To establish a method for related substances determination in timolol maleate by ultra-high performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry（UPLC-Q/Orbitrap HRMS），by which the impurities both in active pharmaceutical ingredients（APIs） and preparations can be recognized and determined.

An ACE Excel3 C₁₈-AR column（150 mm×4.6 mm，3 μm）was used for the separation and a mixture of 0.01 mol·L^-1 ammonium acetate solution with 0.02% formic acid and methanol was employed as the mobile phase by gradient elution，at a flow rate of 0.6 mL·min^-1. The detection wavelength for UV detector was 295 nm，an HESI（heated ESI）ion source was employed in both the positive mode and negative mode. The possible fragmentation patterns prediction was conducted with the help of Mass Frontier 8.0 and Compound Discover 3.3. The related substances could be recognized and determined by means of the forced degradation of the APIs，with the calibration by the correction factors and confirmation by the mass spectrum data from UPLC-Q/Orbitrap HRMS.

The timolol impurity B[3-(tert-butylamino)-2-(4-morpholino-1，2，5-thiadiazol-3-yloxy)propan-1-ol]，timolol impurity D(4-morpholino-1，2，5-thiadiazol-2-ol)，timolol impurity E((S，Z)-4-(｛1-(tert-butylamino)- 3-[(4-morpholino-1，2，5-thiadiazol-3-yl) oxy] propan-2-yl｝oxy)-4-oxobut-2-enoic acid maleate salt) and timolol impurity C[N-(tert-butyl)-2，3-bis (4-morphloline-1，2，5-thiadiazol-3-yloxy) propan-1-amine maleate] were produced from the APIs under selected conditions and separated well in the specified HPLC condition，the limit of quantitation was 0.05 μg·mL^-1 and the limit of detection was 0.015 μg·mL^-1 for HPLC-UV. The contents of individual impurities were between 0.000 4%-0.09% and the results of total impurities were between 0.02%-0.12% for the samples from 4 different manufactures. The probable chemical structures of the 6 unspecified impurities were speculated according to the fragmentation pattern of fragment ions，combined with the fragment information，chemical structure of API and the references.

The system solution can be obtained by the degradation of the API，and be implied in the impurity analysis for the timolol maleate. The results can be used as a reference for the quality control of timolol maletae.

To compare the in vitro bioactivity between a generic lactobacillin granules drug and three commercial lactobacillin granules drugs, this study investigated inhibition on pathogenic bacteria, the growth promoting effect on probiotics, and establish a method to evaluate the in vitro bioactivity consistency of drugs that regulate gut microbiota.

Two culture systems were set up to investigate the inhibitory effect on pathogenic bacteria and the growth promoting effect on probiotics by lactobacillin granules. The in vitro bioactivity consistency of four products was evaluated by microbial growth curve and analyzed by two-way analysis of variance with Dunnett-t test.

No significant difference (P>0.05) was observed on inhibition of Staphylococcus aureus and growth promotion of Lactobacillus rhamnosus between the generic lactobacillin granules and the commercial lactobacillin granules.

This method could be used to evaluate the in vitro bioactivity of drugs that regulate gut microbiota, and provided guidance on relevant drug development and quality evaluation.

To establish a method for determining molecular weight and distribution of raw mannatide and its preparation with SEC-RI-MALLS.

Specificity，accuracy, precision and robustness of SEC-RI-MALLS method was verified by performing on a Shodex OHpak SB-804 HQ column with 0.05 mol·L^-1 sodium sulfate buffer as mobile phase at a flow rate of 0.5 mL·min^-1 and a comparison between SEC-RI-MALLS and GPC was studied too.

The tablet excipient starch had no interference to the test. The relative accuracy error between the measured value and the labeled value of dextran standard was less than 3.0%. RSD of precision was 0.40% when the sample concentration was 2 mg·mL^-1 and the RSD of reproducibility and robustness were less than 5.0%. There was no significant difference between Shodex OHpak SB-804 HQ column and TSK-GEL G4000 PW_XL column by comparing the results of 79 batches of samples. Compared with the national standard method (GPC) for molecular weight determination, the molecular weight of SEC-RI-MALLS method was 19 509 Da higher on average, and the molecular weight distribution was more concentrated.

SEC-RI-MALLS method can determine the molecular weight and distribution of mannatide without standard with good accuracy and robustness. Compared with the existing method, SEC-RI-MALLS method is more conducive to the safety and effectiveness control of the variety.

To establish a bridging enzyme-linked immunosorbent assay (ELISA) method for the determination of anti-drug antibody(ADA) and a competitive ELISA method for the determination of neutralizing antibody(NAb) in cynomolgus monkey serum, and to conduct methodological validation.

The steps of bridging ELISA method were as follows: the 96-well plates were precoated with telitacicept(RC18) which could combine with anti-RC18 antibody in the samples to form a complex, then were sequentially added biotinylated RC18(Biotin-RC18), horseradish peroxidase conjugated streptavidin (SA-HRP), and tetramethylbenzidine (TMB) substrate solution for color development. After terminating the reaction, the absorbance was read at a wavelength of 450 nm/630 nm on an ELISA reader. The procedures of competitive ELISA method were as follows: the 96-well plates were precoated with B-cell activation factor of the TNF family (BAFF) or a proliferation inducing ligand (APRIL) protein and then were added the samples which was pre-mixed with Biotin-RC18 to form BAFF or APRIL anti-RC18-antibody and Biotin-RC18 complex. SA-HRP and TMB substrate solution for color development were added sequentially. After terminating the reaction, the absorbance was read at an ELISA reader with dual wave length.

The precision of linear range of bridging ELISA method was less than 12.32%, the sensitivity was 50 ng·mL^-1, the critical threshold of screening was 0.937, and the critical threshold of confirmation was 23.62%. The precision of the linear range of competitive ELISA method was less than 20%, the sensitivity was 312.50 ng·mL^-1, and the threshold for determining the activity of NAb against the target BAFF and APRIL was 0.79 and 0.69, respectively. On BAFF and the method of research targets respectively can tolerate 2.5 μg·mL^-1 and 5 μg·mL^-1 RC18 in serum.

The results of method validation indicate that both bridging ELISA and competitive ELISA meet the requirements of preclinical immunogenicity studies of biological products, and can be used for analysis of the concentrations of ADA and NAb in cynomolgus monkey serum.

To improve the liquid chromatographic determination method of cefixime granules related substance.

High performance liquid chromatography was used, YMC-Triart C₁₈ column (250 mm×4.6 mm, 5 μm) was selected, 0.05 mol·L^-1 ammonium formate solution (pH 4.7)-methanol was used as mobile phase, flow rate was 1 mL·min^-1, and gradient washing was carried out.The injection volume was 10 μL. The detection wavelength was 254 nm.

This chromatographic condition was applied to the detection of cefixime granules. The differences between this method, the pharmacopeial method and the method of USP PF 2018 were compared, and the systematic methodological verification of specificity, linearity, accuracy, precision and durability were completed. Using pharmacopeial methods, baseline separation of degradation impurities A1~A4 or impurities B1~B4 cannot be reached, and current methods cannot be used to determine polymer B and polymer D. The method proposed in this article can make the resolution between cefixime and each specific impurities meet the requirements (R ≥1.5), and can detect and quantify polymer B and polymer D at the same time, and the resolution was better than the current method.

This method improves the separation between cefixime and impurities, more impurities is detected and can accurate quantify specific impurities. This method has high sensitivity and good repeatability, and is suitable for the quality control of cefixime.

To determine the polysaccharide hydrolysates from Polygonati Rhizoma under glycoside enzymatic hydrolysis’ HPLC fingerprint. Examine the variations among the polysaccharides produced by the various varieties of Polygonati Rhizoma and provide references for the assessment of the polysaccharide quality.

After the polysaccharide from Polygonati Rhizoma was hydrolyzed by fructose enzymes, its fingerprint was established by HPLC-ELSD. The fingerprint was then analyzed using similarity analysis (SA), hierarchical cluster analysis (HCA), and principal component analysis (PCA) to determine the differences between the polysaccharides from various Polygonati Rhizoma varieties.

Polysaccharides from various strains of Polygonati Rhizoma had different HPLC-ELSD fingerprints, and a total of 17 distinct oligosaccharide fragments were discovered, all of which contained fructose, glucose, and sucrose. Analysis revealed that there are significant intra species differences, minor differences, and high levels of similarity between the three types of Polygonati Rhizoma. The real Polygonati Rhizoma differs significantly from the imitation in several important ways.

Polygonati Rhizoma can be successfully classified according to varieties, using the fingerprint of polysaccharide hydrolysates of the various varieties of Polygonati Rhizoma and its adulterants. The developed HPLC method can be used for the differential analysis of polysaccharides in Polygonati Rhizoma and is straightforward, precise, and repeatable.

To comprehensively evaluate the quality of Poriae Cutis, and to establish a dual wavelength switching HPLC method for comparing the characteristic spectra of Poriae Cutis and studying the content of 11 triterpenoid components, to provide reference for the qualitative and quantitative research of Poriae Cutis.

Agilent 5 HC-C₁₈(2) column(250 mm×4.6 mm, 5 μm) was adopted. Acetonitrile solution (contain 3% tetrahydrofuran) (A) and 0.1% formic acid aqueous solution (B) were used as the mobile phase with gradient elution at a flow rate of 1.0 mL·min^-1. The column temperature was 30 ℃ and the injection volume was 20 μL. The detection wavelengths were 210 and 243 nm.

The feature profiles developed were effective in identifying the 18 shared peaks. RSD for precision, repeatability and stability (48 h) tests were all less than 3.72%(n=6). The 11 chemical components to be measured were well separated, with good linearity in the mass range examined (all r ≥ 0.999 6). The average recovery rate was 95.4%-105.5%, and the RSD was 1.0%-3.1%. The RSDs of precision, repeatability, and stability (48 h) tests were all less than or equal to 3.0%(n=6). The results of similarity analysis showed that most of the origins of Poriae Cutis were very similar to each other. The results of content determination showed that among the 11 triterpenoid constituents, poricoic acid A accounted for the highest percentage in all batches of Poriae Cutis. In addition, the content of five components, poricoic acid A, dehydrotrametenolic acid, poricoic acid B, dehydroeburicoic acid and trametenolic acid, fluctuated relatively more, while the other components fluctuated more gently. No significant geographic variation in samples from different origins.

A method for the determination of Poriae Cutis characteristics and multi-component content was established, which laid the foundation for quality control of Poriae Cutis.

To explore the minimum effective inhibitory concentration of bacteriostatic agent in the prescription of human interferon α1b spray, and to determine the reasonable dosage of bacteriostatic agent.

Biological activity detection method was used to screen bacteriostatic agents in the prescription of human interferon α1b spray preparation, determine bacteriostatic agents. The bacteriostatic efficacy was determined according to the bacteriostatic efficacy test method of general principles 1121 of the Chinese Pharmacopoeia 2020 edition to determine the minimum effective bacteriostatic concentration of bacteriostatic agents. Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger were used as test strains to test the applicability of colony counting method. The dose screening test of bacteriostatic agent was designed to investigate the bacteriostatic effect of bacteriostatic agent with different concentrations on four kinds of experimental bacteria, and the reasonable dosage of bacteriostatic agent was selected.

Benzalkonium bromide was screened out as the bacteriostatic agent in the prescription of human interferon α1b spray. When the concentration of Benzalkonium bromide was 0.05-0.1 mg·mL^-1, its bacteriostatic efficacy against Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger met the grade A criteria.

The minimum effective bacteriostatic concentration of benzalkonium bromide in the prescription of human interferon α1b spray is determined to be 0.05 mg·mL^-1.

To establish an ion chromatography method for determination the content of sodium caprylate in human blood albumin products.

The samples were precipitated with eluent, the suspension was centrifuged and filtered, the filtrate was injected to IC, and heptanoic acid was used as the internal standard. A Dionex InPac^TM NS1 Analytical Column (250 mm×4mm, 10 μm) and a Dionex InPac^TM NG1 Guard Column (35 mm×4 mm, 10 μm) were used，the flow rate was 1.0 mL·min^-1. The conductivity detector and ASRS 300 membrane suppressor were used, and the regenerant solution was 5 mmol·L^-1 tetrabutylsodium hydroxide solution; the column temperature was 30 ℃ and the injection volume was 25 μL.

The resolution between the peaks of sodium caprylate and the internal standard was greater than 1.5, and the linearity of concentration of sodium caprylate was good in the range of 0.38-2.52 mmol·L^-1, r=0.999 5 (n=6). The RSD of the repeatability test was 1.1% (n=6). The average recovery was 97.4% and RSD was 1.8% (n=9). The limits of quantification and detection were 0.19 nmol and 0.09 nmol, respectively. The determination results of the content of sodium octanoate in 20 batches of human blood albumin samples from 7 enterprises at home and abroad ranged from 0.073-0.163 mmol·g^-1.

The method established in this study is simple to operate, accurate in results, high in sensitivity and good in repeatability, can be used for the determination of sodium caprylate content in human blood albumin products and provide a method guarantee for its quality control.

To establish an HPLC method for the determination of potentially genotoxic impurity E, impurity I, and 2-chloromethyl-4-methoxy-3，5-dimethlpyridine in esomeprazole sodium.

The chromatographic conditions were as follows: impurity E, YMC-Triart C₁₈ column (250 mm×4.6 mm, 5 μm), mobile phase A 0.05 mol·L^-1 monopotassium phosphate buffer, mobile phase B was acetonitrile, the gradient elution program was used at the flow rate of 1.0 mL·min^-1, the detection wavelength was 302 nm and the column temperature was 30 ℃. Impurities I, Agilent Microspher C₁₈ column (100 mm×4.6 mm, 3 μm), mobile phase A was water-phosphate buffer (pH 7.6)-acetonitrile(80∶10∶10), mobile phase B was acetonitrile-phosphate buffer (pH 7.6)-water (80∶1∶19), the flow rate was 1.0 mL·min^-1, the detection wavelength was 302 nm and the column temperature was 30 ℃. 2-chloromethyl-4-methoxy-3，5-dimethlpyridine, GL Inertsil ODS-3 column (250 mm×4.6 mm, 5 μm), mobile phase A was 0.01 mol·L^-1 disodium phosphate solution (pH 6.5), mobile phase B was acetonitrile, the flow rate was 1.0 mL·min^-1, the detection wavelength was 265 nm and the column temperature was 30 ℃.

The linear ranges of impurity E, impurity I and 2-chloromethyl-4-methoxy-3，5-dimethlpyridine were 0.025 1-0.200 7, 0.020 2-0.302 7, 0.126 6-2.110 0 μg·mL^-1. The LOQ of impurity E, impurity I, 2-chloromethyl-4-methoxy-3，5-dimethlpyridine were 0.50, 0.40, 2.53 ng, and the LOD were 0.15, 0.12, 0.84 ng. The average recovery rate ranged from 96% to 104%, and the RSD was less than 2%. No potential toxic impurities were detected in the samples.

The method has the advantages of good repeatability, high precision, high accuracy and good linearity, and the analysis method are simple and efficient.