Improved Sample Handling Research Articles

The power of modern extraction techniques: A breakthrough in vitamin K extraction from human serum

As a representative of the lipophilic vitamins, vitamin K is one of the most difficult vitamins to analyze. Even modern approaches using mass spectrometry detection cannot be carried out without multi-step, time-consuming, economically, and ecologically demanding sample treatment. These findings suggest an opportunity to develop new modern trends that could shorten and simplify the whole procedure, whereby emphasizing the processing of a large number of samples with an additional focus on more environmentally friendly techniques. We selected and compared the main types of both, modern and conventional approaches for vitamin K analysis in human serum including liquid–liquid extraction and solid-phase extraction on the one hand, as well as supported liquid extraction, Phree™ phospholipid removal plate, miniaturized liquid–liquid extraction, and dispersive pipette extraction on the other hand. The different techniques were compared from an analytical perspective, including parameters such as recovery, limits of detection, and matrix effects, as well as from a non-analytical perspective, including time consumption, cost effectiveness, and manual labor required to perform the procedure. The modern techniques seem to have great advantages. Our results indicate that the Phree™ phospholipid removal plate, and the miniaturized version of the liquid–liquid extraction are the best techniques. This work was also carried out to provide a guide for the extraction of vitamin K while determining it using the liquid chromatography-mass spectrometry technique, which may improve sample handling and help to remove potential obstacles in the future.

Black gold rush - Evaluating the efficiency of the Fractionator in separating Hymenoptera families in a meadow ecosystem over a two week period.

In the face of insect decline, monitoring projects are launched widely to assess trends of insect populations. Collecting over long time periods results in large numbers of samples with thousands of individuals that are often just stored in freezers waiting to be further processed. As the time-consuming process of sorting and identifying specimens prevents taxonomists from working on mass samples, important information on species composition remains unknown and taxonomically neglected species remain undiagnosed. Size fractioning of bulk samples can improve sample handling and, thus, can help to overcome the taxonomic impediment. In this paper, we evaluate the efficiency of the fractionator in separating Hymenoptera families from a Malaise trap sample of a meadow ecosystem over a two week interval to make them available for further morphological identification. The fractionator system by Buffington and Gates (2008) was used to separate the sample in two size classes - a large (macro) and a small (micro) fraction - and Hymenoptera specimens were then counted and identified on family level. In total, 2,449 Hymenoptera specimens were found in the macro fraction and 3,016 in the micro fraction (5,465 specimens in total). For 24 out of 34 Hymenoptera families (71%), separation was significant. This study illustrates the efficiency of the fractionator and its potential to improve workflows dealing with specimen-rich Malaise trap samples.

Capillary liquid chromatography coupled with mass spectrometry for analysis of nanogram protein quantities on a wide-pore superficially porous particle column in top-down proteomics

In top-down proteomics experiments, intact protein ions are subjected to gas-phase fragmentation for MS analysis without prior digestion. This approach is used to characterize post-translational modifications and clipped forms of proteins, avoids several “inference” problems associated with bottom-up proteomics, and is well suited to the study of proteoforms. In the past decade, top-down proteomics has progressed rapidly, taking advantage of MS instrumentation improvements and the efforts of pioneering groups working to improve sample handling and data processing. The potential of this technology has been established through its successful use in a number of important biological studies. However, many challenges remain to be addressed like improving protein separation capabilities such that it might become possible to expand the dynamic range of whole proteome analysis, address co-elution and convoluted mass spectral data, and aid final data processing from peak identification to quantification. In this study, we investigated the use of a wide-pore silica-based superficially porous media with a high coverage phenyl bonding, commercially packed into customized capillary columns for the purpose of top-down proteomics. Protein samples of increasing complexity were tested, namely subunit digests of a monoclonal antibody, components of purified histones and proteins extracted from eukaryotic ribosomes. High quality mass spectra were obtained from only 100 ng of protein sample while using difluoroacetic acid as an ion pairing agent to improve peak shape and chromatographic resolution. A peak width at half height of about 15 s for a 45 min gradient time was observed on a complex mixture giving an estimated peak capacity close to 100. Most importantly, efficient separations were obtained for highly diverse proteins and there was no need to make method specific adjustments, suggesting this is a highly versatile and easy-to-use setup for top-down proteomics.

Studying membrane proteins with MicroED.

The structural investigation of biological macromolecules is indispensable in understanding the molecular mechanisms underlying diseases. Several structural biology techniques have been introduced to unravel the structural facets of biomolecules. Among these, the electron cryomicroscopy (cryo-EM) method microcrystal electron diffraction (MicroED) has produced atomic resolution structures of important biological and small molecules. Since its inception in 2013, MicroED established a demonstrated ability for solving structures of difficult samples using vanishingly small crystals. However, membrane proteins remain the next big frontier for MicroED. The intrinsic properties of membrane proteins necessitate improved sample handling and imaging techniques to be developed and optimized for MicroED. Here, we summarize the milestones of electron crystallography of two-dimensional crystals leading to MicroED of three-dimensional crystals. Then, we focus on four different membrane protein families and discuss representatives from each family solved by MicroED.

Automated Trimethyl Sulfonium Hydroxide Derivatization Method for High-Throughput Fatty Acid Profiling by Gas Chromatography-Mass Spectrometry.

Fatty acid profiling on gas chromatography–mass spectrometry (GC–MS) platforms is typically performed offline by manually derivatizing and analyzing small batches of samples. A GC–MS system with a fully integrated robotic autosampler can significantly improve sample handling, standardize data collection, and reduce the total hands-on time required for sample analysis. In this study, we report an optimized high-throughput GC–MS-based methodology that utilizes trimethyl sulfonium hydroxide (TMSH) as a derivatization reagent to convert fatty acids into fatty acid methyl esters. An automated online derivatization method was developed, in which the robotic autosampler derivatizes each sample individually and injects it into the GC–MS system in a high-throughput manner. This study investigated the robustness of automated TMSH derivatization by comparing fatty acid standards and lipid extracts, derivatized manually in batches and online automatically from four biological matrices. Automated derivatization improved reproducibility in 19 of 33 fatty acid standards, with nearly half of the 33 confirmed fatty acids in biological samples demonstrating improved reproducibility when compared to manually derivatized samples. In summary, we show that the online TMSH-based derivatization methodology is ideal for high-throughput fatty acid analysis, allowing rapid and efficient fatty acid profiling, with reduced sample handling, faster data acquisition, and, ultimately, improved data reproducibility.

Fully automated peptide mapping multi-attribute method by liquid chromatography–mass spectrometry with robotic liquid handling system

The multi-attribute method (MAM) based on liquid chromatography (LC)–tandem mass spectrometry is emerging as a powerful tool to directly monitor multiple product quality attributes simultaneously. To better implement MAM, either for product characterization or for quality control (QC), there is a need for a robust, universal, and high-throughput workflow that can be broadly adopted in different laboratories with minimal barriers to implementation. Manual preparation of samples for MAM, however, is labor intensive and produces nontrivial variations across analysts and laboratories. We describe the development of a fully automated peptide mapping procedure with a high-throughput robotic liquid handling system to improve sample handling capacity and outcome reproducibility while saving analyst hands-on time. Our procedure features the automation of a “microdialysis” step, an efficient desalting approach prior to proteolytic digestion that optimizes digestion completeness and consistency each time. The workflow is completely hands-free and requires the analyst only to pre-normalize the sample concentrations and to load buffers and reagents at their designated positions on the robotic deck. The robotic liquid handler performs all the subsequent preparation steps and stores the digested samples on a chiller unit to await retrieval for further analysis. We also demonstrate that the manual and automated procedures are comparable with regard to protein sequence coverage, digestion completeness and consistency, and quantification of posttranslational modifications. Notably, in contrast to a previously reported automated sample preparation protocol that relied on customized accessories, all components in our automation procedure are commercial products that are readily available. In addition, we also present the high-throughput data analysis workflow by using Protein Metrics. The automation procedure can be applied cross-functionally in the biopharmaceutical industry and, given its practicality and reproducibility, can pave the way for MAM implementation in QC laboratories.

A simple method to immunopurify erthyropoiesis stimulating agents from urine, aiming to optimize erythropoietin screening by SAR-PAGE.

The efficiency of the immunopurification step of urinary erythropoietin (EPO) and recombinant forms is important for their optimal detection in antidoping screening. Previous investigations of immunopurification techniques have been done for immunomagnetic beads, EPO Purification Kit (EPK) columns (MAIIA Diagnostics), and enzyme-linked immunosorbent assay (ELISA) microplates (Stemcell Technologies) conjugated/coated with anti-EPO antibodies. In this study, a new immunopurification technique using anti-EPO sepharose gel beads, developed by MAIIA Diagnostics, to simplify and minimize sample handling was evaluated. This EPO Purification Gel Kit (EPGK) was compared with our current routine EPK for limit of detection (LOD). Linearity, recovery, repeatability, sample incubation time, and sample volume were also evaluated for EPGK. The LODs and linearity for EPK and EPGK were comparable to each other and the recovery for BRP, NESP, CERA, and EPO-Fc were within the range of other studies, and concentration of the sample eluate improved the recovery results. Little variation was seen within days, between days, and between operators. A 90 minute incubation of the sample with the sepharose gel beads is sufficient for most of the erythropoiesis stimulating agents (ESAs) tested, with 10 mL being an optimal sample volume for EPGK. The improved sample handling, higher sample throughput and the reduced working time demonstrate that the EPGK is a better alternative to the current MAIIA EPK immunopurification method for urine. The EPO Purification Gel Kit (from MAIIA Diagnostics) was evaluated and validated for immunopurification of endogenous erythropoietin and exogenous erythropoiesis stimulating agents from urine samples. The kit was a better alternative to that currently used (EPO Purification Kit) in many antidoping laboratories because it improves sample handling and increases sample throughput.

A new enzyme immunoassay for the determination of highly sialylated and fucosylated human α1-acid glycoprotein as a biomarker of tumorigenesis

BackgroundUpon initiation and progression of cancer, α1-acid glycoprotein (AGP) possessing highly sialylated and fucosylated glycans appears in the serum, and recently has attracted a great deal of attention, as a potential biomarker of tumorigenesis in humans. MethodsTo establish a rapid and precise method for the quantitative assay of fucosylated AGP in serum samples, we developed an enzyme immunoassay (EIA) bearing an anti-AGP antibody and a fucose-binding lectin, Aleuria aurantia (AAL) with additional endeavor to improved sample handling, and antibody preparations. ResultsThe amounts of fucosylated AGP could be determined by the present method with a good performance feature in all tested samples from both cancer patients and healthy controls. From cancer patients under chemotherapy we show that fucosylated AGP could be a clinically relevant biomarker for cancer progression or prognosis as well as for an early assessment of clinical response and treatment outcomes. Furthermore, in a different setting, fucosylated AGP also showed relevance in patients who received immunotherapy with an anti-programmed cell death-1 (PD-1) antibody. Conclusionsα1,3fucosylated AGP is a potential biomarker of cancer initiation, progression and response to treatment in cancer patients.

Trends and advances in food analysis by real-time polymerase chain reaction.

Analyses to ensure food safety and quality are more relevant now because of rapid changes in the quantity, diversity and mobility of food. Food-contamination must be determined to maintain health and up-hold laws, as well as for ethical and cultural concerns. Real-time polymerase chain reaction (RT-PCR), a rapid and inexpensive quantitative method to detect the presence of targeted DNA-segments in samples, helps in determining both accidental and intentional adulterations of foods by biological contaminants. This review presents recent developments in theory, techniques, and applications of RT-PCR in food analyses, RT-PCR addresses the limitations of traditional food analyses in terms of sensitivity, range of analytes, multiplexing ability, cost, time, and point-of-care applications. A range of targets, including species of plants or animals which are used as food ingredients, food-borne bacteria or viruses, genetically modified organisms, and allergens, even in highly processed foods can be identified by RT-PCR, even at very low concentrations. Microfluidic RT-PCR eliminates the separate sample-processing step to create opportunities for point-of-care analyses. We also cover the challenges related to using RT-PCR for food analyses, such as the need to further improve sample handling.Electronic supplementary materialThe online version of this article (doi:10.1007/s13197-016-2205-0) contains supplementary material, which is available to authorized users.

Sensing lanthanide metal content in biological tissues with magnetic resonance spectroscopy.

The development and validation of MRI contrast agents consisting of a lanthanide chelate often requires a determination of the concentration of the agent in ex vivo tissue. We have developed a protocol that uses 70% nitric acid to completely digest tissue samples that contain Gd(III), Dy(III), Tm(III), Eu(III), or Yb(III) ions, or the MRI contrast agent gadodiamide. NMR spectroscopy of coaxial tubes containing a digested sample and a separate control solution of nitric acid was used to rapidly and easily measure the bulk magnetic susceptibility (BMS) shift caused by each lanthanide ion and gadodiamide. Each BMS shift was shown to be linearly correlated with the concentration of each lanthanide ion and gadodiamide in the 70% nitric acid solution and in digested rat kidney and liver tissues. These concentration measurements had outstanding precision, and also had good accuracy for concentrations ≥10 mM for Tm(III) Eu(III), and Yb(III), and ≥3 mM for Gd(III), gadodiamide, and Dy(III). Improved sample handling methods are needed to improve measurement accuracy for samples with lower concentrations.

Biopsy Misidentification Identified by DNA Profiling in a Large Multicenter Trial

The Reduction by Dutasteride of Prostate Cancer Events (REDUCE) prostate cancer risk reduction study randomly assigned 8,231 men to dutasteride or placebo for 4 years. Protocol-mandated biopsies were obtained after 2 and 4 years. After the discovery of three cases of biopsy sample misidentification in the first 2 years, all protocol-mandated biopsy samples were DNA tested to verify biopsy identity. Biopsy and blood DNA profiling was performed retrospectively for the year 2 scheduled biopsies and prospectively for the year 4 scheduled biopsies. Toward the end of year 2, multiple changes were made to improve sample handling and chain of custody. Of the 6,458 year 2 and 4,777 year 4 biopsies, 26 biopsies reflecting 13 sample handling errors at year 2 (0.4%) and one biopsy reflecting one sample handling error at year 4 (0.02%) were confirmed to be mismatched to the patient for whom they were originally submitted. Of 6,733 reference blood samples profiled, 31 (0.5%) were found to be mismatched to the patient's verified identity profile. Sample identification errors occurred at local research sites and central laboratories. Biopsy misidentification is a potential problem in clinical laboratories and clinical trials. Until now, biopsy misidentification has not been studied in the setting of a large, multinational clinical trial. In the REDUCE study, process improvement initiatives halfway through the trial dramatically reduced biopsy mismatches. The potential for biopsy mismatches in clinical trials and clinical practice is an under-recognized problem that requires rigorous attention to details of chain of custody and consideration of more widespread DNA identity testing.

Shotgun proteome analysis utilising mixed mode (reversed phase‐anion exchange chromatography) in conjunction with reversed phase liquid chromatography mass spectrometry analysis

The 2-D peptide separations employing mixed mode reversed phase anion exchange (MM (RP-AX)) HPLC in the first dimension in conjunction with RP chromatography in the second dimension were developed and utilised for shotgun proteome analysis. Compared with strong cation exchange (SCX) typically employed for shotgun proteomic analysis, peptide separations using MM (RP-AX) revealed improved separation efficiency and increased peptide distribution across the elution gradient. In addition, improved sample handling, with no significant reduction in the orthogonality of the peptide separations was observed. The shotgun proteomic analysis of a mammalian nuclear cell lysate revealed additional proteome coverage (2818 versus 1125 unique peptides and 602 versus 238 proteins) using the MM (RP-AX) compared with the traditional SCX hyphenated to RP-LC-MS/MS. The MM analysis resulted in approximately 90% of the unique peptides identified present in only one fraction, with a heterogeneous peptide distribution across all fractions. No clustering of the predominant peptide charge states was observed during the gradient elution. The application of MM (RP-AX) for 2-D LC proteomic studies was also extended in the analysis of iTRAQ-labelled HeLa and cyanobacterial proteomes using nano-flow chromatography interfaced to the MS/MS. We demonstrate MM (RP-AX) HPLC as an alternative approach for shotgun proteomic studies that offers significant advantages over traditional SCX peptide separations.

Surface plasmon resonance instrument as a refractometer for liquids and ultrathin films

A surface plasmon resonance (SPR) setup in Kretschmann configuration is being utilized as a refractometer for both liquids as well as ultrathin films. The SPR signal detection technology used is based on a goniometer approach providing a wide angular scan range which facilitates highly accurate liquid and gas phase measurements. Attention was paid to improve sample handling and preparation. In order to avoid cross-contamination between measurements an easily removable and exchangeable molded PDMS flow cell was used during the measurements. By careful choice of components for liquid handling the dead volume of the system could be reduced down to some microliters. The angular change and thus the refractive index for sucrose, ethylene glycol (EG) and ethanol solutions with different concentrations, the thickness and refractive index of deposited Langmuir-Blodgett (LB) films, and the interaction kinetics between a biotin containing self-assembled monolayer (SAM) and streptavidin were determined. The measured refractive indices of sucrose, EG and ethanol solutions corresponded well with literature values. LB films were characterized by measuring the complete SPR curve in an angular scan range from 40 to 78°. A two-color SPR approach combined with two-media measurements was successfully employed for simultaneous and unambiguous determination of both refractive index and thickness of stearic acid monolayers. The thickness obtained for the stearic acid monolayer was 2.66 nm, and the refractive indices at 635 and 670 nm were 1.5800 and 1.4138, respectively. The developed sensor-plate holder enabled functionalization of the SPR gold chip outside the instrument, therefore making the sample handling more flexible. The affinity constant obtained for the streptavidin–biotin interaction was 1.01 × 10 −8 M. The total angle SPR method used in this study clearly shows its potential to be used as a refractometer for both liquids and ultrathin films, as well as for traditional liquid phase biomolecular kinetic studies.

Environmental Proteomics: a Paradigm Shift in Characterizing Microbial Activities at the Molecular Level

The increase in sequencing capacity led to a new wave of metagenomic projects, enabling and setting the prerequisite for the application of environmental proteomics technologies. This review describes the current status of environmental proteomics. It describes sample preparation as well as the two major technologies applied within this field: two-dimensional electrophoresis-based environmental proteomics and liquid chromatography-mass spectrometry-based environmental proteomics. It also highlights current publications and describes major scientific findings. The review closes with a discussion of critical improvements in the area of integrating experimental mass spectrometry technologies with bioinformatics as well as improved sample handling.

Fospropofol Assay Issues and Impact on Pharmacokinetic and Pharmacodynamic Evaluation

To the Editor:— Fospropofol disodium (GPI 15715 or Aquavan® Injection; MGI Pharma, Inc., Bloomington, MN) is a water-soluble, phosphono-O -methyl prodrug of propofol for intravenous injection. It has been evaluated for sedation during diagnostic and routine therapeutic procedures. The early evaluation studies were published mostly in Anesthesiology between 2003 and 2005.After intravenous administration, fospropofol is rapidly metabolized by alkaline phosphatase enzymes, releasing propofolFP. Several pharmacokinetic and pharmacodynamic studies have shown that propofolFPdemonstrated differences in pharmacokinetic and pharmacodynamic profiles compared with propofol in a lipid solution.1–5We have recently discovered an assay problem that may have affected the measurement of propofolFPplasma concentrations in previously published studies. In the earlier studies,1–4,6blood samples were collected in tubes containing sodium orthovanadate (SOV; 60 mg added as a solid powder to maintain 10 mg/ml concentration) to prevent further in vitro conversion of fospropofol to propofol by alkaline phosphatase enzymes. This was found to result in incomplete dissolution of the SOV powder and variable concentrations of SOV that affected plasma pH and caused hemolysis of many samples, leading to changes in propofol extraction recovery and storage stability. As a result, the propofolFPconcentrations obtained in previous studies1–4,6could possibly be inconsistent and unreliable, because the impact of the aforementioned factors was neither known nor controlled, and therefore, the originally reported propofol pharmacokinetic and pharmacodynamic results and the derived conclusions could be inaccurate. It was shown that the assay and stability problem was limited to quantitation of propofolFPand that it did not affect the fospropofol concentrations. The new drug application for fospropofol disodium was submitted to the US Food and Drug Administration in September 2007. The propofol assay problem was reported in detail in the New Drug Application, as were details of the revised assay methodology. Subsequent to the discovery of the problem, the sample handling procedure was standardized to reduce variation in SOV concentration (e.g. , SOV was added as a solution), and improved sample handling and processing techniques that resolved the problems were developed and validated. Additional studies were then conducted using an appropriate assay to assess the pharmacokinetics and pharmacodynamics of fospropofol in healthy volunteers and patients. We plan to publish these results shortly, along with an estimate of the degree of error from the previously published studies that reported results using the old assay. We very much regret the magnitude of the originally published incorrect information and the confusion that it has and will cause in the pharmacokinetics of propofol from the use of fospropofol.*MGI Pharma, Inc., Bloomington, Minnesota. ajit.shah@mgipharma.com

PolLux: A new facility for soft x-ray spectromicroscopy at the Swiss Light Source

We report on the successful installation and operation of a scanning transmission x-ray microspectroscope (STXM) at the PolLux facility at the Swiss Light Source. This integration of an advanced STXM with improved sample handling capabilities and a novel beamline provides unique capabilities. PolLux uses linearly or circularly polarized x-rays from a bending magnet with an extended photon energy range (200-1400 eV). It is therefore well suited to determine a sample's quantitative chemical composition, molecular orientation, or thickness of organic as well as condensed matter materials. The local magnetic state of magnetic thin films is accessible through fast helicity switching by steering the electron beam off axis through the bending magnet. Ex vacuo girder movers allow fast and highly reproducible (<1 microm) alignment of the instrument with respect to the photon beam. The present spatial resolution is approximately 20 nm, limited by the zone plates utilized. The instrument has the stability and positional resolution to operate with much higher resolution optics as it becomes available. In addition to characterization experiments, we present several typical examples from materials research and environmental science to exemplify the capabilities.

Characterization of the 5-HT2b Receptor in Evaluation of Aequorin Detection of Calcium Mobilization for Miniaturized GPCR High-Throughput Screening

Fluorescent detection of calcium mobilization has been used successfully to identify modulators of G-protein-coupled receptors (GPCRs); however, inherent issues with fluorescence may limit its potential for high-throughput screening miniaturization. The data presented here demonstrate that the calcium-sensitive photoprotein aequorin (AequoScreen), when compared with FLUO-4 in the same cellular background, allows for miniaturization of functional kinetic calcium flux assays, in which the rank order of potency and efficacy was maintained for a series of diverse small-molecule modulators. Small-volume (<10 microL) 384- and 1536-well aequorin assays were implemented by integration of acoustic dispensing (Echo 550) and kinetic flash luminometry (CyBi Lumax). The enhanced high signal-to-background ratios observed relative to fluorescence were readily manipulated by altering per-well cell densities and yielded acceptable screening statistics in miniaturized format for both agonist and antagonist screening scenarios. In addition, the authors demonstrate the feasibility of using agonist concentrations less than EC(50) in a miniaturized antagonist assay. These features, coupled with improved sample handling, should enhance sensitivity and provide the benefits of miniaturization including cost reduction and throughput gains.

Analysis of primary aliphatic short-chain monoamines by LC in water samples

Several derivatization procedures with o-phthaldialdehyde- N-acetylcysteine (OPA-NAC) were compared for a rapid analysis of primary aliphatic short-chain monoamines in water samples by HPLC using a LiChorospher analytical separation column (100RP 18 125 mm×4 mm i.d., 5 μm). Both the solution and the solid-support assisted off-line derivatization on C 18 SPE cartridges were inadequate options because of beginning degradation processes of the instable isoindol derivatives during their transfer to the analytical column. This problem was precluded with the on-column or solid-support assisted on-line derivatization. In the last mentioned procedure, the derivatization took place in a Hypersil C 18 precolumn ( 20 mm×2.1 mm i.d., 30 μm) connected with an additional preconcentration step resulting in better detection limits (0.002–0.040 μg ml −1 requiring only 150 μl of water sample) than in the on-column procedure (0.08–0.16 μg ml −1). The improved sample handling, the better control of parameters affecting reaction rates, the fully automation of this method with only 10 min analysis time for each sample are further advantageous. The potential of the solid-support assisted on-line derivatization was outlined and applied to water samples from several sources. Recovery values near 100% were obtained.

Detection and identification of human bronchoalveolar lavage proteins using narrow-range immobilized pH gradient DryStrip and the paper bridge sample application method.

The use of two-dimensional gel electrophoresis as a tool for the investigation of human bronchoalveolar lavage fluid (BALF) has been hampered by technical difficulties. In the last decade attempts have been made to establish a two-dimensional (2-D) protein map of BALF samples, resulting in the identification of a number of proteins present in BALF. In this study, we report an improved sample handling and separation protocol for investigation of human BALF proteins. The sample has been analyzed by employing a number of strategies, including the 'paper bridge' sample application method in combination with narrow range immobilized pH gradient (IPG) DryStrips, followed by comparison to the corresponding plasma map. Using peptide mass fingerprinting, we have identified 49 proteins in the narrow pH range 4.5-5.2 from an individual healthy BALF sample. Furthermore, we identified 17 BALF proteins, not detected in plasma. Twelve of these proteins have, to our knowledge, not previously been described in the BALF 2-D map. The mapping of BALF proteins with inclusion of those at low concentration increases the possibility to subsequently screen patient material for disease markers.

Development of a urinary free cortisol assay using solid-phase extraction–capillary electrophoresis

In clinical practice, the measurement of urinary free cortisol (UFC) provides the most sensitive and specific diagnostic information for excess adrenal production of cortisol. The existing methodologies (RIA and HPLC) are time consuming, costly, involve tedious extractions, derivatizations and problems with non-specific interactions with cortisol metabolites in urine. In the present study, we describe the development of an SPE–CE method for the rapid analysis of UFC. UFC was concentrated using SPE C 18 cartridges (3M Empore) under a vacuum and eluted with acetonitrile–SDS. The use of 10% acetone to wash cartridges before final elution with acetonitrile–SDS showed significant improvements in the free cortisol recovery. The complete extraction was accomplished in 10–15 min with a recovery of 89–94%. CE analysis was done on a Beckman P/ACE 5010 with detection at 254 nm using a neutral capillary. Detection limits of free cortisol in urine was improved to 10 μg/l with SPE compared to 500 μg/l without SPE. No interferences either from BSA or other urinary cortisol metabolites affected the free cortisol determinations. The results showed the feasibility of a rapid UFC detection with improved sample handling capacity.