High-performance Liquid Chromatography Methodology Research Articles

24-Hour Pharmacokinetic Relationships for Vancomycin and Novel Urinary Biomarkers of Acute Kidney Injury.

Vancomycin has been associated with acute kidney injury in preclinical and clinical settings; however, the precise exposure profiles associated with vancomycin-induced acute kidney injury have not been defined. We sought to determine pharmacokinetic/pharmacodynamics indices associated with the development of acute kidney injury using sensitive urinary biomarkers. Male Sprague-Dawley rats received clinical-grade vancomycin or normal saline as an intraperitoneal injection. Total daily doses between 0 and 400 mg/kg of body weight were administered as a single dose or 2 divided doses over a 24-h period. At least five rats were utilized for each dosing protocol. A maximum of 8 plasma samples per rat were obtained, and urine was collected over the 24-h period. Kidney injury molecule-1 (KIM-1), clusterin, osteopontin, cystatin C, and neutrophil gelatinase-associated lipocalin levels were determined using Milliplex multianalyte profiling rat kidney panels. Vancomycin plasma concentrations were determined via a validated high-performance liquid chromatography methodology. Pharmacokinetic analyses were conducted using the Pmetrics package for R. Bayesian maximal a posteriori concentrations were generated and utilized to calculate the 24-h area under the concentration-time curve (AUC), the maximum concentration (Cmax), and the minimum concentration. Spearman's rank correlation coefficient (rs ) was used to assess the correlations between exposure parameters, biomarkers, and histopathological damage. Forty-seven rats contributed pharmacokinetic and toxicodynamic data. KIM-1 was the only urinary biomarker that correlated with both composite histopathological damage (rs = 0.348, P = 0.017) and proximal tubule damage (rs = 0.342, P = 0.019). The vancomycin AUC and Cmax were most predictive of increases in KIM-1 levels (rs = 0.438 and P = 0.002 for AUC and rs = 0.451 and P = 0.002 for Cmax). Novel urinary biomarkers demonstrate that kidney injury can occur within 24 h of vancomycin exposure as a function of either AUC or Cmax.

Simplified HPLC methodology for quantifying biological pterins by selective oxidation

Tetrahydrobiopterin (BH4) has become a potential therapeutic tool to treat cardiovascular diseases, since it is an essential cofactor of nitric oxide synthase. In order to quantify the amount of BH4 and its related biopterins, a procedure that involves differential oxidation is currently used, which measures biopterin (the product of the oxidation of BH4 and BH2) at two different pH conditions to calculate the quantity of BH2 and BH4, using high performance liquid chromatography (HPLC). In this work, a method was established in order to quantify BH4 and BH2 by adapting previously described procedures. Several chromatographic conditions were evaluated to define the most convenient methodology. Four types of mobile phases and two different analytical columns were used for HPLC. Additionally, calibration curves were made in acid and basic pH compatible with the differential oxidation method. Each method was suitable for quantification purposes, but the choice was based on an economic factor. The selected condition was a mobile phase of 95% water/5% methanol using a C18 column at 35°C at a flow rate of 0.9mL/min. Then, it was calculated the recovery rate, which was about 80% using the chosen method. The aim of this work was to establish a simplified method of differential oxidation, compatible with matrixes such as cardiac tissue in order to facilitate the assessment of the BH4/BH2 ratio in biological samples.

Antimutagenic evaluation of traditional medicinal plants from South America Peumus boldus and Cryptocarya alba using Drosophila melanogaster

ABSTRACTPeumus boldus Mol. (“Boldo”) and Cryptocarya alba Mol. Looser (“Peumo”) are medicinal shrubs with wide geographical distribution in South America. Their leaves and fruits are commonly used in traditional medicine because they exhibit natural medicinal properties for treatment of liver disorders and rheumatism. However, there are no apparent data regarding potential protective effects on cellular genetic components. In order to examine potential mutagenic and/or antimutagenic effects of these medicinal plants, the Drosophila melanogaster (D. melanogaster) wing-spot test was employed. This assay detects a wide range of mutational events, including point mutations, deletions, certain types of chromosomal aberrations (nondisjunction), and mitotic recombination. Qualitative and quantitative analyses of phenolic and anthocyanin compounds were carried out using biochemical and high-performance liquid chromatography methodologies. In addition, the antioxidant capacity of P. boldus and C. alba leaf extracts was also analyzed. P. boldus and C. alba extracts did not induce significant mutagenic effects in the D. melanogaster model. However, simultaneous treatment of extracts concurrently with the mutagen ethyl methane sulphonate showed a decrease of mutant spots in somatic cells of D. melanogaster, indicating desmutagenic effects in this in vivo model. Flavonoids and anthocyanins were detected predominantly in the extracts, and these compounds exerted significant antioxidant capacity. The observed antimutagenic effects may be related to the presence of phytochemicals with high antioxidant capacity, such as flavonoids and antohocyanins, in the extracts.

Nutritionally Important Pigments in Purslane (Portulaca oleracea) Differ between Cultivars and in Response to Nitrogen

Purslane (Portulaca oleracea) is a succulent weedy annual in much of the United States. In other parts of the world, purslane is grown as a specialty crop, valued for its nutritional quality. As a leafy crop, purslane contributes carotenoid phytochemicals in the typical Mediterranean diet. Nitrogen (N) influences plant growth and alters pigment composition and accumulation. However, little is known about the impact N fertility may have on pigment concentrations in purslane shoot tissues. The objective of this study was to evaluate the influence of N fertility levels on biomass and concentrations of nutritionally important carotenoid and chlorophyll pigments in purslane. Green Leaf and Golden Leaf purslane cultivars were grown in nutrient solution culture under N concentrations of 13, 26, 52, or 105 mg·L−1. Plants were harvested at 45 days after planting (DAP), and measured for concentrations of shoot pigments using high-performance liquid chromatography (HPLC) methodology. There was no influence of N treatment concentration on purslane shoot tissue fresh weight (FW) accumulation. Nitrogen treatment significantly influenced shoot tissue β-carotene (BC), lutein (LUT), neoxanthin (NEO), total carotenoids, chlorophyll a, chlorophyll b, total chlorophyll, and the chlorophyll a to b ratio in purslane shoot tissues. Concentrations of LUT, NEO, violaxanthin (VIO), chlorophyll b, total xanthophyll cycle pigments, and the chlorophyll a to b ratio differed between the purslane cultivars. Increases in N concentrations acted to increase concentrations of nutritionally important shoot tissue carotenoid pigments in only the Green Leaf purslane cultivar. Therefore, N fertility management and cultivar selection should be considered when producing purslane as a nutritious specialty vegetable crop.

Prevalence of Creatine Deficiency Syndromes in Children With Nonsyndromic Autism.

Creatine deficiency may play a role in the neurobiology of autism and may represent a treatable cause of autism. The goal of the study was to ascertain the prevalence of creatine deficiency syndromes (CDSs) in children with autism spectrum disorder (ASD). In a prospective multicenter study, 443 children were investigated after a confirmed diagnosis of ASD. Random spot urine screening for creatine metabolites (creatine, guanidinoacetate, creatinine, and arginine) with liquid chromatography-tandem mass spectrometry and second-tier testing with high-performance liquid chromatography methodology was followed by recall testing in 24-hour urines and confirmatory testing by Sanger-based DNA sequencing of GAMT, GATM, and SLC6A8 genes. Additional diagnostic tests included plasma creatine metabolites and in vivo brain proton magnetic resonance spectroscopy. The creatine metabolites in spot urine in the autism group were compared with 128 healthy controls controlled for age. In 443 subjects with ASD investigated for CDS, we had 0 events (event: 0, 95% confidence interval 0-0.0068), therefore with 95% confidence the prevalence of CDS is <7 in 1000 children with ASD. The autism and control groups did not vary in terms of creatine metabolites (P > .0125) in urine. Our study revealed a very low prevalence of CDS in children with nonsyndromic ASD and no obvious association between creatine metabolites and autism. Unlike our study population, we expect more frequent CDS among children with severe developmental delay, speech impairment, seizures, and movement disorders in addition to impairments in social communication, restricted interests, and repetitive behaviors.

A sensitive high performance liquid chromatography assay for the quantification of doxorubicin associated with DNA in tumor and tissues

Doxorubicin, a widely used anticancer agent, exhibits antitumor activity against a wide variety of malignancies. The drug exerts its cytotoxic effects by binding to and intercalating within the DNA of tumor and tissue cells. However, current assays are unable to accurately determine the concentration of the intracellular active form of doxorubicin. Thus, the development of a sample processing method and a high-performance liquid chromatography (HPLC) methodology was performed in order to quantify doxorubicin that is associated with DNA in tumors and tissues, which provided an intracellular cytotoxic measure of doxorubicin exposure after administration of small molecule and nanoparticle formulations of doxorubicin. The assay uses daunorubicin as an internal standard; liquid–liquid phase extraction to isolate drug associated with DNA; a Shimadzu HPLC with fluorescence detection equipped with a Phenomenex Luna C18 (2μm, 2.0×100mm) analytical column and a gradient mobile phase of 0.1% formic acid in water or acetonitrile for separation and quantification. The assay has a lower limit of detection (LLOQ) of 10ng/mL and is shown to be linear up to 3000ng/mL. The intra- and inter-day precision of the assay expressed as a coefficient of variation (CV%) ranged from 4.01 to 8.81%. Furthermore, the suitability of this assay for measuring doxorubicin associated with DNA in vivo was demonstrated by using it to quantify the doxorubicin concentration within tumor samples from SKOV3 and HEC1A mice obtained 72h after administration of PEGylated liposomal doxorubicin (Doxil®; PLD) at 6mg/kg IV x 1. This HPLC assay allows for sensitive intracellular quantification of doxorubicin and will be an important tool for future studies evaluating intracellular pharmacokinetics of doxorubicin and various nanoparticle formulations of doxorubicin.

Employment of High-Performance Thin-Layer Chromatography for the Quantification of Oleuropein in Olive Leaves and the Selection of a Suitable Solvent System for Its Isolation with Centrifugal Partition Chromatography.

A high-performance thin-layer chromatographic methodology was developed and validated for the isolation and quantitative determination of oleuropein in two extracts of Olea europaea leaves. OLE_A was a crude acetone extract, while OLE_AA was its defatted residue. Initially, high-performance thin-layer chromatography was employed for the purification process of oleuropein with fast centrifugal partition chromatography, replacing high-performance liquid-chromatography, in the stage of the determination of the distribution coefficient and the retention volume. A densitometric method was developed for the determination of the distribution coefficients, KC = CS/CM. The total concentrations of the target compound in the stationary phase (CS) and in the mobile phase (CM) were calculated by the area measured in the high-performance thin-layer chromatogram. The estimated Kc was also used for the calculation of the retention volume, VR, with a chromatographic retention equation. The obtained data were successfully applied for the purification of oleuropein and the experimental results confirmed the theoretical predictions, indicating that high-performance thin-layer chromatography could be an important counterpart in the phytochemical study of natural products. The isolated oleuropein (purity > 95%) was subsequently used for the estimation of its content in each extract with a simple, sensitive and accurate high-performance thin-layer chromatography method. The best fit calibration curve from 1.0 µg/track to 6.0 µg/track of oleuropein was polynomial and the quantification was achieved by UV detection at λ 240 nm. The method was validated giving rise to an efficient and high-throughput procedure, with the relative standard deviation % of repeatability and intermediate precision not exceeding 4.9% and accuracy between 92% and 98% (recovery rates). Moreover, the method was validated for robustness, limit of quantitation, and limit of detection. The amount of oleuropein for OLE_A, OLE_AA, and an aqueous extract of olive leaves was estimated to be 35.5% ± 2.7, 51.5% ± 1.4, and 12.5% ± 0.12, respectively. Statistical analysis proved that the method is repeatable and selective, and can be effectively applied for the estimation of oleuropein in olive leaves' extracts, and could potentially replace high-performance liquid chromatography methodologies developed so far. Thus, the phytochemical investigation of oleuropein could be based on high-performance thin-layer chromatography coupled with separation processes, such as fast centrifugal partition chromatography, showing efficacy and credibility.

Fermentative biohydrogen production using hemicellulose fractions: Analytical validation for C5 and C6-sugars, acids and inhibitors by HPLC

Hemicellulose fractions derived from lignocellulosic biomass acid pretreatments have been considered promising substrates for H2 production via anaerobic fermentation. In this context, the objective of this study is to develop a high-performance liquid chromatography (HPLC) methodology for the simultaneous determination of glucose, xylose, arabinose, acetic acid, butyric acid, 5-hydroxymethyl-2-furaldehyde (HMF) and 2-furaldehyde (furfural) in the complex matrices used for the H2 production from hemicellulose fractions. HPLC analyses were performed using two columns under different conditions: the Aminex HPX-87H column (mobile phase consisted of 0.005 mol L−1 H2SO4 solution at a flow rate of 0.7 mL min−1 and temperature of 60 °C) and the Rezex RFQ Fast acid (mobile phase consisted of 0.005 mol L−1 H2SO4 solution at a flow rate of 1.0 mL min−1 and temperature of 80 °C). Acids (acetic and butyric acids) and inhibitors (HMF and furfural) were analyzed by UV–Vis detection at 210 nm, and C6 and C5-sugars (glucose, xylose and arabinose) were analyzed using a refractive index detector (RI). Some validation parameters, such as selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and robustness were successfully evaluated. The monitoring of fermentative media for H2 production from different hemicellulose fractions was successfully performed using the proposed methodologies. The acid-pretreated samples from corn cob, cocoa husk, sugarcane straw and bagasse substrates presented a range of xylose contents from 84.85 to 222.98 mmol L−1, which resulted in a maximum production of 2.99 mol H2/mol total carbohydrate.

Separation and Quantification of Phospholipid and Neutral Lipid Classes by HPLC–CAD: Application to Egg Yolk Lipids

In this paper the methods of different lipid classes analysis using high performance liquid chromatography (HPLC) with charged aerosol detector (CAD) are presented. The methods allowed base-line separation of all investigated egg yolk phospholipids (phosphatidylcholine, PC; phosphatidylethanolamine, PE; sphingomyelin, SM; and their lyso forms) and neutral lipid classes (triacylglycerol, TAG; cholesterol, Chol). Total lipids extraction was carried out by the Folch method. The polar and neutral lipid classes were separated by using normal-phase (silica) SPE cartridges. Two different HPLC methodologies (one for polar and one for neutral lipids) for the separation and quantification of lipid classes on a diol column were applied. The methods were optimized and the response and sensitivity of CAD was studied. In terms of linearity, precision, and sensitivity the methods were validated.

Application of one- and two-dimensional high-performance liquid chromatography methodologies for the analysis of C-glycosylflavones from fenugreek seeds

The composition of C-glycosylflavones in fenugreek seeds is complicated and comprises of flavone mono- and di-C-glycosides, derivatives of both apigenin and luteolin as well as their ester forms. In this paper the application of one- and two-dimensional HPLC techniques: off-line heart-cutting and on-line comprehensive for the analysis of flavone C-glycosides in fenugreek seeds is presented. For the identification of flavone C-glycosides two types of detectors: diode array detector and mass detector, coupled to HPLC were used. 18 flavones were separated and identified in the methanol extract from fenugreek seeds. The best separation was achieved in off-line heart-cutting mode. The presence of two new compounds in fenugreek seeds was shown, namely: vicenin-3 and isoschaftoside. Furthermore, five unknown C-glycosides, possibly derivatives of apigenin were revealed.

Eicosanoids and Docosanoids in Plasma and Aorta of Healthy and Atherosclerotic Rabbits

Eicosanoids and docosanoids have been shown to be involved in atherosclerosis. Polyunsaturated fatty acids (PUFAs) are important nutrients that are metabolized by lipoxygenases and cyclooxygenases to various mono-hydroxy metabolites which can be further metabolized by specific enzymes to more complex eicosanoids and docosanoids. In this study a high-performance liquid chromatography methodology was established and rabbits were fed with a control or a high-cholesterol diet to induce atherosclerotic lesions to determine pro- or anti-inflammatory lipid mediators in atherosclerotic vessels. In aortic samples from atherosclerotic rabbits we determined for the first time various eicosanoids/docosanoids and observed an increased concentration of 12-lipoxygenase metabolites. Increased levels of 12-hydroxyeicosatetraenoic acid (12-HETE) in high-cholesterol versus control animals as well as increased ratios of 12-HETE/arachidonic acid ratios indicate that 12-lipoxygenase metabolites may have importance in atherosclerosis. In addition, decreased concentrations of the 5-lipoxygenase metabolite leukotriene B4 levels were detected in high-cholesterol animals. A positive correlation of total plaque area with plasma levels of 12-HETE and a negative correlation with aortic levels of endogenous PPARγ-ligand 13-oxo-octadecadienoic acid were found. This study let us conclude that the cholesterol content in the diet might influence atherosclerosis via increased 12-lipoxygenase- and cyclooxygenase-mediated pathways and reduced 5-lipoxygenase pathways.

Clinical Laboratory–Based Assay Methodologies May Underestimate and Increase Variability of Vancomycin Protein Binding in Hospitalized Patients

To evaluate and compare the percent protein binding of vancomycin in hospitalized patients by using a clinical or research laboratory-derived assay methodology, and to evaluate potential patient characteristics accounting for alterations in protein binding. Prospective noninterventional cohort study. Single-center tertiary care medical center. A total of 55 hospitalized adults who were receiving vancomycin for a suspected or documented infection between August and November 2011 and required therapeutic drug monitoring. Vancomycin protein-binding studies were conducted by using ultracentrifugation of 63 blood samples collected from the 55 patients for therapeutic drug monitoring as part of clinical practice. Total and free drug concentrations were assayed in the research laboratory by using high-performance liquid chromatography (HPLC) and in the clinical laboratory by using fluorescence polarization immunoassay (FPIA). Multivariate linear regression analysis was performed to identify patient variables that were predictive of vancomycin protein binding. The average protein binding was statistically significantly lower and more variable when assayed by FPIA compared with HPLC (mean±SD 47.3±13.0% vs 54.6±9.5%, p<0.001). Multivariate analyses showed that after controlling for days of vancomycin therapy, patients in the intensive care unit (ICU) had a protein binding value that was 8.4% lower than non-ICU patients (p=0.005). Using research laboratory-based HPLC methodology, we identified an average vancomycin protein binding of 54.6% with considerably less variability than reported in the literature using clinical-based assay methodologies. Further, we identified patient factors that may likewise have an impact on this value. Future studies of vancomycin protein binding should consider use of a nonclinical assay to minimize methodological-induced variability.

Validation of an HPLC Methodology for the Identification and Quantification of Biogenic Amines in Chicken Meat

This study validated a high performance liquid chromatography (HPLC) method to determine biogenic amines in chicken meat. For the identification of biogenic amines, an isocratic elution system coupled with a UV detector (254 nm) was used after a perchloric acid (5 %) extraction and benzoyl chloride derivatization of the samples. The standards of tyramine, putrescine, cadaverine, spermidine, and spermine were used for the following validation parameters: selectivity, linearity, accuracy, recovery, limit of detection and quantification, and robustness. Finally, chicken meat commercialized in two types of packaging was evaluated. The results showed an excellent selectivity and separation of all amines, r 2 > 0.99, relative standard deviation <5 %, recovery between 64 and 112 %, limits of detection and quantification between 0.03–1.25 and 0.15–5.00 μg L−1, respectively, and appropriate robustness for the proposed methodology. Moreover, both chicken meat commercial packages had similar values for all amines; only tyramine was significantly different (P ≤ 0.05). The proposed method was suitable to detection and quantification of simultaneous five biogenic amines in chicken meat.

Seasonal variation in content of camptothecin from the bark of Nothapodytes nimmoniana (Grah.) Mabb., using HPLC analysis

Objective:To study and compare seasonal variation in camptothecin (CPT) content from bark samples of Nothapodytes nimmoniana obtained from geographically and climatologically isolated populations.Methods:A standard High Performance Liquid Chromatography methodology was used to analyze and quantify CPT from bark samples of N. nimmoniana.Results:Sample collected from Amboli yielded highest CPT content 1.337 g/100 g dry bark powder during the monsoon compared to other localities in study. Monsoon (August) showed to accumulate higher levels of CPT in barks of N. nimmoniana as compared to summer (May). Amboli averaged highest accumulation of CPT compared to other localities under study.Conclusion:These findings indicate season to have control over accumulation of CPT. Locality Amboli has highest CPT content in all seasons and were the elite population during the study. The study also suggests the need for further investigation in lights of biosynthesis in the plant.

Development of an analytical HPLC methodology to study the effects of thymosin β4 on actin in sputum of cystic fibrosis patients

A high-performance liquid chromatography (HPLC) methodology is presented. Using bovine and rabbit F- and G-actin, this methodology results in both fractions as being well-resolved peaks, which were confirmed by dot blot immunoassay and fluorescence microscopy. F- and G-actin were incubated with thymosin β4 (Tβ4) and DNase and then analyzed by HPLC, which indicated that Tβ4 and DNase inhibit G-actin polymerization and that Tβ4 depolymerizes F-actin in a dose- and time-dependent manner. The F- and G-actin content in sputum from healthy controls and cystic fibrosis (CF) patients were measured by HPLC before and after incubation with Tβ4, DNase, and gelsolin. These data demonstrate higher quantities of F-actin in the sputum of CF patients compared to healthy individuals, and also demonstrate a significantly increased F/G-actin ratio in CF sputum. Further, Tβ4, DNase, and gelsolin each increase the depolymerization of F-actin in CF sputum in a dose-dependent fashion that is additive when these agents are combined.

Influência de etapas do processo de vinificação na acidez de vinhos elaborados com uvas Vitis labrusca

Acidity is one of the most important parameters for sensorial profile of wines and is measured through the presence of organic acids, especially malic and tartaric acids, and of cations such as potassium. This study has used a high performance liquid chromatography methodology to evaluate the malic, lactic and tartaric acid concentrations in common wines elaborated with Terci grapes ( Ives ), to verify their influence in wine acidity and pH. Additionally, the potassium content was measured by spectrometry of atomic emission. The generated data were used in the principal component analysis (PCA) and in the determination of the correlation between the analyzed parameters. The occurrence of malolactic fermentation was able to alter wine composition leading to the separation of the samples in two clusters by PCA analysis. The overall samples presented acid characteristics, apart from the fact of completing malolactic fermentation. The correlation analysis made feasible the determination of the influence of malolactic fermentation in the wine acidity and pH, but no influence was noticed related to potassium and tartaric acid concentrations.

Analytical and Preparative Chiral Separation of β-Carboline Derivatives, LDL Oxidation Inhibitors, Using HPLC and CE Methodologies: Determination of Enantiomeric Purity

In the present study, baseline separation of the enantiomers of 16 β-carboline derivatives was successfully achieved using both capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) techniques in short run times (<15 min) and thus permit the determination of enantiomeric excess. In HPLC methodology, cellulose chiral stationary phase (Chiralcel OD-H) was used with a binary mobile phase constituted of n-hexane/ethanol 85/15 leading to a resolution factor of 12.6 in 15 min. Preparative HPLC allowed to obtain pure enantiomers of two compounds. In CE, chiral selectivity was developed with an in-capillary stacking strategy using anionic (highly sulfated-γ) cyclodextrins 5% (w/v) as chiral selectors and a 60 mM phosphate buffer (pH 2.5) resulting in a resolution of 10.26 in 14 min of analysis. The analytical characteristics of the two developed methods were studied in terms of repeatability, limits of detection and limits of quantification showing their suitability to be extended to all the other molecules.

Radiotracer properties determined by high performance liquid chromatography: a potential tool for brain radiotracer discovery

Previously, development of novel brain radiotracers has largely relied on simple screening tools. Improved selection methods at the early stages of radiotracer discovery and an increased understanding of the relationships between in vitro physicochemical and in vivo radiotracer properties are needed. We investigated if high performance liquid chromatography (HPLC) methodologies could provide criteria for lead candidate selection by comparing HPLC measurements with radiotracer properties in humans. Ten molecules, previously used as radiotracers in humans, were analysed to obtain the following measures: partition coefficient (Log P); permeability (P(m)); percentage of plasma protein binding (%PPB); and membrane partition coefficient (K(m)). Relationships between brain entry measurements (Log P, P(m) and %PPB) and in vivo brain percentage injected dose (%ID); and K(m) and specific binding in vivo (BP(ND)) were investigated. Log P values obtained using in silico packages and flask methods were compared with Log P values obtained using HPLC. The modelled associations with %ID were stronger for %PPB (r(2)=0.65) and P(m) (r(2)=0.77) than for Log P (r(2)=0.47) while 86% of BP(ND) variance was explained by K(m). Log P values were variable dependant on the methodology used. Log P should not be relied upon as a predictor of blood-brain barrier penetration during brain radiotracer discovery. HPLC measurements of permeability, %PPB and membrane interactions may be potentially useful in predicting in vivo performance and hence allow evaluation and ranking of compound libraries for the selection of lead radiotracer candidates at early stages of radiotracer discovery.

D/l threo-methylphenidate enantiomers influence on catecholaminergic enzyme activities

Introduction: Racemate of d/l-threo-methylphenidate (MPH; Ritalin) is an effective first-line treatment for the symptoms associated with attention-deficit/hyperactivity disorder (ADHD). Although MPH has long been administered as a racemic mixture of the two enantiomers, d-MPH and l-MPH, converging lines of evidence drawn from investigations using in vitro systems indicate that it is predominantly, d-threo MPH which mediates the pharmacological/therapeutic actions of MPH. Aim: In the present study, we investigated which of the two enantiomers has stronger effects on the tyrosine hydroxylase (TH) and monoamine-oxidase B (MAO-B) enzyme activity in vitro, as both enzymes are important for dopamine efficacy. Methods: Using homogenates of rat pheochromocytoma cells (PC-12) we investigated dose dependent effects of d-threo and l-threo MPH. Homogenates were treated with 0/1/10/100 nM and 1/10/100 µM of each MPH enantiomer. TH activity was detected via high-performance liquid chromatography (HPLC) methodology, while MAO-B activity was measured using fluorescent based enzyme-linked immunosorbent assay (ELISA). Results: We could observe dose dependent higher TH as well as MAO-B activity after treatment with d-threo MPH compared to l-threo MPH. Discussion: This exploratory investigation revealed in vitro pharmacological evidence for a potential difference between MPH enantiomers on dopaminergic enzyme activity. This finding might point to the therapeutic effects in the treatment of ADHD.

The coenzyme Q10 content of food supplements

In the European Union (EU), food supplements are regulated as food and their use is expanding rapidly. There is no enforcement to ensure that good manufacturing practices (GMPs) are followed during production but manufacturers are fully responsible for their products. Recently, the safety and quality of supplements available in the market has come into question. In our surveillance, we examined coenzyme Q10 content of 58 food supplements available in three EU member states or from Internet stores using high-performance liquid chromatography methodology. While some of the tested supplements contained almost exactly the same quantity of active ingredient as labelled, one-third of the products contained <70% of the labelled content. In the food supplements obtained online the medium content was lower than in the products purchased in pharmacies. To protect the consumer and assure the safety and quality of products, the market authorities need to exert better control. In addition, it would make sense to enforce additional requirements to ensure GMPs are followed in the manufacturing process of food supplements.