Liquid Chromatographic Method Research Articles

Evaluation of Quercetin's Bioenhancing Effect on Oral Pharmacokinetics of Rosuvastatin in Wistar Rats Using RP-HPLC Method.

The absolute oral bioavailability of rosuvastatin (RST), a secondgeneration statin, is low i.e. 20% and only 10% is recovered as metabolite N-desmethy l rosuvistatin. Since it is a hydrophilic statin, RST relies on the organic anion transporting polypeptide- 1B1 (OATP-1B1), as the key mechanism for active transport into hepatocytes. Quercetin (QUE) being a bio enhancer and inhibitor of OATP1B1 can augment the bioavailability and pharmacokinetics of RST. The present study includes the development of a simple and validated bioanalytical Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method for the estimation of RST and to study the effect of co-administration of QUE as a bio enhancer on its bioavailability. An analytical column of Kromasil 100, C18 (250 mm × 4.6 mm, 5 μm), was used for chromatographic separationand acetonitrile (ACN): acetic acid buffer pH 3.0 adjusted with glacial acetic acid (55:45 Vol. %) as mobile phase with flow rate 1.0 ml/min monitored at 242 nm. The ACN: methanol (50:50 Vol. %) was employed as the final solvent for extraction. The developed method has been successfully applied in a study on the pharmacokinetics of the drug RST in rats after co-administration of QUE, which was carried out using non-compartmental analysis in order to estimate the blood concentration of the drug. The pharmacokinetics of RST was found to be altered significantly (highest concentration of RST in the blood (Cmax) = 67.3 ng/ml to 122.2 ng/ml) (p < 0.001), area under curve (AUC)0-t (p < 0.0001) and AUC0-inf (p = 0.0005) when co-administered with QUE at 120 min (tmax). The results are in accordance with the fact that QUE increases plasma levels in rats through herb-drug interactions.

Evaluation of Novel HLM Peptide Activity and Toxicity against Planktonic and Biofilm Bacteria: Comparison to Standard Antibiotics.

Antibiotic resistance is one of the main concerns of public health, and the whole world is trying to overcome such a challenge by finding novel therapeutic modalities and approaches. This study has applied the sequence hybridization approach to the original sequence of two cathelicidin natural parent peptides (BMAP-28 and LL-37) to design a novel HLM peptide with broad antimicrobial activity. The physicochemical characteristics of the newly designed peptide were determined. As well, the new peptide's antimicrobial activity (Minimum Inhibitory Concentration (MIC), Minimum Bacterial Eradication Concentration (MBEC), and antibiofilm activity) was tested on two control (Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922) and two resistant (Methicillin-resistant Staphylococcus aureus (MRSA) ATCC BAA41, New Delhi metallo-beta- lactamase-1 Escherichia coli ATCC BAA-2452) bacterial strains. Furthermore, synergistic studies have been applied to HLM-hybridized peptides with five conventional antibiotics by checkerboard assays. Also, the toxicity of HLM-hybridized peptide was studied on Vero cell lines to obtain the IC50 value. Besides the percentage of hemolysis action, the peptide was tested in freshly heparinized blood. The MIC values for the HLM peptide were obtained as 20, 10, 20, and 20 μM, respectively. Also, the results showed no hemolysis action, with low to slightly moderate toxicity action against mammalian cells, with an IC50 value of 10.06. The Biomatik corporate labs, where HLM was manufactured, determined the stability results of the product by Mass Spectrophotometry (MS) and High-performance Liquid Chromatography (HPLC) methods. The HLM-hybridized peptide exhibited a range of synergistic to additive antimicrobial activities upon combination with five commercially available different antibiotics. It has demonstrated the biofilm-killing effects in the same concentration required to eradicate the control strains. The results indicated that HLM-hybridized peptide displayed a broad-spectrum activity toward different bacterial strains in planktonic and biofilm forms. It showed synergistic or additive antimicrobial activity upon combining with commercially available different antibiotics.

Green RP-HPLC method for simultaneous quantification of epigallocatechin-3-gallate and rosmarinic acid in lipid-based nanocarriers and biological fluids: Quality by Design-driven optimization and lean six sigma approach

This study presents the development and validation of a reverse-phase high-performance liquid chromatography (RP-HPLC) method for concurrent quantification of EGCG and RA in prepared lipid-based nanocarriers and biological fluids using a QbD approach, greenness assessment, and Six-Sigma methodology. Initially, variations in critical analytical attributes were identified using the Ishikawa fishbone diagram and Risk Priority Number scores. A Taguchi Orthogonal Array design was employed to screen the critical method parameters influencing method development. Systematic optimization of the RP-HPLC method was subsequently achieved using a Box-Behnken design, with the % organic phase, flow rate, and column temperature as the variables. The responses were retention time, tailing factor, and theoretical plates for both EGCG and RA. Chromatographic separation was accomplished with a methanol and 0.1% formic acid (60:40) isocratic flow system on a Phenomenex Luna C18 column (4.6 × 250 mm, 5 μm) at a 1 mL/min flow rate. The method was rigorously validated according to ICH guidelines. Linearity was observed over a concentration range of 2 to 10 µg/mL for both EGCG and RA, yielding correlation coefficients of 0.998 and 0.999, respectively. The LOD and LOQ were determined to be 0.51 µg/mL and 1.54 µg/mL for EGCG, and 0.35 µg/mL and 1.07 µg/mL for RA. Moreover, the %RSD for all validation parameters were consistently below 2%. Forced degradation studies were conducted under acidic, basic, oxidative, and photolytic conditions to elucidate potential degradation pathways and identify degradation products. The developed method was applied to estimate EGCG and RA in prepared lipid-based nanocarriers and biological fluids like blood plasma and urine. Recovery assays in lipid-based nanocarriers, plasma, and urine samples demonstrated excellent recoveries (96.2–102.1%). The method's greenness was assessed using various tools, and its accuracy was evaluated using Six Sigma methodology. Overall, the developed HPLC method offers a rapid, sensitive, and reliable approach for quantifying EGCG and RA, laying the groundwork for their further investigation as anticancer agents, alone and in combination therapies.

A solid phase extraction of bisphenol derivates for their LC-PDA-MS/MS determination in beverages of the Turkish market and its greenness, whiteness, and blueness assessment

Bisphenols transfer from the materials used to food and beverages packaging and disrupt the endocrine system. A solid phase extraction procedure for quantitatively determining bisphenol A, F, and S was developed here. The quantitative determination of these bisphenols was carried out by Liquid Chromatography with ultraviolet followed by tandem mass spectrometry for all types of food analysis laboratory. Analytes separation was carried out by a SUPELCO Ascentis® Express C18 (100×3.0mm, ID, 2.7µm) column with gradient elution mode. All samples were extracted with using Chromabond C18 (500mg, 6mL)ec cartridges before injection to concentrate the analyte. The working range was 0.5-20µg/mL in bisphenol S and bisphenol F, 1.8-72.0µg/mL for bisphenol A in ultraviolet; and 0.09-3.60µg/mL for bisphenol S, 1.5-60.0µg/mL for bisphenol F and 2.5-72.0µg/mL for bisphenol A in tandem mass spectrometry. The recovery range for the new solid phase extraction with liquid chromatography method developed is between 92.20% and 96.92%. This is the first time, 59 types of beverages, except alcoholic and dairy products, and 16 brands of bottled water, were analyzed in Turkish markets. Bisphenol derivatives were measured at LOD levels in 2 drinking waters and 26 beverages, and above LOQ levels in 6 beverages. The greenness score of the developed method is calculated as 0.6, blueness as 65.0 and whiteness as 90.8 and 92.6 for PDA and tandem mass detector, respectively. As a result, the proposed method is green, sustainable, and applicable.

Rapid dual-modal detection of two types of pesticides in fruits using SERS-based immunoassay

Pesticide residue is a harmful chemical residue produced in agricultural production, posing a threat to human health. To detect mixed pesticide residues in food rapidly and simultaneously, we present a dual-channel dual-modal test strip immunoassay. According to the standard colorimetric bar, the naked-eye test can achieve semi-quantitative detection. By collecting the Raman spectra of the signal probes, surface-enhanced Raman scattering (SERS) detection can achieve accurate quantification of pesticide residues. Specifically, a double-layer 4-mercaptobenzoic acid (4-MBA) molecule-labeled bimetallic core-shell material (AuMBA@AgMBA) was conjugated with target antibodies to obtain signal probes. Here, antibody bonding was achieved by the dehydrated condensation of its amino group with the carboxyl group of the outer 4-MBA. In addition, the Au nanoparticles were connected to rabbit IgG by electrostatic adsorption as indicating probes to ensure the normal operation of the test strips. Under the optimal parameters, the detection ranges of acetamiprid and carbendazim were 0.3 - 2μg/kg and 3 - 30μg/kg, respectively. Furthermore, the recovery experiments showed that the results of the SERS method matched well with the high-performance liquid chromatography method. The SERS immunoassay is characterized by high sensitivity, rapidity and reliability, which has potential to simultaneously detect mixed pesticides in the field.

Quality enhancement of potato chips through acrylamide mitigation and comparison with local potato chips in Bangladesh

Potato chips are popular and frequently consumed ready-to-eat food in the world. Acrylamide is a possible carcinogenic and neurotoxic compound formed in potato chips during heat processing, a public health concern. This study aimed to develop potato chips to mitigate acrylamide formation, followed by Food and Drug Administration (FDA) guidelines with some modifications. A total of 15 local chip samples were collected by a stratified random sampling method and were assessed for nutritional values, sensory attributes, microbial quality, and acrylamide levels. Acrylamide was extracted from potato chips using a novel activated charcoal method, then determined by the High- Performance Liquid Chromatography (HPLC) method and showed that only 40% of local potato chips were above the benchmark level established by the EU 2017/2158 (750 ppb) (parts per billion) and ranged from 461 ppb to 2129 ppb while in the developed chips it was not detected. The microbial results showed that the developed chips are safe according to Bangladesh Standards and Testing Institution (BSTI) standard, while the local chips contain total viable count (TVC) (&gt;104 ) and yeast-mold count in a significant amount (&gt;103 ). Sensory evaluation results showed that the developed potato chip samples had high ratings for all the evaluated attributes. From the nutritional point of view, fat content is lower while protein and fiber content is higher in developed potato chips. Moisture content is significantly higher in local potato chips than in developed potato chips, which is the major concern for microbial quality. As a result of the study's findings, acrylamide formation in potato chips was successfully mitigated by adhering to FDA modification guidelines without compromising quality, despite the fact that many locally produced potato chips are unfit for human consumption.

DEVELOPMENT AND OPTIMIZATION OF HPLC METHODS FOR THE CHIRAL SEPARATION OF ARYLPYRAZOLE, CHLOROACETANILIDE AND ORGANOCHLORINE PESTICIDES: CASE STUDIES OF BENZOBICYCLON, ACETOCHLOR AND CHLORDANE.

This work is centered on the development and optimization of High-Performance Liquid Chromatographic methods for the separation of chiral pesticides, particularly benzobicyclon, chlordane and acetochlor employing Agilent Technologies Infinity II chiral HPLC system equipped with a UV – 2000 detector. The stereoisomers were successively separated using a standard analytical column of dimensions 250 mm X 4.6 mm. The Cis– stereoisomers of chlordane exhibited partial baseline separation while the Trans – enantiomers were fully separated. Benzobicyclon and acetochlor enantiomers attained full baseline separation under optimized conditions. Chlordane and benzobicyclon were analyzed using normal phase chromatography [NP]c with a mobile phase of 90% Normal hexane and 10% Isopropanol, while acetochlor was analyzed by reverse phase chromatography [RP] c using 70% methanol and 30% water. Benzobicyclon was later spiked into soil sample and subsequently extracted using Environmental Protection Agency (EPA) methods and the extracts were further analyzed with a shorter analytical column of dimensions 75 mm X 4.6 mm. The HPLC separation revealed that the longer column provided more efficient separation for the benzobicyclon stereoisomers than the shorter column as reflected by high separation factors (?). The high recovery rate of the stereoisomers of benzobicyclon and the internal standard from the soil extracts confirmed the effectiveness of the method of extraction and the HPLC system employed.

Advancing Targeted Metabolomics Using Cyanopropyl-Based Liquid Chromatography Tandem Mass Spectrometry.

The change of metabolic pathways is recognized as the key to disease discovery prompting the development of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-based quantitative platforms to explore the dynamic metabolite profiles of organisms. In this study, a liquid chromatography method based on cyanopropyl (CN) was developed. By adjusting the pH environment of the column, we achieved the elution of 51 metabolites spanning the most comprehensive set of biological pathways currently known. Offering rapid chromatography, efficient separation, and green chemistry benefits, the method encompasses nucleosides and nucleotides, the oxidative-redox metabolome, the glycolysis pathway, the pentose phosphate pathway, the purine de novo pathway, amino acids, and neurological disorder-related metabolites. The mass spectrometry was equipped with electrospray ionization in both positive and negative modes with scheduled multiple reactions monitoring. The validation of the method involved a comprehensive assessment of linearity, accuracy, precision, and matrix effect. The linear range was from 1.0 to 2000 ng mL-1 with a high correlation coefficient (r > 0.99). The LOD ranged from 0.1 to 10 ng mL-1, and the LOQ ranged from 0.1 to 25 ng mL-1. The overall recovery ranged from 81.3% to 117.8%, with RSD < 15.1%. Subsequently, an analysis of metabolites was conducted in dSH-SY5Y neuroblastoma cells with 6-hydroxydopamine, a commonly used neurotoxin in neurodegenerative diseases. The results demonstrate that neurotoxin-induced mitochondrial damage significantly altered related analytes, corroborating previous estimates and validating the feasibility and reliability of the bioanalytical platform.

Development and Validation of Rapid and Cost-Effective RP-HPLC/UV Method for Determination of Cetirizine/Levocetirizine in Blood Plasma of Smokers and Non Smokers: a Two Cohort Comparative Study.

In this research study, a simple, rapid, sensitive, and cost-effective Reverse Phase High Performance Liquid Chromatography method was developed and validated for determination of Cetirizine and Levocetirizine in human plasma. The drugs were separated on an ACE Generix100-5 C18 RP (250 × 4.6mm, 5μm) column, using Acetonitrile and Distilled water pH7 (59:41v/v) as mobile phase with a flow rate of 1.5mL/min, retention time of 3.39 ± 0.02min, detection wavelength of 230nm, and column oven temperature at 26°C. The drug was extracted from human plasma using Methanol, the percent recovery was 97%. The LOD and LOQ for Cetirizine and Levocetirizine were 0.5ng and 1ng. The method was linear over the concentration range of 0.5-1000ng /mL for Cetirizine and Levocetirizine. The method was applied for analysis of drugs in plasma following oral administration of Cetirizine (10mg) and Levocetirizine (5mg) in smokers and nonsmokers of Pashtun ethnicity belonging from different districts of Khyber Pakhtunkhwa, Pakistan. The difference between Cetirizine plasma levels in smokers and non-smokers was significant with p -value 0.0279.

Novel analytical approach for baclofen quantification in rodent plasma.

A simple, accurate, and robust reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantification of Baclofen in rat plasma. The method demonstrated high degree of linearity (r2 = 0.9936) across a concentration range of 10-50 μg/mL. Precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), and robustness were evaluated according to ICH guidelines. The LOD and LOQ were found to be 0.076197 and 0.23090 μg/mL, respectively. This method provides an efficient approach for Baclofen quantification in plasma, making it suitable for pharmacokinetic and bioavailability studies. The novelty of this study lies in its optimization for routine use in laboratories, ensuring reproducibility with minimal variations across different conditions and analysts.

The efficacy of high pressure liquid chromatography (HPLC) in detecting congenital glycosylation disorders (CDG)

Abstract Objectives Congenital disorders of glycosylation (CDG) are a family of rare inherited metabolic disorders. This study aimed to examine the carbohydrate-deficient transferrin (CDT) screening results of 1,328 patients with suspected CDG by using transferrin- high pressure liquid chromatography (Tf- HPLC) method and to evaluate the performance of the method as a reference diagnostic tool. Methods Relative CDT levels (CDT concentrations expressed as percent of total transferrin) were determined in serum samples by HPLC. The method sensitivity, specificity and positive predictive value (PPV) were further calculated. Results Abnormal transferrin isoform profile consistent with CDG Type-I and CDG Type-II were determined in 50 cases; in 44 cases asiolo-Tf (7.63 ± 5.44 %) and disialo-Tf (36.29 ± 9.04 %), in six cases monosialo-Tf (3.95 ± 0.95 %) and trisialo-Tf (25.05 ± 4.46 %) were determined and decreased tetrasialo-Tf (49.75 ± 11.59 %) was identified in all cases. Two cases having abnormal CDT pattern were molecularly diagnosed with hereditary fructose intolerance and galactosemia and 11 cases diagnosed with CDG based on clinical and molecular analysis showed a normal pattern. The sensitivity, specificity and positive predictive values of Tf-HPLC method were 81.96 %, 99% and 96 %, respectively. Conclusions Tf-HPLC is a useful, highly sensitive, cost-advantageous and reliable method for the detection and preliminary diagnosis of CDG for laboratories working with large sample series.

Multi-Indicator Comprehensive Quality Evaluation of Turpinia arguta (Lindl.) Seem Herbs at Different Harvesting Periods

This study aimed to investigate differences in the leaf biomasses, chemical compositions, and pharmacological activities of Turpinia arguta histocultured seedlings and live seedlings at different harvesting periods (S1, S2, S3, and S4) in order to determine the optimal harvesting period. Eleven indexes, including biomass, key active components, secondary metabolites, nutrient content, and antioxidant activity, were evaluated by high-performance liquid chromatography and colorimetric methods during different harvesting periods. The weights of the 11 indexes were calculated by principal component analysis, and then a comprehensive quality evaluation was performed. The results showed significant differences in leaf biomasses, key active components, secondary metabolites, nutrient contents, and antioxidant activities between the different harvesting periods. The highest quality score was obtained for the S4 period, indicating that the quality of T. arguta was the best at this time and that the S4 period was the most suitable harvesting period. At this period (S4), the medicinal component content, antioxidant activity, and comprehensive quality score of the histocultured seedlings of T. arguta were higher than those of the live seedlings, indicating the importance of tissue culture technology in enhancing the quality of T. arguta. This study provides more novel and abundant information and reference for determining the appropriate harvesting period of T. arguta, with the aim of providing newer scientific guidance for the management of herbs.

Chromatographic analysis of ponatinib and its impurities: method development, validation, and identification of new degradation product

BackgroundPonatinib, a third-generation tyrosine kinase inhibitor, is employed in the management of adult chronic myeloid leukemia. Nevertheless, the presence of process impurities and degradation impurities linked to ponatinib may potentially influence its effectiveness and safety. Therefore, the objective of this research was to establish a robust liquid chromatography method and systematically validate it for the detection of substances related to ponatinib.MethodsThe separation of ponatinib and its impurities was conducted using an Agilent 5HC-C18 chromatographic column (4.6 mm × 250 mm, 5 μm). The mobile phase A comprised a mixture of water and acetonitrile in a 9:1 ratio, with an aqueous solution of pH 2.4 containing 2 mM potassium dihydrogen phosphate and 0.4% triethylamine. Mobile phase B, consisting of acetonitrile, was eluted in a gradient fashion. The flow rate was set at 1.0 mL/min, detection wavelength at 250 nm, column temperature at 40°C, and injection volume at 10 μL.ResultsThe method demonstrated high specificity, sensitivity, solution stability, linearity, precision, accuracy, and robustness. Additionally, this research unveiled a novel compound, imp-B, generated via the oxidative degradation of ponatinib. The molecular structure of the newly discovered product was elucidated through the utilization of nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS).ConclusionIn conclusion, the chromatographic method developed in this study has the potential to be utilized for the detection of ponatinib and its impurities, thereby offering significant insights for quality assessment in ponatinib research.

Early Diagnosis of Tumorigenesis via Ratiometric Carbon Dots with Deep-Red Emissive Fluorescence Based on NAD+ Dependence

The early diagnosis of tumorigenesis is crucial for clinical treatment, but the resolution and sensitivity of conventional short-wavelength biomarkers are not ideal because of the complicated interference in living tissue. Herein, a nicotinamide adenine dinucleotide (NAD+)-responsive probe with deep-red emissive ratiometric fluorescence was synthetized as a promising target for energy metabolism patterns during tumorigenesis. Interestingly, the solvents H3PO4 and 2,2′-dithiodibenzoic acid enhanced the red emission (640 and 680 nm) of o-phenylenediamine-based carbon dots (CDs), leading to the formation of a nanoscale graphite-like skeleton covered with -P=O, -CONH-, -COOH and -NH2 on their surfaces. Meanwhile, this method exhibited high sensitivity to the discriminating target NAD+, with a detection limit of 63 μM due to the inner filter effect and fluorescence resonance energy transfer process between NAD+ and CDs, which is superior to the reported capillary electrophoresis and liquid chromatographic detection methods (the reported detection limit was about 0.2 mM) in complex biological samples and even cancer cells. Encouragingly, NAD+ significantly promoted nucleus-targeting fluorescence and cell migration compared to GSH and pH stimulation, which were gradually eliminated in human hepatocellular carcinoma (HepG2) cells after 2-deoxy-d-Glucose inhibited the glycolytic phenotype. The proposed method holds great potential for the temporal and spatial resolution of NAD+-dependent tumor diagnosis in complex living systems.

Eco-friendly RP-HPLC approach for simultaneously estimating the promising combination of pentoxifylline and simvastatin in therapeutic potential for breast cancer: Appraisal of greenness, whiteness, and Box–Behnken design

Abstract Breast cancer affects millions of women worldwide. This study explores the potential of combining pentoxifylline (PTX) and simvastatin (SIM) as a treatment for breast cancer. We aimed to develop six sustainability tools using green and white metrics to evaluate the environmental impact of reversed-phase high-performance liquid chromatography (RP-HPLC) methods for analyzing and separating PTX and SIM in their pure forms. The tools include analytical GREEnness, green analytical procedure index, Complexgreen analytical procedure index, analytical greenness metric for sample preparation, blue applicability grade index, and the RGB 12 algorithm. For the separation, we used a Novapack C8 column (15 × 0.46 cm, 5 µm) at 25°C. The injection volume was 5.0 µL, the wavelength was set to 210 nm, and the total runtime was 5 min. We identified optimal chromatographic conditions efficiently using the Box–Behnken design with minimal trials. We investigated the effects of three factors on retention time and resolution: acetonitrile ratio, pH, and flow rate. We used overlay plots with a 60:40 ratio (v/v) of acetonitrile and bi-distilled water to forecast the most effective mobile phase. The calibration curves for PTX and SIM showed a correlation value of over 0.999 within the range of 5–60 µg·mL−1. The recovery rates ranged from 99.9% to 100.2%, indicating high accuracy. Our RP-HPLC technique proves to be reliable and efficient for the simultaneous estimation of multiple anticancer drugs. We evaluated the environmental sustainability of this approach using green and white metrics, and the recommended method has been thoroughly validated according to International Council for Harmonisation guidelines, making it highly reliable for analyzing new formulations.

Development and Validation of Stability Indicating RP-HPLC Method for the Estimation of Pirtobrutinib and Characterization of its Degradants by LC - MS

Background: Pirtobrutinib is a novel non-covalent BTK inhibitor used to treat adult patients with relapsed/refractory mantle cell lymphoma and B-cell leukemias. The mechanism of action involves binding and inhibition of Bruton's tyrosine kinase (BTK). Objective: The main goal of the current work was to create a selective liquid chromatographic method (RP-HPLC) that is easy to use, accurate and exact for quantifying Pirtobrutinib and its degradation products, thereby seeking insight into the drug’s degradation behaviour. Methods: Using an isocratic mode and a 50:50 mixture of acetonitrile and buffer solution (0.1% orthophosphoric acid) as the mobile phase, a High Performance Liquid Chromatographic System with a PDA detector and an X-Bridge Phenyl column (150 x 4.6mm, 3.5μm) at a flow rate of 1.0 ml/min was able to achieve good chromatographic separation. At 219 nm, the detection was carried out. A retention time of 2.271 minutes was discovered. To ascertain the drug's degrading properties and stability, forced degradation tests were carried out, leading to the development of the RPHPLC method. The chemical structures of the degradation products were clarified and their fragmentation mechanisms were suggested using LC-MS. Results: The suggested approach demonstrated a linearity within the 25-150% (2.5 to 15μg/mL) concentration range, with a correlation coefficient of 0.9999. The precision of the system (measured by % RSD=0.49) and the method (measured by % RSD=0.86) were all within the acceptable limits set by ICH guidelines, with % RSDs less than 1% and less than 2% for system precision and method precision, respectively. The LOD was 0.3μg/mL, and the LOQ was1μg/mL. Pirtobrutinib underwent rigorous tests for forced degradation under the specified conditions outlined in ICH Q1 (R2) guidelines. Pirtobrutinib was primarily broken down in acidic, alkaline, peroxide, and thermal conditions. It was found to remain stable under reduction, photolytic, and hydrolytic conditions. Through LC-MS analysis, the chemical structures of the resulting degradation by-products were identified, along with the proposed pathways of their fragmentation. Conclusion: The current study gives insight into Pirtobrutinib’s degradation behaviour. Pirtobrutinib was stable in reduction, photolytic, and hydrolytic conditions but degraded more readily in acidic, alkaline, peroxide, and thermal environments. The degradation products were characterized as 4-carbamoyl- 5-chloro-3(4-((5-fluoro-2-methoxybenzamido)methyl)phenyl)-4,5-dihydro-1H-pyrazol-5-aminium (acid impurity, DP1), N-(4-(4,5-diamino-1H-pyrazol-3-yl)benzyl)-5-fluoro-2-hydroxybenzamide (alkali impurity, DP2), 5-amino-3-(4-((s-fluro-2-methoxybenzamido)methyl))-4,5dihydro-1H-pyrazol-5-aminium (peroxide impurity, DP3) and N-(4-(4-acetyl-5-amino-4,5-dihydro-1H-pyrazol-3-yl)benzyl)-5-fluoro-2- methoxybenzamide compound with λ1-oxidane (1:1) (thermal impurity, DP4) and their fragmentation pathways were proposed. This study presents the first ever reported method for the quantification of Pirtobrutinib. It ensures precise quantitation of Pirtobrutinib, a new Bruton's Tyrosine Kinase inhibitor, and its degradation products. The method's enhanced sensitivity and regulatory compliance ensure its consistent use in the quantification of Pirtobrutinib.

Acute change in resting energy expenditure and vital signs in response to white tea consumption in females: a pilot study

BackgroundWhite tea, derived from the Camellia sinensis plant like other teas, uses tender buds and young leaves and undergoes minimal processing. This results in higher levels of antioxidants and bioactive substances, which may enhance thermogenesis more effectively than other teas. This first human study aimed to investigate the acute effects of white tea consumption on resting energy expenditure (REE) and some vital signs, including blood pressure (BP), heart rate (HR), and body temperature (BT).MethodsThirty-two healthy female volunteers with normal initial BP and whose caffeine intakes were < 300 mg/d were enrolled in the study. The caffeine and total phenolic content of white tea samples were determined by the high-performance liquid chromatography method and the Folin-Ciocalteu colorimetric method, respectively. After baseline measurements, participants consumed white tea containing 6 mg of caffeine per kilogram of lean body mass, and the white tea was prepared with bottled drinking water at 80 °C and brewed for 3 min. REE, BP, and BT were assessed at various intervals (baseline, 30 min, 120 min, and 180 min) post-consumption of the white tea.ResultsThe results revealed a significant increase in REE by 8.7% at 180 min after the consumption. In particular, there was a substantial difference in both values between the intervals of 30 min to 180 min and baseline to 180 min for REE (p < 0.05). Maximal oxygen consumption and BT also increased significantly over time (p < 0.05) and the observed increment in BT suggests a thermogenic effect associated with white tea consumption. However, systolic BP, diastolic BP, and heart rate showed no significant difference.ConclusionsThese findings suggest white tea consumption may acutely enhance REE and maximal oxygen consumption, so the results are promising for body weight management. This study is the first human study in the literature about the effects of white tea on energy expenditure and vital signs.

Integrated approach to predicting habitat suitability and evaluating quality variations of Notopterygium franchetii under climate change.

The impact of climate change on medicinal plants has significantly altered their suitable environments, thereby affecting the quality of herbal medicines. Notopterygium franchetii (N. franchetii), a medicinal plant intricately linked to its natural habitat, exhibits substantial quality variations influenced by the ecological conditions of its native region. In this study, comprehensive field surveys were conducted to gather occurrence records and samples of N. franchetii. The Maxent model and ArcGIS software were employed to predict the suitable habitats of N. franchetii during different time periods. A high-performance liquid chromatography (HPLC) method was developed to establish the chemical fingerprints of 21 sample batches. Fourteen common peaks were subjected to cluster analysis, principal component analysis, and orthogonal partial least squares-discriminant analysis. The findings revealed quality variations correlated with their geographic origins, identifying peaks 10, 1, 14, 3, and 4 as crucial for quality differentiation. The study indicates that precipitation, temperature, and altitude significantly influence the distribution of N. franchetii. Under current climate conditions, the suitable habitat area for N. franchetii is estimated to be approximately 94,637.33 km2. However, projections under three future climate scenarios suggest a declining trend in suitable habitat areas. A quality zoning map of N. franchetii was developed, integrating a correlation model between chemical composition and environmental variables with the spatial analysis and visualization capabilities of ArcGIS. The high-quality regions for N. franchetii are predominantly located in the Gannan, Linxia, Dingxi, Longnan, and Wuwei districts. These research outcomes offer a valuable reference for identifying suitable cultivation areas and assessing the quality of N. franchetii in Gansu Province.

Method development and validation of an analytical quality by design ultrafast liquid chromatographic method for the determination of bedaquiline from pharmaceutical bulk and nanoemulsions.

Bedaquiline (BDQ) is a drug used to treat multidrug-resistant tuberculosis (MDR-TB). It exhibits exposure-dependent efficacy in eliminating Mycobacterium tuberculosis (Mtb). An easy, efficient and precise reverse-phase ultrafast liquid chromatography (RP-UFLC) method was developed to validate the free base of the antitubercular medication BDQ. BDQ was separated using a 10:90 v/v mobile phase of ammonium acetate buffer solution (pH = 5.4) and high-performance liquid chromatography-grade methanol, with a flow rate of 1.5mL/min and a UV detection wavelength of 226 nm. By using the Box-Behnken design (BBD) and response surface methodology (RSM), the method was optimised by varying critical analytical attributes (CAA) and critical performance attributes (CPAs) namely ammonium acetate fraction (%), flow rate (ml/min), buffer system molarity (M) and pH. BDQ was eluted at 7.5min utilising isocratic elution. The method was linear in the concentration range of 0.5-300 μg/mL with limit of detection values of 0.039 μg/mL and limit of quantification of 0.12 μg/mL. The results indicate that this validated method can be used as an alternative method for assay of BDQ.

From historical drugs to present perils: UHPLC-QqQ-MS/MS determination of methaqualone and its designer analogs (NPS) with comprehensive fragmentation pathways study (QTOF)

Methaqualone, introduced in the 1960s as a sedative-hypnotic alternative to barbiturates, was withdrawn from the market due to its side effects and growing recreational use. Despite this, interest in methaqualone and its analogs remains high, raising concerns about potential abuse in the future. An ultra-high-performance liquid chromatography method coupled with triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) was developed to determine nine methaqualone-related compounds simultaneously. Biological samples were prepared using liquid-liquid extraction with ethyl acetate at pH 9, quantification was performed in blood using multiple reaction monitoring (MRM) mode. Methaqualone-d7 served as an internal standard. The limit of quantification (LOQ) ranged from 0.1 to 0.2 ng/mL, with precision and accuracy within 20%. Recovery ranged from 84.2% to 113.7%. The developed method allowed chromatographic separation of all compounds tested, including two structural isomers: methtylmethaqualone and etaqualone. The mass spectra aquired from quadrupole time-of-flight mass spectrometer allowed for the elucidation of comprehensive fragmentation study of methaqualone derivatives. The described situation poses a significant problem from the analytical point of view, as well as interpretation and forensic toxicological expertise. The developed method will contribute to increased analytical capabilities and enhanced detection of compounds from the methaqualone group that may appear on the illicit market.