Silica Column Research Articles

DNA Extraction with TRIzol Reagent Using a Silica Column.

TRIzol is a monophasic solution of phenol and guanidine isothiocyanate used for the extraction of RNA, DNA and proteins from tissues or cells. However, few studies have described its application to DNA extraction due to its time-consuming procedure. We present a TRIzol-modified method of extracting DNA from tissues using the TRIzol reagent and a silica column, which requires only one-third of the time required for the classic extraction procedure. Spectrophotometric analysis showed that the 260/280 and 260/230 nm optical density ratio of the DNA extracted using the TRIzol-modified method is ideal and equal to that obtained by the classic method and commercial DNAiso methods. The DNA extracted by the TRIzol-modified method had the same performance in a restriction enzyme digestion and quantitative PCR as that extracted using the classic method. Using the TRIzol-modified method saves time, simplifies the DNA extraction procedure, and facilitates various molecular biology assays.

SYNTHESIS, ANALYSIS AND SEPARATION OF ISOMERIC INDENO- AND INDOLOQUINOXALINES

Due to the increasing number of respiratory diseases with developed inflammatory effects epidemics, the importance of finding a highly effective broad spectrum antiviral drug in combination with the anti-inflammatory activity is growing. Structurally similar derivatives of indolo[2,3-b]quinoxaline and indeno[1,2-b]quinoxalin-11-one exhibit a wide and partially similar spectrum of action, which makes them promising potential antiviral agents. These compounds were synthesized via substituted 1,2-diaminobenzenes and isatin (in case of 1) or ninhydrin (in case of 2) condensation and with no farther purification (indeno[quinoxalinone derivatives) or with washing with hot diluted alkali solution (to remove the major impurities of indoloquinoxaline derivatives). Thus, the aimed compounds were isolated as a mixture known, for a more detailed study of biological activity (especially in the case of ligandenzyme interactions), individual isomers should be investigated. This work is dedicated to the regio-isomeric composition investigation of synthesized indolo- and indenoquinoxalines and their separation. Considering minimal differences between isomeric compounds structure and chemical behavior, jnly chromatographic separation methods were required. Combination of poor solubility in low polar solvents and low separation effect for both of compound sets (indeno- and indoloquinoxalines) made column silica gel purification unacceptable. Indolo[2,3-b]quinoxaline isomers were separated by preparative TLC method using chloroform and ethyl-acetate as eluent. Separation was confirmed by UV-HPLC and MS-MS-UPLC methods. Indeno[1,2-b]quinoxalin-11-one derivatives were not separated in preparative quantities using silica gel column/thin layer methods.

Efficiency of DNA Isolation Methods Based on Silica Columns and Magnetic Separation Tested for the Detection of Mycobacterium avium Subsp. Paratuberculosis in Milk and Faeces.

Timely and reliable detection of animals shedding Mycobacterium avium subsp. paratuberculosis (MAP) should help to effectively identify infected animals and limit infection transmission at early stages to ensure effective control of paratuberculosis. The aim of the study was to compare DNA extraction methods and evaluate isolation efficiency using milk and faecal samples artificially contaminated by MAP with a focus on modern instrumental automatic DNA isolation procedures based on magnetic separation. In parallel, an automatic and manual version of magnetic separation and two methods of faecal samples preparation were compared. Commercially available DNA isolation kits were evaluated, and the selected kits were used in a trial of automatic magnetic beads-based isolation and compared with the manual version of each kit. Detection of the single copy element F57 was performed by qPCR to quantify MAP and determine the isolation efficiency. The evaluated kits showed significant differences in DNA isolation efficiencies. The best results were observed with the silica column Blood and Tissue kit for milk and Zymo Research for faeces. The highest isolation efficiency for magnetic separation was achieved with MagMAX for both matrices. The magnetic separation and silica column isolation methods used in this study represent frequently used methods in mycobacterial diagnostics.

A simple liquid chromatography/differential ion mobility spectrometry tandem mass spectrometry method for the determination of trimethylamine-N-oxide in human serum: An application in dialysis patients.

Trimethylamine-N-oxide (TMAO) is a potential indicator of cardiovascular disease and chronic kidney disorders. It is important to monitor the TMAO level in plasma or serum of hemodialysis patients. A simple liquid chromatography/differential ion mobility spectrometry tandem mass spectrometry (HPLC/DMS-MS/MS) method was established and validated for the determination of TMAO in the serum of hemodialysis patients. Chromatographic separation was performed on a Waters Atlantis HILIC silica column (2.1 × 50 mm, 3 μm). The gradient mobile phase consisted of 10 mM ammonium formate buffer and acetonitrile with 0.1% formic acid in both solvents. The serum sample was precipitated with acidic acetonitrile prior to HPLC/DMS-MS/MS analysis and TMAO-d9 was used as the internal standard. Data acquisition was performed in positive ion mode with a DMS system before the electrospray ionization source. The selected reaction monitoring transitions were m/z 76.0 → 58.0 and m/z 85.2 → 66.1 for TMAO and the internal standard, respectively. Excellent linearity was observed over the calibration range 0.05-20 μg/mL (r2 > 0.995). The method was validated for good specificity and sensitivity. The inter-run and intra-run precision and accuracy were less than 13.6% and 10.7%, respectively. We established a novel and robust HPLC/DMS-MS/MS method for the quantification of TMAO in human serum samples. The validated assay was simple, rapid, sensitive and reliable. The developed method could be applied to the assay of serum samples from patients with kidney disease who are undergoing hemodialysis.

Selection of calibration compounds for selectivity evaluation of siloxane-bonded silica columns for reversed-phase liquid chromatography by the solvation parameter model

For the faster evaluation of selectivity in reversed-phase liquid chromatography of siloxane-bonded silica columns using the solvation parameter model a minimal set of calibration compounds is described suitable for mobile phase composition from 20-70% (v/v) methanol-, acetonitrile-, or tetrahydrofuran-water. The Kennard-Stone uniform mapping algorithm is used to select the calibration compounds from a larger database of compounds with known retention properties used earlier for column selectivity evaluation. Thirty-five compounds are shown to be necessary to minimize the standard deviation of the system constants and to minimize the difference between the system constants determined by conventional calibration and the values obtained for the reduced calibration compounds. The models for SunFire C18 with methanol-, acetonitrile- and tetrahydrofuran-water mobile phase compositions and XBridge Shield RP18, XBridge C8, XBridge Phenyl and Discovery HS F5 with methanol- and acetonitrile-water mobile phase compositions had an average coefficient of determination of 0.996 (standard deviation = 0.003, n = 11) and average standard error of the estimate 0.025 (standard deviation = 0.005, n = 11) for the reduced calibration compounds. Some octadecylsiloxane-bonded silica stationary phases with a high bonding density and methanol-water mobile phase compositions containing ≤ 30% (v/v) methanol exhibit extreme retention factors (log k > 2.5) for the low-polarity, two-ring aromatic compounds in the thirty-five compound calibration set. Alternative calibration compounds with more favorable retention properties are suggested as replacements in these cases. The predictive capability of the calibration models is validated using external test sets characterized by the average error, average absolute error and root mean square error of prediction. For the thirty-five calibration compounds sets the average absolute error 0.026 (standard deviation = 0.009, n = 11) and root mean square error of prediction 0.032 (standard deviation = 0.010, n = 11) confirm the suitability of the calibration models for column selectivity evaluation. System maps for XBridge Shield RP18 for 20-70% (v/v) methanol-water and Synergi Hydro-RP and 50% (v/v) methanol-water at temperatures from 25-65 °C together with a correlation diagram for XBridge Shield RP18 and SunFire C18 are presented as representative applications of the reduced calibration compounds for column selectivity evaluation.

Imidazolium ionic liquid bonded silica stationary phases. Part II. 1-Ethylimidazolium stationary phase

In this second research report (i.e., Part II), an ionic liquid (IL)-bonded silica column having an anion-exchange characteristic is described and characterized chromatographically with test acidic solute probes and by physical measurements such as Fourier transform infrared (FTIR) and thermogravimetric analysis (TGA). The IL anion-exchange stationary phase consists of a short alkyl chain IL stationary phase that has been synthesized by covalently immobilizing 1-ethylimidazole on a silica substrate through chloropropyltrimethoxysilane tether. The retention characteristics of the resulting stationary phase have been examined with acidic solutes such as aromatic acid derivatives and nucleotides. The test results show good resolution and separation efficiency indicating the presence of different interaction mechanisms such as π–π, electrostatic and hydrophobic interactions to a certain extent. Abbreviations: ABs: alkylbenzenes; ADP: adenosine 5′-diphosphate; AMP: adenosine 5′-monophosphate; CPTMS: 3-chloropropyltrimethoxysilane; ILs: ionic liquids; UMP: uridine 5′-monophosphate; UDP: uridine 5′-diphosphate; UTP: uridine 5′-triphosphate.

Thermochromatographic behavior of iodine in fused silica columns when evaporated from lead\u2013bismuth eutectic

As a step toward licensing a lead–bismuth eutectic (LBE) based reactor design, the adsorption behavior of iodine evaporated from LBE on fused silica was examined. Using inert and reducing carrier gases, depositions with an adsorption enthalpy of − 95 to − 113 kJ mol−1 were observed. These depositions are compatible with a single species, tentatively identified as bismuth monoiodide, BiI. When introducing oxidizing conditions, multiple iodine species with higher volatility form, with adsorption enthalpies ranging from − 67 to − 86 kJ mol−1. Based on an empirical correlation one of these species was identified as monatomic iodine. This work provides fundamental understanding of the LBE/iodine gaseous chemistry and related adsorption deposition behavior.

A comprehensive evaluation of two sample treatment procedures for the determination of emerging and historical halogenated flame retardants in biota

Two different sample preparation protocols for the determination of 37 emerging and historical halogenated flame retardants (HFRs) in marine tissues were compared with regards to extraction recovery, lipid removal efficiency, repeatability, reproducibility, and ability to measure sub-ng g−1 (dry weight) concentrations in marine biota. One method involved a purification step using gel permeation chromatography (GPC) followed by a HPLC fractionation step on a Partisil amino-cyano normal phase (GPC-Partisil procedure) and the other more traditional method was based on sulphuric acid treatment followed by silica column fractionation (H2SO4-silica procedure). The samples were analysed by gas chromatography (GC) and liquid chromatography (LC) tandem mass spectrometry (MS/MS). Sample fractionation in both methods enabled unique sample preparation procedures to isolate the GC from the LC amenable compounds. Both methods could remove > 99% of the lipids which was necessary prior to GC- and LC-MS/MS analyses. The majority of the target compounds (70%) had acceptable recoveries between 60–120% for both methods. However, the sulphuric acid treatment resulted in the degradation of the TBP-AE and the silica column fractionation resulted in the loss of BEH-TEBP and the elution of PBB-Acr and TBBPA-BME in the unsuitable fraction. High recoveries of DBE-DBCH (α+β), EHTBB, BTBPE, BEH-TEBP, and PBB-Acr were attributed to matrix effects, suggesting the need to use isotope-labelled surrogate standards of the target compounds. The optimisation of the silica column chromatography, GPC, and Partisil fractionation is described and discussed to afford easy implementation of the method. The method using GPC followed by Partisil fractionation is more efficient and more reproducible than the sulphuric acid-silica procedure. The application of this method to marine biota reference materials revealed the presence of relatively high concentrations of DBE-DBCH isomers and BDE-47 in fish samples. The method detection limits comply with the recommendations of the European Commission.

Qualitative analysis of zolpidem and its metabolites M-1 to M-4 in human blood and urine using liquid chromatography–tandem mass spectrometry

The aim of this study was to develop the method for the qualitative analysis of zolpidem and its metabolites M-1 to M-4 using liquid chromatography–tandem mass spectrometry (LC–MS/MS) in human blood and urine. To obtain analytical standards, a method for synthesizing zolpidem metabolites M-1 to M-4 was developed. A combination of a copper-catalyzed three-component coupling reaction and pyrrolidine-catalyzed imine formation was adopted to synthesize the methyl esters of M-1 (M-1-Me) and M-2 (M-2-Me), which were hydrolyzed to afford M-1 and M-2, respectively. M-3 and M-4 were also synthesized. The LC–MS/MS conditions were optimized using authentic standards. M-1 to M-4 were analyzed in human blood and urine. M-1 to M-4 were successfully synthesized. The mass spectra of M-1 and M-2 were almost identical, but their peaks were chromatographically separated using a octadecyl silica column. Likewise, mass spectra of M-3 and M-4 were similar, but their peaks were chromatographically separated. The peak intensities of the metabolites in human blood and urine were M-1 > M-2, and M-4 > M-3; M-3 was detected only in urine. The presence of other hydroxyzolpidems was also revealed. Chromatographic separation of M-1 and M-2 and that of M-4 and other hydroxyzolpidems is necessary to analyze zolpidem metabolites in biological matrices because their mass spectra are quite similar. This study is the first to analyze zolpidem and M-1 to M-4 simultaneously using LC–MS/MS, which can provide more compelling evidence of zolpidem intake.

A sensitive liquid chromatography-tandem mass spectrometry method for quantitative determination of dasotraline in human plasma and its clinical application

This manuscript describes a highly sensitive liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the determination of dasotraline in human plasma. Dasotraline and the internal standard (IS) d4-13C4-dasotraline were extracted from the 0.500-mL plasma pre-mixed with 0.20-mL of 0.5 M sodium bicarbonate solution by a 3-mL of hexane containing 0.7 % sec-butyl alcohol. The organic extract, after dried down, was reconstituted in 150 μL acetonitrile containing 0.1 % formic acid. Forty (40) μL of the resulted sample was injected into LC–MS/MS for analysis. Chromatographic separation was on a Betasil Silica column. MS/MS detection was by monitoring m/z 275→159 and 283→160 for dasotraline and IS, respectively. Peak area ratio of analyte/IS was used for constructing calibration curve and calculating sample concentration. The retention time was ∼3.1 min for both dasotraline and IS. The validated linear range was 5–5000 pg/mL with correlation coefficient r ≥ 0.999. Intra-run precision and accuracy were ≤ 7.3 % CV (n = 6) and 94.4–101.0 % of nominals. Inter-run precision and accuracy were ≤ 4.7 % CV (n = 18) and 96.1–99.8 % of nominals. Plasma sample was confirmed stable for 8 cycles of freeze/thaw, 29 h on bench-top, and up to 977 days of storage at both −20 °C and −70 °C. This method was successfully applied to analyze pharmacokinetic (PK) samples from a single ascending dose (SAD) clinical study with healthy subjects. PK results indicated that dasotraline was slowly absorbed (tmax: 10–12 h) and slowly eliminated (terminal elimination half-life, i.e. t1/2: 47–77 h) with dose proportional Cmax but slightly greater than dose proportional AUC with increase of dosed amount.

Determination of biodiesel and used cooking oil in automotive diesel/green diesel fuels through high-performance liquid chromatography.

This study reports a simple and convenient analytical method for the simultaneous determination of biodiesel and vegetable oils or used cooking oils in petrodiesel and green diesel (hydrotreated vegetable oils or paraffinic diesel). The approach is based on normal-phase high-performance liquid chromatography with refractive index detection. It employed silica stationary phase, n-hexane mobile phase with isopropanol modifier to achieve optimum separation between hydrocarbons (petrodiesel or green diesel), fatty acid methyl esters (biodiesel) and triglycerides (vegetable oils and used cooking oil). In addition to determining vegetable oils or used cooking oils as adulterants in diesel, this method is also proposed as a better alternative to the standard method ASTM D7371, which is currently recommended for determining fatty acid methyl esters in petrodiesel. The method development involved screening of various stationary and mobile phases, with and without modifiers, to achieve acceptable chromatographic resolutions between analytes. Under the optimized method conditions, silica column, and n-hexane containing 0.6% isopropanol as the mobile phase provided the best results. The real-world scenario was simulated for the method validation carried out by fortifying Jatropha seed oil, soybean oil, and used cooking oil in the biodiesel blended petrodiesel and green diesel. Measurement of all analytes was accompanied by high precision, low limit of detection/quantification and linear response range of 0.05 to 50% for biodiesel, and 0.05 to 30% for vegetable oils.The proposed method is simple, fast (runtime 7min), and does not require sample pre-treatment and backflushing.

Identification and Structural Analysis of Spirostanol Saponin from Yucca schidigera by Integrating Silica Gel Column Chromatography and Liquid Chromatography/Mass Spectrometry Analysis.

Yucca schidigera Roezl (Mojave), a kind of ornamental plant belonging to the Yucca genus (Agavaceae), whose extract exhibits important roles in food, beverage, cosmetic and feed additives owing to its rich spirostanol saponins. To provide a comprehensive chemical profiling of the spirostanol saponins in it, this study was performed by using a multi-phase liquid chromatography method combining a reversed phase chromatography T3 column with a normal phase chromatography silica column for the separation and an ESI-Q-Exactive-Orbitrap MS in positive ion mode as the detector. By comparing the retention time and ion fragments with standards, thirty-one spirostanol saponins were identified. In addition, according to the summary of the chromatographic retention behaviors and the MS/MS cleavage patterns and biosynthetic pathway, another seventy-nine spirostanol saponins were speculatively identified, forty ones of which were potentially new ones. Moreover, ten novel spirostanol saponins (three pairs of (25R/S)-spirostanol saponin isomer mixtures) were targeted for isolation to verify the speculation. Then, the comprehensive chemical profiling of spirostanol saponins from Y. schidigera was reported here firstly.

Development and validation of an analytical method for quantification of potential genotoxic impurity 4‐dimethylaminopyridine in linagliptin active pharmaceutical ingredient using Hydrophilic Interaction Liquid Chromatography

AbstractA sensitive analytical method was developed to estimate the potential genotoxic impurity 4‐dimethylaminopyridine in linagliptin pharmaceutical ingredient. 4‐Dimethylaminopyridine is a reactive reagent used in synthesis of linagliptin. Sensitivity of the studied method was excellent at a level of 30 ppm of 4‐dimethylaminopyridine relative to linagliptin when samples of linagliptin were prepared at 10.0 mg/mL. A silica (150 mm × 4 mm, 3 µm) column was used to analyze the samples in hydrophilic interaction liquid chromatography mode. A mobile phase of water–acetonitrile (15:85, v/v) containing 10.0 mM ammonium acetate was prepared and adjusted to pH 6.2. Flow rate of the method was 1.3 mL/min and the ultraviolet detector was set at 258 nm. This method was validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines in terms of detection limit, quantitation limit, linearity, precision, accuracy, specificity, and robustness. The method had excellent linearity in the concentration range of 15–450 ppm with a correlation coefficient of 0.999. Detection limit and quantitation limit were 4.46 and 14.87 ppm, respectively.

Extraction of Small RNAs by Titanium Dioxide Nanofibers.

MicroRNAs (miRNAs) are small RNAs, that bind to mRNA targets and regulate their translation. Functional study of miRNAs and exploration of their utility as disease markers require miRNA extraction from biological samples, which contain large amounts of interfering compounds for downstream RNA identification and quantification. The most common extraction methods employ either silica columns or TRIzol reagent, but these approaches afford low recovery for small RNAs, possibly due to their short strand lengths. Here, we describe the fabrication of titanium dioxide nanofibers and the optimal extraction conditions to improve miRNA recovery from biological buffers, cell lysate, and serum.

Intrinsic difference between phenyl hexyl- and octadecyl-bonded silicas in the solute retention selectivity in reversed-phase liquid chromatography with aqueous mobile phase.

For choosing an optimal column for a particular separation by reversed-phase liquid chromatography (RPLC), it is essential to quantitatively understand the effects of the chemical structure of hydrophobic bonded layer derived onto silica particles on the distribution equilibrium of a solute compound at the interface between the aqueous mobile phase and the packing material. However, there is still a lack of understanding of the solute distribution equilibrium in RPLC separation due to the complexities of the chemistry at the interface between the mobile phase and the bonded layer. We successfully determined the distribution coefficients of various organic compounds concerning to their accumulation onto the water/bonded layer interface and into the bonded layer from bulk water using surface-bubble-modulated liquid chromatography with octadecyl- and phenyl hexyl-bonded silica columns. The water/phenyl hexyl-bonded layer interface accumulates organic compounds much less than the water/octadecyl-bonded layer interface due to its lower interfacial tension, and this result suggests that phenyl hexyl group orient their benzene ring facing toward water. On the other hand, aromatic moiety of phenyl hexyl group enhances partitioning of the organic compounds into the bonded layer. Experimental findings in the present work demonstrated that the water/bonded layer interface and the bonded layer itself have independent contributions to the solute distribution and the water/phenyl hexyl-bonded layer interface shows quite different solute retention selectivity from the water/octadecyl-bonded layer interface.

Multiplexed SNP Typing Of DNA After Demineralization And Organic Extraction From A Bone

Analysis of DNA from bones and teeth still suffers from low efficiency of extraction methods and poor reliability of results during forensic typing, because of co-extraction of microbiological contaminants and DNA degradation. Here we tested 15 variants of DNA extraction from old human bone using silica columns, organic phenol-chloroform-isoamyl alcohol solution, and combination of those two, with additional decalcification of different duration time. Multiplex PCR and single nucleotide polymorphism genotyping were performed to estimate the efficiency of extraction process and the level of DNA degradation. In comparison with silica-based method, the organic extraction gave more complete profiles with shorter time of decalcification, also less allele drop-outs and higher fluorescence level, independently of the additional cleaning. The minimal duration of successful decalcification was 48h.

Stability-indicating LC-MS/MS and LC-DAD methods for robust determination of tasimelteon and high resolution mass spectrometric identification of a novel degradation product

Novel stability-indicating HPLC methods have been presented for the analysis of Tasimelteon (TSM) besides its main degradation products in bulk and pseudo tablet formulations in this study. For the LC-DAD method; quantitation of TSM and its separation with other degradation products were achieved on an Ascentis ExpressTM pentafluorophenylpropyl (F5)-bonded fused-core silica particle column (2.7 μm particle size 100 × 4.6 mm, Supelco) using the mobile phase consisted of acetonitrile: acetate buffer (0.025 M, pH 4.5): water (40:10:50, v/v/v); the elution was performed at 0.8 mL min−1 flow rate, detecting the compounds at 281 nm. In addition to regular in- house validation studies, the developed method was tested on another UPLC instrument to assess its ruggedness for possible method transfer demands. For the LC–MS/MS method, analyte quantitation was achieved on a second-generation monolithic silica column (Chromolith™ High-Resolution RP-18e, 100 × 4.6 mm from Merck KGaA, Germany); the mobile phase was a mixture with 0.1 % (v/v) formic acid in water and 0.1 % (v/v) formic acid in acetonitrile (60: 40 (v/v), pH = 2.5). The instrumental and analytical performances of all three instrumental systems were compared with each other in terms of the working range, LOD, and LOQ. In addition to the above, a new degradation product was identified using LCMS-IT-TOF system. It can be concluded that among the similar ones in the literature, this study is the most comprehensive method optimization and validation study to date about TSM, in which a novel degradation product was also reported.

The utility of silica hydride-based stationary phases for dual-mode ultra high performance liquid chromatography separation of synthetic cathinone positional isomers.

Emerging drugs usually mimic the effects of traditional drugs, but are not always controlled due to the chemically altered structures. Positional isomers of emerging drugs are difficult to analyze because they challenge the separation and detection techniques commonly employed by forensic laboratories. The utility of silica hydride stationary phases for the ultra-high performance liquid chromatography separation of synthetic cathinone positional isomers was studied in this manuscript. SiH phases are operable under both reversed phase and aqueous normal phase chromatographic conditions without the need to change solvent reservoirs. The separation of eight positional isomers of the synthetic cathinone, pentedrone, was investigated using five silica hydride phases, and compared to a classical dual column reversed phase, hydrophilic interaction chromatography system, and to a bimodal pentaflurophenyl column. Significant selectivity differences were observed using either a combination of a classical reversed phase C18 and NP Silica columns or the various bimodal columns. The silica hydride silica-C column, which contains no derivatized ligands attached to the silica hydride backbone, not only gave the most orthogonal separation of the bimodal columns, but provided a unique separation of all eight positional isomers (resolution ≥ 1) using the combination of reverse phase and aqueous normal phase chromatographic conditions.

Simple liquid chromatography-electrospray ionization ion trap mass spectrometry method for the quantification of galacto-oxylipin arabidopsides in plant samples

A simple and sensitive method to quantify five different arabidopsides by HPLC—ion trap mass spectrometry in complex plant samples was developed and validated. Arabidopsides are oxidized galactolipids first described in Arabidopsis thaliana but also produced by other plant species under stress conditions. External calibration was performed using arabidopsides purified from freeze-thawed Arabidopsis leaves. Lipids were extracted and pre-purified on an SPE silica column before HPLC–MS analysis. Arabidopsides were separated on a C18 column using a gradient of mQ water and acetonitrile:mQ water (85:15) supplemented with formic acid (0.2%) and ammonium formate (12 mM). The method was validated according to European commission decision 2002/657/CE. LOD, LOQ, linearity, intra-day and inter-day precision and accuracy, selectivity, matrix effects and recoveries were determined for the five metabolites. The established method is highly selective in a complex plant matrix. LOD and LOQ were, respectively, in the range 0.098–0.78 and 0.64–1.56 µM, allowing the arabidopside quantification from 25.6–62.4 nmol/g fresh weight. Calibration curve correlation coefficients were higher than 0.997. Matrix effects ranged from -2.09% to 6.10% and recoveries between 70.7% and 109%. The method was successfully applied to complex plant matrixes: Arabidopsis thaliana and Nasturtium officinale.

Streptococcus mutans Secreted Products Inhibit Candida albicans Induced Oral Candidiasis.

In the oral cavity, Candida species form mixed biofilms with Streptococcus mutans, a pathogenic bacterium that can secrete quorum sensing molecules with antifungal activity. In this study, we extracted and fractioned culture filtrate of S. mutans, seeking antifungal agents capable of inhibiting the biofilms, filamentation, and candidiasis by Candida albicans. Active S. mutans UA159 supernatant filtrate components were extracted via liquid-liquid partition and fractionated on a C-18 silica column to resolve S. mutans fraction 1 (SM-F1) and fraction 2 (SM-F2). We found anti-biofilm activity for both SM-F1 and SM-F2 in a dose dependent manner and fungal growth was reduced by 2.59 and 5.98 log for SM-F1 and SM-F2, respectively. The SM-F1 and SM-F2 fractions were also capable of reducing C. albicans filamentation, however statistically significant differences were only observed for the SM-F2 (p = 0.004). SM-F2 efficacy to inhibit C. albicans was confirmed by its capacity to downregulate filamentation genes CPH1, EFG1, HWP1, and UME6. Using Galleria mellonella as an invertebrate infection model, therapeutic treatment with SM-F2 prolonged larvae survival. Examination of the antifungal capacity was extended to a murine model of oral candidiasis that exhibited a reduction in C. albicans colonization (CFU/mL) in the oral cavity when treated with SM-F1 (2.46 log) and SM-F2 (2.34 log) compared to the control (3.25 log). Although both SM-F1 and SM-F2 fractions decreased candidiasis in mice, only SM-F2 exhibited significant quantitative differences compared to the non-treated group for macroscopic lesions, hyphae invasion, tissue lesions, and inflammatory infiltrate. Taken together, these results indicate that the SM-F2 fraction contains antifungal components, providing a promising resource in the discovery of new inhibitors for oral candidiasis.