Temperature-Responsive Magnetic Nanoparticles for Bioanalysis of Lysozyme in Urine Samples

Highly selective multifunctional magnetic nanoparticles containing a thermoresponsive polymer shell were developed and used in the sample pretreatment of urine for the assessment of lysozymuria in leukemia patients. Crosslinked poly(N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) was grown onto silica-coated magnetic nanoparticles by reversible addition fragmentation chain transfer (RAFT) polymerization. The lysozyme binding property of the nanoparticles was investigated as a function of time, protein concentration, pH, ionic strength and temperature and their selectivity was assessed against other proteins. High-abundant proteins, like human serum albumin and γ-globulins did not interfere with the binding of lysozyme even at elevated concentrations characteristic of proteinuria. A sample cleanup procedure for urine samples has been developed utilizing the thermocontrollable protein binding ability of the nanoparticles. Method validation was carried out according to current bioanalytical method validation guidelines. The method was highly selective, and the calibration was linear in the 25 to 1000 µg/mL concentration range, relevant in the diagnosis of monocytic and myelomonocytic leukemia. Intra- and inter-day precision values ranged from 2.24 to 8.20% and 1.08 to 5.04%, respectively. Intra-day accuracies were between 89.9 and 117.6%, while inter-day accuracies were in the 88.8 to 111.0% range. The average recovery was 94.1 ± 8.1%. Analysis of unknown urine samples in comparison with a well-established reference method revealed very good correlation between the results, indicating that the new nanoparticle-based method has high potential in the diagnosis of lysozymuria.

Utilizing a Rapid Multi-Plug Filtration Cleanup Method for 72 Pesticide Residues in Grape Wines Followed by Detection with Gas Chromatography Tandem Mass Spectrometry

A convenient and fast multi-residue method for the efficient identification and quantification of 72 pesticides belonging to different chemical classes in red and white grape wines has been developed. The analysis was based on gas chromatography tandem quadrupole mass spectrometric determination (GC–MS/MS). The optimization strategy involved the selection of the amount of multi-walled carbon nanotubes (MWCNTs) and the number of cleanup procedure cycles for multi-plug filtration cleanup (m-PFC) to achieve ideal recoveries and reduce the sample matrix compounds in the final extracts. The optimized procedure obtained consistent recoveries between 70.2 and 108.8% (70.2 and 108.8% for white wine, and 72.3 and 108.4% for red wine), with relative standard deviations (RSDs) that were generally lower than 9.2% at the three spiking levels of 0.01, 0.05 and 0.1 mg/kg. The linearity was studied in the range between 0.002 and 0.1 mg/kg using pesticide standards prepared both in pure solvent and in the presence of the matrix, showing coefficients of determination (R2) higher than 0.9495 for all the pesticides. To improve accuracy, matrix-matched calibration curves were used for calculating the quantification results. Finally, the method was used successfully for detecting pesticide residues in commercial grape wines.

Eco-Friendly UPLC-MS/MS Quantitation of Delafloxacin in Plasma and Its Application in a Pharmacokinetic Study in Rats

A novel UPLC-MS/MS assay was developed for rapid quantification of delafloxacin (a novel fluoroquinolone antibiotic in plasma samples by one step sample cleanup procedure. Delafloxacin (DFX) and internal standard (losartan) were separated on a UPLC BEH C18 column (50 × 2.1 mm; 1.7 μm) by using gradient programing of a mobile phase containing 0.1% formic acid in acetonitrile and 0.1% formic acid in water. The quantification was performed by a using triple-quadrupole mass detector at an electrospray ionization interface in positive mode. The precursor to the product ion transition of 441.1 → 379.1 for the qualifier and 441.1 → 423.1 for the quantifier was used for DFX monitoring, whereas 423.1 → 207.1 was used for the internal standard. The validation was performed as per guidelines of bioanalytical method validation, and the evaluated parameters were within the acceptable range. The greenness assessment of the method was evaluated by using AGREE software covering all 12 principles of green analytical chemistry. The final score obtained was 0.78, suggesting excellent greenness of the method. Moreover, Deming regression analysis showed an excellent linear relationship between this method and our previously reported method, and it is suitable for high-throughput analysis for routine application. The proposed method was effectively applied in a pharmacokinetic study of novel formulation (self-nanoemulsifying drug delivery systems) of DFX in rats.

Development of a method for assessing the accumulation and metabolization of antidepressant drugs in zebrafish (Danio rerio) eleutheroembryos

Antidepressant drugs are widely used for the treatment of common mental or other psychiatric disorders such as depression, which affect about 121 million people worldwide. This widespread use has contributed to the input of these pharmaceuticals and their metabolites into the environment. The aim of this work was to develop an analytical method to quantify the most widely used antidepressant drugs, selective serotonin reuptake inhibitors (SSRI), and their main metabolites in the environment. For this, a new and reliable miniaturized extraction method based on dispersive SPE cleanup procedure for extraction of SSRI followed by derivatization with n-heptafluorobutyrylimidazole, and detection by GC-MS was developed. The methodology, including a first-order one-compartment model, was then applied to a bioconcentration study in zebrafish (Danio rerio) eleutheroembryos. The results showed low bioaccumulation of these compounds; however, a biotransformation evidence of the parent compounds into their metabolites was observed after 6 h of exposure. These results indicate the need to integrate metabolic transformation rates to fully model and understand the bioaccumulation patterns of SSRI and their metabolites.

A New Method for Improving Extraction Efficiency and Purity of Urine and Plasma Cell-Free DNA

This study assessed three commercially available cell-free DNA (cfDNA) extraction kits and the impact of a PEG-based DNA cleanup procedure (DNApure) on cfDNA quality and yield. Six normal donor urine and plasma samples and specimens from four pregnant (PG) women carrying male fetuses underwent extractions with the JBS cfDNA extraction kit (kit J), MagMAX Cell-Free DNA Extraction kit (kit M), and QIAamp Circulating Nucleic Acid Kit (kit Q). Recovery of a PCR product spike-in, endogenous TP53, and Y-chromosome DNA was used to assess kit performance. Nucleosomal-sized DNA profiles varied among the kits, with prominent multi-nucleosomal-sized peaks present in urine and plasma DNA isolated by kits J and M only. Kit J recovered significantly more spike-in DNA than did kits M or Q (p < 0.001) from urine, and similar amounts from plasma (p = 0.12). Applying DNApure to kit M- and Q-isolated DNA significantly improved the amplification efficiency of spike-in DNA from urine (p < 0.001) and plasma (p ≤ 0.013). Furthermore, kit J isolated significantly more Y-chromosome DNA from PG urine compared to kit Q (p = 0.05). We demonstrate that DNApure can provide an efficient means of improving the yield and purity of cfDNA and minimize the effects of pre-analytical biospecimen variability on liquid biopsy assay performance.

A new method for improving extraction efficiency and purity of urine and plasma cell-free DNA

This study assessed three commercially available cell-free DNA (cfDNA) extraction kits and the impact of a PEG-based DNA cleanup procedure (DNApure) on cfDNA quality and yield. Six normal donor urine and plasma samples, and specimens from four pregnant (PG) women carrying male fetuses underwent extractions with the JBS cfDNA extraction kit (kit J), MagMAX Cell-Free DNA Extraction kit (kit M), and QIAamp Circulating Nucleic Acid Kit (kit Q). Recovery of a PCR product spike-in, endogenous TP53, and Y-chromosome DNA was used to assess kit performance. Nucleosomal-sized DNA profiles varied among the kits, with prominent multi-nucleosomal-sized peaks present in urine and plasma DNA isolated by kits J and M only. Kit J recovered significantly more spike-in DNA compared with kit M or Q (p<0.001) from urine, and similar amounts from plasma (p=0.12). Applying DNApure to kit M- and Q-isolated DNA significantly improved the amplification efficiency of spike-in DNA from urine (p<0.001) and plasma (p≤0.013). Furthermore, kit J isolated significantly more Y-chromosome DNA from PG urine compared to kit Q (p=0.05). We conclude that DNApure provides an efficient means of improving the yield and purity of cfDNA and minimizing effects of pre-analytical biospecimen variability on liquid biopsy assay performance.

The Occurrence of Aflatoxins in Nuts and Dry Nuts Packed in Four Different Plastic Packaging from the Romanian Market

Mycotoxins are secondary metabolites produced by various fungi. A very important category of mycotoxins are aflatoxins, considered to be the most dangerous in humans. Aflatoxin B1, well known as a favorable factor in the occurrence of hepatocellular carcinoma in humans, is the most controversial of all mycotoxins. Aflatoxins, found in naturally contaminated food, are resistant to degradation by heat. Current food processing practices and conventional storage conditions do not completely eliminate aflatoxin contamination from the food supply chain. Long storage food products—such as peanuts, pistachio, nuts in general, and dried fruits—are susceptible to aflatoxins contamination. The type of plastic material can influence the concentration of aflatoxins during storage due to the permeability to gas and moisture exchange with the external milieu. Nuts in general and dried fruits are consumed in large quantities worldwide. Therefore, herein we investigated the effect of plastic material on the total aflatoxins and aflatoxin B1 content in 64 samples of nuts and dried fruits packed and stored in low-density polyethylene (LDPE), polypropylene (PP), polyethylene (PE), and polyethylene terephthalate (PET). The method consisted in a cleanup procedure using immunoaffinity columns coupled with RIDASCREEN FAST immunoenzymatic competitive assays based on the ELISA technique. Collected data were subjected to statistical analysis and multiple comparisons tests were applied. From the total analyzed samples, 14.06% exceeded the maximum admitted European levels for total aflatoxins. The highest concentrations of total aflatoxins were obtained from samples packed in LDPE, followed by PP, PE, and PET. Aflatoxin B1 was detected in all samples packed in LDPE, PP, and PE. Most of the samples packed in PET had concentrations <1 µg/kg. These results indicate that nuts in general packed and stored in LDPE are more prone to contamination with aflatoxins, while PET is more suitable for maintaining the quality and safety of these products.

Comparison of Sin-QuEChERS Nano and d-SPE Methods for Pesticide Multi-Residues in Lettuce and Chinese Chives

In this study, a new rapid cleanup method was developed for the analysis of 111 pesticide multi-residues in lettuce and Chinese chives by GC–MS/MS and LC–MS/MS. QuEChERS (quick, easy, cheap, effective, rugged and safe)-based sample extraction was used to obtain the extracts, and the cleanup procedure was carried out using a Sin-QuEChERS nano cartridge. Comparison of the cleanup effects, limits of quantification and limits of detection, recoveries, precision and matrix effects (MEs) between the Sin-QuEChERS nano method and the classical dispersive solid phase extraction (d-SPE) method were performed. When spiked at 10 and 100 μg/kg, the number of pesticides with recoveries between 90% to 110% and relative standard deviations < 15% were greater when using the Sin-QuEChERS nano method. The MEs of Sin-QuEChERS nano and d-SPE methods ranged between 0.72 to 3.41 and 0.63 to 3.56, respectively. The results verified that the Sin-QuEChERS nano method was significantly more effective at removing pigments and more convenient than the d-SPE method. The developed method with the Sin-QuEChERS nano cleanup procedure was applied successfully to determine pesticide residues in market samples.

Simultaneous Determination of Curcumin and Resveratrol in Lipidic Nanoemulsion Formulation and Rat Plasma Using HPLC: Optimization and Application to Real Samples

Background: Curcumin and resveratrol are naturally occurring polyphenols that are highly effective in inhibiting the growth of cancer cells. A robust reversed-phase HPLC method has been developed for the simultaneous determination of these two natural drugs. Objective: The method was adapted to analyze both drugs in pure forms, in lipidic nanoemulsion formulation as well as in rat plasma. The method was applied to real samples after intravenous (IV) injection of rats. Method: Analysis utilized C18 column using acetonitrile (ACN)–water (pH adjusted to 4.6 by 1% orthophosphoric acid) in the ratio of 55+45 (v/v) at a flow rate of 0.8 mL/min with detection at 425 and 304 nm for curcumin and resveratrol, respectively. Results: Extraction efficiency of curcumin and resveratrol using ACN–methanol was 96.10–101.00% (RSD 2.49) and 95.00–99.87% (RSD 2.59), respectively. The assay was linear from 0.05 to 4.00 μg/mL (correlation coefficient of 0.9989 and 0.9981, respectively) and precise [average interday and intraday precision for curcumin RSD% (0.45, 2.04) and resveratrol RSD% (2.25, 1.71)] in spiked rat plasma. The LOD and LOQ were found to be (0.0085 μg/mL, 0.025 μg/mL) and (0.02, 0.06), respectively. Conclusions: The data presented demonstrate that the method provides rapid, sensitive, and precise determination of curcumin and resveratrol in spiked rat plasma and in nanoemulsion dosage form without tedious cleanup procedure, which was successfully applied for quantitation of both drugs following their IV administration to albino rats. Highlights: Validated chromatographic method has been developed for simultaneous determination of curcumin and resveratrol. Optimization of chromatographic conditions was achieved. Application of the method on nanoemulsion formula, on spiked rat plasma, and pharmacokinetics study.