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 × 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 ∆9-tetrahydrocannabinol (THC), 11-hydroxy-∆9-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THCCOOH), cannabinol (CBN), cannabidiol (CBD), ∆9-tetrahydrocannabinolic acid A (THC-A), 11-nor-9-carboxy-∆9-tetrahydrocannabinol glucuronide (THCCOOH-gluc), and ∆9-tetrahydrocannabinol glucuronide (THC-gluc). Samples were collected using a Quantisal™ 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 Kinetex™ column packed with 2.6-μm 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 5000™ 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 großtechnischen Maßstab steigen nicht nur die Mengen an den Hauptprodukten dieser Prozesse, sondern auch die Mengen der dabei anfallenden Nebenprodukte. Die Entsorgung dieser Nebenprodukte über Flüsse oder Kläranlagen stellte auf Grund deren BOD (biochemical oxygen demand) zunehmend ein Problem dar, dessen Lösung unabdingbar wurde. Eines dieser Nebenprodukte, welches in stets zunehmender Menge anfällt ist Molke, das Hauptnebenprodukt der Käseherstellung. Die produzierte Jahresmenge betrug für das Jahr 2011 alleine in Deutschland geschätzte 11,80 Mio. Tonnen. Das einstige Abfallprodukt Molke wird seit den 1950er Jahren in zunehmendem Maß vorwiegend zu Molkekonzentrat, einer 5 bis 6-fach aufkonzentrierten Molkelösung, und Molkeproteinpulvern mit steigendem Proteinanteil aufgearbeitet. In den 2000er Jahren kamen verstärkt Isolate einzelner Molkeproteine dieser Produktpalette hinzu. Bereits vor 15 Jahren wurde die weltweit produzierte Menge an Molkeproteinkonzentrat auf über 140.000 Tonnen geschätzt. Molkeproteinpulver und alpha-Lactalbumin (ALA) wurden zu dieser Zeit in einer geschätzten Menge von 2.300 Tonnen bzw. 230 Tonnen jährlich hergestellt. Mit der zunehmenden Reinheit der Proteinfraktionen stieg auch deren Marktwert von etwa 1 $/kg für die ersten Molkeproteinpulver in den 1960er Jahren bis auf 600$/kg für Fraktionen einzelner Proteine mit hoher Reinheit. Die überwiegende Mehrheit dieser Produkte findet seinen Einsatz in der Nahrungsmittelindustrie, wie beispielsweise bei der Produktion von Säuglingsnahrung, Sportlerernährung oder als Texturbildner in verschiedensten Nahrungsmitteln des täglichen Konsums. Die am weitesten verbreiteten Prozesse zur großindustriellen Aufarbeitung von Molke sind die Ultrafiltration und die Chromatographie. Andere Verfahren wie Fällungsprozesse oder peptische Hydrolysen kommen mit steigender Prozessgröße 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 Veränderungen 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 müssen keinen kostenintensiven Entsorgungsverfahren zugeführt werden. Im Gegensatz zu Verfahren mit Fällungssalzen 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 Viskosität zu erhöhen. Die eingesetzten Adsorbentien sind kostengünstig (unter 10 €/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 Maßstab nicht oder nur ungenügend möglich ist. Im Prozess werden lediglich einfache Rührkessel und Filtrationseinheiten eingesetzt. Des Weiteren ist der Prozess leicht in bestehende Aufarbeitungsverfahren integrierbar und/oder durch zusätzliche Verfahren, wie beispielsweise vor- oder nachgeschaltete Membranverfahren, ergänzbar. Dadurch besitzt er ein hohes Potential zur Optimierung und weiteren Kostenersparnis. In dieser Arbeit werden zwei Ansätze zur Aufarbeitung verfolgt, miteinander verglichen und ein Prozessentwurf mit Wirtschaftlichkeitsbetrachtung für den Batch-Ansatz vorgestellt. Für einen chromatographischen Ansatz wird ausgehend von Isothermen und Adsorptions- und Desorptionsversuchen in einer Kleinsäule die Produktivität für ein Material aus gamma-Aluminiumoxid ermittelt. Für alle in dieser Arbeit verwendeten Materialien aus Bentonit/Kieselsäure, Siliziumdioxid und gamma-Aluminiumoxid werden die Permeabilität der Säulenpackung, deren maximale Bindekapazität und Stabilität angegeben. Der Einfluss einer Reduktion der Partikelgröße auf die Proteinbindekapazität daraus resultierender Adsorbensschüttungen wird ebenfalls betrachtet. Eine gängige Betriebsweise für viskose Fluide mit teilweise ungelöstem Feststoffanteil, wie Molkekonzentrat, stellt die Expanded Bed Chromatographie dar. Eine rechnerische Abschätzung für das Material aus gamma-Aluminiumoxid ergänzt experimentelle Beobachtungen hinsichtlich der Eignung des Materials für 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. Für den Batch-Ansatz wird aus den Ergebnissen von Adsorptionsisothermen und einstufigen Batchversuchen eine Stoffstromsimulation auf Grundlage eines verfahrenstechnischen Fließbilds erstellt. Der vorgestellte Prozess besteht aus 6 Rührkesselreaktoren, Filtrationsmodulen zur Fest-Flüssig-Phasen Trennung und zwei Sprühtrocknungseinheiten. Eine Prüfung 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 students’ 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 students’ 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 μL). 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 "Société Française des Sciences et des Techniques Pharmaceutiques” (SFSTP). The assay was found to be linear in the concentration ranges 1 to 500ng mL−1 for fluoxetine and norfluoxetine and 20 to 500ng mL−1 for reboxetine and paroxetine. Despite the small sampling volume, the limit of detection was estimated at 20pg mL−1 for all the analytes. The stability of DBS was also evaluated at −20°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 extractive—derivative 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±4.3μg/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, −2.9 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 γ-glutamyl-transferase (γGT). 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 (P < 0.05) 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 γGT). 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