Notes
This 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, %-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 %g L-1, for mono-alkyl phthalates and alkylphenols 1 %g L-1, and for di-alkyl phthalates 5 %g L-1. The linearity of the method was tested in the concentration range from 1 to 200 %g 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 %g 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.