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.

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.

Automated method for quantification of 20 amino acids in cell culture media during biopharmaceutical development.

Herein, a step-by-step protocol for simultaneous detection of 20 amino acids commonly present in cell culture media is described. The protocol facilitates detection of both primary and secondary amino acids through a two-step precolumn derivatization strategy using ortho-phthalaldehyde and 9-fluorenylmethyl chloroformate as derivatizing agents. The separation of derivatized amino acids with varying hydrophobicity is achieved through reverse-phase chromatography. The amino acids are simultaneously detected in a single workflow through the use of Variable Wavelength Detector at 338 and 262 nm. The protocol is applicable for both mammalian and bacterial cell culture matrices with an option for automation of precolumn derivatization.

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.

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.

Naphthalene-2,3-dicarboxaldehyde as a pulsed-post column derivatization reagent; comparison with two alternative o-phthalaldehyde based chemistries for the determination of histamine.

In the present study, naphthalene-2,3-dicarboxaldehyde (NDA) was used in on-line post column derivatization (PCD) coupled to liquid chromatography under the new concept of Pulsed-PCD. In Pulsed-PCD, the reagents are introduced into the flowing stream of the mobile phase under precise timing overlapping the eluted analyte. The consumption of the reagents is minimized to a few microliters, resulting in a significant advantage, that is the use of expensive reagents in PCD. For this reason, NDA-CN chemistry was used for the determination of histamine in food samples, such as eggplant and spinach. Two additional methods were developed based on the reaction of histamine with o-phthalaldehyde (OPA), namely the classic OPA - nucleophilic compound reaction and the specific OPA - histamine reaction in alkaline medium. The chromatographic conditions and the Pulsed-PCD conditions were investigated, while the analytical figures of merit were satisfactory. In all three methods, a pulse of 50 µL was used (OPA/NDA + Buffer), reducing dramatically the consumption of PCD reagents.

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.

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.

Development of an equipment free paper based fluorimetric method for the selective determination of histidine in human urine samples.

A direct fluorimetric method, employing µicro-analytical paper-based devices (µ-PADs) for the selective determination of histidine (HIS) is described. The suggested method exploits the fluorescence emission of histidine after its rapid reaction with o-phthalaldehyde (OPA) at a basic medium (pH = 10) on the surface of a paper device with the application of a UV lamp at 354 nm. The devices are inexpensive and are composed of chromatographic paper and wax barriers. The analytical protocol is easily applicable with minimal technical expertise and without the need of expensive experimental apparatus. The user has to add a test sample, illuminate the device with a UV lamp, and read the fluorescence of the sensing area using a simple imaging device such as a cell-phone camera. The method is free from common interferences likely to affect the measurement of histidine and is selective among all other amino acids. This analytical procedure was optimized and validated, paying special attention to its intended application. The detection limits are as low as 1.8 µM with very satisfactory precision ranging from 6.4% (intra-day) to 8.9% (inter-day). Random urine samples from adult volunteers (n = 5) were successfully analyzed and HIS content ranged between 260 and 1114 µmol L-1 with percentage recoveries in the range of 78.2 and 124.6%.

Development and validation of a direct HPLC method for the determination of salivary glutathione disulphide using a core shell column and post column derivatization with o-phthalaldehyde.

Glutathione disulfide (GSSG) has been monitored in human saliva samples by an optimized and validated method that is based on liquid chromatography coupled to on-line post column derivatization. The analyte was separated from the sample matrix using a 100% aqueous mobile phase through a core-shell reversed phase column. Following optimization of the reaction using Box- Behnken experimental design and validation, GSSG was quantified accurately and selectively in the range of 100-2000 nmol L-1 with a LOD of 20 nmol L-1. GSSG was quantified in 15 out of 20 human saliva samples (75%) with a mean value of 860 nmol L-1 (150-4600 nmol L-1). Blocking of reduced Glutathione with N-ethylmaleimide ensured stability of the samples for at least 72 h at all temperatures examined.

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.

Single run analysis of glutathione and its disulfide in food samples by liquid chromatography coupled to on-line post-column derivatization.

Glutathione and its disulfide were determined in a single run using liquid chromatography with on-line post-column derivatization and fluorimetric detection (340 nm/425 nm). The analytes were separated using a reversed-phase column capable of operating at 100% aqueous mobile phase and detected following direct on-line reaction with o-phthalaldehyde (7.5 mmol L-1) in highly basic medium (0.37 mol L-1 NaOH). The instrumental and chemical variables were carefully investigated towards high sensitivity and throughput, while special attention was paid to validating potential matrix effects. Glutathione and its disulfide could be selectively determined with respective LODs of 0.10 and 0.30 µmol L-1 in the absence of matrix effect (<6%). The endogenous content of the analytes was accurately determined in various food samples with recoveries ranging between 80 and 120% in all cases. The proposed method is reliable and promising as a generic analytical tool for the convenient estimation of the redox status of glutathione in various food matrices.

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.

Simple and sensitive quantification of glutathione hydropersulfide alkylated using iodoacetamide by high-performance liquid chromatography with post-column derivatization.

A novel analytical method was developed for the quantification of glutathione hydropersulfide (G-SSH) in biological samples by high-performance liquid chromatography (HPLC) with post-column derivatization. G-SSH was treated with iodoacetamide as an alkylating agent for 5 min at 37 °C, and the resultant acetamide-labeled G-SSH (G-SS-acetamide) was subjected to HPLC. After separation on a reversed-phase column, G-SS-acetamide was quantified by detection using a post-column reaction with orthophthalaldehyde under alkaline conditions. The standard G-SS-acetamide was synthesized through the S-S exchange reaction between oxidized glutathione and 2-mercaptoacetamide. It should be noted that some types of alkylating agents, including N-ethylmaleimide and monobromobimane, cleave the polysulfide chains of polysulfides that consist of glutathione, resulting in the production of alkylated G-SSHs. We confirmed that iodoacetamide did not enhance the cleavage of acetamide-labeled glutathione trihydropersulfide (G-SSS-acetamide). The lowest quantification limit was estimated to be 25 nM for G-SS-acetamide. This method can be useful for studying the dynamics of sulfane sulfur in glutathione-containing matrices.

Measurement of Glutathione as a Tool for Oxidative Stress Studies by High Performance Liquid Chromatography.

BACKGROUND: Maintenance of the ratio of glutathione in the reduced (GSH) and oxidised (GSSG) state in cells is important in redox control, signal transduction and gene regulation, factors that are altered in many diseases. The accurate and reliable determination of GSH and GSSG simultaneously is a useful tool for oxidative stress determination. Measurement is limited primarily to the underestimation of GSH and overestimation GSSG as a result of auto-oxidation of GSH. The aim of this study was to overcome this limitation and develop, optimise and validate a reverse-phase high performance liquid chromatographic (HPLC) assay of GSH and GSSG for the determination of oxidant status in cardiac and chronic kidney diseases. METHODS: Fluorescence detection of the derivative, glutathione-O-pthaldialdehyde (OPA) adduct was used. The assay was validated by measuring the stability of glutathione and glutathione-OPA adduct under conditions that could affect the reproducibility including reaction time and temperature. Linearity, concentration range, limit of detection (LOD), limit of quantification (LOQ), recovery and extraction efficiency and selectivity of the method were assessed. RESULTS: There was excellent linearity for GSH (r2 = 0.998) and GSSG (r2 = 0.996) over concentration ranges of 0.1 µM-4 mM and 0.2 µM-0.4 mM respectively. The extraction of GSH from tissues was consistent and precise. The limit of detection for GSH and GSSG were 0.34 µM and 0.26 µM respectively whilst their limits of quantification were 1.14 µM and 0.88 µM respectively. CONCLUSION: These data validate a method for the simultaneous measurement of GSH and GSSG in samples extracted from biological tissues and offer a simple determination of redox status in clinical samples.

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.

Fluorimetric Method for the Determination of Histidine in Random Human Urine Based on Zone Fluidics.

In the present study, the determination of histidine (HIS) by an on-line flow method based on the concept of zone fluidics is reported. HIS reacts fast with o-phthalaldehyde at a mildly basic medium (pH 7.5) and in the absence of additional nucleophilic compounds to yield a highly fluorescent derivative (λex/λem = 360/440 nm). The flow procedure was optimized and validated, paying special attention to its selectivity and sensitivity. The LOD was 31 nmol·L-1, while the within-day and day-to-day precisions were better than 1.0% and 5.0%, respectively (n = 6). Random urine samples from adult volunteers (n = 7) were successfully analyzed without matrix effect (<1%). Endogenous HIS content ranged between 116 and 1527 µmol·L-1 with percentage recoveries in the range of 87.6%-95.4%.

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.

Automated fluorimetric sensor for glutathione based on zone fluidics.

A zone-fluidics (ZF) based automated fluorimetric sensor for the determination of glutathione (GSH) is reported. Discrete zones of GSH and o-phthalaldehyde (OPA) mix and react on-line under mild basic pH without the need of additional nucleophillic reagents, to yield a fluorescent isoindole derivative (λex/λem = 340/425 nm). The proposed ZF sensor was optimized (pH, c(OPA), time, instrumental variables) and validated. Cysteine, glutamate, glycine and ammonium were representatively examined in terms of selectivity and were found not to react in 10-fold excess. Linearity was proved in the range of 5-100 µmol L-1 GSH, with an LOD of 1 µmol L-1 at a practical sampling rate of 20 h-1 and RSD < 0.5% (within-day) and 4.2% (day-to-day). The dosage uniformity of commercially available GSH - containing nutraceuticals was evaluated.