A mass spectrometric method for in-depth profiling of phosphoinositide regioisomers and their disease-associated regulation

Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. To enable the comprehensive analysis of phosphoinositide phosphorylation status and acyl chain identity, we develop PRMC-MS (Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry). Using this method, we reveal a severe skewing in acyl chains in phosphoinositides in Pten-deficient prostate cancer tissues, extracellular mobilization of phosphoinositides upon expression of oncogenic PIK3CA, and a unique profile for exosomal phosphoinositides. Thus, our approach allows characterizing the dynamics of phosphoinositide acyl variants in intracellular and extracellular milieus.

Comparative study the essential oil composition of flowers and leaves of Crataegus monogyna L.

The aim of the work was to obtain essential oil from the leaves and flowers of the genus Hawthorn Crataegus monogyna L. and study its component composition to justify the possibility of expanding the use of this herbal drugs in medicine. Materials and methods. The leaves and flowers of Сrataegus monogyna L., which were used to obtain essential oil, were harvested in the western region of Ukraine in 2019. The quantitative content of essential oil in the herbal drugs was determined by hydrodistillation. By used chromato-mass spectrometry method determined the component composition of essential oil of leaves and flowers of Сrataegus monogyna L. The obtained spectra were compared with the library of mass spectra NIST05 and WILEY 2007 with a total number of spectra over 470000 in combination with programs for identification AMDIS and NIST.Results. The essential oil of leaves and flowers of Сrataegus monogyna L. was obtained and its quantitative content was determined, which was 0.15±0.02 % for flowers and 0.11±0.03 % for leaves. By used chromato-mass spectrometric method in the essential oil of flowers of Сrataegus monogyna L. revealed 37 compounds, leaves – 33. In the essential oil of flowers found monoterpenoids, the content of which was 0.44 % of the total, triterpenoids 7.17 %, aromatic compounds 12.61 %. In the essential oil of leaves were found monoterpenoids 37.8 %, aromatic compounds 10.25 %, norterpenoids 3.62 %, sesquiterpenoids 4.02 %, diterpenoids 2.17 %, triterpenoids 5.08 %.Conclusions. Due to the defined component composition of the essential oil of flowers and leaves of Сrataegus monogyna L., we consider its use to create drugs of external action for the treatment of skin diseases of various etiologies caused by staphylococcal and fungal microflora.

Etiological significance of Corynebacterium spp. in the development of diseases of the respiratory tract

Corynebacterium spp. It is associated with inflammatory diseases of the respiratory tract (tracheitis, pharyngitis, rhinosinusitis, bronchitis, pneumonia, etc.). C. pseudodiphtheriticum can be the causative agent of bacterial coinfection in patients with a new coronavirus infection (COVID-19). The aim is to determine the pathogenic properties and resistance to antimicrobial drugs of Corynebacterium spp. strains to establish their etiological significance in the development of inflammatory diseases of the respiratory tract. Strains of Corynebacterium spp. isolated from patients with inflammatory diseases of the respiratory tract (43 pcs.) and practically healthy individuals (29 pcs.). Isolates were identified by mass spectrometric method (MALDI-TOF MS), their cytopathic effect in CHO-K1 cell culture, hemolytic, urease activity, antimicrobial drug resistance were determined. Strains of Corynebacterium spp. isolated from patients in the amount of 105 CFU/ml or more, practically healthy - 104 CFU/ml or less. Isolates of Corynebacterium spp. patients had a more pronounced cytopathic effect (83.7±11.1%) and were more often resistant to antimicrobial drugs than those isolated from practically healthy. To establish the etiological significance of Corynebacterium spp. isolates. in the development of inflammatory diseases of the respiratory tract, it is advisable to determine their amount in biological material (105 CFU/ml or more), the cytopathic effect on CHO-K1 cell culture, as well as the presence of multiple resistance to antimicrobial drugs. Differences in the characteristics of Corynebacterium spp. isolates. from patients with respiratory tract pathology and practically healthy individuals are associated with the strain, not the species, of corynebacteria.

Chromato-mass-spectrometric research in Salvia grandiflora L., Salvia pratensis L. and Salvia verticillata L. aboveground organs

The genus Salvia L. has more than 900 species distributed throughout the globe. 21 species are growing in Ukraine. All species of this genus have essential oils. Salvia officinalis and Salvia sclarea have been used in the culture and are widely used in medical practice. The chemical composition of other species of sage and the possibility of their use in pharmaceutical and medical practice are almost not studied. Taking into account the results of chemotaxonomic studies of species of the flora genus of Ukraine, their prevalence and prospects for introduction into the culture, for further studies were selected raw materials of S. grandiflora, S. pratensis and S. verticillata. The aim. The aim of the study was to conduct a chromato-mass spectrometric study of the aboveground organs of S. grandiflora L., S. pratensis L. and S. verticillata L. to establish the prospects for the use of raw materials of these species in medical and pharmaceutical practice. Materials and methods. The objects of the study were leaves of S. officinalis, leaves, stems and flowers of S. grandiflora, S. pratensis and S. verticillata, which were harvested on the basis of the botanical garden of Ivan Franko National University of Lviv. The research of volatile substances in the objects of the research was carried out by the method of GC-MS on the basis of the Department of Natural Sciences for Foreign Students and Toxicological Chemistry of Zaporizhia State Medical University. Results. As a result of the study, 243 substances were found in the objects of the study, of which 149 were identified. 77 substances were found in the leaves of S. officinalis, 80, 26 and 63 substances in the leaves, stems and flowers of S. grandiflora, respectively, in the leaves , stems and flowers of S. pratensis – 28, 30 and 48 substances, respectively, in leaves, stems and flowers of S. verticillata – 39, 22 and 39 substances, respectively. Dominant compounds among substances of terpenoid nature are: cyclofenchene, camphene, 1,8-cineole, α-thujone, β-thujone, camphor borneol, caryophyllene, humulene, viridiflorol, sabinene, pyranone, β-pinene, phytol, kolavenol, β-copaen, loliolide, pseudolimonene and spatulenol. Among the dominant substances, 8 were detected for the first time in these species: cyclofenchene, viridiflorol, sabinene, pyranone, phytol, kolavenol, loliolide and pseudolimonene. Conclusions. The leaves of S. officinalis, leaves, stems and flowers of S. grandiflora, S. pratensis and S. verticillata of the flora of Ukraine were studied by chromato-mass spectrometric method. As a result of the study, 243 substances were identified, of which 149 were identified. Promising raw materials containing terpene compounds for S. grandiflora there are leaves, and for S. pratensis and S. verticillata – flowers, so they are promising agents for introduction into pharmaceutical practice

Quantification for Antibody-Conjugated Drug in Trastuzumab Emtansine and Application to In Vitro Linker Stability and In Vivo Pharmacokinetic Study in Rat Using an Immuno-Affinity Capture Liquid Chromatography-Mass Spectrometric Method

Trastuzumab emtansine (T-DM1, brand name: Kadcyla®) is the first FDA-approved antibody-drug conjugate (ADC) for metastatic human epidermal growth factor receptor 2 positive (HER2+) breast cancer. It consists of three components: trastuzumab, an anti-HER2 monoclonal antibody, maytansinoid (DM1) as a cytotoxic drug, and maleimidomethyl cyclohexane-1-carboxylate (MCC) as a linker. In particular, the MCC linker is known as a non-cleavable linker and has a feature of being conjugated to DM1 by a covalent thioether bond. In this study, we developed an immuno-affinity capture liquid chromatography-mass spectrometric (LC-MS/MS) assay for quantifying the antibody-conjugated drug (acDrug) component of T-DM1. To quantify acDrug, desulfurated DM1 was prepared using a chemical desulfuration pretreatment and quantified as an acDrug. A quadratic regression (weighted 1/concentration), with equation y = ax2 + bx + c, was used to fit the calibration curves over the concentration range of 17.09~1709.44 ng/mL for the acDrug of T-DM1. The quantification run met the in-house acceptance criteria of ±25% accuracy and precision values for the quality control (QC) samples. In conclusion, an immuno-affinity capture LC-MS/MS assay was successfully developed to quantify acDrug of T-DM1 and applied to evaluate in vitro plasma linker stability and preclinical pharmacokinetic (PK) study in rats. This assay could be helpful when applied to other ADCs with the same linker-cytotoxic drug platform.

Quantification of Tofacitinib in Human Plasma Samples Using Radio-Labeled Internal Standard

A simple, sensitive and accurate liquid chromatography tandem mass spectrometric method has been developed and validated for determination of Tofacitinib in human plasma. The method was developed on Agilent Zorbax SB-C8 150×4.6 mm, 5 µm column using 10mM ammonium formate: acetonitrile (40:60 v/v) mobile phase for Chromatographic separation of Tofacitinib. The Tofacitinib and Tofacitinib- C3 were monitored by electrospray ionization in positive ion multiple reactions monitoring mode to detect the Tofacitinib at mass/charge 313.200/149.200 and Tofacitinib-13C3 (Internal Standard) at 316.500/149.200. Liquid-liquid extraction was employed in the extraction of analytes from human plasma. Both drug and internal standards were stable in plasma samples. The proposed method was validated as per international council of harmonization guidelines over a linear concentration range of 0.2ng/mL to 100.0ng/mL with a correlation coefficient (r) of ≥ 0.9983. Bioavailability and Bioequivalence studies of tofacitinib in biological samples can be achieved by analyzing them using the validated developed method. This study plays a key role in determining routine therapeutic drug monitoring of tofacitinib drug.