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LA (lot. 140107, 99.05%), Sal A (lot. 140710, 99.53%), Danshensu (lot. 141024, 99.10%), Salvianolic acid C (Sal C, lot. 130902, 99.53%) were purchased from Shanghai Winherb Medical S & T Development Co., Ltd. (Shanghai, China). The mixture of Salvianolic acid T and U (Sal T/U) was kindly provided by Tasly Pharmaceutical Co., Ltd. (Tianjin, China). Deuterium oxide, 99.9 atom % D (D2O), contains 0.05 wt. % 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP), Phosphoric acid, Sodium phosphate dibasic dehydrate and Sodium phosphate monobasic dehydrate were bought from sigma-Aldrich Corporate (St. Louis, MO, USA). HPLC grade formic acid was purchased from ROE Scientific Inc. (Newark, DE, USA). HPLC grade acetonitrile and methanol were obtained from Merck KGaA (Darmstadt, Germany). NMR tube (Norell 502–7) and rubber tube cap (Norell SEPTA-5-W) were purchased from Norell, Inc. (Landisville, NJ, USA). Lumbar puncture needle (0.7 mm×170 mm) was purchased from Shanghai SA Medical & Plastic Instruments Co., Ltd. (Shanghai, PR China). High purity argon was purchased from Shanghai Wugang gas Co., Ltd. (Shanghai, PR China). Ultrahigh-purity water was produced using a Millipore Milli-Q System (Milford, MA, USA).
Overall experimental procedures were presented in Fig 2. It consisted of three sections as follows: (1) sample preparation, (2) structure elucidation of degradation products and proposing degradation pathway of LA, (3) degradation kinetic study of LA and Sal A.
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0164421.g002 Schematic representation of experimental procedure. LA was accurately weighted, dissolved using a mixture of 90% 200 mM phosphate buffer and 10% D2O (containing TSP as the internal standard for chemical shift calibration and quantitative analysis). 500 μL of the test solution was added to the NMR tube, and high purity argon was bubbled into the bottom of test solution through a lumbar puncture needle for 2 minutes to vent oxygen. After that, the NMR tube was rapidly sealed up with a rubber cap. Degraded Samples prepared using different methods were shown in Fig 3. All samples were heated in a thermostat bath set at 91°C. LA solution was prepared using the method proposed in this work in sample set 1. LA solution was added into NMR tube without venting oxygen in sample set 2. LA solution was added into tube without venting oxygen in sample set 3. The colors of sample solutions in sample set 1 were much lighter than others. According to the results of Q-NMR and LC-MS, a portion of Sal A were oxidized in sample set 2, while the concentration of Sal A were very low in sample set 3.
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0164421.g003 Degraded Samples prepared using different methods. HPLC analysis was carried out on an 1100 Series HPLC system (Agilent, Waldbronn, Germany) with diode array detector using an XBridge Shield RP18 column (2.1 mm×150 mm, 3.5 μm, Waters). The temperature of column was maintained at 35°C. UV spectra were recorded from 190 to 400 nm and the detection wavelength was set at 280 nm. The flow rate was 0.2 mL/min, and an in-line filter was used before the analytical column. A gradient elution of mobile phase A (0.1% aqueous formic acid in water) and B (acetonitrile containing 0.1% formic acid) was used. The gradient was as follows: started at 98% A and 2% B, then to 85% A and 15% B at 15 min, 80% A and 20% B at 35 min, 75% A and 25% B at 45 min, 55% A and 45% B at 65 min, 10% A and 90% B at 66 min, kept with 10% A and 90% B from 66 to 70 min. After that, the system was restored to initial conditions in 25 min. HPLC/MSn analysis was performed with an Agilent 1100 Series HPLC and LCQ Deca XPplus ion trap mass spectrometer (Thermo Finnigan, San Jose, CA, USA) equipped with electrospray ionization (ESI) source, with Xcalibur 1.3 controlling software. Nitrogen (N2) was used as the sheath and auxiliary gas, and helium (He) was used as the damping and collision gas. HPLC conditions were the same as described above. The key MS conditions were as follows: negative ion mode, mass range m/z 95–1000. The data-dependent MS/MS and MSn events were performed on the most intense ions detected in full scan and MS/MS. ESI parameters were as follows: source voltage, 3 kV; sheath gas, 30 arbitrary units (arb); auxiliary gas, 10 arb; capillary voltage, -15V; capillary temperature, 350°C; tube lens, -30 V.
Quantitative NMR analysis was carried out on a Bruker Avance III (500.13M) NMR spectrometer equipped with a 5 mm 1H/D-BBO probehead (Bruker BioSpin GmbH, Rheinstetten, Germany). All the samples were locked individually on 90% H2O + 10% D2O and were measured at 293.0 K. Gas flow was set at 400 L/h. The spectra were acquired in 32 scans using 32 k data points by noesypr1d pulse program with water signal suppression. The 90° pulse width was set to 14.62 μs and the acquisition time was 2.04 s. The spectra width was set to 8013 Hz. The longitudinal relaxation time T1 was determined for the protons of interest (Table 1, Fig 1). Relaxation delay time (D1) was optimized for the T1 of the longest relaxing TSP nuclei, to ensure maximum recovery of the transverse magnetization, and D1 was set to 15 s.
Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0164421.t001 T1 values of monitored protons (tested at 293 K). The stock solution (5.56 mg/mL) was prepared by dissolving an appropriate amount of LA in 200 mM phosphate buffer (pH 5.29). The calibration curve was made using seven standard solutions of different concentrations (5.00, 4.00, 3.00, 2.00, 1.00, 0.500, 0.100 mg/mL). The standard solutions were prepared by diluting an appropriate volume of stock solution with the phosphate buffer and D2O (containing TSP as the internal standard for chemical shift calibration and quantitative analysis) was added to volume percentage of 10% for each sample. Each solution was analyzed twice. The peak area values were plotted against the corresponding analyte concentrations to obtain the linear calibration. Intraday precision of the method was determined by measuring six sample of LA (2.00 mg/mL) on the same day.
Twelve or more test tubes were laid in a thermostat bath at predefined temperature and were periodically withdrawn during a kinetic run. Withdrawn samples were rapidly cooled in ice to quench the reaction and were stored in an ice bath until analysis within 2 h. Each study was comprised of twelve or more assays spaced to provide change of ~ 0.1 C10 per samples. After NMR analysis, degradation samples were diluted with an equal volume of 0.3 M phosphoric acid. 2 μL diluted samples were injected into LC-MSn system for tentative structure elucidation of the degradation products. The influences of temperature and pH values on degradation of LA were investigated. The influence of temperature on degradation was investigated in phosphate buffer solutions at a pH value of 4.75. The reaction rate constants were calculated at 80, 91 and 100°C, respectively. The effect of pH values on degradation was determined at 91°C in phosphate buffer solutions. Specific experimental conditions were listed in Table 2. All the pH measurements were performed on a pH meter (S40 SevenMulti, Mettler-Toledo GmbH, Greifensee, Switzerland) equipped with combination pH electrode (InLab Expert Pro).
Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0164421.t002 Experimental conditions and kinetic constants for LA degradation. According to the results of preliminary experiment, supposing the degradation of LA followed irreversible first-order reaction kinetics, ordinary differential equation of LA concentration was obtained as follows: dC1dt=−kr1⋅C1(1) where C1 is the concentration of LA and kr1 is the of LA. Therefore, the concentration of LA could be described by Eqs (2) and (3) C1t=C10⋅e−kr1⋅t(2) lnC1tC10=−kr1⋅t(3) where C10 is the initial concentration of LA. For Sal A, it was degraded from LA and would transform to Sal C and other isomers. Assuming all these reactions follow irreversible first-order reaction kinetics, ordinary differential equation of Sal A was obtained as follows: dC2dt=kr2⋅C1−kr3⋅C2(4) where C2 is the concentration of Sal A, kr2 is the rate constant of LA transform to Sal A, kr3 is the rate constant of Sal A transform to other compounds. Because the initial concentration of Sal A was 0, Eq (5) can be derived from Eqs (2) and (4): C2tC10=kr2kr1−kr3⋅(e−kr3⋅t−e−kr1⋅t)(5) The influence of temperature on reaction rate constant was given by Arrhenius equation: ln kr=ln A−EaRT(6) where A represents frequency factor, Ea stands for activation energy, R is ideal gas constant (8.314 J/mol·K) and T is temperature (K). All data fitting were carried out using OriginLab (Pro 8, OriginLab Corp., Northampton, MA).
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