242 research outputs found
Semiautomated High-Performance Liquid Chromatographic Method for the Determination of Benzodiazepines in Whole Blood
A semiautomated method for the determination of five frequently prescribed benzodiazepines (BZD) (clonazepam, diazepam, flunitrazepam, midazolam, and oxazepam) in whole blood samples by reversed-phase high-performance liquid chromatography following simple online enrichment and clean-up on a short precolumn is described. After precipitation of protein and red cells with a mixture of organic solvents (methanol/acetonitrile, 50:50), the aliquot is centrifuged and the organic upper phase evaporated under a gentle stream of nitrogen. The residue is reconstituted by adding 500 pL of a mixture of phosphate buffer (20mM, pH 2.2) and acetonitrile (70:30, v/v). The sample is then directly introduced into the column-switching column. The precolumn is first washed with phosphate buffer at pH 7.2. Compounds retained on the precolumn are then eluted in the back-flush mode and separated on a C8 semi-microcolumn (Lichrospher select B, 125 Ă 3 mm). The BZD studied are determined by a diode-array detector at 254 nm. The method shows excellent linearity between 25 and 1000 ng/mL for clonazepam, flunitrazepam, and midazolam and between 25 and 5000 ng/mt for diazepam and oxazepam. The recoveries are around 80% for clonazepam and oxazepam and around 90% for the three others. Coefficients of variation for between-day and within-day assays are < 15% for low concentrations close to the limit of quantitation and < 5% for high concentration
Analysis of cannabinoids in oral fluid by liquid chromatography-tandem mass spectrometry
A sensitive method was developed for quantifying a wide range of cannabinoids in oral fluid (OF) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These cannabinoids include â9-tetrahydrocannabinol (THC), 11-hydroxy-â9-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-â9-tetrahydrocannabinol (THCCOOH), cannabinol (CBN), cannabidiol (CBD), â9-tetrahydrocannabinolic acid A (THC-A), 11-nor-9-carboxy-â9-tetrahydrocannabinol glucuronide (THCCOOH-gluc), and â9-tetrahydrocannabinol glucuronide (THC-gluc). Samples were collected using a Quantisalâą device. The advantages of performing a liquid-liquid extraction (LLE) of KCl-saturated OF using heptane/ethyl acetate versus a solid-phase extraction (SPE) using HLB copolymer columns were determined. Chromatographic separation was achieved in 11.5min on a Kinetexâą column packed with 2.6-ÎŒm core-shell particles. Both positive (THC, 11-OH-THC, CBN, and CBD) and negative (THCCOOH, THC-gluc, THCCOOH-gluc, and THC-A) electrospray ionization modes were employed with multiple reaction monitoring using a high-end AB Sciex API 5000âą triple quadrupole LC-MS/MS system. Unlike SPE, LLE failed to extract THC-gluc and THCCOOH-gluc. However, the LLE method was more sensitive for the detection of THCCOOH than the SPE method, wherein the limit of detection (LOD) and limit of quantification (LOQ) decreased from 100 to 50pg/ml and from 500 to 80pg/ml, respectively. The two extraction methods were successfully applied to OF samples collected from volunteers before and after they smoked a homemade cannabis joint. High levels of THC were measured soon after smoking, in addition to significant amounts of THC-A. Other cannabinoids were found in low concentrations. Glucuronide conjugate levels were lower than the method's LOD for most samples. Incubation studies suggest that glucuronides could be enzymatically degraded by glucuronidase prior to OF collectio
Einsatz von synthetischen anorganischen Adsorbermaterialien zur Proteinaufreinigung am Beispiel der Molkeproteine
Seit dem Beginn der Produktion und Veredelung von Nahrungsmitteln im groĂtechnischen
MaĂstab steigen nicht nur die Mengen an den Hauptprodukten dieser Prozesse, sondern
auch die Mengen der dabei anfallenden Nebenprodukte. Die Entsorgung dieser Nebenprodukte
ĂŒber FlĂŒsse oder KlĂ€ranlagen stellte auf Grund deren BOD (biochemical oxygen
demand) zunehmend ein Problem dar, dessen Lösung unabdingbar wurde. Eines dieser
Nebenprodukte, welches in stets zunehmender Menge anfÀllt ist Molke, das Hauptnebenprodukt
der KĂ€seherstellung. Die produzierte Jahresmenge betrug fĂŒr das Jahr 2011 alleine in Deutschland geschĂ€tzte 11,80 Mio. Tonnen. Das einstige Abfallprodukt Molke wird seit den 1950er Jahren in zunehmendem MaĂ vorwiegend zu Molkekonzentrat, einer 5 bis
6-fach aufkonzentrierten Molkelösung, und Molkeproteinpulvern mit steigendem Proteinanteil
aufgearbeitet. In den 2000er Jahren kamen verstÀrkt Isolate einzelner Molkeproteine dieser Produktpalette hinzu. Bereits vor 15 Jahren wurde die weltweit produzierte Menge
an Molkeproteinkonzentrat auf ĂŒber 140.000 Tonnen geschĂ€tzt. Molkeproteinpulver und alpha-Lactalbumin (ALA) wurden zu dieser Zeit in einer geschĂ€tzten Menge von 2.300 Tonnen bzw. 230 Tonnen jĂ€hrlich hergestellt. Mit der zunehmenden Reinheit der Proteinfraktionen stieg auch deren Marktwert von etwa 1 /kg fĂŒr Fraktionen einzelner Proteine mit hoher Reinheit.
Die ĂŒberwiegende Mehrheit dieser Produkte findet seinen Einsatz in der Nahrungsmittelindustrie,
wie beispielsweise bei der Produktion von SÀuglingsnahrung, SportlerernÀhrung
oder als Texturbildner in verschiedensten Nahrungsmitteln des tĂ€glichen Konsums. Die am weitesten verbreiteten Prozesse zur groĂindustriellen Aufarbeitung von Molke sind
die Ultrafiltration und die Chromatographie. Andere Verfahren wie FĂ€llungsprozesse oder
peptische Hydrolysen kommen mit steigender ProzessgröĂe immer seltener als alleiniges
Aufarbeitungsverfahren von Molke zum Einsatz. Viele der beschriebenen und angewendeten
Verfahren arbeiten in einem der ersten Schritte mit einer Modifikation des pH-Wertes
des Eduktes Molke. Dies ist in dem hier vorgestellten Verfahren nicht der Fall, das Edukt wird ohne VerÀnderungen oder Modifikationen eingesetzt. Es werden keine einzelnen Komponenten der Molke im Voraus entfernt oder durch z. B. peptische Hydrolyse zerstört. Die
eingesetzten Chemikalien sind ungiftig und mĂŒssen keinen kostenintensiven Entsorgungsverfahren
zugefĂŒhrt werden. Im Gegensatz zu Verfahren mit FĂ€llungssalzen können die
eingesetzten Chemikalien im Produkt durch Neutralisation einfach wieder entfernt werden
oder kommen in nur geringen Konzentrationen vor. Es treten im Gegensatz zu chromatographischen
Methoden nur geringe Fluidströme, z. B. Waschwasser, auf und der Prozess bedarf keiner massiven Temperaturerhöhung des Eduktes, um beispielsweise die ViskositÀt
zu erhöhen. Die eingesetzten Adsorbentien sind kostengĂŒnstig (unter 10 âŹ/kg) und können im Fall von Siliziumdioxid als restproteinbeladener Tierfutterzusatz entsorgt werden. In dem vorgestellten Prozess können die Hauptkomponenten der Molke alpha-Lactalbumin
(ALA), beta-Lactoglobulin (BLG) und Lactose voneinander getrennt werden, was mit den
meisten Membranverfahren im industriellen MaĂstab nicht oder nur ungenĂŒgend möglich
ist. Im Prozess werden lediglich einfache RĂŒhrkessel und Filtrationseinheiten eingesetzt. Des Weiteren ist der Prozess leicht in bestehende Aufarbeitungsverfahren integrierbar und/oder durch zusĂ€tzliche Verfahren, wie beispielsweise vor- oder nachgeschaltete Membranverfahren,
ergÀnzbar. Dadurch besitzt er ein hohes Potential zur Optimierung und weiteren Kostenersparnis. In dieser Arbeit werden zwei AnsÀtze zur Aufarbeitung verfolgt,
miteinander verglichen und ein Prozessentwurf mit Wirtschaftlichkeitsbetrachtung fĂŒr den Batch-Ansatz vorgestellt. FĂŒr einen chromatographischen Ansatz wird ausgehend von Isothermen
und Adsorptions- und Desorptionsversuchen in einer KleinsÀule die ProduktivitÀt
fĂŒr ein Material aus
gamma-Aluminiumoxid ermittelt. FĂŒr alle in dieser Arbeit verwendeten Materialien
aus Bentonit/KieselsÀure, Siliziumdioxid und
gamma-Aluminiumoxid werden die PermeabilitĂ€t der SĂ€ulenpackung, deren maximale BindekapazitĂ€t und StabilitĂ€t angegeben. Der Einfluss einer Reduktion der PartikelgröĂe auf die ProteinbindekapazitĂ€t daraus resultierender
AdsorbensschĂŒttungen wird ebenfalls betrachtet. Eine gĂ€ngige Betriebsweise
fĂŒr viskose Fluide mit teilweise ungelöstem Feststoffanteil, wie Molkekonzentrat, stellt die
Expanded Bed Chromatographie dar. Eine rechnerische AbschĂ€tzung fĂŒr das Material aus gamma-Aluminiumoxid ergĂ€nzt experimentelle Beobachtungen hinsichtlich der Eignung des Materials
fĂŒr die Expanded Bed Chromatographie. Zur Trennung von ALA und BLG wird ein
Ansatz mittels selektiver Adsorption im Durchbruch mit einem Ansatz mittels selektiver Desorption in einem Stufengradienten aus Kaliumphosphat als Eluent verglichen. FĂŒr den
Batch-Ansatz wird aus den Ergebnissen von Adsorptionsisothermen und einstufigen Batchversuchen
eine Stoffstromsimulation auf Grundlage eines verfahrenstechnischen FlieĂbilds
erstellt. Der vorgestellte Prozess besteht aus 6 RĂŒhrkesselreaktoren, Filtrationsmodulen
zur Fest-FlĂŒssig-Phasen Trennung und zwei SprĂŒhtrocknungseinheiten. Eine PrĂŒfung des
vorgestellten Prozesses auf seine Wirtschaftlichkeit wird vor dem Hintergrund möglicher
Produktpreise diskutiert
Direct analysis of dried blood spots coupled with mass spectrometry: concepts and biomedical applications
Because of the emergence of dried blood spots (DBS) as an attractive alternative to conventional venous plasma sampling in many pharmaceutical companies and clinical laboratories, different analytical approaches have been developed to enable automated handling of DBS samples without any pretreatment. Associated with selective and sensitive MS-MS detection, these procedures give good results in the rapid identification and quantification of drugs (generally less than 3min total run time), which is desirable because of the high throughput requirements of analytical laboratories. The objective of this review is to describe the analytical concepts of current direct DBS techniques and to present their advantages and disadvantages, with particular focus on automation capacity and commercial availability. Finally, an overview of the different biomedical applications in which these concepts could be of major interest will be presented. Figure Direct analysis of dried blood spot
How do professional development programs on comparing solution methods and classroom discourse affect students' achievement in mathematics? The mediating role of studentsâ subject matter justifications
Comparing solution methods fosters strategy flexibility in equation solving. Productive classroom discourse such as Accountable Talk (AT) orchestrated by teachers can improve students' justifications during classroom discussions and achievement. Do students' subject matter justifications during classroom discourse mediate the effect of teachers' professional development (PD) programs focused on comparing and AT on studentsâ mathematics achievement? We investigated whether two PD programs (comparing or comparing+AT) compared to a control group increased the number of students justifications, and whether this affected mathematics achievement (strategy flexibility, procedural knowledge, and conceptual knowledge). The study (739 9th and 10th grade students in 39 classes) had an experimental pre-post control group design. Both PD programs significantly increased students justifications compared to the control group. The results of our multilevel path models showed significant small mediation effects in the comparing+AT group on procedural and conceptual knowledge. No mediation effects were found in the comparing group
Quantitative Capillary Supercritical Fluid Chromatography and Supercritical Fluid Extraction of Basic Drugs of Abuse
Capillary SFC of basic drugs of abuse have been studied using two different columns, and the results were compared with GC. These results have proved the ability of supercritical CO2 to solubilize and extract basic drugs of interest. A novel restrictor has been used to control precisely the flow rate through the extraction cell over a long period of time. Optimal conditions for quantitative recoveries of aqueous morphine adsorbated on C18 silica by SFE has been determined. Recoveries of 100% with a variation of 2% was obtained with an extraction time of 25 min. The influence of the pressure, solvent modifier, and flow rate of extraction fluid on the extraction efficiency was determined. These conditions were also found to be applicable for extracting other drugs of abuse
Quantitative Capillary Supercritical Fluid Chromatography and Supercritical Fluid Extraction of Basic Drugs of Abuse
Capillary SFC of basic drugs of abuse have been studied using two different columns, and the results were compared with GC. These results have proved the ability of supercritical CO2 to solubilize and extract basic drugs of interest. A novel restrictor has been used to control precisely the flow rate through the extraction cell over a long period of time. Optimal conditions for quantitative recoveries of aqueous morphine adsorbated on C18 silica by SFE has been determined. Recoveries of 100% with a variation of 2% was obtained with an extraction time of 25 min. The influence of the pressure, solvent modifier, and flow rate of extraction fluid on the extraction efficiency was determined. These conditions were also found to be applicable for extracting other drugs of abuse
Use of the dried blood spot sampling process coupled with fast gas chromatography and negative-ion chemical ionization tandem mass spectrometry: application to fluoxetine, norfluoxetine, reboxetine, and paroxetine analysis
The objective of this work was to combine the advantages of the dried blood spot (DBS) sampling process with the highly sensitive and selective negative-ion chemical ionization tandem mass spectrometry (NICI-MS-MS) to analyze for recent antidepressants including fluoxetine, norfluoxetine, reboxetine, and paroxetine from micro whole blood samples (i.e., 10 ÎŒL). Before analysis, DBS samples were punched out, and antidepressants were simultaneously extracted and derivatized in a single step by use of pentafluoropropionic acid anhydride and 0.02% triethylamine in butyl chloride for 30min at 60°C under ultrasonication. Derivatives were then separated on a gas chromatograph coupled with a triple-quadrupole mass spectrometer operating in negative selected reaction monitoring mode for a total run time of 5min. To establish the validity of the method, trueness, precision, and selectivity were determined on the basis of the guidelines of the "SociĂ©tĂ© Française des Sciences et des Techniques Pharmaceutiquesâ (SFSTP). The assay was found to be linear in the concentration ranges 1 to 500ng mLâ1 for fluoxetine and norfluoxetine and 20 to 500ng mLâ1 for reboxetine and paroxetine. Despite the small sampling volume, the limit of detection was estimated at 20pg mLâ1 for all the analytes. The stability of DBS was also evaluated at â20°C, 4°C, 25°C, and 40°C for up to 30days. Furthermore, the method was successfully applied to a pharmacokinetic investigation performed on a healthy volunteer after oral administration of a single 40-mg dose of fluoxetine. Thus, this validated DBS method combines an extractiveâderivative single step with a fast and sensitive GC-NICI-MS-MS technique. Using microliter blood samples, this procedure offers a patient-friendly tool in many biomedical fields such as checking treatment adherence, therapeutic drug monitoring, toxicological analyses, or pharmacokinetic studie
Restoration of euthyroidism accelerates bone turnover in patients with subclinical hypothyroidism: a randomized controlled trial
This study evaluated the effect of physiological l-thyroxine (L-T4) treatment on bone metabolism in patients with subclinical hypothyroidism. Sixty-six women with subclinical hypothyroidism (TSH 11.7±0.8mIU/l) were randomly assigned to receive L-T4 or placebo for 48weeks. Sixty-one of 66 patients completed the study. Individual L-T4 replacement (mean dosage 85.5±4.3ÎŒg/day) was performed targeting euthyroid thyroid-stimulating hormone (TSH) levels. The primary outcome measure was 24- and 48-week change in markers of bone formation (total and bone alkaline phosphatase [ALP, bone ALP], osteocalcin [OC]) and resorption (pyridinoline [PYD] and deoxypyridinoline [DPD], C-terminal cross-linking telopeptide type I [CTX]). Secondary outcomes were 48-week changes in bone mineral density (BMD) of the lumbar spine and hip, measured by dual-energy X-ray absorptiometry. Compared with placebo, l-thyroxine (n=31) resulted in significant activation of bone turnover. Overall, a significant treatment effect was observed for DPD (between-group difference 16.0%; 95%CI, 10.9 to 21.1), CTX (29.9%; 95%CI, 23.3 to 36.5), and bone ALP (13.2%; 95%CI, 6.6 to 19.7) after 24weeks. At the end of the study, lumbar BMD in the both treatment groups differed by 1.3% (95%CI, â2.9 to 0.5) with lower levels in l-thyroxine treated women. Significant difference in BMD between groups was also observed at the trochanter. We conclude that physiological l-thyroxine treatment accelerates bone turnover reflecting early activation of bone remodeling units in the initial replacement of subclinical hypothyroidism. The observed bone loss could be interpreted as an adaptive mechanism on decreased bone turnover in preexistent hypothyroidism, and not as l-thyroxine-induced clinically important bone loss. However, long-term studies are needed to confirm this assumptio
Diagnostic performance of ethyl glucuronide in hair for the investigation of alcohol drinking behavior: a comparison with traditional biomarkers
Background: Ethyl glucuronide (EtG) in hair has emerged as a useful biomarker for detecting alcohol abuse and monitoring abstinence. However, there is a need to establish a reliable cutoff value for the detection of chronic and excessive alcohol consumption. Methods: One hundred and twenty-five subjects were classified as teetotalers, low-risk drinkers, at-risk drinkers, or heavy drinkers. The gold standard for subjects' classifications was based on a prospective daily alcohol self-monitoring log. Subjects were followed for a 3-month period. The EtG diagnostic performance was evaluated and compared with carbohydrate-deficient transferring (CDT) and the activities of aspartate aminotransferase, alanine aminotransferase, and Îł-glutamyl-transferase (ÎłGT). Results: A cutoff of >9pg/mg EtG in hair, suggesting an alcohol consumption of >20/30g (at-risk drinkers), and a cutoff of >25pg/mg, suggesting a consumption of >60g (heavy drinkers), were determined by receiver operating characteristic analysis. The EtG diagnostic performance was significantly better (Pâ<â0.05) than any of the traditional biomarkers alone. EtG, as a single biomarker, yielded a stronger or similar diagnostic performance in detecting at-risk or heavy drinkers, respectively, than the best combination of traditional biomarkers (CDT and ÎłGT). The combination of EtG with traditional biomarkers did not improve the diagnostic performance of EtG alone. EtG demonstrated a strong potential to identify heavy alcohol consumption, whereas the traditional biomarkers failed to do so. EtG was not significantly influenced by gender, body mass index, or age. Conclusion: Hair EtG definitively provides an accurate and reliable diagnostic test for detecting chronic and excessive alcohol consumption. The proposed cutoff values can serve as reference for future cutoff recommendations for clinical and forensic us
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