To optimize an LC-Q TOF MS method for the determination of target proteins expressed in recombinant cells from a cell bank.

The recombinant Chinese hamster ovary (CHO) cell line expressing antibodies was selected. The supernatant of recombinant CHO cell suspensions was retained after 12 500 r·min^-1 centrifuging for 10 min and treated with 50 mmol·L^-1 NH₄HCO₃ solution. Chymotrypsin was added for enzymatic digestion, and the comparative analyses were carried out using two LC-Q TOF MS with Advance Bio Peptide(100 mm×2.1 mm, 2.7 μm) column, the Sciex TRIPLE TOF 5600+ and the Agilent Q TOF G6545, respectively. To establish the database, the amino acid sequences of the characteristic peptides of the protein heavy and light chains were entered into SCIEX BioPharmaView Version 3.0 and Agilent MassHunter BioConfirm 12.0 software, respectively. The peptides detected in the samples were searched to identify the target peptides.

The samples were analysed by a Sciex TRIPLE TOF 5600+ instrument to obtain 8 characteristic peptides with 100%amino acid sequence coverage and the samples were analysed by an Agilent Q TOF G6545 instrument to obtain 12 characteristic peptides with 100% amino acid sequence coverage.

The method established in this study is simple to operate, has high coverage and good stability, can be used to identify the expression of target proteins in recombinant CHO cell lines. It also provides a reference for the expression identification of target proteins in similar cell lines.

To confirm the validity of solvent/detergent (S/D) treatment and dry heating for inactivation of virus in C1 esterase inhibitor (C1-INH).

The Sindbis virus in samples with S/D was inactivated by the S/D method, and the virus titer before and after inactivation was detected using the plaque assay. Dry heating method was used to inactivate encephalomyocarditis virus (EMCV) and porcine parvovirus (PPV), and cytopathic assay was used to detect the virus titer before and after inactivation.

After inactivation by the S/D method, the reductions of Sindbis virus in three batches of smaples with S/D were > 4.35 lgPFU·mL^-1, > 4.51 lgPFU·mL^-1,> 4.64 lgPFU·mL^-1, respectively. After dry heating, the reductions of EMCV in three batches of samples without S/D were ≥5.38 lgTCID₅₀/0.1 mL、≥5.12 lgTCID₅₀/0.1 mL、≥5.25 lgTCID₅₀/0.1 mL, respectively, and the reductions of PPV in three batches of samples without S/D were 4.57 lgTCID₅₀/0.1 mL、4.18 lgTCID₅₀/0.1 mL、4.68 lgTCID₅₀/0.1 mL, respectively.

With evaluation on the inactivation capability of the indicator viruses, it is proved that the S/D treatment and dry heating has good inactivation and removal effect on the virus in C1-INH.

To establish a atomic absorption spectroscopy (AAS) method for detecting calcium content in recombinant human coagulation factor Ⅷ for injection, and evaluate its uncertainty.

A calcium hollow cathode lamp light source was used. Air acetylene flame atomizer. Detection wavelength 422.7 nm. 8.9% lanthanum oxide solution was used as an ionization inhibitor. Conduct inspections on specificity, accuracy, precision, quantification limit, linearity and range, stability, etc. in accordance with the validation guidelines for drug quality standard analysis methods in Part 9101 of the 2020 edition of the Chinese Pharmacopoeia. The established method was applyed to detect the content of 6 batches of samples.

There was a good linear relationship between calcium element and absorbance in the concentration range of 0.0-5.0 μg·mL^-1, with r=0.999 8. The detection line was 0.071 6 μg·mL^-1; The quantification limit was 0.216 9 μg·mL^-1. The recovery rate of the spiked sample was 100.8% to 103. 2%. The repeatability RSD was 0.64%, and the solution was stable within 8 hours. The content determination results of 6 batches of samples all complied with the regulations. The uncertainty assessment result was (117.12±4.82) μg·mL^-1, k=2.

AAS method is established for the detection of calcium content in recombinant human coagulation factor Ⅷ for injection. This method has the advantages of high sensitivity, fast analysis speed, good repeatability, high accuracy, and cost savings. At the same time, the uncertainty of this method is evaluated, which can provide quantitative evaluation indicators for the quality control of this variety.

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 method to determination of the related substances and polymer impurities in cefminox sodium for injection.

Cefminox sodium was degraded in high temperature to prepare degradation solution. An RP-HPLC method for the related substances analysis was established with a Kromisil C₁₈ column (250 mm×4.6 mm, 5 μm), using 10 mmol·L^-1 phosphate buffer solution (pH 2.0)-acetonitrile (98：2) (A)-acetonitrile (B) with gradient elution at a flow rate of 1.0 mL·min^-1. The column temperature was maintained at 25 ℃, the detection wavelength was set at 254 nm, and the injection volume was 20 μL. The specificity of RP-HPLC method and identification of unknown impurities was researched by 2D HPLC-MS/MS.

14 main impurities were characterized in the degradation solution, including 3 cefminox dimmers and isomers which were characterized firstly. The impurities were determined in 7 batches of samples by principal component self-control with correction factor, the contents of impurity 3 were 0.01%-0.13%, the contents of impurity 4 were 0.10%-0.16%, the contents of impurity 5 were 0.02%-0.07%, the contents of impurity 6 were 0.04%-0.07%, the contents of polymer impurities were 0.03%-0.06%, the maximum single impurity contents were 0.01%-0.03%, while the total impurity contents were 0.28%-0.63%.

Cefminox degradation solution in high temperature can be used to identify related impurities and polymer peaks as the systematic suitability testing solution. The RP-HPLC method was suitable for related substances as well as polymer impurities in cefminox sodium for injection. This work provides useful information for the quality control of cefminox sodium, which can contribute to establishment of reasonable impurity limits.

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 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 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.

To identify the structure and determine the source of unknown impurities present at levels greater than 0.1% in representative pharmaceutical excipient benzalkonium chloride samples, in accordance with ICH Q3A guidelines.

A new LC-MS method suitable for the structure prediction of related substances of benzalkonium chloride was established. The separation was performed on an Acclaim 120 C₁₈ column (250 mm×4.6 mm, 5 μm) using a mobile phase consisting of methanol (mobile phase A) and 20 mmol·L^-1 ammonium formate aqueous solution (pH 3.5, mobile phase B) with gradient elution at a flow rate was 1 mL·min^-1. The post-column split was set to 1 ：3. MS data were collected in positive ion mode using an electrospray ionization (ESI) source. The structures of unknown impurities were inferred using a “Diagnostic fragment ion extension strategy” and confirmed by comparing the chromatographic and mass spectrometric behaviors of the impurities with those of reference substances. Additionally, the source attribution and genotoxicity prediction of the detected impurities were performed.

A total of five unknown impurities were detected in a representative sample of benzalkonium chloride by the newly established LC-MS method, and their structures were inferred. The structures of four impurities were confirmed using two commercially available reference substances and two reference substances synthesized in a directionally oriented manner. Impurity D was identified as N-benzyl-N,N-dimethyl-1-phenylmethanaminium chloride. Impurity E as N-benzyl-N-methyl-1-phenylmethanamine. The impurity G as N-benzyl-N-methyldodecan-1-amine, and impurity H as N,N-dibenzyl-N-methyldodecane-1-ammonium chloride. Impurity F was hypothesized to be an isomer of the formula C₁₅H₁₇N. Predictions using Nexus 2.6.0 software indicated that all the above impurities fell into category 5 and had no genotoxic potential. Furthermore, the method successfully located four unknown impurities detected by the USP method in benzalkonium chloride related substances using impurity reference standards.

This study systematically examines the structure and safety risks of potential impurities in benzalkonium chloride, providing valuable insights for enhancing quality control standards and pharmacopoeia criteria both domestically and internationally.

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.