Browsing by Subject "UHPLC"

The literature review focused on liquid chromatographic-mass spectrometric (LC-MS) methods used to quantify B12 vitamers in food matrices. Various MS methods have been used for the detection of B12, offering more specificity than other commonly used analysis techniques. This thesis aimed to develop a method for quantifying the native forms of B12 in different food matrices and avoiding the commonly used conversion to cyanocobalamin during extraction. In the experimental study, an ultra-high-performance LC-tandem MS (UHPLC-MS/MS) method was developed and validated for selectivity, specificity, recovery, repeatability, reproducibility, trueness, and measurement uncertainty to determine B12 vitamers in fermented plant-based foods and microbial cell supernatants. The development was initiated by setting up mass spectrometer conditions and selecting transitions for multiple reaction monitoring (MRM) to achieve selective and sensitive detection method for individual B12 vitamers. This was followed by developing the UHPLC method utilizing a reversed-phased C18 column and gradient elution with 0.5% formic acid and 0.5% FA in methanol. The vitamers were ionized using electrospray ionization in a positive ion mode and detected in an MRM mode using hydroxocobalamin, cyanocobalamin, adenosylcobalamin, and methylcobalamin. All B12 vitamers were detected and separated with the developed and optimized UHPLC-MS/MS method. The internal standard calibration method was necessary to overcome matrix effects when analyzing food samples. The calibration curve content range was 0.2–200 pg/µL, and the results showed good linearity. The instrumental method was selective, precise, repeatable, and reproducible with detection and quantitation limits of 0.03–0.4 pg/µL and 0.2–2 pg/µL, respectively. The measurement uncertainty of the instrumental method varied between 10% and 20%. For the entire method, recoveries for the B12 vitamers ranged from 40% to 200%, and measurement uncertainties from 40% to 60%. Results for the total B12 content in food samples deviated from those determined using a conventional UHPLC-PDA method: Recovery for tempeh was over 90%, but for fortified bread only 20%. These results indicate the need for further development of sample pretreatment. The instrumental method was successfully validated and separated matrix compounds from B12 vitamers in food samples to some extent. The developed sample pretreatment method is a good starting point for developing more effective sample pretreatment methods in the future.

B12-vitamiini on rakenteeltaan monimutkainen, vesiliukoinen kompleksiyhdiste. Ihmisille aktiivisessa B12-vitamiinin muodossa alempana ligandina on 5,6-dimetyylibentsimidatsoli. B12-vitamiinia saadaan eläinperäisistä elintarvikkeista. Tämän tutkielman kirjallisuusosio käsitteli B12-vitamiinin rakennetta, vitamiiniaktiivisuutta, ruokavalion vaikutusta saantiin ja imeytymisen vaiheita ihmisen elimistössä. Sen lisäksi käsiteltiin B12-vitamiinin analytiikkaa ja pitoisuuksia elintarvikkeissa. Useissa tutkimuksissa B12-vitamiinia on määritetty mikrobiologisella menetelmällä. Menetelmän epäillään nykyisin vääristävän B12-vitamiinipitoisuuksia, koska menetelmässä käytetty testiorganismi reagoi positiivisesti myös B12-vitamiinin kaltaisiin muihin korrinoidiyhdisteisiin. Tämän tutkielman kokeellisen osion tavoitteena oli tutkia kuinka paljon eläinperäiset elintarvikkeet sisältävät B12-vitamiinia. Tutkimukseen valittiin keskeisiä eläinperäisiä elintarvikkeita sekä kaksi elintarvikkeeksi hyväksyttyä hyönteislajia. Tavoitteena oli myös verrata toisiinsa mikrobiologista ja erittäin suuren erotuskyvyn nestekromatografista (UHPLC) menetelmää B12-vitamiinin määrityksessä sekä tutkia eri uuttomenetelmien vaikutusta analysoituihin B12-vitamiinipitoisuuksiin. Uuttokokeiden tulosten perusteella kaikille näytteille ei voitu käyttää samaa esikäsittelymenetelmää. Uutto pepsiinikäsittelyllä paransi määritettyä B12-vitamiinipitoisuutta kirjolohella, keltuaisella, naudan sisäpaistilla ja maidolla, kun taas pankreatiinikäsittely paransi määritettyä B12-vitamiinipitoisuutta juustoilla ja silakalla. Elintarvikkeista odotetusti eniten B12-vitamiinia oli naudan ja porsaan maksan lisäksi silakassa, juustoissa ja naudan sisäpaistissa. Vähiten B12-vitamiinia oli broilerin rintafileessä. Mikrobiologisella ja UHPLC-menetelmällä määritetyissä B12-vitamiinipitoisuuksissa oli merkittävä ero menetelmien välillä. Liha- ja kalanäytteiden B12-vitamiinipitoisuudet olivat 7–64 % pienempiä UHPLC-menetelmällä kuin mikrobiologisella menetelmällä määritettynä, maitotuotteilla ja keltuaisella 20–67 % pienempiä ja myös hyönteisillä eroa oli 71–81 %. Mikrobiologinen menetelmä on herkkä ja edullinen käyttää. Jatkossa elintarvikkeiden B12-vitamiinipitoisuus kannattaa kuitenkin määrittää UHPLC-menetelmällä, joka erottaa ihmiselle aktiivisen B12-vitamiinin muodon muista ei-aktiivisista muodoista eikä anna vastetta muille B12-vitamiinin kaltaisille yhdisteille.

Microalgae are promising raw materials for food- and biotechnology because they contain a lot of proteins, unsaturated fatty acids, pigments, vitamins and minerals. There are few studies on vitamin B in microalgae and some of them are based on partly inaccurate methods. Microalgae in general, analytical methods regarding their analysis and how they use vitamins were discussed in the literature part of this thesis. The structures, chemical properties and occurrence in foods as well as commonly used analytical methods of the vitamins in question were presented. The aim of the experimental part of this thesis was to analyse commercially marketed microalgae supplements (Chlorella sp. and Arthrospira sp. (spirulina)) and laboratory-grown microalga (Euglena gracilis) as potential sources of folate, niacin, vitamin B2 and B12. Contents of vitamin B12, B2 and niacin were analysed using UHPLC method separately validated for each vitamin. The total folate content was analysed microbiologically and folate vitamers by using UHPLC. The vitamin B12 was analysed microbiologically and the active forms of vitamin B12 were confirmed using LC-MS. Acid hydrolysis was used in analysing niacin content. The total folate content in chlorella supplements was of the same order when analysed microbiologically or with UHPLC. Instead, in spirulina supplements the microbiologically analysed total folate content was higher than the total folate content based on the sum of folate vitamers analysed with UHPLC. At most, the total folate content of E. gracilis -sample was 3-fold higher than in commercial microalgae supplements. Especially in spirulina supplements, the vitamin B12 contents were clearly higher when analysed microbiologically than they were when analysed with UHPLC. The difference was most likely due to pseudocobalamin that resembled vitamin B12. On average E. gracilis -samples had higher vitamin B2 content than the commercial supplements. E. gracilis -samples and chlorella supplements contained more niacin than spirulina supplements. According to this thesis, commercially marketed microalgae supplements contained different amounts of vitamin B. Chlorella sp. was proved to be a great source of folate, vitamin B12 and niacin and moderate source of B2. The majority of vitamin B12 in Arthrospira sp. (spirulina) was pseudocobalamin. Despite that, spirulina supplements proved to be a moderate source of vitamin B12. On average, E. gracilis had the highest vitamin B content and it would potentially be an excellent source of vitamin B – if it was accepted for food use.

A common quality issue in fresh-cut vegetables is enzymatic browning. Anti-browning agents regulated by (EU) N:o 1129/2011 can be used in processing and vegetables are usually treated with compounds after washing and cutting. Polyphenol oxidase enzyme (PPO) in vegetables ox-idises phenolic compounds to quinones and brown melanin pigments are formed when reactive quinones polymerize. The literature review focused on phenolic compounds of apples, the mech-anism of action and inhibition of PPO. In addition, the review covered anti-browning agents in-vestigated in previous studies and physical inhibition methods for enzymatic browning. The aim of the experimental study was to find a mixture of anti-browning agents classified as a processing aid for fresh-cut apple with the help of the experimental design. In addition, apples treated with three different concentrations of calcium ascorbate were also investigated. The colour of the treated apples was measured using a colorimeter, the activity of PPO was determined spec-trophotometrically and the concentrations of the most common phenolic compounds of Granny Smith were determined by Ultra-High Performance Liquid Chromatography (UHPLC). The concentrations of anti-browning agents of the mixtures in PLSR model did not have a statis-tically significant effect on colour and the coefficient of determination was satisfactory. The ap-ples from the experimental design maintained their colour over one but less than five days and the apples treated with calcium ascorbate maintained their colour for one week which was the targeted shelf life. The PPO activity of the samples from the experimental design increased during the storage but activity of the samples treated with calcium ascorbate was zero all the time. The standard deviations of concentrations of phenolic compounds were high and no clear trend re-garding the change of concentrations and browning was noticed. The three major phenolic com-pounds of Granny Smith were (-)-epicatechin, (+)-catechin and chlorogenic acid. The concentra-tions of catechins were higher in the peel than in the flesh but for chlorogenic acid it was the opposite. The shelf life of one week was not achieved with the mixtures of the experimental design but calcium ascorbate acted effectively. Also compounds in the mixtures had the ability to inhibit the PPO because the apples did not start to brown immediately as the control samples treated with water. The results confirmed previous results from the literature that calcium ascorbate is an effective anti-browning agent and therefore it is one of the most common anti-browning agents in the fresh-cut industry.

Avenanthramides are hydroxycinnamic acids unique to oat (Avena sativa L., Poaceae). They consist of an anthranilic acid part (anthranilic acid or hydroxylated and/or methoxylated derivative of anthranilic acid) that is conjugated to a cinnamic acid part via an amide bond. More than 25 different avenanthramides are found and identified in oat. However, the most common forms are esters of 5-hydroxyanthranilic acid with caffeic (2c), ferulic (2f) and p-coumaric (2p) acids. Avenanthramides have been shown to possess antioxidant and anti-inflammatory properties. Currently there is a great interest towards oat and bioactive compounds of oat, like avenanthramides. In the previous studies there has been a lot of diversity concerning the extraction method used for analysis of avenanthramides and usually quantitation methods were based on high-performance liquid chromatography (HPLC). The aim of this research was to develop a method based on ultra-high-performance liquid chromatography (UHPLC) for the analysis of the most common forms of avenanthramides (2c, 2f and 2p) in oat and oat products. In addition, also other forms of avenanthramides, like 2fd and 2pd, were identified and quantified using liquid chromatography-mass spectrometry (UHPLC-QTOF-MS). The extraction method of avenanthramides was optimized and the UHPLC-PDA method used for quantitation was validated. Finally, the study measured the differences in concentrations of avenanthramides in different oat products. In this study it was recognized that the concentrations and the extent of variation of the concentrations of avenanthramides were affected by the sample amount, the homogeneity of the samples and the extraction time used. Especially bigger sample amount of oat flour (0.5 g) led to larger and more reproducible results than smaller amount (0.1 g). The ratio of sample and solvent 1:10 worked excellently and as an extraction solvent ethanol:water (80:20) was more efficient than tested ethanol:water (80:20) with a phosphate buffer at pH 2.8. Smaller particle size of oat flour and extraction overnight led to better extractability of avenanthramides than a short extraction of sample with larger particle size. The UHPLC method was optimized using chromatographic parameters. The avenanthramides separated from each other acceptably and were eluted as follows during the 16-minute run: 2c, 2p, 2f. The response of the UHPLC instrument was linear in the tested concentration range for all three avenanthramides. The results were reproducible, and the accuracy of the UHPLC instrument was acceptable. The recovery % for avenanthramides were 99–117%. Also, other forms of avenanthramides, like 2fd, 2pd, 5p, 5f ja 3f, were identified in different oat samples using UHPLC-QTOF-MS technique. The total amount of avenanthramides in analyzed oat samples varied between 3–41.3 µg/g per fresh weight. Oat bran included them the most and oat snack the least. Avenanthramide 2c was dominant in oat flour which included 2p the least. In oat bran, in oat drink, in oat snack, in Nyhtökaura and in Muru the avenanthramide 2f was dominant over the two other forms. The UHPLC method developed in this study can be applied to the analysis of the most common forms of avenanthramides 2c, 2f and 2p in oat and in oat products and can be used in oat research in the future. The method can also be used to identify and quantitate more widely other forms of avenanthramides in different oat products. The optimized extraction was shown to be functional and reproducible to already homogeneous and processed samples, but the extraction of non-homogeneous samples could be optimized further in the future.

Tutkielman tarkoitus oli selvittää kolmen eri valointensiteetin vaikutuksia mikrolevälajien, E.gracilis ja Selenastrum sp., kasvuun ja karotenoidituotantoon. Käytetyt valointensiteetit olivat 0 (pimeä), 200 ja 400 µmol m-2s-1. Levät kasvatettiin kiertovesikalankasvattamon jätevedessä, minkä vuoksi työssä tarkasteltiin myös ravinnepoistumaa. Kasvatukset havaittiin typpirajoitteisiksi, mikä osaltaan heikensi levien kasvua ja siten karotenoidituotantoa. Jätevesi kasvualustana aiheutti E.gracilis -levän kasvatuksiin kontaminaation vieraslevällä. Karotenoidit eristettiin kasvatuskokeen päätyttyä uuttamalla paineistetulla nesteuutolla (Accelerated Solvent Extraction, ASE) etanolin ja 2-metyylitetrahydrofuraanin liuotinyhdistelmällä. Karotenoidien jatkoanalyysiin käytettiin erittäin korkean erotuskyvyn nestekromatografiaa (UHPLC, Ultra-High Performance Liquid Chromatography) yhdistettynä sähkösumutusionisaatioon ja massaspektrometriin. Tunnistettujen karotenoidien määriä analysoitiin niiden signaalivahvuuksiin ja käytettävissä olleisiin malliaineisiin perustuen. Valointensiteeteillä havaittiin olevan vaikutus levien kasvuun, kokonaiskarotenoidi- ja klorofyllipitoisuuksiin sekä astaksantiiniin, luteiiniin ja neoksantiiniin. Kyseiset erikseen tunnistetut pigmentit olivat kaikki ksantofyllejä, joilla on liialta valointensiteetiltä suojaava tehtävä leväsoluissa. Valointensiteetin sietokyky poikkesi kuitenkin levälajeilla eivätkä sen vaikutukset olleet yksiselitteisiä. Pimeässä levien kasvu ja ravinnepoistuma olivat heikkoja. Pigmenttien säilymisen puolesta pimeä osoittautui jopa voimakkainta valointensiteettiä paremmaksi vaihtoehdoksi. Molempien levien ravinteiden kulutus oli tehokasta. Valointensiteetin lisäksi kasvuun ja karotenoidituotantoon vaikutti muutkin tekijät, kuten kokeen aikana saavutettu kasvuvaihe, jatkuva valotus, kontaminoituminen, ravinnerajoitteisuus sekä käytettyjen malliaineiden laatu. Jatkotutkimuksissa tulisi pyrkiä minimoimaan tai poissulkemaan näiden tekijöiden vaikutukset.

Wood-decaying fungi of Basidiomycota are important decomposers of wood and plant biomass in nature. Interactions of five birch-wood decaying fungi (Fomitopsis pinicola, Fp; Fomitopsis betulina, Fb; Phlebia radiata, Pr; Fomes fomentarius, Ff; Schizophyllum commune, Sc) were studied by forming combinations of two, three or five fungal species and cultivating them on low-nitrogen liquid medium, and on three different agar media. Of the fungal species studied, Sc was the most dominant at the metabolic level. Pr was more dominant than Fp, Ff and Fb, while Fp was more dominant than Ff and Fb. This conclusion was supported by measurements from the cultivations on liquid medium (pH, enzyme activities, antioxidant and reduction ability, sugar consumption), non-metric multidimensional scaling (NMDS) analysis and compounds found in extracts from culture fluids and mycelia. Three variations of mycelial interactions were observed when fungal combinations were cultivated on agar media: inhibition and two types of co-existence. Tens of compound peaks were detected in HPLC-chromatograms of the culture fluid and mycelial extracts, but only a part of the compounds could be identified. The presence of two or more fungal species in the cultivations increased the number of peaks in the chromatograms indicating the potential of co-cultivation as a method for production of new natural compounds. Several extracts showed weak antibacterial properties. These results suggest that larger-scale cultivations, more concentrated extracts, and isolation of the numerous compounds in the extracts could facilitate finding and identifying antimicrobial substances produced by fungi.