Solid-phase extraction and GC-MS analysis of potentially genotoxic cleavage products of β-carotene in primary cell cultures

Abstract

A validated method for the simultaneous determination of prominent volatile cleavage products (CPs) of β-carotene in cell culture media has been developed. Target CPs comprised β-ionone (β-IO), cyclocitral (CC), dihydroactinidiolide (DHA), and 1,1,6-trimethyltetraline (TMT). CPs were extracted by solid-phase extraction applying a phenyl adsorbent, eluted with 10% (v/v) tetrahydrofuran in n-hexane, and identified and quantified by gas chromatography-mass spectrometry with electron impact ionization. Method validation addressed linearity confirmation over two application ranges and homoscedasticity testing. Recoveries from culture media were between 71.7% and 95.7% at 1.0 μg/ml. Precision of recoveries determined in intra-day (N = 5) and inter-day (N = 15) assays were <2.0% and <4.8%, respectively. Limit of detection and limit of quantification of the analysis method were <18.0 and <53.0 ng/ml for β-IO, CC, and TMT, whereas 156 and 474 ng/ml were determined for DHA, respectively. Although extractions of blank matrix proved the absence of interfering peaks, statistical comparison between slopes determined for instrumental and total method linearity revealed significant differences. The method was successfully applied in selecting an appropriate solvent for the fortification of culture media with volatile CPs, including the determination of their availability over the incubation period. For the first time, quantification of volatile CPs in treatment solutions and culture media for primary cells becomes accessible by this validated method.FigureCultured primary rat hepatocytes in phase contrast after nuclea staining with DAPI including a chromatogram (GC-MS) of volatile cleavage products of b-carotene, which are presumed to exert genotoxic effects on hepatocytes and pneumocytes

Validation and application of sub-2 μm core–shell UHPLC–UV–ESI–Orbitrap MS for identification and quantification of β-carotene and selected cleavage products with preceding solid-phase extraction

“…Methylisoeugenol (purity >98 %) was obtained from SAFC (St. Louis, MO, USA) and used as internal standard (IS) as outlined previously [47]. β-Apo-4′-carotenal (apo-4′), β-apo-10′-carotenal (apo-10′), β-apo-12′-carotenal (apo-12′), and 5,6-epoxy-carotenal (5,6-epoxy-BC) were received as lyophilized powder from CaroteneNature GmbH (Lupsingen, Switzerland) at 92–99 % purity and stored light protected at −20 °C.…”

Quantitative analysis of trace levels of β-ionone in water by liquid-liquid-phase extraction-gas chromatography-mass spectrometry (LLE-GC-MS)

“…Peak areas are corrected by IS for determination of linearity and precision of the data. Linearity refers to the ability of a method that measures the goodness of corrected peak areas and related concentrations within a given range [17]. The linear regression coefficient R 2 is 0.9997, with a standard deviation (D s ) of 0.0285 a relative standard deviation (D rs ) of 4.74%.…”

An MC3T3-E1 Cell Line Biomembrane Extraction and HPLC–ESI-MS n Method for Simultaneous Analysis of Potential Anti-Osteoporosis Components of Epimedium koreanum

“…Therefore, methods using biomembrane-affinity chromatography and cell membrane chromatography (CMC) have been designed for rapidly predicting bioactive candidates in TCMs [4][5][6]. Furthermore, application of live cell extraction to screen and analyze bioactive components by hyphenated techniques in liquid chromatography (LC)-MS/gas chromatography (GC)-MS as a feasible, rapid method has attracted much attention worldwide [7][8][9][10].…”