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

A validated ultrahigh-performance liquid chromatography method using 1.7 µm core-shell particles is presented for the identification and quantification of ß-carotene (BC) and related cleavage products (CPs) in primary cell culture media. Besides BC, apo-4'-, apo-8'-, apo-10'-, and apo-12'-carotenals, as well as 5,6-epoxy-ß-carotene, were selected as target analytes. Detection was performed via an 80-Hz diode array detector and an electrospray ionization-linear quadrupole ion trap-Orbitrap XL mass spectrometer, both hyphenated in series. Total analysis time was below 6 min with peak widths <12 s. Addition of trifluoroacetic acid and tetrahydrofuran to the mobile phase allowed for the mass spectrometric detection of BC and related CPs and reduced peak tailing due to improved solubility of hydrophobic analytes. Intra-day and inter-day precision for UV and mass spectrometric detection were ≤1.5 % for retention times and ≤5.1 % for peak areas. Instrumental linearity was confirmed by Mandel's fitting test between 0.25 (or 1.00 µg/mL) and 5.00 µg/mL for UV detection. The higher sensitivity of mass spectrometric detection allowed for the coverage of three concentration domains between 0.025 and 5.00 µg/mL in linearity testing. Homoscedasticity was confirmed between 0.10 and 5.00 µg/mL for Orbitrap XL MS. The limits of quantification were between 52.6 and 889.4 ng/mL for UV detection and between 19.3 and 102.4 ng/L for mass spectrometric detection. Offline solid-phase extraction from culture media fortified with BC and CPs provided intra- and inter-day recoveries between 65.8 and 102.4 % with coefficients of variation ≤6.2 %. Primary rat hepatocyte cultures treated with BC and subjected to different oxidative stress conditions contained 5,6-epoxy-BC and apo-4'-carotenal besides residual BC. Apparently, 5,6-epoxy-BC was formed in the medium via autoxidation of BC by ambient oxygen.

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

A validated method for the simultaneous determination of prominent volatile cleavage products (CPs) of ß-carotene in cell culture media has been developed. Target CPs comprised ß-ionone (ß-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 ß-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.