| Guanxinning liquid injection, which was listed in ministerial standard, was a clinical medicine commonly used in China for the treatment of coronary artery disease and angina pectoris. In this paper preparation procedure, quality control method and pharmacokinetics on a few active constitutes of Guanxinning lyophilized powder for injection were studied.The preparation procedure of Guanxinning lyophilized powder for injection was established using different kinds of methods and techniques. The optimal extraction process of Guanxinning lyophilized powder for injection was studied by orthogonal design with the contents of salvianolic acid B and ferulic acid as quality assessment indices. Several factors were studied in this experiment, including the size of herbs, solvent consumption, duration of extraction and times of extraction. Concentration of alcohol, extraction times, acticarbon dosage were tested to optimize the refining process and lyophilization was also studied. The lyophilized powder was yellowish freeze-dried powder and solved easily in water.Pyrogen, asepsis, heave metal, et al were detected according to Chinese Phamacopoeia （2005） in order to improve the safety of the drug. Ultraviolet spectrophotometry methods were used for identification of phenolic acids of Salvia miltiorrhiza Bge. in Guanxinning lyophilized powder for injection. Thin layer chromatography （TLC） method was developed for the identification of ferulic acid in Ligusticum chuanxiong Hort. of Guanxinning. High performance liquid chromatography （HPLC） fingerprint was carried out on a Luna C18 column with a gradient elution programme when the mobile phase was mixed with solvent A （acetonitrile） and solvent B （aqueous phosphoric acid, pH 2.59）. Salvianolic acid B was used as internal standard. HPLC fingerprints of herbs consisted of Guanxinning were studied under the same chromatographic condition, and recommendatory producing area were indentified. HPLC fingerprints were applied to analyze quality of Guanxinning lyophilized powder for injection and its intermediate. There was good correlation among lyophilized powder, herbs and intermediate.The determination of salvianolic acid B and ferulic acid was carried out by using HPLC method. The assay was carried out on a Hypersil ODS2 C18 reserved phase column （250×4.6 mm, 5μm） with methanol-acetonitrile-1.7% carboxylic acid （11:12:77） as mobile phase. The column temperature was set at 28℃. The assay showed a good linearity from 1.70 to 25.47μg·mL-1 for salvianolic acid B and from 0.05 to 0.68μg·mL-1 for ferulic acid. The mean recoveries were 101.1% （RSD=2.9%） and 99.2% （RSD=2.8%）, respectively. A HPLC method was developed for simultaneous determination of danshensu, protocatechuic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, ferulic acid, isoferulic acid and salvianolic acid B in Guanxinning lyophilized powder for injection. The determination was accomplished by a gradient elution system. The linear calibration curves were plotted with satisfied relative coefficients. The average recoveries were in the range of 96.3-104.3% with RSD less than 3.5 %. The assay was performed on a Luna C18 reserved phase column （250×4.6 mm, 5μm） with the column temperature maintained at 35℃when the detection wavelength was 280 nm. The mobile phase was gradient elution which was mixed with solvent A （acetonitrile） and solvent B （aqueous phosphoric acid, pH 2.59）. The gradient program was 0-30 min from A-B （2:98, v/v） to A-B （18:82, v/v）, and next 30-54 min, linear change to A-B （34:66, v/v）. With the same gradient elution system, danshensu, protocatechuic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid, ferulic acid, isoferulic acid and salvianolic acid B were determined in Guanxinning injection. The concentrations were compared from lyophilized powder to injection. Results showed that concentration of total active gradients was higher in lyophilized powder, which indicated that it was necessary to change the injection into lyophilized powder.A HPLC method was developed to quantify ferulic acid and isoferulic acid in rat plasma using precipitation and coumarin as internal standard. The linear ranges were 5.780 - 5780 ng·mL-1 for ferulic acid and 1.740 - 348.0 ng.mL-1 for isoferulic acid. The low quantification limit was 5.780 ng·mL-1 and 1.740 ng·mL-1, respectively. The intra- and inter-day relative standard deviations （RSDs） in the measurement of quality control （QC） samples were no more than 14.9%. The accuracy was between -5.8% - 4.0% in terms of relative error （RE）. The mean extraction recovery was 85.1% （RSD=1.6） and 91.1% （RSD=1.5）, respectively. The corresponding pharmacokinetic parameters were obtained. For ferulic acid, t1/2 was 0.570±0.427 h, Cmax was 315.4±181.1 ng·mL-1 and tmax was 0.033±0 h. For isoferulic acid, t1/2 was 2.571±1.992 h, Cmax was 126.1±71.36 ng·mL-1 and tmax was 0.044±0.027 h. With the same assay method, concentration of ferulic acid and isoferulic acid in rat plasma were determined after i.v. and i.m. administration of Guanxinning lyophilized powder for injection. The values of absolute bioavailability were 82.6% for ferulic acid and 73.2% for isoferulic acid.A sensitive and accurate liquid chromatography-mass spectrometry （LC-MS） method was developed to simultaneously quantify danshensu, protocatechuic acid, protocatechuic aldehyde, chlorogenic acid, caffeic acid and salvianolic acid B in rat plasma. After deproteinized by methanol, the plasma samples were extracted by liquid-liquid extraction with ethyl acetate-ethyl ether （3:1, v/v） and analyzed with galic acid as internal standard using negative electrospray ionization （ESI） mass spectrometry in selected ion monitoring （SIM） mode. The method was with good linearity in the range of 0.342 - 85.0μg·mL-1 for danshensu, 0.0647 - 12.9μg·mL-1 for protocatechuic acid, 0.0933 - 18.7μg·mL-1 for protocatechuic aldehyde, 0.0085 - 3.40 ng·mL-1 for chlorogenic acid, 0.0138 - 2.75μg·mL-1 for caffeic acid and 0.0272 - 810μg·mL-1 for salvianolic acid B. The lower limits of quantification were 0.342μg·mL-1, 0.0647μg·mL-1, 0.0933μg·mL-1, 0.0085μg****mL-1, 0.0138μg·mL-1 and 0.0272μg·mL-1, respectively. The method was shown to be reproducible and reliable with intra-day and inter-day precision below 10.0%, accuracy within -3.7% and 5.0%, and mean extraction recoveries excess of 86.8% （RSD=4.8）, 85.6% （RSD=2.1）, 78.1% （RSD=4.4）, 76.5% （RSD=4.3）, 84.7% （RSD=3.5） and 82.3% （RSD=4.0）, respectively. Danshensu, protocatechuic acid, protocatechuic aldehyde, chlorogenic acid and caffeic acid eliminated quickly in vivo after i.v. administration of Guanxinning lyophilized powder for injection. Compared to them, salvianolic acid B eliminated more slowly, the t1/2 of which was 1.18 h. Bioequiavailability was compared between Guanxinning lyophilized powder and injection in rats after i.v. administration. Results showed that these two formulations were not bioequivalent, and extent of absorption for lyophilized powder was better than the injection. |
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