High-performance Liquid Chromatography Measurements Research Articles

Lycium barbarum extract as an environmentally friendly corrosion inhibitor for AISI 52100 bearing steel in hydrochloric acid medium

This study reports the use of Lycium barbarum extract (LBE), a green and environmentally friendly additive, to inhibit the corrosion of AISI 52100 bearing steel in 1 M hydrochloric acid (HCl) solutions. Corrosion inhibition was experimentally investigated via the weight loss method and electrochemical analyses. The surface morphologies were evaluated by scanning electron microscopy (SEM), and the chemical compositions were analyzed via energy-dispersive X-ray spectroscopy (EDS) and Fourier transform infrared spectroscopy (FTIR). The experimental electrochemical and weight loss results demonstrated that LBE has excellent inhibitory properties, with the highest inhibition efficiencies of 92.68 % and 92.31 % when the concentration is 500 ppm, for steel immersed in HCl solutions. Surface coverage studies revealed that the data for the adsorption of LBE on steel were consistent with the Langmuir isotherm. The initial process was investigated via polarization analysis, and the LBE adsorption process was found to be a hybrid of physical and chemical adsorption. To investigate the effectiveness of LBE in preventing corrosion, the corrosion inhibition mechanism was analyzed via a wettability study, high performance liquid chromatography (HPLC) measurements and theoretical quantum chemical calculations. The results indicated that Ara, Rha, Man and Glu played significant effect on the protection of bearing steel in 1 M HCl solution.

Evaluation of fluorous affinity using fluoroalkyl-modified silica gel and selective separation of poly-fluoroalkyl substances in organic solvents.

In this study, we focused on the fluorous affinity acting among fluorine compounds, and then developed a new separation medium and evaluated their performance. We prepared the stationary phases for a column using silica gel-modified alkyl fluoride and investigated the characteristics of fluorous affinity by comparing them with a typical stationary phase, which does not contain fluorine, using high-performance liquid chromatography (HPLC). In HPLC measurements, we confirmed that while all non-fluorine compounds were not retained, retention of fluorine compounds increased as the number of fluorine increased with the stationary phase. It also revealed that the strength of fluorous affinity changes depending on the types of the organic solvent; the more polar the solvent, the stronger the effect. Additionally, the stationary phase was employed to compare the efficiency of our column with that of a commercially available column, Fluofix-II. The retention selectivity was almost the same, but the absolute retention strength was slightly higher on our column, indicating that the column is available for practical use.

Development of a CaCO3 Precipitation Method Using a Peptide and Microwaves Generated by a Magnetron

Microwave applications, such as microwave ovens and mobile phones, are ubiquitous and indispensable in modern society. As the utilization of microwave technology is becoming more widespread, the effects of microwaves on living organisms and physiological processes have received increased attention. This study aimed to investigate the effects of microwaves on calcium carbonate biomineralization as a model biochemical process. A magnetron oscillator was used to generate 2450 MHz microwaves because magnetrons are relatively inexpensive and widespread. We conducted transmission electron microscopy (TEM), atomic force microscopy (AFM), TEM-electron energy-loss spectroscopy (EELS), dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC) measurements to analyze the calcium carbonate precipitates. Our findings showed the formation of string-like precipitates of calcium carbonate upon microwave irradiation from one direction, similar to those obtained using a semiconductor oscillator, as reported previously. This implied that the distribution of the frequency had little effect on the morphology. Furthermore, spherical precipitates were obtained upon microwave irradiation from two directions, indicating that the morphology could be controlled by varying the direction of microwave irradiation. Magnetrons are versatile and also used in large-scale production; thus, this method has potential in medical and industrial applications.

Magnetic composite – Based dual nanozymes derived from coffee ground waste: A sustainable approach for colorimetric assay and prolonged reuse

Nanozymes serve as an excellent substitute for traditional enzymes across various scientific domains, particularly in the colorimetric sensor array. Nonetheless, their tendency to aggregate into larger clusters and the challenges associated with recovery and reuse have limited their applicability, especially in the context of sustainable development. Based on the 3R principle “Reduce-Recycle-Reuse”, this study introduces a magnetic composite in which two typical nanozymes composed of silver nanoparticles and magnetite nanoparticles are immobilized on the activated carbon derived from spent coffee grounds using one-pot in-situ synthetic approach for easy recovery and long-term reuse. The successful synthesis of the targeted composite was verified through its characterization analysis techniques, including XRD, FT-IR, SEM-EDS, element mapping, HR-TEM, VMS, BET, and TGA. The experimental results revealed that the coexistence of these nanoparticles not only guarantees the magnetic property of the composite but also enhances its peroxide-mimicking activity in the catalytic oxidation of a reliable colorimetric substrate (3,3,5,5-tetramethylbenzidine) through the predominant of •OH radical. The conditions for optimizing the catalytic efficiency of the composite were also presented. Under this condition, the targeted composite showed high accuracy, selectivity, and anti-interference ability in colorimetrically quantifying the ascorbic acid concentration, as confirmed by high-performance liquid chromatography measurement. Notably, the catalytic activity, magnetic property, and fundamental characteristics of the proposed composite remained almost unchanged even after a series of at least 6 consecutive assays. This observation substantiates its remarkable capacity for prolonged reuse, a unique attribute not present in recently developed nanozymes.

Photolysis mechanism of Di(tert‐butylphenyl)iodonium salt using 2‐isopropylthioxanthone as a sensitizer

AbstractDiaryliodonium salts (Ar2I+X−) are used as a photosensitive initiator that generates acid or radical species by ultraviolet light irradiation. Recently, sensitization of Ar2I+X− has gained importance owing to the escalating demand for high‐sensitive initiators with longer wavelength absorption such as 365 and 436 nm. However, the mechanism of photolysis of Ar2I+X− has not been strictly elucidated. This paper shows discussions of the details of its mechanism. Herein, we analyzed the photosensitization of Ar2I+X− with 2–isopropylthioxanthone (ITX) based on transient absorption techniques. As a result, it was revealed that electron transfer occurred from a triplet excited state of ITX to Ar2I+X− with an electron transfer rate constant of 4.2 × 109 s−1. Furthermore, high performance liquid chromatography measurements found the quantum yield of the photolysis was determined to be 0.48.

Portable paper-based probe for on-site ratiometric fluorescence determination of total flavonol glycosides in plant extract using smartphone imaging.

A smartphone-assisted, paper-based ratio fluorescence probe is presented for the rapid, low-cost and on-site quantification of total flavonol glycosides in Ginkgo biloba extracts (GBE). The Al3+/Eu-MOF/paper-based probe utilizes lanthanide metal-organic framework (Ln-MOF) nanoparticles immobilized on Whatman filter paper along with Al3+ for detecting flavonols, which are the hydrolyzed products of flavonol glycosides. The color change of the paper-based fluorescence image from red to orange depends on the concentration of the target analyte in the sample solution. The smartphone equipped with a red, green, blue (RGB) color detector measured the fluorescence signal intensity on the paper substrate after adding flavonol. The analytical variables affecting the performance of the probe, including the addition sequence of the aluminum nitrate solution, its concentration, that of the Ln-MOF solution, the drying time of the paper probe, the reaction time and the sensitivity parameters of the mobile phone camera (ISO), were optimized. Under optimal conditions, the Al3+/Eu-MOF/paper-based probe has good linear response in the concentration range 7 ~ 80µg mL- 1 and a lower detection limit of 2.07µg mL- 1. The results obtained with the paper-based ratio fluorescence probe and smartphone combination were validated by comparing them with high-performance liquid chromatography (HPLC) measurements. This study provides a potential strategy for fabricating Al3+/Eu-MOF/paper-based probe used for total flavonol glycosides determination.

The effect of casing and gypsum on the yield and psychoactive tryptamine content of Psilocybe cubensis (Earle) Singer

Psychedelic fungi have experienced a surge in interest in recent years. Most notably, the fungal secondary metabolite psilocybin has shown tremendous promise in the treatment of various psychiatric disorders. The mushroom species that produce this molecule are poorly understood. Here we sought to examine for the first time, the response of a psilocybin-producing species Psilocybe cubensis to casing (peat moss and vermiculite) and supplementation with gypsum (calcium sulfate dihydrate), two common practices in commercial mushroom cultivation. Mycelial samples of genetically authenticated P. cubensis were used to inoculate popcorn grain bags. The fully colonized bags of popcorn grain (0.15 kg) were transferred to bins of 0.85 kg pasteurized horse manure, with or without 1 cm thick layer of casing and/or 5 % gypsum. Our results indicate that the use of a casing layer significantly increases the biological efficiency (161.5 %), by approximately four fold, in comparison to control (40.5 %), albeit with a slight delay (∼2 days) for obtaining fruiting bodies and a somewhat reduced total tryptamine content (0.85 %) as gauged by High Performance Liquid Chromatography measurements. Supplementation with both casing and gypsum, however, appears to promote maximal yields (896.6 g/kg of dried substrate), with a biological efficiency of 89.6 %, while also maintaining high total tryptamine expressions (0.95 %). These findings, revealing methods for maximizing yield of harvest and expressions of psychoactive tryptamines, may prove useful for both home growers and commercial cultivators of this species, and ultimately support the growth of a robust industry with high quality natural products.

Twin-screw extrusion of vitamin D3/iron-blend granules in corn and lentil composite flours: Stability, microstructure, and interaction of vitamin D3 with human osteoblast cells.

Vitamin D3 (VD3) and iron-blend granules were blended with corn and lentil composite flour (75/25, w/w) and fed into a pilot-scale twin-screw extruder to produce ready-to-eat snacks. The morphology and microstructure of extruded snacks were examined using scanning electron microscopy with energy-dispersive X-ray (SEM-EDX), X-ray powder diffraction, and FT-IR. Differential scanning calorimetry and thermogravimetric analysis measured the melting temperature and thermal stability of the extrudates. SEM and FT-IR analysis demonstrate that micronutrients are mixed well in formulations used in extrudates at high shear and high temperatures. The SEM-EDX exhibited the presence of iron, whereas high performance liquid chromatography measurements confirmed the significant retention of VD3 in the extruded snacks. The interaction between VD3 and human osteoblast cells was studied using live imaging and the MMT assay. Overall, for the first time, VD3 and Fe2+ blend granules have been used in an extrusion platform, which has significant potential for the intervention of VD3 and iron deficiencies. PRACTICAL APPLICATION: For the first time, we reported the use of VD3/iron-blend granules in extruded products. The findings of this work demonstrated the thermal stability and capability of providing adequate quantities of VD3 and iron in corn flour/lentil flour/VD3-iron blend extruded snacks. Furthermore, the interaction of VD3 with osteoblast cells highlights the potential health benefits of the extrudates.

Flexible Screen-Printed Amperometric Sensors Functionalized With Spray-Coated Carbon Nanotubes and Electrodeposited Cu Nanoclusters for Nitrate Detection

In this work, we present a novel, sensitive, easy-to-fabricate, flexible amperometric sensor constituted by screen-printed silver (Ag) electrodes functionalized with a copper (Cu) film electrodeposited on top of a spray coated network of single-walled carbon nanotubes (SWCNTs). The Cu/SWCNTs/Ag electrode showed excellent catalytic activity towards the electro-reduction of nitrate ions at neutral pH with a significant increase in cathodic peak currents in comparison with the electrode without SWCNTs (Cu/Ag). The developed Cu/SWCNTs/Ag sensor showed a wide linear detection range from 0.5 μM to 6.0 mM (0.31 mg/l to 372.02 mg/l) with good sensitivity (18.39 μA/mM) and a calculated limit of detection (LOD) of 0.166 nM (10.29 μg/l). It also showed a good selectivity (maximum standard deviation (SD) was 3.25 μA) towards different interfering ions (Fe2+, Na+, Cu2+, SO42-, CH3COO-, Cl-, NO2- and HCO3-), as well as good reproducibility, mechanical durability, time and temperature stability. In real sample analysis (tap and river water), the sensor exhibited good agreement with the compared outcome of high-performance liquid chromatography (HPLC) measurements, proving to be a promising analytical tool for the detection of nitrate in water.

Enhancing Antioxidant Retention through Varied Wall Material Combinations in Grape Spray Drying and Storage.

The encapsulation of bioactive compounds, which spans phytochemicals, vitamins, antioxidants, and other precious substances, has risen to prominence as a crucial area of interest spanning various domains, including food, pharmaceuticals, and cosmetics. This investigation delved into the efficacy of distinct wall materials-whey protein isolate, high methoxy pectin, and gum arabic-when employed individually or in combination to encapsulate and preserve phenolic compounds and antioxidants during storage. The encapsulation process involved spray-drying bioactive compounds extracted from grapes. Over a span of 120 days, the stability of these encapsulated compounds was meticulously evaluated, encompassing assessments via different antioxidant capacity assays, phenolic content analyses, and high-performance liquid chromatography measurements. The modeling of retention kinetics during storage facilitated the comprehension of the release mechanisms. Notably, the findings underscore the pivotal role of wall materials in preserving these bioactive compounds, with each material or combination of materials exhibiting varying degrees of protective capacity. Remarkably, the synergistic blend of whey protein, pectin, and gum arabic showcased the utmost retention of bioactive compounds over this study's period. The amassed data distinctly show that an amalgamation of wall materials can indeed considerably enhance the stability of encapsulated bioactive compounds, presenting promising applications within the realms of both the food and pharmaceutical industries.

Enzymatic valorization of cellulosic and hemicellulosic-based biomasses via the production of antioxidant water-soluble hydrolyzate of maize stalks and the green bio-deinking of mixed office waste paper

AbstractBio-valorization of various biomasses provides a sustainable promising approach for the eco-friendly production of variable value-added products. Herein, the current study devoted to the enzymatic valorization of two widely available biomasses, namely, maize stalks and waste paper. The cellulytic and hemicellulytic-rich cocktail was produced through the fermentation of rice straw by a locally isolated fungal strain Aspergillus terreus. The potential applicability of the produced cocktail for the enzymatic hydrolysis of the polysaccharide constituents of maize stalks was evaluated under various strategies. The reported results indicated that the microwave pretreatment of the biomass yielding a water-soluble hydrolyzate rich in cellobiose and xylobiose, sustained by thin layer (TLC) and high-performance liquid chromatographic (HPLC) measurements, in addition to phenolic compounds. Moreover, the enzymatic hydrolysis of the extracted hemicellulosic fraction from maize stalks was rich in xylooligosaccharides and phenolic compounds higher than that released from the hydrolysis of commercial xylan. The estimated antioxidant activity of the resulted hydrolyzate was also monitored by the scavenging of 1,1-diphenyl-2-picrylhydrazyl free radical spectrophotometrically at 515 nm. Moreover, the potential applicability of the produced enzymatic cocktail was examined for the bio-deinking of waste paper. The physical, chemical, and surface morphological characteristics of the treated paper sample was compared to a blank one regarding the whiteness index, ash content, scanning electron microscopy (SEM), energy dispersive X-ray (EDX), and Fourier transform infrared spectroscopy (FTIR). On the base of the estimated results, the produced enzymatic cocktail possessed efficient dislodgement ability for the printed ink from the paper surface.

Efficient electrospray deposition of surfaces smaller than the spray plume

Electrospray deposition (ESD) is a promising technique for depositing micro-/nano-scale droplets and particles with high quality and repeatability. It is particularly attractive for surface coating of costly and delicate biomaterials and bioactive compounds. While high efficiency of ESD has only been successfully demonstrated for spraying surfaces larger than the spray plume, this work extends its utility to smaller surfaces. It is shown that by architecting the local “charge landscape”, ESD coatings of surfaces smaller than plume size can be achieved. Efficiency approaching 100% is demonstrated with multiple model materials, including biocompatible polymers, proteins, and bioactive small molecules, on both flat and microneedle array targets. UV-visible spectroscopy and high-performance liquid chromatography measurements validate the high efficiency and quality of the sprayed material. Here, we show how this process is an efficient and more competitive alternative to other conformal coating mechanisms, such as dip coating or inkjet printing, for micro-engineered applications.

A study on the pesticides-loading capacity of dendritic fibrous nano silica synthesized from 1-pentanol-water microemulsion with a low oil-water ratio.

Dendritic fibrous nano silica (DFNS) represents an optimal carrier material for pesticide constituents, due to its radial accessibility channels and high specific surface area. A low-energy methodology for synthesizing DFNS at a low volume ratio of oil to water is provided by employing 1-pentanol as the oil solvent in the microemulsion synthesis system, renowned for its remarkable stability and exceptional solubility. The DFNS@KM nano-pesticide was fabricated using a diffusion supported loading (DiSupLo) method and kresoxim-methyl (KM) as the template drug. Findings from Fourier-transform infrared spectroscopy, XRD, thermogravimetric, differential thermal analysis, and Brunauer-Emmet-Teller analyzes revealed the physical adsorption of KM onto the synthesized DFNS without any chemical bonding, with KM mainly existing in an amorphous state within the channels. High-performance liquid chromatography measurements demonstrated that only the loading amount of DFNS@KM was primarily dependent on the KM to DFNS ratio, with minimal effects observed from loading temperature and time. The loading amount and encapsulation efficiency of DFNS@KM were found to be 63.09% and 84.12%, respectively. Furthermore, DFNS effectively prolonged the release of KM, with a cumulative release rate of 85.43% over 180 h. The successful loading of pesticide components into DFNS synthesized with a low oil-to-water ratio provides theoretical support for the industrialization of nano-pesticides, with significant implications for enhancing pesticide utilization, reducing pesticide dosage, augmenting agricultural efficiency, and promoting sustainable agricultural development.

Identification and Quantification of the Component Composition of the Active Substance Gentamicin Sulfate by <sup>1</sup>Н and <sup>13</sup>С NMR Spectroscopy

Gentamicin sulfate is a broad-spectrum bactericidal aminoglycoside antibiotic produced by Micromonospora purpurea and used in clinical practice as an active substance containing a mixture of gentamicins C1 and C2. Gentamicin content is a standardised parameter controlled as part of a pharmaceutical product review. Currently, the quality control of the active substance gentamicin sulfate involves the use of high-performance liquid chromatography (HPLC) for the indirect identification of the component composition by comparison with a reference standard and for its quantification by calibration against the reference standard.The aim of the study was to develop a procedure for identifying and quantifying the components of the active substance gentamicin sulfate using nuclear magnetic resonance (NMR) spectroscopy that would not require reference standards.Materials and methods. The authors studied a sample of the active substance gentamicin sulfate using an Agilent DD2 NMR spectrometer operating at 600 MHz to record NMR spectra and an Agilent 1200 HPLC system equipped with a multiple wavelength detector (MWD) to obtain chromatograms.Results. Having determined the spectral properties of the C1, C1A, C2, C2A and C2B gentamicins comprising the active substance, the authors observed characteristic signals of the same type that corresponded to each of the gentamicins and did not overlap with other signals in the 1H and 13C spectra. These characteristic signals provided spectral markers indicative of the gentamicins of interest in the sample, with normalised integrated intensities equal to the mole fractions of the analytes. The authors compared NMR and HPLC measurements of the active substance composition.Conclusions. The authors developed a procedure for identifying and quantifying the constituents of the active substance gentamicin sulfate by 1H and 13C NMR spectroscopy, which requires less effort than HPLC procedures and no reference standards for the intended applications.

Global estimation of phytoplankton pigment concentrations from satellite data using a deep-learning-based model

Based on a global matchup between satellite observations and high performance liquid chromatography (HPLC) measurements, we developed a deep-learning-based model (DL-PPCE model) for globally estimating concentrations of 17 different phytoplankton pigments. The model adopted a fusion architecture of residual and pyramid networks to achieve robust estimation performance. The model inputs include three different data types: essential ocean color parameters, satellite-derived environmental parameters, and the slope of above-surface remote-sensing reflectance (Rrs). We compared the model performances with various input parameters to determine the most effective inputs. The results showed that Rrs in the essential ocean color parameters and sea surface temperature (SST) in the environmental parameters were the most critical input parameters. The estimation of phytoplankton pigment concentrations was validated against HPLC data using the leave-one-out cross-validation method. Except for three pigments, 19′-butanoyloxy-fucoxanthin, prasinoxanthin, and lutein, the estimated pigment concentrations and in-situ observations were strongly correlated for all other pigments (an average relative root-mean-square error of 0.59, R2≥0.60, and regression slopes close to 1). In addition, a time series analysis was performed on the MODIS retrieved global pigment concentrations during 2003–2021 using the established DL-PPCE model to explore the relationship between the distribution of phytoplankton groups and El Niño in the western equatorial Pacific. Our findings revealed that the prokaryotes-dominated area extended eastward from180°E to 150°W during the 2015/2016 El Niño event. From 2003 to 2021, prokaryotic abundance was positively correlated with El Niño intensity (R=0.65,P≪0.01) but negatively correlated with the abundance of the entire phytoplankton community (R=−0.53,P≪0.01). These results demonstrate that the DL-PPCE model presents a novel approach for estimating the concentration of 17 pigments worldwide, and the estimated pigment concentrations are advantageous for analyzing the phytoplankton community dynamics on a large spatiotemporal scale.

Combining orthogonal measurements to unveil diclofenac encapsulation into polymeric and lipid nanocarriers

The quantification of the drug associated to nanoparticle carriers, often expressed in terms of encapsulation efficiency, is a regulatory requirement. The establishment of independent methods to evaluate this parameter provides a means for measurement validation, which is critical in providing confidence in the methods and enabling the robust characterization of nanomedicines. Chromatography is traditionally used to measure drug encapsulation into nanoparticles. Here, we describe an additional independent strategy based on analytical centrifugation. The encapsulation of diclofenac into nanocarriers was quantified based on the mass difference between placebo (i.e. unloaded) and loaded nanoparticles. This difference was estimated using particle densities measured by differential centrifugal sedimentation (DCS) and size and concentration values measured by particle tracking analysis (PTA). The proposed strategy was applied to two types of formulations, namely poly(lactic-co-glycolic acid) (PLGA) nanoparticles and nanostructured lipid carriers, which were analysed by DCS operated in sedimentation and flotation modes, respectively. The results were compared to those from high performance liquid chromatography (HPLC) measurements. Additionally, X-ray photoelectron spectroscopy analysis was used to elucidate the surface chemical composition of the placebo and loaded nanoparticles. The proposed approach enables the monitoring of batch-to-batch consistency and the quantification of diclofenac association to PLGA nanoparticles from 0.7 ng to 5 ng of drug per 1 μg of PLGA, with good linear correlation between DCS and HPLC results (R2 = 0.975). Using the same approach, similar quantification in lipid nanocarriers was possible for a loading of diclofenac ≥1.1 ng per 1 μg of lipids, with results in agreement with the HPLC method (R2 = 0.971).Hence, the strategy proposed here expands the analytical tools available for evaluating nanoparticles encapsulation efficiency, being thus significant for increasing the robustness of drug-delivery nanocarriers characterization.

HPLC Analysis of Polyphenols Derived from Hungarian Aszú from Tokaj Wine Region and Its Effect on Inflammation in an In Vitro Model System of Endothelial Cells.

Many studies have been published in recent years regarding the fact that moderate wine consumption, as a part of a balanced diet can have a beneficial effect on human health. The biologically active components of wine continue to be the subject of intense research today. In this study, the bioactive molecules of Hungarian aszú from the Tokaj wine region were analyzed using high-performance liquid chromatography (HPLC) and investigated in an in vitro model system of endothelial cells induced by bacterial-derived lipopolysaccharide. The HPLC measurements were performed on a reversed phased column with gradient elution. The non-cytotoxic concentration of the active substance was determined based on 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT)-, apoptosis, and necrosis assays. The antioxidant effect of the extract was determined by evaluating its ability to eliminate ROS. The expressions of the interleukin-(IL)1α, IL1-β, IL-6, and IL-8 pro-inflammatory cytokines and nitric oxide synthase (eNOS) at the mRNA level were evaluated using a quantitative polymerase chain reaction (qPCR). We found that the lipopolysaccharides (LPS)-induced increases in the expressions of the investigated cytokines were significantly suppressed by Hungarian aszú extract, excluding IL-6. In our experimental setup, our treatment had a positive effect on the eNOS expression, which was impaired as a result of the inflammatory manipulation. In our experimental model, the Hungarian aszú extract decreased the LPS-induced increases in the expression of the investigated cytokines and eNOS at the mRNA level, which presumably had a positive effect on the endothelial dysfunction caused by inflammation due to its strong antioxidant and anti-inflammatory effects. Collectively, this research contributes to a more thorough understanding of the bioactive molecules of aszú from the Tokaj wine region.

In-Vial Detection of Protein Denaturation Using Intrinsic Fluorescence Anisotropy.

The conventional quality control techniques for identifying the denaturation of biopharmaceuticals includes sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identifying fragmentation, ion exchange chromatography and isoelectric focusing for identifying deamidation, reverse-phase high-performance liquid chromatography (HPLC) for identifying oxidation, and size-exclusion HPLC for identifying aggregation. These stability assessments require essential processes that are destructive to the product tested. All these techniques are lab based and require sample removal from a sealed storage vial, which can breach the sterility. In this work, we investigate the heat- and surfactant-induced denaturation of an in-vial-stored model protein, bovine serum albumin (BSA), by analyzing its intrinsic fluorescence without removing the sample from the vial. A lab-based bespoke setup which can do the measurement in vial is used to demonstrate the change in fluorescence polarization of the protein to determine the denaturation level. The results obtained are compared to circular dichroism and size-exclusion HPLC measurements. The results prove that in-vial fluorescence measurements can be performed to monitor protein denaturation. A cost-effective portable solution to provide a top-level overview of biopharmaceutical product stability from manufacture to the point of patient administration can be further developed using the same technique.

Light Increases Astaxanthin Absorbance in Acetone Solution through Isomerization Reactions.

Astaxanthin quantitative analysis is prone to high variability between laboratories. This study aimed to assess the effect of light on the spectrometric and high-performance liquid chromatography (HPLC) measurements of astaxanthin. The experiment was performed on four Haematococcus pluvialis-derived astaxanthin-rich oleoresin samples with different carotenoid matrices that were analyzed by UV/Vis spectrometry and HPLC according to the United States Pharmacopoeia (USP) monograph. Each sample was dissolved in acetone in three types of flasks: amber glass wrapped with aluminium foil, uncovered amber glass, and transparent glass. Thus, the acetone solutions were either in light-proof flasks or exposed to ambient light. The measurements were taken within four hours (spectrometry) or three hours (HPLC) from the moment of oleoresin dissolution in acetone to investigate the dynamics of changes in the recorded values. The results confirm the logarithmic growth of astaxanthin absorbance by 8-11% (UV/Vis) and 7-17% (HPLC) after 3 h of light exposure. The changes were different in the samples with different carotenoid matrices; for instance, light had the least effect on the USP reference standard sample. The increase in absorbance was accompanied with the change of isomeric distribution, namely a reduction of 13Z and an increase of All-E and 9Z astaxanthin. The greater HPLC values' elevation was related not only to the increase of astaxanthin absorbance, but also to light-dependent degradation of internal standard apocarotenal. The findings confirm a poor robustness of the conventional analytical procedure for astaxanthin quantitation and a necessity for method revision and harmonization to improve its reproducibility.

Selective determination of formaldehyde by high-performance liquid chromatography with porous graphitic carbon column using N,N′-bis(9-anthrylmethyl)propane-1,3-diamine as derivatizing reagent

Aromatic compounds containing two secondary amino groups were designed and prepared as new derivatizing reagents for aldehydes. One of them, N,N'-bis(9-anthrylmethyl)propane-1,3-diamine (APD), could achieve selective determination of formaldehyde (FA) on a porous graphitic carbon (PGC) column using xylenes, chlorobenzene, and 1-chloronaphthalene as mobile phases by high-performance liquid chromatography (HPLC). The APD-FA derivative was eluted from the PGC column, while the other APD-aldehyde derivatives remained on the column during the HPLC measurements. This specific elution was not observed using mobile phases such as acetonitrile, 1,4-dioxane, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, chloroform, benzene, toluene, benzyl alcohol, 2-ethyl-1-hexanol, and pyridine. The APD-FA derivative had a six-membered ring of two tertiary amines identified using 1H NMR spectroscopy. When the π-π interaction of the solvent molecule of the mobile phase with PGC overcame that between the APD-FA derivative and PGC, the APD-FA derivative could be eluted from the column. The best resolution between the peak of the APD-FA derivative and that of free APD was observed when using o-xylene. The optimum derivatization and the HPLC conditions for selective HPLC determination of FA were to conduct the derivatization of FA by heating in an aqueous phase with APD in o-xylene at 100°C. In this method, FA could be derivatized with APD at a mildly neutral pH of 6.7, unlike the low pH required for the derivatization of aldehydes with 2,4-dinitrophenylhydrazine (DNPH), which is commonly used for the derivatization of aldehydes. The detection and quantification limits of FA were 0.8 and 3.5ngmL-1 in this HPLC method with fluorescent detection, respectively. This selective HPLC method could be applied to the determination of FA in various water samples. It was found that only APD among the derivatizing reagents containing two secondary diamines was useful for the selective determination of FA.