To develop the national standard materials of progesterone in frozen human serum and to evaluate the accuracy and standardization of serum progesterone detection kits.

Low and high concentrations of serum were from normal people and pregnant women individuals respectively，with a clear appearance，no obvious hemolysis，jaundice and lipid blood. After all tests for four infectious diseases were negative，thrombin，anhydrous calcium chloride and anhydrous sodium carbonate were added according to the proportion of 1 kU·L^-1，2.219 6 g·L^-1 and 2.65 g·L^-1 respectively，and centrifuged (4 000 r·min^-1，4 ℃，25 min) after fully mixing. After twice of multiple filtration and sterilization，serum pools were packed in ampoules to prepare candidates Ⅰ and Ⅱand stored at -70 ℃. Single factor variance method and linear regression method were used to evaluate the homogeneity and stability of the candidate. Five labs working together used reference method (isotope dilution liquid chromatography tandem mass spectrometry) to assign value for candidate. The uncertainty was calculated. At the same time，the commutability was evaluated.

Through statistical analysis, the F values of homogeneity for candidate Ⅰ and Ⅱ were 0.570 9 and 1.200 9, respectively, which were all less than F_0.05. Candidate Ⅰ and Ⅱ could be stable at least 8 h at 20-25 ℃. At 2-8 ℃, candidate Ⅰ and Ⅱ could be stable at least 33 d and 7 d respectively. Candidate Ⅰ and Ⅱ cound be stable at least 33 d at -20 ℃. Candidate Ⅰ and Ⅱ remained stable after repeated freeze-thaw cycles for 6 times. The assigned values were：candidate Ⅰ：(4.44±0.24) ng·mL^-1 (k=2), Ⅱ：(16.79±0.82) ng·mL^-1 (k=2). The determination results of candidate Ⅰ and Ⅱ were all within the 95% confidence interval of the fitting regression line established by the determination values of fresh serum samples.

Progesterone candidate had good homogeneity，stability，interoperability. It had accurate and reliable determination value，which could be used as national standard. The establishment of this national standard was of great significance for promoting the consistency and standardization of progesterone testing results.

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 establish an HPLC method for simultaneous determination of seven components(narirutin, naringin, hesperidin, neohesperidin, naringenin, nobiletin and atractylenolide Ⅲ) in Zhizhu granules. And to provide reference for its quality evaluation by using chemometric analysis.

The chromatographic column Waters Symmetry C₁₈(250 mm×4.6 mm，5 μm) was adopted using HPLC-DAD. The mobile phase consisted of acetonitrile (A)-0.1% phosphoric acid (B) in gradient elution with flow rate of 1.0 mL·min^-1, the detection wavelength were 220 nm (atractylenolide Ⅲ), 280 nm (narirutin, naringin, hesperidin, neohesperidin and naringenin) and 332 nm (nobiletin). The column temperature was set at 30℃ and the injection volume was 10 μL. Cluster analysis, principal component analysis and orthogonal partial least squares-discrimination analysis were used to distinguish the content determination results.

The resolution of each component in 24 batches of Zhizhu granules was good, and the linear relationship between concentration and peak area was good (r>0.999 9). The average recoveries were within 87.6%-114.3%. There were some differences in 24 batches of Zhizhu granules, and 4 different components were screened out, which were naringin, hesperidin, neohesperidin and naringin. The contents of narirutin, naringin, hesperidin, neohesperidin, naringenin, nobiletin and atractylenolide Ⅲ in 24 batches of Zhizhu granules samples were 0.378 4-1.380 1 mg·g^-1, 5.125 8-18.137 6 mg·g^-1, 0.283 9-1.195 8 mg·g^-1, 4.490 3-22.585 0 mg·g^-1, 0.022 5-0.349 8 mg·g^-1, 0.063 3-0.211 4 mg·g^-1 and 0.054 7-0.137 5 mg·g^-1, respectively.

The established method is accurate, reliable and reproducible, which can provide reference for the quality control of Zhizhu granules.

To establish a method of UPLC fingerprint and quantitative analysis of gallic acid, protocatechuic acid, p-hydroxycinnamic acid, myricitrin and quercitrin for Frucius Aceris Fabri, and to provide a reference for the quality control of Frucius Aceris Fabri.

The determination was performed on a Waters CORTECS UPLC T3 column (150 mm×2.1 mm, 1.6 μm), with mobile phase consisting of acetonitrile -0.1% phosphoric acid by gradually elution at a flow rate of 0.20 mL·min^-1. The column temperature was 30 ℃, and detection wavelength was set at 300 nm. The quality of 10 batches of Frucius Aceris Fabri was evaluated by similarity analysis, CA and TOPSIS analysis of fingerprints.

The UPLC fingerprint of Frucius Aceris Fabri was established, and 14 peaks were selected as the characteristic fingerprint peaks. Five chemical components were identified, which were gallic acid, protocatechuic acid, p-hydroxycinnamic acid, myricitrin and quercitrin. And a quantitative method for the determination of the 5 chemical components was established. Good similarities were found in the established fingerprint through similarity analysis. CA and TOPSIS analysis showed that 10 batches of Frucius Aceris Fabri samples could be clustered into 3 groups. The sample S9 from Jiangxi was classified as class Ⅰ with best quality. The samples from Guangxi and Guangdong were classified as Class Ⅱ with medium quality. The samples S1, S4 and S6 from Jiangxi were classified as Ⅲ with worst quality. The linear relationship of the 5 chemical components was good, and the r values were all above 0.999. The contents of the 5 chemical components in 10 batches of samples were 0.236-0.356 mg·g^-1, 0.118-0.398 mg·g^-1, 0.108-0.141 mg·g^-1, 0.146-0.222 mg·g^-1 and 0.046-0.104 mg·g^-1, respectively.

The established UPLC fingerprint and quantitative analysis methods of Frucius Aceris Fabri are precise and stable, which can be used for evaluating and controlling the quality of Frucius Aceris Fabri.

To establish an HPLC method for simultaneous determination of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythiaside A, 3，4-O-dicaffeoylquinic acid, 3，5-O-dicaffeoylquinic acid, 4，5-O-dicaffeoylquinic acid, forsythin, andrographalide and dehydroandrographalide in compound Shuanghua tablets.

The samples were extracted with 50% methanol solution under ultrasonic condition, and were performed on Agilent Zoabax SB-C₁₈ column (250 mm×4.6 mm, 5 μm) by gradient elution of acetonitrile -0.15% phosphoric acid solution at a flow rate of 1.0 mL·min^-1. The column temperature was 30 ℃, the injection volume was 10 μL, and the detection wavelength were set at 327 nm (detecting neochlorogenic acid，chlorogenic acid, cryptochlorogenic acid, forsythiaside A，3，4-O-dicaffeoylquinic acid，3，5-O-dicaffeoylquinic acid and 4，5-O-dicaffeoylquinic acid) and 226 nm (detecting forsythin, andrographalide and dehydroandrographalide).

The linear ranges of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid，forsythiaside A，3，4-O-dicaffeoylquinic acid, 3，5-O-dicaffeoylquinic acid, 4，5-O-dicaffeoylquinic acid, forsythin, andrographalide and dehydroandrographalide were 4.071-40.71 μg·mL^-1(r=0.999 9), 20.16-201.6 μg·mL^-1(r=0.999 9), 4.730-47.30 μg·mL^-1(r=0.999 9), 4.536-45.36 μg·mL^-1(r=0.999 8), 1.817-18.17 μg·mL^-1(r=0.999 9), 2.266-22.66 μg·mL^-1(r=0.999 7), 3.321-33.21 μg·mL^-1(r=0.999 9), 3.462-34.62 μg·mL^-1(r=0.999 6), 2.111-21.11 μg·mL^-1(r=0.999 9) and 2.290-22.90 μg·mL^-1(r=0.999 7), respectively. The average recoveries (n=6) were 101.3%(2.1%)，103.0%(1.5%)，100.9%(2.0%)，101.1%(2.0%)，98.4%(1.6%)，102.2%(2.4%)，98.2%(1.3%)，97.8%(2.0%)，99.0%(2.0%) and 96.4%(1.1%), respectively. The contents of the 10 components in 4 bacthes of Fufang Shuanghua tablets were in the range of 1.685-2.649 mg·g^-1, 12.202-13.780 mg·g^-1, 2.415-2.594 mg·g^-1, 1.340-1.919 mg·g^-1, 0.501-0.791 mg·g^-1, 0.891-1.342 mg·g^-1, 1.299-2.105 mg·g^-1, 1.147-1.504 mg·g^-1, 0.654-0.694 mg·g^-1, 0.846-1.151 mg·g^-1, respectively.

This simple accurate reproducible method can be used for the quality control and evaluate of compound Shuanghua tablets.

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 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.