To establish a quality evaluation method of Erdi Xiaoke mixture based on HPLC fingerprint, multi-component determination and chemometric analysis.

The separation was performed on a 30 ℃ thermostatic Acutfex PW-C₁₈ (250 mm×4.6 mm, 5 μm) column, with the mobile phase comprising of acetonitrile -0.1% phosphoric acid flowing at 1.0 mL·min^-1 in a gradient elution manner, and the detection wavelength was set at 285 nm to establish HPLC fingerprint of Erdi Xiaoke mixture. Contents of 11 components [5-hydroxymethyl furfural(5-HMF), salvianic acid A, chlorogenic acid, caffeic acid, calycosin-7-glucoside, acteoside, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid, salvianolic acid B and formononetin] in 12 batches of Erdi Xiaoke mixture were determined, and similarity evaluation, cluster analysis, principal component analysis and orthogonal partial least squares discriminant analysis were used to comprehensively evaluate the quality of Erdi Xiaoke mixture.

The 11 constituents showed good linear relationships within their respective ranges(r≥0.999 6), with average recovery rates of 95.3%-101.5%, and the RSDs were in the range of 1.1%-2.3%. There were 18 common peaks in the fingerprints for 12 batches of samples with the similarities above 0.960. The range of the content of the above 11 components in the 12 batches of samples were as follows as 43.40-65.70, 219.43-274.70, 119.00-164.78, 11.87-18.17, 42.80-56.86, 10.01-20.71, 8.21-14.73, 40.15-57.39, 43.29-60.29, 755.22-1 208.50, 2.47-4.44 μg·mL^-1. And 12 batches of Erdi Xiaoke mixture could be clustered into three categories with some differences in sample quality among different batches. In addition, 5-HMF, isochlorogenic acid B and chlorogenic acid were the main potential markers affecting the quality of Erdi Xiaoke mixture.

This method is stable, reliable, accurate and sensitive, and can be used to control and comprehensively evaluate the quality of Erdi Xiaoke mixture.

To establish an HPLC-CAD method for the determination of related substances in peramivir injection and to analyze the impurity profile.

Welch Ultimate Polar RP column (250 mm×4.6 mm, 3.5 μm) was used with 5 mmol·L^-1 ammonium formate solution (pH adjusted to 4.6 with formic acid) -acetonitrile (95∶5) as mobile phase A and mobile phase A-acetonitrile (50∶50) as mobile phase B under gradient elution at the flow rate of 0.8 mL·min^-1. Column temperature was 30 ℃. CAD atomizer temperature was 50 ℃, acquisition frequency was 5 Hz and filter was 3.6 s. The degradation pathway and impurity structure of peramivir were estimated by LC-MS/MS.

The separation between peramivir and the impurities was good. The mass concentration of peramivir and 7 impurities showed good linear relationships with the peak area in the ranges of 0.2-12 μg·mL^-1. The limits of quantitation were 4.32-12.8 ng, and the limits of detection were 2.16-6.44 ng. The average recovery (n=9) of impurity Ⅲ was 96.2%, RSD was 1.8%. The control solution and the test product solution were stable for 102 hours at room temperature. After fine-tuning the parameters of liquid chromatography, there was no influence on the detection results of related substances. Based on LC-MS/MS, the structure and possible degradation pathway of the degraded related substances were deduced.

This method is accurate, reliable and specific for the determination of related substances in peramivir injection, and provides a basis for the establishment of quality standards for this variety.

To establish the HPLC fingerprint and multi-component quantitative analysis of Hanchuang Zupa granules, and evaluate the quality of multiple batches of Hanchuang Zupa granules by chemical pattern recognition technology.

The sample pretreatment conditions and chromatographic analysis conditions of Hanchuang Zupa granules were optimized, and the optimal HPLC fingerprint and multi-component quantitative analysis method were established as follows: stationary phase was YMC-Pack ODS-A column (250 mm×4.6 mm, 5 μm, 12 nm) was adopted, and the mobile phase was acetonitrile-water (containing 0.1% phosphoric acid) with gradient elution, the detection wavelength was 220 nm, the column temperature was 30 ℃, the flow rate was 1.0 mL·min^-1. Hierarchical cluster analysis (HCA), principal components analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were applied to evaluate the quality of 17 batches of Hanchuang Zupa granules.

Methodological investigation of HPLC fingerprint and content determination were well verified and met the analysis requirements. A total of 25 common peaks were obtained by full peak matching, and eight of them were identified by comparing with the retention time of mixed reference substances. The similarities of 17 batches of samples were above 0.90, which showed good consistency and stability between the samples. Seventeen samples could be classified into three clusters. Three principal components from 21 common peaks were extracted by PCA. Six quality differential compounds were presented in the fingerprints by OPLS-DA, including rutoside, gallic acid, ammonium glycyrrhizinate, chlorogenic acid and so on. The resolution and linear relationship of eight components in quantitative analysis were good. The average recovery rates were 98.0%-99.1% with RSD≤2.0%.

In this study, the qualitative analysis of HPLC fingerprint and quantitative analysis of multiple index components is specific, simple and accurate, which can provide a reference for the quality control and quality evaluation of Hanchuang Zupa granules.

To develop a rapid, specific and sensitive UPLC-MS/MS method for the determination of vonoprazan in human plasma and its application in a bioequivalence study of two types of tablets.

A single dose, two-cycle, two products, and self-cross controlled trial design on bioequivalence was used. Plasma samples were collected from healthy human volunteers at different time points after oral administration with the test or reference product of 20 mg fumarate vonoprazan tablets under both fasting and fed conditions, respectively. The plasma samples were treated by acetonitrile protein precipitation and then analyzed by UPLC-MS/MS. Chromatographic separation of vonoprazan was achieved using a Waters ACQUITY UPLC® BEH C₁₈（50 mm × 2.1 mm, 1.7 μm）column at 40 ℃. The mobile phase consisted of water (containing 0.1% formic acid) for eluent A and acetonitrile (containing 0.1% formic acid) for eluent B under a gradient elution. An electrospray ionization (ESI) with multiple reaction monitoring (MRM) mode was used to monitor the precursor-product ion transitions of m/z 346.1→315.4 for vonoprazan and m/z 350.1→316.2 for vonoprazan-d₄.

The rang of linearity was 0.30-50.00 ng·mL^-1（r>0.998 9）, and the LLOQ was 0.30 ng·mL^-1. Intra- and inter-day precision values were within 5.7%, and intra- and inter-day accuracy values were ranged from -2.15% to 0.82%. Recovery, specificity, matrix effect and stability met the guiding principles. This method has been successfully applied to study the bioequivalence of vonoprazan fumarate tablets. The C_max of the test product in postprandial and fasting tests were (29.08±11.59) ng·mL^-1 and (26.87±8.14) ng·mL^-1, respectively, and the AUC_0-t was (258.90±87.71) h·ng·mL^-1 and (223.08±43.27) h·ng·mL^-1, respectively. The C_max of the reference product in postprandial and fasting tests were (28.73±10.25) ng·mL^-1 and (26.93±8.09) ng·mL^-1, respectively, and the AUC_0-t was (250.33±73.13) h·ng·mL^-1 and (227.56±46.26) h·ng·mL^-1, respectively. In the postprandial trial, the 90% CIs for the geometric mean ratios of C_max, AUC_0-t and AUC_0-∞ of the test and reference products were 88.64%-112.28%, 96.1%-108.2% and 96.6%-108.7%, respectively. And in the fasting trail, the 90% CIs for the geometric mean ratios of C_max, AUC_0-t and AUC_0-∞ of the test and reference products were 94.01%-106.23%, 94.71%-102.03% and 95.18%-102.47%, respectively.

This validated method has the advantages of simplicity, rapid, and high sensitivity. Test vonoprazan fumarate tablets are bioequivalent to the reference product.

To investigate the metabolites and metabolic pathways of WJ-14, a novel anti-depressant compound, in rats and human liver microsomes by HPLC-MS/MS.

Samples of rat plasma and urine, as well as in vitro incubation system of human liver microsomes were analyzed by HPLC-MS/MS. Chromatographic conditions: the chromatographic conditions were as follows: Hypersil BDS C₁₈ (100 mm×4.6 mm, 3 μm) column, with 0.1% formic acid aqueous solution (A)-methanol (B) as the mobile phase, gradient elution flow rate 0.4 mL·min^-1, column temperature 40 ℃, and injection volume 5 μL. The identification of metabolites and speculation on the metabolic pathways of WJ-14 were conducted by comparing the fragmentation mode and characteristic fragment ions of WJ-14.

A total of 12 metabolites of WJ-14 were identified, including 6 in rat and 6 in human liver microsomes, with two metabolites were detected in both rat and human liver microsomes. The major metabolic pathways of WJ-14 were identified as hydroxylation, demethylation, hydration, glucuronidation and methylation.

In this work, we investigated the metabolites and metabolic pathways of WJ-14 using HPLC-MS/MS for the first time. The metabolism of WJ-14 occurred in phase Ⅰ and phase Ⅱ metabolic reactions, in which hydroxylation in phase Ⅰ is dominant. The results of the current study may provide valuable information for the formulation development and application of WJ-14.

To establish a quantitative analysis of multi-components by single marker (QAMS) method to simultaneously determine the contents of kalangin, diphenylheptane A, galangin, kaempferin and galangin-3-O-methyl ether.

Hypersil ODS2 C₁₈ (250 mm×4.6 mm, 5 μm) was used by high performance liquid chromatography (HPLC) and was eluted with 0.3% acetic acid in water (A) and acetonitrile (B) (0-2 min, 10%B→15%B；2-5 min, 15%B→20%B；5-20 min, 20%B→35%B；20-30 min, 35%B→40%B；30-55 min, 40%B；55-60 min, 40%B→100%B), the column temperature was 30 ℃, the volume flow rate was 0.8 mL·min^-1, and the detection wavelength was 254 nm. The relative correction factors of each chemical component were calculated with gingerin as the internal reference, and the content was determined, which was compared with the results of the external standard method, and the quality of galangal from different origins was evaluated by chemical pattern recognition.

Kaempferol, diphenylheptane A, galangin, kaempferin and galangin-3-O-methyl ether had a good linear relationship in the ranges of 0.005 5-0.110 0, 0.140 0-2.800 0, 0.149 6-2.992 0, 0.021 5-0.430 0 and 0.022 2-0.444 0 μg, respectively, and the average sample recoveries were 100.7%, 101.4%, 99.9%, 100.9% and 101.7%, respectively. The RSDs were 1.3%, 2.8%, 0.83%, 1.4% and 1.8%, respectively. Quantitative analysis of multicomponents by single marker method and external standard method was no significant difference in the results.

This method is rapid, accurate and specific, which can provide a reference for the quality control of galangal.

To determine the contents of 28 elements in Xianzhuli under processes of dry distillation and fire preparation by inductively coupled plasma mass spectrometry (ICP-MS), and evaluate the rationality of the current temperature used in the dry distillation process as well as whether the preparation process of commercial samples conforms to the traditional or modern one.

The samples of Xianzhuli were pre-treated with nitric acid before microwave digestion. The contents of 28 elements in 59 batches of Xianzhuli samples were determined by inductively coupled plasma mass spectrometry(ICP-MS), and the methodology was investigated.

The standard curve of 28 elements had a good linear relationship with r≥0.999 2. The detection limits were 0.007 1-1.249 5 ng·mL^-1, the RSDs for precision, repeatability and repeatability tests were 0.30%-3.5%, 0.69%-6.4% and 1.1%-3.3%, respectively and the recovery rates were 88.6%-105.5% with RSDs ranged from 1.0% to 3.2%. The contents of Na, Mg, K, Ca, Mn, Fe, Zn and Rb in 59 batches of Xianzhuli were high, and the contents of heavy metal elements did not exceed the limit requirements. The results of cluster analysis showed that the samples prepared by dry distillation and fire preparation were clustered into one class. By principal component analysis, 7 principal components were obtained, and the cumulative variance rate was 75.6%. Mg, K, Ca, Cr, Fe, Co, Cu, Rb, Cd, Ba, Tl and Pb were identified as the characteristic elements of Xianzhuli.

Based on the contents of beneficial and harmful elements, it is found that the contents of toxic and harmful elements in the sample processed with 160 ℃ dry distillation are lower than processed with fire preparation. And the contents of beneficial elements are similar in two kinds of samples. This temperature is consistent with the dry distillation process temperature approved by provincial drug regulatory authorities for currently certified enterprises. Most of the commercially available samples meet the requirements of traditional or modern processes, and a few may have quality differences due to different processes. The determination and analysis of heavy metals and other elements in Xianzhuli under different processes can provided scientific basis for safe production and rational use of Xianzhuli.

To identify the risks and provide a scientific basis for the formulation of the limit standards by the determination and risk assessment of pesticide residues in Yixintong tablets and hawthorn leaf extracts.

The samples were extracted with acetonitrile by high speed homogenater, and purified by solid - phase extraction(SPE) column of envi-carb/NH₂. 262 pesticides in hawthorn leaf extracts and Yixintong tablets were detected by GC-MS/MS and LC-MS/MS. Moreover, the acute and chronic intake risks were calculated using the deterministic risk assessment model.

25 and 31 types of pesticides were detected in hawthorn leaf extracts and Yixintong tablets, respectively. 18 types of pesticides were detected in all of them. The contents were 0.004-1.457 mg·kg^-1, 0.007-2.1 mg·kg^-1, respectively. The results of risk assessment revealed that the short-term hazard index(HIa) of hexaconazole was 1 and the risk was needed to be paid more attention. HIa and long-term hazard index(HIc) of other pesticides detected in Yixintong tablets and hawthorn leaf extracts were much lower than 1, indicating an acceptable risk.

In the present study, the pesticide residues were explored, and their health risks were assessed；therefore our study provided technical support for the improvement of risk assessment applicable to traditional Chinese medicines (TCMs) and the formulation of relevant pesticide residue limit standards.

To evaluate the differences of aqueous extract protein content and kinds among Hudilong origins in order to provide reference for the identification and quality evaluation.

The protein content of aqueous extract was determined, and the differences among the origins were compared by nonparametric tests. The protein fingerprints were established by SDS-PAGE, and the band matrix was constructed based on the molecular weight of the bands. The differences of the protein kinds among the 3 origins were compared and analyzed by CA and OPLS-DA.

There was no statistically significant difference in the aqueous extract protein content between the P. vulgaris and P. guillelmi, but both of them differed significantly from that of P. pectinifera (P<0.01). Aqueous extract protein content. from P. pectinifera was higher than from P. vulgaris or P. guillelmi. The results of SDS-PAGE showed that there were differences in the molecular masses and quantities of the protein bands of the 3 origins with relative molecular masses ranging from 10×10³ to 250×10³. The results of the CA and OPLS-DA showed that each of the origin of Hudilong could be differentiated. The differences of the protein relative molecular masses were 133.7×10³, 10.4×10³, 66.9×10³, 51.9×10³, 13.4×10³, 7×10³, 29.4×10³, 14.6×10³, 62.2×10³, 81.1×10³, 89.2×10³, 7.7×10³, and 101.7×10³, respectively.

There are differences in the aqueous extract protein among 3 origins of Hudilong. The P. vulgaris and P. guillelmi have high similarity in the aqueous extract protein components content and kinds. SDS-PAGE technique could be used to provide reference for the identification and quality evaluation.