Research On Quality Control Method Of The Multi-index Components In Salvia Miltiorrhiza And Its Compound Preparations

Salvia miltiorrhiza is a common Chinese herb. It is considered in Chinese tradition medicine theory that Salvia miltiorrhiza is micro-chilly and bitter in taste, belongs to heare and liver theoty. It has the efferciency of removing blood stasis and alleviate pain, activating blood of promote menstruation, clearing away heart-fire and eliminating annoy. According to statistics, there has been numerous of Salvia miltiorrhiza preparations. The major chemical composition of Salvia miltiorrhiza include both lipid-soluble diterpene quinine and water-soluble phenolic acid.Quality control for drug is an essential attributes and the basis on security and validity of medicine. Through looking up large number of literature, it can be found that quality control methods of many Salvia miltiorrhiza preparations have been reported. However, the quality control of some Salvia miltiorrhiza preparations has not been reported, or only one or two active components for quality control has been reported. It can not totally eveluate the quality of these drugs. Furthermore, some quality control methods reported before need to be improved.This research selected six kinds of Salvia miltiorrhiza preparations, such as Xinkening capsule, Xinkeshu capsule, Guanxinning injection, Danshen dripping solution,Danxiangguanxin injection,Danshen powder injection and Salvia miltiorrhiza pieces. The multi-index components of Salvia miltiorrhiza and other drugs were used as the quality control indices. The quality control methods of drug was systematic studied by RP-HPLC. The author established the assay of RP-HPLC to determine the six components in Xinkening capsule, Xinkeshu capsule and Guanxinning injection, the assay of RP-HPLC to determine the five water-soluble components in Danshen dripping solution and Danxiangguanxin injection, the assay of RP-HPLC to determine the four water-soluble components in Danshen dripping solution and Danshen powder injection, and the assay of RP-HPLC to determine the five water-soluble components in Salvia miltiorrhiza pieces. The methods is special, sensitive and accurate. Therefore, The methods can be used for the quality control of the above mentioned Salvia miltiorrhiza compound preparations and Salvia miltiorrhiza pieces.Part one Research on Quality Control Method of the Multi-index Components in Salvia Miltiorrhiza Compound Capsule Preparations(1) Determination of Six Components in Xinkening Capsule by RP-HPLC simultaneouslyObjective:To study the contents and determination method of danshensu, protocatechuic aldehyde, salvianolic aicd B, cryptotanshinone, tanshinoneⅠand tanshinoneⅡA in Xinkening capsule.Methods: (1) The extraction method: different extraction solvents and extration time were tested for optimization of the extraction efficacy of Xinkening capsule. The most efficacy extration method was selected for the extraction of the major chemical components of Xinkening capsule. (2) Optimization of the chromatographic conditions: chromatographic conditions such as stationary phase, mobile phase systems, gradient programs and detection method were tested for optimization of the chromatographic conditions,so that the chromatographic peaks of components to be measured were completely separated with other peaks. (3) System suitability test: under the above conditions, the resolution, theoretical plate number and symmetry factor of the six chemical components was studied. (4) Constructed the calibration curve; testing the limits of detection (LOD), precision, stability and reproducibility. (5) Testing the recovery. (6) Determinating the samples.Results: (1) The extraction method: Xinkening capsule was extracted with 75% methanol by a sonifier at room temperature for 30 min. The extraction method of the major chemical components of Xinkening capsule was efficacy. (2) The chromatographic condition: the procedure of RP-HPLC was performed on a column of DiamonsilTM C18 (250 mm×4.6 mm, 5μm) using methanol -1% acetic acid glacial water solution as the mobile phase in gradient elution. The flow rate was 1.0 mL/min. The column temperature was 30℃. The detection wavelength was 270 nm. (3) System suitability test: under the above conditions, the peaks of components wear separated well with the resolution of more than 1.5, and the theoretical plate number of danshensu was larger than 5000, of cryptotanshinone was larger than 10000. (4) The calibration curve: the sample size of danshensu, protocatechuic aldehyde, salvianolic aicd B, cryptotanshinone, tanshinoneⅠand tanshinoneⅡA showed respectively a good linear relationship at range of 219.0~3285, 35.08~526.2, 438.0~6570, 16.68~250.2, 10.05~150.8 and 18.65~279.8 ng (r=0.9999, 0.9999, 0.9999, 0.9998, 0.9997 and 0.9999) . (5) Recovery test: the average recovery (n=6) was 100.1%, 100.8%, 98.7%, 100.5%, 100.0% and101.8%(RSD=1.5%, 0.96%, 0.59%, 1.2%, 1.3% and 0.58%)respectively. (6) Determinating the samples: the established method was applied for determination of the major chemical components in Xinkening capsule of different production batches and different manufactures. The content of each chemical component was calculated according to reference substances .Conclusions: The method is peculiar with a good reproducibility, which can be used for the qualitative evaluation of Xinkening capsule. (2) Determination of Six Components in Xinkeshu Capsule by RP-HPLC simultaneouslyObjective: A RP-HPLC was established for the determination of six effective components: danshensu, protocatechuic aldehyde, puerarin, salvianolic aicd B, cryptotanshinone and tanshinoneⅡA in Xinkeshu capsule.Methods: The procedure of RP-HPLC was performed on a column of DiamonsilTM C18 (250 mm×4.6 mm, 5μm) using methanol -1% acetic acid glacial water solution as the mobile phase in gradient elution. The flow rate was 1.0 mL/min. The detection wavelength was 270 nm. The column temperature was 30℃.Results: The sample size of danshensu, protocatechuic aldehyde, puerarin, salvianolic aicd B, cryptotanshinone and tanshinoneⅡA showed respectively a good linear relationship at the range of 219.0~3285 ng, 14.03~210.5 ng, 114.0~1710 ng, 438.0~6570 ng, 10.42~156.4 ng and 9.325~139.9 ng (r=0.9999, 0.9999, 0.9999, 0.9996, 0.9997 and 0.9997). The average recovery (n=6) was 100.4%, 99.5%,98.0%, 98.6%, 101.7% and 100.9% (RSD=1.6%, 1.8%, 0.80%, 0.79%, 0.64 % and 1.5%) respectively.Conclusions: The method is peculiar with a good reproducibility, which can be used for the qualitative evaluation of Xinkeshu capsule.Part two Research on Quality Control Method of the Multi-index Components in Salvia Miltiorrhiza Compound Injection Preparations(1) Simultaneous Determination of Danshensu, Protocatechuic aicd, Protocatechuic aldehyde, Ferulic aicd, Rosmarinic acid and Salvianolic aicd B in Guanxinning Injection by High Performance Liquid Chromatography with Diode Array DetectionObjective: A high performance liquid chromatographic method with diode array detection was established for simultaneous determination of six water-soluble compounds danshensu, protocatechuic aicd, protocatechuic aldehyde, ferulic aicd, rosmarinic acid and salvianolic aicd B in Guanxining injection.Methods: The column was DiamonsilTM C18 (250 mm×4.6 mm, 5μm). The mobile phase was methanol and 5% acetic acid glacial with gradient elution and the flow rate was 1.0 mL/min, the column temperature was 30℃, the detection wavelength was 286 nm.Results: The linearity was obtained over the range of 87.68~438.40 ng (r=0.9993) for danshensu with average recovery rate reaching 98.2% (RSD=0.27%), 29.68~148.40 ng (r=0.9997) for protocatechuic acid with average recovery rate reaching 98.4% (RSD=1.2%), 14.03~70.16 ng (r=1.0000) for protocatechuic aldehyde with average recovery rate reaching 99.2% (RSD=1.3%), 30.40~152.00 ng (r=0.9999) for ferulic acid with average recovery rate reaching 98.9% (RSD=0.96%), 103.70~518.40 ng (r=0.9996) for rosmarinic acid with average recovery rate reaching 98.1% (RSD=0.82%), 193.20~966.00 ng (r=0.9996) for salvianolic aicd B with average recovery rate reaching 99.3% (RSD=0.31%).Conclusions: The validated method has the advantages of simplicity, precision and reliability, allowing its intrinsic quality control of Guanxinning injection. (2) Simultaneous Determination of 5 Water-soluble Components in Containing Salvia Miltiorrhiza Injections by RP-HPLC with Diode Array DetectionObjective: To develop a reversed-phase high performance liquid chromatography with diode array detection for the simultaneous determintion of 5 water-soluble components, danshensu, protocatechuic aicd, protocatechuic aldehyde, rosmarinic acid and salvianolic acid B in Salvia miltiorrhiza -containing injections.Methods: The column was DiamonsilTM C18 (250 mm×4.6 mm, 5μm). The mobile phase was methanol and 5% acetic acid glacial with gradient elution and the flow rate was 1.0 mL/min , the column temperature was 30℃, the detection wavelength was 298 nm.Results: The linearity was obtained over the range of 0.5480~3.836μg (r=0.9992) for danshensu with average recovery rate reaching 101.2% (RSD=2.3%), 0.01484~0.1039μg (r=1.0000) for protocatechuic acid with average recovery rate reaching 97.8% (RSD=0.16%), 0.01052~0.07367μg (r=1.0000) for protocatechuic aldehyde with average recovery rate reaching 102.4% (RSD=0.83%), 0.04536~0.3175μg (r=0.9999) for rosmarinic acid with average recovery rate reaching 101.6% (RSD=1.2%), 0.1546~1.082μg (r=1.0000) for salvianolic aicd B with average recovery rate reaching 102.5% (RSD=0.26%).Conclusions: The validated method has good accuracy and a simple procedure. It may be practical to evaluate and control the quality of Salvia miltiorrhiza -containing injections. (3) Simultaneous Determination of 4 Water-soluble Components in Danshen Dripping Solution by High Performance Liquid Chromatography with Diode Array DetectionObjective: A high performance liquid chromatographic method with diode array detection was established for simultaneous determination of 4 water-soluble compounds of danshensu, protocatechuic aldehyde, rosmarinic acid and salvianolic aicd B in danshen dripping solution.Methods: The column used was DiamonsilTM C18 (250×4.6 mm, 5μm). The mobile phase was methanol and 5% glacial acetic acid with gradient elution and the flow rate was 1.0 mL/min, the column temperature was 30℃, and the detection wavelength was 286 nm.Results: The linearity was obtained over the range of 0.2192~1.934μg (r=0.9999) for danshensu with average recovery of 102.6% (RSD=0.55%), 0.0351~0.2456μg (r=1.0000) for protocatechuic aldehyde with average recovery of 103.5% (RSD=0.42%), 0.2592~1.814μg (r=1.0000) for rosmarinic acid with average recovery of 99.8% (RSD=0.68%), 0.3864~2.705μg (r=0.9999) for salvianolic aicd B with average recovery of 102.8% (RSD=0.49%), respectively.Conclusions: The method is rapid, accurate and reliable, and can be used to control the quality of Danshen dripping solution.(4) Simultaneous Determination of 4 Water-soluble Components in Danshen Powder Injection by RP-HPLC with Diode Array DetectionObjective: A high performance liquid chromatographic method with diode array detection was established for simultaneous determination of 4 water-soluble compounds danshensu, protocatechuic aicd, protocatechuic aldehyde and rosmarinic acid in danshen powder injection.Methods: The column was DiamonsilTM C18 (250 mm×4.6 mm, 5μm). The mobile phase was methanol and 5% acetic acid glacial with gradient elution and the flow rate was 1.0 ml/min, the column temperature was 30℃, the detection wavelength was 298 nm.Results: The linearity was obtained over the range of 0.1644~2.630μg (r=0.9999) for danshensu with average recovery rate reaching 101.6% (RSD=1.1%), 0.007420~0.1187μg (r=1.0000) for protocatechuic aicd with average recovery rate reaching 102.2% (RSD=1.8%), 0.008770~0.1403μg (r=1.0000) for protocatechuic aldehyde with average recovery rate reaching 101.3% (RSD=2.0%), 0.01944~0.3110μg (r=1.0000) for rosmarinic acid with average recovery rate reaching 100.9% (RSD=1.6%).Conclusions: The method is rapid, accurate and reliable ,and can be used to control the quality of Danshen powder injection.Part three Simultaneous Determination of 5 Water-soluble Components in Salvia Miltiorrhiza Pieces by Reversed-phase High Performance Liquid Chromatography with Diode Array DetectionObjective: To develop a reversed-phase high performance liquid chromatography with diode array detection for the simultaneous determintion of 5 water-soluble components,danshensu, protocatechuic aicd, protocatechuic aldehyde, rosmarinic acid and salvianolic acid B in Salvia miltiorrhiza pieces.Methods: The column was DiamonsilTM C18 (250 mm×4.6 mm, 5μm). The mobile phase was methanol and 5% acetic acid glacial with gradient elution and the flow rate was 1.0 mL/min, the column temperature was 30℃, the detection wavelength was 298 nm.Results: The linearity was obtained over the range of 0.5480~3.836μg (r=0.9992) for danshensu with average recovery rate reaching 100.7% (RSD=0.96%), 0.0148~0.1039μg (r=0.9999) for protocatechuic acid with average recovery rate reaching 100.8% (RSD=1.0%), 0.0105~0.0737μg (r=0.9999) for protocatechuic aldehyde with average recovery rate reaching 100.6% (RSD=0.60%), 0.0454~0.3175μg (r=0.9999) for rosmarinic acid with average recovery rate reaching 100.7% (RSD=1.0%), 0.1546~1.082μg (r=0.9999) for salvianolic aicd B with average recovery rate reaching 101.3% (RSD=0.88%).Conclusions: The validated method has good accuracy and a simple procedure. It may be practical to evaluate and control the quality of Salvia miltiorrhiza pieces.