Therapeutic oligonucleotides (OGNs) drugs are artificially synthesized single or double stranded short nucleic acids, typically 15 to 30 base pairs in length. OGNs have been rapidly developed as new therapeutic drugs with increasing attention in the discovery and development of drugs concerning various disease fields. Compared with Europe and America, there are currently no other OGNs drugs listed in China, except for Spinraza, which has been approved for marketing as an orphan drug. The development of OGNs in China started relatively late and is still in its early stages of development. However, the OGNs drug market in China is anticipated to grow quickly due to the country's large population, high patient demand, ongoing support for the development of oligonucleotide drugs in the future, and the steady maturation of related technologies by domestic businesses. Because of their special physicochemical characteristics, OGNs drugs are challenging to design biological analysis techniques. Currently, there are few reports on quantitative analysis methods for oligonucleotide drugs in China. Therefore, the development of sensitive and reliable bioanalysis methods for oligonucleotides is the key to investigate oligonucleotides' pharmacokinetic and pharmacodynamic properties. Liquid chromatography-mass spectrometry (LC-MS) can quantify OGNs and their metabolites concurrently, compared with traditional ELISA approaches. Numerous benefits come with using LC-MS, in particular, the extensive use of high-resolution mass spectrometry allows for the identification of metabolites, which provides details on base composition and sequence structure, in addition to quantitative information about target oligonucleotides. It has now emerged as the go-to technique for OGN quantitative analysis. The application of LC-MS in the identification of therapeutic oligonucleotide medicines is the primary focus of this paper, which also discusses its benefits and drawbacks. Lastly, it looks at the LC-MS development trend for oligonucleotide detection, which includes a lower detection level and potential general methods.

Objective: To compare the quality of wild and cultivated Arnebiae Radix,using macroscopic investigation and chemometric analysis of the different components in wild and cultivated Arnebiae Radix from three different habitats. Methods: Wild and cultivated Arnebiae Radix were collected and their macroscopic features were compared. Using the ACQUITY UPLC BEH C₁₈ (2.1 mm×100 mm, 1.7 μm) column, with acetonitrile-0.05% formic acid water as the mobile phase, the contents of D-shikonin, acetylshikonin, β-acetoxyisovalerylshikonin, isobutyrylshikonin, β,β’-dimethylacrylalkannin and isovalerylshikonin in 48 batches of wild and cultivated Arnebiae Radix were determined. The detection wavelength was 275 nm and the flow rate was 0.2 mL·min^-1. PCA and OPLS-DA were performed to reveal the differential components of wild and cultivated Arnebiae Radix. Results: There were great differences in macroscopic features of wild and cultivated Arnebiae Radix, and the linear relationship between the contents of six naphthoquinone components was good. The correlation coefficients were above 0.999, the average recovery rates were 93.4%-102.9%, and the RSDs were less than 3.0%. The contents of six components in different batches of wild and cultivated Arnebiae Radix were quite different, and the contents of D-shikonin and acetylshikonin in wild products were significantly higher than those in cultivated products, indicating that there were still certain differences between wild products and cultivated products. The PCA model established could distinguish wild products and cultivars, and two differentiating components in wild products and cultivars were revealed by OPLS-DA, namely isobutyryl shikonin, β,β’-dimethylacrylalkannin. Conclusion: By comparing the core size, cork curl degree and specific odor of wild and cultivated products, the two can be identified. The established content determination method is repeatable, specific, stable and feasible. The differential components in wild and cultivated Arnebiae Radix in three different regions are identified, which provides a basis for the quality control of Arnebiae Radix and provides ideas for expanding the source of Arnebiae Radix.

Thyroid-stimulating hormone (TSH) is a glycoprotein hormone produced by the anterior pituitary. It can regulate the synthesis and secretion of thyroid hormone in thyroid follicular cells, which has important physiological significance. As a drug, it has important application value. Biological activity detection is an effective and necessary to evaluate its quality. This article discusses the signal transduction mechanism of thyroid stimulating hormone, the clinical diagnosis and treatment of thyroid stimulating hormone, and the determination method of biological activity.

Ongoing procedure performance verification (OPPV) of an analytical procedure is the process of ensuring it continues to meet its intended use after completion of validation. On the basis of previous studies, this paper further discussed the indicators of ongoing procedure performance verification (including system suitability indicators and reported values, etc.) and the implementation of analysis tools (control charts), and demonstrated the specific operation steps of ongoing procedure performance verification with examples. It is hoped that this paper will provide new ideas for the accurate and standardized procedure verification in the pharmaceutical field, especially in the enterprises and regulatory departments.

Objective: To establish an HPLC method of the content and related substances of compound amino acid injection(3AA). Methods: RP-HPLC was adopted to determine compound amino acid injection(3AA), combining with the use of two-dimensional column switching-LC/MSⁿ method was applied to separate and identify the impurities. The determination was performed on Capcell PAK AQ C₁₈(250 mm×4.6 mm, 3 μm) column with 0.2 mol·L^-1 sodium dihydrogen phosphate solution (adjusted pH to 2.8 with phosphoric acid) -acetonitrile (98∶2) as mobile phase at the flow rate of 1.0 mL·min^-1. The column temperature was 40 ℃, and the detection wavelength was 210 nm. And the injection volume was 20 μL. The LC/MSⁿ method was performed on a Thermo Accucore AQ C₁₈ (100 mm×4.6 mm, 2.6 μm) column with 0.1% formic acid solution as mobile phase A, 0.1% formic acid solution-acetonitrile as mobile phase B, at a flow rate of 0.4 mL·min^-1, and at a column temperature of 40 ℃. The mass spectrometry conditions were performed using an ESI ionisation source in the positive-ion scanning mode with a scan range of m/z 100-1 000, and the secondary mass spectrum was carried out by data-dependent scanning. Results: The related substances were completely separated from the main constituents in RP-HPLC. The standard curve of valine was linear over the range of 1.263-5.050 mg·mL^-1, with the average recovery of 99.0% (n=9). The standard curve of isoleucine was linear over the range of 1.350-5.402 mg·mL^-1, with the average recovery of 99.4%(n=9). The standard curve of leucine was linear over the range of 1.647-6.588 mg·mL^-1, with the average recovery of 99.5%(n=9). The main impurities in the three batches of samples were all process impurities introduced from the raw materials, with methionine content of 4.344 μg·mL^-1, 3.751 μg·mL^-1, 4.503 μg·mL^-1, respectively, phenylalanine content of 4.636 μg·mL^-1, 4.889 μg·mL^-1, 4.753 μg·mL^-1, respectively. The maximum single impurity contents were 0.01%, 0.02% and 0.01%, respectively. Conclusion: The method is proved by the methodology validation that it can be used for the quality control of compound amino acid injection(3AA).

Objective: To establish the UPLC-MS/MS method for simultaneous determination of 9 components (nicotinic acid, kaempferol, swertisin, quercetin, luteolin, rutin, vitexin, spinosin, salicylic acid) in Desmodium caudatum (Thunb.) DC. and construct a back propagation(BP) neural network model to predict the origin of Desmodium caudatum (Thunb.) DC. from different habitats. Methods: The chromatographic separation was achieved on an Agilent Zorbax SB C₁₈ column (50 mm×3.0 mm,1.8 μm). The mobile phase consisted of methanol-0.1% acctic acid (containing 0.02 mol·L^-1 ammonium acetate) at a flow rate of 0.3 mL·min^-1 with gradient elution, the MS analysis were performed by multiple reaction monitoring (MRM) under ESI⁺ and ESI. A correlation analysis was conducted on the contents of each component, and a BP neural network model was constructed to distinguish Desmodium caudatum (Thunb.) DC. from different habitats. Results: Under the optimized conditions, 9 components(nicotinic acid, kaempferol, swertisin, quercetin, luteolin, rutin, vitexin, spinosin, salicylic acid) showed good linear relationships in the ranges of 0.388 8-38.88 ng·mL^-1, 10.07-1 006.6 ng·mL^-1, 34.22-34 221.6 ng·mL^-1, 3.944-394.4 ng·mL^-1, 2.124-212.4 ng·mL^-1, 4.344-434.4 ng·mL^-1, 46.50-4 650.1 ng·mL^-1, 1.649-164.9 ng·mL^-1, 4.880-488.0 ng·mL^-1, respectively (r>0.995 1), whose average recoveries were 96.9%-103.9% (RSDs<1.9%). The contents of the above nine components in 40 batches of Desmodium caudatum (Thunb.) DC. were 1.657-7.407 μg·g^-1, 15.801-64.488 μg·g^-1, 1 068.348-4 270.780 μg·g^-1, 10.608-123.228 μg·g^-1, 3.897-16.802 μg·g^-1, 1.269-97.834 μg·g^-1, 405.285-1 955.796 μg·g^-1, 13.614-36.124 μg·g^-1, 4.417-87.509 μg·g^-1, respectively. According to correlation analysis, four components (swertisin, rutin, spinosin, and luteolin) in Desmodium caudatum (Thunb.) DC. showed a highly linear positive correlation, indicating that these four components had a certain synergistic effect in Desmodium caudatum (Thunb.) DC.. The BP neural network model was constructed to predict Desmodium caudatum (Thunb.) DC. from different habitats, and the accuracy of the test set reached 92.3%. Conclusion: The method is simple, sensitive and efficient, and can be used for the rapid determination of the components in Desmodium caudatum (Thunb.) DC.. Using the BP neural network model to predict the habitats plays a significant role in tracing the origin of Desmodium caudatum (Thunb.) DC..

Objective: To compare the regulatory effects of authentic and counterfeit Arnebiae Radix on intestinal flora in mice based on metagenomic sequencing. Methods: Firstly, 24 clean grade female BLAB/C mice were randomly divided into 3 groups:blank control group, A1 (Arnebia euchroma) group and A2 (Arnebiae Radix whose origins were not included in Chinese Pharmacopoeia) group. After gavage arrived at the specified time, colon contents (feces), ileal contents (feces) and small intestinal contents (feces, except ileal parts) were extracted for intestinal flora analysis. Genomic DNA was extracted and amplified by PCR from the extracted mouse intestinal contents. The PCR products were mixed and purified. Then library was constructed and sequenced. After quality control of sequencing data and removal of chimera sequence, the final effective data was obtained. Operational taxonomic unit(OTU) clustering and species annotation were performed on the obtained valid data, and sample diversity analysis was conducted. Results: In this study, both A1 (Arnebia euchroma) group and A2 (Arnebiae Radix non-pharmacopoeia) group reduced the diversity of mice colon microbiota. At the phylum level, group A1 significantly increased the abundance of Firmicutes in the small intestine and ileum, and groups A1 and A2 significantly increased the relative abundance of Bacteroidetes in the colon. At the genus level, group A1 significantly increased the relative abundance of Lactobacillus in the small intestine of mice, and group A2 significantly increased the relative abundance of Lactobacillus in the ileum of mice. Group A1 increased the relative abundance of Lactobacillus, one of the dominant bacteria in the colon of mice, and group A2 increased the relative abundance of Bacteroides. Alistipes mainly existed in the colon, A2 group significantly reduced the relative abundance of Alistipes in the colon of mice, while Alistipes in the A1 group was cultivated. Conclusion: According to the results of the regulation effect of intestinal flora, the intestinal flora regulation effect in the two experimental groups of the authentic Arnebiae Radix and its confusion products from markets are not consistent, the results of this study can provide a theoretical basis for further exploring the mechanism of Arnebiae Radix.

Objective: To establish an innovative analytical method based on high resolution sampling two-dimensional chromatography (HiRes 2D-LC) for determination of the content of vitamin D₃ in vitamin D drops. Methods: Two-dimensional liquid chromatography was used. Thermo HYPERSIL Gold Silica (100 mm×2.1 mm, 1.9 μm) column was used in the first dimension with n-hexane-n-amyl alcohol (996∶4) as mobile phase. The flow rate was 0.2 mL·min^-1. The samples were injected and tested at the wavelength of 265 nm. The column temperature was 40 ℃. In the second dimension liquid chromatography, ShimPack Velox Hilic (50 mm×2.1 mm, 2.7 μm) was used as the column with n-hexane-n-pentanol-isopropanol (98∶1∶1) as the mobile phase. The flow rate was 0.5 mL·min^-1. The samples were injected and tested at the wavelength of 265 nm. The column temperature was 40 ℃. A six-position 14-way valve and was equipped with 2 multi-center cutting valves was equipped to make multiple consecutive cuts of the pre-vitamin D₃ peak and vitamin D₃ peak. Results: The calibration curves showed a good linearity at the range of 1.018 4-5.092 mg·mL^-1(r≥0.999 8). The precision test showed the RSDs of the peak area of pre-vitamin D₃ and vitamin D₃ were 0.95% and 0.40%, respectively. The repeatability test showed the RSD of vitamin D₃ content was 0.41%. The average recovery rate (n=9) was 101.4%. The test solution was stable at 4 ℃ and 10 ℃ for 12 h, and the RSDs were 0.58% and 0.66%, respectively. The contents of vitamin D₃ in the samples of vitamin D drops measured by this method were 100.4%, 101.6%, 100.9%, 101.6%, 102.7% and 101.6%, which was basically consistent with the results measured by the fourth method in General Chapter 0722 of ChP 2020 Vol Ⅳ. Conclusion: This method has a good specificity and high sensitivity to accurately determine the content of vitamin D₃ in vitamin D drops.

Objective: 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. Methods: 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. Results: 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. Conclusion: 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.

Objective: 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. Methods: 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. Results: 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%. Conclusion: 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.

Objective: To analyze and optimize stir-baked with vinegar technology of Genkwa Flos decoction pieces by multi-index weighted scoring method based on orthogonal experiment, and to provide technical index on standardization of stir-baked with vinegar technology of Genkwa Flos decoction pieces. Methods: The orthogonal experiment was employed to analyze and evaluate the four key factors related to stir-baked with vinegar technology including the amount of vinegar, dampening time, temperature and stir-baked time by the six main indicators including the total contents of chlorogenic acid, tiliroside, luteolin, apigenin, hydroxygenkwanin and genkwanin, the content of alcohol extractive and appearance characters. Results: The amount of vinegar and stir-baked temperature had notable effects, while the dampening time and stir-baked time had no notable effects on the test results. Considering comprehensively the effects of four key factors, the optimized stir-baked with vinegar technology was as follows: add 0.3 kg of vinegar to 1 kg of Genkwa Flos, and fry at 160 ℃ for 10 min after dampening for 15 min. Conclusion: This study offers guidance and reference for standardizing the stir-baked with vinegar technology of Genkwa Flos decoction pieces.

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

Objective: To establish high-performance liquid chromatography method using cyclodextrin as a mobile phase additive for the determination of ursolic acid and oleanolic acid in Corni Fructus, providing a basis for quality control of Corni Fructus. Methods: Molecular design and response surface method were used to optimize the determination method of ursolic acid and oleanolic acid in Corni Fructus. Agilent Eclipse XDB C₁₈ column (150 mm×4.6 mm, 5 μm) was adopted as the stationary phase, the mobile phase was 3 mmol·L^-1 γ-cyclodextrin solution (contained 0.05% phosphoric acid)-acetonitrile (60∶40), the flow rate was 1.0 mL·min^-1, the column temperature was 25 ℃ and the detecting wavelength was 210 nm. Results: The resolution of chromatographic peaks of ursolic acid and oleanolic acid was 3.81. Linearities were good in the range of 400-4 000 ng for ursolic acid and in the range of 200-2 000 ng for oleanolic acid. The RSDs for precision, reproducibility, and stability were less than 1.4%, 1.8%, and 1.7%, respectively. The recovery rates were 98.5% (RSD=1.7%) and 99.1% (RSD=2.0%). The contents of ursolic acid in three batches of Corni Fructus were 2.66 mg·g^-1, 2.35 mg·g^-1 and 2.91 mg·g^-1, and those of oleanolic acid were 0.83 mg·g^-1, 0.78 mg·g^-1and 1.12 mg·g^-1, respectively. Conclusion: This method is simple, accurate, reliable, and environmental friendly, and can be used as a quality control for Corni Fructus.

The change of analytical procedure, a part of the entire lifecycle of a method, is increasingly valued with the emergence of new technologies, the increasing demand for product quality, 3R, environmental protection, and cost reduction requirements. This paper refers to the latest progress in the study of counterpart law at home and abroad, systematically classifies changes of analytical procedures from the perspective of methodology, and discusses the differences between different types of changes and the evaluation criteria and implementation approaches of analytical procedures after procedure changes.

Bioassays play a key role in the development and quality control of biological products, and the research on them has also made great progress in recent years. Here, the historical process of bioassays was reviewed. Then- the current contents about bioassays in pharmacopoeia and technical guidelines weresorted out. At last, the latest progress related to bioassays was summarized and analyzed including the introduction of new concepts such as analytical procedure, method life cycle and analytical target profile.This paper might provide a systematical reference for the people work in the biopharmaceutical field to fully understand the bioassays and related guidelines. Meanwhile, it can also provide a theoretical support for drug researchers to establish scientific and reliable bioassays.

Objective: To establish a method for determination of potential anti-vascular disease active components of Semen raphani based on cell membrane chromatographic (CMC) model of human umbilical vein cell (HUVEC). Methods: The HUVEC cell membrane chromatography coupled with HPLC-ESI IT TOF MS system were used to screen the active components of Semen raphani. The selected active components were further applied to ox-LDL induced HUVEC to verify their protective effects. Results: Two retained components were selected from Semen raphani by this method. One component was identified as erucinic acid by comparing with the reference material. Compared with the model group, the cell survival rates of the erucinic acid pretreatment groups increased significantly. The amount of ICAM-1 and VCAM-1 decreased in a dose-dependent manner. Bcl-2 protein levels decreased and Bax protein levels increased, with statistical significance (P<0.05). Conclusion: The method can rapidly obtain active ingredients from complex traditional Chinese medicines. It provides a reference for the application of cell membrane chromatography and the development of Semen raphani.

Objective: To establish a quantitative analysis of multi-components by single marker (QAMS) for simultaneous determination of 7 flavanoids (5-hydroxy-6, 7, 3’, 4’-tetramethoxyflavone, apigenin, hispidulin, kaempferol, jaceosidin, eupatilin and casticin) in Artemisiae Argyi Folium. Methods: The HPLC system consisted of the Agilent ZORBAX SB-C₁₈ column (150 mm×4.6 mm, 5 μm) column with gradient elution of acetonitrile and 0.2% phosphoric acid as the mobile phase at a flow rate of 1.0 mL·min^-1, a detection wavelength of 350 nm, and a column temperature of 30 ℃. Eupatilin was selected as the internal reference substance, the relative correction factors between eupatilin and the other 6 flavanoids were established, and the contents of these 7 constituents in samples were calculated to realize QAMS. At the same time, compared with the external standard method to verify the accuracy and feasibility of the QAMS method. Results: Within a certain linear range, the relative correction factors between eupatilin and 5-hydroxy-6, 7, 3’, 4’-tetramethoxyflavone, apigenin, hispidulin, kaempferol, jaceosidin as well as casticin were 0.958, 1.387, 1.000, 0.950, 0.957 and 1.297, respectively (RSDs of RCFs were less than 2.0%). The contents of 5-hydroxy-6, 7, 3’, 4’-tetramethoxyflavone, apigenin, hispidulin, kaempferol, jaceosidin, eupatilin, casticin in 20 batches of Artemisiae Argyi Folium were 0.031 4-0.623 5 mg·g^-1, 0.000 9-0.092 6 mg·g^-1, 0.020 6-0.170 7 mg·g^-1, 0.011 0-0.184 7 mg·g^-1, 0.011 7-0.864 0 mg·g^-1, 0.253 2-2.555 0 mg·g^-1 and 0.015 6-0.250 7 mg·g^-1, respectively. Conclusion: Using eupatilin as the internal reference, QAMS method for 7 flavanoids is established. The method is accurate and reliable, and can be used for quality control and quantitative analysis of Artemisiae Argyi Folium.

In recent years, the domestic gene therapy industry has developed rapidly, and innovative gene therapy products are emerging constantly. How to effectively control the quality of such products has become a highly concerned issue in the industry. This article draws inspiration from relatively mature, effective, and practical methods in the field of quality control of gene therapy products. Taking recombinant adeno-associated virus gene therapy products as an example, starting from the selection of testing items and methods, precautions, and formulation of quality standards, it elaborates on their quality control inspection items and related technical requirements. We hope this article can provide reference for practitioners to conduct quality control research on such products, and through subsequent communication and discussion, a certain consensus can be reached, thereby accelerating the development of gene therapy industry in China.

Scientific design of experiment (DOE) is the main tool to ensure the accuracy of test results and the reliability of conclusions. It is discussed the validation design for bioassay with many influencing factors and great variation, and the calculation method of sample size in method validation design is elaborated in detail here. It is hoped that it could provide theoretical reference for bioassay valiadation to accurately understand the experimental design of method validation.

Objective: To establish an HPLC-MS/MS method for the simultaneous determination of six characteristic components of Scutellariae Radix including baicalein, baicalin, wogonin, wogonoside, chrysin and oroxylin A in rat plasma, and to compare their pharmacokinetic profiles after oral administration of Scutellariae Radix and Niuhuang Jiedu tablets (NHJDT) to rats. Methods: Rats were given 250 mg·kg^-1 of NHJDT suspension or the prescribed equivalent amount of Scutellariae Radix aqueous extract, and plasma samples were collected at different time intervals. Naringenin was used as internal standard. After precipitated with methanol, the plasma samples were separated on an Inertsil C₈-3 (150 mm×4.6 mm, 5 μm) column by linear gradient elution with 0.1% formic acid solution(A)-0.1% formic acid-methanol(B) as mobile phase. The flow rate was 1.0 mL·min^-1, and the column temperature was 35 ℃. The analytes were detected by tandem mass spectrometry with the electrospray ionization (ESI) source combined with multiple reaction monitoring(MRM) mode in positive ion mode. The pharmacokinetic parameters were calculated and statistically analyzed. Results: The six characteristic components of Scutellariae Radix were all in good linear relationships in the range of 5-500 ng·mL^-1. The RSDs for accuracy test were in the range of 88.43%-108.6%, and the RSDs for inter-batch and intra-batch precision tests were all below 15%. The matrix effect and plasma stability met the requirements of methodology validation in biological sample analysis. Baicalin and wogonoside were the major components detected in rat plasma after oral gavage of Scutellariae Radix aqueous extract and NHJDT suspension. The AUC_0-t of wogonoside was significantly increased in NHJDT group compared with Scutellariae Radix group. Furthermore, the C_max of wogonoside and baicalin were significantly increased while the T_max was decreased after NHJDT suspension administration. Conclusion: This method is specific and sensitive for the determination of six characteristic components of Scutellariae Radix, and suitable for pharmacokinetic study of rat plasma. NHJDT with co-existing components enhances the absorption and influences the pharmacokinetic behaviors of active ingredients of Scutellariae Radix.

Objective: To determine the cytokine levels in immunity evaluation of dental restoration membrane of xenogeneic acellular dermal matrix using cytometric beads array（CBA）. Methods: The dental restoration membrane of xenogeneic acellular dermal matrix was selected as a test sample and implanted into Balb/C mice IL-6, IL-10, IL-17A, IL-23, TNF-α, IFN-γ, IFN-β, MCP-1 and GM-CSF were measured by CBA. The data of the test group and the control group were analyzed statistically. Results: Compared with the negative control group, there was no significant difference in the IL-10、IL-23、IFN-β content in the positive control group, while the IL-1α, IL-1β, IL-6, IL-17A, TNF-α, IFN-γ, MCP-1 and GM-CSF contents were significantly higher than the sham control group（P<0. 01）. In the sample groups, except for the high dose group of MCP-1 and TNF-α, which was significantly higher than the sham operation group（P<0. 01 and P<0. 05, respectively）. The contents of IL-6, IL-10, IL-17A, IL-23, TNF-α, IFN-γ, IFN-β, MCP-1, GM-CSF were not changed significantly（P>0. 05）. Conclusion: The dental restoration membrane of xenogeneic acellular dermal matrix have no significant effect on cytokines, as show at the levels of cytokines measured using CBA method.

Objective: To provide reference for quality control and authenticity identification of Arnebiae Radix medicinal materials and decoction pieces in the market. By studied on the ITS2 sequences’ characters of imported Arnebiae Radix, based on DNA barcoding and PCR-RFLP technologies. Methods: The ITS2 region was selected as the DNA barcode sequence for comparison and identification of imported Arnebiae Radix and reference medicinal materials. The ITS2 sequences of imported Arnebiae Radix from different sources with reference medicinal materials were compared based on DNA barcoding and PCR-RFLP technologies. Results: After the restriction endonucliase AluI enzyme digestion, the agarose-gel electrophoresis results of 39 imported Arnebiae Radix samples showed that, only DH3 had bands at around 500 bp, and none bands between 100 bp and 300 bp. And the results of other imported Arnebiae Radix samples had two or three obvious bands between 100 bp and 300 bp. The ITS2 sequences of imported Arnebiae Radix samples were compared with the reference medicinal materials, among which DH3 had the largest differences of 15 bases compared to the reference medicinal materials, the ITS2 sequence of F2 was same to the reference medicinal materials, and other imported Arnebiae Radix samples had 1-9 bases difference compared to the reference medicinal materials. The clustering results showed that the imported Arnebiae Radix sample DH3 was clearly distinguished from other imported Arnebiae Radix samples and reference medicinal materials which was in a single branch. There were 14 samples, which were clustered together with the reference medicinal materials in one branch with support rate ≥50%. Conclusion: The ITS2 region is selected to compare the similarities and differences of ITS2 sequences between imported Arnebiae Radix samples and reference medicinal materials based on DNA barcode and PCR-RFLP technologies, which provids a reference for effective identification of Arnebiae Radix medicinal materials and decoction pieces, and a strong guarantee for market supervision of Arnebiae Radix medicinal materials.

Objective: To establish an UPLC quantitative analysis method for the simultaneous determination of 13 components in Yankening tablets, including phellodendrine, coptisine, baicalin, palmatine, berberine, wogonoside, baicalein, aloe-emodin, rhein, wogonin, emodin, chrysophanol, physcion. Methods: Agilent Eclipse Plus C₁₈(100 mm×2.1 mm, 1.8 μm) column was used with acetonitrile-0.1% phosphoric acid as mobile phase, and gradient elution at a flow rate of 0.3 mL·min^-1. The detection wavelengths were 210 nm and 254 nm. The column temperature was 40 ℃. Results: Phellodendrine hydrochloride, coptisine hydrochloride, palmatine hydrochloride, berberine hydrochloride, baicalin, wogonoside, baicalein, aloe-emodin, rhein, wogonin, emodin, chrysophanol, physcion showed good linear relationships within their concentration range of 0.97-48.63 μg·mL^-1, 0.95-47.52 μg·mL^-1, 0.86-43.15 μg·mL^-1, 0.86-43.19 μg·mL^-1, 0.89-44.37 μg·mL^-1, 1.00-49.84 μg·mL^-1, 1.02-51.01 μg·mL^-1, 0.97-48.31 μg·mL^-1, 0.99-49.50 μg·mL^-1, 1.04-51.80 μg·mL^-1, 1.00-50.04 μg·mL^-1, 1.00-49.80 μg·mL^-1, 1.01-50.64 μg·mL^-1. The average recoveries(n=6) were 95.2%, 96.7%, 95.9%, 98.3%, 94.1%,97.6%, 99.2%, 96.6%, 95.5%, 97.2%, 97.0%, 97.8%, 98.7%, RSD values were all less than 2.0%. The contents of the 13 chemical components in 3 batches of samples were 1.367-1.488 mg·g^-1(calculated as phellodendrine hydrochloride), 0.378-0.412 mg·g^-1(calculated as coptisine hydrochloride), 4.611-5.505 mg·g^-1, 0.324-0.407 mg·g^-1(calculated as palmatine hydrochloride), 3.665-3.878 mg·g^-1(calculated as berberine hydrochloride), 1.107-1.682 mg·g^-1, 0.392-0.941 mg·g^-1, 0.076-0.105 mg·g^-1, 0.097-0.116 mg·g^-1, 1.059-1.213 mg·g^-1, 0.149-0.167 mg·g^-1, 0.213-0.239 mg·g^-1, 0.047-0.059 mg·g^-1. Conclusion: The method is accurate, high analysis efficiency, good repeatability, it can be used to control the quality of Yankening tablets.

Objective: To optimize the content determination method of morphine sulfate suppositories, which had high toxicity of extraction solvent and poor specificity and generality of chromatographic conditions. Methods: The optimization of the preparation method for the test solution was to prepare the test solution by ice bath freezing and n-heptane extraction, and to compare the content determination results of the test solution prepared by the United States Pharmacopeia method (extracted with trichloromethane). The optimization of chromatographic conditions for content determination was based on the comprehensive comparison of United States Pharmacopeia, British Pharmacopeia and YBH specification. Symmetry C₁₈ column (150 mm×4.6 mm, 5 μm) was finally used. The mobile phase was 0.01 mol·L^-1 potassium dihydrogen phosphate solution containing 0.202% sodium heptane sulfonate (containing 0.1% triethylamine, adjusted to pH 2.5 with phosphoric acid) -methanol (70∶30). The flow rate was 1.0 mL·min^-1. The column temperature was 30 ℃. The detection wavelength was 284 nm. The injection volume was 20 μL. Results: The content measured by ice bath freezing method was relatively low, while the content measured by n-heptane extraction and trichloromethane extraction was basically consistent, indicating that n-heptane could be used as extraction solvent instead of halogenated alkane. The linear range of morphine sulfate was 2.12-105.94 μg·mL^-1(r=0.999 9), and the LOQ was 10.60 ng. The RSD values of system precision, repeatability, intermediate precision and stability were all less than 2%. The average recoveries were 98.6%-99.6% and RSD was 0.21%-0.66%. After forced degradation, the main peak of the tested solution could be separated from the degraded impurities, with good specificity. By changing the flow rate, column temperature, pH value, mobile phase ratio and column brand, the main peak could be well separated from each impurity peak, with good robustness. Conclusions: This study optimized the determination method for morphine sulfate suppositories. The preparation of the test solution used n-heptane instead of the highly toxic haloalkane, and meanwhile improved the chromatographic conditions, enhanced the environmental protection, specificity and applicability of the method. It can be used for the content determination of morphine sulfate suppositories.

Objective: To investigate the effect of allogeneic bone on viability and proliferation of human peripheral blood lymphocyte by CCK-8 method and CFSE method. Methods: The lymphocyte viability was detected by CCK-8 method after co-culture of human fresh peripheral blood lymphocytes and the sample extraction (decalcified bone matrix and freeze-dried tissue engineered bone). Also, the lymphocyte proliferation was determined by flow cytometry after co-culture of fresh peripheral blood lymphocytes labeled with CFSE and the sample extraction. Results: The result of CCK-8 method showed that there was no significant difference in absorbance value between decalcified bone matrix and medium control. But there was a significant increase of absorbance value in freeze-dried tissue engineered bone compared with that in medium control (P<0.05), suggesting that freeze- dried tissue engineered bone improved lymphocyte viability. The result of CFSE method showed that there was no significant difference in Dil, PF, EI and RI of lymphocyte, and Dil of lymphocyte subtype between freeze-dried tissue engineering bone and medium control (P>0.05). The results suggested that freeze-dried tissue engineered bone had no significant effect on the proliferation of human peripheral blood lymphocyte. Conclusion: Compared with medium control, decalcified bone matrix has no significant effect on human peripheral blood lymphocyte viability. Freeze-dried tissue engineered bone improves the human peripheral blood lymphocyte viability, but dose not significantly affect the proliferation of human peripheral blood lymphocyte. CCK-8 method mainly reflects the cell viability, but CFSE method could reflect the cell proliferation more reasonable.

Objective: To determine the color of Arnebiae Radix, and the contents of six main purple pigment components (acetylshikonin, β-acetoxyisovalerylalkannin, deoxyshikonin, isobutylshikonin, β, β’-dimethylacrylalkannin and isovalerylshikonin) in Arnebiae Radix, and to study on the correlation between the color of Arnebiae Radix and the contents of six main purple pigment components. Methods: The L, a, and b values of the sample powder were determined using a spectrophotometer to characterize the color of Arnebiae Radix. The International Commission on Illumination (CIE) had developed a Lab color model, which was a digital description of human vision. A higher L value indicated greater brightness, a higher a value indicated redness and a lower a value indicates greenness, and a higher b value indicated yellowing and a lower b value indicates blueness. The contents of purple pigment components were determined using high performance liquid chromatography (HPLC), and the correlation between L, a, and b values and the contents of six main purple pigments was calculated using SPSS software. Results: The contents of acetylshikonin in 135 batches of samples ranged from 0.01% to 3.39%. The contents of β-acetoxyisovalerylalkannin ranged from 0.00% to 1.95%. The contents of deoxyshikonin ranged from 0.00% to 0.23%. The contents of isobutylshikonin ranged from 0.01% to 1.13%. And the contents of isovalerylshikonin ranged from 0.02% to 2.88%. The contents of β, β’-dimethylacrylalkannin ranged from 0.01% to 2.17%. There was a significant negative correlation between the contents of acetylshikonin, β-acetoxyisovalerylalkannin and isobutylshikonin and the L (black_white) chromaticity value of Arnebiae Radix, with a Spearman correlation coefficients between -0.138 and -0.222. The chromaticity value of a (red_green) was related to the five components other than acetylshikonin, which were β-acetoxyisovalerylalkannin, deoxyshikonin, isobutylshikonin, β, β’-dimethylacrylalkannin, and isovalerylshikonin, with a spearman correlation coefficients between 0.176 and 0.355; b (blue_yellow) chromaticity value was related to the five components other than β-acetoxyisovalerylalkannin, which were acetylshikonin, deoxyshikonin, and isobutylshikonin, β, β’-dimethylacrylalkannin. β, β’-dimethylacrylalkannin was positively correlated with a coefficient of 0.290, and negatively correlated with the other four components with a coefficients between -0.325 and -0.633. Conclusion: It is recommended that the assay limits of Arnebiae Radix be revised to β, β’-dimethylacrylalkannin not less than 0.30% and isovalerylshikonin not less than 0.29%.

Objective: To establish a UPLC-MS/MS method for the simultaneous determination of 47 pesticide residues in Sucrose. Methods: The residues in the sample were extracted by acidic acetonitrile, concentrated by blow dry with nitrogen gas and then analyzed by using LC-MS/MS with multiple reaction monitoring(MRM). Blank matrix standard curve with internal standard method was used to determine the residue contents. Results: Each substance to be tested had good linearity relationship within certain concentration (r>0.995). The limits of quantification (LOQ) were 0.01-2.00 μg·kg^-1. The recoveries of 47 pesticide residues at three levels of 10, 25 and 50 μg·kg^-1 were from 71.0% to 106.0% with the RSD of 0.90%-8.5% in sucrose. 2 batches in 15 batches of sucrose samples were detected thiamethoxam and thiamethoxam, while the rest were not detected. Conclusion: The method is simple, rapid and characterized with acceptable sensitivity and accuracy to meet the requirements of the determination of analysis, and this developed procedure is suitable for the determination of pesticide residues in Sucrose, can be used for quality control of sucrose.

Objective: To establish a dual-wavelength method for the determination of amylose and amylopectin in potato starch in Tibet. Methods: The determination wavelengths of amylopectin and amylopectin in Tibetan potato were 623 and 557 nm, respectively, and the reference wavelengths were 498 and 729 nm. The contents in 11 batches of starch samples collected were determined and analyzed. Results: The results showed that the mass concentration of amylose and amylopectin showed a good linear relationship (r=0.999 9, r=0.999 8) in the range of 0- 66 μg·mL^-1 and 0-90 μg·mL^-1, respectively. The average recoveries of amylose and amylopectin were 102.8% (RSD=1.8%) and 98.5% (RSD=2.2%). The precision (RSD=0.071%, RSD=0.31%), repeatability (RSD=0.26%, RSD=2.4%) and stability (RSD=0.14%, RSD=1.4%) were all in compliance with regulations. The contents of amylose and amylopectin in Tibetan potato starch were 36.74% and 45.87%, respectively. Compared with the commercially available potato starch, the amylose content of Tibetan potato starch was significantly increased (P<0.001), while the amylopectin content was significantly decreased (P<0.05). Conclusion: The method is proved to be suitable for the quality evaluation of potato starch in Tibet. There are significant differences in the contents of amylopectin and amylopectin between potato starch in Tibet and potato starch in the market, which provides an experimental basis for the establishment of quality standards of potato starch in Tibet and the development of high-value products.

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

Liposome is a drug delivery system which has been successfully transformed for clinical application. In the past few decades, liposome as a drug delivery system has been extensively and deeply studied in the field of therapy. A variety of liposome drugs have been marketed at home and abroad, but there are few reports on the systematic quality detection and evaluation methods. In this paper, the preparation technology and systematic quality evaluation methods of liposome drugs were reviewed based on domestic and foreign literature and relevant technical guidelines, so as to provide reference for the development and evaluation of liposome drug delivery technology.

Objective: To establish a method for identifying Fritillariae Cirrhosae Bulbus and its adulterant Fritillariae Ussuriensis Bulbus. Methods: 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. Results: 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. Conclusion: The method had good specificity and sensitivity, and could be used for the detection of adulterated Fritillariae Cirrhosae Bulbus with Fritillariae Ussuriensis Bulbus.

Objective: To determine the contents of Cd and As in Isatidis Radix and to explore the application of target-organ toxicity dose(TTD) modification of hazard index (HI) method in assessing the risk of combined exposure to heavy metals and harmful elements in traditional Chinese medicines (TCMs). Methods: According to the monitoring data of Cd and As in Isatidis Radix, the exposure doses were calculated. The health risk caused by combined exposure to Cd and As was preliminary screened by HI method. Moreover, a more accurate TTD method was used. Results: The qualified rates of Cd in 29 batches of Isatidis Radix were 100%. However, 5 batches of As contents exceeded the limit standard. The HQ values of As in 5 batches of Isatidis Radix were more than 1. The results of TTD method showed that for the end points of cardiovascular system, blood, nervous system, the HI values of 5 batches of Isatidis Radix were more than 1, and the health risk was not acceptable. Conclusion: The cumulative risk assessment of heavy metals in TCMs is proposed in this study, which offers novel ideas for the development of risk assessment methods of exogenous harmful residues in TCMs, and provides technical support for formulating scientific limit standards.