A novel chiral thiol reagent for automated precolumn derivatization and high-performance liquid chromatographic enantioseparation of amino acids and its application to the aspartate racemase assay

A novel optically active thiol compound, N-(tert-butylthiocarbamoyl)-L-cysteine ethyl ester (BTCC), is synthesized as a chiral derivatization reagent. This compound and o-phthalaldehyde react with amino acid enantiomers to produce fluorescent diastereomers that are readily separable on a reverse-phase column by HPLC. Enantioseparation of acidic amino acids in particular is markedly improved using BTCC. In this study, the HPLC method for enantioseparation with the novel compound is applied to the aspartate (Asp) racemase assay. Derivatized D-Asp is eluted before the L-Asp derivative. Consequently, a small amount of D-Asp produced by the activity of racemase on a large quantity of L-Asp substrate may be quantified accurately, even at very low activity. Since the derivatization reaction proceeds rapidly at room temperature, a fully automated system is established for derivatization and sample injection. The automated method is practical and successfully applied to the archaeal Asp racemase assay. We presume that the procedure is additionally applicable to the enantioseparation of other amino acids, amino alcohols, and catecholamines.     

Scalable transcriptome preparation for massive parallel sequencing

Background

The tremendous output of massive parallel sequencing technologies requires automated robust and scalable sample preparation methods to fully exploit the new sequence capacity.

Methodology

In this study, a method for automated library preparation of RNA prior to massively parallel sequencing is presented. The automated protocol uses precipitation onto carboxylic acid paramagnetic beads for purification and size selection of both RNA and DNA. The automated sample preparation was compared to the standard manual sample preparation.

Conclusion/Significance

The automated procedure was used to generate libraries for gene expression profiling on the Illumina HiSeq 2000 platform with the capacity of 12 samples per preparation with a significantly improved throughput compared to the standard manual preparation. The data analysis shows consistent gene expression profiles in terms of sensitivity and quantification of gene expression between the two library preparation methods.     

Semi-automatic liquid chromatographic analysis of olpadronate in urine and serum using derivatization with (9-fluorenylmethyl)chloroformate

The semi-automatic bioanalytical assays for olpadronate [(3-dimethylamino-1-hydroxypropylidene)bisphosphonate] involves a protein precipitation with trichloroacetic acid and a double co-precipitation with calcium phosphate for serum samples and a triple calcium co-precipitation for urine samples. These manual procedures are followed by an automated solid-phase extraction on a cation-exchange phase. The procedure is continued either directly, at high olpadronate levels in urine, or after off-line evaporation under nitrogen and reconstitution in water on the same robotic workstation. The continued automatic procedure comprehends derivatization with (9-fluorenylmethyl)chloroformate, ion-pair liquid–liquid extraction and ion-pair HPLC with fluorescence detection at 274/307 nm. The intra- and inter-day precisions for urine and serum samples are typically in the 5–8% range for different olpadronate concentrations [levels near the lower limit of quantification (LLQ) excluded]. The LLQ is 5 ng/ml olpadronate for a 2.5-ml urine sample and 10 ng/ml for a 1-ml serum sample, respectively.     

Use of automated microscopy for the detection of disseminated tumor cells in bone marrow samples

The use of automated microscopy has reached the maturity necessary for its routine use in the clinical pathology laboratory. In the following study we compared the performance of an automated microscope system (MDS) with manual method for the detection and analysis of disseminated tumor cells present in bone marrow preparations from breast carcinoma patients. The MDS System detected rare disseminated tumor cells among bone marrow mononuclear cells with higher sensitivity than standard manual microscopy. Automated microscopy also proved to be a method of high reproducibility and precision, the advantage of which was clearly illustrated by problems of variability in manual screening. Accumulated results from two pathologists who had screened 120 clinical slides from breast cancer patients both by manual microscopy and by use of the MDS System revealed only two (3.8%) missed by the automatic procedure, whereas as many as 20 out of 52 positive samples (38%) were missed by manual screening.     

Development and validation of a high-performance liquid chromatography–tandem mass spectrometry assay for the determination of sanfetrinem in human plasma

A rapid, selective and accurate high-performance liquid chromatography–tandem mass spectrometry assay for the quantification of sanfetrinem in human plasma has been developed and validated. The performance of manual and automated sample preparation was assessed; 50 μl of plasma sample was deproteinized with acetonitrile, followed by dilution with water and injection onto the LC system. Chromatographic separation was achieved on a Phenomenex Luna C₁₈(2), 50×2.0 (5 μm) column with a mobile phase consisting of water–acetonitrile with 0.1% formic acid followed by detection with a Perkin-Elmer API3000 mass spectrometer in multiple reaction monitoring mode. The lower limit of quantification was improved by five times compared to the UV method previously reported. A range of concentration from 10 ng/ml to 5 μg/ml was covered. The method was applied to the quantification of sanfetrinem in human plasma samples from healthy volunteers participating in a clinical study.     

A comparison of fluorescent stains for the assessment of viability and metabolic activity of lactic acid bacteria

Lactic acid bacteria (LAB) are used as starter or probiotic cultures in the food and pharmaceutical industries and, therefore, rapid and accurate methods for the detection of their viability are of practical relevance. In this study 10 LAB strains, belonging to the genera Enterococcus, Lactococcus, Leuconostoc, Lactobacillus, Streptococcus and Weissella, were subjected to heat and oxidative stresses and cell injury or death was assessed comparing different fluorescent probes (Syto 9; Propidium Iodide, PI; 4,6-diamidino-2-phenylindole, DAPI; 5,(6)-carboxyfluorescein diacetate, cFDA) to identify the stain combination which most reliably allowed the detection of live/metabolically active and dead cells. Protocols for specimen preparation and staining were optimized and a simple procedure for automated cell counts was developed using NIH ImageJ macros. Cysteine and semi-solid agar solution were efficiently used as anti-fading agent and mounting medium, respectively. The double staining cFDA-PI apparently offered the best and most versatile indication of both cell metabolic activity and membrane integrity. An excellent correlation between manual and automated cell counts for the majority of strain/stain combinations was found. This work provides a simple protocol for specimen preparation and staining based on the use of safe, easy to prepare and inexpensive reagents as compared to other methods. Additionally, the automated cell count procedure developed can be applied to several bacterial species and allows an increase in the number of experimental trials and the reproducibility and sensitivity of the analysis.     

Phosphorylation sites in riboflavin-binding protein characterized by fast atom bombardment mass spectrometry

The Lowry method for quantitation of protein was adapted to automated flow injection analysis. The procedure was developed using two different pure proteins: bovine serum albumin and hepatitis B surface antigen. The system was optimized for reagent concentration, pH, gain, temperature, sample volume, and output. The response of each protein was affected differently by temperature. The reaction slopes and absorbance values of the proteins were similar at 90 degrees C to allow quantitation of hepatitis surface antigen against bovine serum albumin. Advantages of the automated flow injection analysis Lowry procedure include: rapid analyses (90 samples/h), small sample volume (30 microliters, 100 microliters), fast response (20 s), reproducibility (less than or equal to 2% CV within an assay and 3 to 6% CV among assays), sensitivity (5 micrograms), and high correlation (99.8%) with manual assay. After a 30-min set-up period, the analyzer was available to assay protein on demand throughout the day, making it suitable for process and quality control testing.     

Automation of a Hemagglutination-Inhibition Test for Parainfluenza 3 Antibodies in Bovine Sera

An automated hemagglutination-inhibition (HI) test for the "shipping fever" strain (SF-4) of parainfluenza 3 antibody in bovine sera was developed and compared to manual tube and microtiter test procedures. The automated system operating at 60 samples per hr provided the most test results per specified time period, and the manual tube test provided the least. The manual microtiter test and the automated system at 40 samples per hr, falling between the two above procedures, were comparable in the number of sera that could be titrated in 1 day by one technician. There was little difference between automated and manual test reproducibility when measured at the twofold titer one-dilution difference level. However, the automated system titrated a higher number of sera at the same titer on repeat runs than either of the manual test procedures. The automated one-quartile difference reproducibility (each twofold dilution subdivided into 4 units-"quartiles") was equal to the manual test one-dilution difference reproducibility. The standard deviation of the per cent variation from the mean of paired serum titers for 40-sample-per-hr runs ranged from +/-3.49 to +/-5.36%. The manual and automated systems were of comparable sensitivity in their detection of negative sera.     

Nanoflow liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry: sample preparation, data analysis, and application to the analysis of complex peptide mixtures

We report the development of a robust interface for off-line coupling of nano liquid chromatography (LC) to matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) and its application to the analysis of proteolytic digests of proteins, both isolated and in mixtures. The interface makes use of prestructured MALDI sample supports to concentrate the effluent to a small sample plate area and localize the MALDI sample to a predefined array, thereby enriching the analyte molecules and facilitating automated MALDI-MS analysis. Parameters that influence the preparation of MALDI samples from the LC effluent were evaluated with regard to detection sensitivity, spectra quality, and reproducibility of the method. A procedure for data processing is described. The presented nano LC MALDI-MS system allowed the detection of several peptides from a tryptic digest of bovine serum albumin, at analyzed amounts corresponding to one femtomole of the digested protein. For the identification of native proteins isolated from mouse brain by two-dimensional gel electrophoresis, nano LC MALDI-MS increased the number of detected peptides, thereby allowing identification of proteins that could not be identified by direct MALDI-MS analysis. The ability to identify proteins in complex mixtures was evaluated for the analysis of Escherichia coli 50S ribosomal subunit. Out of the 33 expected proteins, 30 were identified by MALDI tandem time of flight fragment ion fingerprinting.     

Fully-automated systematic toxicological analysis of drugs,poisons, and metabolites in whole blood,urine, and plasma by gas chromatography–full scan mass spectrometry

The availability of automated, rapid and reliable methods for the systematic toxicological analysis (STA) of drugs and poisons in biosamples is of great importance in clinical and forensic toxicology laboratories. Gas chromatography–continuous scan mass spectrometry (GC–MS) possesses a high potential in STA because of its selectivity and identification power. However, in order to develop a fully automated STA method based on GC–MS two main obstacles have to be overcome: (a) sample preparation is rather sophisticated owing to the need to isolate analytes from the aqueous matrix and to allow a correct GC repartition of polar analytes; (b) the large amount of information collected within a single analysis makes it difficult to isolate relevant analytical information (mass spectra of analytes) from the chemical noise. Using a bench-top GC–MS system equipped with a laboratory robot for sample preparation (the Hewlett-Packard 7686 PrepStation) and an original method for mass spectral purification, a fully automated STA procedure was developed involving isolation of drugs from the sample (whole blood with minimal pretreatment, plasma, urine) by means of solid-phase extraction, derivatization (trimethylsilylation) of the acidic–neutral and of the basic extracts, GC–MS analysis, processing of data, and reporting of results. Each step of the procedure, and the method for data analysis in particular, can be easily integrated with other existing STA methods based on GC–MS.     

Semi-automatic liquid chromatographic analysis of pamidronate in serum and citrate plasma after derivatization with 1-naphthylisothiocyanate

The semi-automatic method for the determination of the bisphosphonate pamidronate in serum and citrate plasma involves a manual protein precipitation with trichloroacetic acid and a manual coprecipitation of the bisphosphonate with calcium phosphate, followed by an automated solid-phase extraction on anion-exchange columns. After off-line evaporation of the extract under nitrogen and reconstitution in water, the automatic procedure is continued by automatic derivatization with 1-naphthylisothiocyanate, ion-pair liquid–liquid extraction and a treatment with hydrogen peroxide, prior to analysis by ion-pair HPLC and fluorescence detection at 285/390 nm. The intra- and inter-day precisions are 1.3 and 7%, respectively, for a standard of 100 ng ml⁻¹ pamidronate in serum; the average accuracy for this standard is 107%. The lower limit of quantification is 20 ng ml⁻¹ pamidronate in 1 ml of human serum.     

On-line coupling of sequential injection with liquid chromatography for the automated derivatization and determination of gamma-aminobutyric acid in human biological fluids

The principle of sequential injection analysis (SIA) was exploited to develop a rapid fully automated and efficient pre-column derivatization procedure coupled on-line to liquid chromatography (HPLC). Using the SIA-HPLC derivatization protocol gamma-aminobutyric acid (GABA) was determined fluorimetrically in human biological fluids with o-phthaldialdehyde (OPA) as derivatization reagent and minimum sample pretreatment. A lab-built SIA system was used to handle samples, standard solutions and OPA reagent. Appropriate volumes of the reagents were introduced in the holding coil of the SIA system and were mixed on propulsion to the HPLC loop through a suitable reaction coil. The chemical (pH, c(OPA), c(mercaptoethanol)) and instrumental variables (volumes of sample and reagent, reaction time) of the reaction were studied and optimized in terms of maximum sensitivity. The chromatographic variables (gradient composition of the eluent and flow rate) were studied for optimum selectivity and peak characteristics. The developed experimental configuration facilitated fully-automated operation thus minimizing errors in handling. Additionally the method as a whole provided very satisfactory sensitivity, precision and accuracy. Direct determination of GABA in human urine and cerebrospinal fluid (CSF) at microg L(-1) (ppb) levels was accomplished, with minimum sample pretreatment.     

A new automated method for isolation of Chlamydia trachomatis from urine eliminates inhibition and increases robustness for NAAT systems

Chlamydia trachomatis is a leading cause of sexually transmitted infection. Diagnostic methods with easy non-invasive sample collection are important to increase testing and hence to reduce the spread of this infection. To enable more use of urine samples in C. trachomatis diagnostics, automation is an absolute requirement since obtaining high-quality DNA from urine specimens involves extensive processing.

Here, we present a study in which a new automated sample preparation method, BUGS'n BEADS™ STI (BnB STI), was used up-front of the BDProbeTec™ ET end point analysis and compared with the full BDProbeTec™ ET method to analyze C. trachomatis in 1002 urine samples.

The BnB STI system represents a new concept within magnetic sample preparation in which bacteria are first isolated from the sample material followed by purification of bacterial nucleic acid using the same magnetic particles. Similar sensitivity and specificity were obtained with both methods. None of the samples processed with BnB STI inhibited the BDProbeTec™ ET test whereas 1.8% showed inhibition when processed according to the manual BDProbeTec™ ET DNA preparation method. Moreover, the average MOTA scores obtained with the BnB STI system were 48% higher for all amplification controls and 57% higher for positive samples, compared to the manual sample preparation. Based on these results and the significant reduction in hands-on-time for urine sample processing, the automated BnB STI sample preparation method was implemented for routine analysis of C. trachomatis from urine samples.     

Fast and sensitive liquid chromatography-mass spectrometry assay for seven androgenic and progestagenic steroids in human serum

A fast and sensitive LC-MS/MS method for the quantitative analysis of seven steroid hormones in 150 μl of human serum was developed and validated. The following compounds were included: 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, testosterone, pregnenolone, and progesterone. Individual stable isotope-labeled analogues were used as internal standards. Sample preparation was performed by liquid-liquid extraction, followed by oxime derivatization to improve the ionization efficiency of the analytes. In contrast to the common derivatization-based methods, the reaction was incorporated into the sample preparation process and the only additional step due to the derivatization was a short heating of the autosampler vials before the sample injection. Chromatographic separation was achieved on a reversed-phase column using a methanol-water gradient. For the analyte detection, a triple quadrupole instrument with electrospray ionization was used. Total run time was 7.0 min and the lower limits of quantification were in the range of 0.03-0.34 nM (0.01-0.10 ng/ml), depending on the analyte. The method was validated using human serum samples from both sexes and applied for the serum steroid profiling of endometriosis patients.     

Semi-automatic liquid chromatographic analysis of pamidronate in urine after derivatization with 1-naphthylisothiocyanate

An existing sensitive chromatographic assay for pamidronate in urine has considerably been automated. Using the same sample processor, the solid-phase extraction (SPE) was automated separately from the derivatization with 1-naphthylisothiocyanate, the two-fold ion-pair liquid–liquid-extraction and the treatment with hydrogen peroxide for the 2–20 ng/ml concentration range. The automatic procedure was preceded by a triple calcium precipitation and interrupted by evaporation of the SPE eluate under nitrogen. For the 0.5–5 μg/ml concentration range one automatic sequence was used by avoiding evaporation during the sample treatment. In addition to the labour-saving of the semi-automatic procedure, the daily sample-throughput was improved compared to the existing manual assay. Further, the validation showed marginal improvements in the precision, accuracy and lower limit of quantification.     

Liquid ultraviolet matrix-assisted laser desorption/ionization -- mass spectrometry for automated proteomic analysis

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet -- matrix-assisted laser desorption/ionisation -- mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. The low-femtomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydroxybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and low-mass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.     

Towards standardized automated immunomonitoring: an automated ELISpot assay for safe and parallelized functionality analysis of immune cells

The ELISpot assay is used for the detection of T cell responses in clinical trials and vaccine evaluations. Standardization and reproducibility are necessary to compare the results worldwide, inter- and intra-assay variability being critical factors. To assure operator safety as well as high-quality experiment performance, the ELISpot assay was implemented on an automated liquid handling platform, a Tecan Freedom EVO. After validation of the liquid handling, automated loading of plates with cells and reagents was investigated. With step by step implementation of the manual procedure and liquid dispensing optimization on the robot platform, a fully automated ELISpot assay was accomplished with plates remaining in the system from the plate blocking step to spot development. The mean delta difference amounted to a maximum of 6%, and the mean dispersion was smaller than in the manual assay. Taken together, we achieved with this system not only a lower personnel attendance but also higher throughput and a more precise and parallelized analysis. This platform has the potential to guarantee validated, safe, fast, reproducible and cost-efficient immunological and toxicological assays in the future.     

Determination of d- and l-aspartate in cell culturing medium,within cells of MPT1 cell line and in rat blood by a column-switching high-performance liquid chromatographic method

HPLC fluorometric methods have been used to analyze trace amounts of d-amino acids in biological samples. In this study, we established an expedient column-switching fluorometric HPLC system that would improve the analysis of d-amino acids, in particular d-aspartate (Asp). Our system consists of the fluorogenic derivatization of amino acids with NBD-F and two chromatographic steps, one that separates individual amino acids in reverse phase mode and another that separates the chiral forms of each amino acid in normal-phase mode. The two separation steps are linked through a trapping column by an automated column-switching system. In addition, sample preparation is simplified and improved, where trichloroacetic acid is used for deproteinization, and borate buffer, pH 9.5 is employed for the fluorescent derivatization. The detection limit for d-Asp in culturing medium is 5 nM. The resulting peak heights correlated well with concentrations that ranged from 12.5 to 250 nM for both d- and l-Asp. The present method was applied to determine d- and l-Asp levels in cell culturing medium, and within cells of MPT1 cell line. The detected cellular levels of d- and l-Asp agree with those detected by our previous method. In addition, this method was used to measure d- and l-Asp levels in rat blood samples, and the results are consistent with the reported values.     

Increased Throughput by Parallelization of Library Preparation for Massive Sequencing

Background

Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing.

Methodology/Principal Findings

In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling.

Conclusions/Significance

We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsize sequencing centers.     

Sample preparation with an automated robotic workstation for organic acid analysis by gas chromatography-mass spectrometry

We attempted to automate sample preparation for analysis of organic acids by gas chromatography-mass spectrometry using a computer-controlled, automated robotic workstation that is integrated and connected to the gas chromatography-mass spectrometry (HP-5890/5971) system. Of the two methods developed, one employed solvent extraction, while the other utilized a silica, solid-phase extraction cartridge. Both automated methods were compared to a manual, solvent extraction procedure used routinely in our laboratory. Normal, spiked urine, and urine from patients with a variety of metabolic abnormalities were analyzed. The robotic workstation did not meet all our requirements for a rapid, reliable, laboratory device. Recoveries with the automated procedure were less than with the manual method, and some organic acids important in the diagnosis of inborn errors of metabolism were not detected. Additionally, the robotic device had mechanical and design problems that made it slower and less reliable than the manual procedure.