To develop an HPLC method for the determination of related substances and assay of levamisole hydrochloride tablets.

The chromatographic separation of related substances was performed on a Thermo Hypsil C₁₈ column(100 mm×4.6 mm, 3 μm). A gradient elution was applied with a mobile phase composed of 0.75% monobasic ammonium phosphate solution(adjusted at pH 6.5 with diiopropylamine) and acetonitrile. The assay analytical column was packed with IntertSustain C₁₈ column(150 mm×4.6 mm, 5 μm). The mobile phase was 70 volumes of 0.75% monobasic ammonium phosphate solution(adjusted at pH 7.0 with diiopropylamine) with 30 volumes of acetonitrile. The wavelength detection was set at 215 nm, the injection volumn was 10 μL, and the flow rate was 1.0 mL·min^-1.

Levamisole and its impurities were separated well by related substances HPLC method above. Impurities A, B, C, D, E showed the good linearities in the concentration ranges of 10.87-25.37 μg·mL^-1, 11.62-27.10 μg·mL^-1, 12.38-28.90 μg·mL^-1, 30.89-72.07 μg·mL^-1, 12.41-28.95 μg·mL^-1 (r>0.999). The average correction factors of impurities A, B, C, D, E determined by three columns and liquid chromatographies were 1.6, 1.4, 2.6, 1.2, 2.4, respectively, the recovery rates(n=9) were 98.1%, 99.0%, 98.6%, 100.1%, 99.9% and the RSDs were 0.63%, 0.79%, 0.92%, 0.96%, 0.33%. The separation of levamisole and impurity C was good by assay HPLC method. Levamisole showed the good linearity in the concentration range of 0.10 14-0.304 3 mg·mL^-1 (r=0.999 9). The average recovery rate(n=9) of levamisole was 100.2%, RSD=0.52%. Ten batches levamisole hydrochloride tablets from five domestic pharmaceutical enterprises were determined by the above related substances and assay HPLC method, the total impurities mass were 0.05%-0.62%, and the assays were 99.5%-104.3%.

The established method is high selection and accurate. It is suitable applied for determination of related substances and assay of levamiole hydrochloride tablets.

To establish a method for quality evaluation of Artemisiae Scopariae Herba [Artemisia capillaris Thunb. (Mianyinchen)] dispensing granules by combining characteristic chromatogram, quantitative analysis of multi-components by single marker (QAMS) and chemical pattern recognition analysis.

The high performance liquid chromatography (HPLC) characteristic chromatogram was established by 15 batches of Artemisiae Scopariae Herba [Artemisia capillaris Thunb. (Mianyinchen)] standard decoctions and 3 batches of dispensing granules. The contents of 6 components were determined by QAMS. The chromatographic separation was achieved on a Acclaim^TM RSLC 120 C₁₈ column (100 mm×2.1 mm, 2.2 μm), with the mobile phase comprising of acetonitrile -0.05% phosphoric acid flowing at 0.4 mL·min^-1 in a gradient elution manner. And the detection wavelength was set at 327 nm. The similarity evaluation system of fingerprint of traditional Chinese medicine was used to determine the common peak for similarity evaluation. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were applied for chemical pattern recognition. The transfer rates of the 6 components from decoction pieces to standard decoctions and dispensing granules were calculated.

The similarities of characteristic chromatograms of 15 batches of Artemisiae Scopariae Herba [Artemisia capillaris Thunb. (Mianyinchen)] standard decoctions and 3 batches of dispensing granules were all above 0.85. And 8 common characteristic peaks were identified. The results of HCA and PCA indicated the similarity of ingredients in formula granules to those in standard decoctions. The contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C in standard decoctions were 1.87-5.23, 7.44-15.26, 2.85-8.18, 3.05-6.14, 0.99-3.93 and 3.23-10.38 mg·g^-1 and the transfer rates of these components from decoction pieces to standard decoction were 23.85%-37.28%, 19.57%-31.93%, 28.15%-45.88%, 22.34%-36.59%, 16.64%-28.36% and 21.81%-39.19%, respectively. The contents and transfer rates of these 6 compounds in dispensing granules were close to that of standard decoctions.

The characteristic chromatogram and QAMS method established can be used for quality control and process research of Artemisiae Scopariae Herba [Artemisia capillaris Thunb. (Mianyinchen)] dispensing granules.

To investigate the metabolites of Periploca forrestii in plasma, urine, and feces of normal and adjuvant arthritis（AA） rats, and explore the effect of rheumatoid arthritis（RA） on the metabolism of active components of P. forrestii.

AA rat model was made by means of Freund’s complete adjuvant. Plasma, urine and feces of normal and AA rats were analyzed by UPLC-Q-TOF-MS^E method using ACQUITY UPLC BEH C₁₈ (100 mm×2.1 mm, 1.7 μm) column with 0.01% formic acid water -0.01% formic acid acetonitrile as mobile phase gradient elution at a flow rate of 0.25 mL·min^-1, and electrospray ion source under negative ion mode.

Two and three prototype components, 32 and 35 metabolites were detected in normal rats and AA model rats. The metabolic pathways mainly include monocaffeoylquinic acid reduction, methylation, ring-opening cleavage, glucuronidation, dicaffeoylquinic acid reduction, methylation, acetylation, isomerization, sulfation glucuronidation, etc.

The metabolites in plasma and urine of AA model rats are more diverse than those in normal rats, and the disease state of RA may affect the metabolic pathway of effective components of P. forrestii in the body.

To determine seven impurities in oxytocin for injection and investigate the limit values.

HPLC and principal component self-control with correction factor were adopted. The determination was performed on a Waters Xbridge C₁₈ column(150 mm×4.6 mm, 5 μm). The mobile phase consisted of 0.1 mol·L^-1 dihydrogen phosphate solution (adjusted to pH 5.4)-acetonitrile (90∶10, phase A), and acetonitrile (phase B) with gradient elution at a flow rate of 1.5 mL·min^-1. The column temperature was maintained at 32 ℃, and the detection wavelength was set at 220 nm. The injection volume was 100 μL. The linear equations of oxytocin, impurities Ac-Oxy, Oxy[Glu⁴], Oxy[+Gly¹⁰], Oxy[-NH₂], Oxy[trisulfide], Oxy[cis-dimer] and Oxy[trans-dimer] were drawn. The correction factors of each impurity related to oxytocin were calculated by slope. The contents of impurities in 3 batches of oxytocin for injection were determined and compared with the results of impurity reference method.

The limits of quantification for seven impurities were 2.75-5.66 ng, while the detection limits were 1.38-2.83 ng. The linear ranges of seven impurities were 0.03-3.40 μg·mL^-1 with good linearity(r>0.999). The correction factors of Ac-Oxy, Oxy[Glu⁴] and Oxy[-NH₂] were 1.1, while the correction factors of Oxy[+Gly¹⁰] and Oxy[trisulfide] were 1.2 and 0.9, respectively. The correction factors of Oxy[cis-dimer] and Oxy[trans-dimer] were both 1.3. The seven impurities were determined in 3 batches of samples by principal component self-control with correction factor. The contents of impurity Ac-Oxy were 0.96%, 0.93% and 1.01%, respectively. The contents of impurity Oxy[Glu⁴] were 0.07%, 0.06% and 0.08%, respectively. The contents of impurity Oxy[+Gly¹⁰] were 0.07%, 0.04% and 0.04%, respectively. The contents of impurity Oxy[-NH₂] were 0.09%, 0.05% and 0.07%, respectively. The contents of impurity Oxy[trans-dimer] were 0.27%, 0.18% and 0.22%, respectively. The maximum single impurity contents were 0.18%-0.19%, while the total impurity contents were 1.88%-2.06%. Compared the results measured by principal component self-control with correct factor method and the impurity reference method, there was no significant difference between two methods (p>0.05).

The method is proved to be simple, repeatable and accurate for the content determination of related substances in oxytocin for injection.

To study and establish a method based on gas chromatography and chemometrics techniques for distinguishing Artemisiae Argyi Folium and its adulterants Artemisiae Mongolica Folium.

Gas chromatography method was established with Agilent HP-5 19091J (30 m×0.32 mm, 0.25 μm) as chromatographic column, and hydrogen flame ion detector (FID) as detector. After the chemical composition of 21 chromatographic peaks in the chromatogram were identified, and the peak area data of the 21 chromatographic peaks in 29 batches of samples were determined. Similarity analysis, correlation analysis, cluster analysis, principal component analysis and orthogonal partial least squares-discriminant analysis were applied to analyze the chromatographic data.

The results of chemometric analysis indicated that tpeak 20 (chamazulene), peak 3 (1, 8-cineole) and peak 19((1S, 8aα)-decahydro-1, 4aβ-dimethyl-7β-isopropenyl-1-naphthol) were the differential characteristic chromatographic peaks between Artemisiae Argyi Folium and its adulterants Artemisiae Mongolica Folium. The ratios of the peak areas of peak 3 to peak 20 were in the ranges of 54.50-348.39 and 0.16-0.87 respectively, and the ratios of the peak areas of peak 19 to peak 20 were in the ranges of 18.55-128.46 and 0.01-0.14 respectively. These significant differences could be used for the identification of Artemisiae Argyi Folium and its adulterant Artemisiae Mongolica Folium.

The research findings can be used for the identification of Artemisiae Argyi Folium and its adulterant Artemisiae Mongolica Folium, and these have certain reference significance for the research and analysis of Artemisiae Argyi Folium and related drugs.

To evaluate the chemical composition difference of Cassia angustifolia Vahl leaves and Cassia auriculata L. leaves by high-resolution mass spectrometry and omics analysis, so as to establish the detection method of Cassia auriculata L. leaves adulterated in Cassia angustifolia Vahl leaves.

The positive and negative MS^E data of 13 batches of Cassia angustifolia Vahl leaves and 9 batches of Cassia auriculata L. leaves were collected by ultra performance liquid chromatography coupled with electrospray ionization quadrupole time of flight mass spectrometry(UPLC-Q TOF MS). The mobile phase was acetonitrile(A)-1% acetic acid(B). The column temperature was 30 ℃. The flow rate was 0.3 mL·min^-1. Injection volume was 1 μL. The mass range was m/z 50-1 200. The chemical composition difference of Cassia angustifolia Vahl leaves and Cassia auriculata L. leaves were processed by the omics analysis QI software based on the orthogonal partial least-squares discrimination analysis(OPLS-DA) after the negative MS^E data were obtained. One out of seven specific components from Cassia auriculata L. leaves was separated and identified. A method with the specific component as the reference substance for the detection of adulterated Cassia auriculata L. leaves in Cassia angustifolia Vahl leaves was established by UPLC, and it was used in 27 batches of Cassia angustifolia Vahl leaves samples and 3 batches of laboratory-made positive samples.

Cassia angustifolia Vahl leaves and Cassia auriculata L. leaves were significantly different from each other. The specific component separated from Cassia auriculata L. leaves was identified as kaempferol 3-O-(2”-O-apiofuranosyl) rutinoside. In the method for the detection of adulterated Cassia auriculata L. leaves in Cassia angustifolia Vahl leaves by UPLC, the precision(RSD=1.3%), repeatability(RSD=1.3%) and stability(RSD=0.58%) met the requirements. No kaempferol 3-O-(2”-O-apiofuranosyl) rutinoside was detected in 23 out of 27 batches of Cassia angustifolia Vahl leaves samples, but it was detected in 4 batches of Cassia angustifolia Vahl leaves samples and 3 batches of positive samples made in the laboratory.

In this study, the difference of Cassia angustifolia Vahl leaves and Cassia auriculata L. leaves is distinguished clearly based on the technology of UPLC-Q TOF MS and OPLS-DA. The specific component of Cassia auriculata L. leaves is separated and identified. A method for the detection of adulterated Cassia auriculata L. leaves in Cassia angustifolia Vahl leaves is established by UPLC, and it provides the basis for quality control and quality standard improvement of Cassia angustifolia Vahl leaves. The technology is helpful to solve the problem of adulteration identification of traditional Chinese medicine.

To establish a method for the rapid identification of the authenticity of Gastrodiae Rhizoma herbs based on enzymatic recombinase amplification (ERA) technique.

Following the principle of ERA primer design, Oligo 7.0 software was applied to screen and optimize the ERA-specific primers of Tianma based on the ITS2 genome sequences of Gastrodia elata and its common artifacts. Primer Premier 5.0 software was applied to design the specific PCR primers for the identification of Gastrodia elata, and through the optimization of ERA and PCR reaction system, the optimal reaction time for ERA was finally determined to be 17 min, the optimal reaction temperature was 40 ℃, the optimal annealing temperature for PCR was 57 ℃, and the cycle was 32 times, and the established method was verified for sensitivity and specificity, and the samples of asparagus available in the traditional Chinese medicine The sensitivity and specificity of the established method were verified, and the commercially available asparagus samples in the market were selected for testing.

The designed primers for the specific identification of ERA in Gastrodia elata did not cross-react with its common forgeries, and the specificity was good, a repeatability results showed that the three repeatability tests were consistent, with no false positives or false negatives；the sensitivity of this method for Gastrodia elata genomic DNA was 1 pg·μL^-1, which was higher than that of conventional PCR；the ERA technique can be used for the rapid identification of commercially available samples of Gastrodia elata and the results of the assay are the same as those of the PCR method.

The established detection method is simple, rapid, with high specificity and sensitivity, and provides a new means for the authentication of the Gastrodiae Rhizoma.

To establish an UPLC-MS/MS method for simultaneous determination of 11 components(caffeic acid, afzelin, gallic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, salidroside, eleutheroside B, ginsenoside Re, ginsenoside Rg₁, ginsenoside Rd) in Hongwushen capsules.

A Shim-pack GIST C₁₈ chromatographic column(100 mm×2.1 mm, 2 μm) was used. The mobile phase was composed of acetonitrile -0.1% acetic acid -1 mmol·L^-1 ammonium acetate aqueous solution and the gradient elution was applied. The flow rate was 0.3 mL·min^-1, the column temperature was 30 ℃, and the injection volume was 1 μL. Electrospray ionization (ESI) source and multiple reaction monitoring (MRM) mode in both positive and negative were used for ion detections.

The linear relationship of 11 components was good in the concentration range, and the linear correlation coefficients were all above 0.999 0. The RSD values of precision were less than 3%. The repeatability and stability were good, and the RSD values were less than 5%. The average recoveries were in the range of 97.1%-101.5% with RSDs≤3.7%. The contents of caffeic acid, afzelin, gallic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, salidroside, eleutheroside B, ginsenoside Re, ginsenoside Rg₁ and ginsenoside Rd in 10 batches of Hongwushen capsules were 0.010-0.013 mg·g^-1, 0.000 7-0.001 4 mg·g^-1, 0.035-0.038 mg·g^-1, 0.312-0.315 mg·g^-1, 0.413-0.417 mg·g^-1, 0.411-0.416 mg·g^-1, 4.355-4.358 mg·g^-1, 0.030-0.032 mg·g^-1, 0.993-0.999 mg·g^-1, 1.120-1.124 mg·g^-1 and 2.536-2.538 mg·g^-1, respectively.

The method is accurate, sensitive, stable and reproducible, and can be used for the quality control of Hongwushen capsules.

To establish the first national standard for oncolytic activity assay of herpes simplex virus type 1(HSV-1).

According to the requirements in Chinese Pharmacopoeia(Volume Ⅲ, 2020 edition), the liquid and freeze-dried standard for oncolytic activity of HSV-1 were prepared and tested, of which the stability were evaluated by thermal acceleration test. The oncolytic activity of the standard was calibrated collaboratively by U-2 OS cells/CCK-8 method in 3 laboratories. The liquid standard was compared with the freeze-dried standard, and the more suitable one was selected as the national standard.

The prepared standard substance was all qualified, among which the moisture content of the freeze-dried standard was 1.09% and the dispensing accuracy was 0.15%. The results of stability test were calculated by Arrhenius formula. It was preliminarily predicted that it would take 7.7 years for the oncolytic activity of liquid standard to decrease by 10% at -70 ℃, and it would take 6.1×10⁵ years for the oncolytic activity of lyophilized standard to decrease by 10% at -70 ℃. Compared with liquid standard, the stability of lyophilized standard was greatly improved. Twenty-one times of collaborative calibration tests by 3 laboratories showed that the oncolytic activity liquid standard was 7.08×10⁴ U·mL^-1 and the oncolytic activity liquid standard was 1.82×10⁴ U·vial^-1. After lyophilized, the oncolytic activity of the bulk of HSV-1 standard decreased from 7.08×10⁴ U·mL^-1 to 3.03×10⁴ U·mL^-1. However, the good S-shaped dose-response curve still appeared after 100 times of pre-dilution, which did not affect the requirements of its use as a standard. When the liquid standard was used as the sample and the freeze-dried standard was used as the standard for calibration, the geometric coefficient of variation (GCV) of the results of the 3 laboratories decreased from 64.4% to 29.2%, and the precision of the experiment was greatly improved.

The batch of freeze-dried HSV-1 standards for oncolytic activity assay meets the relavant requirements, and is more suitable for use as a national standard than liquid standards. Its oncolytic activity is assigned a value of 1.82×10⁴ U·vial^-1.