Ortho-phthaldialdehyde Research Articles

The Impact of the Adjunct Heat-Treated Starter Culture and Lb. helveticus LH-B01 on the Proteolysis and ACE Inhibitory Activity in Dutch-Type Cheese Model during Ripening.

Simple SummaryAdjunct cultures are used in cheesemaking to improve flavour characteristics and accelerating cheese ripening. Different adjunct cultures are capable of producing enzymes with the specificity to hydrolyze caseins, leading to the release of various bioactive compounds. We studied the effect of adjunct heat-treated starter XT–312 and a cheese culture Lb. helveticus LH-B01 on selected physicochemical, microbiological properties, and on proteolysis in cheese models. Additionally, the effect of adjunct cultures on ACE inhibitory activity during ripening was determined. The application of adjunct cultures may be used as functional ingredients in Dutch-type cheese to maintain sufficient bioactive properties and improve proteolysis.Adjunct cultures are used in cheesemaking to improve the sensory characteristics of the ripened cheeses. In addition, it is known that different adjunct cultures are capable of producing enzymes with the specificity to hydrolyze caseins, leading to the release of various bioactive compounds (bioactive peptides, amino acids, etc.). The objective of this study was to evaluate the effect of adjunct heat-treated starter XT–312 and a cheese culture Lb. helveticus LH-B01 on the proteolytic activity and angiotensin converting enzymes inhibitors (ACE) in cheese models during ripening. Seven different cheese models were evaluated for: proteolytic activity using the spectrophotometric method with ortho-phthaldialdehyde (OPA), soluble nitrogen (SN), trichloroacetic acid-soluble nitrogen (TCA-SN) phosphotungstic acid-soluble nitrogen (PTA-SN), total nitrogen (TN), pH, contents of water, fat, as well as for total bacteria count (TBC), count of Lactococcus genus bacteria, count of Lb. helveticus, and number of non-starter lactic acid bacteria (NSLAB). Presence of adjunct bacterial cultures both in the form of a cheese culture LH-B01 and heat-treated XT–312 starter promoted primary and secondary proteolysis, which resulted in acceleration of the ripening process. ACE inhibitory activity and proteolytic activity was the highest throughout of ripening for cheese model with LH-B01 culture. The cheese models with the adjunct heat-treated starter were characterized by lower TBC, NSLAB and lower count of Lactococcus genus bacteria during ripening, compared to control cheeses.

Optimization and Development of Albumin-Biopolymer Bioconjugates with Solubility-Improving Properties.

Bioconjugation is an emerging field in the food and pharmaceutical industry. Due to its biocompatibility and high ligand binding capacity, albumin is widely used in modern drug delivery systems. However, the protein is sensitive to environmental stresses; albumin conjugates, on the other hand, have improved functional properties. Biopolymers are gaining interest due to their biodegradability and safety, compared to synthetic polymers. In this study, albumin–biopolymer bioconjugates were prepared by nonenzymatic Maillard reaction at 60 °C and 80% relative humidity. This nonenzymatic conjugation takes place between reducing sugars and available amino groups of a protein in certain conditions. The optimal molar ratio and time for the conjugation were studied by several investigation methods, including circular dichroism and fluorescence spectroscopy, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and determination of available amino groups with ortho-phthaldialdehyde (OPA) assay. All of the measurements provided evidence for the covalent bonding of albumin and biopolymers, resulting in bioconjugates. Based on the results, a higher molar ratio and longer time are necessary to complete the reaction with the available amino groups. However, the optimal parameters are specific to each given biopolymer. The rheological behavior of the conjugates is characteristic of the initial biopolymer, which can be useful in drug development. Moreover, both the physical characteristics of albumin and the solubility-improving capacity were enhanced. Therefore, the potential use of albumin–biopolymer bioconjugates in the pharmaceutical industry could be considered.

Method for Reproducible Shipboard Segmented Flow Analysis Ammonium Measurement Using an In-House Reference Material for Quality Control

Ammonium is a fundamental nutrient for phytoplankton growth in seawater and is a key component of the microbial loop. Ammonium measured in parallel with other nutrients is crucial in understanding the small temporal scale changes in oceanographic ecology. Despite the importance of measuring ammonium at sea, owing to its lability, there is no consensus on the best method. The lack of availability of certified reference materials for ammonium in seawater also makes it difficult to assess the accuracy and reproducibility of ammonium measurements. In this study we present a modified segmented flow analysis method using ortho-phthaldialdehyde (OPA) with fluorescence detection to measure ammonium at sea together with four other macro-nutrients (nitrate, nitrite, silicate and phosphate) in near real time. An in-house ammonium quality control (QC) material was produced to improve the accuracy and repeatability of the measurement at sea. The QC was prepared following two different methods and stored in two types of containers. The suitability of the in-house QC’s as a reference material were assessed onboard the RV Investigator in 2018 during two oceanographic voyages, including one on the repeat SR03 CLIVAR transect. This paper describes the production and assessment of the in-house QC for ammonium in seawater, providing groundwork for creating a short-term stable ammonium reference material for sea going voyages. The uncertainty of this method of ammonium measurement was found to be 0.10 μmol/L at ammonium concentration of 1.0 μmol/L. Results show that preparation of the QC inside a laminar flow cabinet and directly into 10 mL polypropylene sample tubes just prior to the commencement of the voyage improved its stability.

Conjugate microgel-stabilized Pickering emulsions: Role in delaying gastric digestion

In this study, a new class of microgels called ‘conjugate microgels’ was designed, where whey protein isolate (WPI) was conjugated with dextran (Dx, 500 kDa) (WPI-Dx) via Maillard reaction before fabricating the microgel particles. Such microgel particles were assessed for their abilities to act as Pickering stabilizers for oil-in-water emulsions and also checked if they offered gastric stability to the Pickering emulsions during in vitro digestion against interfacial pepsinolysis. WPI-Dx conjugates were obtained by controlled dry heating (60 °C, 79% RH, 24–48 h incubation). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and ortho-phthaldialdehyde (OPA) profile revealed that the degree of conjugation ranged from 11.6 to 28.1%. The WPI-Dx conjugates were re-dispersed and heat-treated to form heat-set gels with moduli ranging from ~45 to 250 kPa. Microgel particles (hydrodynamic diameters of 130–150 nm, ζ-potentials of −4.5 to −8.0 mV) were created by controlled shearing of these heat-set gels. Interfacial shear rheology measurements and microscopic examination confirmed that conjugated microgel particles with lower degree of conjugation (WPDx10M) were effective as Pickering stabilizers. When present in an aqueous dispersion, WPDx10M had reduced degree of gastric proteolysis (120–130 μM free NH2) as compared to non-conjugated counterparts (187–205 μM free NH2). When present at the droplet surface, cross-correlation image analysis revealed that WPDx10M was successful in delaying interfacial gastric proteolysis. Insights from this study suggest that conjugate microgel particles might be useful to design gastric-stable Pickering emulsions in the future for effective delivery of lipophilic compounds to the intestines.

In-vitro degradation and toxicological assessment of pulsed electric fields crosslinked zein-chitosan-poly(vinyl alcohol) biopolymeric films

We investigated the in vitro degradation and cytotoxic effects of edible films developed from pulsed electric fields (PEF) treated zein-chitosan-poly(vinyl alcohol) dispersions at specific energy 60–70, 385–400, and 620–650 kJ/kg. The degradation was evaluated using both simulated gastro-intestinal electrolyte solutions (SGES) and enzyme hydrolysis. The results of ortho-phthaldialdehyde (OPA) test indicated that the chemical breakdown of the films in SGES and enzyme increased with degradation time, but the product's features were unmodified. The Fourier Transform Infrared spectroscopy (FTIR) data showed enhancement of zein and chitosan transformation from ordered helices to β-sheet conformation. Relative cell survival rates of Hepa-1c1c7 cells investigated using 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) showed that the survival remained higher than 70% in both the supernatant and the residue of the SGES degraded samples and the supernatant from enzyme hydrolysis, which suggested that there was no significant toxicity of the films in the tested system. Although the residue from pancreatic digestion (240 min) (i.e. undigested films and a cocktail of digestion enzymes) expressed cytotoxicity activity, there was limited evidence of direct toxicity of the films. The findings of the study demonstrate the potential for PEF modified zein-chitosan-poly(vinyl alcohol) films as value-added biomaterials for the application in edible food packaging.

Aminopeptidase Modified Hydrolytic Enzymes to Improve the Efficiency of Sugar Production from Alkaline Pretreated Switchgrass

Cellulases and β-glucosidases (βGSD) are enzymes commonly used in the biofuel industry. In this study, smaller-sized variants were generated with aminopeptidase such that high catalytic capabilities were retained. Under the defined experimental conditions, the degree of hydrolysis was greater using cellulase substrates, compared to βGSD, based on ortho phthaldialdehyde (OPA) assay data (44% versus 15%). Proteolysis of cellulases was also evident based on sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) protein banding patterns seen after peptidase treatment. Residual cellulase activity was retained after peptidase hydrolysis (67% to 73%) based on standard filter paper assays. Peptidase treated cellulases and βGSD were then utilized for hydrolysis of alkaline-pretreated switchgrass (Panicum virgatum). Interestingly, the efficiency of the reaction, defined as milligrams of sugar produced per filter paper unit, was higher using truncated cellulases for bioprocessing reactions (~14%), especially in the absence of sodium azide. Conversely, incubation of βGSD with peptidase revealed minimal proteolysis with low impact on the efficiency of hydrolysis.

Characterisation of heat-induced protein aggregation in whey protein isolate and the influence of aggregation on the availability of amino groups as measured by the ortho-phthaldialdehyde (OPA) and trinitrobenzenesulfonic acid (TNBS) methods.

Whey protein isolate (WPI) solutions, with different levels of aggregated protein, were prepared by heating (5% protein, pH 7, 90°C for 30min) WPI solutions with either 20mM added NaCl (WPI+NaCl), 5mM N-ethylmaleimide (WPI+NEM) or 20mM added NaCl and 5mM NEM (WPI+NaCl+NEM). Gel electrophoresis demonstrated that the heated WPI and WPI+NaCl solutions had higher levels of aggregated protein, due to more covalent interactions between proteins, than the heated WPI+NEM and WPI+NaCl+NEM solutions. There were marked differences in the levels of amino groups between all heated WPI solutions when measured by the OPA and TNBS methods, with lower levels being measured by the TNBS method than by the OPA method. These results demonstrate that the measurement of available amino groups by the OPA method is less impacted than by the TNBS method after heat-induced structural changes, arising from disulfide or sulfhydryl-disulfide bond-mediated aggregation of whey protein molecules.

Hypocholesterolemic and Angiotensin I Converting Enzyme-Inhibiting Activity of Trypsin-Hydrolysed Bovine Casein

Nowadays, the need for new dietary containing some bioactive peptides with specific amino acid sequences increased to express biological functions. We investigated the hypocholesterolemic effect and ACE-inhibiting activity of bovine casein hydrolysates with different molecular weights. The bovine casein was hydrolysed with trypsin for different amounts of time to release bioactive peptides from the casein. Then, crude casein hydrolysate was ultrafiltered using membranes with two different molecular-weight cut offs (1 kDa and 10k Da) to determine the hypocholesterolemic effects and ACE-inhibiting activities of peptides with different molecular weights. The results showed that a higher level of hydrolysis (more active bioactive peptides) was associated with greater hypocholesterolemic and ACE-inhibiting effects in all the tested samples. The unhydrolysed crude casein reduced the cholesterol level in ortho-phthaldialdehyde (OPA) assays by 39.5%, while the 10 kDa and 1 kDa permeates of casein hydrolysed with trypsin for 4 h reduced the cholesterol level by 50.7 and 69.6%, respectively. The unhydrolysed crude casein and the 10 kDa and 1 kDa permeates of casein hydrolysed by trypsin for 4 h inhibited ACE activity by 18.9%, 29.7%, and 51.4%, respectively.

بهینه‌سازی میزان کانژوگه شدن پروتئین‌های ایزوله سویا با دکستران در شرایط مختلف واکنش مایلارد

Introduction: Engineering of food proteins with improved functional properties and higher resistance to heat is the main goal of food scientists. Food technologists are always seeking for innovative, simple and effective methods to manipulate proteins, hence natural modification has had the most attention in last decades. One of the natural ways used for protein modifications isMaillard grafting reaction. Maillard reaction as a consequence of covalentbindingbetween the available amino groups of the proteins and carbonyl containing moiety of the polysaccharides, causes a loss in free amino group content of the mixture that can be measured through different methods. Protein-polysaccharide hybrids, as a result of dry heating of two biopolymers mixture under controlled reaction conditions, cause the emergence of conjugates with novel functionalities.Selecting appropriate reaction conditions has a significant impact on the properties of the final conjugates. Among the factors affecting the degree of conjugation, temperature, pH and incubation time have considerable roles in determining the degree of conjugation. There are various methods by which conjugation degree can be assessed and factors such as accuracy, cost, accessibility and etc. must be considered when selecting a measuring method. Therefoe, in this study the effect of different Maillard reaction conditions on the conjugation degree of soy proteins-dextran heated mixtures have been investigated. In addition, the glycation degree was compared and reported by both OPA assay and UV absorbance methods. Materials and methods: Soy proteins–dextran conjugates were prepared as described later. First, soy proteins and dextran were mixed with phosphate buffer (0.1 M, pH: 8.5 and 7) and 1 to 4 ratio of protein to polysaccharide. After mixing and incubating at ambient temperature for some hours, solutions were frozen at –80 ℃ and freeze dried. Then, the lyophilized powder was incubated at 40 ℃ for 0, 4, 8, 24 hours and 2, 4, 6, 8, 12 days, at 60 ℃ for 0, 1, 2, 3, 4, 6, 8 days and at 80 ℃ for 0, 1, 2, 4, 8, 16, 24, 48 hours, under the 79 percent relative humidity in presence of saturated KBr. For each treatment a non conjugated sample was prepared in the exact same condition. Conjugation of proteins to polysaccharides was monitored by two methods (OPA assay and UV absorbance). Determining degree of glycation by OPA method was done as follows, in this procedure the level of available amino groups was estimated using the ortho- phthaldialdehyde (OPA) reagent, the absorbance was measured at 340 nm and degree of glycation was measured using a formula. To investigate the UV absorption of conjugated proteins, the samples were diluted using distilled water and the absorption was read using a UV-visible spectrophotometer. Results & Discussion: Covalent linkage between amino group of proteins and carbonyl group of polysaccharides causes depletion in the amount of available amino groups. The extent of soy proteins-dextran conjugation under various Maillard reaction conditions was evaluated by the reduction of available amino groups of proteins, the more reduction in amount of amino groups, the more conjugation between protein and polysaccharide. OPA results showed that, in the samples heated at 40 °C and 80 °C (at both pH 7 and 8.5), the amount of free amino groups slightly reduced compared to 60 °C heated samples. The disappearance of available amino groups at 60°C was faster than other temperatures and formation of conjugates in this temperature was more successful. A stepwise reduction in free amino group content observed with increasing incubation time. When soy proteins were incubated at pH 8.5 for 8 days at 60 °C, a considerable decrease in available amino group contents occurred. UV absorption results showed similar trend of changes in OPA method. Increasing UV absorption is due to the intermediate Maillard reaction products (MRP). Increasing incubation time, temperature and pH cause a significant increase in UV absorbance. Increasing UV absorption with increasing heating time indicates the fact that Maillard reaction products (MRP) formation is more favorable at alkaline pH. Data of UV absorption are a proper evidence for OPA assay results. Conclusion: As a conclusion, both OPA assay and UV absorption methods are cost effective, accurate and simple methods which can represent valuable information concerning the Maillard conjugation.

Automating a 96-Well Microtiter Plate Assay for Quick Analysis of Chemically Available Lysine in Foods

A new method for quick analysis of available lysine content in different food products has been developed by automating a 96-well microtiter plate assay. Although manual fluorometric methods validated in order to determinate available lysine content already existed for this compound, the benefits of applying appropriate automation should provide continuous operation, increased precision, an affordable electronic audit trail, and significantly reduced time and reagent consumption. The objective of this work was to adapt the ortho-phthaldialdehyde (OPA) fluorometric method to an automated workstation. Considerable effort went into developing and validating an automated method. The analytical parameters of linearity (r = 0.999), the precision of the method (relative standard deviations = 2.8–3.0 % for the different samples), and the results of the comparison with the corresponding OPA manual fluorometric method show that the studied method is useful for the measurement of available lysine in several food products from different natural origins such as liquid foods (soy, oat, quinoa beverages, and ultra-high temperature/sterilized milk) and powdered samples (powdered adapted, powdered follow-up, and junior milk infant formulas) with reduced time and reagent consumption.

Maillard-conjugate based core–shell co-assemblies for nanoencapsulation of hydrophobic nutraceuticals in clear beverages

Maillard reaction based protein-polysaccharide conjugates (PPC) are typically better emulsifiers than the proteins used to form them. Such conjugates have been proposed as encapsulation agents, but their potential as nanoencapsulation agents in clear beverages has not yet been proposed. In this research we formed casein-maltodextrin (CN-MD) conjugates and co-assembled them with hydrophobic nutraceuticals to create nanovehicles for the enrichment of clear beverages. Conjugation was validated using gel electrophoresis and by ortho-phthaldialdehyde (OPA) assay. Vitamin D (VD) was nano-entrapped by the conjugates, and compared to controls based on an unconjugated CN-MD mixture and unencapsulated VD. The following advantages of the conjugates were shown: the diameters of conjugate-VD nanoparticles was smaller and their solution was more transparent than that of the vitamin dispersion or the protein-polysaccharide mixture with VD. Conjugates conferred better protection against oxidation to both VD and epigallocatechin gallate (EGCG) than the CN-MD mixture. Conjugates were more colloidally stable at the CN original pI, allowing the formation of nanoparticles with mean diameter below 30 nm when mixed with VD, even at the original pI of CN. Conjugates provided protection against degradation at low pH and during shelf life. Entrapment efficiency was measured using Nile red (NR), a fluorescent model for a hydrophobic nutraceutical. Protein affinity to the hydrophobic compound was not diminished due to conjugation, and 90% entrapment efficiency was obtained under the conditions tested. During simulated gastric digestion, Nile red was not released from the conjugates, suggesting potential application in enteric delivery.

A miniature and field-applicable multipumping flow analyzer for ammonium monitoring in seawater with fluorescence detection

In this article, a simple, economic, and miniature flow analyzer for ammonium in seawater based on the solenoid micropumps is presented. A single reagent of sodium tetraborate, ortho-phthaldialdehyde (OPA), and sodium sulfite was used and optimized applying the modified SIMPLEX method. A special-made detection cell for fluorescence detection of the reaction product isoindol-1-sulfonat was made and combined with a commercial photomultiplier tube, a long-pass optical filter, and an UV-LED as excitation light source. A LOD down to 13nmol/L was achieved. The fabrication and application of a miniature reaction coil heating device for reaction rate enhancement is further described. The system featured an injection frequency of 32h−1 at average standard deviation of 3%.

Analysis of the enantiomers of serine by reversed-phase high-performance liquid chromatography with OPA-Boc as pre-column derivatization agent. Critical evaluation of the analysis ofd-serine in rat brain

Summary The enantiomers of serine have been separated after pre-column derivatization with ortho-phthaldialdehyde (OPA) and N-tert-butyloxycarbonyl-l-cysteine (Boc). Analysis of the enantiomers of amino acids after pre-column derivatization with OPA-Boc has been reported in the literature but no critical evaluation has been carried out. In this study, decomposition of the diastereomers, effect of the sample pH, and amount of derivatization agent were studied; the half life of the decomposition was also determined. The method was used for analysis of D and l-serine from three different areas of rat brain (the striatum, prefrontal cortex, and cerebellum).

Optimisation of amino sugar quantification by HPLC in soil and plant hydrolysates

Amino sugars are increasingly used as indicators for the accumulation of microbial residues in soil and plant material. A reverse-phase high-performance liquid chromatography method was improved for the simultaneous determination of muramic acid, mannosamine, glucosamine and galactosamine in soil and plant hydrolysates via ortho-phthaldialdehyde (OPA) pre-column derivatisation and fluorescence detection. The retention time was reduced, and the separation of muramic acid and mannosamine was optimised by modifying the mobile phase. The effects of excitation wavelength, OPA reaction time, tetrahydrofuran concentration and pH value of the mobile phase on the amino sugar separation were tested. Quantification limits were in the range of 0.13 to 0.90 μg ml−1. No interferences exist from amino acids or other primary amines, occurring in soil and plant hydrolysates.

Simultaneous determination of l-arginine and 12 molecules participating in its metabolic cycle by gradient RP-HPLC method : Application to human urine samples

We have developed and described a highly sensitive, accurate and precise reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous determination of l-arginine and 12 molecules participating in its metabolic cycle in human urine samples. After pre-column derivatization with ortho-phthaldialdehyde (OPA) reagent containing 3-mercaptopropionic acid (3MPA), the fluorescent derivatives were separated by a gradient elution and detected by fluorescence measurement at 338 nm (excitation) and 455 nm (emission). l-Arginine (ARG) and its metabolites: l-glutamine (GLN), N G-hydroxy- l-arginine (NOHA), l-citrulline (CIT), N G-monomethyl- l-arginine (NMMA), l-homoarginine (HARG), asymmetric N G, N G-dimethyl- l-arginine (ADMA), symmetric N G, N G′-dimethyl- l-arginine (SDMA), l-ornithine (ORN), putrescine (PUT), agmatine (AGM), spermidine (SPERMD) and spermine (SPERM) were extracted in a cation-exchange solid-phase extraction (SPE) column and after derivatization separated in a Purospher ® STAR RP-18e analytical column. The calibration curves of analysed compounds are linear within the range of concentration: 45–825, 0.2–15, 16–225, 12–285, 0.1–32, 15–235, 0.1–12, 0.1–12, 10–205, 0.02–12, 0.1–24, 0.01–10 and 0.01–8 nmol mL −1 for GLN, NOHA, CIT, ARG, NMMA, HARG, ADMA, SDMA, ORN, PUT, AGM, SPERMD and SPERM, respectively. The correlation coefficients are greater than 0.9980. Coefficients of variation are not higher than 6.0% for inter-day precision. The method has been determined or tested for limits of detection and quantification, linearity, precision, accuracy and recovery. All detection parameters of the method demonstrate that it is a reliable and efficient means of the comprehensive determination of ARG and its 12 main metabolites, making this approach suitable for routine clinical applications. The levels of analysed compounds in human urine can be successfully determined using this developed method with no matrix effect.

Study of a new derivatizing reagent that improves the analysis of amino acids by HPLC with fluorescence detection: application to hydrolyzed rape bee pollen

A simple and sensitive method for evaluating the chemical compositions of protein amino acids, including cystine (Cys)(2) and tryptophane (Try) has been developed, based on the use of a sensitive labeling reagent 2-(11H-benzo[alpha]-carbazol-11-yl) ethyl chloroformate (BCEC-Cl) along with fluorescence detection. The chromophore of the 1,2-benzo-3,4-dihydrocarbazole-ethyl chloroformate (BCEOC-Cl) molecule was replaced with the 2-(11H-benzo[alpha]-carbazol-11-yl) ethyl functional group, yielding the sensitive fluorescence molecule BCEC-Cl. The new reagent BCEC-Cl could then be substituted for labeling reagents commonly used in amino acid derivatization. The BCEC-amino acid derivatives exhibited very high detection sensitivities, particularly in the cases of (Cys)(2) and Try, which cannot be determined using traditional labeling reagents such as 9-fluorenyl methylchloroformate (FMOC-Cl) and ortho-phthaldialdehyde (OPA). The fluorescence detection intensities for the BCEC derivatives were compared to those obtained when using FMOC-Cl and BCEOC-Cl as labeling reagents. The ratios I (BCEC)/I (BCEOC) = 1.17-3.57, I (BCEC)/I (FMOC) = 1.13-8.21, and UV(BCEC)/UV(BCEOC) = 1.67-4.90 (where I is the fluorescence intensity and UV is the ultraviolet absorbance). Derivative separation was optimized on a Hypersil BDS C(18) column. The detection limits calculated from 1.0 pmol injections, at a signal-to-noise ratio of 3, ranged from 7.2 fmol for Try to 8.4 fmol for (Cys)(2). Excellent linear responses were observed, with coefficients of >0.9994. When coupled with high-performance liquid chromatography, the method established here allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids including (Cys)(2) and Try from bee-collected pollen (bee pollen) samples.

Monolithic column-based reversed-phase liquid chromatography separation for amino acid assay in microdialysates and cerebral spinal fluid

The development of a HPLC method using a monolithic C 18 column is described using fluorescence detection for the assay of 21 amino acids and related substances with derivatisation using ortho-phthaldialdehyde (OPA) in the presence of 3-mercaptopropionic acid (3-MPA). The method employs a tertiary gradient and has a run time of 24 min. Linearity ( r 2) for each amino acid was found to be greater than 0.99 up to a 10 μM concentration; reproducibility across all analyses (relative standard deviation (R.S.D.)) was between 0.97 and 6.7% and limit of detection (LOD) between 30 and 300 fmol on column. This method has been applied to the analysis of amino acids in both spinal microdialysis and cerebral spinal fluid samples.

Enhanced cellular uptake of a glutathione selective fluorogenic probe encapsulated innanoparticles

Selective fluorogenic probes for the labelling of intracellular reduced glutathione (GSH),i.e. ortho-phthaldialdehyde (OPA) and naphthalene-2,3-dicarboxaldehyde (NDA), have beenencapsulated in polymeric nanoparticles (NPs) and the ability of the NPs to enhance uptake of theprobe by microbial cells has been evaluated. Preparation of the probe-loaded NPs composed ofEudragit® E was based on an oil-in-water emulsification solvent evaporation method using anultrasonic probe and polyvinyl alcohol as the surfactant. The encapsulation efficiencyof the probes in lyophilized NPs was determined using high performance liquidchromatography (HPLC). A higher encapsulation rate of NDA than OPA was found:47.6 ± 9.9 (n = 6) and 2.1 ± 0.2% (n = 3), respectively. The NDA-loaded particle diameter and zeta potential were224.6 ± 14.7 nmand +40.9 ± 6.5 mV, respectively. After 20 min incubation of cultured Candida albicans yeastcells with either free NDA or NDA-loaded NPs (final NDA concentration100 µM), cells were harvested and corresponding lysates were analysed using HPLC coupled withspectrofluorimetric detection. Incubation of cells with NDA-loaded NPs increasedintracellular levels of NDA-GSH adduct by about nine-fold in comparison with the freeprobe. Adhesion on the cells and the penetration behaviour of NPs loaded with either NDAor fluorescent label (Nile Red) were characterized qualitatively by confocal laser scanningmicroscopy.

Flow-injection analysis with fluorescence detection for the determination of trace levels of ammonium in seawater

A method using flow-injection, gas-diffusion, derivatisation and then fluorescent detection has been established for ammonium ion determination in seawater. The fluorescent derivative formed by reacting ortho-phthaldialdehyde (OPA) and sulfite with ammonia gives high sensitivity while removing potential interferences. This is required to measure the low concentrations of ammonium often seen in the open ocean. The experimental conditions (flow-rate, reagent concentrations, membrane configurations, etc.) were manipulated to improve performance. For a sample throughput of 30 samples h(-1), the limit of detection was 7 nM, the coefficient of variation was 5.7% at 800 nM, and the calibration curve was linear to at least 4 micromol L(-1). Interferences were minimised by a gaseous diffusion step. Volatile small molecular-weight amines as interferents were discriminated against by this method. They neither passed through the membrane as efficiently as ammonia, nor reacted as readily with OPA when sulfite was the reductant. Contamination by ammonia from laboratory and shipboard sources complicates application of the method to natural waters, especially measurement of low concentrations (<100 nM) in open-ocean waters. Steps to overcome contamination are described in detail. Some results are presented for ammonium determination in Southern Ocean and Huon Estuary (Tasmania) waters.

New protamine quantification method in microtiter plates using o-phthaldialdehyde/ N-acetyl- l-cysteine reagent

Protamine is a well-known excipient in pharmaceutics. It represents a peptide consisting of exclusive aliphatic amino acids, hence it cannot be quantified by UV-spectroscopy ( λ max 280 nm). A new and sensitive quantification method based on the derivatisation of protamine with ortho-phthaldialdehyde (OPA) in the presents of 2-mercaptoethanol (ME) or N-acetyl- l-cysteine (NAC) in basic aqueous solution using 96-well microtiter plates are introduced in this report. The resulting isoindol derivatives reveal a fluorescence excitation (maximum λ ex 345 nm) and emission (maximum λ em 450 nm) spectra. Derivatives of OPA/NAC reagent were found to be useful for protamine quantification in pharmaceutical nanoparticle preparation containing DNA. A sufficient stability of the isoindol derivatives was shown. It was possible to determine protamine free base, protamine sulphate and protamine chloride with limits of detection less than 1.1 μg/ml.