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 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 the detection of genetic impurity chloropropanol in hydroxypropyl distarch phosphate (HPDSP).

The method was performed with Agilent VF-WAX capillary column and temperature programmed by GC-MS. The initial temperature was 80 ℃ for 8 min, then increased to 220 ℃ at the rate of 35 ℃·min^-1 and maintained for 5 min with the flow rate of 2 mL·min^-1. The temperature of inlet was 200 ℃, and the injection volume was 1 μL without split. The detector was electron impact (EI) with selected ion monitoring (SIM) mode and ion source temperature at 230 ℃. The m/z of qualitative ions of 1-chloro-2-propanol were 45, 43, 79, 81, and the m/z of quantitative ion was 79. The m/z of qualitative ions of 2-chloro-1-propanol were m/z 58, 62, 63, 64, 65, and m/z of the quantitative ion was 62.

Good linear was achieved in the range of 20.04 ng·mL^-1 to 1 002 ng·mL^-1 (r=0.999 8, n=7) and the recovery rate ranged from 93.7% to 117.1% for 1-chloro-2-propanol. The contents of chloropropanol in 9 batches were within the range of 0.000 006%-0.000 08%.

The method shows the advantages of high sensitivity, good accuracy, linearity and can be used for the detection of genetic impurities in HPDSP.

To prepare and investigate the content determination of Nocardia rubra cell wall skeleton (N-CWS) loading film.

Taking chitosan and polyvinyl alcohol as film-forming materials, N-CWS film was prepared by solution casting method. The film was characterized by scanning electron microscopy(SEM), Fourier transform infrared spectroscopy(FTIR) and universal tensile testing machine. Determination of arabinose in N-CWS film was carried out by HPLC. The Kromasil C₁₈ column(150 mm×4.6 mm, 5 μm) was adopted, with the elution of 50 mmol·L^-1 phosphate buffer (pH 7.0)-acetonitrile (80∶20, v/v). The flow rate was 1.0 mL·min^-1, the detection wavelength was 254 nm, and the column temperature was 40 ℃.

When the mass ratio of chitosan to polyvinyl alcohol was 3∶2 and the mass ratio of membrane material to glycerol was 1∶2, the tensile modulus reached 2.47 mPa and the tensile strain at break was 136.67%. Scanning electron microscopy showed that N-CWS was distributed asymmetrically on the bottom of the film. The linear relationship of D-arabinose in the prepared film was good in the range of 1.1-220 μg·mL^-1, and the average recovery rate was 99.5%. The determination results of three batchs N-CWS films showed that the contents of D-arabinose in the films were (0.988±0.041) mg·g^-1, (1.035±0.049) mg·g^-1, (1.088±0.046) mg·g^-1, respectively.

The preparation process of N-CWS loading film is simple and feasible, and the content determination method is accurate and fast. This study can provide reference for the development of new dosage forms of N-CWS.

To establish an HPLC-MS/MS method for the determination of the genotoxic impurities (S)-5-chloro-N-(3-chloro-2-hydroxypropyl) thiophene-2-amide and 4-(3-oxy-4-morpholine) nitrobenzene in rivaroxaban. The factors affecting impurities separation and the response of mass spectrometry were systematically investigated.

A ZORBAX SB-C₁₈ column(150 mm×2.1 mm, 3.5 μm) was used with 10 mmol·L^-1 ammonium formate solution-methanol as mobile phase at flow rate of 0.4 mL·min^-1. And MRM detection by electrospray positive ionization was used.

(S)-5-chloro-N-(3-chloro-2-hydroxypropyl) thiophene-2-amide and 4-(3-oxy-4-morpholine) nitrobenzene had good linear relationships in the range of 0.008-3.4 ng·mL^-1 and 0.2-3.4 ng·mL^-1, respectively. The average recoveries(n=9) were 97.9% and 98.9%, RSD were 0.94% and 0.68% respectively. The limits of quantitation were 0.008 ng·mL^-1 and 0.2 ng·mL^-1 respectively. The results of genotoxic impurity residue determination in 15 batches of rivaroxaban samples were far below the limit value.

The method is sensitive and specific, and can effectively detect trace genotoxic impurities in rivaroxaban.

To establish an LC-MS/MS method for the determination of the abundance data of dexamethasone-related metabolic enzymes and transporters in the placenta of Chinese pregnant women.

A Shim-pack GISS-HP C₁₈ (100 mm×2.1 mm, 1.9 μm) chromatographic column was used, and the mobile phases were consisted of 0.2% formic acid-water (phase A) and 0.2% formic acid-acetonitrile (phase B) with gradient elution at a flow rate of 0.2 mL·min^-1. The post-column phase C consisted of 0.5% ethylene glycol-acetonitrile was added at a flow rate of 0.1 mL·min^-1, and the ESI source positive ion MRM mode was used for quantitative analysis. The established method was investigated methodologically and the expression levels of 11β hydroxysteroid dehydrogenase 1(11β-HSD1), 11β hydroxysteroid dehydrogenase 2(11β-HSD2), cytochrome P450 3A4 enzyme (CYP3A4) and P-glycoprotein protein (P-gp) were quantitatively analyzed in the placenta of Chinese pregnant women in the third trimester.

The established LC-MS/MS method had a linear range of 0.1-100 nmol·L^-1(r >0.999). The precision and accuracy results met the requirements of the biological sample analysis method verification in Chinese Pharmacopoeia (RSD≤15%), and the stability results showed that the samples were stability. The protein abundance of 11β-HSD2, 11β-HSD1, CYP3A4 and P-gp were (84.46±59.97) pmol·g^-1, (11.44±3.73) pmol·g^-1, (8.83±2.78) pmol·g^-1 and (7.94±4.10) pmol·g^-1, respectively. Besides, the results of the study also showed that there was no significant difference in the distribution of related metabolic enzymes and transporters in different parts of the placenta. However, there was a significant difference in the abundance of P-gp in the placenta between Chinese people (7.94±4.10) pmol·g^-1 and white people (4.41±2.46) pmol·g^-1.

The LC-MS/MS method established in this study has high accuracy and sensitivity and is suitable for detecting the abundance values of dexamethasone-related metabolic enzymes and transporters in human placenta.

To predict potential quality markers(Q-Marker) of Taxus media Rehd. based on fingerprint and network pharmacology methods, and establish an evaluation method of Taxus media Rehd. based on Q-Marker, as so to provide basis for the establishment of quality standard and quality evaluation system of Taxus media Rehd..

Waters SymmmetryShield^TM RP18(250 mm×4.6 mm, 5 μm) chromatographic column was used for separation, and the wavelength was 254 nm. The mobile phase was acetonitrile -0.01% trifluoroacetic acid aqueous solution for gradient elution at a flow rate of 1.0 mL·min^-1, and the column temperature was 30 ℃. The injection volume was 10 μL. The fingerprints of 24 batches of Taxus media Rehd. with 8 kinds of cultivation years were established and the common peaks were identified. Hierarchical clustering analysis (HCA) was used for classification, orthogonal partial least square-discriminant analysis (OPLS-DA) was used to screen out the main marker components that cause differences between different years. Combined with network pharmacology, the core targets and key pathways were constructed a “component-target-pathway” network map through corresponding databases. Q-Markers were further verified and quality of Taxus media Rehd. comprehensively evaluated by HCA and PCA.

A total of 25 common peaks were obtained in the HPLC fingerprint of Taxus media Rehd., 18 compounds were identified, and their similarities were all above 0.900, including taxanes, flavonoids, alkaloids, steroids and phenols. The results of HCA showed that there were some differences among samples with different cultivation years. Eleven different markers were screened by OPLS-DA, and they were paclitaxel, 10-deacetylbaccatin Ⅲ (10-DAB), baccatin Ⅲ, cephalomannine, deacetyltaxol (10-DAT), ferulic acid, kaempferide, rutin, amentoflavone-4’, 4", 7-trimethyl ether, skyrin, 4-hydroxybenzaldehyde. Based on the difference markers, network pharmacology was applied to analyze them from the perspective of effectiveness. Paclitaxel, 10-DAB, bakatine Ⅲ, cephalomannine and 10-DAT were preliminstly predicted as Q-Markers of Taxus media Rehd.. They could regulate cancer pathway, AGE-RAGE signaling pathway in diabetic complications and other signaling pathways by PIK3R1, AKT1, EGFR, HRAS and MAPK1 targets, and play the role of reducing swelling and menstruation diuresis clearing. The results of HCA verified the rationality of Q-Marker, and the comprehensive evaluation results of PCA showed that sample which was cultivated for ten years and harvested in spring bore the best quality.

Paclitaxel, 10-DAB, bakatine Ⅲ, cephalomannine and 10-DAT are selected as Q-Markers based on fingerprint and network pharmacological methods. The quality of Taxus media Rehd. in different cultivation years is sorted comprehensively by combining with chemical recognition pattern method. The 10-year-old sample harvested in spring gets the highest score and the quality is good. The study provides new method for establishing of quality standard and the quality evaluation of Taxus media Rehd..

To investigate the accumulation pattern of metabolites in Glycyrrhiza uralensis by qualitative and quantitative analyses of metabolic constituents in Glycyrrhiza uralensis with different cultivation years, and to search for its differential metabolites.

The separation was performed on an Agilent SB-C₁₈ (100 mm×2.1 mm, 1.8 μm) column with 0.1% formic acid aqueous solution as the mobile phase A and 0.1% formic acid acetonitrile solution as the mobile phase B. The gradient elution was carried out at a flow rate of 0.35 mL·min^-1, and the column temperature was 40 ℃ with an injection volume of 4 μL. The mass spectrometry was performed with positive and negative ions scanning in multiple reaction monitoring mode. The mass spectrometry was performed in multi-response monitoring mode with positive and negative ion scanning. The qualitative and quantitative analyses of the metabolites in Glycyrrhiza uralensis were carried out on the basis of the self-constructed secondary mass spectrometry database, and the multivariate statistical analyses of the metabolites of Glycyrrhiza uralensis with different cultivation were combined with principal component analysis(PCA), orthogonal partial least squares discriminant analysis(OPLS-DA), and cluster heat map analyses.

(1) A total of 1 038 metabolites were detected from the samples of Glycyrrhiza uralensis with different cultivation years, among which 201 differential metabolites existed between annual and biennial Glycyrrhiza uralensis, 125 up-regulated and 76 down-regulated; 223 differential metabolites existed between biennial and three years old Glycyrrhiza uralensis, 64 up-regulated and 159 down-regulated; 185 differential metabolites existed between annual and three years old Glycyrrhiza uralensis, 59 up-regulated and 126 down-regulated. Four metabolites specific to annual Glycyrrhiza uralensis, six to biennial and one to three-year old were found. (2) K-mean cluster analysis was performed on the differential metabolites, and the differential metabolites were classified according to the different accumulation trends, and it was found that most of the metabolites such as flavonoids, phenolic acids, terpenes, lignans, and coumarins peaked in biennial Glycyrrhiza uralensis, and most of the metabolites such as alkaloids, amino acids and their derivatives peaked in annual Glycyrrhiza uralensis, and a part of the flavonoids, phenolic acids and other metabolites reached peaks in three years old Glycyrrhiza uralensis, suggesting that the metabolism of Glycyrrhiza uralensis in the body reached the peaks. peak value, suggesting that there was a certain pattern of metabolite content changes in Glycyrrhiza uralensis. (3) 160 differential metabolites annotated in Kyoto Encyclopedia of Genes and Genomes (KEGG) datebase and flavonoids, amino acids and their derivatives, and organic acids were the differential metabolites that accounted for a relatively large number of them. A total of 79 differential metabolic pathways were enriched among different comparison groups, among which 6 differential metabolic pathways were highly significantly enriched (P<0.01) and 23 significantly enriched (P<0.05), and the distributions of compounds involved in the above pathways were basically the same as before enrichment in comparison of different cultivation year.

The present study elucidate the differences between the metabolic components of Glycyrrhiza uralensis with different cultivation years, and further analyse the metabolic pathways that might cause the differences through the differential metabolites, which can provide a certain reference basis for the determination of the harvesting year of Glycyrrhiza uralensis and the study of the quality formation mechanism.

To analyze the chemical constituents of Quyusanjie capsules by LC/MS, and establish a method for the determination of active ingredients in Quyusanjie capsules.

Using UPLC-Q TOF MS/MS technology, the Hypersil Gold C₁₈ column(100 mm×2.1 mm, 1.9 μm) was used, the mobile phase was acetonitrile(A) and 0.1% formic acid in water(B) with gradient elution, at a flow rate of 0.4 mL·min^-1, the the column temperature was 40.0 ℃, and the mass spectrometry data was collected by negative ions mode scanning. Through database matching, elemental composition and fragment structure analysis, the main chemical substances in Quyusanjie capsules were identified. HPLC was used to qualitatively analyze the chemical components of Quyusanjie capsules. The Ultimate^® AQ-C₁₈ column(250 mm×4.6 mm, 5 μm) was used, the mobile phase was acetonitrile(A) -0.1% phosphoric acid(B) with gradient elution at the flow rate of 1.0 mL·min^-1, the column temperature was 25 ℃, and the detection wavelength was 203 nm. The content of naringin, neohesperidin, notoginsenoside R₁, ginsenoside Rg₁, and ginsenoside Rb₁ in 11 different batches of Quyusanjie capsules were determined using external standard method. QAMS method was established using ginsenoside Rg₁ as the internal reference.

Twenty-nine compounds were identified from Quyusanjie capsule. The contents of naringin, neohesperidin, notoginsenoside R₁, ginsenoside Rg₁ and ginsenoside Rb₁ measured by external standard method were 0.484-1.097 mg·g^-1, 0.341-0.618 mg·g^-1, 1.685-2.399 mg·g^-1, 5.748-8.386 mg·g^-1, 3.868-5.898 mg·g ^-1, respectively. Measured with the QAMS method, the contents of naringin, neohesperidin, notoginsenoside R₁ and ginsenoside Rb₁ were 0.516-1.153 mg·g^-1, 0.372-0.667 mg·g^-1, 1.794-2.580 mg·g^-1, 4.373-6.690 mg·g^-1, respectively. The relative error between the calculated values of the QAMS method and the measured value of the external standard method was less than 8.9%.

UPLC-Q TOF MS/MS method can quickly identify the chemical components of Quyusanjie capsules. The established external standard method is stable and reliable, and can be used for the quality control of Quyusanjie capsules. The method of QAMS has good feasibility and is suitable for the determination of the daily production of Quyusanjie capsules.

To develop the first batch of traditional Chinese medicine chemical reference substance 2-acetate regaloside A used in standard of Baihe (Juandan) Peifang Keli.

The structure was elucidated by nuclear magnetic resonance spectrometry, mass spectrometry, ultraviolet spectroscopy and infrared spectroscopy. Purity analysis, determination of related substance and investigation of other physical and chemical parameters were carried out by using analytical different techniques such as high performance liquid chromatography, gas chromatography, coulometry and ignition residue method. On the above basis, the mass balance was used for value determination, and the ¹H quantitative nuclear magnetic resonance method was applied for further verification.

The structure of the traditional Chinese medicine chemical reference substance was determined as 2-acetate regaloside A, and it was identified as a new compound by SciFinder search. The calibration value by mass balance method was 99.31%, and result of ¹H quantitative nuclear magnetic resonance method was 100.06% (n=3, RSD=0.47%).

The accurate development and calibration of the first batch of 2-acetate regaloside A fully ensures the implementation of the standard for Baihe (Juandan) Peifang Keli.