Solvent-Free Microwave Extraction of Thymus mastichina Essential Oil: Influence on Their Chemical Composition and on the Antioxidant and Antimicrobial Activities.

Solvent-free microwave extraction (SFME) is a combination of microwave heating and dry distillation performed at atmospheric pressure without the addition of water or organic solvents that has been proposed as a green method for the extraction of essential oils from aromatic and medicinal herbs. In this work, SFME and the conventional techniques of steam distillation (SD) and hydrodistillation (HD) were compared with respect to the extraction and antioxidant and antimicrobial activities of Thymus mastichina essential oil. The main constituent of essential oils obtained using different methods was 1,8-cineole (eucalyptol). The results showed that the essential oils extracted by means of SFME in 30 min were quantitatively (yield) and qualitatively (aromatic profile) similar to those obtained using conventional HD over 120 min. In addition, SFME generates less waste and less solvent, consumes less energy, and provides a higher yield for a shorter extraction time, which is advantageous for the extraction of the T. mastichina essential oil compared to SD. The antioxidant and antimicrobial activities of the T. mastichina essential oil obtained from either SFME or conventional extraction methods (SD or HD) showed a similar pattern. Large-scale experiments using this SFME procedure showed a potential industrial application.

Thymus mastichina: Composition and Biological Properties with a Focus on Antimicrobial Activity.

Thymus mastichina has the appearance of a semishrub and can be found in jungles and rocky lands of the Iberian Peninsula. This work aimed to review and gather available scientific information on the composition and biological properties of T. mastichina. The main constituents of T. mastichina essential oil are 1,8-cineole (or eucalyptol) and linalool, while the extracts are characterized by the presence of flavonoids, phenolic acids, and terpenes. The essential oil and extracts of T. mastichina have demonstrated a wide diversity of biological activities. They showed antibacterial activity against several bacteria such as Escherichia coli, Proteus mirabilis, Salmonella subsp., methicillin-resistant and methicillin-sensitive Staphylococcus aureus, Listeria monocytogenes EGD, Bacillus cereus, and Pseudomonas, among others, and antifungal activity against Candida spp. and Fusarium spp. Additionally, it has antioxidant activity, which has been evaluated through different methods. Furthermore, other activities have also been studied, such as anticancer, antiviral, insecticidal, repellent, anti-Alzheimer, and anti-inflammatory activity. In conclusion, considering the biological activities reported for the essential oil and extracts of T. mastichina, its potential as a preservative agent could be explored to be used in the food, cosmetic, or pharmaceutical industries.

Determination of opiates in whole blood using microextraction by packed sorbent and gas chromatography-tandem mass spectrometry.

In Portugal, and worldwide, the abuse of psychoactive substances is increasing, with a significant incidence of deaths related to their consumption. Opiates are one of the most prevalent group of substances in that context, and they are responsible for a significant impact of the mentioned harms. Therefore, it becomes necessary to equip labs with faster and effective methods to identify and quantify these substances. This work describes the development and validation of a novel analytical method for the simultaneous determination of morphine, codeine and 6-monoacetylmorphine in blood samples by gas chromatography-tandem mass spectrometry (GC-MS/MS), using microextraction by packed sorbent (MEPS) for sample preparation. Before the MEPS procedure, a precipitation step with acetonitrile was performed. The MEPS parameters were optimized using the fractional factorial planning (2k-1) and surface response methodology. The final optimized conditions were: number of strokes (20), amount of formic acid in the washing solution (3.36%), number of washes of the sorbent (1), amount of ammonium hydroxide in the elution solution (2.36%) and number of elution cycles (11). After the extraction procedure, the analytes were derivatized with MSTFA with 5% TMS. Using a sample volume of 250 µL, the method was validated according to internationally accepted standards. The method proved to be linear in the range of 5-1000 ng/mL with coefficients of determination greater than 0.99 for all analytes. Intra-and inter-day precision and accuracy were in accordance with the above-mentioned criteria, presenting coefficients of variation ≤15% and relative errors within a range of ± 15% of the theoretical concentration. The absolute recoveries ranged from 6 to 23%. The validated method was applied to the analysis of real samples, being an advantageous tool for the detection of those substances in blood. This is the first time that GCMS/MS with MEPS was used for the determination of these compounds in biological fluids.

Determination of methadone and EDDP in oral fluid using the dried saliva spots sampling approach and gas chromatography-tandem mass spectrometry.

The present work describes the development and validation of a novel approach to determine methadone (MTD) and its main metabolite (EDDP) in oral fluid samples, using the dried saliva spots (DSS) sampling approach and gas chromatography-tandem mass spectrometry (GC-MS/MS). Oral fluid samples (50 µL) were applied into Whatman™ 903 protein saver filter paper cards and were allowed to dry overnight. The extraction was carried out by immersion of the spot in 1 mL of isopropyl alcohol with agitation for 1 min. Afterwards, the extract was centrifuged for 15 min at 3500 rpm and the supernatant evaporated to dryness and reconstituted with 50 µL of methanol. The procedure was considered linear in the range of 10 to 250 ng/mL for both compounds, with determination coefficients greater than 0.99. Intra- and inter-day precision and accuracy revealed coefficients of variation (CVs) lower than 15% at the studied concentrations, with mean relative errors within ± 15% of the nominal concentrations. Recoveries ranged from 45 to 74%. The limits of detection and quantification were 5 and 10 ng/mL respectively for both analytes. All studied parameters complied with the defined criteria and the method enabled the successful determination of MTD and EDDP in oral fluid samples from patients undergoing opiate substitution/maintenance therapy.

Flow system for the automatic screening of the effect of phenolic compounds on the luminol-hydrogen peroxide-peroxidase chemiluminescence system.

In this work, an automated flow-based procedure for the screening of the effect of the different phenolic compounds on the chemiluminescence (CL) luminol-hydrogen peroxide-horseradish peroxidase (HRP) system is presented. This procedure involves the combination of multisyringe flow injection analysis (MFSIA) and sequential injection analysis (SIA) techniques and exploits the ability of the different subgroups of phenols, such as cholorophenols, nitrophenols, methylphenols and polyphenols, to enhance or inhibit the described CL system. The implementation of this reaction in the SIA-MSFIA system enabled favourable and precise conditions to evaluate the effect of phenolic compounds, as it involves an in-line reaction between the phenolic derivative, hydrogen peroxide and peroxidase and subsequent oxidized HRP intermediates generation prior to the fast reaction with the chemiluminogenic reagent. Several studies were then performed with the aim of establishing the appropriate flow system configuration and reaction conditions. It was shown that phenol and chlorophenols produce an enhanced CL response and nitrophenols, methylphenols and polyphenols are inhibitors within the range of concentrations studied (1-100 mg/L). Based on these studies, the developed method was applied to the determination of total polyphenol and phenol content in wine/grape seeds and water samples, respectively, and the results obtained showed good agreement with those furnished by the corresponding Folin-Ciocalteu and 4-aminoantipyrine reference methods. The developed approach is further pursued by designing an automated generic tool for performing studies of peroxidase-catalysed CL reactions of luminol focused on the detection of compounds that will affect the rate of those reactions.

Determination of total and oxidized glutathione in human whole blood with a sequential injection analysis system.

This work reports the development of a simple, robust, automated sequential injection analysis (SIA) system for the enzymatic determination of total (tGSH) and oxidized (GSSG) glutathione in human whole blood. The reduced (GSH) glutathione concentration is then obtained as the difference between the tGSH and GSSG concentrations. The determination was based on the DTNB-GSSG reductase recycling assay, which couples the specificity of the GSSG reductase (GR) with an amplification of the response to glutathione, followed by spectrophotometric detection of the 2-nitro-5-thiobenzoic acid (TNB) formed (lambda=412 nm). The implementation of this reaction in a SIA flow system with an in-line dilution strategy permitted the necessary distinct application ranges for tGSH and for GSSG. It also guaranteed the exact timing of fluidic manipulations and precise control of the reaction conditions. The influence of parameters such as reagents concentration, temperature, pH, flow rate of the carrier buffer solution, as well as reaction coil length, etc., on the sensitivity and performance of the SIA system were studied and the optimum reaction conditions subsequently selected. Linear calibration plots were obtained for GSH and GSSG concentrations up to 3.00 and 1.50 microM, with detection limits of 0.031 and 0.014 microM, respectively. The developed methodology showed good precision, with a relative standard deviation (R.S.D.)<5.0% (n=10) for determination of both glutathione forms. Statistical evaluation showed good compliance, for a 95% confidence level, between the results obtained with the SIA system and those furnished by the comparison batch procedure.