Simultaneous detection of seven phenolic acids in Danshen injection using HPLC with ultraviolet detector§

Simultaneous detection of seven phenolic acids in Danshen injection using HPLC with ultraviolet detector § § first-author corresp ‡Corresponding Author ‡Corresponding Author †E-mail:quhb@zju.edu.cn †E-mail:quhb@zju.edu.cn quhb@zju.edu.cn Department of Chinese Medicine Science and Engineering, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China Department of Chinese Medicine Science and Engineering, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China A high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) detector had been developed for simultaneous quantification of danshensu, protocatechuic aldehyde, caffeic acid, salvianolic acid D, rosmarinic acid, salvianolic acid B and salvianolic acid A in Danshen injection. According to the UV spectra of these components, three detection wavelengths have been selected as follows: 280 nm for danshensu and protocatechuic aldehyde, 326 nm for caffeic acid, salvianolic acid D and rosmarinic acid, 286 nm for salvianolic acid B and salvianolic acid A. The limit of detection (LOD) was improved to be in the range of 0.008~0.160 μg/ml. Moreover, excellent linear behavior over the investigated concentration range was observed, with R > 0.999 for all the analytes. High-performance liquid chromatography (HPLC) Danshen injection (DSI) Phenolic acids Multi-wavelengths Quantification INTRODUCTION 1 Danshen (DS), the dried root of Salvia miltiorrhiza , is a famous traditional Chinese medicine and widely used to treat coronary heart disease, cerebrovascular disease, bone loss, hepatitis, hepatocirrhosis and chronic renal failure (Wasser et al., 1998 1998 ; Liu et al., 2000 2000 ; Ji et al., 2003 2003 ; Chae et al., 2004 2004 ; Ling et al., 2005 2005 ). There are a number of traditional Chinese medicinal preparations containing DS, such as Danshen tablets, Danshen dripping pills, Danshen injection (DSI) and Xiangdan injection. DSI is made from the aqueous extract of DS and widely used in clinics as a treatment for coronary heart disease, heart-stroke and cerebrovascular disease. The chemical constituents of DS include both lipophilic and hydrophilic components. The major hydrophilic components are phenolic acids including danshensu (DSS), protocatechuic aldehyde (PA), rosmarinic acid (RA) and salvianolic acids, which are also major pharmacologically active constituents in DSI. Taking the phenolic acids as target compounds, many high-performance liquid chromatographic (HPLC) methods have been established for the quality control of DS and its related traditional Chinese medicinal preparations. For instance, HPLC coupled with ultraviolet (UV) detection (Zhang et al., 2005 2005 ) or diode-array detection (DAD) (Hu et al., 2005 2005 ; Liu et al., 2007 2007 ) together with liquid chromatography-tandem mass spectrometry (LC-MS n ) method and HPLC with a coulometric electrode array system (HPLC-CEAD) (Ma et al., 2007 2007 ) have been established for chemical fingerprint analysis of DS. HPLC-UV methods have been used in the quantification of both four phenolic acids and three lipophilic components in DS (Yuan et al., 2005 2005 ) and seven active components including salvianolic acids, tanshinones and ginsenosides in the Fufang Danshen tablet (Wei et al., 2007 2007 ). An HPLC method with photodiode-array UV detection has been developed to quantify three major lipophilic components (cryptotanshinone, tanshinone I and tanshinone IIA) and three major hydrophilic components (DSS, PA and salvianolic acid B (SaB)) (Fig. 1 1 ) of DS (Zhou et al., 2006 2006 ). An HPLC method with DAD has been established for simultaneous quantification of six phenolic acids (DSS, PA, RA, lithospermic acid, SaB and salvianolic acid A (SaA)) in DS and its related traditional Chinese medicinal preparations (Liu et al., 2006 2006 ). All of the abovementioned papers have indicated that HPLC is a feasible method for quality control of DS and its related preparations including DSI. On the other hand, we realized that the contents of some phenolic acids, such as caffeic acid (CA) and salvianolic acid D (SaD) (Fig. 1 1 ), are so low that they are difficult to be quantified. The present study aimed at developing a more sensitive HPLC method with UV detector for simultaneous quantification of those components which are in low content in the DSI. In the analytical process three wavelengths of 280 nm, 326 nm and 286 nm have been selected to detect DSS and PA, CA, SaD and RA, SaB and SaA, respectively, and the limit of detection (LOD) was improved to be in the range of 0.008~0.160 µg/ml. This method has been applied in determining the amount of these seven compounds, from different batches and producers, in the DSI. MATERIALS AND METHODS 1 Chemicals and reagents 2 DSIs were supplied by a Chinese pharmaceutical manufacturer (Qingchunbao, Zhejiang, China). Standard substances including DSS, PA, CA, RA and SaB were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). SaD and SaA were isolated from aqueous extract of DS. These isolated salvianolic acids were identified on the basis of mass spectrum (MS) data, purities of which were all over 97% by HPLC analysis. The HPLC-grade acetonitrile and formic acid were purchased from Merck (Darmstadt, Germany) and the Tedia Company (Fairfield, USA), respectively. Deionized water used throughout the experiments was produced using a Mill-Q academic water purification system (Milford, MA, USA). Instrumentation and analytical conditions 2 The HPLC system HP 1100 series (Agilent Technologies, Waldbronn, Germany) was equipped with Chemstation Software (Agilent Technologies) and was comprised of a quaternary pump, an online vacuum degasser, an autosampler and a thermostated column compartment. A UV detector was used for the chromatographic analysis and a DAD detector was used to obtain UV spectra of seven standard substances. All separations were carried out on a Tigerkin C 18 column (200 mm×4.6 mm i.d., 5.0 µm particle size) from Dalian Sipore Co., Ltd. (Dalian, China). A linear gradient elution of Eluents A (0.5% (v/v) aqueous formic acid) and B (0.5% (v/v) formic acid in acetonitrile) was used to run the separation. The elution programme was well optimized and conducted as follows: the first linear gradient was 5%~20% Eluent B in the range of 0~10 min, the second one was 20%~25% Eluent B in the range of 10~17 min, and the last one was 25%~55% Eluent B in the range of 17~35 min. Then the system was restored to initial conditions after 5 min. The solvent flow rate was 1.0 ml/min, the injection volume was 20 µl, and the column temperature was maintained at 30 °C. The chromatograms were recorded at 280 nm in the range of 0~13 min, 326 nm in the range of 13~23.5 min, and 286 nm in the range of 23.5~35 min. Solution preparation 2 1. Standard solution 3 The standard stock solutions of DSS (4.05 mg/ml), PA (5.10 mg/ml), CA (1.0 mg/ml), SaD (1.15 mg/ml), RA (2.70 mg/ml), SaB (1.00 mg/ml) and SaA (1.10 mg/ml) were prepared in phosphoric acid-methanol-water (0.5/80/19.5, v/v/v) and stored away from light at 4 °C. Working solutions of the lower concentration were prepared by an appropriate dilution of the stock solution. 2. Sample solution 3 DSI of 200 µl was diluted to 1 ml with deionized water and the dilution was injected into the HPLC system. RESULTS AND DISCUSSION 1 Chromatographic separation 2 An aqueous acetonitrile solvent system was often used in the HPLC analysis of phenolic acids in DS (Wasser et al., 1998 1998 ; Liu et al., 2000 2000 ; Zhang et al., 2005 2005 ; Zhou et al., 2006 2006 ; Ma et al., 2007 2007 ) and the modifiers were formic acid, acetic acid, or phosphoric acid. Water (0.5% (v/v) formic acid) and acetonitrile (0.5% (v/v) formic acid) were selected as the mobile phases in the experiment. From the UV spectra of these seven phenolic acids (Fig. 2 2 ), 280 nm for DSS and PA, 326 nm for CA, RA and SaD, and 286 nm for SaB and SaA, were selected as detective wavelengths. Five column temperatures (15, 20, 25, 30 and 35 °C) were investigated and the separation of CA, SaD and SaB was found to be improved when the temperature stepped up. However, base-line separation of DSS was obtained at 30 °C (Fig. 3 3 ). As a result, the column temperature was determined as 30 °C. The most suitable flow rate was found to be 1.0 ml/min. Finally the chromatograms, after optimization of the mixture of standard solution and the real sample solution, were described as in Fig. 4 4 . Calibration curves and limits of detection 2 Integrated chromatographic peak areas ( Y ) were plotted against the corresponding concentrations ( X , μg/ml) of the seven constituents in the standard solutions to obtain calibration curves based on linear regression analysis. The regression curves were obtained from six concentration levels and all the analytes had good linearity ( R > 0.999) in the investigated ranges (Table 1 1 ). Limit of quantification (LOQ) was defined as the lowest concentration level resulting in a peak height of 10 times the baseline noise (the signal-to-noise ratio ( S / N ) is 10). The minimum concentration, which could be calculated at S / N =3, was considered to be the LOD. The LOD values for seven target components were in the range of 0.008~0.160 µg/ml (Table 1 1 ), which were better than those (0.04~0.43 µg/ml) in a previous report (Wasser et al., 1998 1998 ). LOQ : Limit of quantification; LOD : Limit of defection Compounds Retention time (min) Test ranges (µg/ml) Calibration curves R LOQ (µg/ml) LOD (µg/ml) DSS 7.85±0.05 40.50~607.50 Y =14.605 X −31.357 0.9999 0.400 0.130 PA 11.50±0.05 10.20~183.60 Y =110.45 X +95.782 0.9998 0.100 0.030 CA 14.05±0.05 1.00~15.00 Y =124.96 X −0.2877 0.9990 0.025 0.008 SaD 19.96±0.05 3.45~34.50 Y =24.469 X −1.8991 0.9994 0.160 0.050 RA 22.48±0.05 16.20~113.40 Y =80.06 X −102.55 0.9993 0.150 0.050 SaB 24.31±0.05 10.00~90.00 Y =23.653 X −11.357 0.9998 0.500 0.160 SaA 26.02±0.05 1.10~22.00 Y =62.356 X −4.9326 0.9993 0.500 0.160 Repeatability, precision and stability 2 The analysis repeatability was examined by using six duplicate samples from the same batch of DSI, treated according to the sample preparation procedure and analyzed with the established HPLC method. The stabilities of these seven compounds were tested at six time points (0, 2, 4, 8, 16 and 24 h) during 24 h. The result showed that DSS, PA, CA, SaD, RA and SaB were stable in the testing period, except for SaA with a stability of just 4 h (Table 2 2 ). According to this decomposing speed, it was almost certain that SaA in DSI became unstable just after opening the bottle of injections. After 4 h, the chromatographic peak of SaA in DSI had almost disappeared, while SaA in stock solution was found to be stable. It was thought to relate to their different pH values (about pH 7 in DSI and pH 3 in stock solution). The instability of SaA was thought to indicate that its relative standard deviation ( RSD ) value of repeatability analysis was more than 5% (Table 2 2 ). Stability of SaA in 4 h and stability of others in 24 h; RSD : Relative standard deviation Compounds RSD for repeatability (%) RSD for stability * * (%) DSS 1.160 0.775 PA 1.379 0.541 CA 0.757 1.806 SaD 2.476 3.401 RA 0.712 0.763 SaB 0.773 1.334 SaA 9.178 5.743 The intra- and inter-day instrument precisions were determined by analyzing three different concentration solutions (low, medium and high) of authentic standards. The intra- and inter-day experiments were tested by injecting three times in a single day and three times on six successive days, respectively. The results are shown in Table 3 3 . Due to its instability, the maximum RSD values in the precision test were obtained from SaA. Accuracy (%)=(mean of measured concentration)/(nominal concentration)×100 2 Exp. 2 Comp. 3 Low concentration 3 Medium concentration 3 High concentration Found (µg/ml) RSD (%) Accuracy * * (%) Found (µg/ml) RSD (%) Accuracy (%) Found (µg/ml) RSD (%) Accuracy (%) 7 Intra-day ( n =3) DSS 41.97±0.016 0.038 103.62 203.06±0.115 0.057 100.28 322.84±0.153 0.047 88.57 PA 9.76±0.003 0.032 95.71 52.48±0.046 0.087 102.91 93.25±0.089 0.096 101.58 CA 0.96±0.092 0.097 95.59 4.36±0.004 0.094 109.00 7.087±0.008 0.113 101.24 SaD 3.37±0.006 0.185 97.69 9.47±0.012 0.125 102.90 16.87±0.039 0.231 97.80 RA 17.24±0.031 0.178 106.42 38.25±0.037 0.097 101.20 64.07±0.048 0.075 98.88 SaB 10.08±0.066 0.656 100.84 29.38±0.049 0.166 97.94 52.43±0.015 0.029 104.85 SaA 1.03±0.032 3.080 93.99 4.15±0.048 1.161 94.34 15.24±0.074 0.485 92.35 11 7 Inter-day ( n =6) DSS 42.12±0.182 0.432 104.02 203.34±0.919 0.452 100.42 321.24±1.485 0.462 88.13 PA 9.81±0.068 0.689 96.19 52.76±0.416 0.789 103.45 93.55±0.687 0.734 101.91 CA 0.96±0.007 0.742 96.15 4.39±0.037 0.844 109.79 7.10±0.041 0.578 101.44 SaD 3.42±0.048 1.408 99.21 9.60±0.114 1.192 104.32 16.87±0.067 0.395 97.81 RA 17.40±0.177 1.015 107.64 38.48±0.261 0.679 101.79 64.14±0.288 0.449 98.98 SaB 10.32±0.220 2.130 113.42 29.62±0.204 0.688 98.74 52.50±0.365 0.695 105.00 SaA 0.98±0.091 9.296 89.05 4.01±0.235 5.862 91.08 15.15±0.538 3.552 91.80 Recovery test 2 In the experiment, three different quantities (low, medium and high) of authentic standards were added to sample solutions. Then the mixture solutions were analyzed using the developed HPLC-UV method mentioned above and the quantity of each component was subsequently calculated from the corresponding calibration curve. The result (Table 4 4 ) showed that the recovery of these six phenolic acids, except SaA, ranged from 94.32%~106.05%. In previous experiments, SaA was found to be unstable in DSI and relatively stable in phosphoric acid-methanol-water (stock solution). Due to this fact, the recoveries of SaA that were obtained were much higher than 100% at low concentration and lower than 100% at medium and high concentrations. These recoveries were tested from low concentration to high concentration. Recovery (%)=(amount found)/(amount spiked)×100 Comp. Phenolic acid spiked (µg/ml) Found (µg/ml) RSD (%) Recovery * * (%) DSS 114.82 120.52 0.361 104.97 143.17 150.21 0.395 104.92 170.10 163.39 0.150 96.06 PA 22.03 23.05 0.112 104.62 27.85 28.74 0.259 103.21 33.15 32.59 0.087 98.32 CA 1.47 1.41 1.643 95.85 1.85 1.96 1.474 106.05 2.25 2.16 1.719 96.16 SaD 4.40 4.20 1.370 95.42 5.52 5.27 0.880 95.55 6.67 6.32 1.960 94.79 RA 11.02 10.39 0.248 94.32 13.50 13.46 0.081 99.70 16.20 15.66 0.278 96.66 SaB 6.50 6.28 0.871 96.61 8.20 8.09 0.265 98.70 9.80 9.31 0.718 94.97 SaA 1.60 1.91 0.819 119.65 1.98 1.64 1.610 82.92 2.39 1.93 1.216 80.74 Sample analysis 2 The developed method was applied to the simultaneous quantification of DSS, PA, CA, SaD, RA, SaB and SaA in DSIs from different batches. The result (Table 5 5 ) showed that as the unique target component for quality control of DSI, the content of PA approximated to 250 µg/ml in all of the samples, while contents of other components differed greatly among batches from the same company. The variety may result from the low level of quality standards. It is well known that the preparations of traditional Chinese medicines generally include multiple bioactive constituents while only one component (e.g. PA in DSI) was considered as the target for quality control. It meant that only the target compound was well monitored during the manufacturing process and other components were ignored. 2 Batch No. 7 Content (µg/ml) DSS PA CA SaD RA SaB SaA 0610261 1232.70 260.36 18.34 37.57 95.23 62.65 16.23 0607282 1448.54 288.97 18.57 76.14 173.64 86.94 31.11 0611291 1296.28 275.80 20.82 52.10 122.59 76.92 23.09 0611301 1245.27 254.05 16.37 54.92 133.73 100.60 28.11 0612011 1153.68 253.37 17.75 46.18 120.19 80.26 18.34 0612031 956.39 232.31 15.31 35.07 100.46 49.11 14.62 0610201 1352.65 263.70 16.94 54.18 163.86 70.07 21.15 0701051 1239.32 240.19 19.31 43.44 124.76 83.88 24.19 0612051 1066.27 256.42 19.28 32.96 110.59 55.54 14.02 0612061 1363.77 275.05 20.74 52.49 123.91 97.36 26.57 0703221 450.22 224.64 15.20 16.31 58.19 14.72 6.94 0612021 1352.92 263.58 22.40 50.21 129.32 87.37 23.37 0610251 1426.40 277.45 18.60 55.05 135.90 81.75 19.90 0610241 1446.65 225.00 16.25 65.85 171.05 94.10 24.20 CONCLUSION 1 In the present study, an HPLC method with ultraviolet detector has been developed for the simultaneous detection of seven phenolic acids of DSS, PA, CA, SaD, RA, SaB and SaA in DSI. 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Improved quality control method for Danshen products—consideration of both hydrophilic and lipophilic active components The papers for the Postdoctoral Fellows Association of Zhejiang Province, China 10.1631/jzus.B0820095 Project supported by the National Basic Research Program (973) of China (No. 2005CB523402) and the Program for the New Century Excellent Talents in University of China (No. NCET-06-0515) 2008-03-20 2008-04-24 2022-01-05 Copyright © 2008, Journal of Zhejiang University Science 2008 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 09 09