 2.9927 2.9836 2.9735 M18 ND 0.3534 0.4045 0.5495 0.5432 0.5427 0.5389 0.5433 Sum ND 376.5922 427.5872 733.6385 732.4671 731.7937 731.5560 730.8129 a b ‘ND’ in the ‘Compound’ column expressed under LOQ. Total content of the 18 investigated compounds. Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 9 of 10 http://www.jast-journal.com/content/5/1/34 Table 6 Contents of 18 analytes in 12 parent plants (mg/g) Compound Zhuye Zhizi Paicao Danggui Shannai Chenpi Juhua Sharen Tanxiang Gongdingxiang Guangmuxiang Linglingxiang M1 ND 2.1057 ND ND ND ND ND ND ND ND ND 19.8802 M2 ND ND ND ND ND ND ND ND ND 4.7841 ND 0.0790 M3 ND ND ND ND ND ND ND ND 0.0038 ND ND ND M4 ND ND ND ND ND ND ND ND ND 5.4547 ND 0.0498 M5 ND 19.9996 ND ND ND ND ND ND ND ND ND ND M6 ND 1.6513 ND ND ND 0.2580 ND ND ND ND ND ND M7 ND 43.3886 ND 2.5174 ND ND ND ND ND ND ND ND M8 ND 5.0078 ND ND ND 1.0468 ND ND ND ND ND ND M9 ND ND ND 0.6170 ND 0.0805 ND ND ND ND ND ND M10 0.3268 ND ND ND ND ND ND ND ND ND ND ND M11 0.4161 ND ND ND ND ND ND ND ND ND ND ND M12 ND 0.0111 ND ND ND 3.6198 0.2104 ND ND ND 0.0217 ND M13 ND ND ND ND ND 4.6719 0.3139 ND ND ND ND ND M14 ND 8.6950 0.0302 ND ND ND ND 0.0292 0.0025 ND ND ND M15 ND ND ND ND ND 0.1017 0.0472 ND ND ND ND ND M16 0.0638 0.0750 ND 0.2479 17.7933 0.6813 0.4755 ND ND ND ND 0.3214 M17 ND 1.9330 ND ND ND 0.8970 1.4942 ND ND ND ND ND M18 ND 0.0648 ND ND ND 0.5263 0.1625 ND ND ND ND ND Sum 0.8067 82.9319 0.0302 3.3823 17.7933 11.8833 2.7037 0.0292 0.0063 10.2388 0.0217 20.3304 a b ‘ND’ in the ‘Compound’ column expressed under LOQ. Total content of the 18 investigated compounds. Gao et al. Journal of Analytical Science and Technology 2014, 5:34 Page 10 of 10 http://www.jast-journal.com/content/5/1/34 Sample analysis Author details School of Traditional Chinese Materia Medica 49#, Shenyang Pharmaceutical The HPLC analytical method described above was subse- University, Wenhua Road 103, Shenyang 110016, People’s Republic of China. quently used to simultaneously quantify 18 compounds in School of Pharmacy, Shihezi University, Shihezi 832002, People’s Republic of seven commercial products and 12 parent plants supplied China. Shanxi Xinghuacun Fen Jiu Group Co., Ltd, Shanxi 450000, People’s Republic of China. by Shanxi XinghuaCun Fen Wine Group Co., Ltd. (Shanxi, China). Generally, the 18 compounds were authenticated Received: 19 December 2013 Accepted: 12 June 2014 by comparison of their retention times and MS spectra with those of reference standards. The representative References HPLC chromatograms of mixed standard solution and Ballester AR, Lafuente MT, De Vos RCH, Bovy AG, González-Candelas L (2013) sample solutions are shown in Figure 4. The analytical re- Citrus phenylpropanoids and defence against pathogens. Part I: metabolic profiling in elicited fruits. Food Chem 136:178–185 sults are summarized in Tables 5 and 6. According to the Chen QC, Youn UJ, Min BS, Bae KH (2008) Pyronane monoterpenoids from the chromatographic results shown in Table 5, there was no fruit of Gardenia jasminoides. J Nat Prod 71:995–999 any constituents to be detected in 45° FenJiu (solvent of Dinda B, Debnath S, Banik R (2011) Naturally occurring iridoids and secoiridoids. An updated review, part 4. Chem Pharm Bull 59:803–833 ZYQL). Moreover, the concentration of compounds M1 Esteban MD, González Collado L, Macías FA, Massanet GM, Rodríguez Luis F to M18 in 45° ZYQL were higher than those in 42° and (1986) Flavonoids from Artemisia lanata. Phytochemistry 25:1502–1504 38°, which showed that with the increase of alcoholicity, Gao HY, Huang J, Wang HY, Du XW, Cheng SM, Han Y, Wang LF, Li GY, Wang JH (2013) Protective effect of Zhuyeqing liquor, a Chinese traditional health the content of bioactive constituents increased as well. In liquor, on acute alcohol-induced liver injury in mice. J Inflamm 10:30 addition, there was no content difference between the suc- Han Y (2007) Investigation on the immunoregulation functions of Zhuyeqing cessive 5 years of 45° ZYQL. This indicated that the qual- Liquor. Liq Making Sci Technol 2:117–119 Hòrie T, Ohtsuru Y, Shibata K, Yamashita K, Tsukayama M, Kawamura Y (1998) C ity of 45° ZYQL was stable for at least 5 years. NMR spectral assignment of the a-ring of polyoxygenated flavones. Table 6 showed the content of compounds in 12 par- Phytochemistry 47:865–874 ent plants, which exhibited that the major bioactive con- Huang Y, Chang RJ, Jin HZ, Zhang WD (2012) Phenolic constituents from Tsoongiodendron odorum chun. Tianran Chanwu Yanjiu Yu Kaifa 24:176–178 stituents were mainly from Gardenia jasminoides Ellis Ke Y, Jiang Y, Luo SQ (1999) Chemical constituents from Clinopodium chinense (Zhizi), Kaempferia galanga L. (Shannai), Citrus reticu- (Benth.) O. Ktze. Chin Traditional Herbal Drugs 30:8–10 lata Blanco (Chenpi), and Lysimachia foenum-graecum Kumarasamy Y, Byres M, Cox PJ, Delazar A, Jaspars M, Nahar L, Shoeb M, Sarker SD (2004) Isolation, structure elutiondition, and biological activity of flavone Hance (Linglingxiang). And this result was greatly useful 6-C-glycosides from Alliaria petiolata. Chem Nat Compd 40:122–128 and helpful for the quality control and further formula Lin LB, Fu XW, AI CH, Shen J, Wei K, Li W (2006) Studies on chemical constituents optimization of the technical study of Zhuyeqing Liquor. in leaves of Mallotus furetianus. China J Chin Materia Medica 31:477–479 Liu XM, Jiang Y, Sun YQ, Xu XW, Tu PF (2011) Chemical constituent study of Herba Cistanches. Chin J Pharm 46:1053–1058 Conclusions Ma ZT, Yang XW, Zhong GY (2009) A new flavonoid glucoside from Huanglian jiedutang decoction. China J Chin Materia Medica 34:1097–1100 An HPLC-PDA method has been developed for the sim- Miyake Y, Mochizuki M, Okada M, Hiramitsu M, Morimitsu Y, Osawa T (2007) ultaneous determination of 18 major compounds ex- Isolation of antioxidative phenolic glucosides from lemon juice and their tracted from ZYQL for the first time. The validation suppressive effect on the expression of blood adhesion molecules. Biosci Biotech Biochem 71:1911–1919 data indicated that this method is reliable and can be ap- Okamurα N, Hine N, Tateyamα Y, Nakazawα M, Fujiokα T, Mihashi K, Yagi A plied to determine the contents of the 18 compounds in (1998) Five chromones from Aloe vera leaves. Phytochemistry 49:219–223 different ZYQL products. This valuable information con- Rayyan S, Fossen T, Solheim Nateland H, Andersen ØM (2005) Isolation and identification of flavonoids, including flavone rotamers, from the herbal drug cerning the concentration of these bioactive constituents ‘crataegi folium cum flore’ (hawthorn). Phytochem Anal 16:334–341 in ZYQL could be of great importance for the quality as- Wang QJ, Wang YS, He L, Lou ZP, Zang S (2010) Study on chemical constituents sessment and should therefore be useful for the guidance from Ipomoea Pescaprae (L.) Sweet. Chin J Marine Drugs 29:41–44 Yang HP (2007) Zhuyeqing liquor in different historical period. Liquor Making Sci of development of the new health care products. Fur- Technol 2:110–112 thermore, this HPLC-PDA assay supplies a rapidness Yoo SW, Kim JS, Kang SS, Son KH, Chang HW, Kim HP, Bae KH, Lee CO (2002) and effectiveness method for the simultaneous determin- Constituents of the fruits and leaves of Euodia daniellii. Arch Pharm Res 25:824–830 ation of multiple constituents in ZYQL. Zhang YW, Chen YW (1997) Isobiflorin, a chromone C-glucoside from cloves (Eugenia Caryophyllata). Phytochemistry 45:401–403 Competing interests The authors declare that they have no competing interests. doi:10.1186/s40543-014-0034-1 Cite this article as: Gao et al.: Simultaneous quantification of major Authors' contributions bioactive constituents from Zhuyeqing Liquor by HPLC-PDA. Journal of HYG carried out the whole experiment, SYW participated in the sample Analytical Science and Technology 2014 5:34. preparation, HYG and JHW performed the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript. Acknowledgements Grateful acknowledgement is made to the Shanxi XinghuaCun Fen Jiu Group Co., Ltd. (Shanxi Province, China) and National Key Technology R&D Program (2012BAI30B02) for financial support of this work. The authors acknowledge Waters Co. Ltd. for Cooperation Laboratory.

Journal

"Journal of Analytical Science and Technology"Springer Journals

Published: Dec 1, 2014

Keywords: Analytical Chemistry; Characterization and Evaluation of Materials; Monitoring/Environmental Analysis

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