To employ adeno-associated viruses as vectors to design and prepare pseudoviruses comprising multiple DNA viral gene fragments. These were used as positive controls for the detection of human viruses in the field of molecular testing, thereby compensating for the absence of wild-type viral controls in viral molecular testing methods.

The adeno-associated viral vectors served as the backbone of the pseudoviruses, with a total of four target gene fragments from three viruses incorporated: human papillomavirus type 16 (HPV16), type 18 (including HPV18-1 and HPV18-2) and human polyomavirus (HPyV) were, respectively, inserted into one viral vector, and the adeno-associated viral pseudoviruses were packaged by multiple plasmid cotransfection. The infectious activity of the pseudoviruses was confirmed by a cell infection assay, and a quantitative analysis of the pseudoviruses genome was conducted using fluorescence quantitative PCR.

The titers of HPV16, HPV18-1, HPV18-2, and HPyV genomes in the pseudoviruses were 7.77×10⁸ copies·mL^-1,6.77×10⁸ copies·mL^-1, 7.04×10⁸ copies·mL^-1, and 1.24×10⁹copies·mL^-1, respectively, as determined by fluorescence quantitative PCR. Following a 48 h period of infection in 293T/17 cells, the viral gene sequences of HPV16, HPV18-1, HPV18-2, and HPyV were successfully identified using fluorescence quantitative PCR with the intracellular pseudoviruses. The copy numbers were 1.30×10⁶ copies·mL^-1, 6.59×10⁵ copies·mL^-1,6.27×10⁵ copies·mL^-1, and 4.17×10⁶ copies·mL^-1. Following the infection of 293T/17 cells with the reporter gene pseudoviruses for a period of 48 hours, a distinct green fluorescence was evident under a fluorescence microscope, thereby confirming the infectious activity of the pseudoviruses. The pseudoviruses was employed as a positive control for the verification of applicability in human mesenchymal stem cells, and the recoveries of the four viral gene fragments by fluorescence quantitative PCR for nucleic acid extraction were 94.4%, 70.7%, 83.1%and 90.9%, respectively.

The present study demonstrates that the adeno-associated virus packaging method can be employed to produce a multiviral gene pseudoviruses with infectious activity. The pseudoviruses can be employed as a positive control for the replacement of multiple wild-type viruses in the viral fluorescence quantitative polymerase chain reaction (PCR) of stem cell samples.

This method not only reduces the financial burden associated with the preparation of positive controls, but it also offers enhanced biosafety and improves the efficiency and quality control of the qPCR method, from the extraction of genes to their amplification.

To explore the fragmentation patterns of etomidate analogues by mass spectrometry to provide reference for the identification and structural analysis of these substances.

Etomidate, metomidate, propaxate and isopropaxate were analyzed by gas chromatography-quadrupole time-of-flight mass spectrometry(GC-Q TOF MS) and liquid chromatography high-resolution mass spectrometry (LC-Q Orbitrap MS) under EI and ESI positive ionization modes. And the EI-MS and ESI-MS/MS fragmentation patterns of etomidate analogues were deduced.

Etomidate and its analogues had similar fragmentation pathways. In EI-MS mode, the fragmentation pathway was mainly focused on the breaking of carbon-nitrogen bonds, and the specific fragment ions (m/z 105 and m/z 77) were formed. In ESI-MS/MS positive ion mode, the fragmentation pathway was mainly focused on the fracture of carbon-nitrogen bond, resulting in the formation of the characteristic fragment ion with styrene as neutral loss, Subsequently, the neutral fragments R₁ and H₂O were lost in turn, and the specific fragment ions (m/z 113 and m/z 95) was formed. Moreover, the neutral loss of R₁ in ESI-MS/MS positive ion mode could more intuitively indicate the substituents on the parent nucleus and further determine the structure of etomidate and its analogues.

The fragmentation patterns of etomidate and its analogues are helpful to analyze and infer the structure of these substances, and provide reference for the identification and structural analysis of these substances in forensic laboratory.

To establish a simple and efficient method for detecting telomerase activity for evaluating tumorigenic risk of cell products.

Specific primers and probes were designed for the conserved domain of telomerase catalytic subunit (TERT), and the primers and probes of TERT gene were optimized and screened, and the primers and probes of internal reference genes were set up. Multiple quantitative PCR reaction was performed using two-color fluorescent probes in a reaction system, and RT-qPCR probe method was established. The expression of TERT gene in human mesenchymal stem cells HMSC was used to determine whether telomerase activity existed in the cells indirectly.

TERT gene of 293T/17 positive control cells could be stably and specifically detected by this method, with a mean Ct value of 23.96 and a RSD of 1.5. The internal reference gene GAPDH could be detected successfully, the mean Ct value was 14.13, the RSD was 1.3%. The reference gene GAPDH in MRC-5 could be detected in negative control cells, the mean Ct value was 12.81, the RSD was 0.46%, the TERT gene was not detected, and the telomerase activity was negative. The reference gene GAPDH in HMSC could be detected, the Ct mean value was 13.01, and the RSD was 3.8%, whereas, the telomerase activity of HMSC was negative.

The real-time fluorescent quantitative RT-qPCR probe method established in this study can accurately detect the expression of catalytic subunit mRNA in telomerase positive cells with good repeatability and high specificity. It can be used to analyze telomerase activity of stem cells and indirectly evaluate tumorigenic risk of cell products derived from stem cells.

To identify the related substances in desloratadine using two-dimensional liquid chromatography-mass spectrometry (2D LC-MS) technology.

A Waters Symmetry C₈ column (250 mm×4.6 mm, 5 μm) was employed. The mobile phase A consisted of a mixture containing 1% triethylamine and 10 mmol·L^-1 potassium dihydrogen phosphate solution adjusted to pH 2.0 with phosphoric acid, acetonitrile, and methanol(80：10：10), while mobile phase B contained 1% triethylamine, 10 mmol·L^-1 potassium dihydrogen phosphate solution(pH adjusted to 2.0 with phosphoric acid), acetonitrile, and tetrahydrofuran (30：70：5). A linear gradient elution was used for the first-dimension liquid chromatography separation of related substances in desloratadine. Individual separated components were captured through a multi-channel switching valve into retention vessels, then transferred to a Thermo BDS Hypersil C₁₈ column (100 mm×4.6 mm, 3 μm). After a second-dimension gradient elution with a mobile phase composed of 10 mmol·L^-1 ammonium acetate solution and methanol to achieve rapid desalting, electrospray ionization positive ionization-quadrupole-time-of-flight high-resolution mass spectrometry was employed to measure the accurate masses and elemental compositions of parent ions and their daughter ions of the related substances. Structural identification was achieved through spectral analysis.

Under the established analytical conditions, desloratadine and its related substances were well separated. A total of 12 related substances were detected and identified in desloratadine and its stressed degradation samples. Among these 12 degradants, three were known impurities, while the others are newly identified impurities.

2D LC-MS can effectively provide specific identification of desloratadine’s related substances under non-volatile mobile phase separation conditions. The findings from this study serve as a reference for controlling the quality of desloratadine.

To establish an HPLC-MS method for determination of three diastereoisomers in nirmatrelvir API.

The analytical column was an InfinityLab Poroshell SB-C₁₈ (150 mm×3.0 mm，2.7 μm), the two columns connected in series in series. The mobile phase was buffer (0.1% formic acid) (A) -acetonitrile (B), the diluent was water-acetonitrile-methanol (61 ：19.5 ：19.5). The whole run carried out by gradient elution. The column temperature was 80 ℃，the injection volume was 2 μL, and the sample temperature was 5 ℃.

Nirmatrelvir was separated completely from three diastereoisomers (the resolution>1.5). The test solution was stable for at least 3 d. The LOQ of diastereoisomer 1 was 0.04%, the LOQ of diastereoisomer 2 was 0.04%, the LOQ of diastereoisomer 3 was 0.05%. The linear correlation coefficient of diastereoisomer 3 was >0.99. The linear range was LOQ-150% of specification. The average recovery (n=9) of diastereoisomer 3 was 97.2%, RSD = 3.1%. The repeatability and intermediate precision completely met the requirements. The three diastereoisomers contents in three batches of nirmatrelvir API 24 months long-term stability test were all completely met the requirements, respectively.

This method is simple, rapid, sensitive and specific to be used for the determination of three diastereoisomers in nirmatrelvir API.

To identify the related substances of progesterone sustained-release vaginal gel by liquid chromatography mass spectrometry (LC-MS).

The separation was carried out on YMC-Triart C₁₈ (150 mm×4.6 mm，3 μm) column by gradient elution using acetonitrile and water as the mobile phases. The accurate mass and elemental composition of the parent ions and their product ions of related substances were determined by electrospray-ionization quadrupole time-of-flight high resolution mass spectrometry (ESI Q TOF MS) at collision energies of 5-45 eV. The structures of the related substances were identified by spectral analysis.

Under the established analytical condition, progesterone and its related substances were adequately separated, and 18 major related substances were detected and identified by hyphenated techniques in progesterone sustained-release vaginal gel and their stressed samples. Among them, 7 were the impurities listed in the EP or previous reported, and the others were unknown related substances identified for the first time in this paper. The established LC-MS technique was effective for the separation and identification of the related substances of progesterone sustained-release vaginal gel.

Progesterone is unstable under acid, alkaline, and high-temperature stress conditions, and prone to produce oxidation degradants. The research results can provide a reference for the quality control of progesterone sustained-release vaginal gel.

To establish an in vitro permeation method for crisaborole ointment and to provide a reference for the bioequivalence of crisaborole ointment.

The HPLC method was used to determine the permeability of crisaborole ointment, and the vertical Franz diffusion pool method was used to study the release characteristics of the ointment in vitro. The chromatographic column was Thermo BDS Hypersil-C₁₈(150 mm×4.6 mm, 5 μm), with 0.1% phosphoric acid solution-acetonitrile (60：40) as the mobile phase, flow rate of 1.5 mL·min^-1, column temperature of 30 ℃, detection wavelength of 254 nm, and injection volume of 10 μL.

The peak shape of crisaborole in the chromatogram of the test sample was good. There was no peak at the position of crisaborole in the negative sample chromatogram, which did not interfere with the determination. The linear relationship of crisaborole was good within the range of 0.026-21.02 μg·mL^-1 (r≥0.999). In six repeated experiments, the mean maximum transmittance (J_max) of the self-made product and the reference formulation were compared (odds ratio: 0.94). Simultaneously calculate the confidence interval for J_max, with a 90% confidence interval ranging from 87.9% to 102.0%. The mass conservation percentage of each sample was 94.1%-101.1%(n=12). The reference solution was left at room temperature for 26 h, with a peak area RSD of 0.44%. The self-made product and reference preparation were left in a water bath at room temperature and 32 ℃ for 26 h, with peak area RSD ranging from 0.78% to 1.1%, indicating good stability of the solution. For the three batches of samples tested, the ratio of the mean J_max values of the self-made product and the reference formulation was within the range of 0.92-0.97. Simultaneously calculate the confidence interval for J_max, with a 90% confidence interval ranging from 84.8%-100.7%.

This method is simple and convenient to operate, with high selectivity, strong exclusivity and high accuracy.