| Hulisan tablet is a compound preparation of traditional Chinese medicine,which is made of Acointum Kusnezoffu Reichb.,Panax notoginseng(Burk.)F.H.Chen,Cynanchum wallichii Wight and Campanumoea javanica Blume.The current quality standard is a single page standard.The content of each standard is quite different,the detection items are complex,the method operation is cumbersome and the accuracy is poor.It is unable to objectively and accurately evaluate the quality of Hulisan tablet and effectively control the product quality.In recent years,there are few studies on the quality of Hulisan tablets.Therefore,the purpose of this project is to improve the quality standard of Hulisan tablets.Based on the current quality standard,the project is to improve and optimize the quality control of Hulisan tablets by using Microscopic Identification,TLC,HPLC,ICP-MS,HPLC-Q-TOF-MS/MS Objective to provide the basis for the internal quality control and evaluation of the preparation.1.Identification of Hulisan tablets(1)Microscopic identification:referring to the Chinese Pharmacopoeia(2020 Edition)and consulting the literature,the characters and microscopic identification of four pieces of Prepared Acointum Kusnezoffu Reichb.,Panax notoginseng(Burk.)F.H.Chen,Cynanchum wallichii Wight and Campanumoea javanica Blume were carried out.Based on the microscopic characteristics of the four drugs,the microscopic identification items in the original standard of Hulisan tablets were modified by observing and analyzing the powder.The gelatinized starch granules were observed under the microscope to be light yellow,with different shapes and sizes,irregular texture and fine particles on the surface.The stone cells are colorless,and the cells connected with the metacortical cells are brown.They are square like,rectangular like,round like,fusiform or long strip like,with a diameter of 20～133(234)μm and a length of 465 μm.the thickness of the wall is different.The striation is obvious in those with thick wall,and the striated pores are fine.Some of them contain brown substance.The epigenetic cortical cells were brown,square like or polygonal on the surface,with uneven wall thickening,and some protruded into the cell cavity in the form of tumor(Acointum Kusnezoffu Reichb.).Starch grains are compound and consist of more than 2～10 grains.The fragments of resin canal contained yellow secretion(Panax notoginseng(Burk.)F.H.Chen).The diameter of calcium oxalate cluster crystals is 20～30μm(Cynanchum wallichii Wight).The diameter of the fragments of the milk tube was 15～25μm,containing pale yellow granules.The starch grains are single,umbilical,round,oblong,etc.,with a diameter of 3～12 μm(Campanumoea javanica Blume.)(2)Thin layer identification:based on the current quality standard of Hulisan tablets,the thin layer identification of Acointum Kusnezoffu Reichb.(benzoylneoaconitine,benzoylhypaconitine,benzoylaconitine)and Panax notoginseng(Burk.)F.H.Chen(notoginsenoside R1,ginsenoside Rg1,ginsenoside Rb1)in the preparation was studied.Firstly,the preparation method of the test solution was optimized and systematic investigation was carried out to determine the initial concentration Then,the systematic methodology investigation is carried out.The results showed that the established method could be used for the identification of Hulisan tablets.HPLC method was established for the identification of caudatin in Cynanchum wallichii Wight and lobetyolin in Campanumoea javanica Blume.The results showed that the specificity was good,the negative had no interference,and it had good reproducibility and durability.2.Inspection of Hulisan tablets(1)According to the inspection regulations of five harmful heavy metals in Chinese Pharmacopoeia(2020 Edition),lead,cadmium,arsenic,mercury and copper in Hulisan tablets were detected by ICP-MS.the results showed that the average contents of heavy metals in all batches were far lower than the corresponding standards.(2)Objective to optimize the limit test of diester alkaloids in the current standard.The experimental method is the same as the content determination item of Acointum Kusnezoffu Reichb..Through systematic methodological investigation,the three components of diester alkaloids-neoaconitine,hypaconitine and aconitine show a good linear relationship,with good precision,stability and repeatability.According to the limit of 120%of the average total amount of diester alkaloids from the highest manufacturer,the content of diester alkaloids in this product should not exceed 167.0 μg·g-1 based on the total amount of neoaconitine,hypaconitine and aconitine.3.Determination of Hulisan tablets(1)Determination of Acointum Kusnezoffu Reichb:to optimize the current standard,kromasil 100-5-C18(4.6 mm × 250 mm,5μm)column was used;acetonitrile(A)-0.2%acetic acid solution(triethylamine adjusted pH to 6.20)(B),flow rate:1.0 mL·min-1;gradient elution,detection wavelength:235 nm;column temperature:35℃;injection volume:10 μL.After systematic methodological study,benzoylmesaconine,benzoylhypacoitine and benzoylaconitine were negative without interference and showed a good linear relationship with good precision,stability and repeatability.The total content of Radix Aconiti Preparata in 12 batches of samples is 320.735～1152.215 μg·g-1,which is in line with the limit range of Chinese Pharmacopoeia(2020 Edition).(2)Content determination of Panax notoginseng(Burk.)F.H.Chen:the content determination items of notoginseng in the current standard were optimized,and three components of notoginseng R1,Ginsenoside Rg1 and ginsenoside Rb1 were detected.The chromatographic column was Agela Durashell C18(4.6 mm × 250 mm,5 μm),with acetonitrile as mobile phase(A)and water as mobile phase(B).the detection wavelength was 203 nm.The average content of notoginseng R1,Ginsenoside Rg1 and ginsenoside Rb1 in Panax notoginseng(Burk.)F.H.Chen powder was 3.386～5.166.mg·g-1,and the mean is 4.430 mg·g-1.Due to the limited number of samples collected,the limit is tentatively determined to be 80%of the current average minimum content of Hulisan tablets.Results the content limit of the sample is as follows:the total content of notoginsenoside R1,ginsenoside Rg1 and ginsenoside Rb1 should not be less than 2.708 mg·g-1.4.Fingerprints of Aconitum alkaloidsIn order to evaluate the quality of Hulisan tablets more comprehensively and show its overall characteristics,the HPLC method was used to establish the fingerprints of Aconitum alkaloids in Hulisan tablets.The experimental method was the same as the content determination of Acointum Kusnezoffu Reichb..The fingerprints peaks were determined ten common.Through the determination of 12 batches of samples,the control fingerprint was generated.The similarity of all batches was above 0.90,and the results were satisfactory 0.937.The results showed that the method had good precision,repeatability and stability,and could be used for the quality control of Hulisan tablets.On the basis of this,the chromatographic peaks of the fingerprint of Huli San were qualitatively analyzed by high performance liquid chromatography-mass spectrometry(HPLC-Q-TOF)and electrospray(ESI)under positive ion detection mode.It is speculated that the identities of the three unknown peaks are neoline,talatizamine,benzoyldeoxyaconine. |
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