Sample Preparation Conditions Research Articles

Development and validation of an analytical procedure for the determination of residual protein A in active substances of therapeutic monoclonal antibodies

SCIENTIFIC RELEVANCE. An important quality-control issue for therapeutic monoclonal antibodies (mAbs) is the determination of residual protein A leaching from the carrier during the purification of mAbs by affinity chromatography. However, unrelated compounds (immunoglobulins) present in the sample can complicate the immunoenzymatic detection of protein A (matrix effect), potentially leading to false-negative test results. To increase the efficiency of enzyme-linked immunosorbent assay (ELISA), it is necessary to develop a sample preparation step that can irreversibly break the bond in the protein A–mAb complex.AIM. This study aimed to develop and validate an analytical procedure for the determination of residual protein A in active substances of therapeutic mAbs by ELISA with a test kit comprising in-house reagents.MATERIALS AND METHODS. Recombinant protein A was used as an antigen. Polyclonal antibodies to protein A were produced by immunising chickens, selecting immunised eggs, and isolating antibodies from these eggs. Protein A-specific antibodies were purified by affinity chromatography. Residual protein A was determined using sandwich ELISA with preliminary sample preparation. The analytical procedure was validated in accordance with the requirements of the State Pharmacopoeia of the Russian Federation (Validation of Analytical Procedures, OFS.1.1.0012).RESULTS. The authors obtained, isolated, and purified chicken IgY antibodies to recombinant protein A. The authors selected sample preparation conditions for the determination of residual protein A by ELISA and optimum compositions of buffer solutions, including the composition of a denaturing buffer to disrupt the protein A–mAb complex. The developed analytical procedure was validated. According to the measurements of protein A, the accuracy of the analytical procedure ranged within 83–108% of the nominal value, the interlaboratory precision ranged within 96–116%, and the repeatability was up to 13%. The lower limit of quantitation was 10 ng/mL with a minimum required dilution of 1:10. The analytical range extended from 10 to 40 ng/mL. The analytical procedure showed results comparable to those obtained with a similar test kit from an international manufacturer.CONCLUSIONS. The developed analytical procedure for the determination of residual protein A by ELISA with a test kit comprising in-house reagents can minimise the matrix effect in therapeutic mAbs. This analytical procedure will alleviate import substitution and reduce quality control costs for Russian immunobiologicals.

Doxorubicin, doxorubicinol, cardiotoxicity, breast cancer, volumetric absorptive microsampling, LC-MS/MS.

<b>Background and Objective:</b> Doxorubicin is an anticancer therapy belonging to the anthracycline class, which has clinical activity in breast cancer. Doxorubicin can cause cardiotoxic effects due to the formation of doxorubicinol as its main metabolite. The purpose of this study was to obtain the optimum sample preparation conditions for the analysis of doxorubicin in VAMS and as a form of therapeutic drug monitoring (TDM) in patients with cancer breasts. <b>Materials and Methods:</b> Analyze doxorubicin and doxorubicinol levels with Volumetric Absorptive Microsampling (VAMS) in patients' cancer breasts receiving doxorubicin in their therapeutic regimen. The sample was analyzed using Ultra Performance Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS). The method uses deep linear range concentrations of 8-200 ng/mL for doxorubicin and 3-100 ng/mL for doxorubicinol. <b>Results:</b> Multiple reaction monitoring (MRM) value set at m/z 544.22>396.9 for doxorubicin; m/z 546.22>398.9 for doxorubicinol and m/z 528.5>362.95 for daunorubicin. The LLOQ value obtained was 8 ng/mL for doxorubicin and 3 ng/mL for doxorubicinol with linearity of 0.9904 for doxorubicin and 0.9902 for doxorubicinol. Analysis results show doxorubicin levels were in the range of 9.47 ng/mL to 87.84 ng/mL and doxorubicinol range between 4.24 and 54.02 ng/mL. <b>Conclusion:</b> Dosage cumulative doxorubicin ranges between 47.93 and 346.09 mg/m<sup>2</sup>; with this, the risk of cardiomyopathy in the patients surveyed is under 4%, according to the literature.

Identify the effect of Fe2O3 nanoparticles on mechanical and microstructural characteristics of aluminum matrix composite produced by powder metallurgy technique

Abstract Aluminum is a highly valuable structural metal utilized in various industrial sectors; particularly, it is utilized in considerable quantities in the nautical, aeronautical, and automotive industries. Aluminum is additionally utilized in small amounts in several other industrial sectors. The composite materials are now extensively utilized in various applications after their introduction. In this research, they prepared composite samples of aluminum with adding hematite nanoparticles with different ratio (2, 4, 6, and 8) wt% by powder metallurgy technology, and the sample preparation conditions was (mixing time reach to 2 h for every sample; the compaction loads is 6 tons and sintering temperature equal to 600°C). The tests conducted were XRD, SEM, EDS, green density, green porosity, microhardness, compression, and wear. The results illustrate that the hardness and wear values increase when increasing the hematite percentage.

Multiparameter flow cytometric detection and analysis of rare cells in in vivo models of cancer metastasis.

Rapid and reliable circulating tumor cell (CTC) and disseminated tumor cell (DTC) detection are critical for rigorous evaluation of in vivo metastasis models. Clinical data show that each step of the metastatic cascade presents increasing barriers to success, limiting the number of successful metastatic cells to fewer than 1 in 1,500,000,000. As such, it is critical for scientists to employ approaches that allow for the evaluation of metastatic competency at each step of the cascade. Here, we present a flow cytometry-based method that enables swift and simultaneous comparison of both CTCs and DTCs from single animals, enabling evaluation of multiple metastatic steps within a single model system. We present the necessary gating strategy and optimized sample preparation conditions necessary to capture CTCs and DTCs using this approach. We also provide proof-of-concept experiments emphasizing the appropriate limits of detection of these conditions. Most importantly, we successfully recover CTCs and DTCs from murine blood and bone marrow. In Supplemental materials, we expand the applicability of our method to lung tissue and exemplify a potential multi-plexing strategy to further characterize recovered CTCs and DTCs. This approach to multiparameter flow cytometric detection and analysis of rare cells in in vivo models of metastasis is reproducible, high throughput, broadly applicable, and highly adaptable to a wide range of scientific inquiries. Most notably, it simplifies the recovery and analysis of CTCs and DTCs from the same animal, allowing for a rapid first look at the comparative metastatic competency of various model systems throughout multiple steps of the metastatic cascade.

Greening of the method for simultaneous determining the enisamium iodide and tilorone dihydrochloride using GC-FID assay

Pharmaceutical companies in Ukraine aspire to develop their innovative medicinal products and successfully introduce them to the global market. However, along with the prospects of increased usage of these pharmaceuticals, there arises a challenge of heightened waste production, making them a part of the over twenty million tons of PPCPs produced annually. Consequently, one of the tasks in producing new pharmaceuticals is the development of methodologies and approaches not only for quality control but also for their determination in the environment matrices.
 The aim. Develop and validate GC-FID chromatographic method for the simultaneous determination of Enisamium iodide and Tilorone dihydrochloride, evaluate their applicability, and compare their "greenness" with the previously developed HPLC method.
 Materials and methods. The determination of the Tilorone dihydrochloride and Enisamium iodide was carried out by gas chromatography with a flame ionization detector using the Rxi-5 ms (30 m long, 0.25 mm outer diameter and 0.25 μm liquid stationary phase thickness)
 Results. Chromatographic GC-FID methods have been developed for the simultaneous determination of Enisamium iodide and Tilorone dihydrochloride. Optimal sample preparation conditions were established, and a validation process was conducted. A comparison with the previously developed HPLC method was made regarding "greenness."
 Conclusions. The developed GC-FID methodology is accurate and more environmentally friendly compared to the previously established methods. It can be recommended to determine Enisamium iodide and Tilorone dihydrochloride in the environmental matrices. It is considered environmentally friendly based on the overall GREENness (AGREE) scale, scoring 0.73 (>0.70), which demonstrates the environmentally favourable nature of the proposed analytical approach

Deep eutectic solvent extraction combined with magnetic bead ligand fishing for identification of α-glucosidase inhibitors from Pueraria lobata.

In this study, a deep eutectic solvent (DES) extraction combined with a magnetic bead ligand affinity analytical method was developed and used for α-glucosidase inhibitor identification from Pueraria lobata. Several critical parameters affecting the analysis performance, including the type of DES, molar ratio, water amount, pH, salt concentration, and volume of DES, were investigated. The selected analytical sample preparation conditions were as follows. The composition of DES is choline chloride-1,4-butanediol (1:3), the water content is 40%, pH is 7.0 and the volume of extraction solution is 2 mL. The obtained sample extraction solution was analyzed directly using α-glucosidase immobilized magnetic beads (GMBs). Three α-glucosidase inhibitors in Pueraria lobata, including puerarin, daidzin, and daidzein, were identified. Luteolin was used as a positive control to evaluate the method's selectivity. Results showed it could selectively bond to the GMBs in the DES. As the affinity analysis was performed directly in a DES, the solution-removing process could be avoided. The intra-day and inter-day precisions of the method are 5.21% and 6.38%, respectively. The solvent amount was 1/50-1/2000 of that used in traditional methods.

Dispersive solid phase extraction based on cross-linked hydroxypropyl β-cyclodextrin polymers for simultaneous enantiomeric determination of three chiral triazole fungicides in water.

Anefficient method is presentedfor simultaneous enantioselective determination of three chiral triazole fungicides (namely paclobutrazol, hexaconazole, and diniconazole) in water samples by DSPE-HPLC-UV. The perfect chiral separation of the enantiomers was achieved on a Chiralpak IH column within 15min. In order to adsorb and enrich the analytes from water matrices, a cross-linked hydroxypropyl β-cyclodextrin polymer was synthesized. The prepared material exhibited good adsorption capacity, which was assessed by adsorption kinetic and adsorption thermodynamic experiments. One-variable-at-a-time and the response surface methodology were used to optimize the extraction parameters. Under the optimum sample preparation conditions, good linearity (2.0 ~ 800µg L-1, R2 ≥ 0.9978), detection limits (0.6 to 1.0µg L-1), quantitation limits (2.0 to 3.2µg L-1), recoveries (86.7 ~ 105.8%), and the relative standard deviation (intra-day RSD ≤ 3.7%, inter-day RSD ≤ 5.1%) were obtained, satisfying the requirements of pesticides residues determination. These results demonstrated that the proposed method was applicable for routine determination of chiral triazole fungicide residues in water samples.

Improving the Signal Intensity of Cryosections Using a Conductive Adhesive Film in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

The matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique was used to obtain the molecular images of cryosections without labeling. Although MALDI-MSI has been widely used to detect small molecules from biological tissues, issues remain due to the technical process of cryosectioning and limited mass spectrometry parameters. The use of a conductive adhesive film is a unique method to obtain high-quality sections from cutting tissue, such as bone, muscle, adipose tissue, and whole body of mice or fish, and we have reported the utilization of the film for MALDI-MSI in previous. However, some signal of the small molecules using the conductive adhesive films was still lower than on the indium tin oxide (ITO) glass slide. Here, the sample preparation and analytical conditions for MALDI-MSI using an advanced conductive adhesive film were optimized to obtain strong signals from whole mice heads. The effects of tissue thickness and laser ionization power on signal intensity were verified using MALDI-MSI. The phospholipid signal intensity was measured for samples with three tissue thicknesses (5, 10, and 20 μm); compared to the signals from the samples on the ITO glass slides, the signals with conductive adhesive films exhibited significantly higher intensities when a laser with a higher range of power was used to ionize the small molecules. Thus, the technique using the advanced conductive adhesive film showed an improvement in MALDI-MSI analysis.

Study of sample preparation influence on bacterial lipids profile in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Lipids are one of the cell components therefore it is important to be able to accurately assess them. One of the analytical techniques used to study lipid profiles is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The present study attempted to select optimal conditions for sample preparation and MALDI MS analysis of bacterial lipidome in both positive and negative ion modes using different extraction protocols-Folch, Matyash, and Bligh & Dyer, solvents used to apply samples, and matrices such as 9-aminoacridine (9-AA), α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), 2-mercaptobenzothiazole (MBT), and 2,4,6-trihydroxyacetophenone (THAP). The obtained results allowed concluding that DHB or CHCA matrices are suitable for lipid analysis in the positive mode, and in the negative mode THAP or 9-AA. The most appropriate protocol for extracting lipids from bacterial cells was the Bligh & Dyer method in both ionization modes. The use of the solvent TA30, which was a mixture of acetonitrile and 0.1% trifluoroacetic acid in water, provided on the spectra a significant number of signals from lipids in all groups analyzed, such as fatty acyls, glycerolipids, and glycerophospholipids.

Feasibility of Optical Genome Mapping from Placental and Umbilical Cord Sampled after Spontaneous or Therapeutic Pregnancy Termination

Optical genome mapping (OGM) is an alternative to classical cytogenetic techniques to improve the detection rate of clinically significant genomic abnormalities. The isolation of high-molecular-weight (HMW) DNA is critical for a successful OGM analysis. HMW DNA quality depends on tissue type, sample size, and storage conditions. We assessed the feasibility of OGM analysis of DNA from nine umbilical cord (UC) and six chorionic villus (CV) samples collected after the spontaneous or therapeutic termination of pregnancy. We analyzed quality control metrics provided by the Saphyr system (Bionano Genomics) and assessed the length of extracted DNA molecules using pulsed-field capillary electrophoresis. OMG data were successfully analyzed for all six CV samples. Five of the UC samples did not meet the Saphyr quality criteria, mainly due to poor DNA quality. In this regard, we found that DNA quality assessment with pulsed-field capillary electrophoresis can predict a successful OGM analysis. OGM data were fully concordant with the results of standard cytogenetic methods. Moreover, OGM detected an average of 14 additional structural variants involving OMIM genes per sample. On the basis of our results, we established the optimal conditions for sample storage and preparation required for a successful OGM analysis. We recommend checking DNA quality before analysis with pulsed-field capillary electrophoresis if the storage conditions were not ideal or if the quality of the sample is poor. OGM can therefore be performed on fetal tissue harvested after the termination of pregnancy, which opens up the perspective for improved diagnostic yield.

Unveiling the Magnetic and Structural Properties of (X2YZ; X = Co and Ni, Y = Fe and Mn, and Z = Si) Full-Heusler Alloy Microwires with Fixed Geometrical Parameters

We studied Ni2FeSi-, Co2FeSi-, and Co2MnSi-based full-Heusler alloy glass-coated microwires with the same geometric parameters, i.e., fixed nucleus and total diameters, prepared using the Taylor–Ulitovsky method. The fabrication of X2YZ (X = Co and Ni, Y = Fe and Mn, and Z = Si)-based glass-coated microwires with fixed geometric parameters is quite challenging due to the different sample preparation conditions. The XRD analysis showed a nanocrystalline microstructure for all the samples. The space groups Fm3¯m (FCC) and Im3¯m (BCC) with disordered B2 and A2 types are observed for Ni2FeSi and Co2FeSi, respectively. Meanwhile, a well-defined, ordered L21 type was observed for Co2MnSi GCMWs. The change in the positions of Ni, Co and Mn, Fe in X2YSi resulted in a variation in the lattice cell parameters and average grain size of the sample. The room-temperature magnetic behavior showed a dramatic change depending on the chemical composition, where Ni2FeSi MWs showed the highest coercivity (Hc) compared to Co2FeSi and Co2MnSi MWs. The Hc value of Ni2FeSi MWs was 16 times higher than that of Co2MnSi MWs and 3 times higher than that of Co2FeSi MWs. Meanwhile, the highest reduced remanence was reported for Co2FeSi MWs (Mr = 0.92), being about 0.82 and 0.22 for Ni2FeSi and Co2MnSi MWs, respectively. From the analysis of the temperature dependence of the magnetic properties (Hc and Mr) of X2YZ MWs, we deduced that the Hc showed a stable tendency for Co2MnSi and Co2FeSi MWs. Meanwhile, two flipped points were observed for Ni2FeSi MWs, where the behavior of Hc changed with temperature. For Mr, a monotonic increase on decreasing the temperature was observed for Co2FeSi and Ni2FeSi MWs, and it remained roughly stable for Co2MnSi MWs. The thermomagnetic curves at low magnetic field showed irreversible magnetic behavior for Co2MnSi and Co2FeSi MWs and regular ferromagnetic behavior for Ni2FeSi MWs. The current result illustrates the ability to tailor the structure and magnetic behavior of X2YZ MWs at fixed geometric parameters. Additionally, a different behavior was revealed in X2YZ MWs depending on the degree of ordering and element distribution. The tunability of the magnetic properties of X2YZ MWs makes them suitable for sensing applications.

Metabolomic studies for medicinal plant raw materials

Every year, medicinal plant raw materials are increasingly found in the production of medicines. Despite a number of positive properties of plants and raw materials obtained from them, there are also negative aspects of the introduction of native (natural) drugs. For example, biological active substances, their quantity and activity, depend on the season of the year and the climatic conditions of the region. These factors are highly variable, and correlations of biologically active substances with weather are difficult to predict. Thus, only with the help of modern analytical instruments and validated methods can it be possible to create statistically reliable algorithms for searching for biologically active substances.The polythematic abstract and bibliographic database has been analyzed. The main attention was paid to such scientometric databases as: Web of Science Core Collection, Medline, PubMed, RSCI, as well as data from the eLIBRARY.ru portal over the past 10 years. An assessment was made of the statistical data and publication activity of the authors regarding the request for metabolomics of medicinal plant raw materials in medicine and veterinary medicine.Metabolomic studies of medicinal plants aim to provide a complete and accurate study of all small molecule metabolites and complement other omics technologies such as genomics, transcriptomics and proteomics.Based on the literature reviewed, it can be concluded that there are two main approaches to collecting metabolomic data: targeted and untargeted analysis. Targeted and untargeted metabolomics analyzes are similar in sample collection, preparation, and chromatographic conditions (stationary phase and mobile phase). However, targeted metabolomics studies mainly focus on multiple metabolites from the same or different pathways.

Simple and robust LC–MS/MS method for quantification of colistin methanesulfonate and colistin in human plasma for therapeutic drug monitoring

A rapid, simple, and robust LC–MS/MS method was developed and validated for the quantitation of colistin and colistin methanesulfonate (CMS) in human plasma. The method also prevented overestimation of colistin concentration by establishing the stability of CMS under sample preparation conditions, including blood and plasma storage conditions. Polymyxin B1 was used as an internal standard, and positive-ion electrospray ionization in multiple reaction monitoring mode was used for quantification. Chromatographic separation was achieved using a Zorbax eclipse C18 column (3.5 µm, 2.1 mm i.d. × 100 mm), with a flow rate of 0.5 mL/min, 5 μL injection volume, and gradient elution with a mixture of acetonitrile-water (containing 0.1 % trifluoroacetic acid). The method had a quantifiable range of 0.043–8.61 and 0.057–11.39 μg/mL for colistin A and B in human plasma, respectively, under a total runtime of 6.0 min. Further, it demonstrated appropriate extraction efficiency, no significant interference from co-eluting endogenous compounds, and satisfactory intraday and interday precision and accuracy. The proposed procedure for sample preparation successfully addressed the issue of CMS instability, consequently diminishing the probability of overestimating the concentration of colistin. Therefore, this simple and robust LC-MS/MS method for CMS and colistin quantification in human plasma is a valuable tool for clinicians to accurately monitor colistin treatment in patients with infections caused by multidrug-resistant (MDR) Gram-negative bacteria.

Characterizing lignins from various sources and treatment processes after optimized sample preparation techniques and analysis via ESI-HRMS and custom mass defect software tools

Sample preparation of complex, natural mixtures such as lignin prior to mass spectrometry analysis, however minimal, is a critical step in ensuring accurate and interference-free results. Modern shotgun-MS techniques, where samples are directly injected into a high-resolution mass spectrometer (HRMS) with no prior separation, usually still require basic sample pretreatment such as filtration and appropriate solvents for full dissolution and compatibility with atmospheric pressure ionization interfaces. In this study, sample preparation protocols have been established for a unique sample set consisting of a wide variety of degraded lignin samples from numerous sources and treatment processes. The samples were analyzed via electrospray (ESI)-HRMS in negative and positive ionization modes. The resulting information-rich HRMS datasets were then transformed into the mass defect space with custom R scripts as well as the open-source Constellation software as an effective way to visualize changes between the samples due to the sample preparation and ionization conditions as well as a starting point for comprehensive characterization of these varied sample sets. Optimized conditions for the four investigated lignins are proposed for ESI-HRMS analysis for the first time, giving an excellent starting point for future studies seeking to better characterize and understand these complex mixtures.Graphical

Estimation of measurement uncertainty for the determination of loss on drying of biologicals

Scientific relevance. GOST ISO/IEC 17025-2019 requires testing laboratories to evaluate the measurement uncertainty of their results. Estimating the uncertainty of analytical methods intended for biologicals is a challenging task that requires time, effort, and a special approach. Measurement uncertainty estimation is of particular interest in the case of measuring loss on drying (LOD) for biologicals, since LOD testing procedures involve analysing measurements of a physical value, i.e. mass.Aim. This study aimed to estimate the measurement uncertainty of LOD determination in biological medicinal products.Materials and methods. The study examined a powdered active substance intended for a Bifidobacterium product (test sample). The authors conducted the LOD test in accordance with the State Pharmacopoeia of the Russian Federation (OFS.1.2.1.0010.15). Statistical processing of the results was performed using Microsoft Excel. To estimate the measurement uncertainty, the authors employed the bottom-up approach or used the standard deviation from testing results.Results. The authors identified the uncertainty components that affected the LOD determination results. When calculated using the bottom-up approach, the expanded uncertainty was 0.34% (coverage factor, k=2; approximate confidence level, 95%). In particular, the largest contributor to the expanded uncertainty was the uncertainty of measuring the mass of weighing bottles containing dried test samples (0.147%), whereas the smallest contributor was the uncertainty of weighing empty bottles (0.003%). When calculated using the standard deviation, the uncertainty of two parallel measurements amounted to 0.32%.Conclusions. Both approaches to calculating LOD measurement uncertainty yield comparable results. According to the uncertainty budget analysis, the uncertainty of measuring the mass of weighing bottles with dried test samples is the major contributor to the test result. For this reason, the conditions of sample preparation should be carefully controlled. The study results confirm that the LOD measurement uncertainty can be calculated using the standard deviation. Testing laboratory teams may benefit from the methods for identifying the factors influencing LOD test results and the methods for calculating the uncertainty of measurement described in this study.

Suspect Screening of Lupin-Produced Phytotoxins in Environmental Samples

Phytotoxins, secondary metabolites synthesized by plants, are now recognized as a new category of environmental micropollutants. Only a limited number of phytotoxins have been detected and reported in terrestrial and aquatic environments, partially due to analytical challenges. In this article, a robust, reliable, and efficient approach, namely Source Supported Suspect Screening (4S), is introduced for high-throughput analysis of phytotoxins from their plant origin to the downstream environmental compartments. The approach was established on an ultrahigh-performance liquid chromatography–electrospray ionization–quadrupole‑time‑of‑flight-mass spectrometry (UHPLC–ESI–QTOF-MS) analytical platform where optimal sample preparation and chromatographic conditions were investigated and a new suspect screening protocol was suggested.

Evaluating the use of locked nucleic acid capture probes in hybrid LC-MS/MS analysis of siRNA analytes.

Background: Hybrid LC-MS assays for oligonucleotides rely on capture probes to develop assays with high sensitivity and specificity. Locked nucleic acid (LNA) probes are thermodynamically superior to existing capture probes, but are not currently used for hybrid LC-MS assays. Materials & methods: Using two lipid-conjugated double-stranded siRNAcompounds as model analytes, hybrid LC-MS/MS assays using LNA probes were developed. Results: The workflows demonstrated the superiority of the LNA probes, optimized sample preparation conditions to maximize analyte recovery, evaluated the need for analyte-specific internal standards, and demonstrated that advanced mass spectrometric technology can increase assay sensitivity by up to 20-fold. Conclusion: The workflow can be used in future bioanalytical studies to develop effective hybrid LC-MS/MS methods for siRNA analytes.

Optimisation of the analysis procedure of polybrominated diphenyl ethers (PBDEs) in plastic waste from household products

Polybrominated diphenyl ethers (PBDEs) are one group of flame retardants used as additives in many household products, in-cluding plastic products. Determining the residues of PBDEs in household plastic products is required to control their effects on human health and leach into the environment after disposal. This study was conducted to optimise sample preparation conditions for analysing PBDEs in plastic waste from household products. The study results are given that the instrument detection and quantification limits (IDL and IQL) ranged from 4.19 to 28.4 ppb and 14.0 to 94.8 ppb, respectively. In addition, the method detec-tion limit (MDL) and method quantification limit (MQL) were the lowest at 2.20 and 7.35 ng/g (BDE 99) and the highest at 24.1 and 80.3 ng/g (BDE 209), respectively, while the recovery val-ues for spiking samples at different concentrations ranged from 89.1 to 104.5%. The results of the method validation can confirm that the soxhlet extraction and solid phase extraction (SPE) tech-niques combined with gas chromatography mass spectrometry (GC/MS) equipment can be applied to determine the PBDEs in plastic waste samples.

Preparation of a Single-Cell Suspension from Tumor Biopsy Samples for Single-Cell RNA Sequencing.

An essential requirement for single-cell RNA sequencing in cancer is the preparation of high-quality single-cell suspensions from the tumor tissue. In this work, various methods of dissociation of tumor biopsy specimens were analyzed and developed to obtain a cell suspension with at least 80% viability. It was found that the optimal conditions for sample preparation are mechanical dissociation followed by incubation with a collagenase/hyaluronidase mixture with addition of DNAase I for 60 min. Thus, we optimize the approach for preparing single-cell suspensions from the tumor biopsy tissue for single-cell RNA sequencing.

Relevant Issues of Standardisation of Herbal Drugs and Herbal Drug Preparations Containing Essential Oils Samara State Medical University

Essential oils are a common group of bioactive compounds, yet their standardisation remains relevant.The aim of the study was to compare pharmacopoeial approaches to the standardisation of herbal drugs and herbal drug preparations containing essential oils using the examples of the State Pharmacopoeia of the Russian Federation (14th edition) and the European Pharmacopoeia (10th edition).The advantages of Russian pharmacopoeial approaches include testing for additional groups of bioactive compounds that contribute significantly to the pharmacotherapeutic effect of extractive preparations. The main compendial method for the component analysis of essential oils is gas chromatography with a flame ionisation detector; its limitations include the need for a considerable number of reference standards. The essential oil content limits established by the two pharmacopoeias for the same types of herbal drugs are not the same. The differences may be associated with differences in sample preparation conditions and analytical procedures for the determination of essential oil content, as well as with growth conditions of plants used to obtain data sets for the standardisation of this quality attribute. As a particular medicinal plant may have several chemotypes differing in the essential oil composition, it seems relevant to consider this composition in the studies of pharmacological activity of herbal drugs associated with essential oils.