μL Injection Research Articles

Virtual Instruments for Peak-Overlapping Studies to Determine Low- and High-Concentration Components with Ion Chromatography: Potassium and Sodium.

We developed the LabVIEW-based virtual instruments (VIs) to bridge a gap in commercial software and to enable systematic peak-overlapping studies to recognise the concentration levels enabling reliable simultaneous determination of major and minor constituents in samples with wide concentration proportions. The VIs were applied to a case study of the ion chromatographic determination of potassium as minor and sodium as a major ion with an IonPac CS12A column and 50 μL injection loop. Two successive studies based on multilevel two-factorial response surface experimental designs, (1) a model peak-overlapping study based on single-ion injections, and (2) an accuracy and precision study, provided guidelines for real sample analyses. By adjusting sample dilutions so that the sodium mass concentration was set to 340 mg/L, the simultaneous determination of potassium in the presence of sodium was possible in samples with sodium over potassium concentration ratios between 14 and 341. The relative expanded uncertainty associated with potassium ion determination was between 0.52 and 4.4%, and the relative bias was between -3.8 and 1.9%. We analysed Ringer's physiologic solutions, standard sea, trisodium citrate anticoagulant, and buffered citrate anticoagulant solutions. We confirmed that the VI-supported peak-overlapping studies contributed to the quality of results by enabling the evidence-based choices of concentration levels adjusted by a dilution.

Piperine exerts anti-inflammatory effects and antagonises the properties of celecoxib and ketoprofen: in vivo and molecular docking studies

This study evaluates the anti-inflammatory effects of a natural product, piperine (PPN), using in vivo and in silico methodologies. In the in vivo segment, inflammation was induced in the right hind paw of young chicks via a formalin (50 μL) injection. PPN was orally administered at doses of 25 and 50 mg/kg with or without celecoxib (CXB) and/or ketoprofen (KPN) (42 mg/kg). The vehicle acted as the negative control group (NC). The in silico analysis predicted the drug-likeness, pharmacokinetics, and toxicity profile of PPN, along with evaluating its binding affinity and ligand-receptor interactions. Results indicate that PPN significantly (p < 0.05) reduced licking frequency and paw edoema in a dose-dependent manner. However, in combination therapy, PPN diminished the effects of both CXB and KPN. PPN showed high affinity (–8.6 kcal/mol) towards the COX-2 enzyme. Therefore, PPN exerts anti-inflammatory effects in chicks through COX-2 inhibition pathways and antagonises CXB and KPN activities.

Development and validation of a stability-indicating HPLC method for assay of tonabersat in pharmaceutical formulations

A stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed to assay tonabersat and assess its stability in pharmaceutical formulations. Chromatographic separation was achieved using a Kinetex® C18 column (2.6 µm, 150 x 3 mm, 100 Å) at 50 °C, with a 20 µL injection volume. A linear gradient of acetonitrile in water (5 – 33.5 %) was applied for 1 min, followed by a gradual increase to 100 % over 26 min at a flow rate of 0.5 mL/min. Tonabersat and its degradation products were detected at 275 nm and 210 nm, respectively. The optimized method was used to evaluate the stability of tonabersat in lipid-based pharmaceutical formulations at 5 ± 3 °C, 25 ± 2°C/60 ± 5 % RH, and 40 ± 2 °C/75 ± 5 % RH over 3 months. The method was validated as per ICH guidelines and demonstrated linearity in the range of 5 – 200 µg/mL (R2 = 0.99994) with good accuracy (98.25 – 101.58 % recovery) and precision (% RSD < 2.5 %). The limits of detection and quantitation were 0.8 µg/mL and 5 µg/mL, respectively. Forced degradation studies showed significant degradation on exposure to alkaline (90.33 ± 0.80 %), acidic (70.60 ± 1.57 %), and oxidative stress (33.95 ± 0.69 %) at 70 °C, but no degradation was observed on exposure to thermal or photolytic stress. No chemical degradation was observed in either formulation on storage. Thus, the method was sensitive, specific, and suitable for stability testing of tonabersat in pharmaceutical formulations.

A Quality by Design (QBD) Approach for the Development and Validation of RP-HPLC Method for the Quantification of Silymarin Tablet

This study emphasizes the crucial role of Quality by Design (QbD) in developing pharmaceutical procedures, particularly in risk assessment. It demonstrates how QbD principles were applied to create a precise and effective HPLC method for Silymarin Tablets, ensuring consistent quality within specified criteria. The optimized method, developed using a Design of Experiment approach, employs a C18 column (150 mm x 4.6 mm, 5μm) with isocratic elution using a 95:25 ratio of acetonitrile to orthophosphoric acid buffer (pH 3). Peaks were detected using a PDA detector calibrated at 287 nm, with a flow rate of 1.0 mL/min. The column oven temperature was maintained at 25°C, and a 10 μL injection volume was used. Thorough validation, adhering to USP &lt;1225&gt; and ICH Q2 (R1) standards, ensures the method's reliability. Key factors such as accuracy, precision, robustness, limit of detection (LOD), and limit of quantification (LOQ) were comprehensively assessed. The method exhibits exceptional sensitivity, selectivity, efficiency, precision, accuracy, and cost-effectiveness, making it ideal for pharmaceutical analysis of Silymarin tablets. It has been validated to effectively differentiate between marketed products, including those closely resembling the original. This method is intended for routine quality control analysis in the pharmaceutical industry, highlighting its suitability and reliability for ongoing use.

Development and Validation of a Simple HPLC–UV‐Based Bioanalytical Method for Estimation of Acalabrutinib in Rat Plasma and Its Application in Evaluation of Drug‐Loaded Nanocrystal Formulation

ABSTRACTA robust and reproducible HPLC–UV‐based bioanalytical method for acalabrutinib (ACP) was developed and validated in rat plasma using prednisone as internal standard. The samples were prepared by single‐step protein precipitation method using methanol with 1% v/v formic acid. A polar C18 stationary phase was used with a combination of 10 mM ammonium acetate (pH 3.5) and methanol (60:40 ratio), flow rate 1.1 mL/min, and 50 µL injection volume. ACP and internal standard were retained at ∼5.96 and 8.29 min, respectively. The method was validated according to the United States Food and Drug Administration and ICH M10 bioanalytical method validation guidelines. It was linear in the calibration range (0.08–5 µg/mL) with as 0.9989; accurate (% bias ≤ 0.50 ± 0.67), and precise (%RSD ≤ 0.986) for any quality control standard. Further, the method was applied in estimating ACP in rat plasma samples collected during in vivo pharmacokinetic study of the bulk drug as well as a nanocrystal (NC) suspension formulation. ACP‐NCs were formulated by bottom‐up solvent–antisolvent precipitation with mean particle size 266.32 ± 29.90 nm, 0.25 ± 0.05 PDI, with 92.7% ± 2.3% yield. The NC formulation improved both in vitro dissolution and in vivo absorption, supporting a further development of ACP‐loaded nanodrug delivery systems.

Development and Validation of HPLC-UV Method for the Determination of Levothyroxine in Orodispersible Minitablets for Pediatric Application

Background: Thyroid hormones play an important role in cognition and brain development. The determination of the content of levothyroxine, as well as related substances and dissolution test analysis, should be carried out by methods that are selective and highly sensitive due to the low concentration used in low dose orodispersible minitablets Objectives: This study aims to develop and validate an analytical method by HPLC-UV for the quantification of levothyroxine and its related substances in pharmaceutical formulations; looking forward to being this method suitable for a future dissolution test analysis using tandem mass spectrometry detector. Methods: A Hypersil GOLD C18 (100 x 2.1 mm, 3 μm) column was used with 25°C column temperature, 5 μL injection volume, 0.3 mL/min flow rate and detection at 225 nm. The mobile phase consisted of methanol: 0.05% formic acid (55:45). The developed method was validated for specificity, linearity, precision, accuracy and robustness. Results: The method is linear within the range of 2-20 μg mL-1 (R2=0.9982), which makes the method suitable for the evaluation of levothyroxine in pharmaceuticals formulations. LOQ was 0.17 μg/mL (0.85 ng on column) and LOD 0.05 μg/mL (0.25 ng on column) of LT4. Therefore, in terms of efficiency (1671), retention factor, k (6.79), Tailing factor, T (1.09) and resolution, Rs (5.11) the chromatographic method was found to be suitable according to USP 43. Conclusion: The HPLC UV method was found to be linear, specific, precise, accurate and robust, therefore it is suitable for the quality control of levothyroxine in pharmaceutical ODMTs..

Utilization of Matrix Effect for Enhancing Resolution in Cation Exchange Chromatography.

In ion chromatography studies, the matrix effect of other inorganic ions present in the sample is a well-known phenomenon. In this work, the behavior of inorganic and organic ions was studied in a system overloaded with ammonium ions. The ammonium ions came from a solution of ammonium hydroxide in various concentrations (0.25-1.25%). In this system, which was significantly overloaded with ammonium ions, the behavior of three ions were tested (lithium, tris, and sodium cations). The measurements were performed at different eluent concentrations (6-17 mM), chromatographic column temperatures (25-40 °C), and injected volumes (15-40 µL). The retention times of sodium and lithium ions increased with increasing amounts of injected ammonium, while tris remained essentially unchanged, indicating that the resolution of these ions can be influenced by varying the concentration of the matrix. The results suggested that the observed effect was due to a combination of the pH change caused by the injected matrix, the dissociation of tris ions, the dissociation of the carbocylic ion-exchange groups of stationary phase, the change in buffer capacity, and the amount of ammonium ion introduced. It has been shown that in a well-designed experiment, the addition of ammonium hydroxide to the sample at concentrations greater than 1% can improve the efficiency of organic and inorganic cation separation. It was found that 8 mM methanesulfonic acid eluent, 30 °C, 1% ammonium hydroxide matrix concentration, and 25 µL injection were optimal for the baseline separation of tris and sodium ions on the high-capacity Dionex CS16 column. These ions could not be separated on this column without the presence of the ammonium matrix.

The Development of One New Normal Phase Liquid Chromatography Method and Thermodynamic Investigation of Olodaterol Hydrochloride Enantiomer.

In order to improve and replace the enantiomer method outlined in the olodaterol hydrochloride draft monograph (From the European Pharmacopoeia forum), one new, simple, and fast enantioselective normal phase high-performance liquid chromatography chiral method was developed on polysaccharide-based Chiral MX (2) (4.6 × 250 mm, 5 μm) column. n-Hexane, ethanol, and diethylamine in the ratio of 40:60:0.1 (V/V/V) were selected as mobile phase at a flow rate of 0.8 mL/min, and the detection was performed on a photodiode array detector at 225 nm with 5 μL injection volume. The column temperature was set at 40°C for better peak shape and sensitivity. The analysis time can be shortened to 15 min, whereas the resolution between enantiomer and olodaterol was found to be even more than 10.0, which was far better than that obtained with the reported method in this draft monograph. The developed chiral method was validated in accordance with ICH Q2 (R1), including specificity, LOD&LOQ, precision, linearity, accuracy, and robustness. Thereby, the proposed method was demonstrated to be suitable for the determination of enantiomer inolodaterol hydrochloride bulk drug and drug product. Besides, the thermodynamic parameters were evaluated on the basis of Van't Hoff plots that was used to explain correlative chiral recognition mechanisms with the chiral stationary phase.

Effects of Sodium-Glucose Cotransporter 2 Inhibitors on Transcription Regulation of AgRP and POMC Genes.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors regulate plasma glucose levels in patients with type 2 diabetes mellitus (T2DM) by inhibiting renal glucose reabsorption. This study investigated the impact of empagliflozin (EMPA), an SGLT2 inhibitor, on hypothalamic energy regulation. To directly investigate the role of SGLT2 inhibitors in the hypothalamus, we administered EMPA through intracerebroventricular (i.c.v.) injections into the murine ventricles. After dental cementing the i.c.v. cannula onto the skull, the mice were given 5 days to recover before receiving vehicle or EMPA (50 nM/2 μL) injections. In a high-fat diet (HFD)-induced obesity model, we determined the gene expression levels of agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) in the hypothalamus. Additionally, we assessed FoxO1 expression, which regulates AgRP and POMC gene transcription in hypothalamic cell lines. We found that EMPA directly influenced the expression of endogenous mRNA of POMC and AgRP, which are critical for energy homeostasis, and modulated their transcription in high-fat diet-induced obese mice. Additionally, EMPA affected the expression of FoxO1, a key transcriptional regulator of glucose homeostasis, thereby regulating the transcriptional activity of POMC and AgRP. These results indicate that EMPA significantly influences hypothalamic energy homeostasis, highlighting its potential as a regulator in obesity and T2DM management.

Development of an RP-HPLC Method for the Simultaneous Quantification of Encorafenib and Cetuximab in their Bulk Form

The primary goal of this investigation was to develop and validate a high-performance liquid chromatograophy (HPLC) method that is straightforward, reliable, sensitive, and accurate in quantifying the concentrations of encorafenib and cetuximab in bulk form. A Waters HPLC system with an autosampler and a PDA detector was used to create the HPLC technique. With a particle size of 5 μ and dimensions of 250 x 4.6 mm, a Waters X-Terra RP-18 column was used for the separation. A combination of acetonitrile and 0.1% triethylamine (TEA) at a pH of 2.5 makes up the mobile phase. Acetonitrile to TEA is 40:60 in ratio. A 240 nm wavelength, a 10 μL injection volume, and a flow rate of 1.0 mL/min were used for the chromatographic procedure. Analyte serial dilutions will be evaluated in order to establish linearity. The stock solutions with concentrations ranging from 1.25 to 7.50 μg/mL for cetuximab and 18.75 to 112.50 μg/mL for encorafenib were used to generate these dilutions. The dilutions were made by adding the mobile phase to the stock solutions to create a series. Encorafenib and cetuximab were found to have limit of detection (LoD) and limit of quantitation (LoQ) values of 0.225 and 0.015 g/mL and 0.75 and 0.05 g/mL, respectively. The method that was developed showed a high level of accuracy, as evidenced by the relative standard deviation (RSD) being less than 2%. The developed analytical method for estimating encorafenib and cetuximab is both straightforward and accurate.

LC-MS/MS Bioanalytical Procedure for Atrasentan in Human Plasma: Method Development and Validation

11ABSTRACTObjectives: The objective of this work is to establish a bioanalytical technique for the quantification of atrasentan. The employed methodology for the detection and quantification of atrasentan in human plasma has a high degree of specificity, sensitivity, and simplicity. Methods: Shimadzu pumps were utilized in conjunction with an Autosampler Agilent Zorbax XDB C18 2.1 × 50 mm, 5 μm column employed as a stationary phase to achieve the chromatographic separation. Acetonitrile (0.1% formic acid) and 5 mM ammonium formate made up the mobile phase, which was optimized to have a 70:30% v/v ratio. Isocratic elution was employed in the experiment, with a flow rate of 0.150 mL/min. The entire cycle took 3.0 minutes, using a 10 μL injection volume. Results: The detection procedure is executed utilizing positive ion mode electrospray ionization (ESI) mass spectrometry under atmospheric pressure. For atrasentan, the precursor to product ion transitions used for quantification was m/z 511.623 to 354.15 and for the internal standard, verapamil, m/z 455.22 to 165. 02. The retention times for atrasentan and verapamil were found to be 1.68 and 0.96 minutes, respectively.Conclusion: In accordance with the recommendations of the United States Food and Drug Administration (USFDA) and EMA, it was ascertained that the remaining validation parameters remained within the specified range.

Microvolume analysis of 226Ra by inductively coupled plasma mass spectrometry: Environmental applications to high-resolution profile of wetland soil pore waters

BackgroundThe release to the environment of natural radionuclides like 226Ra due to human activities have raised concerns about human and ecosystem exposure. 226Ra’s resemblance to calcium and involvement in biological processes accentuate the importance of understanding its geochemical behavior and migration pathways. Soil and sediment pore waters provide valuable information but have proved to be challenging to analyze, requiring to push the limits of current sample preparation and detection methods often not scaled for low concentrated microvolumes of samples. A more efficient and accurate analytical method is therefore needed to get reliable data to support hydro-geochemical modeling. ResultsWe describe here a novel comprehensive approach for accurate 226Ra quantification in pore waters of a wetland located downstream of the former Rophin uranium mine. In order to highlight the migration of 226Ra between the different soil layers, microvolumes of soil pore waters were in situ sampled up to 50 cm depth employing Diffusive Equilibrium in Thin-Films (DET) probes. The workflow included spiking DET extracts with an in-house 228Ra tracer, specific solid-phase extraction with 50 mg of AnaLig Ra-01® resin, and elution fraction calcination. This process eliminated interferences and simplify the analysis matrix. Quantification relied on isotope dilution after measurement by ICP-MS hyphenated to a desolvator module and to a microsampler (150 µL injection). The resulting 1 cm-high-resolution profile revealed unusual 226Ra enrichment with depth, signs of Ra mobility from the solid contamination source. These elements are essential for assessing 226Ra distribution coefficients across soil horizons and subsequent wetland remobilization considerations. Significance and noveltyThis first methodological advance for 226Ra constitutes a significant step to understand 226Ra behavior i.e., to quantify its activity level, fluxes inside of wetland soils or at the interface overlying waters/sediments. Its potential extension to studies involving other elements underscores its broader applicability in environmental research. The findings contribute to evidence-based decision-making for environmental protection and management, aiding in the preservation of ecological integrity and human health.

Formation of Multinucleated Giant Cells after Experimental Intracerebral Hemorrhage: Characteristics and Role of Complement C3.

Hematoma clearance is critical for mitigating intracerebral hemorrhage (ICH)-induced brain injury. Multinucleated giant cells (MGCs), a type of phagocyte, and the complement system may play a pivotal role in hematoma resolution, but whether the complement system regulates MGC formation after ICH remains unclear. The current study investigated the following: (1) the characteristics of MGC formation after ICH, (2) whether it was impacted by complement C3 deficiency in mice and (3) whether it also influenced hematoma degradation (hemosiderin formation). Young and aged male mice, young female mice and C3-deficient and -sufficient mice received a 30 μL injection of autologous whole blood into the right basal ganglia. Brain histology and immunohistochemistry were used to examine MGC formation on days 3 and 7. Hemosiderin deposition was examined by autofluorescence on day 28. Following ICH, MGCs were predominantly located in the peri-hematoma region exhibiting multiple nuclei and containing red blood cells or their metabolites. Aging was associated with a decrease in MGC formation after ICH, while sex showed no discernible effect. C3 deficiency reduced MGC formation and reduced hemosiderin formation. Peri-hematomal MGCs may play an important role in hematoma resolution. Understanding how aging and complement C3 impact MGCs may provide important insights into how to regulate hematoma resolution.

Development of HPLC method for isomeric impurities of key starting material of novel oral anticoagulant drug; Edoxaban Tosylate Monohydrate

Reverse-phase high-performance liquid chromatography method has been developed for the determination of EDO-S1 stereoisomeric impurities such as isomer 1, isomer 2, isomer 3, isomer 4, isomer 5, isomer 6 and isomer 7 with good resolution using the column, Bakerbond C18 (150 × 4.6 mm; 3 μm). The separation was achieved with mobile phase-A (10 mM dipotassium hydrogen phosphate pH-7.0 with 10% orthophosphoric acid solution in Milli-Q water) and mobile phase-B (n-Propanol: Acetonitrile ratio of 20:30 % V/V), which consisted of mobile phase mixture in the combination of moilephase-A: mobile phase-B (85:15). The total run time was 30 min at 0.8 mL/min flow rate, 20 µL injection volume and 30 ℃ column oven temperature. The column eluate was monitored at 210 nm to quantify the impurities The method showed adequate specificity, sensitivity, linearity, accuracy, precision, and robustness inline to ICH tripartite guidelines. The limit of detection and quantification limits were 0.1 and 0.3 μg mL−1, respectively, for all isomeric impurities and EDO-S1. The developed method was found to be linear over the concentration range of LOQ to 150% of specification range for isomeric impurities with a correlation coefficient &gt;0.999. The method was precise (%RSD &lt; 5.0), robust, and accurate (with 85%–115% recovery).

Crosstalk Inhibition Between 5-HT2A and Adenosine 2A Receptor-Induced Phrenic Motor Facilitation

Episodic spinal serotonin receptor activation elicits phrenic motor facilitation (pMF), a form of respiratory motor plasticity. Although both intermittent 5-HT2 and 5-HT7 receptor activation elicit pMF, they do so via distinct cellular mechanisms known as the Q (5-HT2A/B) and S (5-HT7) pathways. The Q and S pathways interact via powerful crosstalk inhibition; thus, concurrent activation of both receptor subtypes diminishes/abolishes pMF. At least some of the molecules giving rise to mutually inhibitory interactions between 5-HT2 and 5-HT7 receptors have been identified. Although episodic spinal adenosine 2A (A2A) receptor activation also elicits pMF via the S pathway, we do not know: 1) if 5-HT2 and A2A receptors interact via similar crosstalk inhibition, nor 2) if they do so via the same signaling mechanisms. Thus, we tested the hypothesis that concurrent 5-HT2 and A2A receptor activation diminishes pMF via crosstalk inhibition. Selective 5-HT2A (DOI) and A2A receptor agonists (CGS21680) were delivered to the C4 intrathecal space (3, 6 μl injections, 5-min intervals) in urethane anesthetized, artificially ventilated, vagotomized and paralyzed Sprague Dawley rats. Integrated phrenic nerve burst activity was measured before (baseline), during and 90 minutes post-drug injection. Spinal 5-HT2A and A2A receptor activation both increased phrenic nerve burst amplitude when delivered alone (i.e., pMF); however, pMF was no longer apparent with concurrent receptor activation. The magnitude of pMF, expressed as a % change in phrenic burst amplitude from baseline to 90-min post-injection, was significantly lower with concurrent receptor activation (5-HT2A + A2A= 15 ± 7.7%; n=5) versus 5-HT2A (84 ± 12%; n=5, p=0.0047) and A2A (88 ± 16%; n=5, p=0.0085) alone. Thus, 5-HT2A and A2A receptors interact via crosstalk inhibition similar to 5-HT2 and 5-HT7 receptors. However, the mechanism underlying this crosstalk inhibition remains unknown. NIH HL148030. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Continuous Flow with Reagent Injection on an Inlaid Microfluidic Platform Applied to Nitrite Determination.

A continuous flow with reagent injection method on a novel inlaid microfluidic platform for nitrite determination has been successfully developed. The significance of the high-frequency monitoring of nutrient fluctuations in marine environments is crucial for understanding our impacts on the ecosystem. Many in-situ systems face limitations in high-frequency data collection and have restricted deployment times due to high reagent consumption. The proposed microfluidic device employs automatic colorimetric absorbance spectrophotometry, using the Griess assay for nitrite determination, with minimal reagent usage. The sensor incorporates 10 solenoid valves, four syringes, two LEDs, four photodiodes, and an inlaid microfluidic technique to facilitate optical measurements of fluid volumes. In this flow system, Taylor-Aris dispersion was simulated for different injection volumes at a constant flow rate, and the results have been experimentally confirmed using red food dye injection into a carrier stream. A series of tests were conducted to determine a suitable injection frequency for the reagent. Following the initial system characterization, seven different standard concentrations ranging from 0.125 to 10 µM nitrite were run through the microfluidic device to acquire a calibration curve. Three different calibrations were performed to optimize plug length, with reagent injection volumes of 4, 20, and 50 µL. A straightforward signal processing method was implemented to mitigate the Schlieren effect caused by differences in refractive indexes between the reagent and standards. The results demonstrate that a sampling frequency of at least 10 samples per hour is achievable using this system. The obtained attenuation coefficients exhibited good agreement with the literature, while the reagent consumption was significantly reduced. The limit of detection for a 20 µL injection volume was determined to be 94 nM from the sample intake, and the limit of quantification was 312 nM. Going forward, the demonstrated system will be packaged in a submersible enclosure to facilitate in-situ colorimetric measurements in marine environments.

Impurity assessment, development and validation of an RP-HPLC method for the determination of eleven potential impurities of eltrombopag precursor

Eltrombopag is an oral non-peptide thrombopoietin receptor (TPO-R) agonist indicated for the treatment of thrombocytopenia in patients with persistent or chronic immune thrombocytopenia (idiopathic thrombocytopenic purpura, ITP) or chronic hepatitis C infection and the treatment of severe aplastic anemia. The purpose of this research was to assess the possible impurities that may carry over to eltrombopag from its precursor Eltro-1 (3′-amino-2′-hydroxy-[1,1′-biphenyl]-3-carboxylic acid) and to develop a specific analytical method for the determination of these impurities. Eltro-1 samples synthesized by two different synthesis routes were investigated during the evaluation and method development studies. Besides the expected process-related impurities (Eltro-1A – Eltro‐1J), e.g., starting materials, intermediates, and/or compounds formed from their further reactions, an unknown impurity detected above 0.10% was identified by LC-MS, synthesized and fully characterized by NMR, MS and FTIR (Eltro‐1K). Accordingly, an HPLC-RP method for the determination of eleven impurities (Eltro-1A – Eltro‐1K) in Eltro-1 was developed and validated according to ICH Q2. The control limits for impurities in Eltro-1 were set at ≤ 0.15% for Eltro-1A – Eltro‐1J and ≤ 1.0% for Eltro‐1K based on fate, spike-purge and carryover studies and in accordance with the ICH M7 classification for impurities in drug substance. Eltro-1 and eleven impurities at the specification limit were separated from each other and the diluent peaks with sufficient resolution without interference. Separation was performed on a Waters XBridge C18 column (150 × 4.6 mm, 3.5 μm) at 40 °C with a 10 µL injection volume at a detection wavelength of 220 nm and 15 °C sample temperature. The gradient elution is performed at a flow rate of 1.0 mL/min for 40 min with mobile phase A (0.1% orthophosphoric acid in water) and B (acetonitrile) according to the following program: Time (min) / Acetonitrile (%): 0/0, 35/70, 36/0, 40/0. Test and standard solutions were prepared at a concentration of 1.0 mg/mL and 1.0 µg/mL, respectively, using a mixture of mobile phase A and acetonitrile (75/25) as diluent. This is the first specific, selective, sensitive, linear, precise, accurate, and robust HPLC method for the determination of Eltro-1A – Eltro‐1K in Eltro-1, which showed no significant degradation under thermal stress, photostability (UV and VIS), and standard accelerated and long-term stability conditions.

Development of a Stability Indicating UPLC Method for the Determination of Tirbanibulin in Bulk and Its Pharmaceutical Dosage Form.

The primary goal of this study was to create and validate a simple, precise, sensitive, and accurate ultra-performance liquid chromatography (UPLC) method for estimating tirbanibulin in pure and dosage form. A UPLC technique was developed using a Waters Acquity UPLC Phenyl (100 x 2.1 mm, 1.7 µm) column. The developed technique was validated in accordance with the International Conference on Harmonization (ICH) guidelines. Tirbanibulin was separated chromatographically with high resolution using the mobile phase acetonitrile: buffer (30:70 v/v) at 0.5 mL/min, 5 µL injection volume, and 220 nm wavelength. The validated technique was found to be linear in the 1-15 µg/mL range. The detection and quantification limits for tirbanibulin were 0.03 and 0.1 µg/mL, respectively. The percentage relative standard deviation was less than 2%, demonstrating the precision of the developed technique. Furthermore, the recovery rate was nearly 100%, confirming the accuracy of the method. Minor modifications to the chromatographic conditions demonstrated the robustness of the method. The developed analytical method was precise, simple, reproducible, and sensitive. Consequently, it can be used to determine tirbanibulin.

Improved Efficacy of Delayed Treatment with Human Bone Marrow-Derived Stromal Cells Evaluated in Rats with Spinal Cord Injury.

The treatment of spinal cord injury (SCI) with uncultivated human bone marrow-derived stromal cells (bmSCs) prepared by negative selection has been proposed to be therapeutically superior to treatment with stem cells that were expanded in vitro. To explore their use in clinical trials, we studied the functional effects of delayed application at 7 days after SCI by testing different doses of bmSCs. Spinal cord contusion injury was induced in adult male Wistar rats at the thoracic level T9. Human bmSCs were prepared by negative selection without expansion in vitro (NeuroCellsTM). Treatment consisted of one 150 µL injection into the cisterna magna containing 0.5 or 2.5 million fresh bmSCs or 2.5 million bmSCs. The recovery of motor functions was evaluated during a surveillance period of six weeks (6 W), during which spinal cords were assessed histologically. Treatment resulted in a significant, dose-dependent therapeutic effect on the recovery of motor performance. The histological analysis revealed a lower degree of axonal degeneration and better survival of neurons and oligodendrocytes in bmSCs treated rats. Our results support delayed intrathecal application of bmSCs prepared by negative selection without expansion in vitro as a treatment of SCI.

Development of a rapid, accurate and efficient HPLC method for simultaneous determination of catechins and theaflavins in black tea

Catechins and theaflavins, as the primary bioactive constituents of black tea, have a significant impact on its color, aroma, and flavor profile.We developed a rapid and precise method using HPLC to detect catechins and theaflavins in black tea. The method employed 0.1% formic acid-acetonitrile (phase A) and 1% formic acid-water (phase B) as mobile phases. Separation occurred on an Agilent SB-C18 column (4.6 mm × 250 mm, 5 µm), with 20 μL injection volume. Detection took place at 30 °C with a flow rate of 1 mL/min for 55 min.Quantitative determination utilized the external standard method, exhibiting high sensitivity (limit of detection: 0.19 to 5.14 μg/mL) and a strong linear correlation (R2 ≥ 0.9993) between concentration range and peak area. Precision, repeatability, and stability were excellent, with relative standard deviations (RSDs) below 5% (n = 5). The average recovery rate of standard addition ranged from 98.02% to 102.57%, with an RSD of less than 3.17% (n = 5). In summary, this straightforward, efficient, and precise approach is well-suited for both qualitative and quantitative analysis of catechins and theaflavins in tea. It overcomes the limitations of previous methods, providing a valuable tool for evaluating the quality and composition of black tea.