| Dahuoluo Pill is a traditional combined medicine used for long time in our country, which was documented in the “experience recipe” of Ming Dynasty. The contents of Dahuoluo Pill have been modified during the following decades and it is widely applied in clinics for its good therapeutic effects. Currently there are 109 pharmacy companies producing Dahuoluo Pill with a total of 111 approval numbers. The implementation standards were recorded in the seventeenth album and sixth album of traditional Chinese medicine prescription preparations. Quality detection of Dahuoluo Pill from Tongrentang companies was based on standards of WS3-B-3184-98 and other companies used standards of WS3-B-1082-92 to perform quality detection of the pills. However these standards were used for a long time and were almost not modified since its establishing.Therefore we need to add some necessary detection items to better control its quality for the efficacy, safety and controllability of clinical use. In this thesis, we established microscopic and TLC identification methods to detect its contents; applied HPLC method to detect the concentration of ephedrine hydrochloride and pseudoephedrine hydrochloride, which were the main constituents of Dahuoluo Pill. Besides, we also adopted the method of quantitative analysis of multi-components by single marker(QAMS) to simultaneously detect the concentration of 6 constituents in the pills and used GC method to determine the concentration of its volatile substances.I. The identification of Dahuoluo PillFirstly, the microscopic identification method has been established to indentify Poria, Radix Clematidis, scorpio, Bombyx Batryticatus, Herba Ephedrae, Flos Caryophylli, Radix Rehmanniae Preparata, HerbaPogostemonis, Amoni Fructus Rotundus, Radix Polygoni Multiflori, Radix Linderae, Rhizoma Coptidis, Moschus, Agkistrodon, Rhizoma Gastrodiae and Radix Puerariae16 kinds of medicinal materials, the results showed that microscopic characteristics of each flavor ingredients obvious.Secondly, TLC was used to distinguish all Chinese herbal medicine in the prescription which contains Radix Angelicae Sinensis, Radix et Rhizoma Rhei, Radix Aucklandiae, Radix Glycyrrhizae, Rhizoma Coptidis, Calculus Bovis, Rhizoma Gastrodiae, Radix Polygoni Multiflori. The results revealed strong specificity for the herds above since the prepared sample could be separated effectively and no interference substance was detected. Thus, the established methods of Microscopic and TLC identification are simple and reliable, which can be used for the quality control of Dahuoluo Pill.II. Determination of non-volatile substances in Dahuoluo PillHPLC method has been established to determine the contacts of ephedrine hydrochloride and pseudoephedrine hydrochloride simultaneously. Chromatographic conditions: column was Agilent ZORBX SB-C18(4.6 mm×250 mm, 5 μm), the mobile phase was A(0.1%phosphoric acid solution)-B(acetonitrile). Gradient elution: 0~30min,5%~7%(B); 30~40min, 7%~95%(B); 40~50min, 95%~95%(B); 50~60min,5%~5%(B), at the flow rate of 1.0 m L ?min-1, detection wavelength was207 nm; column temperature was 25℃; injection volume was 10 μL. A good linear correlation was observed within the range of 1.7024~51.072 μg? m L-1 for ephedrine hydrochloride(r=1), the average recovery rate was102.45%, RSD was 1.55%(n=6); pseudoephedrine hydrochloride has a good linear relationship from 1.8368μg?m L-1 to 55.104 μg?m L-1(r = 1), the average recovery rate was 102.50%, RSD was 1.52%(n=6). Then we used this method to determine their concentions of 34 batches of samples, and our results suggested the method is accurate, feasible and reproducible,which can provide a reference for improving quality standards of Dahuoluo Pill.The method of quantitative analysis of multi-conponents by single marker(QAMS) has been adopted to determine hesperidin, puerarin,berberine hydrochloride, palmatine chloride, benzoic acid and baicalin simultaneously. Hesperidin was selected as the internal reference substance and the relative correction factors(RCFs) of puerarin, berberine hydrochloride, palmatine chloride, benzoic acid, baicalin with reference to hesperidin were 4.56 、 2.01 、 1.71 、 10.6 、 7.27. Using different high performance chromatographs and columns to study the relative correction factors’(RCFs) reproducibility we found that relative correction factors(RCFs) value of RSD in different instruments and columns were less than5.0%, indicating the good reproducibility of relative correction factors(RCFs). Using the relative retention time to position each component, the values of puerarin, berberine hydrochloride, palmatine chloride, benzoic acid, baicalin were 2.20, 0.89, 0.94, 0.85, 0.78, RSD were 4.4%, 2.3%,2.2%, 3.1%, 3.4%. We used the method of QAMS to determine the 6corresponding contents of Dahuoluo Pill from 34 batches, comparred the results with those calculated using external standard method, there were no difference between them. Hence, this method can be used to control the quality of Dahuoluo Pill since it is simple, cheap and efficient.III. Determination of volatile substances in Dahuoluo PillIn this part we used gas chromatography(GC) to determine Dahuoluo Pill’s volatile components of Dahuoluo Pill such as borneol, isoborneol and eugenol simultaneously. Method: Agilent HP-5 capillary column(30m?0.32 mm, 0.25 ?m); temperature program as follows: the initial column temperature was 70?C maintained for 5 min, then 5?C?min-1 up to 150 ?C,20?C?min-1 up to 280?C maintained for 5. Injection port temperature was200?C; detector temperature was 300?C; the inject volume was 1 ?L; split ratio was 5:1; running time was 32 min. A good linear correlation was observed within the range of 1.672~83.64 μg?m L-1 for borneol(r=0.9997),the average recovery rate was 99.83%, RSD was 0.98%(n=6); thecalibration curve for isoborneol was found to be linear from 1.6048 μg?m L-1 to 80.24 μg ? m L-1, r=0.9999, the average recovery was99.22%(RSD=1.02%, n=6); a good linear correlation was observed within the range of 1.5747~78.736 μg?m L-1 for eugenol(r=0.998), the average recovery rate was 101.79%, RSD was 1.14%(n=6). We used the method to determine the contents of borneol, isoborneol and eugenol form 34 batches and our results revealed this method is accurate and reliable, simple and feasible, which could provide a basis reference for quality control of Dahuoluo Pill volatiles. It can comprehensively control its quality and ensure the effectiveness of its clinical use. |
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