Qualitative and quantitative determination of selected isothiocyanates using GC-MS/MS : diploma thesis

Izotiocijanati su razgradni produkti hidrolize glukozinolata, fitokemikalija koje se pretežno nalaze u biljkama iz porodice kupusnjača. Izotiocijanati su potencijalno biološki aktivni, zbog čega se razvijaju analitičke metode za njihovo određivanje. U ovom diplomskom radu razvijena je metoda za određivanje 2-metoksifenil- izotiocijanata, 3-metoksifenil-izotiocijanata i 4-metoksifenil-izotiocijanata. Spregnuta tehnika plinska kromatografija-tandemska spektrometrija masa (GC-MS/MS) korištena je za odjeljivanje odabranih izotiocijanta iz smjese te njihovo kvalitativno i kvantitativno određivanje. Za pripremu otopina korišteni su standardi 2-, 3- i 4-metoksifenil-izotiocijanata te otapalo n-heksan. Plinska kromatografija optimizirana je promjenom brzine povećanja temperature peći (20 °C min -1 ). Spektrometar masa s trostrukim kvadrupolom, u MRM načinu snimanja, korišten je za praćenje specifičnih reakcija prijelaza (kvantitativni i dva kvalitativna za svaki spoj). Spektrometrija masa je optimizirana određivanjem energije sraza s dušikom za svaku specifičnu reakciju prijelaza. Prilikom izrade krivulje umjeravanja istraženi su različiti načini pripreme radnih otopina standarda (odabrano je klasično pipetiranje mehaničkim pipetama) te različita područja koncentracija otopina (odabran je raspon 2,5 μg mL -1 – 20,0 μg mL -1 ). Ispitani parametri vrednovanja: točnost, preciznost, specifičnost, linearnost, granica dokazivanja, granica određivanja i stabilnost, pokazali su se zadovoljavajućima. Metoda je uspješno primijenjena u analizi sintetskog uzorka poznate koncentracije. Navedenim je osigurana prikladnost metode za pojedinačno određivanje 2-, 3- i 4-metoksifenil-izotiocijanata u smjesi.Isothiocyanates are degradation products of glucosinolate hydrolysis. Glucosinolates are phytochemicals found mostly in plants from the cabbage family. Isothiocyanates are potentially biologically active, which is why methods for their determination are being developed. In this thesis, a method for the determination of 2-methoxyphenyl isothiocyanate, 3-methoxyphenyl isothiocyanate and 4-methoxyphenyl isothiocyanate was developed. Gas chromatography-tandem mass spectrometry (GC-MS/MS) was applied for the separation of selected isothiocyanates and qualitative and quantitative determination of separated compounds. Standards of 2-, 3- and 4-methoxyphenyl isothiocyanate and solvent n-hexane were used for the preparation of solutions. Gas chromatography was optimized by changing the oven temperature program rate (20 °C min -1 ). Triple quadrupole mass spectrometer, in MRM scan mode, was used for monitoring specific fragmentation reactions (quantifier and two qualifiers for each compound). Mass spectrometry was optimized by determination of the collision energy with nitrogen for each specific reaction of fragmentation. Different approaches of preparing the calibration curve working solutions were examined (forward pipetting with mechanical pipettes was selected) as well as different concentration ranges (range 2,5 μg mL -1 – 20,0 μg mL -1 was selected) were examined for making calibration curves. The validation parameters tested: accuracy, precision, specificity, linearity, the limit of detection, the limit of quantification and stability, were proven satisfactory. The method was successfully applied in the analysis of a synthetic sample of known concentration. This ensures the suitability of the method for the individual determination of 2-, 3- and 4-methoxyphenyl isothiocyanates in the mixture