The Resistance of Bacillus Spores: Implications for the Strain-Specific Response to High-Performance Disinfectants.

Bacterial spores in materials and equipment pose significant biosecurity risks, making effective disinfection crucial. This study evaluated Ortho-phthalaldehyde (OPA) and a quaternary ammonia-glutaraldehyde solution (AG) for inactivating spores of Bacillus thuringiensis (BT), B. cereus (BC), and two strains of B. velezensis (BV1 and BV2). Spores of BV1 and BT were treated with 22.5 mg/m3 OPA by dry fumigation or 1 mg/mL AG by spray for 20 min, according to the manufacturer's recommendation. As no sporicidal effect was observed, OPA was tested at 112.5 mg/m3 for 40 min, showing effectiveness for BT but not for BV1. Minimum bactericidal concentration (MBC) tests revealed higher MBC values for glutaraldehyde, prompting an overnight test with 112.5 mg/m3 OPA by dry fumigation and 50 mg/mL AG by spray, using formaldehyde as a control. AG reduced all Bacillus strains, but with limited sporicidal effect. OPA was sporicidal for BT and BV1 but not for BC and BV2, indicating a strain-dependent effect. Formaldehyde performed better overall but did not completely inactivate BV2 spores. Our findings suggest that OPA and AG have potential as formaldehyde replacements in wet disinfection procedures.

Analysis of Gizzerosine in Foodstuffs by HPLC Involving Pre-column Derivatization with o-Phthaldialdehyde.

Gizzerosine [2-amino-9-(4-imidazolyl)-7-azanonanoic acid] is a toxic amino acid formed from histamine and lysine at high temperatures, and may be present in foodstuffs (e.g., fishmeal and meat-bone meal) for animals including cats and dogs. Here we developed a simple, rapid, sensitive, specific, and automated method for the analysis of gizzerosine in foodstuffs by high-performance liquid chromatography (HPLC) involving pre-column derivatization with o-phthaldialdehyde (OPA) in the presence of N-acetylcysteine (instead of the usual 2-mercaptoethanol or ethanethiol reagent). OPA reacted immediately (within 1 min) with gizzerosine in an autosampler at room temperatures (e.g., 20-25 °C), and their derivative was directly injected into the HPLC column. The highly fluorescent gizzerosine-OPA derivative was well separated from the OPA derivatives of all natural amino acids known to be present in physiological fluids (e.g., plasma), proteins and foodstuffs, and was detected at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The total time for chromatographic separation (including column regeneration) was 20 min per sample rather than 40 min and longer in previous HPLC methods. The detection limit for gizzerosine was at least 6 pmol/ml in an assay solution (HPLC vial) or at least 0.09 pmol per injection into the HPLC column. The analysis of gizzerosine was linear between 1 and 100 pmol per injection. When gizzerosine was extracted from foodstuffs, its detection limit was at least 875 pmol/g foodstuff or at least 0.21 mg/kg foodstuff. Our routine HPLC technique does not require any cleanup of samples or the OPA derivatization products (including the OPA-gizzerosine adduct), and is applicable for the analysis of gizzerosine in both foodstuffs and animal tissues.

Analytical determination of ethylenediamine impurity in tripelennamine hydrochloride by gas chromatography-mass spectrometry using phthalaldehyde as the derivatizing agent.

In the pharmaceutical industry, effective risk management and control strategies for potential genotoxic impurities are of paramount importance. The current study utilized GC-MS to evaluate a precise, linear, and accurate analytical method for quantifying ethylenediamine present in tripelennamine hydrochloride using phthalaldehyde as a derivatizing agent. When phthalaldehyde is sonicated for 10 min at room temperature, it reacts with ethylenediamine to form (1z,5z)-3,4-dihydrobenzo[f][1,4]diazocine. This approach minimizes matrix interference issues and resolves sample preparation difficulties encountered during ethylenediamine identification in GC-MS. In this method, helium serves as the carrier gas, while methanol acts as the diluent. The stationary phase consists of a DB-5MS column (30 m × 0.25 mm × 0.25 µm) with a flow rate of 1.5 mL/min. The retention time of (1z,5z)-3,4-dihydrobenzo[f][1,4]diazocine was determined to be 6.215 min. The method validation demonstrated limits of detection and quantification for (1z,5z)-3,4-dihydrobenzo[f][1,4]diazocine at 0.4 and 1.0 ppm, respectively, with a linearity range spanning from 1 to 30 ppm concentration with respect to the specification level. System suitability, precision, linearity, and accuracy of the current method were assessed in accordance with guidelines, yielding results deemed suitable for the intended use.

One-pot derivatization spectrofluorimetric method for detection of aspartame in sweetener commercial tablets and soft drinks: Greenness assessments of the methodology.

Aspartame is an artificial sweetener used in drinks and many foods. International Agency for Research on Cancer classified aspartame as possibly carcinogenic to humans (IARC Group 2B). In this study, a sensitive and selective spectrofluorimetric method was developed to detect aspartame. The method is based on switching on the fluorescence activity of aspartame upon its condensation with O-phthalaldehyde (Roth's reaction) in the presence of 2-mercaptoethanol. The reaction product was detected fluorometrically at λem of 438 nm after λex of 340 nm. All reaction conditions required to yield the optimal fluorescence intensity were observed and investigated. Furthermore, the approach was validated according to ICH guidelines. Upon plotting the concentrations of aspartame against their associated fluorescence intensity values, the relationship between the two variables was linear within the range of 0.5-3.0 µg/mL. Furthermore, the method was employed to analyze the quantity of aspartame in commercial packages and soft drinks with an acceptable level of recovery. In addition, the Green Solvents Selecting Tool, Complementary Green Analytical Procedure Index, and the Analytical Greenness Metric tool were used to evaluate the sustainability and the greenness of the developed methodology.

O-Phthalaldehyde Derivatization for the Paper-Based Fluorometric Determination of Glutathione in Nutritional Supplements.

Herein, a new, direct paper-based fluorimetric method is described for the quantitative determination of glutathione (GSH) molecules in nutritional supplements. Briefly, the proposed analytical method is based on the fluorescence emission resulting from the direct and selective chemical reaction of GSH molecules with the derivatization reagent that is o-phthalaldehyde (OPA) in acidic conditions at room temperature. The intensity of the emitted fluorescence on the surface of the analytical paper devices after irradiation with a lamp at 365 nm is proportional to the concentration of GSH and is measured using a smartphone as the detector. This methodology, which is suitable for measurements in laboratories with limited resources, does not require specialized instrumentation or trained personnel. The protocol governing the proposed method is simple and easily applicable. Essentially, the chemical analyst should adjust the value of pH on the surface of the paper by adding a minimal amount of buffer solution; then, after adding a few microliters of the derivatization reagent, wait for the surface of the paper to dry and, finally, add the analyte. Subsequently, the irradiation of the sensor and the measurement of the emitted fluorescence can be recorded with a mobile phone. In the present study, several parameters affecting the chemical reaction and the emitted fluorescence were optimized, the effect of interfering compounds that may be present in dietary supplements was examined, and the stability of these paper sensors under different storage conditions was evaluated. Additionally, the chemical stability of these paper devices in various maintenance conditions was studied, with satisfactory results. The detection limit calculated as 3.3 S/N was 20.5 µmol L-1, while the precision of the method was satisfactory, ranging from 3.1% (intra-day) to 7.3% (inter-day). Finally, the method was successfully applied to three different samples of dietary supplements.

Flavipin from fungi as a potential inhibitor of rheumatoid arthritis signaling molecules.

Flavipin, a fungal lower molecular weight biomolecule (MW 196.16 g/mol), has not been yet extensively studied for beneficial preclinical and clinical applications. In recent years, various preclinical mouse models including adjuvant-induced arthritis (AIA) were employed to understand mechanisms associated with Rheumatoid arthritis (RA) and to develop new therapeutic drugs. In the current study, we studied the inhibitory effect of Flavipin on major signaling molecules involved in the inflammatory response during RA using both in-silico virtual interaction and in vivo mouse model of AIA. Our in-silico results clarified that Flavipin interacts with the tumor necrosis factor alpha (TNF-α) through conventional hydrogen binding (H-H) at one of TNF-α critical amino acids tyrosine residues, Tyr119, with binding energy (b.e.) -5.9. In addition, Flavipin binds to ATP-binging sites of the Jesus kinases, JAK1, JAK2 and JAK3, through H-H (b. e. between -5.8 and -6.1) and then it may inhibit JAKs, regulators of RA signaling molecules. Moreover, our molecular dynamics stimulation for the docked TNF-α/Flavipin complex confirmed the specificity and the stability of the interaction. In vitro, Flavipin is not toxic to normal cells at doses below 50 µM (its IC50 in normal fibroblast cell line was above 100 µM). However, in vivo, the arthritis score and hind paw oedema parameters were modulated in Flavipin treated mice. Consistent with the in-silico results the levels of the TNF-α, the nuclear transcription factor kappaB (NF-κB) and the signal transduction and activator of transcription (STAT3, downstream of JAKs) were modulated at joint tissues of the hind-paw of Flavipin/AIA treated mice. Our data suggest Flavipin as a potential therapeutic agent for arthritis can inhibit RA major signaling molecules.

An integrative analytical approach designed for feasible tranexamic acid assay using o-phthalaldehyde as a fluorogenic probe: applications to tablets, ampoules, and urine.

Antifibrinolytic tranexamic acid (TRX) suppresses plasminogen activation to plasmin in a competitive way. TRX is approved for the management of heavy menstrual periods, hereditary angioedema, hemophilia, postpartum hemorrhage, surgery, tooth extraction, and severe blood loss after acute trauma. Here, the practical use of an isoindole derivative was established for a novel, easy-to-use, and affordable TRX assay. In the presence of a molecule containing a sulfhydryl group (2-mercaptoethanol) 0.02% v/v, the primary amine moiety in TRX allows its combination with o-phthalaldehyde to produce a luminous product. Excitation (338.8 nm) and emission (433.9 nm) wavelengths were used to monitor the isoindole fluorophore yield, and each operational variable was carefully examined and adjusted. The calibration graph was constructed with fluorescence intensity versus TRX concentration, excellent linearity was observed at concentrations between 40 and 950 ng/ml, and limit of detection and limit of quantitation were 41.3 and 13.6 ng/ml, respectively. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines were used to validate the method. The developed method for TRX assay in various dosage forms and urine was successfully implemented and was shown to be an effective, simple, and quick replacement for the TRX assay in clinical trials and quality control.

A new application of isoindole fluorophore derivative in sitagliptin antidiabetic medication assay: Application to dosage forms and biological fluid evaluation.

Dipeptidyl peptidase-4 enzyme suppressant is a unique category of oral antidiabetic medication. Sitagliptin (STG) is a perfect member of this category and is pharmaceutically marketed alone or in combination with metformin. Here, the ideal application of an isoindole derivative for STG assay was developed using a feasible, easy-to-use, economic, and affordable method. STG as an amino group donor can form a luminescent derivative: isoindole on interaction with o-phthalaldehyde and the existence of (2-mercaptoethanol) 0.02% (v/v) as a thiol group donor. Excitation (339.7 nm) and emission (434.6 nm) wavelengths were used to monitor the isoindole fluorophore yield; moreover, each experimental variable was carefully investigated and adjusted. The calibration graph was constructed by plotting fluorescence intensities against STG concentrations, and controlled linearity was observed at concentrations ranging from 50 to 1000 ng/ml. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines were analyzed in depth to prove the technique validation. The implementation of the present technique was extended successfully to the evaluation of various types of STG dose forms and spiking samples of human plasma and urine. The developed technique was shown to be an effective, simple, and quick replacement for quality control and clinical study evaluation of STG.

Smartphone-Based High-Throughput Fluorimetric Assay for Histidine Quantification in Human Urine Using 96-Well Plates.

A high-throughput fluorimetric assay for histidine was developed, using a 96-well plates platform. The analyte reacts selectively with o-phthalaldehyde under mild alkaline conditions to form a stable derivative. Instrumental-free detection was carried out using a smartphone after illumination under UV light (365 nm). The method was proved to be linear up to 100 µM histidine, with an LLOQ (lower limit of quantification) of 10 µM. The assay was only prone to interference from glutathione and histamine that exist in the urine samples at levels that are orders of magnitude lower compared to histidine. Human urine samples were analyzed following minimum treatment and were found to contain histidine in the range of 280 to 1540 µM. The results were in good agreement with an HPLC corroborative method.

In Vitro Permeability Study of Homotaurine Using a High-Performance Liquid Chromatography with Fluorescence Detection Pre-Column Derivatization Method.

Homotaurine (HOM) is considered a promising drug for the treatment of Alzheimer's and other neurodegenerative diseases. In the present work, a new high-performance liquid chromatography with fluorescence detection (HPLC-FLD) (λex. = 340 nm and λem. = 455 nm) method was developed and validated for the study of substance permeability in the central nervous system (CNS). Analysis was performed on a RP-C18 column with a binary gradient elution system consisting of methanol-potassium phosphate buffer solution (pH = 7.0, 0.02 M) as mobile phase. Samples of homotaurine and histidine (internal standard) were initially derivatized with ortho-phthalaldehyde (OPA) (0.01 M), N-acetylcysteine (0.01 M) and borate buffer (pH = 10.5; 0.05 M). To ensure the stability and efficiency of the reaction, the presence of different nucleophilic reagents, namely (a) 2-mercaptoethanol (2-ME), (b) N-acetylcysteine (NAC), (c) tiopronin (Thiola), (d) 3-mercaptopropionic acid (3-MPA) and (e) captopril, was investigated. The method was validated (R2 = 0.9999, intra-day repeatability %RSD < 3.22%, inter-day precision %RSD = 1.83%, limits of detection 5.75 ng/mL and limits of quantification 17.43 ng/mL, recovery of five different concentrations 99.75-101.58%) and successfully applied to investigate the in vitro permeability of homotaurine using Franz diffusion cells. The apparent permeability (Papp) of HOM was compared with that of memantine, which is considered a potential therapeutic drug for various CNSs. Our study demonstrates that homotaurine exhibits superior permeability through the simulated blood-brain barrier compared to memantine, offering promising insights for enhanced drug delivery strategies targeting neurological conditions.

Bioconjugation via Hetero-Selective Clamping of Two Different Amines with ortho-Phthalaldehyde.

Amino groups are common in both natural and synthetic compounds and offer a very attractive class of endogenous handles for bioconjugation. However, the ability to differentiate two types of amino groups and join them with high hetero-selectivity and efficiency in a complex setting remains elusive. Herein, we report a new method for bioconjugation via one-pot chemoselective clamping of two different amine nucleophiles using a simple ortho-phthalaldehyde (OPA) reagent. Various α-amino acids, aryl amines, and secondary amines can be crosslinked to the ϵ-amino side chain of lysine on peptides or proteins with high efficiency and hetero-selectivity. This method offers a simple and powerful means to crosslink small molecule drugs, imaging probes, peptides, proteins, carbohydrates, and even virus particles without any pre-functionalization.

HPLC method with post-column derivatization for the analysis of endogenous histidine in human saliva validated using the total-error concept.

Histidine (His) is an essential amino acid that plays an important biological role and associated with various pathological conditions. A simple and reliable method for the determination of endogenous histidine in human saliva was optimized and validated. The analyte was separated from the saliva matrix by cation exchange chromatography and detected fluorimetrically (λex/λem = 360/440 nm) after online, specific post-column derivatization (PCD) reaction with o-phthalaldehyde. The chemical and instrumental variables of the post-column reaction were optimized using Box-Behnken experimental design to achieve maximum sensitivity. Method validation was carried out employing the total-error concept. Histidine could be analyzed reliably in the range of 0.5-5.0 µΜ, with an LOD (S/N = 3) of 50 nM. Monte Carlo simulations and capability analysis were used to investigate the ruggedness of the PCD reaction. The sampling strategy, sample preparation and stability were also investigated. Seventeen saliva samples were successfully analyzed with histidine levels being in the range of 2.7-19.5 µΜ.

DNA-Compatible ortho-Phthalaldehyde (OPA)-Mediated 2-Substituted Isoindole Core Formation and Applications.

The incorporation of the isoindole core into the DNA-encoded chemical library is highly desirable for the great potential pharmacological characters exampled by molecules like lenalidomide. Herein, we reported a DNA-compatible protocol for the OPA-mediated transformation of amines into drug-like moieties represented by isoindolinone and thio-2-isoindole, respectively. The high conversion and wide substrate-scope property of our protocol render its feasibility in the manipulation of terminal amines on oligonucleotide conjugates, including "cap-and-catch" purification, sequential synthesis during DEL construction, and on-DNA macrocyclization.

HPLC Determination of Colistin in Human Urine Using Alkaline Mobile Phase Combined with Post-Column Derivatization: Validation Using Accuracy Profiles.

In this study, the development, validation, and application of a new liquid chromatography post-column derivatization method for the determination of Colistin in human urine samples is demonstrated. Separation of Colistin was performed using a core-shell C18 analytical column in an alkaline medium in order (i) to be compatible with the o-phthalaldehyde-based post-column derivatization reaction and (ii) to obtain better retention of the analyte. The Colistin derivative was detected spectrofluorometrically (λext/λem = 340/460 nm) after post-column derivatization with o-phthalaldehyde and N-acetyl cysteine. The post-column derivatization parameters were optimized using the Box-Behnken experimental design, and the method was validated using the total error concept. The ß-expectation tolerance intervals did not exceed the acceptance criteria of ±15%, meaning that 95% of future results would be included in the defined bias limits. The limit of detection of the method was adequate corresponding to 100 nmol·L-1. The mean analytical bias (expressed as relative error) in the spiking levels was suitable, being in the range of -2.8 to +2.5% for both compounds with the percentage relative standard deviation lower than 3.4% in all cases. The proposed analytical method was satisfactorily applied to the analysis of the drug in human urine samples.

Toxicity of Ortho-phthalaldehyde Aerosols in a Human In Vitro Airway Tissue Model.

Ortho-phthalaldehyde (OPA) is a chemical disinfectant used for the high-level sterilization of heat-sensitive medical instruments. Although OPA is considered a safer alternative to glutaraldehyde, no exposure limits have been established for respiratory exposures to ensure the safety of OPA sterilization and the safe use of OPA-treated medical instruments. In order to address data gaps in the toxicological profile of OPA, we treated human in vitro air-liquid-interface (ALI) airway cultures at the air interface with various concentrations of OPA aerosols for 10 consecutive days. Temporal tissue responses were evaluated at multiple time points during the treatment phase as well as 10 days following the last exposure. The disturbance of glutathione (GSH) homeostasis occurred as early as 20 min following the first exposure, while oxidative stress persisted throughout the treatment phase, as indicated by the sustained induction of heme oxygenase-1 (HMOX-1) expression. Repeated exposures to OPA aerosols resulted in both functional and structural changes, including the inhibition of ciliary beating frequency, aberrant mucin production, decreases in airway secretory cells, and tissue morphological changes. While OPA-induced oxidative stress recovered to control levels after a 10 day recovery period, functional and structural alterations caused by the high concentration of OPA aerosols failed to fully recover over the observation period. These findings indicate that aerosolized OPA induces both transient and relatively persistent functional and structural abnormalities in ALI cultures under the conditions of the current study.

OPA-Based Bifunctional Linker for Protein Labeling and Profiling.

Lysine residues have been considered as a routine conjugating site for protein chemical labeling and modification. The commercially available lysine-labeling agents have several limitations in labeling efficiency, stability, and cost. To pursue alternative protein lysine-labeling strategies, herein, we report the development of an ortho-phthalaldehyde (OPA)-based bifunctional linker suitable for protein chemical labeling and profiling. Among three designed OPA-based bifunctional linkers, OPA-NH-alkyne 5 was proved to be optimal for protein labeling with minimal protein turbidity. We further demonstrated OPA-NH-alkyne 5 was applicable for immediate capture of protein or proteome chemical labeling.

Simultaneous Amino Acid Analysis Based on 19F NMR Using a Modified OPA-Derivatization Method.

To provide alternative methods of analyzing amino acids without liquid chromatography, 19F NMR-based simultaneous and individual detection methods for amino acids using o-phthalaldehyde (OPA)-based 19F labeling have been developed. Since the chemical shifts of almost all 19F-labeled amino acids differ from each other, and they can be discriminated on the 19F NMR spectrum, simultaneous detection of amino acids has been successfully demonstrated. The discrimination pattern of the peak identical to that of the 19F-labeled amino acids was largely dependent on the chemical structure of the thiols having 19F nuclei, strongly suggesting that there is a large potential for clearer discrimination of amino acids by optimizing the thiol structure and/or combined use of thiols.

Homocyclic o-dicarboxaldehydes: Derivatization reagents for sensitive analysis of amino acids and related compounds by capillary and microchip electrophoresis.

Amino acids are essential compounds for living organisms, and their determination in biological fluids is crucial for the clinical analysis and diagnosis of many diseases. However, the detection of most amino acids is hindered by the lack of a strong chromophore/fluorophore or electrochemically active group in their chemical structures. The highly sensitive determination of amino acids often requires derivatization. Capillary electrophoresis is a separation technique with excellent characteristics for the analysis of amino acids in biological fluids. Moreover, it offers the possibility of precapillary, on-capillary, or postcapillary derivatization. Each derivatization approach has specific demands in terms of the chemistry involved in the derivatization, which is discussed in this review. The family of homocyclic o-dicarboxaldehyde compounds, namely o-phthalaldehyde, naphthalene-2,3-dicarboxaldehyde, and anthracene-2,3-dicarboxaldehyde, are powerful derivatization reagents for the determination of amino acids and related compounds. In the presence of suitable nucleophiles they react with the primary amino group to form both fluorescent and electroactive derivatives. Moreover, the reaction rate enables all of the derivatization approaches mentioned above. This review focuses on articles that deal with using these reagents for the derivatization of amino acids and related compounds for ultraviolet-visible spectrometry, fluorescence, or electrochemical detection. Applications in capillary and microchip electrophoresis are summarized and discussed.

Society for Maternal-Fetal Medicine Special Statement: Reducing the risk of transmitting infection by transvaginal ultrasound examination.

Recent surveys have shown widespread lapses in the procedures used to reduce the risk of transmitting infection via medical devices. Transvaginal ultrasound examination has the potential to transmit vaginal infections, including human papillomavirus. Areas of particular concern are the use of probe covers with high rates of leakage, disinfectants that are not effective against human papillomavirus, and coupling gel from multiple-use containers. We reviewed these issues, and we recommend 4 steps to reduce the risk of transmitting infection. First, during every transvaginal ultrasound exam, the probe should be covered with a sterile, single-use "viral barrier" cover or a condom. Second, sterile, single-use ultrasound gel packets should be used. Third, after every examination, the probe should be cleaned to remove any visible gel or debris. Finally, after cleaning, the probe should undergo high-level disinfection using an agent with proven efficacy against the human papillomavirus, including hydrogen peroxide, hypochlorite, or peracetic acid. Glutaraldehyde, orthophthalaldehyde, phenols, and isopropyl alcohol have virtually no efficacy against the human papillomavirus.

Selective reaction of homocysteine with o-phthalaldehyde under flow conditions in highly alkaline medium: fluorimetric determination using zone fluidics.

In the present study we report the reaction between homocysteine and o-phthalaldehyde under flow conditions. Homocysteine reacts on-line with the derivatization reagent in a strong alkaline medium and in the absence of nucleophilic reagents to yield a fluorescent derivative (λex /λem = 370/480 nm). The reaction variables were investigated using the concept of zone fluidics. Selectivity factors against other compounds were calculated at 10-fold excess. The findings formed the basis of an automated proposed method that was found to be linear in the range 0.1-1.5 µmol L-1 , with a limit of detection of 20 nmol L-1 and relative standard deviation < 0.5% (within-day) and 3.2% (between-day). The method proved to be rapid, offering a practical sampling rate of 24 h-1 and accurate following application to an artificial urine matrix with minimum dilution.