Inter-day Repeatability Research Articles

A novel molecularly imprinted solid phase based on SiO2-Ag2O double-nucleation doped with graphene oxide for extraction and determination of flavonoids from Acanthaceae ebracteatus via HPLC-MS/MS

(Objectives) A novel method was developed for extracting and determining flavonoids from marine mangrove medicinal plants. This approach utilizes surface molecular imprinting to increase selectivity. Graphene oxide doped with a SiO2–Ag2O dual core structure served as a scaffold for synthesizing multilayer surface-imprinted polymers. The surface of the SiO2-Ag2O@GO composite was modified through self-polymerization. (Methods) Acacetin molecularly imprinted polymers (AMIPs) were created via surface-initiated precipitation polymerization with acacetin as the template. These AMIPs were synthesized and characterized via ultraviolet (UV) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), and transmission electron microscopy (TEM). Recycling experiments demonstrated the stability of the AMIPs. Analysis of the adsorption mechanism via the Langmuir and Freundlich models indicated that the Langmuir model was more suitable for predicting AMIPs. (Results) Scatchard plots showing two different binding sites of AMIPs for acacetin adsorption at various concentrations. The class selectivity of AMIPs for flavonoids was found in Acanthaceae ebracteatus. Compared with other analogs, competitive binding experiments revealed that the polymer had greater selectivity for acacetin. After AMIP solid-phase extraction (AMISPE), the linearity, LOD, LOQ, intraday and interday variabilities, recovery, and repeatability of AMIPs combined with HPLC–MS/MS were validated, confirming the method’s selectivity, precision, accuracy, and reproducibility. The contents of acacetin, apigenin, kaempferol, luteolin, quercetin, and genistein in Acanthaceae ebracteatus were determined. (Conclusions) These results indicate that AMISPE coupled with HPLC–MS/MS is a new, simple, and practical method for extracting, purifying, and enriching flavonoids from complex samples, such as those of herbs and other natural marine plants.

Maximizing sensitivity and selectivity in LC × LC-HRMS for pesticide analysis via exploitation of per-aqueous liquid chromatography

Accurate monitoring of pesticide residues at minimal concentrations is imperative for adherence to stringent regulatory standards in numerous countries. This study presents an innovative methodology employing comprehensive two-dimensional liquid chromatography coupled with high-resolution mass spectrometry (LC × LC-HRMS). The approach ensures high sensitivity and selectivity in detecting targeted compounds. A pivotal component of this methodology is the utilization of per-aqueous liquid chromatography (PALC) as the first dimension, facilitating the use of water-based mobile phases and addressing solvent mismatch issues. The second dimension employs reversed-phase liquid chromatography (RPLC), enhancing the separation of compounds. PALC proves instrumental in refocusing and enables the practical application of narrow-diameter columns (1.5 mm I.D.). This column design permits a direct split-free connection of the LC × LC to an electrospray-based mass spectrometer (ESI-MS), contributing to heightened sensitivity. The MS acquisition is performed in a targeted single-ion monitoring mode, ensuring reliable quantification and identification of the pesticide compounds. A comprehensive evaluation of key performance metrics, including signal-to-noise ratio, limit of detection, and response linearity, is conducted. The methodology achieves a limit of detection below the ng mL−1 and exhibits response linearity within the concentration range of 1–100 ng mL−1. The robustness of the approach is further demonstrated through intra-day and inter-day repeatability validations. Furthermore, the platform is finally tested on a surface water sample. This study not only introduces an advanced analytical methodology for pesticide multi-residue analysis but also underscores the significance of PALC in enhancing sensitivity by facilitating the use of smaller-diameter columns and water-based mobile phases, along with the role of RPLC in enhancing separation. The proposed approach showcases promising results in achieving detection limits that match the stringent regulatory standards and reliable quantification for effective pesticide residue monitoring.

Stacking in electrophoresis by electroosmotic flow-assisted admicelle to solvent microextraction.

An in-line sample concentration method for capillary electrophoresis called admicelle to solvent microextraction was proposed. In this technique, analytes were trapped in the cetyltrimethylammonium bromide admicelles formed in situ on the negatively charged capillary surface. A solvent plug was then partially injected hydrodynamically to collapse the admicelles, which liberated and focused the analytes at the solvent front. Voltage was applied across the capillary, completing the stacking process. Various solvents, namely, methanol, ethanol, and acetonitrile, were investigated. The optimal solvent for solvent to admicelle microextraction was 30% acetonitrile in 24mM sodium tetraborate (pH 9.2). Sample injection time and solvent to sample injection ratio were also optimised. For this demonstration, the optimum sample injection time and solvent to sample injection ratio were 320s and 1:2, respectively. Using the optimum conditions, UV detection sensitivity was enhanced 132-176-fold for the model anions. The LOQ, %intra-/inter-day (n = 6/n = 12, 2days) repeatability, and linearity (R2) of admicelle to solvent microextraction were 0.08-2µg/mL, 1.9-3.9%, 2.8-4.9%, and 0.992, respectively. Admicelle to solvent microextraction was applied to the analysis of various fortified water samples, with good repeatability (%RSD = 0.5-3.6%), and no matrix interferences.

Validation and optimisation of reduced glutathione quantification in erythrocytes by means of a coulometric high-performance liquid chromatography analytical method.

Glutathione (GSH), a tripeptide that consists of cysteine, glutamate and glycine, is present in all mammalian tissues in the millimolar range. Besides having numerous cellular functions, GSH is an important antioxidant and is considered a valuable biomarker in evaluating oxidative stress. This paper provides a sensitive analytical method using HPLC-ECD to quantify GSH in erythrocytes, validated using the ICH guidelines for Bioanalytical Method Validation. The sample preparation was optimised using centrifugal filtration and a hypotonic phosphate buffer for extracting GSH from erythrocytes. HPLC-ECD parameters were adjusted to allow a fast, reversed phase, isocratic separation in 10min. The detector response was linear between 0.3 and 9.5μg/mL with a satisfactory regression coefficient and a LOQ of 0.11 μg/mL. Intra- and inter-day repeatability ranged between 1.10% and 8.57% with recoveries ranging from 94.3% to 106.0%. Dilution integrity, benchtop, freeze-thaw and long-term stability were investigated. Samples were stable for up to 6months at -80°C. This method has a good linear response and is repeatable, precise and accurate. It minimises GSH auto-oxidation using a centrifugal filter during sample preparation, instead of acidification. Therefore, this analytical method is suitable for quantifying GSH in erythrocytes as a marker of oxidative stress.

Preparation and evaluation of polymeric ionic liquid functionalized microparticles as the efficient adsorbent for pipette tip solid phase extraction of sulfonamides

Sulfonamides (SAs) residues are commonly found in environmental samples, which is highly concerning for public safety and environmental protection. The detection of SAs is quite important but challenging. In this work, a kind of polymeric ionic liquid functionalized microparticles (PIL-FM) was synthesized via a simple free radical polymerization reaction, and applied as the adsorbent for pipette tip solid phase extraction (PT-SPE) of three SAs from river water, milk powder and honey samples. The adsorption performance of PIL-FM on SAs were evaluated using adsorption kinetics and adsorption isotherms, and the results showed that PIL-FM had the best selectivity for SMZ with the maximum adsorption capacity was as high as 45.05 mg·g−1. The extraction process was optimized and the adsorption mechanism was preliminarily discussed. PIL-FM has the excellent selectivity for SAs mainly because of the strong π-π interaction, and the electrostatic repulsion between them in the elution step also has a positive impact. Under optimal conditions, combined with HPLC, this PT-SPE method had wide linearity (0.2–100 μg·L−1, R2 ≥ 0.9998) and low limits of detection (0.06–0.08 μg·L−1). The intra-day and inter-day repeatability were found to be 3.7 %–4.6 % and 4.5 %–6.2 %, respectively. The suggested PT-SPE method is simple, low cost, rapid, and efficient to extract SAs, and can be applied for the monitoring of SAs residues in the aquatic environment and in food.

Preparation of DNA nanoflower-modified capillary silica monoliths for chiral separation.

Innovative chiral capillary silica monoliths (CSMs) were developed based on DNA nanoflowers (DNFs). Baseline separation of enantiomers such as atenolol, tyrosine, histidine, and nefopam was achieved by using DNF-modified CSMs, and the obtained resolution value was higher than 1.78. To further explore the effect of DNFs on enantioseparation, different types of chiral columns including DNA strand containing the complementary sequence of the template (DCT)-modified CSMs, DNF2-modified CSMs, and DNF3-modified CSMs were prepared as well. It was observed that DNF-modified CSMs displayed better chiral separation ability compared with DCT-based columns. The intra-day and inter-day repeatability of model analytes' retention time and resolution kept desirable relative standard deviation values of less than 8.28%. DNF2/DNF3-modified CSMs were able to achieve baseline separation of atenolol, propranolol, 2'-deoxyadenosine, and nefopam enantiomers. Molecular docking simulations were performed to investigate enantioselectivity mechanisms of DNA sequences for enantiomers. To indicate the successful construction of DNFs and DNF-modified CSMs, various charaterization approaches including scanning electron microscopy, agarose gel electrophoresis, dynamic light scattering analysis, electroosmotic flow, and Fourier-transform infrared spectroscopy were utilized. Moreover, the enantioseparation performance of DNF-modified CSMs was characterized in terms of sample volume, applied voltage, and buffer concentration. This work paves the way to applying DNF-based capillary electrochromatography microsystems for chiral separation.

Evaluation of the SpotChecks contrast sensitivity test in healthy adults.

SpotChecks is a new contrast sensitivity (CS) test designed for self-monitoring of vision. This study assessed the test-retest repeatability of take-home SpotChecks, in-office SpotChecks and near Pelli-Robson charts in healthy adults. One eye of 61 healthy adults with near visual acuity (VA) of 6/9 or better (age range 22-84, mean 49 [18] years) was tested during two office visits (mean 10 [8] days apart). Each visit included high-contrast VA, then 12 randomly ordered CS tests (6 different SpotChecks and 6 different Pelli-Robson) under the same lighting (luminance 110 cd/m2), all at near in the same eye with habitual correction. The same eye was self-tested with take-home SpotChecks once a day on 6 days between the office visits. SpotChecks was scored by the logCS at the highest line with ≥2 errors. Pelli-Robson was scored by [0.05 × number of letters read correctly - 0.15]. Repeatability of logCS was defined as 1.96 Sw, Sw representing within-subject standard deviation. Comparison for repeatability was performed with Bootstrap hypothesis test. SpotChecks and Pelli-Robson showed similar intra-session or inter-visit repeatability (p = 0.14-0.81). Inter-day repeatability for take-home SpotChecks was 0.18 logCS, the same as that from the first measurements of two office visits with SpotChecks or Pelli-Robson. Inter-visit repeatability improved to 0.15 by using the average of two repeated measurements for SpotChecks (p = 0.02) or three repeated measurements for Pelli-Robson (p = 0.04). Age showed a small effect on logCS (-0.015/decade, p = 0.02) for both SpotChecks and Pelli-Robson. Mean logCS was 0.05 lower in those ≥50 years (SpotChecks 1.84 [0.10] and Pelli-Robson 1.77 [0.10]) compared with those <50 years of age (SpotChecks 1.89 [0.07] and Pelli-Robson 1.83 [0.07]). SpotChecks showed good repeatability with take-home and in-office testing in healthy adults, making it a promising tool for monitoring disease progression at home.

Stressing the Relevance of Differentiating between Systematic and Random Measurement Errors in Ultrasound Muscle Thickness Diagnostics

BackgroundThe majority of studies that explore changes in musculature following resistance training interventions or examine atrophy due to immobilization or sarcopenia use ultrasound imaging. While most studies assume acceptable to excellent reliability, there seems to be unawareness of the existing absolute measurement errors. As early as 1998, methodological research addressed a collective unawareness of the random measurement error and its practical indications. Referring to available methodological approaches, within this work, we point out the limited value of focusing on relative, correlation-based reliability indices for the interpretability in scientific research but also for clinical application by assessing 1,512 muscle thickness values from more than 400 ultrasound images. To account for intra- and inter-day repeatability, data were collected on two consecutive days within four testing sessions. Commonly-stated reliability values (ICC, CV, SEM and MDC) were calculated, while evidence-based agreement analyses were applied to provide the accompanied systematic and random measurement error.ResultsWhile ICCs in the range of 0.832 to 0.998 are in accordance with the available literature, the mean absolute percentage error ranges from 1.34 to 20.38% and the mean systematic bias from 0.78 to 4.01 mm (all p ≤ 0.013), depending on the measurement time points chosen for data processing.ConclusionsIn accordance with prior literature, a more cautious interpretation of relative reliability values should be based on included systematic and random absolute measurement scattering. Lastly, this paper discusses the rationale for including different measurement error statistics when determining the validity of pre-post changes, thus, accounting for the certainty of evidence.

An ultrasound assisted dispersive micro solid-phase extraction and a composite ionic liquid-metal organic framework for sixteen polycyclic aromatic hydrocarbons analysis in fruit juice and environmental water samples

Aluminum-based metal organic framework composite containing ionic liquid was prepared and used as sorbent for extraction of sixteen polycyclic aromatic hydrocarbons in list of priority pollutants of United States Environmental Protection Agency before their analysis by gas chromatography/mass spectrometry. The dispersive micro solid-phase extraction method, known as a simple and fast method, was preferred as the extraction method. The optimized parameter conditions were 5 mL of sample solution, 10 min sonication by ultrasonic bath, 30 mg of sorbent, 30 °C extraction temperature, 0.1 mL of hexane as elution solvent with 5 min elution time. The suggested method presented that limit of detection and limit of quantification were in the range of 0.01–0.10 μg l-1, and 0.04–0.33 μg L-1, respectively. The intra-day and inter-day repeatability were within the ranges of 1.18–4.88 % and 1.02–5.06 %, respectively. The recoveries for polycyclic aromatic hydrocarbons in peach juice, cherry juice, tap water and rain water samples were obtained in the range of 84.9–99.9 % for spiked 5, 50 and 100 μg l-1 standard polycyclic aromatic hydrocarbons solution.

Reliability of Ultrasound Assessment of Hamstring Morphology, Quality, and Stiffness Among Healthy Adults and Athletes: A Systematic Review.

The incidence and recurrence rate of hamstring strain injuries remain persistently high, with recurrent injuries leading to increased time lost during play and extended recovery periods compared with initial injury. Ultrasound imaging assesses important factors such as hamstring fascicle length (FL), pennation angle (PA), cross-sectional area (CSA), muscle thickness (MT), echo intensity (EI), and shear wave elastography (SWE), all impacting athletic performance. However, its reliability must be established before employing any measurement tool in research or clinical settings. To determine the reliability and measurement error of ultrasound for assessing hamstring FL, PA, CSA, MT, EI, and SWE among healthy adults and athletes; to synthesize the information regarding the operationalization of ultrasound. A systematic literature search was done from January 1990 to February 5, 2023, to identify reliability and validity studies of hamstring ultrasound assessment published in peer-reviewed journals with identifiable methodology of outcome measures. Intraclass correlation coefficient measurement of 14 included studies reported moderate to excellent intrarater, interrater, and test-retest reliabilities of FL, PA, and MT regardless of the site of muscle testing, probe size, and setting, state of muscle, and use of different techniques in the extrapolation of FL. Good to excellent test-retest reliability rates for all hamstring anatomic CSA along midmuscle and different percentages of thigh length using panoramic imaging. Good intrarater reliability of EI regardless of gender and orientation of the probe but with excellent intrarater reliability in transverse scan using maximum region of interest. Good intrarater, interrater, and interday repeatability on SWE with the muscle in a stretched position. Evidence from studies with a predominantly low risk of bias shows that ultrasound is a reliable tool to measure hamstring FL, PA, CSA, MT, EI, and SWE in healthy adults and athletes under various experimental conditions.

Development of a capillary electrophoresis method with laser-induced fluorescence detection for the characterization of oligosaccharides in human milk

Breast milk is the most appropriate food for an infant in the first months of life - it provides the essential nutrients required for proper growth, with breast milk oligosaccharides (HMOs) being the third most abundant component. HMO concentrations vary based on factors like lactation stage and maternal health. They play crucial roles in infant health, acting as prebiotics, antimicrobials, and immune modulators. Analytical methods like capillary electrophoresis and liquid chromatography are the most often used in HMOs qualitative and quantitative analysis. Continuing research aims to enhance analytical techniques for comprehensive HMOs analysis, this work presents the development of a capillary electrophoresis method with laser-induced fluorescence detection along with derivatization and solid-phase extraction steps for the characterisation of major oligosaccharides in colostrum samples. Validation parameters, such as linearity (0.9949–0.9989), limits of detection (5.49–16.40 ng cm−3) and quantification (18.30–54.67 ng cm−3), intra- and inter-day precision (2.41–9.78% and 1.27—14.5%, respectively), trueness (− 8.4 to − 2.0%), and recovery (70.02–113.5%) and repeatability (2.22–10.58%) for solid-phase extraction stage were assessed. The method was evaluated using RGB additive colour model regarding the analytical and practical effectiveness and greenness of the method (achieving a brilliance score of 67.1%). Moreover, the developed method was successfully applied to determine three oligosaccharides (DSLNT, 3’SL, and 6’SL) in colostrum samples. Considering its effectiveness, the method has promise for this type of application.Graphical abstract

Development and validation of a HPLC-UV method for the quantification of major phospholipids in milk

Phospholipids have gained increased interest in the past decades because of their numerous health benefits. Due to the amphiphilic nature of phospholipids, their quantitative analysis requires lengthy procedures. Hydrophilic interaction liquid chromatography (HILIC) with UV detector was used in the present study to effectively separate the major classes of phospholipids found in milk. Phospholipids were effectively separated on a diol-HILIC column using a gradient elution method using acetonitrile and 10 mM ammonium acetate (pH 5 adjusted using acetic acid). Good chromatographic separation of major phospholipid classes: phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sphingomyelin (SM) was possible at 203 nm. The developed method was validated for both phospholipid standards and milk fat. The correlation coefficient for phospholipids using a linear regression equation for 1–50 μg/mL standard solutions and milk fat were >0.99 and >0.98, respectively. The relative standard deviation for intra-day and inter-day repeatability of phospholipids were less than 6 %, and recoveries fell within the 75-120% range. The developed method was applied for the quantification of phospholipids (PC, PE, and SM) in milk from indigenous and crossbred cattle. Gir, Sahiwal, and Karan Fries cows had no significant difference in PE and PC, while Gir cows exhibited significantly higher levels of SM.

2D-network of boron-functionalized N-doped graphene quantum dots for electrochemical sensing of dopamine

A glassy carbon electrode (GCE) modified by boron-functionalized N-doped graphene quantum dots (B-GQDs) was employed for the electrochemical determination of dopamine (DA), a neurotransmitter. The B-GQDs were obtained by hydrothermal etching of GO with H2O2 and NH3 in the presence of H3BO3. By using various techniques, including transmission electron microscopy, atomic force microscopy, molecular absorption spectrometry, fluorescence, ICP-MS, and zeta potential analysis, it was demonstrated that B-GQDs are self-organized in 2D-network, being N-doped GQDs nanoparticles crosslinked by H3BO3. The GCE modification was carried out using the drop-casting method with B-GQDs in Nafion®, previously dispersed through ultrasonication. For the first time, the synthesized nanomaterial was tested in the electroanalysis of neurotransmitters. The B-GQDs/Nafion®/GCE modification led to a significant increase in peak currents in the presence of DA. The electrochemical and interfacial characteristics of the sensors were evaluated by cyclic voltammetry (CV). Under optimized square wave voltammetry and supporting electrolyte conditions, a linear dependence between the anodic peak current and DA concentration was observed in the range of 0.7 to 188 μmol L−1 (R2 = 0.9991). The limit of detection (LOD) was estimated at 0.05 μmol L−1. Selectivity was confirmed in the presence of potential interferents found in urine, with interference response below 12 %. The sensor exhibited excellent intra-day and inter-day repeatability, with a relative standard deviation of 4.12 % and 4.66 %, respectively. Electrochemical determination of DA was conducted in artificial urine samples (Surine®), with recovery values of 90–101 %. The new analytical platform B-GQDs/Nafion®/GCE demonstrated promising characteristics for application in biological and/or pharmaceutical analysis.

High-performance liquid chromatography determination of polyamine contents in poultry based on precolumn benzoyl chloride derivatization

Polyamines are widely present in animal cells and perform important physiological and biochemical functions. Moderate intake of polyamines contributes to good health. Poultry is one of the common sources of polyamines in daily life. However, different tissues from poultry contain different amounts of polyamine. In this study, an efficient and accurate method for the determination of polyamines was developed by high-performance liquid chromatography (HPLC) with precolumn benzoyl chloride derivatization. The linearity of the polyamines was good between 10–400 nmol/mL, with the limit of detection (LOD, S/N = 3) and limits of quantification (LOQ, S/N = 10) were 0.37 and 1.23 nmol/mL. The recoveries of the method ranged from 94.73 % to 106.58 % with high accuracy (RSD < 7 %) and no significant matrix effects or interferences. The RSD of intra-day, inter-day and inter-batch repeatability in the range of 0.07–1.57 %, 0.08–1.03 % and 1.46–4.57 %. The method was successfully used to detect polyamines in poultry tissue, with putrescine, spermidine and spermine levels ranging from 3.24 to 29.85 mg/kg, 0.46 to 164.11 mg/kg and 63.39 to 489.57 mg/kg, respectively. Polyamine levels were higher in the spleen, thymus and bursa of fabricius and lower in the heart and muscle. Furthermore, the method was employed to study the effect of exogenous spermine on polyamine deposition in different tissues of geese. In summary, the method is suitable for the detection of polyamine in animal tissues.

Development of a Multiplexing Injector for Gas Chromatography for the Time-Resolved Analysis of Volatile Emissions from Lithium-Ion Batteries.

Multiplex sampling, so far mainly used as a tool for S/N ratio improvement in spectroscopic applications and separation techniques, has been investigated here for its potential suitability for time-resolved monitoring where chromatograms of transient signals are recorded at intervals much shorter than the chromatographic runtime. Different designs of multiplex sample introduction were developed and utilized to analyze lithium-ion battery degradation products under normal or abuse conditions to achieve fast and efficient sample introduction. After comprehensive optimization, measurements were performed on two different GC systems, with either barrier discharge ionization detection (BID) or mass spectrometric detection (MS). Three different injector designs were examined, and modifications in the pertinent hardware components and operational conditions used. The shortest achievable sample introduction time was 50 ms with an interval of 6 s. Relative standard deviations were lower than 4% and 10% for the intra- and inter-day repeatability, respectively. The sample introduction system and column head pressure had to be carefully controlled, as this parameter most critically affects the amount of sample introduced and, thus, detector response. The newly developed sample introduction system was successfully used to monitor volatile degradation products of lithium-ion batteries and demonstrated concentration changes over the course of time of the degradation products (e.g., fluoroethane, acetaldehyde and ethane), as well as for solvents from the battery electrolyte like ethyl carbonate.

Hydroxylated hierarchical flower-like COF for solid-phase extraction of adrenergic receptor agonists in milk.

A new detection platform based on a hydroxylated covalent organic framework (COF) integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was constructed and used for detecting adrenergic receptor agonists (ARAs) residues in milk. The hydroxylated COF was prepared by polymerization of tris(4-aminophenyl)amine and 1,3,5-tris(4-formyl-3-hydroxyphenyl)benzene and applied to solid-phase extraction (SPE) of ARAs. This hydroxylated COF was featured with hierarchical flower-like morphology, easy preparation, and copious active adsorption sites. The adsorption model fittings and molecular simulation were applied to explore the potential adsorption mechanism. This detection platform was suitable for detecting four α2- and five β2-ARAs residues in milk. The linear ranges of the ARAs were from 0.25 to 50 µg·kg-1; the intra-day and the inter-day repeatability were in the range 2.9-7.9% and 2.0-10.1%, respectively. This work demonstrates this hydroxylated COF has great potential as SPE cartridge packing, and provides a new way to determine ARAs residues in milk.

Exploring a Lux® i-Amylose-3 column in normal phase and polar-organic mode for chiral separation of cathinone derivatives and pyrovalerones using high-performance liquid chromatography.

Each year, new psychoactive substances appear on the global drug market leading to constant changes. Most of these compounds with stimulating effect possess a chiral center, thus leading to two enantiomers with presumably different pharmacological properties. Among them, synthetic cathinones, often misleadingly traded as "bath salts," play an important role. There is little knowledge about the distinct effect of the enantiomers. The aim of this study was to test a commercially available Lux® i-Amylose-3 column by HPLC-UV for enantiorecognition of cathinone derivatives. Overall, 80 compounds were tested in normal phase mode, where 75 substances were separated under initial conditions. After method optimization, at least partial separation was achieved for the remaining compounds. The same set of substances was measured in polar-organic mode, where 63 analytes were resolved into their enantiomers under initial conditions with very short retention times. Both modes showed complementary results for the individual compounds. Furthermore, the tested methods proved to be suitable for differentiation of positional isomers, which can be useful for drug checking programs. All measurements were carried out under isocratic conditions, and intraday and interday repeatability tests were performed.

Development of an Inhibition-Based Colorimetric Method For Glutathione Determination

Objectives: Glutathione (GSH, L-γ-glutamyl-L-cysteinyl-glycine), one of the major cellular antioxidants, is an important non-protein intracellular physiological antioxidant with sulphhydryl groups for detoxification of reactive oxygen species (ROS) in all living organisms. GSH deficiency has been shown to be associated with many human diseases, including cardiovascular, immune and ageing diseases, arthritis and diabetes. Therefore, the development of an accurate, reliable and sensitive method for the determination of GSH in biological fluids is essential for the understanding of GSH homeostasis in medicine and biochemical research Material and Methods: In this study, a very inexpensive, practical, rapid, sensitive, and highly specific colorimetric method for the determination of glutathione (GSH) that can be detected by the naked eye was developed. This method is based on the inhibition of horseradish peroxidase (HRP) by GSH. As the concentration of glutathione increases, a pink coloured compound consisting of 4-chlorophenol, H2O2 and 4-aminoantipyrine (4-AAP) decomposes as a result of the reaction catalyzed by HRP, thus reducing the intensity of the colour. Results: While the linear range of the developed method was found to be between 15.6-1000 mM, the intra- and inter-day repeatability % coefficient of variation values of the method were less than 15%. The effect of potential interfering substances on the developed method was tested, and no interference was found, except for cysteine. Cysteine increased GSH response by 10%. The developed method was used for the determination of GSH in commercial serum samples, and results were obtained between 91-106%. Conclusion: In conclusion, this study has developed a very simple, inexpensive and unique colourimetric method for the determination of GSH.

Smartphone-based digital image colorimetry for the determination of total capsaicinoid content in chili pepper extracts

A simple smartphone-based digital image colorimetry was proposed for the determination of total capsaicinoid content and the assessment of chili pepper pungency. The biobased solvent D-limonene was used for the first time to isolate analytes. Capsaicinoids were efficiently separated from chili pepper by solid–liquid extraction with D-limonene followed by partitioning of the analytes into the ammonium hydroxide solution to eliminate the matrix interference effect. For colorimetric detection of total capsaicinoid content, a selective chromogenic reaction was performed using Gibbs reagent (2,6-dichloroquinone-4-chloroimide). Measurements were performed using a smartphone-based setup and included image analysis with the program ImageJ. The limit of detection of the proposed procedure was 0.15 mg g−1. The intra-day repeatability did not exceed 10.0 %. The inter-day repeatability was less than 16.5 %. The comparison of the smartphone-based procedure with high-performance liquid chromatography showed satisfactory results.

Accurate Detection of G-Series Agent Simulants by Pseudo-Multiple Reaction Monitoring on Miniature LIT-MS.

Rapid and accurate on-site detection of chemical warfare agents (CWAs) could defend military and civilian populations against current and emerging chemical weapons. With the development of ambient ionization and linear ion trap technology, the rapid and accurate quantitative determination method of CWAs based on direct ionization and multistage mass spectrometry has attracted widespread attention. In this study, a microliter electrospray ionization-miniature linear ion trap mass spectrometry (LIT-MS) instrument was designed and constructed, and the effects of quadrupole enhanced dipole resonance excitation on the resolution and sensitivity were investigated; consequently, the parameters of CWAs detection were optimized. Based on the broad time-frequency ion excitation technology, accurate multiple reaction monitoring (MRM) quantitative analysis of DMMP (G-series agent simulants, m/z 125 → m/z 93) was obtained. The linear correlation coefficient in the concentration range of 1 to 20 μg/mL could reach 99.02%, and the relative standard deviations (RSD) of continuous repeatability, interday repeatability, and intraday repeatability were all less than 10%. The results showed that the accurate pseudo-MRM detection method based on miniature linear ion trap mass spectrometry for CWAs detection was feasible.