Fluorometric Assessment of Sulfhydryl Oxidase Activity: Optimization by Response Surface Methodology

Sulfhydryl oxidase was studied using a spectrofluorometric assay. The current protocol operates by using a combination of hemoglobin (HB) and hematin (HT) as a peroxidase mimic to catalyze the H2O2-dependent oxidation of thiamine. The response surface methodology is used to optimize the new method (RSM). The current method is very accurate, sensitive, and linear up to 200 IU. When compared to the colorimetric method, the method produced a satisfactory correlation. The novel protocol is being used to evaluate asthenospermic patients' and fertile men's seminal SHO activity. The current protocol was used to determine reference values for seminal sulfhydryl oxidase activity. Due to the fact the newly developed spectrofluorometric method is more sensitive and precise than other colorimetric methods, and because thiamine is less expensive than other types of probes used in colorimetric and spectrofluorometric methods, it is likely to find widespread use among scientists studying SHO activity in biological tissues. The present method's analytical recovery yielded high specific findings.

Ciprian Porumbescu. Mică poveste despre rochia Berthei (o incursiune printre manuscrise, ediții etc.)

A young Romanian composer from the Austrian Bucovina, Ciprian Porumbescu (1853-1883) is an important figure of the local cultural mythology. This fact, we believe, is due to the association of his image with some ill-fated aspects of his life, such as an impossible love, his premature death, his exultant talent, maybe even his ardent patriotism. In my attempts at recovering the past cultural life of Bucovina, I discovered the journal of Ciprian Porumbescu (that probably includes a rich correspondence as well), written around 1878-1883. I am not talking about a journal on creation, nor about one related to political life. That, in spite of the fact that he was arrested for being the president of a student’s society that had a nationalist nature, yet he didn’t hesitate afterwards being one of the leaders of an international German society. Unfortunately, the above mentioned journal is absent from the public space and the history of its recovery and editing is in itself a history of a failure. That is why, this study is an analysis of the different existing editions of it – that could be described as fragmented, or improvised. In what regards the title of the article, it hints at the imprecisions in transcribing the original text, the difficulties posed by the German existing versions and also at the deficiency of the Romanian translations. Yet, besides all of these, the journal of Ciprian Porumbescu is an exceptional biographical document. Thus, the critique of the existing editions could represent a fist attempt at a textual and analytical recovery of the actual journal.

Voltammetric Determination of Phenylalanine Using Chemically Modified Screen-Printed Based Sensors

This paper describes the sensitive properties of screen-printed carbon electrodes (SPCE) modified by using three different electroactive chemical compounds: Meldola’s Blue, Cobalt Phthalocyanine and Prussian Blue, respectively. It was demonstrated that the Prussian Blue (PB) modified SPCE presented electrochemical signals with the highest performances in terms of electrochemical process kinetics and sensitivity in all the solutions analyzed. PB-SPCE was demonstrated to detect Phe through the influence it exerts on the redox processes of PB. The PB-SPCE calibration have shown a linearity range of 0.33–14.5 µM, a detection limit (LOD) of 1.23 × 10−8 M and the standard deviation relative to 3%. The PB-SPCE sensor was used to determine Phe by means of calibration and standard addition techniques on pure samples, on simple pharmaceutical samples or on multicomponent pharmaceutical samples. Direct determination of the concentration of 4 × 10−6–5 × 10−5 M Phe in KCl solution showed that the analytical recovery falls in the range of 99.75–100.28%, and relative standard deviations in the range of 2.28–3.02%. The sensors were successfully applied to determine the Phe in pharmaceuticals. The validation of the method was performed by using the FTIR, and by comparing the results obtained by PB-SPCE in the analysis of three pharmaceutical products of different concentrations with those indicated by the producer.

IDENTIFICATION AND QUANTIFICATION OF VERAPAMIL IN BLOOD AND URINE

Purpose: To adapt and validate an HPLC method for verapamil determination in blood and urine samples. Materials/Methods: Identification of verapamil and its metabolites was made by means of gas-chromatography, using Agilent 7890B/5977A GC-MS system featuring a DB-1701 column. Quantification was done by means of liquid chromatography on Agilent 1260 series HPLC, equipped with Zorbax Extend-C18 column and both diode-array and fluorescent detection modules. Blood and urine specimens were taken from patients of the Clinic for intensive treatment of acute intoxications and toxicoallergies within the course of their treatment. Results: GC-MS identification of verapamil and its metabolites was carried out after simple liquid-liquid extraction of samples without further chemical derivatization. Adapted HPLC method for quantification require isocratic conditions and mobile phase, consisted of phosphate buffer (pH 2.7; 10 mM) containing 1.5 ml L–1 triethylamine – acetonitrile (70:30, v/v) at 20oC, flow-rate 1.0 mL/min and FLD detection (excitation: 203 nm, emission: 320 nm). The method was demonstrated to be linear within the whole region of interest (4.6-4600 ng mL–1) with excellent accuracy (101.7-102.2%) and inter-day precision (5.81%) as well as good analytical recovery (81.2%) and LOQ (7.0 ng mL–1). Conclusion: A precise and easy to use method for verapamil detection and quantification is developed. The method is applicable as a routine procedure in the Laboratory of analytical toxicology for both diagnosis clarification in cases of acute intoxications and therapeutic drug monitoring.

The Synergy of Thermally Reduced Graphene Oxide in Amperometric Urea Biosensor: Application for Medical Technologies

Thermally reduced graphene oxide (TRGO) is a graphene-based nanomaterial that has been identified as promising for the development of amperometric biosensors. Urease, in combination with TRGO, allowed us to create a mediator-free amperometric biosensor with the intention of precise detection of urea in clinical trials. Beyond simplicity of the technology, the biosensor exhibited high sensitivity (2.3 ± 0.1 µA cm−2 mM−1), great operational and storage stabilities (up to seven months), and appropriate reproducibility (relative standard deviation (RSD) about 2%). The analytical recovery of the TRGO-based biosensor in urine of 101 ÷ 104% with RSD of 1.2 ÷ 1.7% and in blood of 92.7 ÷ 96.4%, RSD of 1.0 ÷ 2.5%, confirmed that the biosensor is acceptable and reliable. These properties allowed us to apply the biosensor in the monitoring of urea levels in samples of urine, blood, and spent dialysate collected during hemodialysis. Accuracy of the biosensor was validated by good correlation (R = 0.9898 and R = 0.9982) for dialysate and blood, utilizing approved methods. The advantages of the proposed biosensing technology could benefit the development of point-of-care and non-invasive medical instruments.

Characterizing the Distribution of Ppm Gluten in Gluten Free Oatmeal Servings Contaminated with a Barley Kernel

Oats are regularly contaminated with gluten-containing grains like wheat, barley and rye. For producers of gluten free oatmeal, contamination potential makes it prudent to understand the consequences in terms of gluten dosing, as labeling requirements specify a gluten maximum. To do this, statistical simulation has been used to produce virtual oat servings (40g) contaminated with either two row or six row spring barley. The results are probability distributions for ‘actual’ ppm gluten (free of measurement influences) and ‘as measured’ via R5 ELISA. Findings show ‘actual’ ppm gluten to be normally distributed with 57 ppm and 41 ppm gluten averages with 14 and 12 ppm standard deviations (stdevs) for two and six row barley, respectively. ‘As measured’ ppm gluten results are lognormally distributed with 61 and 44 ppm gluten averages with 63 and 47 stdevs for two and six row barley, respectively, employing an 80% analytical recovery rate and a multiplier of 1. These analyses show that ‘as measured’ results possess false negative probabilities (relative to a < 20 ppm gluten regulatory requirement) from 0.14 to 0.34 depending on recovery rate and barley type. This work highlights the need for non-homogenous grinding issues to be addressed in whole grain gluten assessment, for analytical recovery rates to be defined for gliadin in oats, and for appropriate conversion factors to be determined in order to attain capable measurement of gluten in oats due to barley kernel contamination.

Competitive pseudo-ELISA based on molecularly imprinted nanoparticles for microcystin-LR detection in water

Microcystins (MCs) are dangerous cyanotoxins for the public health, and microcystin-LR (MC-LR) is one of most toxic, dangerous, and frequently found in water bodies. Typically, the detection of MCs is carried out by means of competitive ELISAs which, however, need special precautions for handling and storage, due to the stability of the antibodies used in this test. Molecularly imprinted nanoparticles (nanoMIPs) represents more robust and cost-effective alternative to antibodies. In this work, we developed a competitive pseudo-ELISA based on nanoMIPs (which are used in place of natural antibodies), for the detection of microcystin-LR (MC-LR). This pseudo-ELISA showed a linear response towards MC-LR, showing high affinity and low cross-reactivity against another analogue toxin (microcystin-YR). The analytical recovery of MC-LR in the analysis of water samples by the proposed pseudo-ELISA was 96 %–130 % and the limit of detection was 2.64 × 10−4 nM. The obtained results suggest that this competitive pseudo-ELISA could have high potential in the detection of toxins, due to its rapid, sensitive and accurate detection of toxin in water samples.

Development of a Novel Biosensor Based on Tyrosinase/Platinum Nanoparticles/Chitosan/Graphene Nanostructured Layer with Applicability in Bioanalysis

The present paper describes the preparation and characterization of a graphene, chitosan, platinum nanoparticles and tyrosinase-based bionanocomposite film deposited on the surface of a screen-printed carbon electrode for the detection of L-tyrosine by voltammetry. The redox process on the biosensor surface is associated with the enzymatic oxidation of L-tyrosine, which is favoured by graphene and platinum nanoparticles that increase electrical conductivity and the electron transfer rate. Chitosan ensures the biocompatibility between the tyrosinase enzyme and the solid matrix, as well as a series of complex interactions for an efficient immobilization of the biocatalyst. Experimental conditions were optimized so that the analytical performances of the biosensor were maximal for L-tyrosine detection. By using square wave voltammetry as the detection method, a very low detection limit (4.75 × 10−8 M), a vast linearity domain (0.1–100 μM) and a high affinity of the enzyme for the substrate (KMapp is 53.4 μM) were obtained. The repeatability of the voltammetric response, the stability, and the reduced interference of the chemical species present in the sample prove that this biosensor is an excellent tool to be used in bioanalysis. L-tyrosine detection in medical and pharmaceutical samples was performed with very good results, the analytical recovery values obtained being between 99.5% and 101%. The analytical method based on biosensor was validated by the standard method of analysis, the differences observed being statistically insignificant at the 99% confidence level.

Cannabinoids determination in bronchoalveolar lavages of cannabis smokers with lung disease

Background Cannabis smoke affects the lungs similarly to tobacco smoke, causing symptoms such as increased cough, sputum, hyperinflation and chronic bronchitis. Chronic use can also cause serious lung diseases and airway obstruction. We developed and validated a method for the identification and quantification of cannabinol (CBN), cannabidiol (CBD), Δ-9-tetrahydrocannabinol (THC) and its metabolites 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THC-COOH) in bronchoalveolar lavages (BALs) from hospitalized former or current tobacco smoking patients with lung disease and a long history of cannabis consumption and limited current tobacco use. Methods For the extraction of cannabinoids from BALs, a 1 mL sample was added with 300 µL of 0.1 N NaOH and 3 mL of hexane/ethyl acetate (9:1). The solvent was then evaporated to dryness. Trimethylsilyl derivatives were prepared and then analyzed by gas chromatography/mass spectrometry. Results The method was linear for the analytes under investigation with coefficients of determination of at least 0.99. Absolute analytical recovery was always better than 80%, imprecision and inaccuracy was always under 15%. Six cases out of 15 were positive for THC, CBN and CBD. In two BALs samples, the presence of 11-OH-THC was also measured while THC-COOH was not detected. In the six positive cases, the last cannabis smoking occurred in the previous 2–14 days. Conclusions This is the first time that cannabinoids have been detected in BALs, demonstrating the presence of a drug with its metabolites in a target organ of consumers who present with a lung disease. This occurrence let us hypothesize a role of cannabinoids in the development of the disease and prompted an investigation on possible associations between cannabis smoking and clinical outcomes in patients with lung disease and eventually evaluate a cytotoxic effect of cannabinoids themselves.