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Introduction: Global metabolomics analyses using body fluids provide valuable results for the understanding and prediction of diseases. However, the mechanism of a disease is often tissue-based and it is advantageous to analyze metabolomic changes directly in the tissue. Metabolomics from tissue samples faces many challenges like tissue collection, homogenization, and metabolite extraction., Objectives: We aimed to establish a metabolite extraction protocol optimized for tissue metabolite quantification by the targeted metabolomics Absolute IDQ ™ p180 Kit (Biocrates). The extraction method should be non-selective, applicable to different kinds and amounts of tissues, monophasic, reproducible, and amenable to high throughput., Methods: We quantified metabolites in samples of eleven murine tissues after extraction with three solvents (methanol, phosphate buffer, ethanol/phosphate buffer mixture) in two tissue to solvent ratios and analyzed the extraction yield, ionization efficiency, and reproducibility., Results: We found methanol and ethanol/phosphate buffer to be superior to phosphate buffer in regard to extraction yield, reproducibility, and ionization efficiency for all metabolites measured. Phosphate buffer, however, outperformed both organic solvents for amino acids and biogenic amines but yielded unsatisfactory results for lipids. The observed matrix effects of tissue extracts were smaller or in a similar range compared to those of human plasma., Conclusion: We provide for each murine tissue type an optimized high-throughput metabolite extraction protocol, which yields the best results for extraction, reproducibility, and quantification of metabolites in the p180 kit. Although the performance of the extraction protocol was monitored by the p180 kit, the protocol can be applicable to other targeted metabolomics assays., Competing Interests: Compliance with ethical standardsThe authors declare that they have no conflict of interest and no conflict of financial interest. This study was not sponsored by Biocrates Life Sciences AG.According to the providers’ statement, the human plasma purchased from Sera Laboratories was collected from consented donors under IRB approved protocols at facilities located in the United States. All applicable international, national, and institutional guidelines for the care and use of animals were followed. Animal experiments were approved by the Upper Bavarian Government (Gz.55.2-1-54-2531-70-07, 55.2-1-2532-153-11).

In order to overcome many limitations of immunoassays, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has the potential to find its place in the clinical laboratory medicine for quantification of steroid hormones. A prerequisite for the application of a new analytical procedure in clinical diagnostics is standardization to minimize analytical intra- and interlaboratory variability and inaccuracy. We evaluate a newly standardized HPLC-MS/MS assay in kit-format, developed for routine determination of 16 steroid hormones in human serum samples. Fifteen metabolites can be measured quantitatively, which include aldosterone, androstenedione, androsterone, corticosterone, cortisol, cortisone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 17β-estradiol (E2), estrone (E1), etiocholanolone, 17α-hydroxyprogesterone (17OHP), progesterone, and testosterone. 11-Deoxycorticosterone is the only compound rated as semi-quantitative in this kit. The sample preparation is performed by solid phase extraction (SPE) on a 96-well plate. The standardized assay has been validated for human serum in terms of lower and upper limit of quantification (LLOQ 0.01-32 ng/mL, ULOQ 5-8000 ng/mL), linear correlation coefficient of calibration (R(2)>0.9966), intra- and inter-day precision (intra-day 1.1-8.8%, inter-day 5.2-14.8% and 8.2-18.6% for 11-deoxycorticosterone), accuracy (intra-day 88.3-115.5% and 109.3-128.2% for 11-deoxycorticosterone, inter-day 91.4-117.2% and 102.3-137.1% for 11-deoxycorticosterone), analytical total error (3.6-17.8%), proficiency test accuracy (85.4-113.4%), recovery (68-99%), and metabolite stability (freeze/thaw stability 95.5-108.1%, short term stability 86.9-107.2%). Inter-assay comparison with a routine reference HPLC-MS/MS assay and seven immunoassays demonstrates the outstanding high performance of this HPLC-MS/MS based kit by improvements in accuracy for progesterone, androstenedione, and 17OHP. Finally, results of two metyrapone tests demonstrate the potential of the standardized HPLC-MS/MS assay for the analysis of a comprehensive steroid hormone profile in clinical diagnostics., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The failure of about half of the drug candidates is associated with poor pharmacokinetic properties leading to a huge loss of time and money [1]. Early profiling of drug like properties provides important information in order to screen out insoluble, poorly absorbed and toxic compounds. Today, large compound libraries have to be screened, and of course the total number of compounds will rise over the next years leading to a growing demand for fully automated assays. A balance between quality, speed, throughput, cost and information content can be accomplished by the careful selection of assays and experimental conditions. Here we describe a novel 384 well format assay for two important ADME related descriptors (lipophilicity and serum protein binding) as input parameters for a precise prediction of fraction absorbed, blood/organ distribution coefficients and permeability, in order to maximize the information about a compound at an early stage of discovery.

Purpose: Lipid-water partitioning of 187 pharmaceuticals has been assessed with solid-supported lipid membranes (TRANSIL) in microwell plates and with multilamellar liposomes for a data comparison. The high-throughput potential of the new approach was evaluated., Methods: Drugs were incubated at pH 7.4 with egg yolk lecithin membranes either on a solid support (TRANSIL beads) or in the form of multilamellar liposomes. Phase separation of lipid and water phase was achieved by ultracentrifugation in case of liposomes or by a short filtration step in case of solid-supported lipid membranes., Results: Lipid-water partitioning data of both approaches correlate well without systematic deviations in the investigated lipophilicity range. The solid-supported lipid membrane approach provides high-precision data in an automated microwell-plate setup. The lipid composition of the solid-supported lipid membranes was varied to study the influence of membrane change on lipid-water partitioning. In addition, pH-dependent measurements have been performed with minimal experimental effort., Conclusions: Solid-supported lipid membranes represent a valuable tool to determine physiologically relevant lipid-water partitioning data of pharmaceuticals in an automated setup and is well suited for high-throughput data generation in lead optimization programs.

The determination of partition coefficients is crucial for the biochemical analysis of membrane-based processes, but requires tedious procedures. We have facilitated this analysis using a silica gel coated with a single phospholipid bilayer (TRANSIL) as the membranous phase. We demonstrate the validity of this method using MARCKS-related protein, a 20-kDa member of the MARCKS family (an acronym for myristoylated alanine-rich C kinase substrate). The partition coefficients describing the association of unmyristoylated and myristoylated MARCKS-related protein with membranes of different phospholipid composition are in agreement with previous work with vesicles and show that both the myristoyl moiety and the basic effector domain of MARCKS-related protein mediate the binding. However, no significant cooperativity is observed between these two domains. Interestingly, MARCKS-related protein binds to TRANSIL membranes more strongly at temperatures below their phase-transition temperature. Taking advantage of this property, MARCKS-related protein was purified by phase-transition chromatography, loading Escherichia coli lysates on a TRANSIL column at 4 degrees C and eluting MRP at room temperature. In conclusion, TRANSIL is a versatile tool to determine the affinity of compounds for phospholipid membranes and to purify membrane-bound proteins. TRANSIL should also enable functional studies of protein-ligand and protein-protein interactions at the surface of membranes., (Copyright 1999 Academic Press.)