Multi-Class Procedure for Analysis of 50 Antibacterial Compounds in Eggshells Using Ultra-High-Performance Liquid Chromatography–Tandem Mass Spectrometry

In this work, for the first time, Ultra-High-Performance Liquid Chromatography–Tandem Mass Spectrometry (UHPLC–MS/MS) method was developed for qualitative and quantitative analysis of veterinary antibiotics (cephalosporins, diaminopyrimidines, fluoro(quinolones), lincosamides, macrolides, penicillins, pleuromutilins, sulfonamides, tetracyclines, and sulfones) in hen eggshells. The sample preparation method is based on a liquid–liquid extraction with a mixture of metaphosphoric acid, ascorbic acid, EDTA disodium salt dihydrate, and acetonitrile. The chromatographic separation was performed on Luna^® Omega Polar C18 10 column in gradient elution mode and quantitated in an 8 min run. Validation such as linearity, selectivity, precision, recovery, matrix effect, limit of quantification (LOQ), and limit of detection (LOD) was found to be within the acceptance criteria of the validation guidelines of the Commission Decision 2002/657/EC and EUR 28099 EN. Average recoveries ranged from 81–120%. The calculated LOQ values ranged from 1 to 10 µg/kg, the LOD values ranged from 0.3 to 4.0 µg/kg, depending on analyte. The developed method has been successfully applied to the determination of antibacterial compounds in hen eggshell samples obtained from different sources. The results revealed that enrofloxacin, lincomycin, doxycycline, and oxytetracycline were detected in hen eggshell samples.     

Determination of Methylene Blue and Its Metabolite Residues in Aquatic Products by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

A sensitive and reliable method was developed to determine methylene blue (MB) and its metabolite residues, including azure A (AZA), azure B (AZB), and azure C (AZC) in aquatic products by HPLC–MS/MS. The samples were extracted by acetonitrile and cleaned up by alumina-neutral (ALN) cartridges. The analytes were separated on a Sunfire C18 column (150 mm × 2.1 mm, 5 µm). The method was validated according to the European criteria of Commission Decision 2002/657/CE. Good linearity between 1–500 µg/L was obtained with correlation coefficients (R²) greater than 0.99. The limit of quantification (LOQ) was 1.0 µg/kg. The average recoveries at three levels of each compound (1, 5, and 10 µg/kg) were demonstrated to be in the range of 71.8–97.5%, with relative standard deviations (RSDs) from 1.05% to 8.63%. This method was suitable for the detection of methylene blue and its metabolite residues in aquatic products.     

Determination of 77 Multiclass Pesticides and Their Metabolitesin Capsicum and Tomato Using GC-MS/MS and LC-MS/MS

A quick, sensitive, and reproducible analytical method for the determination of 77 multiclass pesticides and their metabolites in Capsicum and tomato by gas and liquid chromatography tandem mass spectrometry was standardized and validated. The limit of detection of 0.19 to 10.91 and limit of quantification of 0.63 to 36.34 µg·kg⁻¹ for Capsicum and 0.10 to 9.55 µg·kg⁻¹ (LOD) and 0.35 to 33.43 µg·kg⁻¹ (LOQ) for tomato. The method involves extraction of sample with acetonitrile, purification by dispersive solid phase extraction using primary secondary amine and graphitized carbon black. The recoveries of all pesticides were in the range of 75 to 110% with a relative standard deviation of less than 20%. Similarly, the method precision was evaluated interms of repeatability (RSDr) and reproducibility (RSD_wR) by spiking of mixed pesticides standards at 100 µg·kg⁻¹ recorded anRSD of less than 20%. The matrix effect was acceptable and no significant variation was observed in both the matrices except for few pesticides. The estimated measurement uncertainty found acceptable for all the pesticides. This method found suitable for analysis of vegetable samples drawn from market and farm gates.     

Development of One-Step Non-Solvent Extraction and Sensitive UHPLC-MS/MS Method for Assessment of N-(n-Butyl) Thiophosphoric Triamide (NBPT) and N-(n-Butyl) Phosphoric Triamide (NBPTo) in Milk

N-(n-butyl) thiophosphoric triamide (NBPT) is a urease inhibitor utilised in urea-based fertilizers. In Ireland, fertilizer treated with NBPT is applied to pasture to mitigate both ammonia and nitrous oxide emissions, but concerns arise as to the potential for residues in milk products. A quick ultrafiltration extraction and ultra-high performance liquid chromatography coupled with mass spectrometry triple quadrupole (UHPLC-MS/MS) quantitation method was developed and validated in this study. The method was applied in the analysis of samples collected from a field study investigating potential transfer of NBPT residues into milk. NBPT and NBPTo residues, were extracted from fortified milk samples and analysed on a UHPLC-MS/MS with recoveries ranging from 74 to 114%. Validation of the UHPLC-MS/MS method at low (0.0020 mg kg⁻¹) and high (0.0250 mg kg⁻¹) concentration levels in line with SANTE/12682/2019 showed overall trueness in the range of 99 to 104% and precision between 1 and 10%, RSD for both compounds. The limit of quantitation (LOQ) was 0.0020 mg kg⁻¹ and other tested parameters (linearity, sensitivity, specificity, matrix effect, robustness, etc.) satisfied acceptance criteria. Stability assessment using spiked samples revealed the compounds were stable in raw and pasteurised milk for 4 weeks at –80 °C storage temperature. Maintaining samples at pH 8.5–9.0 further improved stability. Analysis of 516 milk samples from the field study found that NBPT and NBPTo concentrations were below the LOQ of 0.0020 mg kg⁻¹, thus suggesting very low risk of residues occurring in the milk. The method developed is quick, robust, and sensitive. The method is deemed fit-for-purpose for the simultaneous determination of NBPT and NBPTo in milk.     

Application of a Liquid-Liquid Microextraction Method Based on a Natural Hydrophobic Deep Eutectic Solvent for the Extraction of Plastic Migrants from Kombuchas

A vortex-assisted liquid-liquid microextraction, based on a natural hydrophobic deep eutectic solvent made from the monoterpene thymol and octanoic fatty acid, was employed for the analysis of 11 phthalate esters and one adipate in kombucha (a tea-based fermented beverage). Separation and determination were performed using an ultra-high performance liquid chromatography (UHPLC) system coupled to a single quadrupole mass spectrometer. Confirmatory analyses were carried out through UHPLC tandem mass spectrometry. The full method was validated in terms of matrix effect, matrix-matched calibration, sensitivity, recovery, limits of detection and quantification and repeatability. Satisfactory determination coefficients for quadratic calibration curves (≥0.9938), recovery values (67–120%) and limits of detection (0.07–5.45 µg/L) were obtained. Analysis of 26 kombucha samples reported concentrations for dibutyl phthalate and dimethyl phthalate in the range between the limit of quantification (LOQ) and 16.18 ± 1.14 µg/L, although these phthalates were also detected under the LOQ in some of the analyzed samples. Only one of the samples bottled in plastic containers (7) did not present residues while only five of the 19 samples in glass bottles contained any plasticizer. However, the highest concentration was found in a kombucha bottled in food-grade glass. This work represents the first application in which phthalates and adipates are analyzed in kombuchas.     

A Stability-Indicating Ultra Performance Liquid Chromato-Graphic (UPLC) Method for the Determination of a Mycophenolic Acid-Curcumin Conjugate and Its Applications to Chemical Kinetic Studies

A simple, precise, and accurate reversed-phase ultra-performance liquid chromatographic (UPLC) method was developed and validated for the determination of a mycophenolic acid-curcumin (MPA-CUR) conjugate in buffer solutions. Chromatographic separation was performed on a C18 column (2.1 × 50 mm id, 1.7 µm) with a gradient elution system of water and acetonitrile, each containing 0.1% formic acid, at a flow rate of 0.6 mL/min. The column temperature was controlled at 33 °C. The compounds were detected simultaneously at the maximum wavelengths of mycophenolic acid (MPA), 254 nm, and curcumin (CUR), or MPA-CUR, at 420 nm. The developed method was validated according to the ICH Q2(R1) guidelines. The linear calibration curves of the assay ranged from 0.10 to 25 μg/mL (r² ≥ 0.995, 1/x² weighting factor), with a limit of detection and a limit of quantitation of 0.04 and 0.10 μg/mL, respectively. The accuracy and precision of the developed method were 98.4–101.6%, with %CV < 2.53%. The main impurities from the specificity test were found to be MPA and CUR. Other validation parameters, including robustness and solution stability, were acceptable under the validation criteria. Forced degradation studies were conducted under hydrolytic (acidic and alkaline), oxidative, thermal, and photolytic stress conditions. MPA-CUR was well separated from MPA, CUR, and other unknown degradation products. The validated method was successfully applied in chemical kinetic studies of MPA-CUR in different buffer solutions.     

Determination of Anthelmintic and Antiprotozoal Drug Residues in Fish Using Liquid Chromatography-Tandem Mass Spectrometry

The objective of this study is to develop a comprehensive and simple method for the simultaneous determination of anthelmintic and antiprotozoal drug residues in fish. For sample preparation, we used the “quick, easy, cheap, effective, rugged, and safe” (QuEChERS) method with a simple modification. The sample was extracted with water and 1% formic acid in acetonitrile/methanol (MeCN/MeOH) (95:5, v/v), followed by phase separation (salting out) with MgSO₄ and NaCl (4:1, w/w). After centrifugation, an aliquot of the extract was purified by dispersive solid-phase extraction (d-SPE) prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was validated at three concentration levels for all matrices, in accordance with the Codex guidelines (CAC/GL-71). Quantitative analysis was performed using the method of matrix-matched calibration. The recoveries were between 60.6% and 119.9%, with coefficients of variation (CV) <30% for all matrices. The limit of quantitation (LOQ) of the method ranged from 0.02 μg kg⁻¹ to 4.8 μg kg⁻¹ for all matrices. This comprehensive method can be used for the investigation of both anthelmintic and antiprotozoal drugs belonging to different chemical families in fishery products.     

Simultaneous Determination of Multi-Class Pesticide Residues and PAHs in Plant Material and Soil Samples Using the Optimized QuEChERS Method and Tandem Mass Spectrometry Analysis

New analytical approaches to the simultaneous identification and quantification of 94 pesticides and 13 polycyclic aromatic hydrocarbons (PAHs) in five representative matrices (pepper, apple, lettuce, wheat, and soil) were developed. The analyses were based on gas chromatography coupled with triple quadrupole tandem mass spectrometry (GC-MS/MS). The procedure was optimized by changing the solvent used during the extraction, from acetonitrile to the acetone: n-hexane mixture at a volume ratio of 1:4 (v/v), as well as the use of a reduced amount of water during the extraction of compounds from cereals. An additional modification was the use of florisil instead of GCB in the sample cleanup step. A full method validation study was performed, at two concentration levels (LOQ and 1000 × LOQ), which showed satisfactory results for all analytes from the PAHs group, with recoveries ranging from 70.7–115.1%, and an average RSD of 3.9%. Linearity was tested in the range of 0.001–1.000 mg/kg and showed coefficients of determination (R²) ≥ 0.99 for all PAHs. Satisfactory recovery and precision parameters (LOQ and 100 × LOQ) were achieved for almost all analytes from the pesticide group in the range of 70.1–119.3% with the mean RSD equal to 5.9%. The observed linearity for all analytes in the concentration range of 0.005–1.44 mg/kg was R² ≥ 0.99, with the exception of famoxadone, chizalofop-p-ethyl, prothioconazole, spirodiclofen, tefluthrin, and zoxamid. The extended uncertainties were estimated, using a top-down approach of 9.9% (average) and 15.3% (average) for PAHs and pesticides samples, respectively (the coverage factor k = 2, the 95% confidence level). Ultimately, the method was successfully applied to determine pesticide residues in commercial samples of fruit, vegetables and grain, and soil samples for PAHs, which were collected from selected places in the Podkarpacie region. A total of 38 real samples were tested, in which 10 pesticides and 13 PAHs were determined. Proposed changes allow us to shorten the sample preparation time (by 20%) and to reduce the consumption of organic solvents (by 17%). The use of florisil for sample cleanup, instead of GCB, improves the recovery of compounds with flat particles.     

Characterisation of Engineered Nanomaterials in Nano-Enabled Products Exhibiting Priority Environmental Exposure

Analytical limitations have constrained the determination of nanopollution character from real-world sources such as nano-enabled products (NEPs), thus hindering the development of environmental safety guidelines for engineered nanomaterials (ENMs). This study examined the properties of ENMs in 18 commercial products: sunscreens, personal care products, clothing, and paints—products exhibiting medium to a high potential for environmental nanopollution. It was found that 17 of the products contained ENMs; 9, 3, 3, and 2 were incorporated with nTiO₂, nAg, binaries of nZnO + nTiO₂, and nTiO₂ + nAg, respectively. Commonly, the nTiO₂ were elongated or angular, whereas nAg and nZnO were near-spherical and angular in morphology, respectively. The size ranges (width × length) were 7–48 × 14–200, 34–35 × 37–38, and 18–28 nm for nTiO₂, nZnO, and nAg respectively. All ENMs were negatively charged. The total concentration of Ti, Zn, and Ag in the NEPs were 2.3 × 10⁻⁴–4.3%, 3.4–4.3%, and 1.0 × 10⁻⁴–11.3 × 10⁻³%, respectively. The study determined some key ENM characteristics required for environmental risk assessment; however, challenges persist regarding the accurate determination of the concentration in NEPs. Overall, the study confirmed NEPs as actual sources of nanopollution; hence, scenario-specific efforts are recommended to quantify their loads into water resources.     

Complex Activity and Sensor Potential toward Metal Ions in Environmental Water Samples of N-Phthalimide Azo-Azomethine Dyes

Herein, the spectral and electrochemical characterizations of three different substituted N-phthalimide azo-azomethine (NAA) dyes (L) containing an o-hydroxy group and their NAA-M(II) chelates [M(II): Cu, Ni, Co, Pb] were reported by using UV–Vis and fluorescence spectroscopy and potentiometric and voltamperometric techniques. The pK value of the dyes as well as the stoichiometry and stability of the NAA-metal chelates were studied, and the stoichiometry was found to be mostly 1:2 (ML₂) with high complex stability constant values. The sensor activity of N-phthalimide azo-azomethine derivatives toward pH and metal ions has been also investigated and tested for indicator application in acid–base analysis and detection of Cu(II) ions in real samples of surface river water using voltamperometric detection. The results showed that one of the ligands possesses the highest electrochemical response upon binding to copper ions and could be successfully used in the analysis of copper in water at a concentration range of the analyte from 3.7 × 10⁻⁷ to 5.0 × 10⁻⁶ mol L⁻¹, with analytical characteristics of the method being Sr = 1.5%, LOD = 3.58 µg L⁻¹ and LOQ =11.9 µg L⁻¹     

New Strategies for the Simple and Sensitive Voltammetric Direct Quantification of Se(IV) in Environmental Waters Employing Bismuth Film Modified Glassy Carbon Electrode and Amberlite Resin

An analytical procedure regarding the determination of selenium(IV) by anodic stripping voltammetry exploiting the in situ plated bismuth film electrode is described. Since organics are commonly present in untreated natural water samples, the use of Amberlite XAD-7 resin turns out to be quite important to avoid problems such as the adsorption of these compounds on the working electrode. The optimum circumstances for the detection of selenium in water using differential pulse voltammetry techniques were found to be as follows: 0.1 mol L⁻¹ acetic acid, 1.9 × 10⁻⁵ mol L⁻¹ Bi(III), 0.1 g Amberlite XAD-7 resin, and successive potentials of −1.6 V for 5 s and −0.4 V for 60 s, during which the in situ formation of the bismuth film on glassy carbon and the accumulation of selenium took place. The current of the anodic peak varies linearly with the selenium concentration ranging from 3 × 10⁻⁹ mol L⁻¹ to 3 × 10⁻⁶ mol L⁻¹ (r = 0.9995), with a detection limit of 8 × 10⁻¹⁰ mol L⁻¹. The proposed procedure was used for Se(IV) determination in certified reference materials and natural water samples, and acceptable results and recoveries were obtained.     

Development and Comprehensive SPE-UHPLC-MS/MS Analysis Optimization,Comparison, and Evaluation of 2,4-Epibrassinolide in Different Plant Tissues

A determination method for trace 24-epibrassinolide (EBL) in plant tissues was developed using ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). The plant tissue samples were extracted using a methanol–formic acid solution, and the corresponding supernatant was purified with ODS C18 solid-phase extraction column. The extracts were separated using a Zorbax Eclipse Plus C18 (2.1 mm × 50 mm, 1.8 μm) column with methanol and 0.1% formic acid as the mobile phase. The ion source for the mass spectrometry was an electrospray ionization source with positive ion mode detection. The linear range of the target compound was 0.7~104 μg/kg, the limit of detection (LOD) was 0.11~0.37 μg/kg, the limit of quantification (LOQ) was 0.36~1.22 μg/kg, the recovery rate was 84.0~116.3%, and the relative standard deviation (RSD%) was 0.8~10.5. The samples of maize plumule, brassica rapeseed flower, and marigold leaf were detected using the external standard method. The optimization of the extraction method and detection method of EBL improved the detection sensitivity, laid a foundation for the artificial synthesis of EBL, improved the extraction rate of EBL, and provided a theoretical basis for the study of EBL in many plants.     

Method Validation and Evaluation of Safrole Persistence in Cowpea Beans Using Headspace Solid-Phase Microextraction and Gas Chromatography

Bioinsecticides are regarded as important alternatives for controlling agricultural pests. However, few studies have determined the persistence of these compounds in stored grains. This study aimed at optimizing and validating a fast and effective method for extraction and quantification of residues of safrole (the main component of Piper hispidinervum essential oil) in cowpea beans. It also sought to assess the persistence of this substance in the grains treated by contact and fumigation. The proposed method used headspace solid-phase microextraction (HS-SPME) and gas chromatography with a flame ionization detector (GC/FID). Factors such as temperature, extraction time and type of fiber were assessed to maximize the performance of the extraction technique. The performance of the method was appraised via the parameters selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy. The LOD and LOQ of safrole were 0.0057 and 0.019 μg kg⁻¹, respectively and the determination coefficient (R²) was >0.99. The relative recovery ranged from 99.26 to 104.85, with a coefficient of variation <15%. The validated method was applied to assess the persistence of safrole residue in grains, where concentrations ranged from 1.095 to 0.052 µg kg⁻¹ (contact) and from 2.16 to 0.12 µg kg ⁻¹ (fumigation). The levels measured up from the fifth day represented less than 1% of the initial concentration, proving that safrole have low persistence in cowpea beans, thus being safe for bioinsecticide use. Thus, this work is relevant not only for the extraction method developed, but also for the possible use of a natural insecticide in pest management in stored grains.     

Determination of N-Acetyl-l-cysteine Ethyl Ester (NACET) by Flow Injection Analysis and Spectrophotometric Detection Using Different Thiol-Sensitive Ligands

A new flow injection spectrophotometric method for the determination of N-acetyl-l-cysteine ethyl ester (NACET) was developed and validated. The method is based on the reduction of Cu(II)-ligand complexes to chromophoric Cu(I)-ligand complexes with the analyte. The studied ligands were neocuproine (NCN), bicinchoninic acid (BCA) and bathocuproine disulfonic acid (BCS). The absorbance of the Cu(I)-ligand complex was measured at 458, 562 and 483 nm for the reactions of NACET with NCN, BCA and BCS, respectively. The method was validated in terms of linear dynamic range, limit of detection and quantitation, accuracy, selectivity, and precision. Experimental conditions were optimized by a univariate method, yielding linear calibration curves in a concentration range from 2.0 × 10⁻⁶ mol L⁻¹ to 2.0 × 10⁻⁴ mol L⁻¹ using NCN; 2.0 × 10⁻⁶ mol L⁻¹ to 1.0 × 10⁻⁴ mol L⁻¹ using BCA and 6.0 × 10⁻⁷ mol L⁻¹ to 1.2 × 10⁻⁴ mol L⁻¹ using BCS. The achieved analytical frequency was 90 h⁻¹ for all three ligands. The method was successfully employed for NACET determination in pharmaceutical preparations, indicating that this FIA method fulfilled all the essential demands for the determination of NACET in quality control laboratories, as it combined low instrument and reagent costs with a high sampling rate.     

Simple and Equipment-Free Paper-Based Device for Determination of Mercury in Contaminated Soil

This work presents a simple and innovative protocol employing a microfluidic paper-based analytical device (µPAD) for equipment-free determination of mercury. In this method, mercury (II) forms an ionic-association complex of tetraiodomercurate (II) ion (HgI₄²⁻_(aq)) using a known excess amount of iodide. The residual iodide flows by capillary action into a second region of the paper where it is converted to iodine by pre-deposited iodate to liberate I_2(g) under acidic condition. Iodine vapor diffuses across the spacer region of the µPAD to form a purple colored of tri-iodide starch complex in a detection zone located in a separate layer of the µPAD. The digital image of the complex is analyzed using ImageJ software. The method has a linear calibration range of 50–350 mg L⁻¹ Hg with the detection limit of 20 mg L⁻¹. The method was successfully applied to the determination of mercury in contaminated soil and water samples which the results agreed well with the ICP-MS method. Three soil samples were highly contaminated with mercury above the acceptable WHO limits (0.05 mg kg⁻¹). To the best of our knowledge, this is the first colorimetric µPAD method that is applicable for soil samples including mercury contaminated soils from gold mining areas.     

Chip-Based Spectrofluorimetric Determination of Iodine in a Multi-Syringe Flow Platform with and without In-Line Digestion—Application to Salt,Pharmaceuticals, and Algae Samples

In this work, a flow-based spectrofluorimetric method for iodine determination was developed. The system consisted of a miniaturized chip-based flow manifold for solutions handling and with integrated spectrofluorimetric detection. A multi-syringe module was used as a liquid driver. Iodide was quantified from its catalytic effect on the redox reaction between Ce(IV) and As(III), based on the Sandell–Kolthoff reaction. The method was applied for the determination of iodine in salt, pharmaceuticals, supplement pills, and seaweed samples without off-line pre-treatment. An in-line oxidation process, aided by UV radiation, was implemented to analyse some samples (supplement pills and seaweed samples) to eliminate interferences and release iodine from organo-iodine compounds. This feature, combined with the fluorometric reaction, makes this method simpler, faster, and more sensitive than the classic approach of the Sandell–Kolthoff reaction. The method allowed iodine to be determined within a range of 0.20–4.0 µmol L⁻¹, with or without the in-line UV digestion, with a limit of detection of 0.028 µmol L⁻¹ and 0.025 µmol L⁻¹, respectively.     

Lead Assays with Smartphone Detection Using a Monolithic Rod with 4-(2-Pyridylazo) Resorcinol

A monolithic rod of polyurethane foam–[4-(2-pyridylazo) resorcinol] (PUF–PAR) as a simple chemical sensor for lead assays with smartphone detection and image processing was developed. With readily available simple apparatus such as a plastic cup and a stirrer rod, the monolithic PUF rod was synthesized in a glass tube. The monolithic PUF–PAR rod could be directly loaded by standard/sample solution without sample preparation. A one-shot image in G/B value from a profile plot in ImageJ for a sample with triplicate results via a single standard calibration approach was obtained. A linear single standard calibration was: [G/B value] = −0.038[µg Pb²⁺] + 2.827, R² = 0.95 for 10–30 µg Pb²⁺ with a limit of quantitation (LOQ) of 33 µg L⁻¹. The precision was lower than 15% RSD. The proposed method was tested by an assay for Pb²⁺ contents in drinking water samples from Bangkok. The results obtained by the proposed method agree with those of ICP-OES and with 100–120% recovery, demonstrating that the method is useful for screening on-site water monitoring.     

Validated Stability-Indicating GC-MS Method for Characterization of Forced Degradation Products of Trans-Caffeic Acid and Trans-Ferulic Acid

When dealing with simple phenols such as caffeic acid (CA) and ferulic acid (FA), found in a variety of plants, it is very important to have control over the most important factors that accelerate their degradation reactions. This is the first report in which the stabilities of these two compounds have been systematically tested by exposure to various different factors. Forced degradation studies were performed on pure standards (trans-CA and trans-FA), dissolved in different solvents and exposed to different oxidative, photolytic and thermal stress conditions. Additionally, a rapid, sensitive, and selective stability-indicating gas chromatographic-mass spectrometric method was developed and validated for determination of trans-CA and trans-FA in the presence of their degradation products. Cis-CA and cis-FA were confirmed as the only degradation products in all the experiments performed. All the compounds were perfectly separated by gas chromatography (GC) and identified using mass spectrometry (MS), a method that additionally elucidated their structures. In general, more protic solvents, higher temperatures, UV radiation and longer storage times led to more significant degradation (isomerization) of both trans-isomers. The most progressive isomerization of both compounds (up to 43%) was observed when the polar solutions were exposed to daylight at room temperature for 1 month. The method was validated for linearity, precision as repeatability, limit of detection (LOD) and limit of quantitation (LOQ). The method was confirmed as linear over tested concentration ranges from 1−100 mg L⁻¹ (r²s were above 0.999). The LOD and LOQ for trans-FA were 0.15 mg L⁻¹ and 0.50 mg L⁻¹, respectively. The LOD and LOQ for trans-CA were 0.23 mg L⁻¹ and 0.77 mg L⁻¹, respectively.     

Determination of the LOQ in real-time PCR by receiver operating characteristic curve analysis: application to qPCR assays for Fusarium verticillioides and F. proliferatum

Real-time PCR (qPCR) is the principal technique for the quantification of pathogen biomass in host tissue, yet no generic methods exist for the determination of the limit of quantification (LOQ) and the limit of detection (LOD) in qPCR. We suggest using the Youden index in the context of the receiver operating characteristic (ROC) curve analysis for this purpose. The LOQ was defined as the amount of target DNA that maximizes the sum of sensitivity and specificity. The LOD was defined as the lowest amount of target DNA that was amplified with a false-negative rate below a given threshold. We applied this concept to qPCR assays for Fusarium verticillioides and Fusarium proliferatum DNA in maize kernels. Spiked matrix and field samples characterized by melting curve analysis of PCR products were used as the source of true positives and true negatives. On the basis of the analysis of sensitivity and specificity of the assays, we estimated the LOQ values as 0.11 pg of DNA for spiked matrix and 0.62 pg of DNA for field samples for F. verticillioides. The LOQ values for F. proliferatum were 0.03 pg for spiked matrix and 0.24 pg for field samples. The mean LOQ values correspond to approximately eight genomes for F. verticillioides and three genomes for F. proliferatum. We demonstrated that the ROC analysis concept, developed for qualitative diagnostics, can be used for the determination of performance parameters of quantitative PCR.     

Fast and Sensitive Screening of Oxandrolone and Its Major Metabolite 17-Epi-Oxandrolone in Human Urine by UHPLC—MS/MS with On-Line SPE Sample Pretreatment

Oxandrolone, a synthetic testosterone analog, is used for the treatment of several diseases associated with weight loss. Unfortunately, oxandrolone is abused by many athletes and bodybuilders due to its strong anabolic effect. We have developed and validated a highly sensitive and rapid on-line SPE-UHPLC-MS/MS method for the determination of oxandrolone and simultaneous identification of its major metabolite 17-epi-oxandrolone in urine matrices. Enrichment of the analytes via an integrated solid-phase extraction was achieved using an Acquity UPLC BEH C18 Column. Subsequently, the chromatographic separation of the on-line preconcentrated sample fraction was achieved using an Acquity HSS T3 C18 Column. For the structural identification of these analytes, a high-resolution mass spectrometer Synapt-G2Si coupled to the Acquity M-class nano-LC system with ionKey source was used. A highly sensitive determination of oxandrolone was achieved using a tandem quadrupole mass spectrometer XEVO TQD. The method was successfully validated in the linear range of oxandrolone from 81.63 pg·mL⁻¹ (limit of quantification, LOQ) to 5000 pg·mL⁻¹ in the human urine matrix. It was applied to the analysis of real urine samples obtained from a healthy volunteer after the oral administration of one dose (10 mg) of oxandrolone. Concentration vs. time dependence was tested in the time interval of 4 h–12 days (after oral administration) to demonstrate the ability of the method to detect the renal elimination of oxandrolone from the human body. Favorable performance parameters along with successful application indicate the usefulness of the proposed method for its routine use in antidoping control labs.