Improved quality evaluation of Radix Salvia miltiorrhiza through simultaneous quantification of seven major active components by high-performance liquid chromatography and principal component analysis

A novel method, using high-performance liquid chromatography combined with principal component analysis, was developed for the quality evaluation of danshen through simultaneous determination of seven components, namely danshensu, protocatechuic acid, protocatechuic aldehyde, salvianolic acid B, tanshinone I, cryptotanshinone and tanshinone IIA. These seven components were simultaneously separated on a Zorbax SB-C(18) column. The mobile phase consisted of 0.05% phosphoric acid water and methanol:acetonitrile (1:1) with a gradient elution, and the detection wavelength was set at 280 nm. Thirty samples of danshen and its substitutes from different sources were investigated by the established method. The results showed that the content of each analyte varied considerably in different danshen samples. Among the seven components tested, salvianolic acid B, tanshinone IIA, cryptotanshinone, tanshinone I, danshensu and protocatechuic aldehyde were proved suitable and representative as chemical markers for the quality control of danshen except for protocatechuic acid. Moreover, principal component analysis was used for the similarity evaluation of different samples, and it could be straightforward and reliable to differentiate danshen samples of different origins. In conclusion, simultaneous quantification of multiple components by high-performance liquid chromatography combined with principal component analysis would be a better strategy for the quality evaluation of danshen.     

Simultaneous determination and pharmacokinetic study of water-soluble and lipid-soluble components of danshen in rat plasma using HPLC-UV method

A sensitive and specific HPLC-UV method was developed for the simultaneous determination of major active components of danshen in rat plasma. Both water-soluble and lipid-soluble compounds were included, i.e. danshensu, salvianolic acid B and tanshinone IIA. Protocatechuic aldehyde and diazepam were used as internal standards. The chromatographic separation was achieved on a reversed-phase C(18) column by gradient elution using acetonitrile and 0.025% (v/v) phosphoric acid solution as mobile phase, at a flow rate of 1.0 mL/min. Salvianolic acid B, danshensu and internal standards were detected at 281 nm, while the detection of tanshinone IIA was carried out at 272 nm. All calibration curves showed good linearity (r(2) > 0.999) within test ranges. The limit of detection and the limit of quantification for danshensu, salvianolic acid B and tanshinone IIA in plasma were 0.065, 0.043, 0.022, 0.131, 0.085 and 0.044 microg/mL, respectively. This is the first report on the determination and pharmacokinetic study of danshensu, salvianolic acid B and tanshinone IIA in rat plasma and the results indicated that this method was reliable for the determination of the major active components of danshen in rat plasma.     

Simultaneous Determination of Seven Lipophilic and Hydrophilic Components in Salvia miltiorrhiza Bunge by LC-MS/MS Method and Its Application to a Transport Study in a Blood-Brain-Barrier Cell Model

Salvia miltiorrhiza Bunge (SM) has been extensively used in Alzheimer’s disease treatment, the permeability through the blood-brain barrier (BBB) determining its efficacy. However, the transport mechanism of SM components across the BBB remains to be clarified. A simple, precise, and sensitive method using LC-MS/MS was developed for simultaneous quantification of tanshinone I (TS I), dihydrotanshinone I (DTS I), tanshinone IIA (TS IIA), cryptotanshinone (CTS), protocatechuic aldehyde (PAL), protocatechuic acid (PCTA), and caffeic acid (CFA) in transport samples. The analytes were separated on a C18 column by gradient elution. Multiple reaction monitoring mode via electrospray ionization source was used to quantify the analytes in positive mode for TS I, DTS I, TS IIA, CTS, and negative mode for PAL, PCTA, and CFA. The linearity ranges were 0.1–8 ng/mL for TS I and DTS I, 0.2–8 ng/mL for TS IIA, 1–80 ng/mL for CTS, 20–800 ng/mL for PAL and CFA, and 10–4000 ng/mL for PCTA. The developed method was accurate and precise for the compounds. The relative matrix effect was less than 15%, and the analytes were stable for analysis. The established method was successfully applied for transport experiments on a BBB cell model to evaluate the apparent permeability of the seven components.     

高效液相色谱-二极管阵列检测器法测定复方丹参片中12种成分及掺伪鉴别

采用高效液相色谱-二极管阵列检测器法测定复方丹参片中12种成分并判定是否掺有三七茎叶. 流动相为乙腈-0.1%磷酸水溶液（梯度洗脱）,通过变换波长结合紫外光谱扫描定性的方式对丹参素钠、原儿茶酸、原儿茶醛、迷迭香酸、丹酚酸B、三七皂苷R₁、人参皂苷Rg₁、人参皂苷Re、人参皂苷Rb₁、人参皂苷Rb₃、丹参酮I、丹参酮II_A进行分析. 12种成分的线性关系良好（r≥0.999 2）,精密度（RSD<2.0%）、稳定性（RSD<2.0%）、回收率（96.1%~99.8%）均符合方法学要求,可用于复方丹参片的质量控制.     

One single LC-MS/MS analysis for both phenolic components and tanshinones in Radix Salviae Miltiorrihizae and its medicinal products

A LC-MS/MS method was developed for the separation and simultaneous determination of phenolic components including danshensu, protocatechuic acid, protocatechuic aldehyde and caffeic acid as well as tanshinones including cryptotanshinone, tanshinone I and tanshinone IIA in samples of Radix Salviae Miltiorrhizae and Salviae Miltiorrhizae tablet. Triple quadrupole mass spectrometry was optimized in both positive and negative ion multiple reaction monitoring modes for the simultaneous quantitative analysis of the two different types of active components by using a time-segment program. The method gave recoveries of 85.4-106.4% with relative standard deviations of 2.4-8.0% for the spiked herb samples. The limits of detection were 0.30-0.83 μg/g for the analysis of 1.0 g Radix Salviae Miltiorrhizae or tablet samples.     

Simultaneous Determination of Seven Active Compounds in Radix Salviae Miltiorrhizae by Temperature-Controlled Ultrasound-Assisted Extraction and HPLC

A simple and effective high-performance liquid chromatographic (HPLC) method has been developed for simultaneous quantification of three phenolic acids (3,4-dihydroxyphenyllactic acid (Chinese name danshensu), protocatechuic aldehyde, and salvianolic acid B) and four diterpenes (dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone II_A) in radix salviae miltiorrhizae. Chromatography was performed on a 250 mm × 4.6 mm i.d., 5-μm particle size, C₁₈ column. The mobile phase was a linear gradient prepared from 0.1% (v/v) aqueous formic acid and acetonitrile at a flow-rate of 1.0 mL min⁻¹. All the target components were well separated with high resolution and without interference. Good linearity (R ² > 0.999) was observed over the concentration ranges investigated, and intra-day and inter-day precision were high. Temperature-controlled ultrasound-assisted extraction was used to prevent hydrolysis of thermally unstable components during the sample-extraction procedure, and the extraction conditions were carefully optimized. Recovery of the seven components was from 98.45 to 100.63% and relative standard deviations were always <1.5%. The validated method was successfully used for simultaneous quantification of the three phenolic acids and the four diterpenes in radix salviae miltiorrhizae of different geographic origins.     

Pharmacokinetic comparison of five tanshinones in normal and arthritic rats after oral administration of Huo Luo Xiao Ling Dan or its single herb extract by UPLC‐MS/MS

A fast, sensitive and reliable ultra performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed and validated for simultaneous quantitation and pharmacokinetic study of five tanshinones (tanshinone I, tanshinone IIA, tanshinone IIB, dihydrotanshinone I, cryptotanshinone), the bio‐active ingredients of Huo Luo Xiao Ling Dan (HLXLD) in rat plasma. After liquid–liquid extraction, chromatographic separation was accomplished on a Shim‐pack XR‐ODS column (75 × 3.0 mm, 2.2 µm particles) and eluted with a mobile phase consisting of acetonitrile–0.05% formic acid aqueous solution (80:20, v/v) at a flow rate of 0.4 mL/min, and the total run time was 7.0 min. The detection was performed on a triple quadrupole tandem mass spectrometry equipped with an electrospray ionization source in positive ionization and multiple reaction monitoring mode. The lower limits of quantification were 0.050–0.400 ng/mL for all the analytes. Linearity, precision and accuracy, the mean extraction recoveries and matrix effects all satisfied criteria for acceptance. This validated method was successfully applied to a comparative pharmacokinetic study of five bio‐active components in rat plasma after oral administration of HLXLD or Salvia miltiorrhiza extract in normal and arthritic rats. The results showed that there were different pharmacokinetic characteristics among different groups. Copyright © 2016 John Wiley & Sons, Ltd.     

Determination and pharmacokinetic analysis of salvianolic acid B in rat blood and bile by microdialysis and liquid chromatography

Salvianolic acid B is an herbal ingredient isolated from Salvia miltiorrhiza. An in vivo microdialysis sampling method coupled to high-performance liquid chromatography has been developed for continuous monitoring of protein-unbound salvianolic acid B in rat blood and bile. Microdialysis probes were inserted into the jugular vein/right atrium and bile duct of Sprague-Dawley rats, and a dose of 100 mg/kg salvianolic acid B was then administered via the femoral vein. Dialysates were collected and directly injected into a liquid chromatographic system. Salvianolic acid B was eluted using a microbore reversed-phase ODS 5 microm (150 mm x 1 mm I.D.) column. Isocratic elution of salvianolic acid B was achieved within 10 min using the liquid chromatographic system. The chromatographic mobile phase consisted of acetonitrile-methanol-20 mM monosodium phosphoric acid (pH 3.5) (10:30:60, v/v/v) containing 0.1 mM 1-octanesulfonic acid with 0.05 ml/min. The wavelength of the UV detector was set at 290 nm. Salvianolic acid B in both blood and bile dialysates was adequately determined using the liquid chromatographic conditions described, although the blank bile pattern was more complex. The retention times of salvianolic acid B in rat blood and bile dialysates were found to be 7.2 min. Peak-areas of salvianolic acid B were linear (r2 > 0.995) over a concentration range of 0.1-50 microg/ml. In vivo recoveries of microdialysis probes of salvianolic acid B in rat blood and bile averaged 22 +/- 2% and 41 +/- 1%, respectively. This study indicates that salvianolic acid B undergoes hepatobiliary excretion.     

A solid-phase extraction method for high-performance liquid chromatographic determination of salvianolic acid B in rabbit plasma: application to pharmacokinetic study

A sensitive solid-phase extraction/high-performance liquid chromatographic method with ultraviolet detection was established for the analysis of salvianolic acid B in rabbit plasma. The analyte was separated on a reversed-phase column with trifluoroacetic acid-methanol-acetonitrile (70:10:20, v/v/v) as mobile phase at a flow rate of 1 mL/min, and ultraviolet detection at 315 nm. The calibration curve for salvianolic acid B was linear over the range 35-1400 microg/L with coefficients of correlation >0.999. The inter-day and intra-day precisions of analysis were <15%, and assay accuracy ranged from 95.3 to 109.1%. This method is suitable for determining salvianolic acid B in plasma and thus investigating the pharmacokinetics of salvianolic acid B.     

Simultaneous determination of tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone in rat plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study of a standardized fraction of Salvia miltiorrhiza, PF2401-SF

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the simultaneous determination of tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone, the active components of Salvia miltiorrhiza in rat plasma, was developed. After liquid-liquid extraction with tariquidar as an internal standard, tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone were eluted from an Atlantis dC18 column within 5 min with a mixture of methanol and ammonium formate (10 mm, pH 6.5; 85:15, v/v). The analytes were detected by an electrospray ionization tandem mass spectrometry in the selected reaction monitoring (SRM) mode. The standard curves were linear (r=0.999) over the concentration range of 0.25-80 ng/mL for tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone in rat plasma. The coefficients of variation and the relative errors of tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone for intra- and inter-assay at four quality control (QC) concentrations were 1.1-5.1% and -4.0-6.0%, respectively. The lower limit of quantification for tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone was 0.25 ng/mL from 100 microL of plasma. This method was successfully applied to the pharmacokinetic study of tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone after oral administration of PF2401-SF, the standardized fraction of Salvia miltiorrhiza enriched with tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone to male Sprague-Dawley rats.     

Quantitative analysis of the marker compounds in Salvia miltiorrihiza root and its phytomedicinal preparations

A HPLC method is described in order to separate and identify 4 phenolic compounds including lithospermic acid B, 3,4-dihydroxyphenyllactic acid (danshensu), rosmarinic acid, and protocatechuic aldehyde as well as 3 lipophilic ones (tanshinone I, tanshinone II(A) and cryptotanshinone) from the roots of Salvia miltiorrihiza or the herbal product containing S. miltiorrihiza available in Chinese or Japanese market. The influence of extractive conditions, such as method, solvents and time, on the 4 phenolic marker components in S. miltiorrihiza is well discussed. In order to investigate the stability of lithospermic acid B, the preparation samples of an injection and a granule were analyzed after being stored at 4 degrees C, 20 degrees C and 40 degrees C for up to six months.     

Development and Validation of Liquid Chromatography–Tandem Mass Spectrometry for Simultaneous Determination of the Four Active Components of Danhong Freeze‐dried Powder Injection in Human Plasma

Abstract

A liquid chromatography‐tandem mass spectrometry was developed and validated for the simultaneous determination of protocatechuic aldehyde, danshensu, salvianolic acid B, and hydroxysafflor yellow A, with rutin as internal standard. Electrospray ionization patterns and efficiency of the analytes, along with their fragmentation, were investigated to achieve sensitive electrospray tandem mass spectrometry (ESI‐MS/MS) detection. Plasma samples were extracted by solid‐phase extraction columns, and the analytes were separated on a Dikma Diamonsil C₁₈ (200 mm×4.6 mm i.d., 5 µm) column using a mobile phase comprised of methanol:acetonitrile: 0.5% formic acid (20∶25∶55, v/v/v) at a flow rate of 1 ml/min. Detection was performed on a Finnigan TSQ ripple quadrupole tandem mass spectrometer in negative ion‐selected monitoring mode using electrospray ionization. Good linearity over the range 10–1000 ng/ml for danshensu, salvianolic acid B, and hydroxysafflor yellow A, and 5–500 ng/ml for protocatechuic aldehyde with acceptable accuracy and precision, respectively. All the validation data, such as accuracy, precision, and stability, were within the required limits. It was a potential platform for the pharmacokinetic and absorption, distribution, metabolism, and excretion (ADME) study of multiple constituent traditional Chinese medicine.     

Improved LC Method for the Simultaneous Determination of Five Active Components in Danshen and Its Preparations

A novel LC method for the simultaneous determination of tanshinone IIA, tanshinone I, cryptotanshinone, dihydrotanshinone, and salvianolic acid B in Danshen and its preparations was developed on a 3 μm particle-sized ODS short column. The five analytes were well separated within 45 min with good linearities (r ² > 0.9999), precises (<4.0%) and recoveries (97.9–105.3%) by a gradient elution of acetonitrile-0.1% aqueous trifluoroacetic acid (v/v). In addition, ultrasonic extraction procedures for sample preparation were optimized using an orthogonal array design. Quantitative determinations on nine herb samples and four preparations demonstrated the developed approaches were suitable for standardization of this herb and its preparations.

     

高效液相色谱二极管阵列检测法同时测定丹参滴注液中四种水溶性成分的含量

建立了高效液相色谱二极管阵列检测(HPLC-DAD)法同时测定丹参滴注液中丹参素、原儿茶醛、迷迭香酸和丹酚酸B四种水溶性成分的含量。采用DiamonsilTMC18色谱柱(250×4.6 mm,5μm),以甲醇和5%冰乙酸为流动相进行梯度洗脱,流速为1.0mL/min,柱温30℃,检测波长为286 nm。在此色谱条件下四种水溶性成分可完全分离。丹参素、原儿茶醛、迷迭香酸和丹酚酸B的线性范围分别为0.2192~1.934μg(r=0.9999),0.03508~0.2456μg(r=1.0000),0.2592~1.814μg(r=1.0000),0.3864~2.705μg(r=0.9999)。平均回收率丹参素为102.6%,相对标准偏差(RSD)为0.55%;原儿茶醛为103.5%,RSD为0.42%;迷迭香酸为99.8%,RSD为0.68%;丹酚酸B为102.8%,RSD为0.49%。该方法简单、快速,四组分分离良好,可用于丹参滴注液的质量控制。     

复方丹参片中四种成分的高效液相色谱-电化学检测-紫外检测法分析

建立了电化学检测器与二极管阵列检测器联用的液相色谱(HPLC-ECD-DAD)同时分离和测定复方丹参片中原儿茶酸、原儿茶醛、咖啡酸和丹参酮ⅡA 等4种成分的分析方法。采用Zorbax SB-C18柱(150 mm×4.6 mm i.d.,5.0 μm),以甲醇和0.4%磷酸为流动相(流速1.0 mL/min)进行梯度洗脱;ECD检测电压0.7 V,DAD检测波长270 nm,柱温30 ℃。实验结果表明,原儿茶酸、原儿茶醛、咖啡酸和丹参酮ⅡA质量浓度分别为0.3～9.0 mg/L,1.1～54.0 mg/L,1.1～11.1 mg/L和5.2～52.0 mg/L时与其峰面积呈良好的线性关系(线性相关系数均高于0.999),原儿茶酸、原儿茶醛、咖啡酸和丹参酮ⅡA的平均回收率(n=6)分别为97.8%(相对标准偏差(RSD)为2.15%),98.2%(RSD为2.07%),97.6%(RSD为2.18%)和97.2%(RSD为2.07%)。该法同时利用了ECD和DAD的优点,是一种快速、灵敏、准确的分析方法,可以为复方丹参片的质量控制提供科学依据。     

Evaluation of the antioxidant potential of Salvia miltiorrhiza ethanol extract in a rat model of ischemia-reperfusion injury

The present study was undertaken to evaluate the protection potential of ethanol extract of Salvia miltiorrhiza (SMEE) against oxidative injury in the ischemia-reperfusion (I/R) model of rats in vivo. Rats were divided into six groups of 10 rats each. Group I/R model and sham were fed with a standard rat chow, groups SMEE I and SMEE II were fed with a standard rat chow and 400 or 800 mg/kg b.w. ethanol extract for 12 days before the beginning of I/R studies. Positive control group was fed with a standard rat chow and salvianolic acid B (55 mg/kg b.w.) or tanshinone II-A (55 mg/kg b.w.) for 12 days before the beginning of I/R studies. To produce I/R, the left anterior descending artery (LAD) was occluded in anesthetized rats for 15 min, followed by 120 min reperfusion. Infarct sizes were found significantly decreased in SMEE-treated and positive control groups compared to I/R model group. Serum AST, LDH and CK-MB activities were significantly reduced and myocardium Na+-K+ ATPase, Ca2+-Mg2+ ATPase activities and antioxidant enzyme activities (SOD, CAT, GSH-Px) were markedly increased in SMEE-treated and salvianolic acid B or tanshinone II-A positive control groups compared to the I/R model group. Pretreatment of S. miltiorrhiza ethanol extract and salvianolic acid B or tanshinone II-A dose-dependently reduced significantly myocardium MDA level, ROS and NOS activities and enhanced myocardium GSH level in I/R rats compared to I/R rats model. In conclusion, we clearly demonstrated that S. miltiorrhiza ethanol extract pretreatment can decrease oxidative injury in rats subjected to myocardial I/R.     

Determination of tanshinone I in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies

This paper describes a rapid and sensitive high-performance liquid chromatographic (HPLC) method for the determination of the concentration of tanshinone I in rat plasma, and applies the method to pharmacokinetic study. The plasma is deproteinized with acetonitrile containing an internal standard (estradiolbenzoate). The HPLC assay is carried out using a Cosmosil C18 column. The mobile phase is acetonitrile, 0.05 mol/L(-1) ammonium acetate buffer with 1% acetic acid (66:34, v/v). The flow rate is 1.0 mL/min. The detection wavelength is set at 263 nm. The assay accuracy is better than 92%, and the precision of tanshinone I at low to high concentrations is better than 9% and 11% for intra-day and inter-day assays, respectively. The recovery of the method exceeds 88.3% for tanshinone I. The assay shows good linearity (r = 0.9998) over a relatively wide concentration range from 0.05 to 10.0 microg/mL. The method is used to determine the concentration-time profiles of tanshinone I in plasma following an intravenous injection of tanshinone I solution, and the pharmacokinetic parameters of tanshinone I are calculated for the first time by the Drug and Statistics 1.0 program. This assay is successfully applied to the determination of tanshinone I in rat plasma, and the developed method is applied to pharmacokinetic studies for the first time.     

Multiple on‐line HPLC coupled with biochemical detection methods to evaluate bioactive compounds in Danshen injection

On‐line high performance liquid chromatography (HPLC) coupled with three biochemical detection (BCD) methods was applied to evaluate bioactive components in Danshen injection. On‐line HPLC‐photo‐diode array–fluorescence detection based on the fluorogenic substrate 7‐acetoxy‐1‐methyl quinolinium iodide, was built to search acetylcholinesterase (AChE) inhibitors in Danshen injection. On‐line HPLC coupled with the scavenging assay of 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) and 2,2′‐azinobis (3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) free radicals was developed to screen antioxidants. The three active profiles were obviously different. Radical scavenging profiles revealed seven strong peaks in the chromatographic fingerprint possessing obvious free radical inhibition effects, while some minor peaks exhibited stronger AChE inhibition activities. The main radical scavengers and AChE inhibitors were identified by HPLC‐MS. Several unknown ingredients showing strong AChE inhibition activities needed further identification except protocatechuic aldehydrate, salvianolic acid H or I and lithospermic acid. The on‐line multiple on‐line HPLC‐BCD methods will provide powerful tools in the field of pharmacognosy for fast‐track identification of interesting and/or novel bioactive compounds.     

Simultaneous determination of seven phenolic acids in three Salvia species by capillary zone electrophoresis with β‐cyclodextrin as modifier

A capillary zone electrophoresis method was developed for the simultaneous determination of seven phenolic acids, including protocatechuic aldehyde ( 1 ), salvianolic acid C ( 2 ), rosmarinic acid ( 3 ), salvianolic acid A ( 4 ), danshensu ( 5 ), salvianolic acid B ( 6 ), and protocatechuic acid ( 7 ), in Danshen and related medicinal plants. A running buffer composed of 20 mM sodium tetraborate adjusted to pH 9.0, and containing 12 mM β‐cyclodextrin as modifier. Baseline separation was achieved within 17 min running at the voltage of 20 kV, temperature of 25°C and detection wavelength of 280 nm. The relative standard deviations of migration time ranged from 0.2 to 0.7% and the peak area ranged from 1.5 to 3.7% for the seven analytes, indicating the good repeatability of the proposed method. The method was extensively validated by evaluating the linearity (R² ≥ 0.9992), limits of detection (0.14–0.36 μg/mL), limits of quantification (0.47–1.19 μg/mL), and recovery (96.0–102.6%). Under the optimum conditions, samples of Danshen and related medicinal plants were analyzed using the developed method with high separation efficiency.