Online solid-phase extraction-liquid chromatography-mass spectrometry to determine free sterols in human serum.

An automated method for analyzing free non-cholesterol sterols in human serum using online solid phase extraction-liquid chromatography-mass spectrometry is proposed herein. The method allows the determination of three phytosterols (sitosterol, stigmasterol and campesterol) and two cholesterol precursors (desmosterol and lanosterol). The analysis of sterols in human serum is critical in the study of cholesterol-related disorders, such as inherited familial hypercholesterolemias. Special effort was made to isolate the analytes from the serum lipoproteins, their natural conveyance through the bloodstream. The sample treatment consisted of a Bligh-Dyer extraction followed by dilution of the extract. This treatment allowed the sample to be injected into the online system and ensured the correct detection of the analytes, while avoiding the matrix effects commonly related to serum samples. The analytical performance showed linear ranges that covered two orders of magnitude, with correlation coefficients above 0.99. Limits of detection and quantification ranged from 0.2 ng/mL to 13 ng/mL and from 1.0 ng/mL to 43 ng/mL, respectively. Recovery when spiking serum with a half or a tenth of the average concentration reported in human serum ranged from 99% to 111% and from 102% to 120%, respectively. Intra-day precision and inter-day precision were below 20%.

The additive properties of Oxygen Radical Absorbance Capacity (ORAC) assay: the case of essential oils.

The ORAC assay is applied to measure the antioxidant capacity of foods or dietary supplements. Sometimes, the manufacturers claim antioxidant capacities that may not correspond to the constituents of the product. These statements are sheltered by the general understanding that antioxidants might exhibit synergistic properties, but this is not necessarily true when dealing with ORAC assay values. This contribution applies the ORAC assay to measure the antioxidant capacity of ten essential oils typically added to foodstuffs: citronella, dill, basil, red thyme, thyme, rosemary, oregano, clove and cinnamon. The major components of these essential oils were twenty-one chemicals in total. After a preliminary discrimination, the antioxidant capacity of eugenol, carvacrol, thymol, α-pinene, limonene and linalool was determined. The results showed that 72-115% of the antioxidant capacity of the essential oils corresponded to the addition of the antioxidant capacity of their constituents. Thus, the ORAC assay showed additive properties.

Exposure to phthalic acid, phthalate diesters and phthalate monoesters from foodstuffs: UK total diet study results.

Phthalates are ubiquitous in the environment and thus exposure to these compounds can occur in various forms. Foods are one source of such exposure. There are only a limited number of studies that describe the levels of phthalates (diesters, monoesters and phthalic acid) in foods and assess the exposure from this source. In this study the levels of selected phthalate diesters, phthalate monoesters and phthalic acid in total diet study (TDS) samples are determined and the resulting exposure estimated. The methodology for the determination of phthalic acid and nine phthalate monoesters (mono-isopropyl phthalate, mono-n-butyl phthalate, mono-isobutyl phthalate, mono-benzyl phthalate, mono-cyclohexyl phthalate, mono-n-pentyl phthalate, mono-(2-ethylhexyl) phthalate, mono-n-octyl phthalate and mono-isononyl phthalate) in foods is described. In this method phthalate monoesters and phthalic acid are extracted from the foodstuffs with a mixture of acidified acetonitrile and dichloromethane. The method uses isotope-labelled phthalic acid and phthalate monoester internal standards and is appropriate for quantitative determination in the concentration range of 5-100 µg kg⁻¹. The method was validated in-house and its broad applicability demonstrated by the analysis of high-fat, high-carbohydrate and high-protein foodstuffs as well as combinations of all three major food constituents. The methodology used for 15 major phthalate diesters has been reported elsewhere. Phthalic acid was the most prevalent phthalate, being detected in 17 food groups. The highest concentration measured was di-(2-ethylhexyl) phthalate in fish (789 µg kg⁻¹). Low levels of mono-n-butyl phthalate and mono-(2-ethylhexyl) phthalate were detected in several of the TDS animal-based food groups and the highest concentrations measured corresponded with the most abundant diesters (di-n-butyl phthalate and di-(2-ethylhexyl) phthalate). The UK Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment (COT) considered the levels found and concluded that they did not indicate a risk to human health from dietary exposure alone.

Pattern recognition of peach cultivars (Prunus persica L.) from their volatile components.

The volatile compounds of four peach cultivars (Prunus persica L.) were studied: Sudanell, San Lorenzo, Miraflores and Calanda (two clones, Calante and Jesca). 17-23 Samples of each cultivar with the same maturity level were analyzed, measuring color, firmness, and soluble solids content. The pulp was crushed and mixed with water prior to HS-SPME analysis, and GC-MS was used to determine the volatile compounds. Sixty-five compounds were identified using spectral library matching, Kovat's indices and, when available, pure standards. The main components were lactones and C6 compounds. From the distribution of these compounds, Principal Component Analysis led to the clustering of the samples according to their different cultivars. Finally, Canonical Component Analysis was used to create a classification function that identifies the origin of an unknown sample from its volatile composition. The results obtained will help to avoid fraud and protect the European Designation of Origin 'Melocotón de Calanda'.

Ambient ionization-accurate mass spectrometry (AMI-AMS) for the identification of nonvisible set-off in food-contact materials.

Set-off is the unintentional transfer of substances used in printing from the external printed surface of food packaging to the inner, food-contact surface. Ambient ionization-accurate mass spectrometry (AMI-AMS) detected and identified compounds from print set-off not visible to the human eye. AMI mass spectra from inner and outer surfaces of printed and nonprinted food packaging were compared to detect and identify nonvisible set-off components. A protocol to identify unknowns was developed using a custom open-source database of printing inks and food-packaging compounds. The protocol matched print-related food-contact surface ions with the molecular formulas of common ions, isotopes, and fragments of compounds from the database. AMI-AMS was able to detect print set-off and identify seven different compounds. Set-off on the packaging samples was confirmed using gas chromatographic-mass spectrometric (GC-MS) analysis of single-sided solvent extracts. N-Ethyl-2(and 4)-methylbenzenesulfonamide, 2,4-diphenyl-4-methyl-1(and 2)-pentene, and 2,4,7,9-tetramethyl-5-decyne-4,7-diol were present on the food-contact layer at concentrations from 0.21 to 2.7 ± 1.6 µg dm⁻², corresponding to nearly milligram per kilogram concentrations in the packaged food. Other minor set-off compounds were detected only by AMI-AMS, a fast, simple, and thorough technique to detect and identify set-off in food packaging.

Study of the antioxidant mechanisms of Trolox and eugenol with 2,2'-azobis(2-amidinepropane)dihydrochloride using ultra-high performance liquid chromatography coupled with tandem mass spectrometry.

The study of antioxidant mechanisms is a difficult task that involves the monitoring and identification of unknown intermediate and final products. Most of the time, the lifetime of intermediates is too short to allow their isolation and subsequent identification by nuclear magnetic resonance (NMR). The developments of ultra-high performance liquid chromatography (UHPLC) coupled with the advances in the acquisition rates of mass spectrometry could facilitate the research on antioxidant mechanisms. This work is based on the reaction involved in the Oxygen Radical Antioxidant Capacity (ORAC) and Total Radical trapping Antioxidant Parameter (TRAP) assays. Hence, the reaction between 2,2'-azobis(2-amidinepropane)dihydrochloride (AAPH) radicals and an antioxidant was carried out in the thermostatized autosampler of a chromatographic device. Then, the reaction media were injected every six minutes, and the compounds were separated by UHPLC and detected by mass spectrometry in scan mode. Nine consecutive injections were registered in a unique file, then the evolution of the reaction for one hour in a single run was monitored. In this way, the reaction mechanisms of Trolox and eugenol with AAPH were studied, leading to the detection of nine and thirteen different compounds, respectively. An exhaustive analysis of the spectra obtained in product ion scan mode led to the identification of the compounds. Most of them were species previously found in the literature, but others have never been reported, so tentative structures were suggested. All this allowed the proposal of several steps within the antioxidant mechanisms of Trolox and eugenol, showing the great performance of UHPLC-MS/MS to complement the use of NMR in antioxidant mechanistic studies.