Epäspesifisten ja spesifisten vuorovaikutuksien hyödyntäminen testosteronin ja sen sukuisten steroidien kapillaarielektromigraatioanalyyseissä

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.Testosteronin ja sen sukuisten steroidien määrittäminen kehon nesteistä on keskeistä monien sairauksien diagnosoinnissa sekä doping-valvonnassa. Perinteiset steroidien analyysimenetelmät kärsivät usein joko riittämättömästä spesifisyydestä tai työläästä näytteiden esikäsittelystä. Tässä työssä tutkittiin testosteronin ja sen sukuisten steroidien määrittämistä kapillaarielektromigraatiotekniikoilla. Kyseiset tekniikat ovat varsin uusi lähtökohta steroiditutkimuksille. Kapillaarielektromigraatiotekniikat perustuvat molekyylien erottamiseen toisistaan voimakkaassa sähkökentässä. Nestemäinen näyte syötetään ohueen kapillaariin, jonka halkaisija on tyypillisesti 20 100 mikrometriä. Kapillaari on ennen näytteensyöttöä täytetty elektrolyyttinesteellä, ja näytteensyötön jälkeen sen molemmat päät asetetaan elektrolyyttiastioihin yhdessä elektrodien kanssa. Erotuksen aikaansaamiseksi elektrodien välille kytketään voimakas sähkökenttä. Sähköisesti varautuneet molekyylit (ionit) erottuvat sähkökentässä niiden erilaisten elektroforeettisten liikkuvuuksien mukaisesti. Steroidien sähköisestä varauksettomuudesta johtuen niillä ei ole elektroforeettista liikkuvuutta sähkökentässä. Kapillaarielektromigraatiotekniikoiden soveltaminen steroidien erottamiseen edellyttääkin ionisoituvien orgaanisten lisäaineiden käyttöä elektrolyyttiliuoksessa. Lisäaineilla voi olla joko spesifisiä tai epäspesifisia vuorovaikutuksia steroidien kanssa. Tässä työssä testosteronin ja sen sukuisten steroidien määrittämistä tutkittiin epäspesifisellä misellisellä sähkökineettisellä kromatografialla (MEKC). Elektrolyyttiliuoksessa käytettiin lisäaineina tensidejä sekä syklodekstriinejä. Kapillaari täytettiin vain osittain misellisellä elektrolyyttiliuoksella, minkä ansiosta steroidit voitiin tunnistaa sekä ultraviolettispektrofotometrisesti (UV) että sähkösumutus-ionisaatiomassaspektrofotometrisesti (ESI-MS). Osittaistäyttöön (PF) perustuva PF-MEKC UV osoittautui herkemmäksi, tehokkaammaksi ja toistettavammaksi menetelmäksi kuin PF-MEKC ESI-MS. Tutkimuksessa havaittiin, että ESI-MS-liitäntä asettaa huomattavia kemiallisia rajoituksia paitsi ionisaatio- ja detektointitapahtumille myös erotustapahtumalle. Uusilla PF-MEKC UV-menetelmillä steroidistandardit voidaan analysoida nopeasti, tehokkaasti ja kvantitatiivisesti. Herkin PF-MEKC UV-menetelmä soveltuu testosteronin nopeaan ja spesifiseen määrittämiseen miesten virtsanäytteistä miniatyrisoidun immunoaffiniteettikiinteäfaasiuuton (IA-SPE) jälkeen. Tutkimuksessa kehitetty IA-SPE perustuu vuorovaikutuksiin testosteronin ja sille spesifisen rekombinantin Fab-vasta-ainefragmentin välillä. IA-SPE on yksinkertainen ja nopea näytteen esikäsittelymenetelmä, jonka avulla tutkittava yhdiste voidaan konsentroida ja eristää muista näytemolekyyleistä. IA-SPE:tä ei ole aikaisemmin sovellettu testosteronin eristämiseen. Uutta tietoa steroidien sekä ihmisen ja naudan seerumin albumiiniproteiinien välisistä vuorovaikutuksista saatiin affiniteettikapillaarielektroforeesin avulla. Tutkimuksessa kehitettiin uusi algoritmi, jonka avulla voidaan laskea proteiinien ja neutraalien ligandien välisiä sitoutumisvakioita. Testosteronin ja sen sukuisten steroidien sitoutumisessa albumiiniproteiineihin havaittiin merkittäviä eroja

Advanced systems for the rapid detection of anthelmintic drugs in food

Several surface plasmon resonance (SPR) biosensor assays were developed and validated for the detection of anthelmintic veterinary drugs in liver tissue and milk using a QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction procedure. The first screening assay was developed to detect 11 benzimidazole carbamates in milk and liver. In bovine milk the assay showed a limit of detection (LOD) of 2.7 μg kg-1 and a detection capability (CCβ) of 5 μg kg-1. Analyte recovery was in the range 81 to 116% and the assay was found to be fit for purpose when its performance was compared to UPLC-MS/MS analyses of milk from cows treated with benzimidazole products. In bovine liver the LOD (32 μg kg-1) and the CCβ (50 μg kg-1) were determined and the analyte recovery was in the range 77-132%. All non-compliant samples were identified when the assay performance was tested by analysing liver from animals treated with benzimidazole drugs and comparing the results with a UPLC-MS/MS confirmatory method. A screening assay was developed for four amino-benzimidazoles in liver. The LOD (41 μg kg-1) and the CCβ (75 μg kg-1) were determined and the analyte recovery was in the range 103-116%. A screening assay for thiabendazole and 5-hydroxy-thiabendazole in ovine liver tissue using a novel recombinant antibody fragment (Fab) was developed. The LOD (12.3 μg kg-1), the CCβ (20 μg kg-1) and analyte recovery (86-107%) satisfied the criteria required for thiabendazole screening in liver tissue. A biosensor to detect triclabendazole residues in liver tissue was developed through the immobilization of amino-triclabendazole via a glutaraldehyde homo-bifunctional crosslinker. Several experiments were required to reduce non-specific binding in this assay. An LOD of 105 μg kg-1 was determined which was close to the maximum residue limit (MRL) in liver matrix (100 μg kg-1).A biochip array was developed and validated to screen orange juice for fungicide and pesticide residues. The LOD for carbendazim (20 μg kg-1), 2-aminobenzimidazole (4.0 μg kg-1), thiabendazole (4.2 μg kg-1) and ivermectin (10.2 μg kg-1) residues were determined. The CCβ for carbendazim (50 μg kg-1), 2-aminobenzimidazole (10 μg kg- 1), thiabendazole (10 μg kg-1) and ivermectin (20 μg kg-1) residues were sufficient for the analysis of orange juice. When orange juice from retail outlets in the greater Dublin area (n = 15) Two samples contained thiabendazole residues above the CCβ (260 and 181 μg kg-1) however these concentrations were below the maximum residue limit

Development of Mass Spectrometric Methods for Analysis of Anabolic Androgenic Steroids

Anabolic-androgenic steroids (AAS) are synthetic compounds that are structurally related to testosterone. AAS are an important class of drugs that are commonly abused in sport and are classified as prohibited substances according to the World Anti-Doping Agency (WADA). AAS are metabolized extensively in the human body and excreted to urine mostly as glucuronides or sulphates. These conjugated compounds are present in human specimens in low concentrations and for a limited period. Sensitive and specific analytical methods are thus highly important to be able to detect these compounds for an extended time period in athletes. These compounds are usually analyzed from biological matrixes after cleavage of the glucuronide or sulfonate moiety. Direct analysis of intact conjugated steroid metabolites is an attractive option that involves more straightforward sample preparation. Nonetheless, the commercial availability of the conjugated reference material is limited. The first aim of this study was to produce glucuronide-conjugated metabolites of AAS to be used in the development of methods for doping control. Glucuronide-conjugated metabolites of anabolic androgenic steroids were produced by in vitro enzyme-assisted synthesis. The method offers a simple and stereospecific procedure of producing small amounts of reference material, which are needed in the development and application of analytical methods for AAS metabolites. Secondly, these AAS glucuronides were utilized in the development of a liquid chromatography – mass spectrometry (LC-MS) method for detection of glucuronide-conjugated AAS metabolites in human urine. Novel method was validated, and the robustness and transferability of the developed method was studied in an interlaboratory comparison among seven laboratories. The developed method offers simpler and a more straightforward as well as sensitive approach to the analysis of exogenous steroids, without a hydrolysis process and time-consuming sample preparation. Another approach for a more sensitive and specific method for analysis of anabolic steroids from urine, was to combine gas chromatography (GC) featuring good resolving power to softer ionization in atmospheric pressure (API). A microchip-based miniaturized heated nebulizer provides easy interfacing for GC-API-MS with high ionization efficiency. Two sensitive and selective gas chromatography - microchip atmospheric pressure photoionization - tandem mass spectrometry (GC-µAPPI-MS/MS) methods were developed, validated and successfully applied to the analysis of anabolic steroids in authentic excretion urine samples. These methods offer valuable tools for anti-doping laboratories where new analytical strategies are needed to be able to detect an increasing number of prohibited compounds with various physico-chemical properties.Anaboliset steroidit ovat synteettisiä testosteronin johdannaisia, joiden käytön uskotaan tehostavan lihasmassan kasvua sekä urheilusuorituksesta palautumista. Anaboliset steroidit ovat yksi eniten väärinkäytetty dopingaineryhmä kilpaurheilussa. Anabolisten steroidien laboratorioanalytiikka on haastavaa, sillä mittaukset tehdään biologisesta matriisista, tutkittavia aineita on paljon ja mitattavat ainemäärät ovat pieniä. Tässä tutkimuksessa selvitettiin uudenlaisten massaspektrometristen mittausmenetelmien soveltuvuutta anabolisten steroidien dopinganalytiikkaan tarkoituksena parantaa nykyisten analyysien herkkyyttä ja tehokkuutta. Elimistön aineenvaihduntareaktiot muokkaavat steroideja, jotka erittyvät virtsaan pääasiassa glukuronihappoon konjugoituneena. Nämä konjugaatit esiintyvät virtsassa pieninä pitoisuuksina ja rajoitetun ajan. Anabolisten steroidien väärinkäytön toteaminen vaatiikin herkkiä ja spesifisiä analyysimenetelmiä. Perinteiset menetelmät steroidien dopingkäytön toteamiseksi perustuvat pääosin metaboliittien entsymaattiseen hajottamiseen ja epäsuoraan määrittämiseen. Nestekromatografis-massaspektrometrisilla (LC-MS) -menetelmillä on mahdollista analysoida myös suoraan steroidiglukuronideja. Suora määrittäminen nopeuttaa analyysejä näytteen esikäsittelyvaiheiden vähentymisen myötä. Uusien menetelmien kehittämiseen tarvitaan kuitenkin vertailuaineita ja metaboliittien kaupallinen saatavuus voi olla rajoitettua. Tämän työn ensimmäisessä vaiheessa tuotettiin entsyymiavusteisella synteesimenetelmällä anabolisten steroidien glukuronidikonjugaatteja ja kehitettiin LC–MS-menetelmä näiden yhdisteiden määrittämiseksi ihmisen virtsasta. Kehitetty menetelmä validoitiin ja testattiin useassa laboratoriossa. Menetelmä osoittautui erittäin toimivaksi ja laboratorioiden välisen testin perusteella sen siirtäminen toisiin laboratorioihin onnistuu hyvin. Työn toisessa vaiheessa testattiin kuumasumutus-mikrosirua, jonka avulla voitiin helposti yhdistää kaasukromatografi ilmanpaineionisaatio-massaspektrometriin. Ionisaatiomenetelmänä käytettiin hellävaraista ja herkkään fotoionisaatiota normaalissa ilmanpaineessa. Laiteyhdistelmällä kehitettiin kaksi uutta herkkää analyysimenetelmää anabolisten steroidien määrittämiseksi virtsanäytteistä. Menetelmien toimivuutta testattiin autenttisilla virstanäytteillä ja saatuja tuloksia verrattiin myös perinteisten analyysimenetelmien tuloksiin. Kehitetyt analyysimenetelmät osoittautuivat erittäin käyttökelpoisiksi dopinganalytiikassa ja niiden avulla pystytään tehostamaan anabolisten steroidien toteamista virtsanäytteistä

Analysis of steroids in urine by gas chromatography-capillary photoionization-tandem mass spectrometry

A new heated capillary photoionization (CPI) ion source design was developed to photoionize analytes inside a transfer capillary between a gas chromatograph (GC) and a mass spectrometer (MS). The CPI setup included a wide, oval-shaped vacuum-ultraviolet (VUV) transparent magnesium fluoride (MgF2) window to maximize photoionization efficiency and thus sensitivity. The source contained a nitrogen housing around the ionization chamber inlet to avoid undesirable hydrolysis and oxidation reactions with ambient air and to maximize the proportion of formed molecular radical cations of analytes. The feasibility of the ion source was studied by analyzing 18 endogenous steroids in urine as their trimethylsilyl (TMS) derivatives with gas chromatography-tandem mass spectrometry (GC-MS/MS). The method was validated and applied to human urine samples. To our best knowledge, this is the first time that a capillary photoionization ion source has been applied for quantitative analysis of biological samples. The GC-CPI-MS/MS method showed good chromatographic resolution (peak half-widths between 3.1 to 5.3 s), acceptable linearity (coefficient of determination between 0.981 to 0.996), and repeatability (relative standard deviation (RSD%) between 5 to 18%). Limits of detection (LOD) were between 2 to 100 pg mL(-1) and limits of quantitation (LOQ) were between 0.05 to 2 ng mL(-1). In total, 15 steroids were quantified either as a free steroid or glucuronide conjugate from the urine of volunteers. The new CPI source design showed excellent sensitivity for analysis of steroids in complex biological samples. (C) 2019 Elsevier B.V. All rights reserved.Peer reviewe

Development of On-Line High Performance Liquid Chromatography (HPLC)-Biochemical Detection Methods as Tools in the Identification of Bioactives

Biochemical detection (BCD) methods are commonly used to screen plant extracts for specific biological activities in batch assays. Traditionally, bioactives in the most active extracts were identified through time-consuming bio-assay guided fractionation until single active compounds could be isolated. Not only are isolation procedures often tedious, but they could also lead to artifact formation. On-line coupling of BCD assays to high performance liquid chromatography (HPLC) is gaining ground as a high resolution screening technique to overcome problems associated with pre-isolation by measuring the effects of compounds post-column directly after separation. To date, several on-line HPLC-BCD assays, applied to whole plant extracts and mixtures, have been published. In this review the focus will fall on enzyme-based, receptor-based and antioxidant assays

Analysis of anabolic steroids in urine by gas chromatography-microchip atmospheric pressure photoionization-mass spectrometry with chlorobenzene as dopant

Corrigendum to J. Chromatogr. A 1312 (2013), p. 111-117: Support by one project was inadvertently not mentioned in the Acknowledgements. The Acknowledgement should read:Funding from the Academy of Finland (projects 251575 and 257316) and the Finnish Funding Agency for Technology and Innovation(project 1291/31/084) is acknowledged. L. Hintikka is grateful to the CHEMSEM graduate school for financial support. Dr. Ville Saarela isthanked for the design and fabrication of the microchips. J. Chromatogr. A 1448 (2016) p. 128.Peer reviewe

Annual banned-substance review: Analytical approaches in human sports drug testing 2019/2020.

Analytical chemistry-based research in sports drug testing has been a dynamic endeavor for several decades, with technology-driven innovations continuously contributing to significant improvements in various regards including analytical sensitivity, comprehensiveness of target analytes, differentiation of natural/endogenous substances from structurally identical but synthetically derived compounds, assessment of alternative matrices for doping control purposes, and so forth. The resulting breadth of tools being investigated and developed by anti-doping researchers has allowed to substantially improve anti-doping programs and data interpretation in general. Additionally, these outcomes have been an extremely valuable pledge for routine doping controls during the unprecedented global health crisis that severely affected established sports drug testing strategies. In this edition of the annual banned-substance review, literature on recent developments in anti-doping published between October 2019 and September 2020 is summarized and discussed, particularly focusing on human doping controls and potential applications of new testing strategies to substances and methods of doping specified the World Anti-Doping Agency's 2020 Prohibited List

Exploiting bioluminescence to enhance the analytical performance of whole-cell and cell-free biosensors for environmental and point-of-care applications

The routine health monitoring of living organisms and environment has become one of the major concerns of public interest. Therefore, there has been an increasing demand for fast and easy to perform monitoring technologies. The current available analytical techniques generally offer accurate and precise results; however, they often require clean samples and sophisticated equipment. Thus, they are not suitable for on site, real-time, cost-effective routine monitoring. To this end, biosensors represent suitable analytical alternative tools. Biosensors are analytical devices integrating a biological recognition element (i.e. antibody, receptor, cell) and a transducer able to convert the biological response into an easily measurable analytical signal. These tools can easily quantify an analyte or a class of analytes of interest even in a complex matrix, like clinical or environmental samples, thanks to the specificity of the biological components. Whole-cell biosensors among others offer unique features such as low cost of production and provide comprehensive functional information (i.e. detection of unclassified compounds and synergistic effects, information about the bioavailable concentration). During this PhD, several bioengineered whole-cell biosensors have been developed and optimized for environmental and point-of-care applications. Analytical performance of biosensors have been improved (i.e. low limit of detection, faster response time and wider dynamic range) thanks to synthetic biology and genetic engineering tools. Bacterial, yeast and 3D cell cultures of mammalian cell lines have been tailored at the molecular level to improve robustness and predictivity. Several reporter genes, i.e. colorimetric, fluorescent and bioluminescent proteins, have been also profiled for finding the best candidate for each point-of-need application. Furthermore, spectral resolution of different optical reporter proteins has been exploited and multiplex detection has been achieved. The inclusion of viability control strains provided a suitable tool for assessing non-specific effects on cell viability, correcting the analytical signal and increasing the analytical performance of ready-to-use cartridges

Characterization of metabolites in urine samples from individuals with a lethal co-administration of cocaine and ethanol

In this paper, cocaine and eleven of its metabolites have been detected and identified in urine samples. The technique was based in a simple liquid-liquid extraction with detection by gas chromatography coupled to mass spectrometry (GC-MS). The method was validated in terms of repeatability, specificity, recovered, linearity, limit of detection, contamination between samples and robustness, all validation parameters met the acceptance criteria established in the study. The percentages of recovered for cocaine and benzoylecgonine were 120 and 110% respectively. The method’s repeatability (CV %) remained below 15% in all assays. The technique was shown to be linear over the range studied (r2 = 0.999) and sensitive, with detection limits below 60 ng/mL for both analytes. In the analysis of two real samples 11 metabolites of cocaine were detected, benzoylecgonine and cocaetilene were the majorities. Furthermore, were detected and characterized metabolites which only are observed in the urine in overdose cases such as hydroxylated and metoxyhydroxylated metabolites of cocaine, benzoylecgonine and cocaethilene. The technique allowed detecting the cinnamoilbenzoilecgonina which is a metabolite of cinnamoilcocaína. The cinnamoilcocaína is one of the alkaloids in coca plants so the presence of this metabolite showed us natural cocaine consumption and not synthetic