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 an HPLC fingerprint of Zuoyu’an San and conduct quality control by combining chemometrics and quantitative analysis of multi-components with a single-marker（QAMS）.

HPLC was used to establish the fingerprint spectra of 12 batches of Zuoyu’an San and perform similarity evaluation. Cluster analysis (CA)，principal component analysis (PCA)，and orthogonal partial least squares discriminant analysis (OPLS-DA) were combined to perform chemical pattern recognition analysis on different batches of Zuoyu’an San. At the same time，QAMS method was established using rhein as the internal standard reference. The relative correction factors between the internal reference and jatrorrhizine，palmatine，berberine，aloe-emodin，emodin，chrysophanol，and physcion were established. The external standard method (ESM) and QAMS method were used to determine jatrorrhizine，palmatine，berberine，aloe-emodin，rhein，emodin，chrysophanol，and physcion in Zuoyu’an San，respectively. To verify the reliability of QAMS metod results，the contents of 8 components of physcion were determined.

A total of 23 common peaks were calibrated in the fingerprint，and 8 components were identified by comparing with the mixed reference solution. The similarity between the samples and the control spectrum was between 0.952 and 0.994；CA divided 12 batches of Zuoyu’an San samples into 3 categories；PCA showed that there were certain differences in the chemical composition of different batches of Zuoyu’an San，and extracted 5 principal components that affected the quality evaluation of Zuoyu’an San；OPLS-DA screened out 13 potential marker components (VIP>1) among 23 common peaks that caused quality differences between different batches of Zuoyu’an San samples；There was no significant difference between the QAMS method calculated value and the ESM measured value.

The established HPLC fingerprint and multi-index component determination method was accurate and simple，and combined with chemometric methods，could provide reference for the quality control and evaluation of Zuoyu’an San.

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 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 screen the suitable monitoring method applicable to oligotrophs and review the bioburden and microbial species of oligotrophic environments in pharmaceutical water systems and cleanrooms.

The culture conditions and detection methods suitable for microorganisms under oligotrophic conditions were optimized by comparing the counting results of microorganisms in laboratory pure water with R₂A and TSA media at different temperatures，media，and incubation times. In addition，monitoring of oligotrophs in pharmaceutical environments using both the traditional and oligotrophic assays were conducted，combining with the techniques of 16s rDNA sequencing and MALDI-TOF MS for multiphase microbial identification and analysis of the isolated microorganisms under oligotrophic conditions.

The colony counting methods，the type of culture media，and the incubation intervals significantly affected microbial enumeration. The isolation method using oligotrophic medium R₂A was more effective than the TSA medium in this study. The profiles of microorganisms isolated from the R₂A and TSA media were unique. The isolation rate of Gram-negative bacteria in the R₂A medium reached 37.0%，however，that of Gram-negative bacteria in the TSA medium was only 14.0%. In addition，several strains of potential pathogens initially isolated from the R₂A medium grew slowly in the TSA medium and were easily missed by the traditional monitoring method.

With the development of the pharmaceutical industry，the contamination proportion of human-associated Gram-positive cocci might gradually decrease，the oligotrophic culture method as a powerful supplement to traditional monitoring techniques can help to detect potential objectionable microorganism contamination risks at an early stage.

To establish an HPLC method for determination of related substances and purity in pregabalin API.

The analytical column was an Inertsil ODS-3 (150 mm×4.0 mm，3 μm)，the mobile phase A was buffer (7.05 g ammonium dihydrogen phosphate and 1.45 g diammonium hydrogen phosphate dissolved in 1 000 mL water)-methanol-acetonitrile(900：80：20) and the mobile phase B was acetonitrile，the whole carried out by gradient elution at a flow rate of 0.8 mL·min^-1，the detection wavelength was set at 210 nm，the column temperature was 30 ℃ and the injection volume was 50 μL.

Pregabalin was separated completely from the impurities and degradation products (the resolution>2.0). The test solution was stable for at least 48 h. The LOQs of other impurities (verification was performed by using pregabalin)，lactam，4-alkene pregabalin，5-alkene pregabalin，trimer，3-isobutylglutaric acid (PGB-3)，3-isobutyl glutaric acid monoamide (PGB-5)，R-phenethylamine，4-isobutyl-2，6-piperidinedione (PGB-5B)，monoamide phenylethylamine (PGB-5C) and phenylethylamine adipate (PGB-5D) were 0.05%，0.01%，0.01%，0.03%，0.01%，0.05%，0.03%，0.01%，0.01%，0.01% and 0.01%. The linear correlation coefficients of other impurities (verification was performed by using pregabalin)，lactam，4-alkene pregabalin，5-alkene pregabalin，trimer，PGB-3 and PGB-5 were all more than 0.99. The linear ranges were LOQ-150% of impurities’ specification，respectively. The average recoveries (n=9) of lactam，4-alkene pregabalin，5-alkene pregabalin，trimer，PGB-3 and PGB-5 were 100.6% (RSD=0.56%)，100.2% (RSD=0.38%)，100.5% (RSD=0.46%)，101.1% (RSD=1.1%)，100.0% (RSD=0.63%)，100.0% (RSD=0.54%). The repeatability and intermediate precision completely met the requirements. The impurities contents in three batches of pregabalin API 6 months accelerate and 60 months long-term stability test completely met the requirements，respectively.

This method is simple，rapid，sensitive and specific to be used for the determination of related substances and purity in phenethylamine process pregabalin API.

To develop an ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-Q TOF MS) approach to investigate the molecular weight distribution and n value of nonoxynol oligomers，and to study several batches of samples produced domestically or abroad.

The mobile phase was 0.1% trifluoroacetic acid aqueous solution(A)-0.1% trifluoroacetic acid in acetonitrile(B) with linear gradient elution at a flow rate of 0.3 mL·min^-1. The column was Techmate STV SUNSHELL Peptide C₁₈ (100 mm×2.1 mm，2.6 μm，300 Å) and installed in a 60 ℃ column oven. Injection volume was 5 μL. A tandem quadrupole time-of-flight mass spectrometer with Dual ESI as ion source and argon as collision gas were used for detection in positive ion mode with a collection range of m/z 50-3 000. ChemDraw 19.0 software was used to draw the relevant structure，and MassHunter PCDL Manager software was used to build the database and edit the calculation formula.

The weight average molecular weight and number average molecular weight of 6 batches nonoxynol drug substance produced domestically or abroad were determined by the newly established method，and the range of n average value in the structure was obtained. The results showed that the weight average molecular weight and number average molecular weight were different between domestic and foreign products and also between different batches produced by same manufacturer. The n value of structure basis also varies. The weight average molecular weight ranges were 664.6 to 686.8 (n value was about 10)，and number average molecular weight ranges were 677.1 to 715.5 (n value was about 11).

The molecular weight distribution and n value of such oligomer drugs need to be controlled in the production process and quality specifications. The newly established method has high sensitivity，precision and reproducibility，can be qualified for the needs of determining molecular weight distribution and n value of nonoxynol，and provides insight in determination of molecular weight distribution of such oligomers. This study also provides a data supplement for the improvement and revision of the standards of Chinese Pharmacopoeia.