To establish an UPLC-MS method for the analysis of salidroside, methyloleoside, specnuezhenide, acteoside, oleuropein, and G13 in plasma, and the differences in pharmacokinetic profiles of five different concoctions of Ligustri Lucidi Fructus, wine Ligustri Lucidi Fructus, vinegar Ligustri Lucidi Fructus, salt Ligustri Lucidi Fructus and steamed Ligustri Lucidi Fructus in vivo of rats were investigated.

SPF-grade male SD rats were randomly divided into 6 groups, and were given 4.2 g·kg^-1 of aqueous extracts of different concoctions of Ligustri Lucidi Fructus by gavage (for the amount of raw drug), and the plasma samples were methanol-precipitated proteins with geniposide as the internal standard, and the plasma samples were used to determine salidroside, methyloleoside, specnuezhenide, acteoside, oleuropein, and G13 in the plasma of the rats in the different time points by using the negative-ion SIM mode of UPLC-MS/MS. Kinetica 5.1 software was used to calculate the pharmacokinetic parameters, and GraphPad Prism 8.4.0 software was used to analyze the data.

The mass concentrations of salidroside, methyloleoside, specnuezhenide, acteoside and oleuropein were in the range of 2.00-1 385 ng·mL^-1, and the mass concentration of G13 was in the range of 1.30-650 ng·mL^-1, with good linearity, the relative standard deviations of the precision were all less than 10%, the recoveries of the extracts were all in the range of 85%-105%, and the matrix effect and stability were in accordance with the requirements of biological samples. The results of pharmacokinetic study showed that the AUC_0-∞, MRT_0-t, t_1/2, and C_max of acteoside were significantly higher in wine Ligustri Lucidi Fructus (P<0.01) compared to raw Ligustri Lucidi Fructus, which was 751.36 ng·mL^-1·h, 5.87 h, 377.82 h, and 38.11 ng·mL^-1, respectively. C_max, AUC_0-∞, and t_1/2 were significantly higher (P<0.01) for specnuezhenide and G13 in salt Ligustri Lucidi Fructus compared to raw Ligustri Lucidi Fructus, with C_max of specnuezhenide and G13 in salt Ligustri Lucidi Fructus were 66.45 ng·mL^-1 and 204.27 ng·mL^-1, respectively. The AUC_0-∞ were 342.69 ng·mL^-1 and 423.44 ng·mL^-1·h, and t_1/2 were 101.64 h and 15.98 h, respectively. Compared to raw Ligustri Lucidi Fructus, the C_max of oleuropein was significantly higher (P<0.05) in vinegar Ligustri Lucidi Fructus, wine Ligustri Lucidi Fructus, and steamed Ligustri Lucidi Fructus with 66.81 ng·mL^-1, 68.00 ng·mL^-1, and 66.38 ng·mL^-1, respectively. The AUC_0-∞ of salidroside and methyloleoside was the highest in vinegar Ligustri Lucidi Fructus, which was 5 782.74 ng·mL^-1 and 545.26 ng·mL^-1·h, respectively.

This study reveals the changing law of the six active ingredients in different artillery products of Ligustri Lucidi Fructus in vivo and their different characteristics, which provides a basis for the clinical application of different artillery products of Ligustri Lucidi Fructus.

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 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 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.

To analyze the fragmentation rule and pathway of pelargonidin, cyanidin, delphinidin, peonidin, petunidin and malvinidol under UHPLC-QTOF-MS positive mode electric spray, identify the characteristic product ions of six anthocyanins, and provide a theoretical basis for the establishment of mass spectrometry database and detection methods.

The chromatographic conditions were as follows: chromatographic column, Fusion-RPC₁₈ (50 mm×2.0 mm, 4 μm), mobile phase 0.1% formic acid aqueous solution (A) and methanol (B), gradient elution (0-1 min, 95%A; 1-5 min, 95%A→10%A; 5-6 min, 1%A; 6-7 min, 95%A), flow rate 0.3 mL·min^-1, column temperature 40 ℃, and injection volume 10 μL. The mass spectrometry conditions were as follows: TOF MS-IDA MS/MS, curtain gas, 0.20 MPa, collision gas CAD 7 MPa, IS voltage, 5 500 V/-4500 V, ion source temperature, 500 ℃, nebulizer gas, GAS1, 0.38 MPa, auxiliary, GAS2, 0.48 MPa, DP voltage, ±60 V, fragmentation voltage, (35±15) V, and time 0.2 s. Under the condition of positive mode of electrospray, the mass spectrometry data of six anthocyanins were measured. According to the pyrylium ions formed by the 2-phenylchromogenic structure of anthocyanins, and combined with the auxiliary analysis of the mass spectrometer database, the possible product ions were deduced.

The results showed that the six anthocyanins mainly undergo cleavage reactions on the pyranium ring, ultimately generating intermediate ions of pyrogallol and benzyl alcohol. On the other way, it occured α cracking, σ cracking causes the loss of functional groups on the ring, ultimately resulting in the formation of Chain hydrocarbons without functional groups.

The research results can provide support for the mass spectrometry characteristic ion data of six anthocyanins, which can be used to establish ion library data for anthocyanin products and also provide reference for the development and research of detection methods.

To investigate different batches of Runbi Tongqiao drops by fingerprint and multi-index component content determination method, and to provide basis for its quality evaluation.

Acclaim TM-C₁₈ chromatographic column(250 mm×4.6 mm, 5 μm) was used with methanol(A)-acetonitrile(B) -0.2% formic acid aqueous solution(C) as the mobile phase with gradient elution. The flow rate was 0.8-1.0 mL·min^-1. The injection volume was 10 μL and the detection wavelength was 327 nm. The HPLC fingerprint of Runbi Tongqiao drops was established, and the quality consistency of Runbi Tongqiao drops was compared by stoichiometric method. The contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, luteoloside and isochlorogenic acid C were determined by HPLC.

A total of 14 common peaks were calibrated in the fingerprints of 10 batches of samples, and the similarities were ≥0.933. Seven common peaks of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, isochlorogenic acid A, luteoloside and isochlorogenic acid C were identified by reference substances. The contents of seven components in Runbi Tongqiao drops were determined simultaneously, which were 0.131 5-0.133 8 mg·mL^-1, 0.095 5-0.098 9 mg·mL^-1, 0.087 4-0.090 1 mg·mL^-1, 0.012 8-0.015 8 mg·mL^-1, 0.010 3-0.013 7 mg·mL^-1, 0.022 7-0.024 9 mg·mL^-1 and 0.006 8-0.008 6 mg·mL^-1, respectively.

The established fingerprint of Runbi Tongqiao drops is stable and reliable, and the simultaneous determination method of multi-component content is simple and fast. It can be used for the quality control of Runbi Tongqiao drops, which lays a solid foundation for the follow-up study of Runbi Tongqiao drops.

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 study the related substances in salbutamol sulfate inhalation aerosol during national drug sampling and testing, and to compare the impurity content and evaluate the quality between samples from various enterprises.

HPLC external standard method was utilized to simultaneously determine the content of related substances A, B, C, D, E, F, G, H, I, J, N, and other unknown impurities in salbutamol sulfate inhalation aerosol. Thermo Synchronis C₈ chromatography column (250 mm×4.6 mm, 5 μm) was used. Sodium heptane sulfonate solution-acetonitrile was used as the mobile phase. Linear gradient elution was performed and the flow rate was 1.0 mL·min^-1. Column temperature was 40 ℃ and detection wavelength was 220 nm. Injection volume was 20 μL. The sources of impurities through forced degradation experiments were explored. Toxicity prediction software for impurity toxicity assessment was applied.

After method validation, the specificity of the method was good, and the separation degree between each impurity peak was greater than 1.5. RSDs of precision test were 0.30%-1.7%(n=6)；mass concentrations of linear range were from 0.050 to 5.000 μg·mL^-1(r=0.999 9). Limit of quantitative was in the range of 0.025-0.200 μg·mL^-1, limit of detection was in the range of 0.008-0.070 μg·mL^-1. The repeatability RSD of raw materials was 0.80%-3.8%, and the recovery rate was 95.2%-104.8%. The repeatability RSD of inhaled aerosol was 1.2%-2.9%, and the recovery rate was 98.7%-102.8%. The forced degradation test showed that impurities D, F, I, J, and N were all degradation impurities. 110 batches of samples were checked and the results of the relevant substances met the regulations. In the samples of diverse enterprises, impurities C, D, F, and N were detected more frequently, while impurities E, G, and H were not detected. Impurity J was only detected in one batch. The predicted impurity D by QSAR software falls to ICH M7 (R2) level 2.

The established method is sensitive and accurate, and can accurately quantify the content of related substances in salbutamol sulfate aerosol, providing effective technical support for systematic supervision. Further toxicity studies should be conducted on impurity D and reasonable limits should be established.

To establish an HPLC method for determination of related substances in apixaban API.

The analytical column was an ACE Excel3 C₁₈-PFP (150 mm×4.6 mm, 3 μm）. The mobile phase A was buffer(30 mmol·L^-1 ammonium acetate in water)-acetonitrile（90∶10） and the mobile phase B was buffer(30 mmol·L^-1 ammonium acetate in water)-acetonitrile（5∶95）. The whole run carried out by gradient elution at a flow rate of 1.2 mL·min^-1. The detection wavelength was set at 280 nm, the column temperature was 40 ℃ and the injection volume was 10 μL.

Apixaban was separated completely from the impurities and degradation products(the resolution>2.0). The test solution was stable for at least 48 h. The LOQs of apixaban, methyl ester product, ethyl ester product, chlorine impurity, dehydrogenation impurity, bihydrolytic impurity, ringopen methyl ester product, cyclate, impurity D, hydrolytic impurity, ringopen acid impurity, ringopen amide impurity and 5-chlorhexyl chloride derived impurity, were all 0.05%. The linear correlation coefficients of apixaban, methyl ester product, ethyl ester product, hydrolytic impurity, ringopen acid impurity, ringopen amide impurity and 5-chlorhexyl chloride derived impurity were all more than 0.99. The range were from the LOQ for impurity content to 150% of the target concentration. The average recoveries（RSD）(n=9) of methyl ester product, ethyl ester product, hydrolytic impurity, ringopen acid impurity, ringopen amide impurity and 5-chlorhexyl chloride derived impurity were 102.0%（2.7%）, 106.4%（2.2%）, 111.2%（4.0%）, 104.4%（2.9%）, 102.9%（2.7%）, 101.8%（2.9%）. The repeatability and intermediate precision completely met the requirements. The impurities contents in three batches of apixaban API 6 months accelerate stability test completely met the requirements, respectively.

This method is simple, rapid, sensitive and specific to be used for the determination of related substances in apixaban API.

To establish an HPLC method for the determination of related substances in nifedipine.

HPLC was adopted on a PFP column (250 mm×4.6 mm, 5 μm) with a gradient elution system of 20 mmol·L^-1 potassium dihydrogen phosphate solution and methanol, the flow rate was 1.0 mL·min^-1, and the column temperature was maintained at 30 ℃. The detection wavelength was set at 265 nm.

The resolutions were good between the peaks of nifedipine and ten known impurities, including impurity D, 2-nitrobenzaldehyde, monoamide, hydroxy dehydro lactone, impurity C, dehydro-N-oxide, impurity A, impurity B, m-nifedipine, p-nifedi-pine. The resolutions between the known impurity peaks were not less than 1.5, the resolutions between the main peak of nifedipine and it’s front and back impurity peaks were not less than 2.0. The calibration curves of mass concentration of above known impurities were linear respectively in their concentration range of 0.000 2-0.015 mg·mL^-1(r>0.999, n=7). The correlation coefficients of above known impurities were 1.000, 1.000, 1.000, 1.000, 0.999 9, 0.999 9, 0.999 9, 1.000, 0.999 9, 0.999 9, respectively. The average recovery rates of above known impurities were 93.1%(RSD=2.3%), 110.6%(RSD=1.9%), 109.2%(RSD=2.0%), 111.0%(RSD=2.1%), 108.1%(RSD=1.9%), 112.4%(RSD=1.8%), 110.8%(RSD=1.9%), 91.5%(RSD=3.1%), 98.9%(RSD=2.7%), 110.1%(RSD=2.6%), respectively. The detection limit of above known impurities was 0.000 06 mg·mL^-1, the quantification limit of above known impurities was 0.000 2 mg·mL^-1. The impurity determination results of the three batches of nifedipine samples showed that the content of the known impurities and the maximum single unknown impurity were less than 0.1%, the total impurities contents were less than 0.5%.

The method has good sensitivity and specificity, and it is suitable for the quality control of nifedipine.