Showing papers on "Bioanalysis published in 2007"

LC−MS/MS Approach for Quantification of Therapeutic Proteins in Plasma Using a Protein Internal Standard and 2D-Solid-Phase Extraction Cleanup

Abstract: The bioanalysis of plasma samples generated from in vivo studies of therapeutic proteins is of increasing interesting in the biopharmaceutical industry. The conventional ELISA approach has a long assay development time which can limit use in the early discovery and development of protein-based drugs. In this study, an LC−MS/MS bioassay was developed for the quantification of somatropin and a therapeutic human monoclonal antibody. The assay used bovine fetuin as an internal standard and a two-dimensional solid-phase extraction for the cleanup of the plasma digest. Sample extracts were resolved on an analytical size column using a 6 min LC gradient and analyzed using a triple-quadruple mass spectrometer. The linearity of the assay for somatropin was established from 1 to 1000 μg/mL with accuracy and precision within 15%. This LC−MS approach was also applied to a rat pharmacokinetic study of the therapeutic monoclonal antibody with a lower quantitation limit of 0.5 μg/mL. The LC−MS assay had improved accurac...

Strategies for bioanalysis of an oligonucleotide class macromolecule from rat plasma using liquid chromatography-tandem mass spectrometry.

Abstract: Electrospray ionization (ESI) liquid chromatography−tandem mass spectrometry (LC/MS/MS) assays provide high-throughput and selective methods for quantitation of small molecules. Use of LC/MS/MS assays for macromolecules, like oligonucleotides, is challenging due to lack of sensitivity and low analyte recovery from biomatrixes. Due to this fact, the method of choice for oligonucleotides quantitation remains hybridization-based ligand-binding assays. These biological assays usually possess high sensitivity but low selectivity and narrow dynamic range. They also require optimizing suitable “capture and detection” probes, which can be prohibitively time-consuming and expensive in a drug discovery lead−optimization scenario. In this paper, we present a unique LC/MS/MS assay for a model phosphorothioate backbone oligodeoxynucleotide (ODN) drug (7692 amu) from rat plasma. Multiple analytical challenges were encountered. The strategies used to solve these challenges should prove useful to scientists pursuing mass...

Evaluation of ultra-performance liquid chromatography in the bioanalysis of small molecule drug candidates in plasma

Abstract: Ultra-performance liquid chromatography (UPLC) combined with mass spectrometric detection (MS) is used successfully in the bioanalysis of small molecule drug candidates in plasma. UPLC-MS is shown to increase sample throughput by reducing run times over 3-fold, without compromising analytical sensitivity or analyte resolution. The technique is demonstrated to be practical and robust on a commercially available ultra-high pressure system when injecting extracts of plasma and has also shown to be a technique that can be used effectively on a conventional high-performance liquid chromatography system fitted with short columns (

Two-dimensional ultra-thin-layer chromatography and atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry in bioanalysis.

Abstract: The feasibility of ultra-thin-layer chromatography (UTLC) and atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI-MS) for bioanalysis was studied with benzodiazepines as model substances in human urine. Two-dimensional (2D) UTLC was shown to be an efficient technique for the separation of benzodiazepines. Separations occurred in 4-12 min, and the separated compounds were identified by AP-MALDI-MS. The limits of detection with AP-MALDI-MS and AP-MALDI-MS/MS were in the picomole range and thus low enough for bioanalysis. The applicability of the 2D UTLC-AP-MALDI-MS was demonstrated in detection of metabolites with an authentic biological urine sample.

Evaluation of high‐field asymmetric waveform ion mobility spectrometry coupled to nanoelectrospray ionization for bioanalysis in drug discovery

Abstract: The potential of high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to nanoelectrospray ionization (nanoESI) as a method to improve sample throughput for bioanalysis in a discovery pharmaceutical setting was explored in this work. The ability of FAIMS to separate gas-phase ions in the millisecond timescale was exploited to eliminate the need for liquid chromatography. Samples were introduced into the FAIMS electrodes/mass spectrometer using offline nanoESI at 20 nL/min and 1.5 kV. Signals were averaged for 30 s after which the next sample could be analyzed. The separation of simple mixtures, e.g., the removal of metabolite and endogenous interferences from parent drug, was demonstrated. Moreover, the application of nanoESI attenuated the ion suppression effects that normally plague conventional electrospray. On average, approximately two-thirds of the neat sample signal intensity was preserved in extracted plasma samples. Standard curves were prepared for several compounds and linearity was obtained over approximately two to three orders of magnitude. This methodology was further tested with the analysis of plasma samples from a mouse pharmacokinetic study. Concentration values determined using nanoESI-FAIMS were comparable to those determined using conventional LC/MS as demonstrated by percent differences of less than 30%. This work demonstrated the proof of concept that the combination of FAIMS and nanospray ionization can be a potentially useful tool to improve the throughput of discovery bioanalysis.

Impact of Differential Recovery in Bioanalysis: The Example of Bortezomib in Whole Blood

Abstract: A key assumption in pharmaceutical bioanalysis is that spiked standards mimic incurred samples in every analytical aspect. Although deviations from this assumption have been reported in terms of the difference in ion suppression or metabolite interference, the difference of extraction recovery and its impact has been rarely reported and is often characterized as unlikely. In this work, we demonstrated the presence and significance of differential recovery using a real-world example: the assay of bortezomib in whole blood. Recovery differences of up to 10-fold were observed between the spiked standards and the incurred samples when different extraction methods were used. Because of its high impact, it is important that the potential of differential recovery between standards and incurred samples be evaluated during method validation. A simple time course incubation experiment was proposed to screen compounds for potential differential recovery during method validation in heterogeneous matrixes, such as whole blood and tissue. The use of this approach and the interpretation of the results from this experiment were demonstrated using bortezomib in whole blood as an example. The differential recovery of bortezomib is likely to be driven by slow binding to the proteosome present in red blood cells. Spiked samples, however, do not have sufficient time for binding to occur.

Sensitive bioanalysis—combining single-molecule spectroscopy with mono-labeled self-quenching probes

Abstract: Fluorescence single-molecule spectroscopy is an appropriate tool for modern bioanalysis. This technique enables the development of ultra sensitive assays, especially when combined with self-quenching probes. In this review we report novel DNA, enzyme, and antibody assays based on mono-labeled fluorescent probes that are quenched by photoinduced electron transfer (PET).

Bioanalysis works in the IAA AMS facility : Comparison of AMS analytical method with LSC method in human mass balance study

Abstract: Institute of Accelerator Analysis Ltd. (IAA) is the first Contract Research Organization in Japan providing Accelerator Mass Spectrometry (AMS) analysis services for carbon dating and bioanalysis works. The 3 MV AMS machines are maintained by validated analysis methods using multiple control compounds. It is confirmed that these AMS systems have reliabilities and sensitivities enough for each objective. The graphitization of samples for bioanalysis is prepared by our own purification lines including the measurement of total carbon content in the sample automatically. In this paper, we present the use of AMS analysis in human mass balance and metabolism profiling studies with IAA 3 MV AMS, comparing results obtained from the same samples with liquid scintillation counting (LSC). Human samples such as plasma, urine and feces were obtained from four healthy volunteers orally administered a 14C-labeled drug Y-700, a novel xanthine oxidase inhibitor, of which radioactivity was about 3 MBq (85 μCi). For AMS measurement, these samples were diluted 100–10,000-fold with pure-water or blank samples. The results indicated that AMS method had a good correlation with LSC method (e.g. plasma: r = 0.998, urine: r = 0.997, feces: r = 0.997), and that the drug recovery in the excreta exceeded 92%. The metabolite profiles of plasma, urine and feces obtained with HPLC-AMS corresponded to radio-HPLC results measured at much higher radioactivity level. These results revealed that AMS analysis at IAA is useful to measure 14C-concentration in bioanalysis studies at very low radioactivity level.

Evaluation of 384-well formatted sample preparation technologies for regulated bioanalysis.

Abstract: The capabilities and limitations of 384-well formatted sample preparation technologies applied to regulated bioanalysis were evaluated by developing two assays for the simultaneous quantitation of lopinavir and ritonavir, the active ingredients of Kaletra. One method used liquid-liquid extraction (LLE), and the other used solid-phase extraction (SPE). The steps and apparatuses employed by the two methods covered most of those used for bioanalysis. Briefly, the previously validated 96-well formatted assays were adapted to the 384-format with minor modifications. Because the wells of a 384-well plate are clustered together, cross-contamination between adjacent wells was evaluated critically, along with sensitivity, assay throughput, and ruggedness. Samples (35 microL) containing plasma samples (15 microL), internal standard (10 microL), and sodium carbonate (0.5 M, 10 microL to basify the sample) were placed in a 384-well microtiter plate that may contain saquinavir or amprenavir as contamination markers. For LLE preparation, the samples were placed in a deep 384-well plate (300-microL well volume) and extracted with 150 microL of ethyl acetate. Approximately 50 microL of the extracts were removed from each well after phase separation for analysis. For SPE preparation, the fortified samples were transferred to a 384-formatted SPE plate (C18, 5 mg packing). The extracts were eluted from the plate with basified 2-propanol. The LLE or SPE extracts were dried and reconstituted for column-switching high-performance liquid chromatography with tandem mass spectrometric detection (HPLC/MS/MS). The lower limit of quantitation and the assay range were the same as the 96-well formatted assay. If combined with appropriate automation, sample preparation in the 384-well format would be up to five times more efficient than the 96-well format.

Microfluidic devices for the analysis of single cells: leaving no protein uncounted.

Abstract: Microfluidic devices are revolutionizing bioanalysis, and designs capable of detecting single protein molecules are now available. Two recently described microfluidic devices provide information on...

Non-equilibrium migration mechanisms for microfluidic bioanalysis

Abstract: The leitmotif of this work is to exploit Brownian motion for bioanalysis in the context of migration and separation in microfluidic systems operating far from thermal equilibrium. The increasing importance of bioanalysis is based on the fast growing fields of biotechnology and pharmaceutics. They have a great demand of fast, cheap and robust bioanalytical systems. On the one hand, these systems have to provide pure samples, and on the other hand, have to assure the quality of the product. In order to contribute to these demands, fundamental physical phenomena are studied, such as Absolute Negative Mobility, ratchets and diffusion control and their relevance to bioanalytical applications is illustrated. Two possible approaches are pursued: either the phenomena are directly studied with biological samples or with microparticles as models for cells in order to provide a proof of principle.

Efficient and practical syntheses of a stable labelled form of the non‐narcotic analgesic, bicifadine and its metabolites

Abstract: An efficient and practical synthesis of deuterium-labelled bicifadine, together with 3 deuterium-labelled bicifadine metabolites, was developed. The levels of deuterium incorporation are high, and the products were used as internal standards in bioanalytical method development and clinical bioanalysis. Copyright © 2007 John Wiley & Sons, Ltd.

Oxidoreductases for bioanalysis and food controls

Abstract: Most well-known enzymatic methods have been employed in the bioanalysis of food, required by law in a number of countries. Food is a complex sample and hence difficult to analyze. In food analysis, there are many different types of sample, such as: clear or turbid, colorless or colored, neutral or acid, liquids, pastes and solids, that can be analyzed by enzymatic methods. The oxidase - peroxidase system may be used for specific analyses. In the reaction catalyzed by the oxidase, hydrogen is transferred from the substrate to oxygen and hydrogen peroxide is formed as an intermediate. Hydrogen peroxide then reacts with a leuco dye in the presence of the enzyme peroxidase and a color develops which is measured in the visible spectrum. This article is a review of two enzymes, peroxidase (POD) and glycerol-3-phosphate oxidase (GPO), pointing out their principal sources, characteristics and applications