Reversed-phase Solid-phase Extraction Research Articles

Universal Pretreatment Development for Low-input Proteomics Using Lauryl Maltose Neopentyl Glycol

In recent years, there has been a growing demand for low-input proteomics, particularly in the context of single-cell proteomics (SCP). In this study, we have developed a lauryl maltose neopentyl glycol (LMNG)-assisted sample preparation (LASP) method. This method effectively reduces protein and peptide loss in samples by incorporating LMNG, a surfactant, into the digestion solution and subsequently removing the LMNG simply via reversed phase solid-phase extraction. The advantage of removing LMNG during sample preparation for general proteomic analysis is the prevention of mass spectrometry (MS) contamination. When we applied the LASP method to the low-input SP3 method and on-bead digestion in coimmunoprecipitation-MS, we observed a significant improvement in the recovery of the digested peptides. Furthermore, we have established a simple and easy sample preparation method for SCP based on the LASP method and identified a median of 1175 proteins from a single HEK239F cell using liquid chromatography (LC)-MS/MS with a throughput of 80 samples per day.

11-Nor-9-Carboxy-Δ8-Tetrahydrocannabinol, 7-Carboxy Cannabidiol, and 11-Nor-9-Carboxy-Δ9-Tetrahydrocannabinol in Urine by LC-MS/MS.

Δ8-Tetrahydrocannabinol (Δ8-THC) and cannabidiol (CBD) are increasingly popular cannabinoids. Measuring metabolites in urine is an important tool for detecting use and/or exposure as well as for monitoring elimination of these two drugs. Distinguishing between the metabolite 11-nor-9-carboxy-Δ8-tetrahydrocannabinol (Δ8-THC-COOH) and the analogous metabolite of the more common and naturally abundant Δ9-THC: 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THC-COOH) is analytically challenging due to structural similarities between the two compounds. Here, we present a method for separating the positional isomers Δ8-THC-COOH and Δ9-THC-COOH as well as 7-carboxy cannabidiol (CBD-COOH) in urine that includes reverse-phase solid-phase extraction (SPE), followed by liquid chromatographic separation with a perfluorophenyl column, and detection by tandem mass spectrometry (LC-MS/MS).

Optimization of reversed-phase solid-phase extraction for shotgun proteomics analysis.

Reversed-phase solid-phase extraction (SPE) is the method of choice for the purification of proteomics samples. Even though the efficacy of SPE methods is sample type-dependent, the manufacturers' protocols are used in most studies. Using an optimized SPE method can lead to a substantial gain in identification and recovery. In this tutorial, we give a brief introduction to the most important parameters influencing SPE performance, and we present a short workflow (16 measurements) for optimizing the SPE procedure. This is complemented by method performance assessment instructions and a short troubleshooting guide to help users further understand and investigate their SPE methods.

Inaccuracies in plasma oxytocin extraction and enzyme immunoassay techniques

Numerous studies have reported extensive associations between plasma oxytocin (OXT) concentrations and various human physiological and neurobehavioral processes. Measurement of OXT is fraught with difficulty due to its low molecular weight and plasma concentrations, with no consensus as to the optimal conditions for pre-analytical sample extraction, standards for immunoassay validation or the ideal protease inhibitors to prevent OXT degradation. Previous attempts at determining the efficacy of various purification techniques such as solid phase extraction (SPE) or ultrafiltration have only utilized human plasma samples, making it difficult to dissect out whether the effect of interference comes from the extraction process itself or cross-reactivity with other proteins. By testing these on pure OXT solutions, we demonstrate poor recovery efficacy and reliability of reversed phase SPE (maximum 58.1%) and ultrafiltration (<1%) techniques, and the potential for the former to introduce interference into enzyme immunoassay (EIA) measurements. The clonality of antibodies used in EIA kits also potentially contributes to the differences in the readings obtained, and we validate an EIA kit which did not require pre-analytical sample extraction with low cross-reactivity and high reliability (intraclass correlation coefficient 0.980 (95% CI 0.896–0.999). Biochemical techniques used for measuring plasma OXT concentrations must therefore be internally validated prior to translation into clinical studies.

Evaluation of antitumoral and antioxidant activities of the hydroalcoholic extract and fractions obtained from the fruit pericarp of Sapindus saponaria L.

The fruits of Sapindus saponaria L., popularly known as ‘saboeiro’, have been used in medicine. This study evaluated the antioxidant and antitumor activities of the hydroethanolic extract (HAE) and fractions obtained from the fruit pericarp of S. saponaria. The HAE was obtained from the S. saponaria fruit pericarp by maceration; this was followed by fractionation using reversed-phase solid-phase extraction, resulting in fractions enriched with acyclic sesquiterpenic oligoglycosides (ASOG) and saponins (SAP1, and SAP2), confirmed by mass spectrometry with electrospray ionization (ESI-QTOF-MS). The greatest citotoxic activity was observed with the SAP1 fraction against the CaCo2 cell line with a GI50 of 8.1 µg mL−1, while the SAP2 fraction had a GI50 of 13.6 µg mL−1 against CaCo2. The HAE demonstrated the greatest antioxidant activity. S. saponaria has potential therapeutic use in the pharmaceutical industry as a natural anti-oxidant or antitumor product.

Testing of Ion Exchange Solid Phase Extraction Media for Extraterrestrial In Situ Sample Preparation on Liquid Samples

The search for life on other bodies in our solar system is currently focused on Ocean Worlds, those bodies known to contain liquid water, as water is one of the requirements for life as we understand it. In the search for organic biosignatures that would indicate the presence of past or current life, liquid samples from these bodies would utilize an initial sample preparation step in which interfering substances such as salts can be removed before analysis. Previous work on potential sample preparation techniques for these samples evaluated solid phase extraction (SPE) on cation exchange media, but only explored amino acid analytes at low salt to analyte ratios and used high concentration eluents. This work utilized mixed-mode ion exchange solid phases, developing methods for elutions in low concentration solutions and evaluating polar analytes with a range of functionalities. Method development revealed the need for a pre-elution step in the process to decrease the elution volume required. Both anion and cation exchange media were evaluated for the capture of analytes from solutions that simulated Earth’s oceans. The Oasis MCX and Strata X-C cation exchange media provided the best results, with >90% retention of all analytes including amino acids, organic amines, nucleotides, peptides, and an oligonucleotide. These cation exchange media retained even anionic components, including glutamic acid and organic acids, with >90% efficiency. These analytes were released in the wash step, but salt ion removal was completed before release, allowing this technique to be used for desalting of these analytes. Extraction of a 14 component mixed analyte solution also showed retention of all analytes, with testing of analyte concentrations down to 100 nM in 35 g/L simulated ocean solution. Oasis MAX and Strata X-A anion exchange media did not retain glutamic acid, fumaric acid, or dipicolinic acid when the salt concentration was high; these anionic analytes were easily extracted from low salt solutions. The anion exchange media showed a range of functionality for extracting other analytes from the simulated ocean water, capturing >90% of tryptophan and phenylalanine, but retaining <50% of the valine and >20% of the glycine. Irradiation exposure, as a model for the solar irradiation expected during deployment to extraterrestrial locations, did not affect the performance of any ion exchange media tested. A reversed-phase SPE column was directly coupled to a cation exchange column, to investigate removal of nonpolar compounds that might bind to and block the mixed-mode ion exchange media.

Evaluation of the antibacterial and antifungal potentials of peptide-rich extracts from selected Nigerian plants

Introduction: Host defence peptides (HDPs) has gained wide recognition as a potential source of antimicrobial agents because of their abundance in nature, low incidence of reported resistance, and their immunomodulatory properties. This study investigated the presence and antimicrobial activities of bioactive peptides in seven selected plants from the Rubiaceae, Apocynaceae, and Euphorbiaceae families. Methods: The reversed-phase solid-phase extraction was used to obtain the peptide-rich fractions from the crude extracts of the plants, and the circular peptides were chemically detected by a modified G-250 spray on a developed TLC plate. The peptide fraction and crude extracts were screened for their antimicrobial activities using the broth microdilution method. The rate of kill study was conducted for the peptide fractions Euphorbia hirta and Nauclea diderichii Results: The study revealed Nauclea diderichii peptide-rich fraction as the most potent on the tested strains of gram-positive and gram-negative bacteria and fungi (MIC = 7.8, 15.63-62.5, and 7.8 μg/mL respectively). Euphorbia hirta and Nauclea diderichii peptide fractions exhibited bactericidal activity (MIC index ≤4, and >3Log 10 reduction of initial inoculum) and have a broad spectrum of activity, which further validates their ethnomedicinal use in the management of diverse infectious diseases, including enteric and respiratory tract infections. The peptide fractions of all the plants are more potent than the crude extracts. Conclusion: This study established the antimicrobial potentials of the peptide-rich fractions from these plant species, and the basis for further investigation to isolate and characterize the bioactive peptides responsible for their antimicrobial activities.

What are oxytocin assays measuring? Epitope mapping, metabolites, and comparisons of wildtype & knockout mouse urine

Oxytocin has become a popular analyte in behavioral endocrinology in recent years, due in part to its roles in social behavior, stress physiology, and cognition. Urine samples have the advantage of being non-invasive and minimally disruptive to collect, allowing for oxytocin measurements even in some wild populations. However, methods for urinary oxytocin immunoassay have not been sufficiently optimized and rigorously assessed for their potential limitations. Using samples from oxytocin knockout (KO) and wildtype (WT) mice, we find evidence of considerable interference in unextracted urine samples, with similar distributions of measured oxytocin in both genotypes. Importantly, although this interference can be reduced by a reversed-phase solid-phase extraction (SPE), this common approach is not sufficient for eliminating false-positive signal on three immunoassay kits. To better understand the source of the observed interference, we conducted epitope mapping of the Arbor Assays antibody and assessed its cross-reactivity with known, biologically active fragments of oxytocin. We found considerable cross-reactivity (0.5–52% by-molarity) for three fragments of oxytocin that share the core epitope, with more cross-reactivity for longer fragments. Given the presence of some cross-reactivity for even the tripeptide MIF-1, it is likely that many small protein metabolites might be sufficiently similar to the epitope that at high concentrations they interfere with immunoassays. We present a new mixed-mode cation-exchange SPE method that minimizes interference—with knockout samples measuring below the assay’s limit of detection—while effectively retaining oxytocin from the urine of wildtype mice. This method demonstrates good parallelism and spike recovery across multiple species (mice, dogs, sifakas, humans). Our results suggest that immunoassays of urine samples may be particularly susceptible to interference, even when using common extraction protocols, but that this interference can be successfully managed using a novel mixed-mode cation exchange extraction. These findings imply that previous conclusions based on urinary oxytocin measurements—especially those involving unextracted samples—may need to be reassessed.

Solid-Phase Extraction Approaches for Improving Oligosaccharide and Small Peptide Identification with Liquid Chromatography-High-Resolution Mass Spectrometry: A Case Study on Proteolyzed Almond Extract.

Reverse-phase solid-phase extraction (SPE) is regularly used for separating and purifying food-derived oligosaccharides and peptides prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. However, the diversity in physicochemical properties of peptides may prevent the complete separation of the two types of analytes. Peptides present in the oligosaccharide fraction not only interfere with glycomics analysis but also escape peptidomics analysis. This work evaluated different SPE approaches for improving LC-MS/MS analysis of both oligosaccharides and peptides through testing on peptide standards and a food sample of commercial interest (proteolyzed almond extract). Compared with conventional reverse-phase SPE, mixed-mode SPE (reverse-phase/strong cation exchange) was more effective in retaining small/hydrophilic peptides and capturing them in the high-organic fraction and thus allowed the identification of more oligosaccharides and dipeptides in the proteolyzed almond extract, with satisfactory MS/MS confirmation. Overall, mixed-mode SPE emerged as the ideal method for simultaneously improving the identification of food-derived oligosaccharides and small peptides using LC-MS/MS analysis.

In vitro antiviral activity of peptide-rich extracts from seven Nigerian plants against three non-polio enterovirus species C serotypes

BackgroundAs frequent viral outbreaks continue to pose threat to public health, the unavailability of antiviral drugs and challenges associated with vaccine development underscore the need for antiviral drugs discovery in emergent moments (endemic or pandemic). Plants in response to microbial and pest attacks are able to produce defence molecules such as antimicrobial peptides as components of their innate immunity, which can be explored for viral therapeutics.MethodsIn this study, partially purified peptide-rich fraction (P-PPf) were obtained from aqueous extracts of seven plants by reverse-phase solid-phase extraction and cysteine-rich peptides detected by a modified TLC method. The peptide-enriched fractions and the aqueous (crude polar) were screened for antiviral effect against three non-polio enterovirus species C members using cytopathic effect reduction assay.ResultsIn this study, peptide fraction obtained from Euphorbia hirta leaf showed most potent antiviral effect against Coxsackievirus A13, Coxsackievirus A20, and Enterovirus C99 (EV-C99) with IC50 < 2.0 µg/mL and selective index ≥ 81. EV-C99 was susceptible to all partially purified peptide fractions except Allamanda blanchetii leaf.ConclusionThese findings establish the antiviral potentials of plants antimicrobial peptides and provides evidence for the anti-infective use of E. hirta in ethnomedicine. This study provides basis for further scientific investigation geared towards the isolation, characterization and mechanistic pharmacological study of the detected cysteine-rich peptides.

Characterization of allelopathic compounds from the harmful dinoflagellate, Cochlodinium geminatum

In 2006, species of Cochlodinium geminatum were first recorded as new, harmful algal bloom (HAB)-forming dinoflagellate, in the Pearl River Estuary, China. Previous studies indicated that allelopathy (the release of chemical compounds) is an important competitive strategy for C. geminatum in inhibiting the growth of phytoplankton competitors, however, little is known about the allelochemicals produced by this species. In this study, allelopathic compounds were characterized using a series of chromatographic techniques. Methanol extracts from cultures showed significant inhibitory effects towards the co-occurring phytoplankton, Akashiwo sanguinea, and potent hemolytic activity against rabbit erythrocytes. Solid phase extraction (SPE) fractions from a reverse phase SPE column, exhibited inhibitory and synergistic effects on A. sanguinea growth, of which fractions eluted in 80% and 100% methanol showed the strongest allelopathic activity. These fractions were further purified using reverse phase high performance liquid chromatography (HPLC), and six fractions were collected. Three fractions displayed strong inhibitory effects towards A. sanguinea, with 14%–25% inhibitory rates, but a higher 35% inhibitory rate was observed when fractions were combined, suggesting allelochemicals had synergistic allelopathic effects. Allelopathic activity also exhibited time dependent reduction when stored at -20°C. Furthermore, trypsin digestion and dialysis assays indicated C. geminatum produced multiple allelopathic compounds. Our study demonstrated that species of C. geminatum produced and released multiple allelopathic compounds of low polarity and sufficient hydrophilicity, which elicit important competitive advantages by inhibiting co-occurring phytoplankton, and promoting population growth.

Seeking faster, alternative methods for glycolipid biosurfactant characterization and purification.

Biosurfactants have been investigated as potential alternatives for synthetic surfactants in several areas, for example, in environmental and pharmaceutical fields. In that regard, extensive research has been carried out with sophorolipids and rhamnolipids that also present various biological properties with therapeutic significance. These biosurfactants are obtained as complex mixtures of slightly different molecules, and thus when studying these microbial glycolipids, the ability to identify and purify the produced compounds is of extreme importance. This study aimed to develop improved methodologies for the identification, separation, and purification of sophorolipids and rhamnolipids. Therefore, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was modified to ensure faster characterization of both sophorolipids and rhamnolipids, enabling the identification and fragmentation pattern description of 10 and 13 congeners, respectively. The separation and purification of these biosurfactants was achieved with novel reversed-phase solid-phase extraction methods guaranteeing the isolation of different glycolipids, including those considered for their significant biological activity (e.g. antimicrobial, anticancer). It was possible to isolate sophorolipids and rhamnolipids with purity of 94% and 99%, respectively. The methods presented herein can be easily implemented and are expected to make purification of these biosurfactants easier, facilitating the study of their individual properties in further works.

Bioactive peptide isolated from sesame seeds inhibits cell proliferation and induces apoptosis and autophagy in leukemic cells.

Leukemia is the most common type of hematological malignancies. Several natural products including bioactive peptides have been explored and studied for their anti-leukemic activities. In the present study, anti-leukemic peptide, IGTLILM (IM-7), was isolated and identified from the protein hydrolysate of sesame seeds by reverse phase-solid phase extraction, off-gel fractionation and nano LC-MS/MS. The cytotoxic effects of IM-7 were studied in MOLT-4 and NB4 acute leukemic cell lines using an MTT assay. The induction of apoptosis and autophagy was investigated by flow cytometry using Annexin V-FITC/PI staining and anti-LC3/FITC antibodies, respectively. The mRNA alterations of apoptotic and autophagic-related genes were determined by reverse transcription-quantitative PCR. The present study found that IM-7 inhibited the proliferation of MOLT-4 and NB4 cells in dose-dependent manner, but it showed a minimal effect on healthy mononuclear cells. IM-7 activated apoptosis and autophagy through the upregulation of CASP3, ULK1 and BECN1 and the downregulation of BCL2. In addition, IM-7 enhanced the cytotoxic effect of the anti-leukemic drug, daunorubicin. The findings suggested that IM-7 was potent to suppress the proliferation of MOLT-4 and NB4 leukemic cells and induce apoptosis and autophagy through the regulation of caspase 3-Bcl-2 and ULK1-Beclin1, respectively.

CoolTip: Low-Temperature Solid-Phase Extraction Microcolumn for Capturing Hydrophilic Peptides and Phosphopeptides

Reversed-phase solid-phase extraction (SPE) techniques are commonly used for desalting samples before LC/MS/MS in shotgun proteomics. However, hydrophilic peptides are often lost during the desalting step under the standard SPE conditions. Here, we describe a simple protocol in which a stop-and-go extraction tip packed with a poly(styrene-divinylbenzene) copolymer disc is used at 4 °C during sample loading without any organic solvent. Using this method, which we designate as the CoolTip protocol, we identified 2.9-fold more tryptic peptides and 6.1-fold more tryptic phosphopeptides from HeLa lysates than the standard SPE protocol for hydrophilic peptides, with a mobile phase of less than 8% acetonitrile in LC/MS/MS. There was no decrease in the recovery of hydrophobic peptides. CoolTip also provided better quantitative reproducibility in LC/MS/MS analysis. We anticipate that this protocol will provide improved performance in many kinds of shotgun proteomics experiments.

Facilitating serum determination of neuron specific enolase at clinically relevant levels by coupling on-line molecularly imprinted solid-phase extraction to LC-MS/MS

The identification and quantification of biomarkers is essential for the diagnosis, treatment, and long-term monitoring of many human diseases. In the present work, macromolecular synthetic receptors with pre-determined affinity and selectivity for the signature peptide of a prognostically significant small cell lung cancer (SCLC) biomarker - neuron-specific enolase (NSE) – were prepared in a porous polymer microsphere format using a template-directed synthesis strategy performed under precipitation polymerization conditions. The polymer microspheres were packed into short trap columns and then exploited as molecularly selective sorbents in a fully automated, on-line molecularly imprinted solid-phase extraction (MISPE) protocol. The on-line MISPE protocol was optimised with respect to the composition of the loading mobile phase, the flow rate, and the extraction time. The molecularly imprinted polymers (MIPs) showed high affinity and useful selectivity for the peptide target - the hexapeptide ELPLYR - compared to non-imprinted control polymers. The MIPs were able to retain the biomarker on-column for extraction times of up to 20 min, and the on-line MISPE method enabled complete recovery of the biomarker over the linear range 10–100 ng mL−1 when the biomarker was present in spiked ammonium bicarbonate solution (R2 = 0.994). For extractions of ELPLYR from very complex biological matrices, the recoveries of ELPLYR from reversed-phase SPE (RP-SPE)-treated and untreated digested human serum were 100.8 ± 6.2% and 61.6 ± 1.9%, respectively. Extractions of ELPLYR from spiked untreated digested serum were linear in the range of 7.5–375 ng mL−1 (R2 = 0.99). The limit of detection (LOD) and limit of quantification (LOQ) for the biomarker in digested serum were estimated to be 1.8 ng mL−1 and 6.0 ng mL−1, respectively, which is below the median reference level of NSE in humans (8.6 ng mL−1). This work sets in place the basis for a new diagnostic tool for SCLC that is sensitive, robust, automated, and antibody-free, and which works very well with complex human plasma samples.

Improved quantification of lipid mediators in plasma and tissues by liquid chromatography tandem mass spectrometry demonstrates mouse strain specific differences

A liquid chromatography tandem mass spectrometry-based method for the quantitation of 39 lipid mediators in four sample types and in two mouse strains is described. The method builds upon existing methodologies for analysis of lipid mediators by A) utilizing a bead homogenization step for tissue samples; this eliminates the need for homogenization glassware and improves homogenization consistency, B) optimizing the isolation and purification of lipid mediators with polymeric reverse phase SPE columns with lower sorbent masses; this results in lower solvent elution volumes without loss of recovery and C) utilizing an on-column enrichment method to improve analyte focusing before chromatographic separation. The method is linear from 0.25-250 pg on column for low level lipid mediators and from 5-5000 pg on column for high level lipid mediators. The addition of a methyl formate elution step to a previously published method dramatically improved precision and recovery for the cysteinyl leukotrienes. Accuracy and precision for 4 different sample types including human plasma, mouse lung, mouse spleen and mouse liver is demonstrated. Liver samples had extremely high levels of a tentatively identified bile acid which interfered with quantitation of resolvin E1, 11B-prostaglandin F2a and thromboxane A2. Results from 2 different tissue sources from untreated mice (C57BL/6 versus BALB/c) showed dramatically different concentrations of lipid mediators.

Analyses of the brominated vegetable oil in soft drinks using gas chromatography–flame ionization detector and atmospheric pressure gas chromatography–quadrupole/time-of-flight mass spectrometry

Herein, a new method for quantifying the brominated vegetable oil content in commercial soft drinks was developed, which accelerated the sample preparation process and improved analytical efficiency. First, simple and accurate chromatographic separation techniques were performed using a VF-5ht column for both GC-FID (quantitative) and APGC-QTOF (qualitative) analyses. The samples were subjected to chromatography on a reversed-phase solid-phase extraction cartridge. (PoraPak™RxnRP). Transesterification using a boron trifluoride methanol complex in methanol solution was performed. When validating this method, the analyte recovery percentages were between 82.2% and 99.9%, and the recovery and standard deviation of repeatability values were between 1.2% and 3.5%. Using an isotope library, the bromostearic acid methyl esters (9,10-dibromostearic acid methyl ester, 9,10,12,13-tetrabromostearic acid methyl ester, and hexabromostearic acid methyl ester) in the sample mixtures were qualitatively confirmed via APGC-QTOF. A novel aqueous ammonium adduct, which has not been previously reported, was also confirmed. These results indicated that this new method was simple, accurate, and also allowed for precise qualitative and quantitative confirmation as well as high reproducibility.

Development of LC-MS/MS method for simultaneous determination of Canagliflozin and Metformin in human plasma and its pharmacokinetic application in Indian population under fast and fed conditions

A novel, selective and sensitive method is developed for simultaneous estimation of canagliflozin and metformin and successfully applied to fast and fed pharmacokinetic studies in healthy Indian volunteers. The current study reports the development, optimization, and validation of liquid chromatography-mass spectrometry (LC-MS/MS) method for simultaneous quantification of canagliflozin and metformin in human plasma using deuterated canagliflozin D4 and metformin D6 as an internal standard (IS). The solid-phase extraction technique was employed where strata X polymeric reverse phase (30 mg-1 cc) SPE cartridges were used for the extraction of analytes and IS from plasma. The ACE 5 C18 column (50 × 4.6 mm, 5µ) was used to chromatograph the prepared samples. The mobile phase consisted of methanol and 5 mM ammonium trifluoroacetate in water, pH 5 (50:50, v/v) at a flow rate of 0.8 mL/min.Detection was performed by positive ion Turbo ion spray in Multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 461.9 → m/z 191.1 and m/z 461.9 → m/z 267.2, for quantification of canagliflozin. The response of canagliflozin fragments m/z 461.9 → m/z 191.1 and m/z 461.9 → m/z 267.2 was combined. Also, for metformin transitions were monitored at m/z 130.0 → m/z 71.1. Full validation of the method was performed according to the United States Food and Drugs Administration (USFDA) guidelines. Linearity was in the range of 24.95–2806.55 ng/mL for canagliflozin and 24.99–3400.72 ng/mL for metformin. The mean extraction recovery of canagliflozin, canagliflozin D4, metformin, and metformin D6 was 77.240, 84.663, 66.747, and 67.449, respectively across four QC levels. This rapid method with the run time of 2.80 min allows the analysis of more than 400 plasma samples per day.

Palindromic Peptide LfcinB (21‐25)Pal Exhibited Antifungal Activity against Multidrug‐Resistant Candida

AbstractPalindromic peptide LfcinB (21–25)Pal: RWQWRWQWR was synthesized by Solid Phase Peptide Synthesis (SPPS‐Fmoc/tBu), purified by Reverse Phase Solid Phase Extraction (RP‐SPE) and characterized by Reverse Phase High Performance Liquid Chromatography (RP‐HPLC) and Matrix‐Assisted Laser Desorption/Ionization‐Time Of Flight Mass Spectrometry (MALDI‐TOF MS). The antifungal activity of LfcinB (21–25)Pal against both ATCC strains and clinical isolates of C. albicans, C. glabrata, C. krusei, C. auris and C. tropicalis was evaluated. The palindromic peptide exhibited fungistatic and fungicidal activity against all yeast evaluated. The antifungal activity was dependent on peptide concentration in all cases. Additionally, LfcinB (21–25)Pal (25–50 μg/mL) combined with fluconazole exhibited a synergistic antifungal effect against C. tropicalis 883 and C. krusei 6258 (resistant to fluconazole). This study showed that the palindromic peptide derived from Lactoferricin B (LfcinB) exhibited significant antifungal activity against Candida spp, suggesting that this peptide could have a therapeutic application solely or in combination with fluconazole.

A 3D-polyphenylalanine network inside porous alumina: Synthesis and characterization of an inorganic-organic composite membrane.

Surface functionalization of porous materials allows for the introduction of additional functionality coupled with high mechanical stability of functionalized inner pores. Herein, we investigate the surface-initiated ring-opening polymerization (SI-ROP) of phenylalanine-N-carboxyanhydride (PA-NCA) in porous alumina membranes (ALOX-membranes) with respect to different solvent mixtures (tetrahydrofuran (THF) and dichloromethane (DCM)). It was found that increasing the volume fraction of DCM leads to an increasing amount of fibrillar polymer structures within the porous ALOX-membrane. A three-dimensional fibrillar network with intrinsic porosity was formed in DCM, whereas in THF, a dense and smooth polypeptide film was observed. A post-treatment with a mixture of chloroform and dichloroacetic acid leads to rearrangement of the morphology of the grafted polymer films. The analysis by scanning electron microscopy (SEM), near-infrared spectroscopy (NIR) and contact angle measurements (CA) reveals a change in morphology of the grafted polymer films, which is due to the rearrangement of the secondary structure of the polypeptides. No significant loss of the surface-grafted polypeptides was determined by thermogravimetric (TG) measurements, which indicates that the change in morphology of the polymer films is solely a result of a conformational change of the surface-grafted polypeptides. Furthermore, adsorption of a test analyte (chloroanilic acid) was investigated with respect to different polymer functionalization schemes for reversed-phase solid phase extraction applications. The adsorption capability of the functionalized composite membrane was increased from 16.7% to 38.1% compared to the native ALOX-membrane.