Drug Analysis without Primary Reference Standards : Application of LC-TOFMS and LC-CLND to Biofluids and Seized Material

Drug Analysis without Primary Reference Standards: Application of LC-TOFMS and LC-CLND to Biofluids and Seized Material Primary reference standards for new drugs, metabolites, designer drugs or rare substances may not be obtainable within a reasonable period of time or their availability may also be hindered by extensive administrative requirements. Standards are usually costly and may have a limited shelf life. Finally, many compounds are not available commercially and sometimes not at all. A new approach within forensic and clinical drug analysis involves substance identification based on accurate mass measurement by liquid chromatography coupled with time-of-flight mass spectrometry (LC-TOFMS) and quantification by LC coupled with chemiluminescence nitrogen detection (LC-CLND) possessing equimolar response to nitrogen. Formula-based identification relies on the fact that the accurate mass of an ion from a chemical compound corresponds to the elemental composition of that compound. Single-calibrant nitrogen based quantification is feasible with a nitrogen-specific detector since approximately 90% of drugs contain nitrogen. A method was developed for toxicological drug screening in 1 ml urine samples by LC-TOFMS. A large target database of exact monoisotopic masses was constructed, representing the elemental formulae of reference drugs and their metabolites. Identification was based on matching the sample component s measured parameters with those in the database, including accurate mass and retention time, if available. In addition, an algorithm for isotopic pattern match (SigmaFit) was applied. Differences in ion abundance in urine extracts did not affect the mass accuracy or the SigmaFit values. For routine screening practice, a mass tolerance of 10 ppm and a SigmaFit tolerance of 0.03 were established. Seized street drug samples were analysed instantly by LC-TOFMS and LC-CLND, using a dilute and shoot approach. In the quantitative analysis of amphetamine, heroin and cocaine findings, the mean relative difference between the results of LC-CLND and the reference methods was only 11%. In blood specimens, liquid-liquid extraction recoveries for basic lipophilic drugs were first established and the validity of the generic extraction recovery-corrected single-calibrant LC-CLND was then verified with proficiency test samples. The mean accuracy was 24% and 17% for plasma and whole blood samples, respectively, all results falling within the confidence range of the reference concentrations. Further, metabolic ratios for the opioid drug tramadol were determined in a pharmacogenetic study setting. Extraction recovery estimation, based on model compounds with similar physicochemical characteristics, produced clinically feasible results without reference standards.

Defining Adequate Vitamin D Intake : Cross-sectional and Intervention Studies

D-vitamiini ylläpitää normaalia luun kasvua ja uudistumista koko elämän ajan. Suomessa, kuten monissa muissakin länsimaissa, väestön D-vitamiinitilanne on riittämätön – talvisin osalla jopa puutteellinen. Tässä väitöskirjassa on tutkittu, lisääkö D-vitamiini luumassan kertymistä kasvuiässä, ja ylläpitäkö D-vitamiini luuston tasapainoista aineenvaihduntaa aikuisiällä. Nämä vaikutukset saattavat ehkäisi osteoporoosin kehittymistä eri ikäkausina. Väitöskirjatyössä tutkittiin erisuuruisten D-vitamiinilisäysten vaikutuksia kolmessa eri ikäryhmässä, jotka olivat 11-12 -vuotiaat tytöt (N=228), 21-49 -vuotiaat miehet (N=54) ja 65-85 -vuotiaat naiset (N=52). Tutkittavat satunnaistettiin ryhmiin, jotka nauttivat joko lumevalmistetta tai 5-20 µg D3-vitamiinia vitamiinilisänä. Tutkimukset olivat kaksoissokkoutettuja. Tutkimuksen aikana tutkittavilta otettiin paastoveri- ja virtsanäytteitä. Lisäksi he täyttivät tutkimuslomakkeen taustatietojen kartoittamiseksi sekä frekvenssikyselylomakkeen kalsiumin ja D-vitamiinin saannin selvittämiseksi. Tyttöjen luunmineraalitiheys (BMD) mitattiin DXA–laitteella ja miesten volumetrinen luuntiheys pQCT-menetelmällä. Näytteistä määritettiin mm. seerumin 25-hydroksi-D-vitamiinin (=S-25-OHD), lisäkilpirauhashormonin (=S-PTH) ja luun aineenvaihduntaa kuvaavien merkkiaineiden pitoisuuksia. Murrosikäisten tyttöjen poikkileikkaustutkimuksessa S-25-OHD- ja luun muodostusmerkkiaineen pitoisuudet vaihtelivat kuukausien välillä; suurimmat pitoisuudet mitattiin syyskuussa ja pienimmät maaliskuussa, mikä kuvastaa vuodenaikaisvaihtelua. Vastaava vaihtelu havaittiin lannerangan ja reisiluun BMD:ssä. D-vitamiinilisäyksellä oli myönteinen vaikutus tyttöjen luumassan lisääntymiseen. Suurin D-vitamiinilisä (10 µg/vrk) lisäsi luumassaa 17.2% enemmän reisiluussa ja 12.5% enemmän lannerangassa verrattuna lumevalmistetta nauttivien tyttöjen vastaaviin tuloksiin, mutta tulos riippui hoitomyöntyvyydestä. D-vitamiinin vaikutus luustoon välittyi vähentyneen luun hajotuksen kautta. Tutkimustuloksiin perustuen riittävä D-vitamiinin saanti murrosikäisille tytöille on 15 µg/vrk. D-vitamiinilisän vaikutus 65-85 -vuotiaiden naisten S-25-OHD-pitoisuuteen vakioitui kuudessa viikossa annoksen ollessa 5-20 µg/vrk. Näillä D-vitamiiniannoksilla ei saavutettu tavoiteltavaa S-25-OHD-pitoisuutta, joka on 80 nmol/l. Arvioimme, että 60 nmol/l -pitoisuuden, jota esiintyy kesäisin tämän ikäryhmän suomalaisilla, tämän ikäryhmän naiset saavuttaisivat 24 µg:n päivittäisellä D-vitamiinin saannilla. Terveillä miehillä havaittiin vuodenaikaisvaihtelu S-25-OHD- ja S-PTH-pitoisuudessa sekä luun hajotusta kuvaavassa merkkiainepitoisuudessa. Toisaalta vaihtelua ei havaittu radiuksen volumetrisessä luuntiheydessä eikä luun muodostusmerkkiaineen pitoisuudessa. Vuodenaikaisvaihtelu estettiin 17 µg:n päivittäisellä D-vitamiinin saannilla, mutta tämän ei havaittu vaikuttavan radiuksen luuntiheyteen kuusi kuukautta kestävän tutkimuksen aikana. Yhteenvetona todetaan, että D-vitamiinin saanti on edelleenkin riittämätöntä tutkimusten kohderyhmillä. Tämä näkyy S-25-OHD- ja PTH-pitoisuuden sekä luunaineenvaihduntaa kuvaavien merkkiaineiden vuodenaikaisvaihteluna, mikä on haitallista luuston hyvinvoinnille. D-vitamiinin saantia tulisi lisätä, jotta vähintäänkin riittävä D-vitamiinitilanne (S-25-OHD>50 nmol/l) tai mahdollisesti jopa tavoiteltava D-vitaminitilanne (S-25-OHD≥80 nmol/l) saavutettaisiin. Jotta D-vitamiinin saannin lisääminen olisi kaikissa ikäryhmissä mahdollista, on suunniteltava nykyistä enemmän D-vitamiinilla täydennettyjä elintarvikkeita.

Associations of gene polymorphisms and nutrition with calcium homeostasis and bone mineral density : Studies on skeletal nutrigenetics

Heredity explains a major part of the variation in calcium homeostasis and bone strength, and the susceptibility to osteoporosis is polygenetically regulated. Bone phenotype results from the interplay between lifestyle and genes, and several nutritional factors modulate bone health throughout life. Thus, nutrigenetics examining the genetic variation in nutrient intake and homeostatic control is an important research area in the etiology of osteoporosis. Despite continuing progress in the search for candidate genes for osteoporosis, the results thus far have been inconclusive. The main objective of this thesis was to investigate the associations of lactase, vitamin D receptor (VDR), calcium sensing receptor (CaSR) and parathyroid hormone (PTH) gene polymorphisms and lifestyle factors and their interactions with bone health in Finns at varying stages of the skeletal life span. Markers of calcium homeostasis and bone remodelling were measured from blood and urine samples. Bone strength was measured at peripheral and central bone sites. Lifestyle factors were assessed with questionnaires and interviews. Genetic lactase non-persistence (the C/C-13910 genotype) was associated with lower consumption of milk from childhood, predisposing females in particular to inadequate calcium intake. Consumption of low-lactose milk and milk products was shown to decrease the risk for inadequate calcium intake. In young adulthood, bone loss was more common in males than in females. Males with the lactase C/C-13910 genotype may be more susceptible to bone loss than males with the other lactase genotypes, although calcium intake predicts changes in bone mass more than the lactase genotype. The BsmI and FokI polymorphisms of the VDR gene were associated with bone mass in growing adolescents, but the associations weakened with age. In young adults, the A986S polymorphism of the calcium sensing receptor gene was associated with serum ionized calcium concentrations, and the BstBI polymorphism of the parathyroid gene was related to bone strength. The FokI polymorphism and sodium intake showed an interaction effect on urinary calcium excretion. A novel gene-gene interaction between the VDR FokI and PTH BstBI gene polymorphisms was found in the regulation of PTH secretion and urinary calcium excretion. Further research should be carried out with more number of Finns at varying stages of the skeletal life span and more detailed measurements of bone strength. Research should concern mechanisms by which genetic variants affect calcium homeostasis and bone strength, and the role of diet-gene and gene-gene interactions in the pathogenesis of osteoporosis.

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.

Identification of Isoflavonoid Metabolites in Humans

Epidemiological studies have associated high soy intake with a lowered risk for certain hormone-dependent diseases, such as breast and prostate cancers, osteoporosis, and cardiovascular disease. Soy is a rich source of isoflavones, diphenolic plant compounds that have been shown to possess several biological activities. Soy is not part of the traditional Western diet, but many dietary supplements are commercially available in order to provide the proposed beneficial health effects of isoflavones without changing the original diet. These supplements are usually manufactured from extracts of soy or red clover, which is another important source of isoflavones. However, until recently, detailed studies of the metabolism of these compounds in humans have been lacking. The aim of this study was to identify urinary metabolites of isoflavones originating from soy or red clover using gas chromatography - mass spectrometry (GC-MS). To examine metabolism, soy and red clover supplementation studies with human volunteers were carried out. In addition, the metabolism of isoflavones was investigated in vitro by identification of metabolites formed during a 24-h fermentation of pure isoflavones with a human fecal inoculum. Qualitative methods for identification and analysis of isoflavone metabolites in urine and fecal fermentation samples by GC-MS were developed. Moreover, a detailed investigation of fragmentation of isoflavonoids in electron ionization mass spectrometry (EIMS) was carried out by means of synthetic reference compounds and deuterated trimethylsilyl derivatives. After isoflavone supplementation, 18 new metabolites of isoflavones were identified in human urine samples. The most abundant urinary metabolites of soy isoflavones daidzein, genistein, and glycitein were found to be the reduced metabolites, i.e. analogous isoflavanones, a-methyldeoxybenzoins, and isoflavans. Metabolites having additional hydroxyl and/or methoxy substituents, or their reduced analogs, were also identified. The main metabolites of red clover isoflavones formononetin and biochanin A were identified as daidzein and genistein. In addition, reduced and hydroxylated metabolites of formononetin and biochanin A were identified; however, they occurred at much lower levels in urine samples than daidzein or genistein or their reduced metabolites. The results of this study show that the metabolism of isoflavones is diverse. More studies are needed to determine whether the new isoflavonoid metabolites identified here have biological activities that contribute to the proposed beneficial effects of isoflavones on human health. Another task is to develop validated quantitative methods to determine the actual levels of isoflavones and their metabolites in biological matrices in order to assess the role of isoflavones in prevention of chronic diseases.

Studies on CYP2C8-mediated drug interactions

Useiden lääkkeiden yhtäaikainen käyttö on nykyään hyvin yleistä, mikä lisää lääkeaineiden haitallisten yhteisvaikutusten riskiä. Lääkeaineiden poistumisessa elimistöstä ovat tärkeässä osassa niitä hajottavat (metaboloivat) maksan sytokromi P450 (CYP) entsyymit. Vasta aivan viime vuosina on havaittu, että CYP2C8-entsyymillä voi olla tärkeä merkitys mm. lääkeaineyhteisvaikutuksissa. Eräät lääkeaineet voivat estää (inhiboida) CYP2C8-entsyymin kautta tapahtuvaa metaboliaa. Tässä työssä selvitettiin CYP2C8-entsyymiä estävien lääkkeiden vaikutusta sellaisten lääkeaineiden pitoisuuksiin, joiden aikaisemman tiedon perusteella arveltiin metaboloituvan CYP2C8-välitteisesti. Näiden lääkeaineiden metaboliaa tutkittiin myös koeputkiolosuhteissa (in vitro -menetelmillä). Lisäksi CYP2C8-entsyymiä estävän lipidilääke gemfibrotsiilin yhteisvaikutusmekanismia tutkittiin selvittämällä interaktion säilymistä koehenkilöillä gemfibrotsiilin annostelun lopettamisen jälkeen. Yhteisvaikutuksia tutkittiin terveillä vapaaehtoisilla koehenkilöillä käyttäen vaihtovuoroista koeasetelmaa. Koehenkilöille annettiin CYP2C8-entsyymiä estävää lääkitystä muutaman päivän ajan ja tämän jälkeen kerta-annos tutkimuslääkettä. Koehenkilöiltä otettiin useita verinäytteitä, joista määritettiin lääkepitoisuudet nestekromatografisilla tai massaspektrometrisillä menetelmillä. Gemfibrotsiili nosti ripulilääke loperamidin pitoisuudet keskimäärin kaksinkertaiseksi. Gemfibrotsiili lisäsi, mutta vain hieman, kipulääke ibuprofeenin pitoisuuksia, eikä sillä ollut mitään vaikutusta unilääke tsopiklonin pitoisuuksiin toisin kuin aiemman kirjallisuuden perusteella oli odotettavissa. Toinen CYP2C8-estäjä, mikrobilääke trimetopriimi, nosti diabeteslääke pioglitatsonin pitoisuuksia keskimäärin noin 40 %. Gemfibrotsiili nosti diabeteslääke repaglinidin pitoisuudet 7-kertaiseksi ja tämä yhteisvaikutus säilyi lähes yhtä voimakkaana vielä 12 tunnin päähän viimeisestä gemfibrotsiiliannoksesta. Tehdyt havainnot ovat käytännön lääkehoidon kannalta merkittäviä ja ne selvittävät CYP2C8-entsyymin merkitystä useiden lääkkeiden metaboliassa. Gemfibrotsiilin tai muiden CYP2C8-entsyymiä estävien lääkkeiden yhteiskäyttö loperamidin kanssa voi lisätä loperamidin tehoa tai haittavaikutuksia. Toisaalta CYP2C8-entsyymin osuus tsopiklonin ja ibuprofeenin metaboliassa näyttää olevan pieni. Trimetopriimi nosti kohtalaisesti pioglitatsonin pitoisuuksia, ja kyseisten lääkkeiden yhteiskäyttö voi lisätä pioglitatsonin annosriippuvaisia haittavaikutuksia. Gemfibrotsiili-repaglinidi-yhteisvaikutuksen päämekanismi in vivo näyttää olevan CYP2C8-entsyymin palautumaton esto. Tämän vuoksi gemfibrotsiilin estovaikutus ja yhteisvaikutusriski säilyvät pitkään gemfibrotsiilin annostelun lopettamisen jälkeen, mikä tulee ottaa huomioon käytettäessä sitä CYP2C8-välitteisesti metaboloituvien lääkkeiden kanssa.

Pharmacokinetic interactions of pioglitazone

Pioglitazone is a thiazolidinedione compound used in the treatment of type 2 diabetes. It has been reported to be metabolised by multiple cytochrome P450 (CYP) enzymes, including CYP2C8, CYP2C9 and CYP3A4 in vitro. The aims of this work were to identify the CYP enzymes mainly responsible for the elimination of pioglitazone in order to evaluate its potential for in vivo drug interactions, and to investigate the effects of CYP2C8- and CYP3A4-inhibiting drugs (gemfibrozil, montelukast, zafirlukast and itraconazole) on the pharmacokinetics of pioglitazone in healthy volunteers. In addition, the effect of induction of CYP enzymes on the pharmacokinetics of pioglitazone in healthy volunteers was investigated, with rifampicin as a model inducer. Finally, the effect of pioglitazone on CYP2C8 and CYP3A enzyme activity was examined in healthy volunteers using repaglinide as a model substrate. Study I was conducted in vitro using pooled human liver microsomes (HLM) and human recombinant CYP isoforms. Studies II to V were randomised, placebo-controlled cross-over studies with 2-4 phases each. A total of 10-12 healthy volunteers participated in each study. Pretreatment with clinically relevant doses with the inhibitor or inducer was followed by a single dose of pioglitazone or repaglinide, whereafter blood and urine samples were collected for the determination of drug concentrations. In vitro, the elimination of pioglitazone (1 µM) by HLM was markedly inhibited, in particular by CYP2C8 inhibitors, but also by CYP3A4 inhibitors. Of the recombinant CYP isoforms, CYP2C8 metabolised pioglitazone markedly, and CYP3A4 also had a significant effect. All of the tested CYP2C8 inhibitors (montelukast, zafirlukast, trimethoprim and gemfibrozil) concentration-dependently inhibited pioglitazone metabolism in HLM. In humans, gemfibrozil raised the area under the plasma concentration-time curve (AUC) of pioglitazone 3.2-fold (P < 0.001) and prolonged its elimination half-life (t½) from 8.3 to 22.7 hours (P < 0.001), but had no significant effect on its peak concentration (Cmax) compared with placebo. Gemfibrozil also increased the excretion of pioglitazone into urine and reduced the ratios of the active metabolites M-IV and M-III to pioglitazone in plasma and urine. Itraconazole had no significant effect on the pharmacokinetics of pioglitazone and did not alter the effect of gemfibrozil on pioglitazone pharmacokinetics. Rifampicin decreased the AUC of pioglitazone by 54% (P < 0.001) and shortened its dominant t½ from 4.9 to 2.3 hours (P < 0.001). No significant effect on Cmax was observed. Rifampicin also decreased the AUC of the metabolites M-IV and M-III, shortened their t½ and increased the ratios of the metabolite to pioglitazone in plasma and urine. Montelukast and zafirlukast did not affect the pharmacokinetics of pioglitazone. The pharmacokinetics of repaglinide remained unaffected by pioglitazone. These studies demonstrate the principal role of CYP2C8 in the metabolism of pioglitazone in humans. Gemfibrozil, an inhibitor of CYP2C8, increases and rifampicin, an inducer of CYP2C8 and other CYP enzymes, decreases the plasma concentrations of pioglitazone, which can necessitate blood glucose monitoring and adjustment of pioglitazone dosage. Montelukast and zafirlukast had no effects on the pharmacokinetics of pioglitazone, indicating that their inhibitory effect on CYP2C8 is negligible in vivo. Pioglitazone did not increase the plasma concentrations of repaglinide, indicating that its inhibitory effect on CYP2C8 and CYP3A4 is very weak in vivo.

Pharmacokinetic interactions affecting the antidiabetic repaglinide

Introduction Repaglinide is a short-acting drug, used to reduce postprandial hyperglycaemia in type 2 diabetic patients. Repaglinide is extensively metabolised, and its oral bioavailability is about 60%; its metabolites are mainly excreted into bile. In previous studies, the cytochrome P450 (CYP) 3A4 inhibitors itraconazole and clarithromycin have moderately increased the area under the concentration-time curve (AUC) of repaglinide. Gemfibrozil, a CYP2C8 inhibitor, has greatly increased repaglinide AUC, enhancing and prolonging its blood glucose-lowering effect. Rifampicin has decreased the AUC and effects of repaglinide. Aims The aims of this work were to investigate the contribution of CYP2C8 and CYP3A4 to the metabolism of repaglinide, and to study other potential drug interactions affecting the pharmacokinetics of repaglinide, and the mechanisms of observed interactions. Methods The metabolism of repaglinide was studied in vitro using recombinant human CYP enzymes and pooled human liver microsomes (HLM). The effect of trimethoprim, cyclosporine, bezafibrate, fenofibrate, gemfibrozil, and rifampicin on the metabolism of repaglinide, and the effect of fibrates and rifampicin on the activity of CYP2C8 and CYP3A4 were investigated in vitro. Randomised, placebo-controlled cross-over studies were carried out in healthy human volunteers to investigate the effect of bezafibrate, fenofibrate, trimethoprim, cyclosporine, telithromycin, montelukast and pioglitazone on the pharmacokinetics and pharmacodynamics of repaglinide. Pretreatment with clinically relevant doses of the study drug or placebo was followed by a single dose of repaglinide, after which blood and urine samples were collected to determine pharmacokinetic and pharmacodynamic parameters. Results In vitro, the contribution of CYP2C8 was similar to that of CYP3A4 in the metabolism of repaglinide (< 2 μM). Bezafibrate, fenofibrate, gemfibrozil, and rifampicin moderately inhibited CYP2C8 and repaglinide metabolism, but only rifampicin inhibited CYP3A4 in vitro. Bezafibrate, fenofibrate, montelukast, and pioglitazone had no effect on the pharmacokinetics and pharmacodynamics of repaglinide in vivo. The CYP2C8 inhibitor trimethoprim inhibited repaglinide metabolism by HLM in vitro and increased repaglinide AUC by 61% in vivo (P < .001). The CYP3A4 inhibitor telithromycin increased repaglinide AUC 1.8-fold (P < .001) and enhanced its blood glucose-lowering effect in vivo. Cyclosporine inhibited the CYP3A4-mediated (but not CYP2C8-mediated) metabolism of repaglinide in vitro and increased repaglinide AUC 2.4-fold in vivo (P < .001). The effect of cyclosporine on repaglinide AUC in vivo correlated with the SLCO1B1 (encoding organic anion transporting polypeptide 1, OATP1B1) genotype. Conclusions The relative contributions of CYP2C8 and CYP3A4 to the metabolism of repaglinide are similar in vitro, when therapeutic repaglinide concentrations are used. In vivo, repaglinide AUC was considerably increased by inhibition of both CYP2C8 (by trimethoprim) and CYP3A4 (by telithromycin). Cyclosporine raised repaglinide AUC even higher, probably by inhibiting the CYP3A4-mediated biotransformation and OATP1B1-mediated hepatic uptake of repaglinide. Bezafibrate, fenofibrate, montelukast, and pioglitazone had no effect on the pharmacokinetics of repaglinide, suggesting that they do not significantly inhibit CYP2C8 or CYP3A4 in vivo. Coadministration of drugs that inhibit CYP2C8, CYP3A4 or OATP1B1 may increase the plasma concentrations and blood glucose-lowering effect of repaglinide, requiring closer monitoring of blood glucose concentrations to avoid hypoglycaemia, and adjustment of repaglinide dosage as necessary.

Bentsodiatsepiinien ja amfetamiinijohdannaisten seulonta virtsasta mikropilarisähkösumutusmassaspektrometrian avulla

Väärinkäytettyjen aineiden seulontaan käytetyn menetelmän tulee olla herkkä, selektiivinen, yksinkertainen, nopea ja toistettava. Työn tavoitteena oli kehittää yksinkertainen, mutta herkkä, esikäsittelymenetelmä bentsodiatsepiinien ja amfetamiinijohdannaisten kvalitatiiviseen seulomiseen virtsasta mikropilarisähkösumutussirun (μPESI) avulla, mikä tarjoaisi vaihtoehdon seulonnassa käytetyille immunologisille menetelmille, joiden herkkyys ja selektiivisyys ovat puutteellisia. Tavoitteena oli samalla tarkastella mikropilarisähkösumutussirun toimivuutta biologisten näytteiden analyysissa. Esikäsittely optimoitiin erikseen bentsodiatsepiineille ja amfetamiinijohdannaisille. Käytettyjä esikäsittelymenetelmiä olivat neste-nesteuutto, kiinteäfaasiuutto Oasis HLB-patruunalla ja ZipTip®-pipetinkärjellä sekä laimennus ja suodatus ilman uuttoa. Mittausten perusteella keskityttiin optimoimaan ZipTip®-uuttoa. Optimoinnissa tutkittavia yhdisteitä spiikattiin 0-virtsaan niiden ennaltamääritetyn raja-arvon verran, bentsodiatsepiineja 200 ng/ml ja amfetamiinijohdannaisia 300 ng/ml. Bentsodiatsepiinien kohdalla optimoitiin kutakin uuton vaihetta ja optimoinnin tuloksena näytteen pH säädettiin arvoon 5, faasi kunnostettiin asetonitriililla, tasapainotettiin ja pestiin veden (pH 5) ja asetonitriilin (10 % v/v) seoksella ja eluoitiin asetonitriilin, muurahaishapon ja veden (95:1:4 v/v/v) seoksella. Amfetamiinijohdannaisten uutossa optimoitiin näytteen ja liuottimien pH-arvoja ja tuloksena näytteen pH säädettiin arvoon 10, faasi kunnostettiin veden ja ammoniumvetykarbonaatin(pH 10, 1:1 v/v) seoksella, tasapainotettiin ja pestiin asetonitriilin ja veden (1:5 v/v) seoksella ja eluoitiin metanolilla. Optimoituja uuttoja testattiin Yhtyneet Medix Laboratorioista toimitetuilla autenttisilla virtsanäytteillä ja saatuja tuloksia verrattiin kvantitatiivisen GC/MS-analyysin tuloksiin. Bentsodiatsepiininäytteet hydrolysoitiin ennen uuttoa herkkyyden parantamiseksi. Autenttiset näytteet analysoitiin Q-TOF-laitteella Viikissä. Lisäksi hydrolysoidut bentsodiatsepiininäytteet mitattiin Yhtyneet Medix Laboratorioiden TOF-laitteella. Kehitetty menetelmä vaatii tulosten perusteella lisää optimointia toimiakseen. Ongelmana oli etenkin toistoissa ilmennyt tulosten hajonta. Manuaalista näytteensyöttöä tulisi kehittää toistettavammaksi. Autenttisten bentsodiatsepiininäytteiden analyysissa ongelmana olivat virheelliset negatiiviset tulokset ja amfetamiinijohdannaisten analyysissa virheelliset positiiviset tulokset. Virheellisiä negatiivisia tuloksia selittää menetelmän herkkyyden puute ja virheellisiä positiivisia tuloksia mittalaitteen, sirujen tai liuottimien likaantuminen.

Ketoprofeenin biologinen käytettävyys ja farmakokinetiikka sioilla eri antotavoilla ja ketoprofeeniyliannoksen farmakokinetiikka ja vaikutus munuaisiin lampailla

Ketoprofeeni on yleisesti käytetty ei-steroidinen tulehduskipulääke (NSAID) lampaiden ja sikojen kivunlievityksessä. Tietoa ketoprofeenin oikeista annosmääristä eri eläinlajeilla on saatavilla rajallisesti. Oikeaa lääkeainemäärää ei voida luotettavasti ekstrapoloida toisten eläinlajien tai ihmisten perusteella. Epäillyissä tulehduskipulääkemyrkytyksissä ongelmana on tietää, oliko eläimen saama lääkeannos toksinen. Lampailla tehdyn tutkimuksen tavoitteena oli selvittää, muuttuuko ketoprofeenin kinetiikka kymmenkertaisella yliannoksella, tutkia yliannoksen vaikutusta munuaisiin ja löytää yksinkertainen tapa diagnosoida yliannos virtsasta. Sioilla tehdyn tutkimuksen tavoitteena oli selvittää ketoprofeenin biologista käytettävyyttä ja ketoprofeenin farmakokinetiikkaa sioilla intravaskulaarisella, intramuskulaarisella ja peroraalisella annolla. Keskeiset tutkimuksessa määritettävät muuttujat olivat AUC0-_, Cmax ja tmax. Hyötyosuus laskettiin i.v. -annon perusteella. Kuudelle lampaalle annettiin 30 mg/kg i.v. -ketoprofeenia. Ketoprofeenin pitoisuuksia seurattiin 24 tunnin ajan plasmanäytteillä, joiden perusteella määritettiin farmakokineettiset parametrit. Veri- ja virtsanäytteistä tutkittiin muun muassa mahdollisesta munuaisvauriosta kertovia entsyymejä. 24 tunnin kuluttua lääkkeenannosta lampaat lopetettiin ja munuaiset tutkittiin histologisesti. Tutkittaville kahdeksalle sialle annosteltiin 3 mg/kg intravaskulaarista, intramuskulaarista ja oraalista ketoprofeenia sekä 6 mg/kg oraalista ketoprofeenia. Tutkimus suoritettiin satunnaistettuna vaihtovuorotutkimuksena. Ketoprofeenin pitoisuuksia seurattiin plasmanäytteillä 48 tunnin ajan lääkkeenannosta ja kaikille antotavoille laskettiin farmakokineettiset parametrit. Lisäksi tutkittiin valmisteiden biologinen samanarvoisuus. Molempien tutkimusten in vivo -kokeet suoritettiin Eläinlääketieteellisessä tiedekunnassa. Samoin munuaisten histologinen tutkimus ja virtsasta ja verestä tehdyt määritykset, lukuun ottamatta ketoprofeeninpitoisuuden analysointia. Plasman ketoprofeenipitoisuus analysoitiin korkean erotuskyvyn nestekromatografialla (HPLC). Ketoprofeenimääritykset ja farmakokineettinen analyysi suoritettiin Farmasian tiedekunnassa. Lampaiden kymmenkertainen ketoprofeeniyliannos oli toksinen. Seerumin urea- ja kreatiniinipitoisuus nousivat ja histologisissa näytteissä näkyi akuutti munuaistiehyen vaurio. Useiden entsyymien pitoisuus nousi virtsassa. Selvimmin ja nopeimmin nousi virtsan laktaattidehydrogenaasipitoisuus, jonka määrittäminen vaikuttaa potentiaaliselta tavalta diagnosoida ketoprofeenin toksinen annos. Ketoprofeenin eliminaation puoliintumisaika toksisella annoksella oli samaa suuruusluokkaa kuin aiemmissa tutkimuksissa terapeuttisella annoksella, joten yliannos ei muuttanut ketoprofeenin kinetiikkaa. AUC- ja Cmax -arvot olivat suhteessa suurempia kuin terapeuttisella annoksella, joten tutkimuksen perusteella kyseiset arvot eivät nousseet lineaarisesti annoksen noustessa toksiseksi. Sioille annetut ketoprofeenivalmisteet eivät olleet biologisesti samanarvoisia keskenään. Hyötyosuus oli erittäin hyvä kaikilla antotavoilla. tmax oli kaikilla antotavoilla hieman yli tunnin kuluttua lääkkeenannosta. Oraalisen 3 mg/kg -annoksen Cmax oli 5,1 mg/l ja AUC 32 mg l-1 h ja intramuskulaarisen vastaavat arvot olivat 7,6 mg/l ja 37 mg l-1 h. Oraalisen ketoprofeenin annostasojen AUC- ja Cmax -arvot korreloivat keskenään, joten ketoprofeenin kinetiikka oli lineaarista. Intravaskulaarisen ja oraalisen annon puoliintumisajoissa oli tilastollisesti merkitsevä ero. Ketoprofeenin jakautumistilavuudessa ja puhdistumassa ei ollut tilastollisesti merkitsevää eroa eri antotapojen välillä.

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.

Hypertension, kardiotoxicitet och njursvikt i samband med tyrosinkinashämmaren sunitinib

Nybildning av blodkärl från tidigare existerande kärl, angiogenes, är ett väsentligt skede vid tumörtillväxt. Denna process regleras av bland annat tillväxtfaktorer, var av den vaskulära endoteliala tillväxtfaktorn har en central roll. Hämning av angiogenes kan ske antingen extracellulärt med hjälp av humaniserade monoklonala antikroppar eller intracellulärt med hjälp av småmolekylära hämmaren. Sunitinib är en småmolekylär multikinashämmare och inhiberar flera tyrosinkinasreceptorer som påverkar tumörtillväxten och metastasutvecklingen vid cancer. Sunitinibs främsta indikationer är gastrointestinala stromacellstumörer, metastaserad njurcellscancer och neuroendokrina tumörer i bukspottskörteln. Behandling med tyrosinkinashämmare orsakar biverkningar som hypertension, kardiotoxicitet och njursvikt, vilka antas bero på de hämmande effekterna på mål som inte är väsentliga för anti-cancer-aktiviteten (”off-target” biverkningar). Bland annat AMP-aktiverat proteinkinas (AMPK), ett kinas som upprätthåller metabolisk homeostas i hjärtat, inhiberas av sunitinib och antas framkalla kardiovaskulära biverkningar. För att reducera ”off-target” biverkningar strävar man till att hitta alternativ som minskar de skadliga effekterna utan att den terapeutiska aktiviteten försvagas. Bland annat ett begränsat kaloriintag har uppvisat skyddande effekt på hjärtat via mekanismer sammankopplade till ökad resistens mot oxidativ stress, inflammation och mitokondriell dysfunktion, samt avtagande apoptos och autofagi. Detta sker delvis genom aktivering av enzymet Sirt1. Syftet med den här studien var att undersöka ifall kaloribegränsning skyddar mot kardiovaskulära och renala biverkningar inducerade av sunitinib hos råttor. Dessutom studerades vilka signalkedjor i cellen som medverkar. I studien användes 40 spontant hypertensiva råttor samt 10 normotensiva Wistar-Kyoto råttor. Försöksdjuren delades in i fem grupper beroende på behandling; I WKY kontroll, II SHR kontroll, III SHR + kaloribegränsning 70 %, IV SHR + sunitinib 3 mg/kg och V SHR + sunitinib 3 mg/kg + kaloribegränsning 70 %. Behandlingsperioden var åtta veckor. Blodtrycket mättes varje vecka med svansmanchett, urinutsöndringen undersöktes vecka 4 och vecka 8 med metabolismburar, ultraljudsundersökning av hjärtat utfördes sista veckan och blodkärlens respons till acetylkolin och natriumnitroprussid studerades i samband med avlivning. Proteinerna Sirt1 och AMPK analyserades i hjärtat med Western blotting samt förekomsten av makrofagmarkören ED1 i njurarna med immunhistokemi. Studien visade att sunitinibdosen 3 mg/kg är mycket väl tolererbar hos råttor eftersom sunitinib inte orsakade högre blodtryck, kraftigare hypertrofi eller mer omfattande njurskada jämfört med obehandlade SHR- grupper. Utgående från resultaten kan man också konstatera att kaloribegränsningen har positiva kardiovaskulära effekter.

Novel Mass Spectrometric Analysis Methods for Anabolic Androgenic Steroids in Sports Drug Testing

The feasibility of different modern analytical techniques for the mass spectrometric detection of anabolic androgenic steroids (AAS) in human urine was examined in order to enhance the prevalent analytics and to find reasonable strategies for effective sports drug testing. A comparative study of the sensitivity and specificity between gas chromatography (GC) combined with low (LRMS) and high resolution mass spectrometry (HRMS) in screening of AAS was carried out with four metabolites of methandienone. Measurements were done in selected ion monitoring mode with HRMS using a mass resolution of 5000. With HRMS the detection limits were considerably lower than with LRMS, enabling detection of steroids at low 0.2-0.5 ng/ml levels. However, also with HRMS, the biological background hampered the detection of some steroids. The applicability of liquid-phase microextraction (LPME) was studied with metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol. Factors affecting the extraction process were studied and a novel LPME method with in-fiber silylation was developed and validated for GC/MS analysis of the danazol metabolite. The method allowed precise, selective and sensitive analysis of the metabolite and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine without any other steps in sample preparation. Liquid chromatographic/tandem mass spectrometric (LC/MS/MS) methods utilizing electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were developed and applied for detection of oxandrolone and metabolites of stanozolol and 4-chlorodehydromethyltestosterone in urine. All methods exhibited high sensitivity and specificity. ESI showed, however, the best applicability, and a LC/ESI-MS/MS method for routine screening of nine 17-alkyl-substituted AAS was thus developed enabling fast and precise measurement of all analytes with detection limits below 2 ng/ml. The potential of chemometrics to resolve complex GC/MS data was demonstrated with samples prepared for AAS screening. Acquired full scan spectral data (m/z 40-700) were processed by the OSCAR algorithm (Optimization by Stepwise Constraints of Alternating Regression). The deconvolution process was able to dig out from a GC/MS run more than the double number of components as compared with the number of visible chromatographic peaks. Severely overlapping components, as well as components hidden in the chromatographic background could be isolated successfully. All studied techniques proved to be useful analytical tools to improve detection of AAS in urine. Superiority of different procedures is, however, compound-dependent and different techniques complement each other.

Liquid Chromatography-Mass Spectrometry in Studies of Drug Metabolism and Permeability

Poor pharmacokinetics is one of the reasons for the withdrawal of drug candidates from clinical trials. There is an urgent need for investigating in vitro ADME (absorption, distribution, metabolism and excretion) properties and recognising unsuitable drug candidates as early as possible in the drug development process. Current throughput of in vitro ADME profiling is insufficient because effective new synthesis techniques, such as drug design in silico and combinatorial synthesis, have vastly increased the number of drug candidates. Assay technologies for larger sets of compounds than are currently feasible are critically needed. The first part of this work focused on the evaluation of cocktail strategy in studies of drug permeability and metabolic stability. N-in-one liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the multiple component analysis of samples in cocktail experiments. Together, cocktail dosing and LC/MS/MS were found to form an effective tool for increasing throughput. First, cocktail dosing, i.e. the use of a mixture of many test compounds, was applied in permeability experiments with Caco-2 cell culture, which is a widely used in vitro model for small intestinal absorption. A cocktail of 7-10 reference compounds was successfully evaluated for standardization and routine testing of the performance of Caco-2 cell cultures. Secondly, cocktail strategy was used in metabolic stability studies of drugs with UGT isoenzymes, which are one of the most important phase II drug metabolizing enzymes. The study confirmed that the determination of intrinsic clearance (Clint) as a cocktail of seven substrates is possible. The LC/MS/MS methods that were developed were fast and reliable for the quantitative analysis of a heterogenous set of drugs from Caco-2 permeability experiments and the set of glucuronides from in vitro stability experiments. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), was evaluated through comparison with electrospray ionization (ESI), where both techniques were used for the analysis of Caco-2 samples. Like ESI, also APPI proved to be a reliable technique for the analysis of Caco-2 samples and even more flexible than ESI because of the wider dynamic linear range. The second part of the experimental study focused on metabolite profiling. Different mass spectrometric instruments and commercially available software tools were investigated for profiling metabolites in urine and hepatocyte samples. All the instruments tested (triple quadrupole, quadrupole time-of-flight, ion trap) exhibited some good and some bad features in searching for and identifying of expected and non-expected metabolites. Although, current profiling software is helpful, it is still insufficient. Thus a time-consuming largely manual approach is still required for metabolite profiling from complex biological matrices.

Miniaturized mass spectrometric ionization techniques for environmental analysis and bioanalysis

Miniaturized mass spectrometric ionization techniques for environmental analysis and bioanalysis Novel miniaturized mass spectrometric ionization techniques based on atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) were studied and evaluated in the analysis of environmental samples and biosamples. The three analytical systems investigated here were gas chromatography-microchip atmospheric pressure chemical ionization-mass spectrometry (GC-µAPCI-MS) and gas chromatography-microchip atmospheric pressure photoionization-mass spectrometry (GC-µAPPI-MS), where sample pretreatment and chromatographic separation precede ionization, and desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS), where the samples are analyzed either as such or after minimal pretreatment. The gas chromatography-microchip atmospheric pressure ionization-mass spectrometry (GC-µAPI-MS) instrumentations were used in the analysis of polychlorinated biphenyls (PCBs) in negative ion mode and 2-quinolinone-derived selective androgen receptor modulators (SARMs) in positive ion mode. The analytical characteristics (i.e., limits of detection, linear ranges, and repeatabilities) of the methods were evaluated with PCB standards and SARMs in urine. All methods showed good analytical characteristics and potential for quantitative environmental analysis or bioanalysis. Desorption and ionization mechanisms in DAPPI were studied. Desorption was found to be a thermal process, with the efficiency strongly depending on thermal conductivity of the sampling surface. Probably the size and polarity of the analyte also play a role. In positive ion mode, the ionization is dependent on the ionization energy and proton affinity of the analyte and the spray solvent, while in negative ion mode the ionization mechanism is determined by the electron affinity and gas-phase acidity of the analyte and the spray solvent. DAPPI-MS was tested in the fast screening analysis of environmental, food, and forensic samples, and the results demonstrated the feasibility of DAPPI-MS for rapid screening analysis of authentic samples.