To establish a fingerprint of Gualou Guizhi decoction and a method for determination of multi-component to clarify the transfer rule of quantities and quality from decoction pieces and substance benchmarks.

Fifteen batches of Gualou Guizhi decoction substance benchmarks were prepared and analyzed by the method of HPLC. Traditional Chinese medicine (TCM) Chromatographic Fingerprint Similarity Evaluation Software (2012) and hierarchical cluster analysis (HCA), principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) was used to evaluate the quality differences between batches of substance benchmarks, and screen the main chemical compositions that were led to quality discrepancy. The main differential components were determined by HPLC. And their contents, yields and transfer rates were analyzed for transfer rule of quantities and quality.

HPLC fingerprint of Gualou Guizhi decoction was established. A total of 30 common peaks in the fingerprint of Gualou Guizhi decoction substance benchmarks were assigned. Compared with the reference standards, 9 components were identified as gallic acid, albiflorin, paeoniflorin, liquiritin, ononin, liquiritigenin, cinnamic acid, isoliquiritigenin and glycyrrhizic acid. The similarities of 15 batches of Gualou Guizhi decoction substance benchmarks samples were all above 0.920. All of them were divided into two categories by three chemical recognition modes for 5 major differential components, including glycyrrhizic acid, liquiritigenin, paeoniflorin, iquiritin, and ononin. The HPLC was also applied to determine the contents of multi-components and the method validation results were good. The average transfer rates of five index components were 51.65%, 40.46%, 74.74%, 60.06%, and 34.54%, respectively and their average extraction rate was 14.20%.

The critical quality properties of Gualou Guizhi decoction can be stably transferred from decoction pieces to substance benchmarks. The method of fingerprint and content determination is accurate and reliable, which is able to provide technique for quality control of Gualou Guizhi decoction formulation.

To establish a method for identifying Fritillariae Cirrhosae Bulbus and its adulterant Fritillariae Ussuriensis Bulbus.

Chemometrics techniques were used for the analysis of the sample data from determination with Q TOF MS, and characteristic ion pairs were selected as m/z 578.3→164.14 and m/z 578.3→398.31 with triple quadrupole mass spectrometry applied, respectively. Then a specific approach was developed which used Waters Acquisition UPLC CSH (75 mm×2.1 mm, 1.7 μm) as column, and with acetonitrile and 0.1% formic acid solution as the mobile phase, and a flow rate of 0.4 mL·min^-1. Two ion pairs: m/z 578.3→164.14 and m/z 578.3→398.31 were detected by triple quadrupole mass spectrometer with MRM mode.

The validation results through 108 batches of samples indicated that the two characteristic ion pairs, m/z 578.3→164.14 and m/z 578.3→398.31, could effectively distinguish between Fritillariae Cirrhosae Bulbus and its adulterant Fritillariae Ussuriensis Bulbus.

The method had good specificity and sensitivity, and could be used for the detection of adulterated Fritillariae Cirrhosae Bulbus with Fritillariae Ussuriensis Bulbus.

To find out identification characteristics of Baijiangcao from different origins by pharmacognostical study. To discuss the establishment of whole herbs Chinese medicine standard.

Principles of plant taxonomy, macroscopic identification and microscopic identification were used to compare the differences among Baijiangcao from different origins and commercial counterfeit.

The macroscopic characteristics, such as the surface of stem, the cross section of stem, the shape of leaves, hairy on inflorescence and odour. And the microscopic characteristics of transverse section of stem could be used to distinguish Baijiangcao from different origins and commercial counterfeit.

This research establishes the identification method of Baijiangcao from different origins and commercial counterfeit, and provides references for the improvement of quality standard of Baijiangcao from different origins.

To investigate the feasibility of applying for bioequivalence exemption based on parallel artificial membrane permeability assay (PAMPA) data by evaluating the solubility and in vitro permeability of levofloxacin, comparing the prescription differences between the generic and reference formulations, assessing the impact of disparate excipients on the permeation behavior of the active pharmaceutical ingredient (API), and predicting the bioequivalence of the two formulations.

The dissolution of the raw material at pH 1.0 to pH 6.8 was determined using high-performance liquid chromatography. The μFlux^TM system was employed to determine the permeability of the raw material and the mixture of raw material with sodium stearyl fumarate in various media. pH 5.0 fed-state intestinal fluid, pH 6.5 fasted-state intestinal fluid, and pH 7.4 phosphate buffer (16 mL) were precisely added to the donor chamber, while 16 mL of Accepter Sink Buffer was added to the receptor chamber. The rotor speed was set at 200 r·min^-1, and the collection time was 180 min. Determine the permeability of the raw material and the mixture of raw material with sodium stearyl fumarate in various media, as well as the permeability of the raw material in the reference and generic formulations powders. The impact of newly added excipients and other changed excipients on API was evaluated through a two-tailed t-test. The Macro Flux^TM system was used to measure the dissolution-permeation curves of the reference and generic formulations, intestinal simulation fluids at pH 5.0 and pH 6.5 (1 000 mL) were added to the dissolution cup as dissolution media, with a stirring speed of 75 r·min^-1 using a paddle method, 12 mL of Accepter Sink Buffer was added to the receptor chamber, and the stirring speed of the micro-stirring rod was set at 450 r·min^-1, one tablet of each reference or generic formulation was placed in the dissolution cup, and the dissolution-permeation curves of the formulations were measured. The similarity of dissolution curves was compared, and the permeability rate (J_Flux) and cumulative drug permeation amount (AMT) were calculated to predict the bioequivalence of the two formulations, ensuring that the 90% confidence interval for the geometric mean ratio of J_Flux and AMT of the two formulations fell within the range of 80% to 125%.

Levofloxacin solubility ranged from 16.4 mg·mL^-1 to 62.7 mg·mL^-1 across different mediums, its permeability in pH 5.0 fed-state simulated intestinal fluid, pH 6.5 fasted-state simulated intestinal fluid, and pH 7.4 phosphate-buffered saline was 2.92×10^-6 cm·s^-1, 1.01×10^-5 cm·s^-1, and 1.07×10^-5 cm·s^-1, respectively. The addition of sodium stearyl fumarate showed no significant difference in permeability compared to the original API (P<0.05), and there were no significant differences between the powder API of the reference and generic formulations (P<0.05). The dissolution curves of both formulations were similar, with the 90% confidence interval for JFlux and AMT within the predefined range.

Levofloxacin tablets, classified as BCS Class Ⅰ, demonstrated that the altered excipients in the formulation did not impact on the API’s permeability, confirming bioequivalence between the reference and generic formulations. The bioequivalence exemption study based on PAMPA can be utilized for permeability studies of raw materials, excipient screening and optimization, and prediction of formulation bioequivalence, effectively reducing drug development costs and time. This study provides reference data for pharmaceutical companies applying for bioequivalence exemptions.

To establish the RP-UPLC-PDA method for simultaneous determination of triptolide，triptonide, triptophenolide, wilforine, wilforlide A and celastrol in Tripterygii Radix.

Tripterygii Radix were extracted with ethyl acetate and the extracts were dissolved and separated by methanol. The six components were determined by RP-UPLC-PDA method. The chromatographic column was Acquity^TM UPLC BEH C₁₈ column (100 mm×2.1 mm, 1.7 μm), the column temperature was 30 ℃, the injection volumn was 2 μL, the flow rate was 0.4 mL·min^-1 and the mobile phase was acetonitrile (A)-0.1% formic acid (B).

The linear relationship of six components was good (0.999 2≤r≤0.999 7) in the concentration ranges. The average recoveries were 99.2%-103.1% and RSDs were 1.2%-2.9%. The contents in 10 batches of Tripterygii Radix from different habitat were determined. The results showed that the contents of Tripterygii Radix in prepared pieces from different producing areas were different. The highest content of triptolide was 140.2 μg·g^-1, and the lowest content was 103.2 μg·g^-1. The highest content of triptonide was 224.7 μg·g^-1 and the lowest content was 112.2 μg·g^-1. The highest content of triptophenolide was 306.7 μg·g^-1 and the lowest content was 189.6 μg·g^-1. The highest and lowest contents of wilforine were 283.2 μg·g^-1 and 211.2 μg·g^-1. The highest content of wilfhabitat was 31.2 μg·g^-1 and the lowest content was 16.8 μg·g^-1. The highest content of celastrol was 87.6 μg·g^-1, and the lowest content was 52.1 μg·g^-1.

The RP-UPLC-PDA method can simultaneously determine six components in Tripterygii Radix. The method is reliable and stable, which is suitable for quantitative analysis and determination of Tripterygii Radix.

To establish an effective method for detecting the components of Lychas mucronatus and investigating instances of adulteration with whole scorpions in Chinese patent medicines.

Thirty Chinese patent medicines containing whole scorpion components, as documented in the Pharmacopoeia of the People’s Republic of China, were collected. DNA was extracted, and differences in the mitochondrial cytochrome C oxidase subunit Ⅰ(COⅠ) gene sequences were compared. Specific primers for the identification of Mesobuthus martensii and Lychas mucronatus DNA were designed. Annealing temperature, cycle number, Taq polymerase, and DNA concentration were optimized to establish the most suitable PCR identification system and conditions. The optimal PCR conditions for distinguishing the sharp-tailed wolf scorpion were finally determined to be an annealing temperature of 52 ℃, 36 cycles, 2×PCR Mix Taq enzyme, and 1 μL of DNA template. The developed method was applied to identify the presence of Centruroides gracilis components in 30 commercially available Chinese patent medicines.

Gel electrophoresis revealed the presence of authentic DNA bands specific to the Mesobuthus martensii in all 30 whole scorpion-containing Chinese patent medicines. Eighteen medicines (60%) showed a single specific DNA band between 100-250 bp, indicative of Lychas mucronatus, while the blank control exhibited no bands. Sequencing of the PCR products containing the identified Lychas mucronatus -specific bands, after TA cloning, revealed sequences with high similarity (92%-98%) to the reference sequences of Lychas mucronatus. Phylogenetic analysis indicated that the obtained sequences belonged to the same lineage as Lychas mucronatus and were distinct from the Mesobuthus martensii. Therefore, the presence of a single band around 120 bp indicates the identification of Lychas mucronatus.

The presence of Lychas mucronatus components mixed with authentic scorpion materials in Chinese patent medicines indicates a need for strengthened supervision. The method established in this study enables the rapid and accurate detection of Lychas mucronatus components in Chinese patent medicines. This is beneficial for improving the quality control of whole scorpion-containing Chinese patent medicines, providing assurance for their clinical applications.

To study the toxicity of heavy metal cadmium and screen detoxification drugs base on Caenorhabditis elegans.

The microfluidics chip technology was used as the drug screening platform. The 96-well plate exposure pretest was used, and K-medium blank group was set up. The exposure group consisted of nematodes exposed to heavy metal cadmium at the environmental relevant concentration of 0.25-15.0 μg·mL after homogenization. Vitamin C and calcium disodium ethylenediamine tetraacetate were selected as detoxification group. （10±2） nematodes were added into each orifice plate. The number of nematodes was observed and recorded under a microscope, and paid attention to the changes in the structure of the vulva. The experimental data were processed and analyzed by origin 2019b software.

Compared with blank group, the fatality rates of C. elegans in exposed group were respectively 0%, 1.67%, 4.76%, 67.46%, 100% and 100% at 0.25, 1.50, 5.0, 10.0, 12.5, 15.0 μg·mL^-1 concentration, compared with blank group. The structure of the nematode vulva was deformed, from slight protrusion to severe protrusion, and even a tuberous protrusion, and finally ruptured. The mortality of detoxification group was reduced to different degrees compared with exposure group.

Compared with blank control group, with the increase of cadmium exposure concentration and exposure time, cadmium can inhibit the survival of C. elegans, and damage the structure of C. elegans vulva to varying degrees. The results of detoxification group showed that vitamin C had little detoxification effect on cadmium. The complexation of calcium disodium ethylenediamine tetraacetate with cadmium could prolong the survival time of C. elegans to a large extent.

To study different batches of Xiaoer Resuqing granules by combining the method of fingerprint, multi-index component content determination and chemical pattern recognition analysis technology, and to provide the basis for its quality evaluation.

The chromatographic SuperLu C₁₈(250 mm×4.6 mm, 5 μm) column was adopted with mobile phase composed of methanol (A)-0.1% phosphoric acid (B) with gradient elution at a flow rate of 1.0 mL·min^-1. The column temperature was 30 ℃, and the detection wavelength was 280 nm. Eleven characteristic components were identified, and the contents of seven active components, including puerarin, forsythiaside A, phillyrin, baicalin, wogonoside, baicalein and wogonin were determined. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were used to classify 15 batches of samples, and orthogonal least partial squares discriminant analysis (OPLS-DA) was performed. To screen out the difference components between batches of Xiaoer Resuqing granules.

The HPLC fingerprint of Xiaoer Resuqing granules and the content determination method of 7 components were established, the results of the two methods met the requirements. Twenty-eight common peaks were identified, 11 chromatographic peaks were identified, and the similarities of 15 batches of samples ranged from 0.995 to 0.999. HCA and PCA results showed that 15 batches were clustered into two categories, and OPLS-DA screened out 13 markers that caused the differences among the 15 batches of samples. There was little difference in the contents of ingredients.

The established HPLC fingerprint of Xiaoer Resuqing granules and the determination method of 7 components are specific, accurate and reliable. Combined with chemical pattern recognition, it can be effectively used for the quality control of Xiaoer Resuqing granules.

To establish an HPLC-MS/MS method for the determination of FCN-437c in human plasma and its application to the phase I clinical study of FCN-437c.

Following protein precipitation, plasma was injected and measured using HPLC-MS/MS method. The analytes were separated on a YMC Triart PFP column (50 mm×2.1 mm, 5 μm) using 0.5% formic acid (containing 5 mmol·L^-1 of ammonium acetate, A) and acetonitrile (B) as the mobile phase with gradient elution at the flow rate of 0.5 mL·min^-1, the column temperature was set at 35 ℃, the injection amount was 2 μL, and the injector temperature was 10 ℃. MS detection was performed with multiple reaction monitoring (MRM) mode using positive electrospray ionization. The ion transitions were m/z 549.4→449.4 for FCN-437c and m/z 552.3→449.3 for FCN-437-D3, respectively. Other mass spectrometry parameters were TEM, 500 ℃, GS1, 276 kPa, GS2, 207 kPa, DP, 100 V, CXP, 25 V.

The linear range of FCN-437c in human plasma was 5-1 000 ng·mL^-1 (r=0.999 0). The lower limit of quantification was 5 ng·mL^-1. The intra-batch and inter-batch precisions were less than 2.0% and 4.1%, respectively. The average recovery was 104.0% (FCN-437c), 78.6% (FCN-437-D3), and the internal standard normalized matrix factor was 100%-102%. The stock solution of FCN-437c was stable at 4 ℃ for 202 d, the working solution of FCN-437c and internal standard were stable at room temperature for 24 h. FCN-437c in human plasma was investigated to be stable at room temperature for 20 h, four cycles of freeze-thaw, -20 ℃ for 134 d, -80 ℃ for 662 d, as well as for 24 h in the autosampler after treatment. The whole blood samples were stable at room temperature for 4 h. This method was applied to the determination FCN-437c in human plasma, and the deviation between the test results and the initial values of 97.0% ISR samples was within ±20%. The accuracy was 102.0%-108.0% after 10-fold dilution of plasma samples. The cumulative ratio of R_AUC0-24 and R_{C max} was 1.33 times and 1.59 times when FCN-437c was administered continuously compared with single administration.

This method is simple, accurate, robust and specific, which can meet the requirements of quantitative analysis of FCN-437c in human plasma and also can be used to determine FCN-473c in human plasma of the phase I clinical study.

To develop a method for characterizing in vitro release of cyclosporine ophthalmic emulsion.

The Franz diffusion cell and the polyvinylidene fluoride membrane were adopted with buffer-ethanol (40∶60) as receiving media. The sampling time was set at 60, 125, 190, 255, 320, 385 min, respectively.

The in vitro release method showed that the inertia of membrane, specificity, sensitivity and selectivity met the requirements. The validation of HPLC showed that the quantitative limit of the method was 0.07 μg·mL^-1 and a good linear relationship between the concentration range of 0.07-44.62 μg·mL^-1. The average recovery was 98.9%. Compared with the original preparation by FDA guideline, the in vitro release of the self-developed preparation was the same as the reference preparation.

This method is suitable for the in vitro release evaluation of cyclosporine eye drops.