Use of non-specific and specific interactions in the analysis of testosterone and related compounds by capillary electromigration techniques

Determination of testosterone and related compounds in body fluids is of utmost importance in doping control and the diagnosis of many diseases. Capillary electromigration techniques are a relatively new approach for steroid research. Owing to their electrical neutrality, however, separation of steroids by capillary electromigration techniques requires the use of charged electrolyte additives that interact with the steroids either specifically or non-specifically. The analysis of testosterone and related steroids by non-specific micellar electrokinetic chromatography (MEKC) was investigated in this study. The partial filling (PF) technique was employed, being suitable for detection by both ultraviolet spectrophotometry (UV) and electrospray ionization mass spectrometry (ESI-MS). Efficient, quantitative PF-MEKC UV methods for steroid standards were developed through the use of optimized pseudostationary phases comprising surfactants and cyclodextrins. PF-MEKC UV proved to be a more sensitive, efficient and repeatable method for the steroids than PF-MEKC ESI-MS. It was discovered that in PF-MEKC analyses of electrically neutral steroids, ESI-MS interfacing sets significant limitations not only on the chemistry affecting the ionization and detection processes, but also on the separation. The new PF-MEKC UV method was successfully employed in the determination of testosterone in male urine samples after microscale immunoaffinity solid-phase extraction (IA-SPE). The IA-SPE method, relying on specific interactions between testosterone and a recombinant anti-testosterone Fab fragment, is the first such method described for testosterone. Finally, new data for interactions between steroids and human and bovine serum albumins were obtained through the use of affinity capillary electrophoresis. A new algorithm for the calculation of association constants between proteins and neutral ligands is introduced.

A Curious Harpour in Helle : An Edition of the Commentary on the Orpheus Metre of De consolatione philosophiae in MS Thott 304,2

Tutkielmani on tekstieditio keskiaikaisesta kommentaarista, joka löytyy Kööpenhaminan Kuninkaallisen kirjaston käsikirjoituksesta Thott 304,2. Käsikirjoitus voidaan ajoittaa 1400-luvun ensimmäiselle neljännekselle ja se on kirjoitettu keskienglanniksi. Se sisältää John Waltonin runomuotoisen käännöksen Boethiuksen 500-luvun alussa latinaksi kirjoittamasta teoksesta De consolatione philosophiae (Filosofian lohdutus) sekä teosta kommentoivan proosamuotoisen kommentaarin. Käsikirjoitus on vaillinainen, mutta sen säilyneet foliot ovat enimmäkseen erittäin hyväkuntoisia. Itse käsikirjoitusta on tutkittu vain muutamissa artikkeleissa ja yhdessä tutkielmassa; kommentaarista ei ole toistaiseksi tehty kattavaa tutkimusta. Tavoitteeni onkin tuoda kommentaari keskiaikaisen filosofian, keskienglannin ja käsikirjoitusten tutkijoiden käytettäviin. Käsikirjoitus Thott 304,2 on esimerkki epätyypillisestä myöhäiskeskiaikaisesta maallikkomesenaattiudesta Englannissa. Tavanmukaisesti maallikkomesenaatit tukivat uskonnollisten tekstien tuottamista ja kääntämistä, kun taas Thott 304,2 sisältää käännöksen filosofisesta tekstistä. Sen lisäksi mesenaatti oli nainen, aatelistoon kuuluva Elizabeth Berkeley. Varsin harvinaiseksi käsikirjoituksen tekee sen painaminen kirjaksi 1500-luvun alussa. Kirjanpainajan käsikirjoituksen sivuille tekemistä merkinnöistä saadaan korvaamatonta tietoa varhaisesta painotekniikasta, sillä kirjanpainajien käyttämiä käsikirjoituskopioita ei ole säilynyt kovin runsaasti. Waltonin käännös on säilynyt yli kahdessakymmenessä käsikirjoituskopiossa, joista vain Thott 304,2 sisältää laajan kommentaarin. 1500-luvun painoksesta on jäljellä kolme kopiota, ja ne sisältävät saman kommentaarin kuin Thott 304,2. Valitsin editoitavaksi niin kutsuttua Orfeus-runoa kommentoivan osan kommentaarista, sillä se muodostaa ehjän kokonaisuuden ja sopii pituutensa puolesta Pro gradu -tutkielmaan. Orfeus-runo on myös yksi käsikirjoituksen kattavimmin kommentoiduista runoista. Editio on niin sanottu diplomaattinen transkriptio, jossa käsikirjoituksen piirteet on pyritty säilyttämään mahdollisimman tarkasti edition luettavuuden siitä kuitenkaan kärsimättä. Perinteisistä editioista poiketen tutkielmani sisältää myös kommentaarin ja Orfeus-runon transkriptiot, joissa rivijako, lyhenteet ja erikoismerkit on säilytetty. Näiden transkriptioiden toivon auttavan erityisesti käsikirjoituksessa esiintyvien lyhenteiden, erikoismerkkien ja kirjanpainajan merkintöjen tulkinnassa ja tutkimisessa. Editiota ja transkriptioita täydentävät nykyenglanniksi kirjoitettu lyhennelmä kommentaarista ja kuvat käsikirjoituksen sivuista, joilla editoimani kommentaari on. Tutkielmaan sisältyy alkuperäisen tekstin, käännöksen, kommentaarin ja käsikirjoituksen taustaa valottava osuus. Esittelen myös kaikki löytämäni lähteet, joissa käsikirjoitus on mainittu tai joissa sitä on tutkittu. Liitteeksi olen laatinut sanaston helpottamaan kommentaarin tulkitsemista.

Method-MS, final report 2010

Radiometric determination methods, such as alpha spectrometry require long counting times when low activities are to be determined. Mass spectrometric techniques as Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Thermal Ionisation Mass Spectrometry (TIMS) and Accelerator Mass Spectrometry (AMS) have shown several advantages compared to traditional methods when measuring long-lived radionuclides. Mass spectrometric methods for determination of very low concentrations of elemental isotopes, and thereby isotopic ratios, have been developed using a variety of ion sources. Although primarily applied to the determination of the lighter stable element isotopes and radioactive isotopes in geological studies, the techniques can equally well be applied to the measurement of activity concentrations of long-lived low-level radionuclides in various samples using “isotope dilution” methods such as those applied in inductively coupled plasma mass spectrometry (ICP-MS). Due to the low specific activity of long-lived radionuclides, many of these are more conveniently detected using mass spectrometric techniques. Mass spectrometry also enables the individual determination of Pu-239 and Pu-240, which cannot be obtained by alpha spectrometry. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) are rapidly growing techniques for the ultra-trace analytical determination of stable and long-lived isotopes and have a wide potential within environmental science, including ecosystem tracers and radio ecological studies. Such instrumentation, of course needs good radiochemical separation, to give best performance. The objectives of the project is to identify current needs and problems within low-level determination of long-lived radioisotopes by ICP-MS, to perform intercalibration and development and improvement of ICP-MS methods for the measurement of radionuclides and isotope ratios and to develop new methods based on modified separation chemistry applied to new auxiliary equipment.

Diastereomeric Drug Glucuronides: Enzymatic Glucuronidation and Analysis by Capillary Electrophoresis and Liquid Chromatography-Mass Spectrometry

Foreign compounds, such as drugs are metabolised in the body in numerous reactions. Metabolic reactions are divided into phase I (functionalisation) and phase II (conjugation) reactions. Uridine diphosphoglucuronosyltransferase enzymes (UGTs) are important catalysts of phase II metabolic system. They catalyse the transfer of glucuronic acid to small lipophilic molecules and convert them to hydrophilic and polar glucuronides that are readily excreted from the body. Liver is the main site of drug metabolism. Many drugs are racemic mixtures of two enantiomers. Glucuronidation of a racemic compound yields a pair of diastereomeric glucuronides. Stereoisomers are interesting substrates in glucuronidation studies since some UGTs display stereoselectivity. Diastereomeric glucuronides of O-desmethyltramadol (M1) and entacapone were selected as model compounds in this work. The investigations of the thesis deal with enzymatic glucuronidation and the development of analytical methods for drug metabolites, particularly diastereomeric glucuronides. The glucuronides were analysed from complex biological matrices, such as urine or from in vitro incubation matrices. Various pretreatment techniques were needed to purify, concentrate and isolate the analytes of interest. Analyses were carried out by liquid chromatography (LC) with ultraviolet (UV) or mass spectrometric (MS) detection or with capillary electromigration techniques. Commercial glucuronide standards were not available for the studies. Enzyme-assisted synthesis with rat liver microsomes was therefore used to produce M1 glucuronides as reference compounds. The glucuronides were isolated by LC/UV and ultra performance liquid chromatography (UPLC)/MS, while tandem mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR) spectroscopy were employed in structural characterisation. The glucuronides were identified as phenolic O-glucuronides of M1. To identify the active UGT enzymes in (±)-M1 glucuronidation recombinant human UGTs and human tissue microsomes were incubated with (±)-M1. The study revealed that several UGTs can catalyse (±)-M1 glucuronidation. Glucuronidation in human liver microsomes like in rat liver microsomes is stereoselective. The results of the studies showed that UGT2B7, most probably, is the main UGT responsible for (±)-M1 glucuronidation in human liver. Large variation in stereoselectivity of UGTs toward (±)-M1 enantiomers was observed. Formation of M1 glucuronides was monitored with a fast and selective UPLC/MS method. Capillary electromigration techniques are known for their high resolution power. A method that relied on capillary electrophoresis (CE) with UV detection was developed for the separation of tramadol and its free and glucuronidated metabolites. The suitability of the method to identify tramadol metabolites in an authentic urine samples was tested. Unaltered tramadol and four of its main metabolites were detected in the electropherogram. A micellar electrokinetic chromatography (MEKC) /UV method was developed for the separation of the glucuronides of entacapone in human urine. The validated method was tested in the analysis of urine samples of patients. The glucuronides of entacapone could be quantified after oral entacapone dosing.

The challenge of LC/MS/MS multi-mycotoxin analysis - Heracles battling the Hydra?

Mycotoxins are secondary metabolites of filamentous fungi. They pose a health risk to humans and animals due to their harmful biological properties and common occurrence in food and feed. Liquid chromatography/mass spectrometry (LC/MS) has gained popularity in the trace analysis of food contaminants. In this study, the applicability of the technique was evaluated in multi-residue methods of mycotoxins aiming at simultaneous detection of chemically diverse compounds. Methods were developed for rapid determination of toxins produced by fungal genera of Aspergillus, Fusarium, Penicillium and Claviceps from cheese, cereal based agar matrices and grains. Analytes were extracted from these matrices with organic solvents. Minimal sample clean-up was carried out before the analysis of the mycotoxins with reversed phase LC coupled to tandem MS (MS/MS). The methods were validated and applied for investigating mycotoxins in cheese and ergot alkaloid occurrence in Finnish grains. Additionally, the toxin production of two Fusarium species predominant in northern Europe was studied. Nine mycotoxins could be determined from cheese with the method developed. The limits of quantification (LOQ) allowed the quantification at concentrations varying from 0.6 to 5.0 µg/kg. The recoveries ranged between 96 and 143 %, and the within-day repeatability (as relative standard deviation, RSDr) between 2.3 and 12.1 %. Roquefortine C and mycophenolic acid could be detected at levels of 300 up to 12000 µg/kg in the mould cheese samples analysed. A total of 29 or 31 toxins could be analysed with the method developed for agar matrices and grains, with the LOQs ranging overall from 0.1 to 1250 µg/kg. The recoveries ranged generally between 44 and 139 %, and the RSDr between 2.0 and 38 %. Type-A trichothecenes and beauvericin were determined from the cereal based agar and grain cultures of F. sporotrichioides and F. langsethiae. T-2 toxin was the main metabolite, the average levels reaching 22000 µg/kg in the grain cultures after 28 days of incubation. The method developed for ten ergot alkaloids from grains allowed their quantification at levels varying from 0.01 to 10 µg/kg. The recoveries ranged from 51 to 139 %, and the RSDr from 0.6 to 13.9 %. Ergot alkaloids were measured in barley and rye at average levels of 59 and 720 µg/kg, respectively. The two most prevalent alkaloids were ergocornine and ergocristine. The LC/MS methods developed enabled rapid detection of mycotoxins in such applications where several toxins co-occurred. Generally, the performance of the methods was good, allowing reliable analysis of the mycotoxins of interest with sufficiently low quantification limits. However, the variation in validation results highlighted the challenges related to optimising this type of multi-residue methods. New data was obtained about the occurrence of mycotoxins in mould cheeses and of ergot alkaloids in Finnish grains. In addition, the study revealed the high mycotoxin-producing potential of two common fungi in Finnish crops. The information can be useful when risks related to fungal and mycotoxin contamination will be assessed.