DETERMINATION OF ENDOCRINE DISRUPTORS IN URINE USING GC/MS AND CHEMOMETRIC CHARACTERISATION

V doktorski nalogi smo proučevali vpliv nekaterih spojin okoljskih endokrinih motilcev na plodnost moških z namenom, da bi preverili hipotezo ali vplivajo endokrini motilci na kakovost semenčic. V študijo smo vključili 136 parov, ki so se zdravili zaradi neplodnosti. Za analitiko sledov spojin endokrinih motilcev v urinu smo razvili analizno metodo določevanja s plinsko kromatografijo in masno spektrometrijo. Uporabili smo 13C izotopsko označene standardne spojine. Po dodatku internih standardov smo za dekonjugacijo analitov urinu dodali encim β-glukuronidazo. Vzorec smo nato nakisali s HCl na pH 2. Sledila je ekstrakcija z diklorometanom. Čiščenje ekstraktov smo izvedli na SiO2 koloni ter tako odstranili bazične interference, ki jih predstavljajo aminokisline, sečnina, amini in druge bazične spojine. Očiščeni ekstrakt smo odparili do suhega in dodali derivatizacijski reagent N-metil-N-trimetilsilil trifluoroacetamid (MSTFA). Sililirani ekstrakt smo analizirali s plinsko kromatografijo in masno spektrometrijo. Uporabljena je bila tehnika snemanja izbranih masnih fragmentov (SIM). Sočasno smo določali di-alkil ftalate: di-metil ftalat (DMP), di-etil ftalat (DEP), di-butil ftalat (DBP), benzil-butil ftalat (BzBP) ter di-(2-etil-heksil) ftalat (DEHP), njihove metabolite mono-alkil ftalate: mono-etil ftalat (MEP), mono-izo-butil ftalat (MiBP), mono-n-butil ftalat (MnBP), mono-(2-etilheksil) ftalat (MEHP), mono-benzil ftalat (MBzP), mono-izo-nonil ftalat (MiNP), mono-n-oktil ftalat (MnOP), mono-(2-etil-5-oksoheksil)ftalat (MEOHP) ter mono-(2-etil-5-hidroksiheksil)ftalat (MEHHP). Določali smo alkilfenole: 4-terc-oktilfenol (4-tOP), 4-n-oktilfenol (4nOP) ter 4-n-nonilfenol (4nNP). Prav tako smo določali bisfenol A (BPA). Izvedli smo validacijo metode. Meja določanja je bila za bisfenol A 0,1 µg L-1, za mono-alkil ftalate in alkilfenole 1 µg L-1, in za di-alkil ftalate 5 µg L-1. Linearnost metode smo preverili v koncentracijskem območju od 1 do 200 µg L-1. Uporabljena metoda se je pri kvantitativnem določanju spojin endokrinih motilcev v vzorcih urinov izkazala za uspešno, saj je natančna, točna in selektivna. Omogoča sočasno določanje endokrinih spojin z nizko mejo zaznavnosti v območju µg L-1. Z uporabo tehnike snemanja celotnega masnega spektra smo določili še 60 drugih spojin v urinu (maščobne kisline, steroide, mono-aromatske spojine in druge spojine). Kemometrijsko karakterizacijo smo izvedli s pomočjo podatkov pridobljenih z analizami 136 vzorcev urinov moških, ki imajo težave s plodnostjo. Namen raziskave je bil ugotoviti ali obstajajo pomembne razlike med vzorci, ki smo jih uvrstili v dve skupini, glede na normalno ter zmanjšano kakovost semenčic. Skupini se razlikujeta po vrednostih koncentracije in gibljivosti semenčic. Cilj kemometrijske karakterizacije je bil poiskati korelacije med vsebnostjo spojin endokrinih motilcev v urinu, kot so ftalatni estri in njihovi metaboliti, oktil in nonil alkilfenoli in bisfenol A ter kliničnimi parametri kakovosti semenčic kot sta koncentracija in gibljivost semenčic. Dodatno smo iskali korelacije med kakovostjo semenčic in vsebnostjo hormonov: FSH (folikel-stimulativni hormon), LH (luteinizarajoči hormon), estrogen, testosteron, SHBG (spolne hormone vezoči globulin), inhibinB. Izvedli smo korelacijsko analizo, analizo variance (ANOVA), Mann Whitney test, analizo glavnih osi (PCA), hierarhično razvrščanje (CA), linearno diskriminatno analizo (LDA), metodo gradnje regresijskih dreves (CART) in umetne nevronske mreže (ANN). Študija je pokazala, da obstajajo pomembne razlike med obema skupinama moških, ki imajo težave s plodnostjo, predvsem glede na vsebnost di-(2-etilheksil) ftalata (DEHP), mono-(2-etilheksil) ftalata (MEHP) in obeh sekundarnih metabolitov mono-(2-etil-5-oksoheksil) ftalata (MEOHP) ter mono-(2-etil-5-hidroksiheksil) ftalata (MEHHP). V vzorcih urina moških z zmanjšano kakovostjo semenčic so bile izmerjene vsebnosti endokrinih motilcev v povprečju dvakrat višje od vsebnosti v vzorcih urina moškihThis doctoral dissertation investigated the influence of endocrine compounds’ exposure on male fertility and the hypothesis, if endocrine disruptors affect the quality of sperm was tested. This study included 136 couples being treated for infertility. An analytical method for trace analysis of endocrine disruptors in the urine using gas chromatography and mass spectrometry had been developed. The urine was spiked with 13C isotopically labelled internal standards, β-glucoronidase was added for deconjugation of the analytes. The sample solution was then acidified to pH 2 with HCl. Extraction was performed using dichloromethane. The extracts were purified on the SiO2 column and interference compounds were removed such as amino acids, urea, amines and other compounds. Dichloromethane extracts were then evaporated to dryness and N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was added as a derivatisation reagent. An aliquot of the sililated extract was injected into a GC-MS system and a selected ion monitoring (SIM) technique was used. Di-alkyl phthalates: di-methyl phthalate (DMP), di-ethyl-phthalate (DEP), di-butyl phthalate (DBP), benzyl-butyl phthalate (BzBP) and di-(2-ethyl-hexyl) phthalate (DEHP), their metabolites, mono-alkyl phthalates: mono-ethyl phthalate (MEP), mono-iso-butyl phthalate (MiBP), mono-n-butyl phthalate (MnBP), mono-(2-ethylhexyl) phthalate (MEHP), mono-benzyl phthalate (MBzP), mono-iso-nonyl phthalate (MiNP), mono-n-octyl phthalate (MnOP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), alkylphenols: 4-tert-octylphenol (4-tOP), 4-n-octylphenol (4nOP) and 4-n-nonylphenol (4nNP) and Bisphenol A (BPA) were simultaneously determined. Validation of the method was performed. Limit of quantification (LOQ) was for bisphenol A 0.1 µg L-1, for mono-alkyl phthalates and alkylphenols 1 µg L-1, and for di-alkyl phthalates 5 µg L-1. The linearity of the method was tested in the concentration range from 1 to 200 µg L-1. The method was proved to be successful for the quantitative determination of endocrine disrupting compounds in urine samples. It is precise, accurate and selective, and can be used for simultaneous determination of phthalates, their metabolites, alkylphenols, and bisphenol A in trace amounts in human urine. This method allows the determination of endocrine compounds with low detection limit (LOD) in µg L-1. Sixty other compounds in the urine (fatty acids, steroids, mono-aromatic compounds and other compounds) have also been also determined using the full mass spectra (SCAN) technique. Chemometric characterisation was performed using data obtained from all 136 urine samples from men with fertility problems. The purpose of the study was to investigate whether there were any significant differences between samples, which were classified into two groups according to normal and reduced sperm quality. Groups differ in levels of sperm concentration and motility. The aim of the chemometric characterisation was to find correlations between the content of endocrine disrupting compounds in the urine samples, such as phthalate esters and their metabolites, octyl and nonyl alkylphenols and bisphenol A and clinical parameters of semen quality such as sperm concentration and sperm motility. Additionally, correlations between sperm quality and content of hormones: FSH (follicle-stimulating hormone), LH (luteinising hormone), estrogen, testosterone, SHBG (sex hormone binding globulin), and inhibin B were searched. Correlation analysis, analysis of variance (ANOVA), Mann Whitney test, principal component analysis (PCA), cluster analysis (CA), linear discriminant analysis (LDA), classification and regression tree (CART), and artificial neural networks (ANN) were used for chemometric characterization. The study showed that there are significant differences between the two groups of men with fertility problems, particularly with regard to the content of di-(2-ethylhexyl) phthalate (DEHP), mono-(2-ethylhexyl) phthalate (MEHP) and both secondary metabol