Reversed-phase Column Research Articles

Specific determination of (-)- and (+)-gossypol in vegetable oil by ultra-performance liquid chromatography-tandem mass spectrometry with chiral pre-column derivatization.

As the unique ingredient of cottonseed oil, gossypol is toxic and there are differences between the enantiomers. Although the determination of (-)- and (+)-gossypol in cotton plants or cottonseed was performed, the detection about the gossypol enantiomers in vegetable oil was rarely reported. To develop a specific inspection method for (-)- and (+)-gossypol in vegetable oil by ultra-performance liquid chromatography (UPLC) -tandem mass spectrometry with chiral pre-column derivatization. (-)- and (+)-gossypol were separated on an ACQUITY C18 reverse phase column (100 mm × 2.1 mm, 1.7 μm) maintained at 40 °C on an UPLC system with gradient elution. The mobile phases was composed of methanol (solvent A) and water containing 0.1% formic acid (solvent B). Analytes were detected with Electron Spray Ionization source in the positive mode. The contents of (-)- and (+)-gossypol standard derivatives were quantified by Multiple Reaction Monitoring with the transitions of m/z 633/483 for quantitative ion pairs and m/z 633/558 for qualitative ion pairs. The results indicated that the quantitative analysis could be accomplished within 12.5 minutes and the limits of detection of (-)- and (+)-gossypol were 25.55 μg/kg and 14.67 μg/kg, respectively. When this method was applied to olive oil spiked with gossypol standards, good recoveries (95.37%-105.84% for (-)-gossypol, 98.96%-105.14% for (+)-gossypol) and RSDs (3.41%-6.02% for (-)-gossypol, 4.50%-6.94% for (+)-gossypol) were achieved. The present study confirms the feasibility of determining the gossypol enantiomers and achieves trace determination of (-)- and (+)-gossypol in vegetable oil. These results make this method more suitable for the qualitative identification of whether the vegetable oil has been mixed with cottonseed oil. The trace determination of (-)- and (+)-gossypol in vegetable oils has been achieved. This method provides technical support for further identification of whether commercially available vegetable oil has been mixed with cottonseed oil.

Quantitation of the DNA-dependent protein kinase inhibitor peposertib (M3814) and metabolite in human plasma by LC-MS/MS.

The DNA-dependent protein kinase (DNA-PK) is an abundant nuclear protein that mediates DNA double-strand break repair by nonhomologous end joining (NHEJ). As such, DNA-PK is critical for V(D)J recombination in lymphocytes and for survival in cells exposed to ionizing radiation and clastogens. Peposertib (M3814) is a small molecule DNA-PK inhibitor currently in preclinical and clinical development for cancer treatment. We have developed a high-performance liquid chromatography-mass spectrometry method for quantitating peposertib and its metabolite in 0.1 mL human plasma. After MTBE liquid-liquid extraction, chromatographic separation was achieved with a Phenomenex Synergi polar reverse phase (4μm, 2× 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and water over an 8min run time. Mass spectrometric detection was performed on an ABI SCIEX 4000 with electrospray, positive-mode ionization. The assay was linear from 10 to 3000 ng/mL for peposertib and 1-300 ng/mL for the metabolite and proved to be both accurate (97.3%-103.7%) and precise (<8.9%CV) fulfilling criteria from the Food and Drug Administration (FDA) guidance on bioanalytical method validation. This liquid chromatography-tandem mass spectroscopy (LC-MS/MS) assay will support several ongoing clinical studies by defining peposertib pharmacokinetics.

An Ultra-Fast Green UHPLC-MS/MS Method for Assessing the In Vitro Metabolic Stability of Dovitinib: In Silico Study for Absorption, Distribution, Metabolism, Excretion, Metabolic Lability, and DEREK Alerts.

Background and Objectives: Dovitinib (DVB) is a pan-tyrosine kinase inhibitor (TKI) that can be administered orally. In September 2023, the FDA granted Oncoheroes approval to proceed with an Investigational New Drug (IND) application for dovitinib. This application is intended for the treatment of relapsed or advanced juvenile solid tumors, namely, osteosarcoma. Materials and Methods: The target of the present study was to develop a rapid, green, accurate, and sensitive UHPLC-MS/MS method for measuring DVB levels in human liver microsomes (HLMs). The validations of the HLMs were performed via the established UHPLC-MS/MS approach, as stated in the US FDA reported guidelines for the standards of bioanalytical method validation protocol. The StarDrop in silico software package (version 6.6), which involves the DEREK and WhichP450 in silico modules, was used to check the DVB structure for hazardous alerts and metabolic instability. The DVB and encorafenib (EFB), internal standard, and chromatographic peaks were successfully separated using a reversed phase column (an Eclipse Plus Agilent C8 column) and an isocratic mobile phase. The production of DVB parent ions was accomplished by utilizing the positive ionization mode of an ESI source. The identification and measurement of DVB daughter ions were conducted using the MRM mode. Results: The inter-day accuracy and precision exhibited a spectrum of values in the range of -0.56% to 9.33%, while the intra-day accuracy and precision showcased a range of scores between 0.28% and 7.28%. The DVB calibration curve showed a linear relationship that ranged from 1 to 3000 ng/mL. The usefulness of the currently validated UHPLC-MS/MS method was approved by the lower limit of quantification (LLOQ) of 1 ng/mL. The AGREE findings demonstrate that the UHPLC-MS/MS method had a noteworthy degree of ecological greenness. The in vitro half-life (t1/2) and intrinsic clearance (Clint) of DVB were calculated to be 15.48 min and 52.39 mL/min/kg, respectively, which aligned with the findings from the WhichP450 software (version 6.6). Conclusions: Via the usage of in silico software, it has been observed that making small changes to the structure of the aryl piperazine ring and quinolinone moieties, or replacing these groups in the drug design process, shows potential for enhancing the metabolic safety and stability of newly developed derivatives compared to DVB.

B-161 Liquid chromatography/mass spectrometry analysis of ethyl glucuronide and ethyl sulfate using a perfluorophenyl stationary phase

Abstract Background Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are minor metabolites of ethanol that are excreted in urine. These two metabolites are often tested for to determine if an individual has recently consumed alcohol. EtG and EtS are very polar analytes, which make them difficult to chromatograph using reversed-phase chromatography. These analytes often elute early, which can lead to irreproducible results. When EtG and EtS are analyzed by LC/MS with a mobile phase that contains formic acid, the signal of these two analytes can be suppressed. This leads to increased LOQ and cutoffs. We examined the perfluorophenyl (PFP) stationary phase for its utility in analyzing EtG and EtS. This stationary phase has been shown to have greater retention of polar compounds than C18 reversed-phase columns. In addition, we compared the effect of formic acid to acetic acid on the separation. Methods Urine samples containing EtG and EtS were diluted 10:1, using 50mM acetic acid or 0.1% formic acid, into a 96-well microtiter filter plate containing a 0.2micron filter. Internal standards were added to the microtiter plate containing the urine samples. The samples were filtered through the filter plate and analyzed by LC/MS using the PFP column (50 x 2.7micron) with a 50mM acetic acid or 0.1% formic acid/acetonitrile mobile phase. The analytes were detected using negative mode ionization. The total analysis time was 4.5minutes. Results EtG and EtS showed improved retention using the PFP column. Retention times for EtG and EtS were 0.75 and 1.20 minutes respectively using the PFP column, compared to 0.4 and 0.3 minutes using the C18 column. The greater retention time improved the LOQ for both analytes.The LOQ for EtS and EtG were determined to be 125 and 250 ng/mL, respectively. Greater retention of EtG was observed by switching the modifier from formic acid to acetic acid. The retention time increased from 0.45 to 0.75 minutes. There was less signal suppression using acetic acid in place of formic acid. Improvement in peak shape was observed when the injection volume was reduced to 5microliters. The method was validated over a range of 250 to 10000 ng/mL for EtG and 125 to 5000 ng/mL for EtS. Acceptable linearity was obtained for both analytes. Accuracy was performed at three concentration levels. All three levels demonstrated acceptable accuracy with the difference between the measured and expected concentration being with in 20% of each other. Conclusions The PFP stationary can be used to test EtG and EtS. This stationary phase gave greater retention of EtS and EtG. The use of acetic acid improved the limit of detection by decreasing the ion suppression, which decreased the limit of detection. The assay was accurate and precise for both analytes, demonstrated in the validation.

Comparison of piperine content, antimicrobial and antioxidant activity of Piper chaba root and stem

Piper chaba (locally known as “Choi Jhal”) is used traditionally as spices and folk medicine in different parts of Bangladesh. One of the most important bioactive compounds in this plant is piperine. In this study, the amount of piperine in P. chaba root and stem was investigated and the optimal solvent for piperine extraction at room temperature was also studied. High performance liquid chromatography (HPLC) was operated using a reverse phase column where methanol and water (70:30) were used as mobile phase. The detection was performed using photo diode array (PID) detector at a wavelength of 345 nm. The standard piperine showed linearity between 0.005 % and 0.04 % and the correlation co-efficient found for the linearity was 0.9933. The percentage of relative standard deviation (RSD) for both retention time and peak area were less than 2.0 %. The theoretical plate number (N) > 3000 and a tailing factor (T) < 1.5 were found in the acceptable range. The recovery percentage (%) of standard piperine was 99.16 %. Low value of co-efficient of variation and standard deviation are recognized for high precision of the method. The highest amount of piperine was found in root extracted with methanol (MR) amounting to 1.75 % in the root powder. The maximum amount of piperine in the stem was 1.59 % when extracted with methanol (MS). The piperine quantification in other extract like n-hexane root (HR), ethyl acetate root (ER), n-hexane stem (HS), ethyl acetate stem (ES) were 0.76 %, 1.69 %, 0.33 % and 1.46 % respectively. Methanol has given the highest yield of piperine compared to ethyl acetate and n-hexane for both root and stem. The developed method was simple, rapid, economic and validated in terms precision, accuracy and recovery. This selective method is found to be repeatable, accurate and successfully utilized for the Piper extract in marketed and pharmaceutical samples with well chromatographic conditions. The ethyl acetate extract of root and stem showed promising DPPH (1, 1-diphenyl-2-picrylhydrazyl) free radical scavenging activity with an IC50 value of 39.62 ± 0.95 μg/mL and 43.85 ± 1.50 μg/mL respectively. The study reports potential antibacterial activity and antifungal activity of P. chaba root and stem extracts. These outcomes revealed that different extracts of P. chaba may be used as natural preservatives.

Quantification of tricyclic glycopeptide in human plasma by UHPLC-MS3 coupled with counter-extraction follow by protein precipitation to enhance sensitivity

An ultra-high performance liquid chromatography tandem mass spectrometry cubed (UHPLC/MS3) assay coupled with protein precipitation and counter-extraction for detection of tricyclic glycopeptide vancomycin in human plasma was established and validated in this study. After protein precipitation and counter-extraction with dichloromethane, chromatographic separation of vancomycin and norvancomycin were performed on a reversed phase column (XBridge Peptide BEH C18 column, 2.1 × 100 mm I.D, 3.5 μm). The transition (parent ions → fragment ions → further fragment ions) at m/z 725.3 → 144.1 → 100.1 was used for quantification of vancomycin. The transition (parent ions → fragment ions) at m/z 718.3 → 144.2 was used for detection of norvancomycin. The linear range of the developed analytical method for quantification of vancomycin was 0.5–100 µg/mL (r = 0.9989). The range of intra- and inter-day precisions of the assay among low, medium and high concentrations is between 1.88 % and 6.33 %. The sensitivity of the analytical method was significantly improved by using MS3 technique as monitoring mode and counter-extraction with dichloromethane followed by protein precipitation as sample processing assay. The developed UHPLC/MS3 assay was successfully applied for clinical therapeutic drug monitoring (TDM) of vancomycin in 45 human plasma samples.

Liquid chromatography-tandem mass spectrometry analysis of nitrogen mustard degradation products after derivatization with benzoylimidazole

Nitrogen mustards are highly toxic compounds and strictly controlled by the Chemical Weapons Convention. When those agents are used, exposure must be confirmed by analyzing biological samples from victims to allow prompt medication and reduction of secondary damage. In this study, a derivatization-liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method to analyze nitrogen mustard degradation products (ethanolamines; EAs) in human urine was developed. Benzoylimidazole (BzI) was used as a derivatization reagent, and two highly hydrophilic EAs (methyldiethanolamine and ethyldiethanolamine) were analyzed by using a reversed-phase column with sufficient retention. For urine samples, after solid-phase extraction with cation exchange cartridge and derivatization, EAs were analyzed at low limits of detection (5 ng/mL). The calibration curves had a wide linear range (50–1000 ng/mL), and the quantitation results were sufficient for screening. Compared with our previous derivatization-LC-MS/MS method using pentafluorobenzoyl chloride as a derivatization reagent and hydrophilic interaction liquid chromatography (HILIC)-MS/MS method, not only the limits of detection but also the operability and throughput were improved. The performance of the newly developed derivatization reagent was also evaluated using density functional theory (DFT) calculations.

Assay for the quantification of abemaciclib, its metabolites, and olaparib in human plasma by liquid chromatography-tandem mass spectrometry

An isotope-dilution bioanalytical assay for abemaciclib and its metabolites in combination with olaparib was developed and validated in human plasma K2 EDTA. For the quantitative assay, human plasma samples (or human plasma QC samples) were spiked with internal standard solution before a simple protein precipitation with methanol. The extract was injected onto a liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument where it was chromatographically separated by a polar end-capped reversed phase column and guard using gradient elution with water and methanol both modified with 0.2% formic acid (v/v) as the mobile phases. The analytes and internal standards were measured by heated electrospray ionization (HESI) in positive polarity using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. The assay was validated for linear ranges as follows: 0.4 – 1000nM abemaciclib, 0.35 – 1000nM M2 and M18, 0.5 – 1000nM M20, and 0.75 – 1000nM olaparib. The inter-day or between day precision for the quality controls (n = 18) was < 13% and the accuracy was ± 12%, for all analytes, including the lower limit of quantification (LLOQ). The intra-day or within day precision for the quality controls (n = 6) was ≤ 11% and the accuracy was ± 12% for low, mid, and high and < 19% at LLOQ. The recovery in human plasma was determined to be between 92 and 102% for all analytes spanning the linear range. The validated, bioanalytical quantitative assay was designed to measure abemaciclib, its metabolites, and olaparib for pharmacokinetic evaluation of patients in clinical trials for breast, brain, and ovarian cancers.

A validated LC-MS/MS method for the simultaneous quantification of iothalamate and hippuran in serum and urine for non-radioactive kidney function assessment

A novel liquid chromatography-tandem mass spectrometry method is described for the quantitative determination of the kidney function markers iothalamate and hippuran in human serum and urine. It is based on protein precipitation with methanol followed by dilution of the supernatant for serum and simple dilution for urine. The polar analytes are chromatographically separated by a 6.5-min gradient on a low-ligand density reversed-phase column; detection is performed by electrospray ionization tandem mass spectrometry in the positive ion mode against stable-isotope labeled internal standards.The results of a thorough method validation show that iothalamate and hippuran can be simultaneously quantified in the concentration ranges 0.500–30.0 ng/mL and 10.0–5000 ng/mL for serum and urine, respectively, with values for CV and absolute bias not exceeding 10 %, and with sufficient stability in all relevant matrices and solvents. The method was successfully applied for the analysis of serum and urine samples of multiple individuals who received both iothalamate and hippuran.

Advancing Bioanalytical Method Validation: A Comprehensive ICH M10 Approach for Validating LC-MS/MS to Quantify Fluoxetine in Human Plasma and Its Application in Pharmacokinetic Studies.

A fast and sample cleanup approach for fluoxetine in human plasma was developed using protein precipitation coupled with LC-MS-MS. Samples were treated with methanol prior to LC-MS-MS analysis. Chromatographic separation was performed on a reverse phase column with an isocratic mobile phase of methanol and 10 mM ammonium formate pH acidified with formic acid (80:20, v/v) at a flow rate of 0.2 mL/min. The run time was 4 min. Mass parameters were optimized to monitor transitions at m/z [M + H]+ 310 > > 148 for fluoxetine and m/z [M + H]+ 315.1 > > 153 for fluoxetine-d5 as an internal standard. The lower limit of quantification and the dynamic range were 0.25 and 0.25-50 ng/mL, respectively. Linearity was good for intra-day and inter-day validations (R2 = 0.999). The matrix effect was acceptable with CV% < 15 and accuracy% < 15. The hemolytic effect was negligible. Fluoxetine was stable in human plasma for 48 h at room temperature (25 °C), for 12 months frozen at -25 °C, for 48 h in an auto-sampler at 6 °C, and for three freeze/thaw cycles. The validated method was applied in a pharmacokinetic study to determine the concentration of fluoxetine in plasma samples. The study provides a fast and simple bioanalytical method for routine analysis and may be particularly useful for bioequivalence studies. The method was successfully applied to a pharmacokinetic study of fixed-dose fluoxetine in nine healthy volunteers.

RP-HPLC-PDA-Based Simultaneous Estimation of Ramipril and Hydrochlorothiazide in Combined Dosage Forms: A Validated Approach

A rapid, highly sensitive High-Performance Liquid Chromatographic method has been developed for the simultaneous determination of Ramipril (RMP) and Hydrochlorothiazide (HCT) in bulk drug and in tablets. Ramipril (RMP) and Hydrochlorothiazide (HCT) were eluted from an Eclipse plus C18 (250 mm × 4.6 mm I.D.) with particle size 5 µm reversed phase column with a mobile phase consisting of acetonitrile and potassium dihydrogen ortho-phosphate pH 3 (40: 60 v/v) at a flow rate of 1 mL/min with UV detection at 210 nm. The retention time for RMP and HCT was found to be 3.31 ± 0.02 min and 6.64 ± 0.02 min, respectively. The linear response (r2 &lt;0.99) was observed in the range of 2 - 12 μg/mL for RMP and 4 - 24 μg/mL of HCT with low values of limits of detection (LOD) and quantification (LOQ) for both drugs. The method shows good recoveries and intra and inter-day relative standard deviations were less than 1.0%. Validation parameters as specificity, accuracy, ruggedness and robustness were also determined. The proposed method provides accurate and precise quality control tool for routine simultaneous analysis of Ramipril and Hydrochlorothiazide in bulk and in tablet dosage form.

In situ Single-Pass Perfused Rabbit Intestinal Study sample Analysis of Ractopamine by UPLC

In recent years, the prediction of oral drug absorption in humans has been a challenge for researchers and many techniques for permeability studies have been developed for several purposes, including biowaiver processes. The Single-Pass Intestinal Perfusion (SPIP) method performed in rats can provide permeability results closest to in vivo condition. he use of SPIP in rats and calculations for absorption prediction in humans may indicate the transport mechanisms and/or pre-systemic metabolism involved on permeation processes of drugs, since this model is the closest to in vivo conditions. Ractopamine hydrochloride is a commercial beta-adrenergic agonist commonly used as a dietary supplement in cattle production for improved feed efficiency and growth promotion. Currently, regulatory target tissues (as approved in the New Animal Drug Application with Food and Drug Administration) for ractopamine residue testing are muscle and liver. The aim of the study is to develop and validate an UPLC assay method to determine whether detectable and quantifiable levels of perfused ractopamine in small intestine of cattle or not. The separation is carried on Acquity UPLC BEH C18 (150mm x 2.1mm, 1.7 µm) reversed phase column using acetonitrile and 0.01M ammonium formate as the mobile phase in gradient mode at flow rate of 0.5mL/min. The precision, accuracy, extraction recovery, matrix effect, and stability meet the requirements of the guiding principles. In order to verify the sensitivity and selectivity of the method in a real-time situation, the developed method is successfully applied to a perfusion study in small intestine of rabbit and the samples were analyzed by Empower software.

Development of a Simultaneous HPLC Determination Method of Cacao Polyphenols, Caffeine and Theobromine in High Cacao Chocolate by Core-Shell Type Reversed-Phase Columns

Development of a Simultaneous HPLC Determination Method of Cacao Polyphenols, Caffeine and Theobromine in High Cacao Chocolate by Core-Shell Type Reversed-Phase Columns

Determination of ten bisphenols and five parabens in urine by solid supported liquid-liquid extraction and liquid chromatography-tandem mass spectrometry

Bisphenols (BPs) and parabens (PBs) are of great concern for environmental pollution and human health because of their endocrine-disrupting effects and potential health hazards. Urinary biomonitoring of BPs and PBs can provide basic data for human internal exposure evaluation, which is a prerequisite for accurately assessing their health risks. In this study, we developed a new pretreatment procedure based on solid supported liquid-liquid extraction (SLE) for the simultaneous separation of ten BPs and five PBs in human urine, followed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. In the instrumental analysis, the HPLC conditions and MS/MS parameters were comprehensively optimized. Accurate qualitative and quantitative determination of ten BPs and five PBs was achieved by introducing a ternary gradient elution system of water, methanol, and acetonitrile for LC separation. During sample pretreatment, the extraction solvent and elution volume were optimized. Specifically, urine samples were held at room temperature and centrifuged at 3000 r/min for 10 min. The supernatant (2 mL) was then transferred to a glass tube, and the pH was adjusted to 5.0 using HCl (0.5 mL; 0.1 mol/L) and NaAc-HAc buffer (1.5 mL). Thereafter, β-glucuronidase-arylsulfatase (20 μL) and surrogate standard solutions (10 ng;13C12-BPS,13C12-BPAF,13C6-MeP, and 13C6-BuP) were added, and the mixture was incubated in a shaker bath in the dark at 37 ℃ for 16 h. After incubation, the hydrolyzed sample (4 mL) was loaded onto an SLE cartridge and equilibrated for a minimum of 5 min to ensure the solution was completely absorbed by the packing material. Subsequently, the target chemicals were eluted with a mixed ethyl acetate/n-hexane solution (3∶7, v/v; 15 mL). Separation of the targets was performed on a ZORBAX SB-C18 reversed-phase column (250 mm×4.6 mm, 5 μm) using an acetonitrile-methanol-water system as the mobile phase. The method was verified by spiking mixed urine samples at three levels (1, 5, and 50 μg/L), with the recoveries ranging from 84.3% to 119.8%. Except for bisphenols (BPS), whose matrix effect was calculated as -21.8%, the matrix effects of other analytes were lower than 20%, indicating low matrix interference. The linear ranges of the analytes varied from 0.1-500 μg/L to 1-500 μg/L, with correlation coefficients higher than 0.995. The method limits of quantification for target chemicals ranged from 0.03 to 0.30 μg/L, and the relative standard deviations of intra- and inter-day experiments were 1.4%-8.4% and 5.7%-14.6%, respectively, suggesting high stability and reproducibility. The method was successfully applied to the determination of ten BPs and five PBs in 10 urine samples from a general population. The concentrations of target chemicals in the human urine samples varied. Methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), and bisphenol A (BPA) were detected in all samples, with median mass concentrations of 1.10, 0.60, 0.21, and 0.55 μg/L, respectively. The detection rates of the other chemicals were less than 50%, which may be related to the production and use of specific chemicals, their bioavailability, and biological metabolism in humans.

Rapid and simultaneous determination of ultrashort-, short- and long- chain perfluoroalkyl substances by a novel liquid chromatography mass spectrometry method

Per- and Polyfluoroalkyl Substances (PFAS) are a group of persistent organic pollutants that have received considerable attention from public and regulatory groups. Due to regulations of long-chain PFAS, the use of short-chain and ultrashort-chain PFAS is rapidly growing. Thus, there is an urgent need to develop quantitative methods for determining PFAS with different chain lengths in various environmental matrices. This study introduces an innovative liquid chromatography-mass spectrometry (LC-MS) system combining large volume injection (LVI) and online solid phase extraction (SPE). This system incorporates three columns: a reverse-phase (RP) column, a weak anion exchange (WAX) trap column, and a hybrid HILIC/ion-exchange (HILIC/IE) column, controlled by two valves. With valve switching, ultrashort-chain PFAS that are not retained by the RP column are enriched by the trap column, while other PFAS are separated by the RP column. The trapped ultrashort PFAS are then transferred to the HILIC/IE column for further separation. The LVI significantly enhances the method's sensitivity, allowing for rapid and simultaneous determination of ultrashort-, short- and long- chain PFAS in aqueous samples. The matrix effects from various environmental samples were evaluated, and the results indicate that this unique LC-MS method is suitable for analyzing all chain-length PFAS in various matrices, including surface water, sewage effluent, and seawater. Finally, this novel LC-MS method was applied to quantify PFAS in various water samples.

Prolonged release from lipid nanoemulsions by modification of drug lipophilicity

In addition to the solubilization of poorly water-soluble, highly lipophilic drugs, lipid nanoemulsions bear potential for drug targeting approaches. This requires that the drug remains within the emulsion droplets until they reach the site of action. Since drug release is rather controlled by the lipophilicity of the drug than by the formulation, this study systematically investigated the influence of drug lipophilicity on the course of drug transfer in (physiological) acceptor media. An increase in drug lipophilicity, according to ClogD/P values, was achieved by the formation of lipophilic prodrugs of 5-phenylanthranilic acid – a potential pathoblocker. The range of substances was supplemented by orlistat, lumefantrine and cholesteryl acetate as model drugs. Drug transfer from supercooled trimyristin nanodroplets was determined via differential scanning calorimetry by monitoring their onset crystallization temperature, which decreases linearly with increasing drug content. Release of the model (pro)drugs ranged from burst to hardly any release in the order of the ClogD/P values. Except for cholesteryl acetate, the results were in line with the lipophilicity of the model (pro)drugs estimated by their retention times on a reversed-phase HPLC column under isocratic conditions. An approximate prediction of drug release kinetics was, thus, possible by logP calculations and, to a limited extent, also by reversed-phase HPLC. A further finding was the increased drug loading capacity of the lipid nanoemulsion for lipophilic prodrugs, if the structural changes of the parent compound were accompanied by a lower melting point.

Moojecin: The first disintegrin from Bothrops moojeni venom and its antitumor activity in acute myeloid leukemia

Disintegrins are a class of peptides found in snake venom that inhibit the activity of integrins, which are essential cell adhesion receptors in tumor progression and development. In this work, moojecin, a RGD disintegrin, was isolated from Bothrops moojeni snake venom, and its antitumor potential in acute myeloid leukemia (AML) HL-60 and THP-1 cells was characterized. The isolation was performed using a C18 reverse-phase column in two chromatographic steps, and its molecular mass is 7417.84 Da. N-terminal and de novo sequencing was performed to identify moojecin. Moojecin did not show cytotoxic or antiproliferative activity in THP-1 and HL-60 at tested concentrations, but it exhibited significant antimigratory activity in both cell lines, as well as inhibition of angiogenesis in the tube formation assay on Matrigel in a dose-dependent manner. A stronger interaction with integrin αVβ3 was shown in integrin interaction assays compared to α5β1, and the platelet aggregation assay indicated an IC50 of 5.039 μg/mL. Preliminary evaluation of disintegrin toxicity revealed no incidence of hemolysis or cytotoxic effects on peripheral blood mononuclear cells (PBMCs) across the tested concentrations. Thus, this is the first study to report the isolation, functional and structural characterization of a disintegrin from B. moojeni venom and bring a new perspective to assist in AML treatment.

Isolation and characterization of NGFFYamide neuropeptide from Patiria pectinifera pyloric caeca extract

Neuropeptides are small molecules that mediate intercellular signaling and regulate physiological processes. Starfish possess various myoactive neuropeptides, including starfish myorelaxant peptide (SMP) and a calcitonin-type peptide with apical muscle relaxing properties. In this study, we report the purification of a neuropeptide from starfish (Patiria pectinifera) pyloric caeca extract using high-performance liquid chromatography (HPLC) and an in vitro bioassay to screen for fractions and peptides with relaxing effects on P. pectinifera apical muscle preparations. A series of HPLC steps using reversed-phase and cation-exchange columns yielded a purified peptide with muscle-relaxing effects. The purified peptide's structure was determined by LC-MS and Edman degradation, revealing a pentapeptide with an amidated C-terminus (NGFFYamide) and a molecular mass of 646.2930 Da. This is the first report of NGFFYamide purification from P. pectinifera through biochemical methods. The nucleotide sequence encoding the NGFFYamide precursor was determined, showing the presence of a conserved neurophysin domain in the C-terminal region. RT-qPCR results confirmed high expression in radial nerves cord, consistent with previous findings on NG peptides in echinoderms. In vitro pharmacological studies on muscle preparations from P. pectinifera and Asterias amurensis revealed differential relaxing activity of NGFFYamide on apical muscles, while its effects on tube foot preparations were similar in both species. NGFFYamide also induced potent contraction in P. pectinifera cardiac stomach. Comparison of three NG peptides (NGFFYamide, NGFFFamide, and NGIWYamide) on P. pectinifera cardiac stomach revealed varying potency, suggesting class-specific receptor interactions. Tachyphylaxis was observed in P. pectinifera apical muscle but not in A. amurensis, warranting further investigation. Based on these results, it is plausible that NGFFYamide could be involved in regulating locomotion and feeding behavior in P. pectinifera, consistent with findings in Asterias rubens.

Production of rare ginsenosides by biotransformation of Panax notoginseng saponins using Aspergillus fumigatus

Panax notoginseng saponins (PNS) are the main active components of Panax notoginseng. But after oral administration, they need to be converted into rare ginsenosides by human gut microbiota and gastric juice before they can be readily absorbed into the bloodstream and exert their effects. The sources of rare ginsenosides are extremely limited in P. notoginseng and other medical plants, which hinders their application in functional foods and drugs. Therefore, the production of rare ginsenosides by the transformation of PNS using Aspergillus fumigatus was studied in this research. During 50 days at 25 ℃ and 150 rpm, A. fumigatus transformed PNS to 14 products (1–14). They were isolated by varied chromatographic methods, such as silica gel column chromatography, Rp-C18 reversed phase column chromatography, semi-preparative HPLC, Sephadex LH-20 gel column chromatography, and elucidated on the basis of their 1H-NMR, 13C-NMR and ESIMS spectroscopic data. Then, the transformed products (1–14) were isolated and identified as Rk3, Rh4, 20 (R)-Rh1, 20 (S)-Protopanaxatriol, C-K, 20 (R)-Rg3, 20 (S)-Rg3, 20 (S)-Rg2, 20 (R)-R2, Rk1, Rg5, 20 (S)-R2, 20 (R)-Rg2, and 20 (S)-I, respectively. In addition, all transformed products (1–14) were tested for their antimicrobial activity. Among them, compounds 5 (C-K) and 7 [20 (S)-Rg3] showed moderate antimicrobial activities against Staphylococcus aureus and Candida albicans with MIC values of 6.25, 1.25 μg/mL and 1.25, 25 μg/mL, respectively. This study lays the foundation for production of rare ginsenosides.Graphical abstract

A robust method for quantifying 42 phenolic compounds by RP-HPLC/DAD: Columns performance and characterization of Brazilian Citrus peels

Reliable analytical methods are the basis for the elucidation of phenolic compounds in foods. This study aimed to optimize and validate a method for determining 42 phenolics using reverse-phase (RP) high-performance liquid chromatography (HPLC) coupled to diode-array-detector-DAD. The performance of two RP columns was evaluated. The 150x4.6 mm 3-μm column showed superior separation quality, whereas 35 of the 42 phenolics showed a separation resolution ≥1.5. The method's linearity, precision (coefficient variation< 3.09%), recovery (87.5-103.2%), specificity, limits of detection (0.04-0.25 mg/L), and quantification (0.06-0.25 mg/L) had acceptable ranges. Thirty phenolics were quantified in Citrus peels, mainly flavanones, flavanols, flavonols, and phenolic acids, highlighting the high values of hesperidin (535-35070 mg/kg) and naringin (26-36466 mg/kg). Lemon peels named 'Lisboa,' 'Thaiti,' 'Thaiti-2000', and 'Thaiti-2001' presented the main phenolics associated with antioxidant capacity. The presented method was robust for determining 42 phenolic compounds, offering a new approach for bioactive compound quantification in food matrices.