High-performance Liquid Chromatography Research Articles

A novel method for multi-matrix arsenic speciation analysis by anion-exchange HPLC-ICP-MS in the framework of the third (French) total diet study.

This study presents the development and validation of a precise analytical method for the speciation analysis of arsenic (As) compounds, including inorganic species [As(III) and As(V)] and organic species such as monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). The method employs anion-exchange high-performance liquid chromatography (AE HPLC) coupled with inductively coupled plasma-mass spectrometry (ICP-MS). To optimize the sample preparation process, microwave-assisted extraction (MAE) and heat-assisted extraction (HAE) techniques were evaluated and compared. Separation of all four arsenic species was achieved with baseline resolution within 10 min, utilizing an anion-exchange column and a mobile phase gradient of 0.5 and 5 mM ammonium carbonate with 3% (v/v) methanol at a pH of 9.3. The method validation followed the accuracy profile approach, employing six different food matrices analyzed in duplicate over six separate days within a 6-week period. The method demonstrated good intermediate reproducibility, with coefficients of variation (CVR) ranging from 4.7% to 5.5%, while the bias was < 3%. The limit of quantification (LOQ) for the four species was 6.25 μg/kg (dry weight, dw), and the limit of detection (LOD) was 1.88 µg/kg (dw). These results confirm the robustness, accuracy, and suitability of the method for routine As speciation analysis across a variety of food matrices. The method is specifically intended to be applied to the analysis of a panel of food samples as part of the ongoing (French) third total diet study.

Lycopodiaceae herb from Vietnam as a promising medicinal source of natural hupezine and novel huperzine-producing endophytic fungi.

(1) To evaluate the potential of producing huperzine (Hup) and anticholinesterase (AChE) activities of nine native Lycopodiaceae species collected in Vietnam; (2) Isolation, identification and characterization of a novel fungus producing both HupA and HupB isolated from Lycopodium casuarinoides Spring. All methanolic extracts of nine plants showed AChE inhibition from 8.55 to 71.81%. Of note, Huperzia serrata (Thunb.) Trevis, L. casuarinoides, Lycopodium clavatum L., Phlegmariurus squarrosus (G. Forst.), and P. phlegmaria (L.) T. Sen & U. Sen were shown to biosynthesize both HupA and HupB by high-performance liquid chromatography (HPLC). Plants H. serrata, L. casuarinoides and L. clavatum showed the most potent AchE IC50 inhibition. HupA and HupB concentrations from six plants were greater than those of previously reported Lycopodiaceae species. Sixty-four endophytic fungi were isolated from tissue of natural L. casuarinoides and then screened for HupA- and HupB-production by HPLC. Out of 64 fungal strains, only TTD2-2.7 extract could produce both HupA and HupB with the yields of 0.034 and 0.028µg gdcw-1, respectively. Moreover, TTD2-2.7 extract also had inhibitory effects on AChE with the IC50 of 129.76 ± 4.13µgml-1, which was lower than the extract of host plant L. casuarinoides (94.03 ± 4.13µgml-1). The fungus was identified as Aspergillus sp. TTD2-2.7 by morphological characteristics and Internal Transcribed Spacer sequence analysis. These are the first reports of (1) two species L. clavatum and L. casuarinoides producing both HupA and HupB, and (2) L. casuarinoides as novel sources of Hup-producing endophytic fungi as well as (3) fungus Aspergillus as a novel HupA- and HupB-producing endophyte isolated from L. casuarinoides.

Response to azathioprine treatment in autoimmune hepatitis is dependent on glutathione transferase genotypes.

Azathioprine (AZA) is part of the standard treatment for autoimmune hepatitis (AIH). The first step in the complex bioconversion of AZA to active metabolites is mediated by glutathione transferases (GSTs). Elucidate the association between GSTM1 and GSTT1 copy number variation (CNV), genetic variation in GSTA2, GSTP1, and inosine-triphosphate-pyrophosphatase, and the response to AZA in AIH. Genotyping was performed in AIH patients (n = 131) on AZA, and in a Swedish background population (n = 283). Thiopurine metabolites in blood erythrocytes were determined by high performance liquid chromatography. GSTM1 and GSTT1 CNV were associated with treatment response to AZA. Gene deletion of GSTM1-but not of GSTT1-was associated with the liver transaminase levels. None of the studied genetic variants were associated with the thiopurine metabolite concentrations, suggesting non-enzymatic mechanisms of GSTM1 and GSTT1 in the context of AZA efficacy in AIH. The prevalence of GSTM1 and GSTT1 CNV genotypes was similar in AIH and in the background population. This study shows the effects of GSTM1 and GSTT1 CNV on AZA efficacy in AIH, not previously described. It also elaborates on the impact of the definition of treatment response, on the importance of the various GSTs studied. Furthermore, the GSTM1 and GSTT1 CNV frequencies previously reported in European populations were confirmed.

Heterologous expression of a recombinant ACE inhibitory peptide LYPVK and its potential antihypertensive action mechanism.

Enzymatic hydrolysis approach is commonly employed for preparation of active peptides, while the limited purity and yield of produced peptides hinder further development of action mechanisms. This study presents the biotechnological approach for the efficient production of recombinant angiotensin converting enzyme (ACE) inhibitory peptide LYPVK and investigates its potential antihypertensive action mechanism. DNA encoding sequence of recombinant peptide was designed to form in tandem, which was expressed in Escherichia coli BL21 (DE3). The expressed tandem repeat protein with molecular weight of 13.4 kDa was verified by high performance liquid chromatography (HPLC) and amino acid composition. Subsequently, LYPVK was generated following His-tag removal and trypsin-mediated cleavage of the purified protein, which was performed HPLC and liquid chromatography-mass spectrometry (LC-MS) analysis. LYPVK exhibited an IC50 value of 10.6 ± 0.86 μg/mL, demonstrating a non-competitive mode of action and resistance to gastrointestinal enzyme hydrolysis and heat conditions. Molecular docking results showed that LYPVK interacted with ACE through conventional hydrogen bonds and hydrophobic interactions. Except for ACE, ALB, SRC, PPARG, and MMP9 are identified as potential key targets for its antihypertensive activity by network pharmacological analysis. This study provides a promising biotechnological approach for the preparation of active peptides with high purity and yield.

Selective sensing of terbinafine hydrochloride using carbon-based electrodes: a green and sustainable electroanalytical method for pharmaceutical products.

Terbinafine hydrochloride (TBF) is a broad-spectrum antifungal used to treat various dermatophyte infections affecting the skin, hair, and nails. Accurate, sensitive, and affordable analytical methods are crucial for quantifying this drug. In this study, we report on the use of carbon-based electrodes for the electrochemical determination of TBF in pharmaceutical samples, including raw materials and tablets. Notably, for the first time in the literature, we employ screen-printed carbon electrodes (SPCE) for TBF quantification. Additionally, glassy carbon electrodes (GCE) were used to explore the redox behavior of the analyte. Electrochemical performance was evaluated and compared using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Optimized conditions of supporting electrolyte and scan rate studies revealed that the oxidation of TBF involves an equal exchange of protons and electrons. For the GCE, the oxidation process was found to be irreversible, controlled by both diffusion and adsorption, while for the SPCE, it was irreversible and diffusion-controlled. Square wave voltammetry (SWV) was optimized for both electrodes to enhance sensitivity. For SPCE, the calibration curve ranged from 5 to 100 μg mL-1, with an LOD of 1.48 μg mL-1 using a single drop of sample. The calibration curve for GCE was constructed between 2.5 and 30 μg mL-1, with a limit of detection (LOD) of 0.072 μg mL-1. TBF quantification was performed on raw material samples from various suppliers and tablet forms using external calibration with a recovery range within 90-110%. Analysis of the data reveals that the voltammetric method's accuracy aligns well with the chromatographic approach based on high performance liquid chromatography (HPLC). Furthermore, the proposed methodology demonstrated outstanding sustainability, achieving a score of 0.91 in Green Analytical Chemistry (GAC) criteria. Our novel approach combines high analytical efficiency with a reduced environmental impact, establishing itself as a green, cost-effective, and accurate alternative for TBF sensing.

In vitro antischistosomal activity of Artemisia species.

Praziquantel is currently the only effective treatment for schistosomiasis, but several limitations underscore the need for new therapeutic agents. Recent promising in vitro results with Artemisia species and the success of A. annua and its active compound artemisinin in treating parasitic infections warrant the need for further studies. Here we evaluate the in vitro activity of nine Artemisia species, including previously untested species, against newly transformed schistosomula (NTS) and adult Schistosoma mansoni. Three extracts were prepared: an aqueous infusion, a dichloromethane (DCM) fraction of the aqueous infusion, and a DCM extract. All samples were tested for activity at concentrations ranging from 1-25 µg/ml against NTS and the most promising against the adult S. mansoni. All aqueous infusions showed inferior activity against NTS as opposed to the DCM fractions of the infusions and DCM extracts of A. abrotanum, A. arborescens, A. afra, and A. scoparia which displayed superior activity as compared to the positive control praziquantel. The DCM fraction infusions of A. afra (BB) were the most active with IC50 values of 0.35 µg/ml against NTS and 4.5 µg/ml against adult worms. Chemical fingerprinting using High-performance liquid chromatography (HPLC) analysis of the samples revealed some phytochemical similarities and significant differences between the species tested. This study provides supporting evidence of the antischistosomal potential of Artemisia spp. and warrants more in-depth research to identify potential novel therapeutic agents for the treatment of schistosomiasis.

Astragaloside IV ameliorates autism-like behaviors in BTBR mice by modulating Camk2n2-dependent OXPHOS and neurotransmission in the mPFC.

Autism spectrum disorder (ASD) represents a multifaceted set of neurodevelopmental conditions marked by social deficits and repetitive behaviors. Astragaloside IV (ASIV), a natural compound derived from the traditional Chinese herb Astragali Radix, exhibits robust neuroprotective effects. However, whether ASIV can ameliorate behavioral deficits in ASD remains unknown. This work aimed to determine the efficacy and molecular mechanisms of ASIV in ASD. The autistic BTBR T + tf/J (BTBR) mice were used in this study. Behavioral tests were performed to assess the ASD-like phenotypes. The neurotransmitter levels and synaptic transmission were evaluated by high-performance liquid chromatography and whole-cell patch-clamp recordings, respectively. Molecular biological techniques, immunostaining, and RNA-sequencing (RNA-seq) were combined to uncover the underlying molecular mechanisms. Our study showed that both social impairment and repetitive behaviors were significantly improved after ASIV treatment in a dose-dependent manner in BTBR mice. The ASIV treatment normalized the neurotransmitter levels (GABA and glutamate) and their corresponding vesicular transporters (vGAT, vGLUT1) in the medial prefrontal cortex (mPFC). Furthermore, the excitation-inhibition imbalance in layer V of mPFC was reversed after ASIV administration. Mechanistically, bulk RNA-seq and PPI network analysis identified Camk2n2 as the crucial bridging gene regulating oxidative phosphorylation and neurotransmission. Camk2n2 overexpression in the mPFC abolished the beneficial effects of ASIV on autistic symptoms in BTBR mice via the Camk2/CREB pathway. The evidence demonstrates that ASIV may become a promising treatment option for ASD and implies that targeting the Camk2n2/Camk2/CREB axis is warranted to investigate these ASD individuals further.

Lactobacillus acidophilus YL01 and its exopolysaccharides ameliorate obesity and insulin resistance in obese mice via modulating intestinal specific bacterial groups and AMPK/ACC signaling pathway.

Probiotics intervention by Lactobacillus acidophilus has potential effect on alleviating obesity and insulin resistance. However, the limited knowledge of functional substances and potential regulatory mechanisms hinder their widespread application. Herein, L. acidophilus YL01 was firstly isolated from Chinese traditional yogurt, demonstrating inhibitory activities on amylase and glucosidase that are comparable to those of L. rhamnosus LGG. Besides, the oral administration of L. acidophilus YL01 and its EPS significantly reduced body weight in high-fat mice (p < 0.05), as well as fat accumulation in liver and adipocytes. Moreover, they not only reduced fasting blood glucose and glucose/insulin resistance, but also improved dyslipidemia, liver function and inflammation. Further high-performance liquid chromatography analysis and Fourier transform infrared spectroscopy indicated that EPS is an acidic polysaccharide, characterized by a molecular weight of 952 kDa and predominantly composed of glucose. Additionally, the mechanism investigation revealed that the L. acidophilus YL01 and EPS demonstrated limited efficacy in restoring the composition of gut microbiota, but rather exerted an influence on the abundance of specific bacterial groups. The enrichment of the bacterial groups resulted in the increase of acetic acid and butyric acid, which further mediates the gut-liver crosstalk in regulating lipid metabolism by the activation of AMPK/ACC pathway.

Dual effects of exogenous ferulic acid bound in rice starch as 3D printable food ink: Structural fluidity and antimicrobial activity.

Starch-ferulic acid (FA) composites have been developed for medical and food fields, while little focus is caused on their use in functional products by 3D printing. In this work, dynamic high-pressure microfluidization was employed to treat starch at various concentrations, for preparing modified starch-FA composites. The high-performance liquid chromatography results showed that an increased starch concentration was conducive to a high yield of composite with enhanced binding of FA. Compared with pure starch and starch-FA mixture gel, the starch-FA composite gel possessed lower viscosity, with a dramatically reduced extrusion pressure in the 3D printing test. Furthermore, antimicrobial activity tests indicated that the starch-FA composite gel can inhibit the growth of microorganism for achieving a long storage period. Overall, we provide a biomaterial of starch-FA composite that can serve as both a 3D printing food ink and an edible, printable, active, and lightweight packaging ink.

N-acetyl-tryptophan in Acute Kidney Injury after Cardiac Surgery.

Cardiac surgery-associated acute kidney injury is a common serious complication after cardiac surgery. Currently, there are no specific pharmacological therapies. Our understanding of its pathophysiology remains preliminary. A total of 2504 patients with and without acute kidney injury (AKI) following cardiac surgery were enrolled. High-performance liquid chromatography coupled with mass spectrometry was used for untargeted analysis of metabolites in plasma, identifying significant differential metabolites. Subsequently, a tandem liquid chromatography-mass spectrometry-based approach using isotope-labeled standard addition was performed for targeted analysis of the metabolic marker N-acetyl-tryptophan. The function of N-acetyl-tryptophan was determined using different kidney injury mouse models and epithelial cellular models. Transcriptome sequencing, surface plasmon resonance and protein mutation were employed to explore the mechanism of N-acetyl-tryptophan on the kidney. We identified a total of 32 differential metabolites related to AKI occurrence based on a cohort of 1042 patients. Among them, N-acetyl-tryptophan was elevated in plasma of patients with cardiac surgery-associated acute kidney injury compared with those who do not develop AKI after cardiac surgery. The higher level of N-acetyl-tryptophan in plasma was confirmed by accurate targeted quantification. N-acetyl-tryptophan exhibited kidney protective effects in ischemia/reperfusion-, cisplatin-, and unilateral ureteral obstruction-induced kidney injury mouse models. Mechanistically, N-acetyl-tryptophan exerted kidney protective effects by interacting with KEAP1 at 483 and 508 sites, resulting in Nrf2 nuclear translocation and the transcription of proteasome genes. N-acetyl-tryptophan plays a key role in kidney protection.

Screening analysis of doping agents in horse urine and plasma with dilute and shoot using liquid chromatography high resolution mass spectrometry.

Various technical methodologies are required to accurately detect substances of different chemical and pharmacological properties in biological samples, which are increasing in number and variety daily. Therefore, laboratories where many samples and different factors are analyzed simultaneously need methods with easy sample preparation, short analysis times and low analysis costs. In this study, the objective was to scan substances susceptible to chemical degradation, amenable to analysis without hydrolysis, and exhibiting short-term stability by employing a straightforward, expeditious, and cost-efficient method. For this purpose, a high-throughput dilute and shoot screening protocol was developed and validated utilizing high-performance liquid chromatography coupled with high-resolution mass spectrometry to analyze various pharmacological compounds in horse urine and plasma. Over 200 prohibited substances across multiple categories were scanned within a 13 minute run. Chromatographic separation was performed on a C18 column using an elution gradient of mobile phase A, 5 mM ammonium bicarbonate at pH 9, and mobile phase B, methanol, at a flow rate of 0.3 mL min-1. The method was validated according to the specifications of 2002/657/EC multi-screening requirements. The detection capability ranged from ≤1 to 200 ng mL-1 for prohibited substances. The implementation of the screening method in doping analysis, and the analysis of real positive case samples served to underscore the practical applicability of the developed method. To the best of our knowledge, this is a rare method that can be applied to both urine and plasma samples and provides a rapid, practical, broad-spectrum, and high-throughput analysis of prohibited substances in horse plasma and urine cost-effectively.

Exploring Drug-Drug Interactions between Losartan and Carbamazepine: A Pharmacokinetic and Pharmacodynamic Study.

Hypertension, which affects 1.28 billion people globally aged 30 to 79, is characterized by continuously high blood pressure (140/90 or more) and raises the risk of premature death. Losartan, an angiotensin receptor blocker (ARB), is suggested for patients under the age of 55 who cannot take ACE inhibitors as a first treatment option. Epilepsy, a chronic neurological illness marked by repeated seizures, affects more than 50 million individuals worldwide and is the third most common chronic brain disorder. Both hypertension and epilepsy are frequent chronic illnesses, with increased blood pressure greatly raising the risk of epilepsy due to its relationship with cerebrovascular disease, doubling the risk when compared to people with normal blood pressure. The effect on pharmacokinetic and pharmacodynamics of losartan on concomitant administration with carbamazepine was investigated. Wistar rats of either sex, with a minimum of six animals per group, were used in the investigation. The rats were treated with Losartan and Losartan-Carbamazepine for 30 days. Blood samples were taken via retro-orbital plexus at 0, 1, 2, 4, 6, and 12 hours after treatment concluded, and they were subjected to high-performance liquid chromatography for plasma analysis to calculate AUC, t1/2, and Clearance. A pharmacodynamic evaluation was done by inducing hypertension in rats using a 10% fructose solution and the effect of pretreated Losartan and Losartan-Carbamazepine on blood pressure was determined. In the Losartan and Carbamazepine treated group, there was a reduction in the AUC and t1/2 and a reported increase in the clearance value compared to Losartan alone treated rats. In fructose-induced hypertension model to evaluate the effect of losartan and carbamazepine on BP showed an increase in mean arterial pressure, plasma glucose, and a reduction in triglycerides level was noted in comparison to Losartan alone treated rats indicating therapeutic failure of Losartan. Based on these studies, it is concluded that CBZ has reduced the effectiveness of losartan and therefore, co-administration of these drugs should be avoided.

Interaction Analysis between Cationic Lipid and Oligonucleotide with a Lipid Membrane-Immobilized Monolith Silica Column: A High-Performance Liquid Chromatography Approach.

Understanding the interactions between lipid membranes and nucleotide drugs is crucial for nucleic acid therapy. Although several methods have been employed to evaluate nucleotide-lipid membrane interactions, these interactions can be complex; this complexity arises from how external factors, such as ionic strength or temperature, influence the lipid membrane's overall properties. In this study, we prepared a lipid membrane-immobilized monolithic silica (LMiMS) column for high-performance liquid chromatography (HPLC) analysis to understand interactions between the lipid membrane and nucleic acid. First, the Raman shift, zeta potential, fluidity, and polarity of the LMiMS-column membrane were analyzed and compared to dispersed liposomes (not immobilized) with the same lipid composition. The results indicated that the column can effectively imitate the temperature-dependent properties of the lipid membrane, suggesting that the immobilized lipid membrane can be used as a model of dispersed liposomal membranes. Subsequently, HPLC was performed to analyze the interaction between the lipid membrane and oligonucleotides. The retention factor k was determined as an interaction factor between the LMiMS column and nucleic acid models (poly 10, 25, and 50mer dC) at various temperatures and mobile phase salt concentrations. The results revealed that changes in membrane interaction were significant by the phase state and salt concentration. Further analysis of the retention factor k showed that the interaction is weak below the phase transition temperature but strong above the phase transition temperature. The results indicate that membrane properties (rigidity and polarity) are also related to membrane interaction, which can be evaluated with the LMiMS column. This analytical method can provide a new perspective on estimating the interactions between target molecules and the lipid membrane through their temperature-induced dynamics. From these results, our method could be useful for analyzing lipid membrane-oligonucleotide interactions.

Exploring the Physicochemical Compatibility of Minoxidil in Combination with Different Active Pharmaceutical Ingredients in Ready-to-use Vehicles for Alopecia Treatment.

Alopecia is globally known as a distressing medical disorder that affects men and women, and current commercially available minoxidil solutions are formulated with irritant vehicles with frequent complaints of dermatologic adverse effects. This study aimed to investigate further the compatibility of ready-to-use vehicles for the preparation of tailored formulations for alopecia treatment, namely TrichoSol™ (a ready-to-use vehicle for personalized hair solutions) and TrichoFoam™ (a ready-to-use vehicle for personalized foam formulations), in combination with minoxidil and other active pharmaceutical ingredients (APIs), to establish adequate beyond-use dates (BUD) for the given formulations. Products under evaluation were compounded using TrichoSol™ or TrichoFoam™, with direct incorporation of the APIs into these vehicles. Samples were then stored at controlled room temperature for up to 180 days. High-performance liquid chromatography (HPLC) methods were developed and validated, and then utilized to evaluate the compatibility of the APIs in TrichoSol™ and TrichoFoam™. Forced degradation studies were conducted to assess API stability under various stress conditions, and Antimicrobial Effectiveness Testing (AET) was performed at 0 and 180 days after compounding. According to our results, BUDs of up to 90-180 days were obtained for the examined formulations stored at room temperature, considering a degradation of maximum 10% of the nominal concentration of the APIs within them. The formulations exhibited no discernible physical alterations throughout this period and maintained chemical stability within acceptable limits. Microbiological evaluations confirmed the efficacy of the preservative system. Products compounded with TrichoSol™ and TrichoFoam™ showed suitable stability to be used as personalized treatments for alopecia. We can then suggest that the vehicles TrichoSol™ and TrichoFoam™ present effective solutions for compounding personalized hair care treatments.

Species-specific Antimony Isotope Analysis by High Performance Liquid Chromatography Coupled with Multicollector ICPMS Using Hydride Generation as an Interface.

A novel method has been developed for the simultaneous online determination of the isotopic compositions of different antimony (Sb) species in a single analytical run using high-performance liquid chromatography (HPLC) coupled with multicollector inductively coupled plasma mass spectrometry (MC-ICPMS), with hydride generation (HG) serving as the interface. Various parameters affecting the precision of Sb isotope analysis including HG conditions, transient signal processing methods and peak integration windows, were optimized. The linear regression slope method and a 100% peak integration window provided the optimal precision. Under optimized conditions, our method achieved external 2SD precisions better than 0.05‰ for both Sb(III) and Sb(V), with minimal consumption of 0.5 ng for Sb(III) and 5 ng for Sb(V). Furthermore, flow injection (FI) coupled with HG-MC-ICPMS demonstrated precise Sb isotopic analysis with sample requirements as low as 0.25 ng. The proposed methods were validated by analyzing δ123Sb in synthetic solutions and reference materials. Additionally, it was applied to investigate isotopic fractionation during the reduction of Sb(V) by KI, revealing preferential reduction of the light Sb isotope(121Sb). The isotopic compositions of Sb(V) varied from -0.04- 1.18‰, fitting well with a Rayleigh fractionation model and yielding a fractionation factor (αSb(III)-Sb(V)) of 0.99831. In summary, this approach enables high precision isotopic analysis of Sb(III) and Sb(V) simultaneously with reduced sample consumption, providing a powerful tool for investigating Sb isotopic fractionation in various environmental processes and advancing our understanding of the Sb biogeochemical cycle.

Longitudinal observational (single cohort) study on the causes of trypanocide failure in cases of African animal trypanosomosis in cattle near wildlife protected areas of Northern Tanzania.

African animal trypanosomosis (AAT) in cattle is primarily managed through trypanocide administration and insecticide application. Trypanocides can be used for both treatment and prophylaxis, but failure is often reported; this may occur due to resistance, substandard drugs, or inappropriate administration. This study in Tanzania aims to quantify reasons for trypanocide failure. An observational year-long longitudinal study was conducted in high-risk AAT areas in Serengeti District between June 2021-October 2022. Purposive sampling targeted herds with high utilization of the prophylactic trypanocide isometamidium chloride (ISM). When a farmer administered a trypanocide (ISM, diminazine aceturate, homidium), the project veterinarian assessed administration and treatment outcomes were determined based on PCR results from blood samples. A multivariable mixed model was utilized to evaluate risk factors for prophylaxis failure. Quality analysis was performed on trypanocide samples using High Performance Liquid Chromatography. A total of 630 cattle from 21 farms were monitored for a year-long period. A total of 295 trypanocide administrations were reported, predominantly being ISM (56%) used for prophylaxis (87%). One-third of trypanocide administrations were not given adequately, and many trypanocides were given to animals that tested negative for trypanosome infections by PCR. Failures occurred in 7% (95% CI 3.0-14%) of curative treatments, and 44% (95% CI 35-42%) of prophylactic administrations. The brand of ISM was significantly associated with odds of prophylaxis failure (p = 0.011). On quality analysis, two ISM samples had no detectable ISM isomers, but the remainder of ISM and DA samples (n = 46) fell within the range of acceptable levels. Drug counterfeiting, inadequate use of trypanocides, and resistance are all contributing to trypanocide failure, limiting effective AAT control and with implications for human disease risk. In order to curb trypanocide failure a multi-modal approach to managing the use of trypanocides is required to address all contributing factors.

Dry crude pomegranate peel extract as a bio-input for medicinal pharmaceutical gel with healing activity.

The sustainable use of pomegranate peel, a by-product of the food industry, is gaining importance in developing pharmaceutical bio-inputs, aligning with circular economy practices and waste reduction. This study explores the application of dry crude pomegranate peel extract (PPE) as a bio-input for medicinal gels with wound healing properties. PPE was extracted via percolation in ethanol and freeze-dried. High-performance liquid chromatography (HPLC) and mass spectrometry identified key bioactive compounds: gallic acid, punicalagin, punicalin, ellagic acid, and citric acid, recognized for their antioxidant, antimicrobial, and healing properties. In vitro assays revealed low hemolytic activity, non-cytotoxicity, significant fibroblast proliferation, and robust antioxidant activity (95.99% DPPH scavenging at 100 µg/mL). A carbomer gel containing 2.5% w/w of the extract effectively healed wounds in rats, with performance comparable to silver sulfadiazine ointment, while demonstrating no microbial contamination during the process. These findings position PPE as a promising, sustainable, and effective alternative for wound care pharmacotherapy, addressing both medical needs and environmental sustainability.

Development and validation of a high-performance liquid chromatography method with fluorescence detection for the quantification of the resistance protein P-gp in cancer cells.

A method using high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) was developed and validated to quantify the innovative tool LightSpot®-FL-1, a selective permeability-glycoprotein (P-gp)-targeted fluorescent conjugate used to measure P-gp expression in cell samples. Quantifying P-gp is a major challenge in oncology as its overexpression in many cancer cells results in Multidrug Resistance (MDR) associated with chemotherapy failure. To develop the method reported herein, both sample preparation and analysis parameters were investigated. Optimal chromatographic conditions were achieved with 5µL injections at a 1mL/min flow rate on a reversed-phase Zorbax® Eclipse Plus 3.5µm C18 column (150×4.6mm) with isocratic acetonitrile/water (85/15, by volume) elution. Detection was performed with 505nm excitation and 510nm emission wavelengths. Validation studies were designed and performed according to the International Council for Harmonization (ICH) guidelines for bioanalytical method validation. The limit of quantification (LOQ) and limit of detection (LOD) were determined to be 0.5 and 0.2nmol/L, respectively. The linearity range was demonstrated between 10 and 500nmol/L, and the trueness and precision of the method were validated. Good stability was shown in three relevant analytical conditions. The greenness of the developed method was also demonstrated with the AGREE, AGREEprep and MoGAPI tools. Finally, the rapid, precise and sensitive validated analytical method was successfully applied to determine the difference in P-gp expression in three cancer cell lines: DU4475, CCRF-CEM and KG-1a.

A comprehensive evaluation of the quality of Bu Shen Jian Gu oral liquid based on multi-indicator component quantification and chemical pattern recognition.

Bu Shen Jian Gu Oral Liquid (BSJG) is a hospital formulation commonly utilized in the treatment of fractures, joint dislocations, and rheumatoid arthritis. However, there has been no effective and straightforward method to comprehensively assess the quality of BSJG. In this experiment, a fingerprint of various batches of BSJG was established using high-performance liquid chromatography (HPLC). The results indicated that the 13 batches of oral liquid exhibited similarities. A total of 18 consensus peaks were found, and 8 components were identified: protocatechuic acid, chlorogenic acid, 2,3,5,4-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), isochlorogenic acid B, isochlorogenic acid A, naringin, isochlorogenic acid C, and emodin. Hierarchical cluster analysis (HCA), principal component analysis (PCA), and orthogonal partial least squares discrimination (OPLS-DA) were performed on 13 batches of BSJG, which showed differences among different batches. It is worth noting that peak 9 (TSG), peak 1, and peak 13 (naringin) may be the key components affecting the quality of this formulation. The content determination results showed that the eight components exhibit an excellent linear relationship within a specific range, with r > 0.9990. The content of 13 batches of oral liquid is different, and the component with a significant change in content is TSG, which may be related to the transportation and storage of medicinal materials. The fingerprinting method combined with multi-component content determination established in this study can serve as a reference for the quality standards of BSJG.

Systematic identification of the chemical components of leaves and roots of Didymocarpus heucherifolius Hand.-Mazz. based on UHPLC Q-Exactive Orbitrap MS technology coupled with molecular networking strategy

Didymocarpus heucherifolius Hand.-Mazz. is plant of the genus Didymocarpus wall. (Gesneriaceae). Species in this genus have long been used in folk medicine to treat various illnesses, including wounds, kidney stones, inflammations, asthma, influenza, etc. However, there are few studies on chemical components. Crude plant extracts are a complex mixture of several biologically active specialized metabolites. Therefore, a rapid and accurate identification and quantification are critical in phytochemical analysis. An efficient approach based on molecular networking strategy coupled with ultra high-performance liquid chromatography coupled with Q-Exactive Orbitrap tandem mass spectrometry (UHPLC Q-Exactive Orbitrap MS) was used to analyze the chemical components systematically. The chemical was identified using retention time, accurate molecular weight, parent peaks, fragment peaks, fragmentation characteristics, comparative analysis with the literature, reference standards, and standard databases, including mzVault. A total of 108 compounds (32 phenylpropanoids, 14 phenolic acid, 6 fatty acids, 14 organic acid, 9 flavonoid, 15 amino acids, and 28 other components) were identified in the leaves and roots of D. heucherifolius in which 84 and 84 compounds were found in the leaves and roots, respectively. The results showed that 68 components were different in plant roots and leaves, and these compounds were mainly represented in organic acids. Moreover, 50 identical chemical compounds were identified in plant roots and leaves, and these compounds were mainly phenylpropanoid compounds. The UHPLC Q-Exactive Orbitrap MS method was used for the first time to accurately, quickly, and systematically identify the compounds of the roots and leaves of Didymocarpus heucherifolius Hand.-Mazz., thereby providing a theoretical basis for its quality control, in-depth research, and further development.