Stanovení biologicky aktivních látek, zejména karotenoidů, pomocí technik hmotnostní spektrometrie a určení vybraných vlastností luteinu

Ing. Mgr. Barbora HRVOLOVÁ

Doctoral thesis

The analysis of bioactive compounds, especially carotenoids, by mass spectrometry techniques and determination of selected properties of lutein

Abstract:

The bioactive compounds are substances that have an effect on living organisms. In general, the term bioactive compound is usually linked only with positive effects on an organism. The typical representatives of bioactive compounds include phytochemicals, vitamins and minerals. Most of these compounds are associated with beneficial effects to human health due to their antioxidant properties. Diet naturally rich in these bioactive compounds or enriched by food supplements can positively affect human health. In connection with this fact, the need of determination of selected bioactive compounds in different matrices significantly increased. This doctoral thesis deals with simultaneous determination of selected bioactive compounds, specifically carotenoids and fat-soluble vitamins, by mass spectrometry techniques and with determination of selected properties of lutein. Obtained results are summarized in following points: ? A unique HPLC-MS/MS method for the simultaneous quantification of 16 carotenoids and fat-soluble vitamins in human plasma was designed and fully validated according to AOAC criteria. Good quality values of LOD, LOQ, recovery, linearity, matrix effect, accuracy, and precision were obtained by the proposed method. The proposed method was applied to human plasma samples obtained within human dietary intervention study. The method allowed determination of selected analytes in the range of nM. ? The HPLC-MS and FIA method using ESI ionization for determination of lutein was designed. Repeatable, fast and very economical FIA method was used for determination of thermal and photostability of two standards of lutein produced by different companies (ChromaDex, USA and Extrasynthese, France). ? After 336 h of exposure to -20 °C, 6 °C, and 20 °C was approximate decrease in quantity of lutein about 50 % in both cases. In the case of 36 °C and 40 °C not so significant changes were obtained during the time of exposure. ? No statistically significant difference, with exception of exposure to 36 °C and 40 °C, in behaviour under specific thermal conditions between lutein supplied by ChromaDex and by Extrasynthese was obtained. ? In the case of stability to white light at 20 °C the decrease by 16 % in quantity of lutein supplied by ChromaDex was obtained and the quantity of lutein achieved by Extrasynthese decreased by 10.7 %. Statistically significant difference between ChromaDex and Extrasynthese lutein was not obtained. ? Statistically significant differences were obtained in the cases of dependency of quantity of lutein on the time of exposure under aforementioned thermal and light conditions. ? The total antioxidant activity of lutein was determined by ABTS assay. The average total antioxidant activity of lutein supplied by ChromaDex expressed in TROLOX equivalents was 10.98 mg/L. The average total antioxidant activity of lutein supplied by Extrasynthese expressed in TROLOX equivalents was 9.96 mg/L.

Abstract:

The bioactive compounds are substances that have an effect on living organisms. In general, the term bioactive compound is usually linked only with positive effects on an organism. The typical representatives of bioactive compounds include phytochemicals, vitamins and minerals. Most of these compounds are associated with beneficial effects to human health due to their antioxidant properties. Diet naturally rich in these bioactive compounds or enriched by food supplements can positively affect human health. In connection with this fact, the need of determination of selected bioactive compounds in different matrices significantly increased. This doctoral thesis deals with simultaneous determination of selected bioactive compounds, specifically carotenoids and fat-soluble vitamins, by mass spectrometry techniques and with determination of selected properties of lutein. Obtained results are summarized in following points: ? A unique HPLC-MS/MS method for the simultaneous quantification of 16 carotenoids and fat-soluble vitamins in human plasma was designed and fully validated according to AOAC criteria. Good quality values of LOD, LOQ, recovery, linearity, matrix effect, accuracy, and precision were obtained by the proposed method. The proposed method was applied to human plasma samples obtained within human dietary intervention study. The method allowed determination of selected analytes in the range of nM. ? The HPLC-MS and FIA method using ESI ionization for determination of lutein was designed. Repeatable, fast and very economical FIA method was used for determination of thermal and photostability of two standards of lutein produced by different companies (ChromaDex, USA and Extrasynthese, France). ? After 336 h of exposure to -20 °C, 6 °C, and 20 °C was approximate decrease in quantity of lutein about 50 % in both cases. In the case of 36 °C and 40 °C not so significant changes were obtained during the time of exposure. ? No statistically significant difference, with exception of exposure to 36 °C and 40 °C, in behaviour under specific thermal conditions between lutein supplied by ChromaDex and by Extrasynthese was obtained. ? In the case of stability to white light at 20 °C the decrease by 16 % in quantity of lutein supplied by ChromaDex was obtained and the quantity of lutein achieved by Extrasynthese decreased by 10.7 %. Statistically significant difference between ChromaDex and Extrasynthese lutein was not obtained. ? Statistically significant differences were obtained in the cases of dependency of quantity of lutein on the time of exposure under aforementioned thermal and light conditions. ? The total antioxidant activity of lutein was determined by ABTS assay. The average total antioxidant activity of lutein supplied by ChromaDex expressed in TROLOX equivalents was 10.98 mg/L. The average total antioxidant activity of lutein supplied by Extrasynthese expressed in TROLOX equivalents was 9.96 mg/L.

Keywords

karotenoidy, v tucích rozpustné vitamíny, lutein, HPLC-MS/MS, HPLC-MS, FIA, validace, stabilita, antioxidační aktivita, ABTS metoda

Language used: English

Date on which the thesis was submitted / produced: 23. 7. 2018

Thesis defence

• Date of defence: 4. 9. 2018
• Supervisor: doc. RNDr. Jiří Kalina, Ph.D.

University of Ostrava

Faculty of Science

Doctoral programme / field:
Analytical Chemistry / Analytická chemie heterogenních systémů