Metabolite analysis of [<Superscript>18</Superscript>F]Florbetaben (BAY 94-9172) in human subjects: a substudy within a proof of mechanism clinical trial

[¹⁸F]Florbetaben ([¹⁸F]BAY 94-9172) is a promising β-amyloid (Aβ) targeted PET-tracer currently in late stage clinical development. [¹⁸F]Florbetaben can assist in the more accurate diagnosis of Alzheimer’s Disease (AD) by non-invasive, in vivo detection of Aβ in the brain. To determine the arterial input function of the PET tracer—as part of a proof of mechanism (PoM) study—arterial samples were drawn from all subjects at predefined time points post injection (p.i.), and the proportion of unchanged tracer [¹⁸F]Florbetaben was determined by HPLC analysis. Plasma metabolite profiles were investigated following intravenous administration of 300 MBq (±60 MBq) of [¹⁸F]Florbetaben to both, patients with AD and healthy controls (HCs), and various methods for processing the blood samples were evaluated. Addition of acetonitrile to plasma samples (obtained from whole blood by centrifugation) and precipitation of proteins resulted in a recovery of more than 90% of the initial radioactivity in the supernatants. High Performance Liquid Chromatography using a polymer-based column (PRP-1) in conjunction with gradient elution was found to be a suitable method of metabolite analysis of [¹⁸F]Florbetaben. HPLC analyses indicated that [¹⁸F]Florbetaben is rapidly metabolized in vivo with an estimated initial half-life of about 6 min. A polar metabolite fraction, consisting presumably of more than one component, and (to a smaller extent) of the demethylated derivative of [¹⁸F]Florbetaben were time-dependently detected in plasma.     

Evaluation of standard and advanced preprocessing methods for the univariate analysis of blood serum 1H-NMR spectra

Proton nuclear magnetic resonance (¹H-NMR)-based metabolomics enables the high-resolution and high-throughput assessment of a broad spectrum of metabolites in biofluids. Despite the straightforward character of the experimental methodology, the analysis of spectral profiles is rather complex, particularly due to the requirement of numerous data preprocessing steps. Here, we evaluate how several of the most common preprocessing procedures affect the subsequent univariate analyses of blood serum spectra, with a particular focus on how the standard methods perform compared to more advanced examples. Carr–Purcell–Meiboom–Gill 1D ¹H spectra were obtained for 240 serum samples from healthy subjects of the Asklepios study. We studied the impact of different preprocessing steps—integral (standard method) and probabilistic quotient normalization; no, equidistant (standard), and adaptive-intelligent binning; mean (standard) and maximum bin intensity data summation—on the resonance intensities of three different types of metabolites: triglycerides, glucose, and creatinine. The effects were evaluated by correlating the differently preprocessed NMR data with the independently measured metabolite concentrations. The analyses revealed that the standard methods performed inferiorly and that a combination of probabilistic quotient normalization after adaptive-intelligent binning and maximum intensity variable definition yielded the best overall results (triglycerides, R = 0.98; glucose, R = 0.76; creatinine, R = 0.70). Therefore, at least in the case of serum metabolomics, these or equivalent methods should be preferred above the standard preprocessing methods, particularly for univariate analyses. Additional optimization of the normalization procedure might further improve the analyses.     

Hydrogen peroxide induced formation of peroxystannate nanoparticles

Stable, amorphous potassium peroxystannate nanoparticles of controlled average size—in the range 10–100 nm—and of controlled hydrogen peroxide content—in the range of 19–30 wt%—were synthesized by hydrogen peroxide induced polymerization in water–potassium hexahydroxostannate solutions. The sol phase and the precipitate were characterized by vibrational spectroscopies, ¹¹⁹Sn NMR, XPS and XRD using crystalline K₂Sn(OH)₆ and K₂Sn(OOH)₆ reference materials. This is the first study to show that peroxocoordination induces polymerization of a main group element. ¹¹⁹Sn NMR studies show that peroxotin coordination and polymerization took place already in the hydrogen peroxide–water phase. The high abundance of peroxotin bonds revealed by ¹¹⁹Sn MAS NMR, vibrational spectroscopy, and XPS suggests that the particles are predominantly made of peroxo bridged tin networks. Although the particles are highly stable in the dry phase as well as in alcohol solutions and do not lose hydrogen peroxide upon storage, they release their stored hydrogen peroxide content by exposure to water.     

FT-midIR determination of fatty acid profiles, including trans fatty acids, in bakery products after focused microwave-assisted Soxhlet extraction

A study of the feasibility of Fourier transform medium infrared spectroscopy (FT-midIR) for analytical determination of fatty acid profiles, including trans fatty acids, is presented. The training and validation sets—75% (102 samples) and 25% (36 samples) of the samples once the spectral outliers have been removed—to develop FT-midIR general equations, were built with samples from 140 commercial and home-made bakery products. The concentration of the analytes in the samples used for this study is within the typical range found in these kinds of products. Both sets were independent; thus, the validation set was only used for testing the equations. The criterion used for the selection of the validation set was samples with the highest number of neighbours and the most separation between them (H<0.6). Partial least squares regression and cross validation were used for multivariate calibration. The FT-midIR method does not require post-extraction manipulation and gives information about the fatty acid profile in two min. The 14:0, 16:0, 18:0, 18:1 and 18:2 fatty acids can be determined with excellent precision and other fatty acids with good precision according to the Shenk criteria, R ²≥0.90, SEP=1–1.5 SEL and R ²=0.70-0.89, SEP=2–3 SEL, respectively. The results obtained with the proposed method were compared with those provided by the conventional method based on GC-MS. At 95% significance level, the differences between the values obtained for the different fatty acids were within the experimental error. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Some experiences with a mass spectrometry data system validation

Summary An HPLC method with UV and radioactivity detection is reported for analysis of the toxic metabolite of acetaminophen (paracetamol) in human liver samples. Acetaminophen and its oxidative metabolites were separated on an RP-18 column with aqueous NH₄-acetate:methanol mobile phase. The¹⁴C-acetaminophen and its metabolites were monitored at 250 nm and by online-coupled radioactivity detection. HPLC-MS indicated the presence of the toxic metabolite—the new method proving suitable for its serial analysis. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Effect of preparation procedure on the properties of CeO2

The effect of preparation procedure on the physicochemical and catalytic properties of CeO₂ was studied. Differences in the electronic and structural characteristics of CeO₂ depending on preparation procedure and treatment temperature were found using X-ray diffraction analysis, transmission electron microscopy, UV-visible electronic spectroscopy, and X-ray photoelectron spectroscopy. With the use of the temperature-programmed reaction with CO, it was demonstrated that CeO₂ samples with a high concentration of point defects—oxygen vacancies caused by the presence of Ce³⁺—were characterized by an increased mobility of bulk oxygen. The samples of CeO₂ with a high concentration of structural defects—micropores of size 1–2 nm and stepwise vicinal faces in crystallites—exhibited a high catalytic activity in the reaction of CO oxidation.     

Withasteroids of Physalis. IX. Physangulide — The first natural 22S-withasteroid

A new withasteroid — physangulide — has been isolated from the leaves ofPhysalis angulata L. It has been shown that physangulide is the first natural 22S-withanolide. Its structure has been determined as 3β,4β,20,24,25-pentahydroxy-1-oxo-5β,6β-epoxy-20R,22S,24S,25R-withanolide. Its¹H and¹³C NMR spectra, confirming this interpretation, are given. Institute of the Chemistry of Plant Substances, Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 366–371, May–June, 1990.     

Cembrenolides of a soft coral Lobophytum sp. (coelenterata,octocorallia, alcyonacea)

The known cembrenolide sarcophin and its new acetoxy derivative — 13-acetoxy-7,8-epoxycembra-1(15),3,11-trien-2,16-olide — have been isolated from the soft coralLobophytum sp.. The compounds isolated are inhibitors of the activity of Na⁺K⁺-ATPase and are membranotropic agents. The structures of the compounds have been shown on the basis of the results of NMR spectroscopy. Pacific Ocean Institute of Bioorganic Chemistry, Far Eastern Branch, Academy of Sciences of the USSR, Vladivostok. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 762–765, November–December, 1990.     

Determination of arsenic species and arsenosugars in marine samples by HPLC–ICP–MS

Arsenic-speciation analysis in marine samples was performed by high-pressure liquid chromatography (HPLC) with ICP–MS detection. Separation of eight arsenic species—As^III, MMA, DMA, As^V, AB, TMAO, AC and TeMAs⁺—was achieved on a C₁₈ column with isocratic elution (pH 3.0), under which conditions As^III and MMA co-eluted. The entire separation was accomplished in 15 min. The HPLC–ICP–MS detection limits for the eight arsenic species were in the range 0.03–0.23 μg L⁻¹ based on 3σ for the blank response (n=5). The precision was calculated to be 2.4–8.0% (RSD) for the eight species. The method was successfully applied to several marine samples, e.g. oysters, fish, shrimps, and marine algae. Low-power microwave digestion was employed for extraction of arsenic from seafood products; ultrasonic extraction was employed for the extraction of arsenic from seaweeds. Separation of arsenosugars was achieved on an anion-exchange column. Concentrations of arsenosugars 2, 3, and 4 in marine algae were in the range 0.18–9.59 μg g⁻¹. This paper was presented at the European Winter Conference 2005     

Metabolite assignment of ultrafiltered synovial fluid extracted from knee joints of reactive arthritis patients using high resolution NMR spectroscopy

Currently, there are no reliable biomarkers available that can aid early differential diagnosis of reactive arthritis (ReA) from other inflammatory joint diseases. Metabolic profiling of synovial fluid (SF)—obtained from joints affected in ReA—holds great promise in this regard and will further aid monitoring treatment and improving our understanding about disease mechanism. As a first step in this direction, we report here the metabolite specific assignment of ¹H and ¹³C resonances detected in the NMR spectra of SF samples extracted from human patients with established ReA. The metabolite characterization has been carried out on both normal and ultrafiltered (deproteinized) SF samples of eight ReA patients (n = 8) using high-resolution (800 MHz) ¹H and ¹H─¹³C NMR spectroscopy methods such as one-dimensional ¹H CPMG and two-dimensional J-resolved¹H NMR and homonuclear ¹H─¹H TOCSY and heteronuclear¹H─¹³C HSQC correlation spectra. Compared with normal SF samples, several distinctive ¹H NMR signals were identified and assigned to metabolites in the ¹H NMR spectra of ultrafiltered SF samples. Overall, we assigned 53 metabolites in normal filtered SF and 64 metabolites in filtered pooled SF sample compared with nonfiltered SF samples for which only 48 metabolites (including lipid/membrane metabolites as well) have been identified. The established NMR characterization of SF metabolites will serve to guide future metabolomics studies aiming to identify/evaluate the SF-based metabolic biomarkers of diagnostic/prognostic potential or seeking biochemical insights into disease mechanisms in a clinical perspective.     

Alkaloids ofArundo donas VII. A spectroscopic and X-ray structural investigation of arundinine—A new dimeric alkaloid from the epigeal part ofArundo donax

FromArundo donax L. we have isolated the new dimeric alkaloid arundinine, with the composition C₂₃H₂₈N₄O, the structure of which has been established on the basis of its physicochemical characteristics and¹H and¹³C NMR spectroscopy, including 1D and 2D experiments in both homo- and heteronuclear regimes. The proposed structure—3a-(3′-dimethylaminoethylindol-5′-yloxy)-1-methylpyrrolidino[1a,3a-b]indoline — has been confirmed by x-ray structural analysis (XSA). Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (371) 120 64 75. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 790–795, November–December, 1998.     

Element profiles in galvanostatically polarized K+-selective all-solid-state sensors with poly(vinyl chloride)-based membranes

The influence of galvanostatic polarization on ion concentration profiles in all-solid-state ion-selective sensors was studied. As a model system K⁺-selective electrode with poly(vinyl chloride)-based membrane, ionophore–valinomycin and polypyrrole doped by chloride ions as ion-to-electron transducer was selected. The ion exchanger—a typical component of ion-selective membrane—was replaced by lipophilic salt: tetradodecylammonium tetrakis(4-chlorophenyl) borate to avoid spontaneous extraction of potassium ions. Potassium, sodium, and chlorine distribution within the sensor phases were studied using laser ablation micro-sampling followed by inductively coupled plasma mass spectrometry measurements. The experiments revealed accumulation of potassium ions in course of cathodic galvanostatic polarization, with concentration decreasing by moving inside the ion-selective membrane. The surface content of K⁺ ions was found to be linearly dependent on applied current. Influence of sequential anodic galvanostatic polarization or open circuit conditioning applied after cathodic polarization revealed only limited recovery of the initial concentration profiles in the membrane.     

Determination of olanzapine and desmethylolanzapine in the plasma of schizophrenic patients by means of an improved HPLC method with amperometric detection

Summary An improved HPLC method with electrochemical detection has been developed for the determination of olanzapine and its main metabolite, desmethylolanzapine, in human plasma. Chromatographic separation and analysis were performed on a C₈ reversed-phase column with a mixture of methanol, acetonitrile, and pH 3.7 phosphate buffer as mobile phase; 2-methylolanzapine was used as internal standard. Careful pretreatment of the plasma samples was implemented by means of solid phase extraction (SPE). Response was linearly dependent on concentration and precision was satisfactory over the concentration range 0.5–75.0 ng mL⁻¹ for both analytes. The limit of detection was 0.2 ng mL⁻¹ for both analytes. Application to plasma samples of patients treated with Zyprexa tablets gave good results. Because of its sensitivity and selectivity, and the need for small plasma samples, this method seems to be a useful tool for clinical monitoring.     

Spectral elucidation of the acid metabolites of the four geometric isomers of trifloxystrobin

Four geometric isomers of trifloxystrobin (TFS)—namely EE, EZ, ZE, and ZZ—were hydrolyzed by 0.05 M NaOH, resulting in four corresponding acid metabolites. These compounds—namely EE-, EZ-, ZE-, and ZZ-acids—were purified by preparative HPLC and authentically characterized by a combination of infrared, Raman, GC–MS, LC–MS/MS, and NMR spectroscopies. The spectra were found to be very characteristic of the individual isomers, and so they could be used to distinguish the isomers from each other. The detailed spectral features of the individual isomers are presented and compared. EE-acid was identified as being the major metabolite of TFS in soil, which indicates that hydrolysis is the principal route of degradation of TFS. This finding further justifies the importance of the present study in relation to assessing the risk associated with the release of TFS into the environment.     

LC–ESI–MS for Rapid and Sensitive Determination of Aripiprazole in Human Plasma

A rapid, sensitive, and accurate high-performance liquid-chromatographic–mass spectrometric (HPLC–MS) method, with estazolam as internal standard, has been developed and validated for determination of aripiprazole in human plasma. After liquid–liquid extraction the compound was analyzed by HPLC on a C₁₈ column, with acetonitrile—30 mm ammonium acetate containing 0.1% formic acid, 58:42 (v/v), as mobile phase, coupled with electrospray ionization mass spectrometry (ESI-MS). The protonated analyte was quantified by selected-ion recording (SIR) with a quadrupole mass spectrometer in positive-ion mode. Calibration plots were linear over the concentration range 19.9–1119.6 ng mL⁻¹. Intra-day and inter-day precision (CV%) and accuracy (RE%) for quality-control samples (37.3, 124.4, and 622.0 ng mL⁻¹) ranged between 2.5 and 9.0% and between 1.3 and 3.5%, respectively. Extraction recovery of aripiprazole from plasma was in the range 75.8–84.1%. The method enables rapid, sensitive, precise, and accurate measurement of the concentration of aripiprazole in human plasma.     

Detectability of testosterone esters and estradiol benzoate in bovine hair and plasma following pour-on treatment

The abuse of synthetic esters of natural steroids such as testosterone and estradiol in cattle fattening and sports is hard to detect via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. An interesting alternative can be provided by the analysis of the administered synthetic steroids themselves, i.e., the analysis of intact steroid esters in hair by liquid chromatography tandem mass spectrometry (LC/MS/MS). However, retrospective estimation of the application date following a non-compliant finding is hindered by the complexity of the kinetics of the incorporation of steroid esters in hair. In this study, the incorporation of intact steroid esters in hair following pour-on treatment has been studied and critically compared with results from intramuscular treatment. To this end animals were pour-on treated with a hormone cocktail containing testosterone cypionate, testosterone decanoate and estradiol benzoate in different carriers. The animals were either treated using injection and pour-on application once or three times having 1 week between treatments using injection and pour-on application. Animals were slaughtered from 10–12 weeks after the last treatment. Both hair and blood plasma samples were collected and analysed by LC/MS/MS. From the results, it is concluded that after single treatment the levels of steroid esters in hair drop to CCβ levels (5–20 μg/kg) after 5–7 weeks. When treatment is repeated two times, the CCβ levels are reached after 9–11 weeks. Furthermore, in plasma, no steroid esters were detected; not even at the low microgramme per litre level but—in contrast with the pour-on application—after i.m. injection, significant increase of 17β-testosterone and 17β-estradiol were observed. These observations suggest that transport of steroid esters after pour-on application is not only performed by blood but also by alternative fluids in the animal so probably the steroid esters are already hydrolysed and epimerized before entering the blood.     

Elimination of 7-aminoclonazepam in urine after a single dose of clonazepam

The objective of this paper was to determine how long after administration of benzodiazepine clonazepam (CLO), its major metabolite 7-aminoclonazepam (7-ACLO) could be detected in urine collected from 10 healthy volunteers who received a single 3-mg dose of Klonopin (clonazepam). Such data would be of great importance to law enforcement agencies trying to determine the best time interval for urine collection from a victim of drug-facilitated sexual assault in order to reveal drug use. A highly sensitive NCI–GC–MS method for the simultaneous quantitation of CLO and its major metabolite 7-ACLO in urine was developed and validated. The following urine samples were collected from each volunteer: one before CLO administration, and 6 h, and 1, 3, 5, 8, 10, 14, 21 and 28 days after. All urine samples (1 mL) were extracted following addition of the internal standard (D₅-diazepam) and enzymatic hydrolysis (-glucuronidase) using solid-phase extraction columns. Standard curves for CLO (500–4000 pg mL^–1) and 7-ACLO (50–2000 pg mL^–1) were prepared by spiking aliquots of negative urine. The urine from every subject was still positive for 7-ACLO 14 days after administration of the drug. Eight of the ten volunteers had measurable amounts of the metabolite 21 days after administration. One volunteer was still positive 28 days after administration. Six of the volunteers had urine concentrations of 7-ACLO that peaked at 1 day after administration. One volunteer had the highest concentration of 7-ACLO at 3 days, two volunteers at 5 days, and one at 8 days. The range of concentrations detected was from 73.0 pg mL^–1 to 183.2 ng mL^–1. CLO was not detected in any of the samples.     

Water clusters in supramolecular inclusion hydrates. Crystal structure of decaazatricyclo[28.2.2.213.16]tetratriacontane nanohydrate

Inclusion compounds of solvate water molecules in a crystal matrix are investigated by X-ray diffraction analysis of a macrocyclic polyamine decaazatricyclo[28.2.2.2^13.16]tetratriacontane nanohydrate (I). When included in a crystal, water molecules are bonded by water-water (O-H...O) and water—macrocycle (0-H...N and N-H...O) hydrogen bonds, forming a linear cluster. Translated fromZhurnal Strukturnoi Khimii, Vol. 40, No. 5, pp. 993–1001, September–October, 1999.     

An improved high-performance liquid chromatography method for the determination of homocysteine in human plasma

Summary Elevated plasma homocysteine is, a known risk factor in arteriosclerotic vascular disease. To measure homocysteine in a large number of samples, we have developed a rapid, simple, robust and inexpensive reversed-phase HPLC method for routine analysis. Mercaptopro-pionylglycine was used as the internal standard and an external calibration in plasma was performed. Improvement was achieved by the use of gradient elution (using a sodium acetate buffer and methanol) resulting in a higher number of samples analyzed per day. Plasma samples were reduced with tributylphosphine and the proteins were precipitated with perchloric acid before addition of internal standard. The analytes were derivatized by use of 7-fluorobenzofurazone-4-sulfonic acid ammonium salt. For calibration human plasma was spiked with nine different concentrations of homocysteine (range 2–50 μmol L⁻¹). The inter-assay precision of replicate (n=29) analysis of the concentration of homocysteine in a sample of pooled plasma was 3.0%. The limit of detection, defined as three times the signal-to-noise ratio, was 0.25 μmol L⁻¹. The linearity of the assay was confirmed for a plasma concentration range of 2–2000 μmol L⁻¹. The variation of duplicate analyses of 842 plasma samples was 2.6±1.7%.     

The effects of abundant plasma protein depletion on global glycan profiling using NanoLC FT-ICR mass spectrometry

We report the results of abundant plasma protein depletion on the analysis of underivatized N-linked glycans derived from plasma proteins by nanoLC Fourier-transform ion cyclotron resonance mass spectrometry. N-linked glycan profiles were compared between plasma samples where the six most abundant plasma proteins were depleted (n = 3) through a solid-phase immunoaffinity column and undepleted plasma samples (n = 3). Three exogenous glycan standards were spiked into all samples which allowed for normalization of the N-glycan abundances. The abundances of 20 glycans varying in type, structure, composition, and molecular weight (1,200–3,700 Da) were compared between the two sets of samples. Small fucosylated non-sialylated complex glycans were found to decrease in abundance in the depleted samples (greater than or equal to tenfold) relative to the undepleted samples. Protein depletion was found to marginally effect (less than threefold) the abundance of high mannose, hybrid, and large highly sialylated complex species. The significance of these findings in terms of future biomarker discovery experiments via global glycan profiling is discussed.