A validated assay by liquid chromatography-tandem mass spectrometry for the simultaneous quantification of elvitegravir and rilpivirine in HIV positive patients

Because of the large variability in the pharmacokinetics of anti-HIV drugs, therapeutic drug monitoring in patients may contribute to optimize the overall efficacy and safety of antiretroviral therapy. An LC–MS/MS method for the simultaneous assay in plasma of the novel antiretroviral agents rilpivirine (RPV) and elvitegravir (EVG) has been developed to that endeavor. Plasma samples (100 μL) extraction is performed by protein precipitation with acetonitrile, and the supernatant is subsequently diluted 1:1 with 20-mM ammonium acetate/MeOH 50:50. After reverse-phase chromatography, quantification of RPV and EVG, using matrix-matched calibration samples, is performed by electrospray ionization–triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The stable isotopic-labeled compounds RPV-13C6 and EVG-D6 were used as internal standards. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<6.4%), as well as EVG and RPV short and long-term stability in plasma. Calibration curves were validated over the clinically relevant concentrations ranging from 5 to 2500 ng/ml for RPV and from 50 to 5000 ng/ml for EVG. The method is precise (inter-day CV%: 3–6.3%) and accurate (3.8–7.2%). Plasma samples were found to be stable (<15%) in all considered conditions (RT/48 h, +4°C/48 h, −20°C/3 months and 60°C/1 h). Selected metabolite profiles analysis in patients' samples revealed the presence of EVG glucuronide, that was well separated from parent EVG, allowing to exclude potential interferences through the in-source dissociation of glucuronide to parent drug. This new, rapid and robust LCMS/MS assay for the simultaneous quantification of plasma concentrations of these two major new anti-HIV drugs EVG and RPV offers an efficient analytical tool for clinical pharmacokinetics studies and routine therapeutic drug monitoring service.

Multiplex Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Therapeutic Drug Monitoring of Ribavirin, Boceprevir, and Telaprevir

New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy.

A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex.

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.

Campylobacter spp., Yersinia enterocolitica, and Salmonella enterica and their simultaneous occurrence in German fattening pig herds and their environment.

Campylobacter spp., Salmonella enterica, and Yersinia enterocolitica are common causes of foodborne infections in humans with pork as a potential source. Monitoring programs at farm level are, to date, only implemented for S. enterica, while epidemiological knowledge of the other two pathogens is still lacking. This study aimed to assess the pathogen load (in the pigs' environment) in fattening pig herds, their simultaneous occurrence, and the occurrence of Campylobacter spp. and Y. enterocolitica in herds in different Salmonella risk categories. In 50 fattening pig herds in northern Germany, four pooled fecal samples and 10 swab samples from the pigs' direct environment (pen walls, nipple drinkers), indirect environment (hallways, drive boards), and flies and rodent droppings were collected from each herd and submitted for cultural examination. Campylobacter spp. were detected in 38.1% of fecal, 32.7% of direct environment, 5.3% of indirect environment, and 4.6% of flies/pests samples collected, and Y. enterocolitica in 17.1, 8.1, 1.2, and 3.1% and S. enterica in 11.2, 7.7, 4.1, and 1.5%, respectively. For Campylobacter spp., Y. enterocolitica, and S. enterica, 80, 48, and 32% of herds were positive, respectively; 22 herds were positive for both Campylobacter spp. and Y. enterocolitica, 12 for Campylobacter spp. and S. enterica, and 7 for Y. enterocolitica and S. enterica. There was no significant association between the pathogens at herd level. Campylobacter spp. and Y. enterocolitica were found more often in samples from the low Salmonella risk category (odds ratio, 0.51; confidence interval, 0.36 to 0.73, and 0.3, 0.17 to 0.57), and this was also the case for Y. enterocolitica at herd level (odds ratio, 0.08; confidence interval, 0.02 to 0.3). This study provides evidence that the pigs' environment should be accounted for when implementing control measures on farms against Campylobacter spp. and Y. enterocolitica. An extrapolation from the current Salmonella monitoring to the other two pathogens does not seem feasible.

A TEX 86 lake record suggests simultaneous shifts in temperature in Central Europe and Greenland during the last deglaciation

High-resolution quantitative temperature records from continents covering glacial to interglacial transitions are scarce but important for understanding the climate system. We present the first decadal resolution record of continental temperatures in Central Europe during the last deglaciation (similar to 14,60010,600cal. yrB.P.) based on the organic geochemical palaeothermometer TEX86. The TEX86-inferred temperature record from Lake Lucerne (Vierwaldstattersee, Switzerland) reveals typical oscillations during the Late Glacial Interstadial, followed by an abrupt cooling of 2 degrees C at the onset of Younger Dryas and a rapid warming of 4 degrees C at the onset of the Holocene, within less than 350years. The remarkable resemblance with the Greenland and regional stable oxygen isotope records suggests that temperature changes in continental Europe were dominated by large-scale reorganizations in the northern hemispheric climate system.

Development of a bead-based multiplex assay for the simultaneous detection of porcine inflammation markers using xMAP technology

Commercially available assays for the simultaneous detection of multiple inflammatory and cardiac markers in porcine blood samples are currently lacking. Therefore, this study was aimed at developing a bead-based, multiplexed flow cytometric assay to simultaneously detect porcine cytokines [interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor alpha], chemokines (IL-8 and monocyte chemotactic protein 1), growth factors [basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and platelet-derived growth factor-bb], and injury markers (cardiac troponin-I) as well as complement activation markers (C5a and sC5b-9). The method was based on the Luminex xMAP technology, resulting in the assembly of a 6- and 11-plex from the respective individual singleplex situation. The assay was evaluated for dynamic range, sensitivity, cross-reactivity, intra-assay and interassay variance, spike recovery, and correlation between multiplex and commercially available enzyme-linked immunosorbent assay as well as the respective singleplex. The limit of detection ranged from 2.5 to 30,000 pg/ml for all analytes (6- and 11-plex assays), except for soluble C5b-9 with a detection range of 2-10,000 ng/ml (11-plex). Typically, very low cross-reactivity (<3% and <1.4% by 11- and 6-plex, respectively) between analytes was found. Intra-assay variances ranged from 4.9 to 7.4% (6-plex) and 5.3 to 12.9% (11-plex). Interassay variances for cytokines were between 8.1 and 28.8% (6-plex) and 10.1 and 26.4% (11-plex). Correlation coefficients with singleplex assays for 6-plex as well as for 11-plex were high, ranging from 0.988 to 0.997 and 0.913 to 0.999, respectively. In this study, a bead-based porcine 11-plex and 6-plex assay with a good assay sensitivity, broad dynamic range, and low intra-assay variance and cross-reactivity was established. These assays therefore represent a new, useful tool for the analysis of samples generated from experiments with pigs.

Family models of independence/interdependence and their intergenerational similarity in Germany, Turkey, and India

Family change theory suggests three ideal-typical family models characterized by different combinations of emotional and material interdependencies in the family. Its major proposition is that in economically developing countries with a collectivistic background a family model of emotional interdependence emerges from a family model of complete interdependence. The current study aims to identify and compare patterns of family-related value orientations related to family change theory across three cultures and two generations. Overall, N = 919 dyads of mothers and their adolescent children from Germany, Turkey, and India participated in the study. Three clusters were identified representing the family models of independence, interdependence, and emotional interdependence, respectively. Especially the identification of an emotionally interdependent value pattern using a person-oriented approach is an important step in the empirical validation of family change theory. The preference for the three family models differed across as well as within cultures and generations according to theoretical predictions. Dyadic analyses pointed to substantial intergenerational similarities and also to differences in family models, reflecting both cultural continuity as well as change in family-related value orientations.

Simultaneous registration of ECG and cardiac motion by a single esophageal probe

Long-term surface ECG is routinely used to diagnose paroxysmal arrhythmias. However, this method only provides information about the heart's electrical activity. To this end, we investigated a novel esophageal catheter that features synchronous esophageal ECG and acceleration measurements, the latter being a record of the heart's mechanical activity. The acceleration data were quantified in a small study and successfully linked to the activity sequences of the heart in all subjects. The acceleration signals were additionally transformed into motion. The extracted cardiac motion was proved to be a valid reference input for an adaptive filter capable of removing relevant baseline wandering in the recorded esophageal ECGs. Taking both capabilities into account, the proposed recorder might be a promising tool for future long-term heart monitoring.

Ordinal patterns in epileptic brains: Analysis of intracranial EEG and simultaneous EEG-fMRI

Epileptic seizures are associated with high behavioral stereotypy of the patients. In the EEG of epilepsy patients characteristic signal patterns can be found during and between seizures. Here we use ordinal patterns to analyze EEGs of epilepsy patients and quantify the degree of signal determinism. Besides relative signal redundancy and the fraction of forbidden patterns we introduce the fraction of under-represented patterns as a new measure. Using the logistic map, parameter scans are performed to explore the sensitivity of the measures to signal determinism. Thereafter, application is made to two types of EEGs recorded in two epilepsy patients. Intracranial EEG shows pronounced determinism peaks during seizures. Finally, we demonstrate that ordinal patterns may be useful for improving analysis of non-invasive simultaneous EEG-fMRI.

The basel cocktail for simultaneous phenotyping of human cytochrome P450 isoforms in plasma, saliva and dried blood spots.

BACKGROUND AND OBJECTIVE Phenotyping cocktails use a combination of cytochrome P450 (CYP)-specific probe drugs to simultaneously assess the activity of different CYP isoforms. To improve the clinical applicability of CYP phenotyping, the main objectives of this study were to develop a new cocktail based on probe drugs that are widely used in clinical practice and to test whether alternative sampling methods such as collection of dried blood spots (DBS) or saliva could be used to simplify the sampling process. METHODS In a randomized crossover study, a new combination of commercially available probe drugs (the Basel cocktail) was tested for simultaneous phenotyping of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. Sixteen subjects received low doses of caffeine, efavirenz, losartan, omeprazole, metoprolol and midazolam in different combinations. All subjects were genotyped, and full pharmacokinetic profiles of the probe drugs and their main metabolites were determined in plasma, dried blood spots and saliva samples. RESULTS The Basel cocktail was well tolerated, and bioequivalence tests showed no evidence of mutual interactions between the probe drugs. In plasma, single timepoint metabolic ratios at 2 h (for CYP2C19 and CYP3A4) or at 8 h (for the other isoforms) after dosing showed high correlations with corresponding area under the concentration-time curve (AUC) ratios (AUC0-24h parent/AUC0-24h metabolite) and are proposed as simple phenotyping metrics. Metabolic ratios in dried blood spots (for CYP1A2 and CYP2C19) or in saliva samples (for CYP1A2) were comparable to plasma ratios and offer the option of minimally invasive or non-invasive phenotyping of these isoforms. CONCLUSIONS This new combination of phenotyping probe drugs can be used without mutual interactions. The proposed sampling timepoints have the potential to facilitate clinical application of phenotyping but require further validation in conditions of altered CYP activity. The use of DBS or saliva samples seems feasible for phenotyping of the selected CYP isoforms.