Use of a microplate scintillation counter as a radioactivity detector for miniaturized separation techniques in drug metabolism.

In miniaturized separation techniques, such as capillary electrophoresis (CE) or capillary liquid chromatography (LC), conventional on-line radioactivity detection of labeled compounds is restricted, because of insufficient sensitivity. It will be shown that a microplate scintillation counter for 96-well plates (TopCount) can be used as a sensitive and easy-to-handle radioactivity detector for capillary LC and CE. The attractive combination of capillary LC, eluent fractionation, and subsequent off-line counting is described. The new method is applied for rapid and sensitive separation and detection of 3H-labeled parent drug and its metabolites at levels between 25 and 700 cpm in rat urine. The advantages of capillary LC coupled to the TopCount, and combined with LC-MS data, can be of benefit in many analytical areas, including the characterization of metabolites at low concentration within complex biological fluids. With the same setup, the fractionation with subsequent off-line counting is equally applicable to CE. This is demonstrated with electrophoretically separated 14C-labeled impurities, nicely resolved from a negatively charged main compound, at low levels

A microplate solid scintillation counter as a radioactivity detector for high performance liquid chromatography in drug metabolism: validation and applications.

Sensitive radioactivity detection following high performance liquid chromatography (HPLC) separation remains a challenge in many drug metabolism studies with radiolabeled compounds. In this work, solid scintillation counting (SSC) after fraction collection into 96-well plates was evaluated as an off-line radioactivity detection method, in comparison with conventional liquid scintillation counting (LSC). The impact of counting time and biological matrix on the quantification of radiolabeled metabolites and parent drug in samples from animal and human absorption, distribution, metabolism and excretion (ADME) studies was investigated. Three different approaches were used to test whether reliable quantification by off-line SSC detection, which requires an approximately constant counting yield during the entire chromatographic run, can be realized: (i) the measurement of radioactivity-spiked biological blank samples without HPLC separation as an extreme case of biological background, (ii) the measurement of radioactivity-spiked HPLC fractions of biological blank samples and (iii) the comparison of radiochromatograms obtained by off-line SSC and LSC of real samples from ADME studies with radiolabeled compounds. Situations in which variations in SSC yield during an HPLC run are likely to lead to significant errors in quantitation were identified and are discussed. However, examples from a number of animal or human ADME studies showed that in the majority of cases off-line SSC provides very similar quantitative data, compared with the reference method of off-line LSC radioactivity detection. Approaches for validation of the off-line SSC approach in critical cases are discussed. The main advantages of off-line SSC, compared with off-line LSC, are lower detection limits and a substantially higher throughput. Several applications of off-line SSC detection in ADME studies are shown

A microplate solid scintillation counter as a radioactivity detector for high performance liquid chromatography in drug metabolism: validation and applications.

Sensitive radioactivity detection following high performance liquid chromatography (HPLC) separation remains a challenge in many drug metabolism studies with radiolabeled compounds. In this work, solid scintillation counting (SSC) after fraction collection into 96-well plates was evaluated as an off-line radioactivity detection method, in comparison with conventional liquid scintillation counting (LSC). The impact of counting time and biological matrix on the quantification of radiolabeled metabolites and parent drug in samples from animal and human absorption, distribution, metabolism and excretion (ADME) studies was investigated. Three different approaches were used to test whether reliable quantification by off-line SSC detection, which requires an approximately constant counting yield during the entire chromatographic run, can be realized: (i) the measurement of radioactivity-spiked biological blank samples without HPLC separation as an extreme case of biological background, (ii) the measurement of radioactivity-spiked HPLC fractions of biological blank samples and (iii) the comparison of radiochromatograms obtained by off-line SSC and LSC of real samples from ADME studies with radiolabeled compounds. Situations in which variations in SSC yield during an HPLC run are likely to lead to significant errors in quantitation were identified and are discussed. However, examples from a number of animal or human ADME studies showed that in the majority of cases off-line SSC provides very similar quantitative data, compared with the reference method of off-line LSC radioactivity detection. Approaches for validation of the off-line SSC approach in critical cases are discussed. The main advantages of off-line SSC, compared with off-line LSC, are lower detection limits and a substantially higher throughput. Several applications of off-line SSC detection in ADME studies are shown

Pharmacokinetics, distribution, metabolism, and excretion of deferasirox and its iron complex in rats.

Deferasirox (Exjade, ICL670, CGP72670) is an iron-chelating drug for p.o. treatment of transfusional iron overload in patients with beta-thalassemia or sickle cell disease. The pharmacokinetics and disposition of deferasirox were investigated in rats. The animals received single intravenous (10 mg/kg) or p.o. (10 or 100 mg/kg) doses of 14C-radiolabeled deferasirox. Biological samples were analyzed for radioactivity (liquid scintillation counting, quantitative whole-body autoradioluminography), for deferasirox and its iron complex [high-performance liquid chromatography (HPLC)/UV], and for metabolites (HPLC with radiodetection, liquid chromatography/mass spectrometry, 1H and 13C NMR, and two-dimensional NMR techniques). At least 75% of p.o.-dosed deferasirox was absorbed. The p.o. bioavailability was 26% at the 10 mg/kg dose and showed an overproportional increase at the 100 mg/kg dose, probably because of saturation of elimination processes. Deferasirox-related radioactivity was distributed mainly to blood, excretory organs, and gastrointestinal tract. Enterohepatic recirculation of deferasirox was observed. No retention occurred in any tissue. The placental barrier was passed to a low extent. Approximately 3% of the dose was transferred into the breast milk. Excretion of deferasirox and metabolites was rapid and complete within 7 days. Key clearance processes were hepatic metabolism and biliary elimination via multidrug resistance protein 2. Deferasirox, iron complex, and metabolites were excreted largely via bile and feces (total > or = 90%). Metabolism included glucuronidation at the carboxylate group (acyl glucuronide M3) and at phenolic hydroxy groups, as well as, to a lower degree, cytochrome P450-catalyzed hydroxylations. Two hydroxylated metabolites (M1 and M2) were administered to rats and were shown not to contribute substantially to iron elimination in vivo

The Fatty Acid Desaturation Index in Human Plasma: Comparison of Different Analytical Methodologies for the Evaluation of Dietary Effects

Abstract (252 words) Background: Stearoyl-CoA Desaturase (SCD1) plays a role in the development of obesity and related conditions, such as insulin resistance, and potentially also in neurological and heart diseases. The activity of SCD1 can be monitored using the desaturation index (DI), the ratio of product (16:1n-7 and 18:1n-9) to precursor (16:0 and 18:0) fatty acids. Here we analyzed the DI in the plasma total triglyceride fraction and very low density lipoproteins (VLDL) in volunteers on a high carbohydrate diet using a dual ultra-high pressure liquid chromatography – mass spectrometry (UHPLC-MS) strategy. Methods: One assay was based on a simple extraction followed by neutral loss triglyceride – fatty acid analysis, the other on saponification of triglycerides followed by fatty acid analysis (specific for the position of the double bond within monosaturated fatty acids (MUFA). Both assays were applied to the isolated VLDL or to the plasma total triglyceride fraction. Assays were compared using plasma from a study in which 8 lean and obese healthy individuals received a high carbohydrate diet for 3 days. Results: All assays showed acceptable accuracies (75-125) and precisions (<20%) for the analysis of fatty acids in VLDL and plasma. Analysis of human plasma samples revealed increased DI (up to 136% of control, p< 0.05 after a high carbohydrate diet of 3 days. Conclusion: Only the specific fatty acid UHPLC-MS analysis of human plasma VLDL samples reflects the complete biological pathway and showed that a significant increase in DI and associated SCD1 activity –in healthy individuals after just 3 days of high carbohydrate diet

Cross-study and cross-omics comparisons of three nephrotoxic compounds reveal mechanistic insights and new candidate biomarkers

The European InnoMed–PredTox project was a collaborative effort between 15 pharmaceutical companies, 2 small and mid-sized enterprises, and 3 universities with the goal of delivering deeper insights into the molecular mechanisms of kidney and liver toxicity and to identify mechanism-linked diagnostic or prognostic safety biomarker candidates by combining conventional toxicological parameters with “omics” data. Mechanistic toxicity studies with 16 different compounds, 2 dose levels, and 3 time points were performed in male Crl: WI(Han) rats. Three of the 16 investigated compounds, BI-3 (FP007SE), Gentamicin (FP009SF), and IMM125 (FP013NO), induced kidney proximal tubule damage (PTD). In addition to histopathology and clinical chemistry, transcriptomics microarray and proteomics 2D-DIGE analysis were performed. Data from the three PTD studies were combined for a cross-study and cross-omics meta-analysis of the target organ. The mechanistic interpretation of kidney PTD-associated deregulated transcripts revealed, in addition to previously described kidney damage transcript biomarkers such as KIM-1, CLU and TIMP-1, a number of additional deregulated pathways congruent with histopathology observations on a single animal basis, including a specific effect on the complement system. The identification of new, more specific biomarker candidates for PTD was most successful when transcriptomics data were used. Combining transcriptomics data with proteomics data added extra value. Katja A. Matheisa, , , Emmanuelle Comb, c, b, Nelson , Arnd Brandenburge, Hans Gmuendere, Alexandra Sposnyf, Philip Hewittf, Alexander Ambergg, Olaf Boernsend, Bjoern Riefkeh, Dana Hoffmannj, Angela Mallyj, Arno Kalkuhla, Laura Suteri, Frank Dieterled and Frank Staedtler