To establish a specific polymerase chain reaction(PCR) method based on SNP loci for molecular identification of Eupolyphaga Steleophaga formula granules and its counterfeit.

By analyzing the cytochrome oxidase Ⅰ (COⅠ) sequences of Eupolyphaga Steleophaga and its common forgeries，the SNP mutation sites of Eupolyphaga Steleophaga were searched and specifi cprimers were designed，which were TBC-F(5’-TTCTTGTTGGCAAGCAGTATAAT-3’) and TBC-R(5’-AACTACTGCTCAAACAAATAATGGA-3’). Three-step method was used to amplify specific polymerase chain reaction (PCR) and the optimal PCR reaction system was determined by optimizing the PCR reaction procedure. At the same time，in order to ensure the accuracy of the test results，the PCR amplified products were conducted by generation sequencing.

The PCR method with the annealing temperature of 55-57 ℃ and 37 cycles produced a single band at 237 bp for 21 batches of Eupolyphaga Steleophaga，20 batches of standard decoction and 19 batches of its dispensing granules，while it produced no band for the adulterant or the negative control. The experimental result was consistent with the result of Sanger sequencing，which was Eupolyphaga sinensis.

The established specific PCR method can accurately identify the medicinal materials，and standard decoction freeze-dried powder of Eupolyphaga Steleophaga，as well as final products of dispensing granules. It provides a reference for research on the quality standards of Eupolyphaga Steleophaga dispensing granules.

To analyze the variation of volatile components during oxidative rancidity of Scorpion, and identify the main components resulting in the rancid flavor of Scorpion.

Scorpion samples were subjected to accelerated oxidation experiment for 50 d，starting from 0 d，and sensory evaluation of the sample was performed every 10 d. At the same time，the volatile components were detected by solid phase microextraction GC-MS(SPME-GC-MS). Six groups of data were obtained. The volatile components obtained were searched by computer，and the results were matched with the NIST 20 database to determine the chemical structure of that. Meanwhile，the relative content of volatile components was calculated by using 2-octanol as internal standard，and the main components of Scorpion’s rancid flavor were screened out through principal component analysis and cluster analysis.

A total of 51 volatile components such as aldehydes，acids and furans were detected，of which 43 were common to six groups of data. There were significant differences in the content of volatile components of Scorpions in different oxidation time periods，with aldehydes being the highest，with an average content of 101.33 mg·kg^-1，accounting for 54.45%，followed by acids，with an average content of 52.01 mg·kg^-1，accounting for 27.95%. With the prolongation of the oxidation time，the Scorpion appeared to have a rancid flavor，when oxidized for 50 d，a heavy rancid flavor appeared；at the same time，the content of volatile components of all categories showed an increasing trend，among which the content of heptanal，nonanal，n-octanal，2-butyl-2-octenal，4-oxo-2-nonenal，hexanoic acid and 2-n-pentylfuran increased significantly，with an increase of multiples of 4 times to 44 times. Through principal component analysis，three principal components were screened out with eigenvalues above 1 value，among which the eigenvalue of principal component 1 was 40.451，and the contribution rate of that reached a 79.32%，the main influencing factors included aldehydes，acids and furans. Six groups of samples were divided into four categories by cluster analysis，which was consistent with the results of sensory evaluation.

Seven volatile components，heptanal，nonanal，n-octanal，2-butyl-2-octenal，4-oxo-2-nonenal，hexanoic acid，and 2-n-pentylfuran，are the main substances in the production of rancid flavor of Scorpion. This experiment provides a new technology and new method for the development of simple and precise monitoring of the oxidative rancidity process of Scorpion，and to provide a research basis for the improvement of Scorpion quality standard.

To establish an HPLC-principal component self-compare with correction factor method for quantification of two related substances (N-deacetyllappaconitine and ranaconitine) in lappaconitine hydrobromide injection.

The analysis was performed on a Kromasil 300-5-C₁₈ (250 mm×4.6 mm，5 μm) column with a mobile phase composed of 0.04 mol·L^-1 potassium dihydrogen phosphate solution，methanol and acetonitrile (68:17:15). The detection wavelength was 252 nm，the flow rate was 0.8 mL·min^-1，the column temperature was 37 ℃，and the injection volume was 10 μL. The slope of linear equation was used to determine the relative correction factor between the two impurities and lappaconitine hydrobromide. The relative retention time was used to determine the position of related substances. The contents of two impurities in 25 batches of lappaconitine hydrobromide injection produced by four pharmaceutical companies were determined and compared with the results of the external standard method.

Lappaconitine hydrobromide and the impurities were separated well by this method. The relative retention time of N-deacetyllappaconitine and ranaconitine were 1.20 and 1.39，and the correction factors were 1.23 and 0.94，respectively. Lappaconitine hydrobromide，N-deacetyllappaconitine and ranaconitine showed good linearity in the mass concentration ranges of 0.951 7-38.07 μg·mL^-1，1.047-41.87 μg·mL^-1 and 1.001-40.02 μg·mL^-1 with r=1.000，respectively. The average recovery rates of lappaconitine hydrobromide，N-deacetyllappaconitine and ranaconitine were 100.2%，100.5% and 100.5% respectively，with RSD less than 2.0%. The limits of detection (LOD) of lappaconitine hydrobromide，N-deacetyllappaconitine and ranaconitine were 0.095，0.10，0.10 μg·mL^-1 and the limits of quantitation (LOQ) were 0.32，0.35 and 0.33 μg·mL^-1，respectively. The content of N-deacetyllappaconitine in 25 batches of lappaconitine hydrobromide injection was in the range of 0.31%-0.82% and the content of ranaconitine was in the range of 0%-0.09%. It was consistent with the determination result of the external standard method.

The method is proved to be simple，rapid，and accurate for the determination of related substances in lappaconitine hydrobromide injection.

To establish a method for the rapid identification of 22 illegally added drugs in foot-use cold compress gel，and to quantify the content of 4 drugs.

Use ultra-performance liquid chromatography-high resolution mass spectrometry/mass spectrometry (UPLC-HRMS/MS) for qualitative analysis. Using Hypersil GOLD^TM VANQUISH C₁₈ (2.1 mm×100 mm，1.9 μm) chromatographic column，gradient elution was performed with 10 mmol·L^-1 ammonium acetate aqueous solution (A) and methanol (B) as mobile phases，column temperature was 40 ℃，flow rate was 0.2 mmol·L^-1，ESI ion source was used，and qualitative analysis was performed by switching between positive and negative scanning modes. By comparing the retention time，parent ion，and fragment ion information of the standards and sample under the same chromatographic and mass spectrometric conditions confirmed the illegal added drugs in the sample and the suitable methods were used for quantitative analysis.

After qualitative analysis，it was confirmed that the sample contained 4 illegally added drugs：acetaminophen，chloramphenicol，lidocaine hydrochloride and miconazole nitrate. The concentrations of these 4 drugs ranged 2.542-101.7 μg·mL^-1，1.410-169.161 μg·mL^-1，0.002-0.2 μg·mL^-1，5.184-290.304 μg·mL^-1，respectively. The peak areas showed good linear relationships with concentrations，with correlation coefficients of 0.999 9，0.999 7，0.999 9 and 0.999 1，respectively. The limits of detection were 0.015，0.143，0.001，0.833 μg·mL^-1，respectively. At three concentration levels，the recovery rates ranged 98.5%-104.5%，90.2%-99.4%，79.5%-84.9%，102.8%- 104.3%，with the RSD of 0.059%-0.28%，0.20%-0.49%，1.2%-3.1%，0.18%-0.40%，respectively. The contents were 195.05，264.10，1.24，67.00 mg per bottle，respectively.

The qualitative method is simple, rapid, and highly accurate, while the quantitative method is specific, precise, accurate, and stable, making it suitable for the rapid identification of 22 illegally added drugs and the quantification of 4 illegal added drugs in foot-use cold compress gel.

To establish a method for the determination of 17 kinds of anti-allergic compounds in medical cold compress by solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry（SPE-UPLC-MS/MS）. Common medical cold compress matrices were suitable for this new method.

Three pretreatment methods were compared：prime pass-through column purification，SPE purification including HLB and MCX solid-phase extraction. The pretreatment was optimized by focusing on the kind of solid-phase extraction，the type and volume of elution solvents. The analytes were extracted using methanol and then mixed with water at the ratio of 1:9 in volume，purified with an Agilent Bond Elut HLB column. Waters ACQUITY UPLC BEH C₁₈（100 mm×2.1 mm，1.8 μm）column was used with 0.1% formic acid(with 0.5 mmol·L^-1 ammonium acetate)-acetonitrile as mobile phases by gradient elution for separating 17 compounds. Detection was carried out by positive and negative electrospray ionization（ESI）mass spectrometer in multiple reaction monitor (MRM) mode.

All the 17 kinds of compounds showed good linearity in their reasonable ranges（r）>0.99，the limit of detection （LOD） and quantification（LOQ）were 0.01-13.32 μg·g^-1 and 0.02-26.64 μg·g^-1，respectively. The average recoveries of at three spiked levels were in range of 77.7%-108.5%，with RSDs （n=6） were 0.62%-4.7%. The content range of diclofenac sodium in positive samples was 0.09-27.55 mg per tablet.

The method is simple，sensitive and reliable，and is suitable for determination of 17 illegally add chemical drugs in medical cold compress. Moreover，the method might provide technological support for the detection of illegal additions in medical cold compress.

To establish a quantitative nuclear magnetic resonance hydrogen spectroscopy(¹H qNMR) method for determining the moisture content in several pharmaceutical excipients.

Using sodium benzenesulfonate as the internal standard and heavy water as the solvent，the water content in the blank sample was determined by absolute quantification using nuclear magnetic resonance spectroscopy. By comparing with the blank value，a quantitative calculation formula for the water content in the sample was established，and the water content in the sample was tested and calculated.

Methodological studies had shown that this method had a good linear relationship(r=0.999 1)，with a linear range of 0.032-0.561 mg absolute water content in the sample，the precision RSD was 0.039%，repeatability RSD was 0.43%，and the average recovery was 98.7%. The ¹H qNMR method measured the water content in DMSO-1, DMSO-2, sodium benzenesulfonate, and sodium acetate to be 0.23%, 0.28%, 0.46%, and 0.28%, respectively, which were essentially consistent with the results obtained from the method described in the Chinese Pharmacopoeia(2020).

The ¹H qNMR method established is fast，simple to operate，with a small sample size and accurate results. It can be used for the determination of water content in some pharmaceutical excipients.

To explore the feasibility and potential applications of high-resolution cyclic ion mobility separation mass spectrometry (Cyclic IMS) for detecting the conformations (topological isomers) of complex medicines.

Using MccJ25 and Rubrinodin lasso peptides as models，the compounds were subjected to heat treatment at 0，50，and 80 ℃ for 4 h，respectively. The samples were then directly injected for separation and detection using high-resolution Cyclic IMS.

The two model compounds exhibited different topological structural changes at three temperatures tested. MccJ25 showed no significant conformational changes and exhibited a relatively stable topological structure across all temperatures. In contrast，Rubrinodin demonstrated significant conformational changes at each temperature. The cyclic ion mobility cell in Cyclic IMS was able to accurately identify conformational changes，such as folding and unfolding，in the complex drugs.

Cyclic IMS can precisely analyze the conformation (topological isomers) of complex drugs，detect small conformational changes，and offers strong operability，good reproducibility，and high accuracy. It holds potential for use in the conformational determination and quality control of complex drug molecules.

To establish a laser diffraction method for the determination of particle size distribution of budesonide suspension for inhalation.

The Master sizer 3000 laser size analyzer with the Hydro SV wet sampler was used. The sample solution should be immediately measured after ultrasonication. The measurement conditions were as follows：refractive index of budesonide was 1.592，absorbency of budesonide was 0.01，dispersing medium was saturated solution of budesonide containing 0.5% Tween 80 with refractive index of 1.33，stirring rate was 1 000 r·min^-1，and laser obscurations were 6%-12%.

RSD of D₅₀ was lower than 2%，RSDs of D₁₀ and D₉₀ were both lower than 5%．The particle size distribution D₉₀ values of 3 batches of budesonide suspension for inhalation were less than 5 μm.

The method is simple，accurate and producible，which is suitable for the particle size detection of budesonide suspension for inhalation.