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Direct determination of selenium and other trace elements in serum samples by ICP-MS.

Selenium belongs to a group of trace elements of special interest in biological samples for clinical diagnosis. Selenium has antioxidizing functions and is essential for providing the organism with triiodothyronine produced from thyroxine. Among several analytical techniques used to determine the Se concentration in serum, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) has been used in the past because of its high sensitivity. Interference problems originating from different ions on the major Se isotopes have been described to be a limiting factor for the direct determination of Se in these matrices. Standard addition calibration or isotope dilution is often required to overcome carbon-enhanced ionisation effects in biological sample matrices. In most cases, the typical serum sample volume which is available for the analysis is limited to 0.5 ml or less, making multiple sample preparation for standard addition calibration impractical. Isotope dilution requires enriched isotopes and substantial sample preparation. Furthermore, the approximate Se concentration in every sample has to be known to adjust the appropriate amount of spike to each sample. Matrix matching with methanol has been described to overcome ionisation effects but we found limiting factors of this application when other trace elements are also determined within one sample run. This paper describes an effective sample preparation method which allows the direct determination of Se in serum without limiting the analytical capabilities for the additional determination of Al, Cu, Ni, Co, Cd, Mn and Zn in a single sample run by ICP-MS. Optimization procedures are presented and results of the analysis of reference samples are discussed, with a comparison of more than 150 serum data with those obtained by the GF-AAS method.

Animals↗

Vitamin contents of pre-prepared foods sampled from a hospital food service line.

Thiamin, riboflavin, and ascorbic acid in pre-prepared foods sampled from a hospital food service line decreased slightly during a one and one-half hour serving period and differed somewhat according to month of sampling. On a per serving basis, entrée foods supplied 4% to 17% of the RDA for thiamin and 12% to 18% of the RDA for riboflavin for adult men and women. Potato dishes and fruit gelatins furnished 12% to 26% of the RDA for ascorbic acid, but salads were negligible sources. The data suggest a need to furnish additional sources of these vitamins.

Ascorbic Acid↗

Preparation and stability of sixteen murine tissues and organs for flow cytometric cell cycle analysis.

Three different technical protocols were used to prepare samples for flow cytometric (FCM) analysis. Each protocol developed worked best for only certain organs. Protocol I involved mincing small pieces of fresh tissue in the propidium iodide (PI) staining solution and filtering through packed glass wool. The organs that were prepared by protocol I were: submandibular gland, urinary bladder, liver, thymus, bone marrow, spleen, lung, kidney and testis. Protocol II involved exposure of the organ to 0.5% acetic acid for 48 h prior to mincing in the PI. The organs that were prepared by protocol II were: uterus, rectum, colon, ileum, and heart. Protocol III utilized an exposure to 0.5% acetic acid, pepsinization, and then staining with PI. The tissues that were prepared by protocol III were the epithelium of the anterior surface of the cornea and the epithelium of the surface of the tongue. A total of 16 different organs and tissues were successfully prepared. For each organ, averaged DNA histograms were analyzed by nonparametric and parametric programs and the results (phase fractions) are presented in tabular form. Several of the organs used came from animals exposed to 1.0 mg/kg vincristine (VC) for 5-6 h to test the capability of the different protocols to detect the enlargement of the G2 + M compartment by the accumulation of VC-arrested mitotic figures. The stability of the many different sample preparations was tested by comparing averaged DNA histograms obtained on the day of sample preparation to averaged DNA histograms of the same set of samples after storage at 4 degrees C, with or without fixation in 10% phosphate-buffered formalin, for days to weeks. After staining with propidium iodide, fixation of the sample with a final concentration of 2-3% phosphate-buffered formalin, was the procedure adopted to assure sample stability. The demonstration of sample stability permits sample preparation to occur at one site followed by transport of the samples to the FCM laboratory at another geographical location. The major findings of this work were a) technical protocols were developed which resulted in acceptable nuclear suspensions for FCM from 16 different murine organs or tissues, b) the stability of these samples can be assured by fixing the PI stained nuclear suspension with formalin, and c) each different protocol was capable of detecting and preserving at least some of the mitotic figures arrested and collected by vincristine.

Acetates↗

Preparation of thin sections for Fourier Transform-Infrared microscopy: a new embedding medium for cellulosic samples.

Fourier Transform-Infrared [FT-IR] microscopy is a combination of instrumentation from which information can be derived about the structure and composition of materials; however, it presents unique problems for sample preparation. Traditional methods of preparing fiber cross sections employ embedding media such as methacrylates, epoxides and polyvinyl alcohols, all of which have groups in common with the cellulose molecule, and absorb in the same regions of the IR spectrum. Therefore, a new embedding method employing polystyrene has been developed for the preparation of cross and longitudinal sections of cellulosic fibers. Although polystyrene is a strong IR absorbing material, it can be completely removed from specimens prior to analysis. In addition, FT-IR spectra of cross sections have better resolution than conventional preparation methods employing ground samples prepared in a KBr disk.

Cellulose↗

Should Chlamydia trachomatis confirmation make you cross? Performance of collection kits tested across three nucleic acid amplification test platforms.

OBJECTIVE: To investigate the feasibility of confirming initially reactive nucleic acid amplification assays for Chlamydia trachomatis (CT) by cross testing on a second molecular platform. The three platforms investigated were Aptima Combo 2 assay (AC2), Cobas Amplicor CT test (PCR) and ProbeTec ET CT assay (SDA). METHODS: Serial dilutions of a CT culture were prepared in 0.9% saline; used to prepare simulated swab samples for all three platforms, and tested as in the manufacturer's instructions. For the cross testing investigation, 1 ml of the simulated swab samples prepared in each of the three collection kits was transferred into the appropriate collection kit for the second platform. RESULTS: AC2 demonstrated a higher analytical sensitivity than the SDA and PCR assays. Upon cross testing AC2 again demonstrated a superior performance to the SDA and PCR assays even when testing swab samples originally prepared in the SDA and PCR transport medium. The SDA assay was inhibited by the addition of transport medium from both the AC2 and PCR assays. CONCLUSION: The analytical sensitivity of the three assays is not identical, therefore confirming initially reactive samples on a second platform may prove to be difficult. However, the higher sensitivity of the AC2 assay could allow its use as a confirmatory assay for reactive swab samples collected in the SDA and PCR transport medium.

Chlamydia Infections↗

Fast and sensitive environmental analysis utilizing microextraction in packed syringe online with gas chromatography-mass spectrometry: determination of polycyclic aromatic hydrocarbons in water.

A new sensitive, selective, fast and accurate technique for online sample preparation was developed. Microextraction in a packed syringe (MEPS) is a new miniaturised, solid-phase extraction (SPE) technique that can be connected online to GC or LC without any modifications. In MEPS approximately 1mg of the solid packing material is inserted into a syringe (100-250 ml) as a plug. Sample preparation takes place on the packed bed. The bed can be coated to provide selective and suitable sampling conditions. The new method is very promising. It is very easy to use, fully automated, of low cost and rapid in comparison with previously used methods. The determination of polycyclic hydrocarbons (PAHs) in water was performed using MEPS as sample preparation method online with gas chromatography and mass spectrometry (MEPS-GC-MS). The results from MEPS as sample preparation were compared with other techniques such as stir bar sorptive extraction (SBSE) and solid-phase microextraction (SPME). The method was validated and the standard curves were evaluated by the means of quadratic regression and weighted by inverse of the concentration: 1/x for the calibration range 5-1,000 ng/L. The MEPS applied polymer (silica-C8) could be used more than 400 times before the syringe was discarded. The extraction recovery was about 70%. The results showed close correlation coefficients (R>0.998) for all analytes in the calibration range studied. The accuracy of MEPS-GC-MS was between 90 and 113% and the inter-day precision (n=3 days), expressed as the relative standard deviation (RSD%), was 8-16%. MEPS reduced the handling time by 30 and 100 times compared to SPME and SBSE, respectively.

Gas Chromatography-Mass Spectrometry↗

Inorganic, organic, and total mercury in blood and urine: cold vapor analysis with automated flow injection sample delivery.

A cold vapor atomic absorption technique for blood or urine mercury analysis that uses persulfate oxidation to prepare samples for total mercury analysis and acid permanganate oxidation to prepare samples for inorganic mercury analysis is described. The linearity of the procedures ranged from 0.5 to 25 micrograms/L. Precision ranged from 20% at 1 microgram/L to 7% at 20 micrograms/L. Documentation of accuracy is based on analysis of samples prepared by an international proficiency survey program. The development of a two-step digestion procedure followed by automated flow-injection mercury analysis was a necessary precursor to the assessment of inorganic and alkylmercury exposure in a large unexposed human population. Application of this technique to 902 blood and 902 urine samples collected from a normal human population who had no extraordinary mercury exposure generated mean plus two standard deviation skewed confidence-limit ranges of results as follows: blood total mercury, 0-8.4 micrograms/L; blood inorganic mercury, 0-1.7 micrograms/L; blood organic mercury, 0-7.5 micrograms/L; urine total mercury, 0-9.9 micrograms/L; urine inorganic mercury, 0-8.6 micrograms/L; and urine organic mercury, 0-1.8 micrograms/L.

Autoanalysis↗

MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports.

Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacrylamide gel electrophoresis. In an effort to minimize sample handling and increase sample throughput, we have developed a novel in-gel digestion protocol where sample preparation is performed directly on a MALDI probe with prestructured sample support. The protocol consists of few sample-handling steps and has minimal consumption of reagents, making the protocol sensitive, timesaving, and cost-efficient. Performance of the on-probe sample preparation protocol was demonstrated by analysis of a set of rat liver proteins obtained from a fluorescently stained (Cy3 and SyproRuby) two-dimensional polyacrylamide gel. The success rate of protein identification by on-probe tryptic digestion and MALDI peptide mass mapping was 89%. The on-probe in-gel digestion procedure provided superior sensitivity and peptide mass mapping performance as compared to our standard in-gel digestion protocol. The on-probe digestion technique resulted in significantly improved amino acid sequence coverage of proteins, mainly due to efficient recovery and detection of large (>1.5 kDa) hydrophobic peptides. These observations indicate that numerous tryptic peptides are lost when using the standard in-gel digestion methods and sample preparation techniques for MALDI MS. This study also demonstrates that the on-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research, including protein identification and determination of posttranslational modifications.

Animals↗

Evaluation and Optimization of Different Digestion Strategies for In-Depth Proteomic Characterization of Residual Host Cell Proteins in rAAV-Based Gene Therapy Products.

Recombinant adeno-associated virus vectors (rAAVs) are the most important vectors for in vivo gene therapies, yet their safety relies on low levels of residual host cell proteins (HCPs). While mass spectrometry-based proteomics enables sensitive and untargeted HCP profiling, it faces challenges with matrix interferences from purification buffers and the high dynamic range between abundant viral capsids and trace HCPs. Although sample preparation methods that address these challenges are well-established for antibody products, their adaptation to rAAV purification stages and products remains largely unexplored. In this study, we systematically evaluated three widely used proteomic sample preparation workflows─In-Solution, FASP, and SP3─across different stages of rAAV purification. In addition, each workflow was tested under both standard denatured digestion conditions and a "native" digestion strategy designed to reduce dynamic range by preserving capsid integrity while selectively digesting HCPs. This comparison identified the native FASP protocol as the most effective sample preparation method for overcoming matrix interference and dynamic range challenges, consistently outperforming other workflows in HCP identification across purification stages. Further optimization of the native FASP workflow enhanced its performance, achieving the highest host cell (HC) proteome depth, particularly in highly purified drug substance samples. This optimized sample preparation strategy provides a robust and easy-to-use framework for deep characterization of the host cell proteome in rAAV samples. By enabling deeper insights into the HCP profile, this approach supports improved understanding of the rAAV purification process and facilitates the development of targeted strategies to enhance product quality and safety.

Dependovirus↗

Novel strategies for clinical drug analysis with new column technology in liquid chromatography.

Clinical drug analysis has been the focus of much attention and controversy because of the increase in substance abuse testing of addicts, patients, employees and others, in addition to the rapidly growing fields of therapeutic drug monitoring and clinical toxicology. This review focuses on the latest methodological and technological advances in liquid chromatography (LC) as it is applied to clinical drug analysis. Based on the author's experience, key chromatographic parameters such as carbon load, functionalities and temperature are proposed for the separation of structurally similar metabolites and for resolving chromatographic interferences by other drugs and metabolites. Novel sample preparation for cyclosporine and gradient elution of its metabolites are reviewed, followed by an update on monitoring of 3'-azido-3'-deoxythymidine with emphasis on automated sample preparation. Various approaches of direct sample analysis are advocated for increased efficiency as a result of minimal sample preparation and potential advantages such as decreased exposure of personnel to infectious samples. An update of microbore LC indicates that 2-mm columns may be readily used for clinical paediatric and neonatal analysis without dedicated chromatographs. Potential applications of multidimensional-multimodal chromatography include analyses of a cocaine impurity, anticonvulsants, antidepressants, and five ingredients of a common cold medication.

Body Fluids↗

Free amino acid determination in whole peanut seeds.

A method which circumvents the lengthy procedural steps for sample preparation in the analysis of free amino acids (FAA) in the peanut is described. Sample preparation consists of extracting a full fat meal with a lithium citrate sample buffer for 15 min. The sample is centrifuged and filtered with two Millipore filters in series. The resulting extract is analyzed "as is" for FAA via a physiological amino acid procedure. The method significantly reduces sample preparation time and eliminates the use of hazardous chemicals. It is well adapted to the analysis of large numbers of samples or samples of limited quantity. It should be applicable to the analysis of FAA in other oilseeds.

Amino Acids↗

Techniques of preparing plant material for chromatographic separation and analysis.

This paper discusses preparation techniques of samples of plant material for chromatographic analysis. Individual steps of the procedures used in sample preparation, including sample collection from the environment or from tissue cultures, drying, comminution, homogenization, leaching, extraction, distillation and condensation, analyte enrichment, and obtaining the final extracts for chromatographic analysis are discussed. The techniques most often used for isolation of analytes from homogenized plant material, i.e., Soxhlet extraction, ultrasonic solvent extraction (sonication), accelerated solvent extraction, microwave-assisted extraction, supercritical-fluid extraction, steam distillation, as well as membrane processes are emphasized. Sorptive methods of sample enrichment and removal of interferences, i.e., solid-phase extraction, and solid-phase micro-extraction are also discussed.

Chromatography↗

Progress toward a simplified polymerase chain reaction and its application to diagnosis of tuberculosis.

The complexity, expense, and susceptibility to contamination of the polymerase chain reaction (PCR) are all issues which need to be overcome if PCR is to be used outside of research laboratories. We addressed these problems with respect to the diagnosis of tuberculosis. First, we simplified the procedure for extracting Mycobacterium tuberculosis DNA from sputum samples. Two methods of sample preparation were compared: the chaotrope-silica method and a novel, more simple chloroform method. Second, we developed a colorimetric method for product detection. This method was as sensitive and specific as agarose gel electrophoresis for detection of PCR product. By using a one-tube nested protocol, 5 to 50 genome equivalents of M. tuberculosis DNA were detected. The simplified colorimetric PCR was compared with microscopy and culture for detection of M. tuberculosis in clinical specimens of sputum. A total of 171 sputum samples were investigated from 108 patients, 12 of whom were subsequently found to have tuberculosis by culture and/or microscopy. PCR of samples prepared by the chaotrope-silica method had a sensitivity of 75% and a specificity of 100% whereas PCR of samples prepared by the chloroform method had a sensitivity of 92% and a specificity of 99% when compared with the sensitivities and specificities of the combined classical microbiological methods for the diagnosis of tuberculosis. The simplified colorimetric PCR in combination with the chloroform sample preparation method was at least as sensitive as microscopy but had a greater specificity because samples with atypical mycobacteria were not detected by PCR. The sensitivity of the method for detection of smear-negative and extrapulmonary tuberculosis remains to be investigated.

Base Sequence↗

Molecular diagnosis and epidemiology of fungal infections.

A variety of methods are utilized for DNA strain subtyping of Candida spp. because no 'gold standard' exists. Random amplified polymorphic DNA (RAPD) or restriction enzyme analysis (REA) are useful to determine the source of an outbreak, but more reproducible and discriminatory methods such as Southern hybridization and pulsed field gel electrophoresis (PFGE) may be required. When applied to some nosocomial Candida infections, multiple strains and species have been identified. Microevolution of yeast species occurs and epidemiologically related isolates may show minor pattern differences, creating uncertainty as to whether they are distinct strains. Approximately 1000 isolates of Aspergillus fumigatus from environmental and clinical sources were typed by REA probed with an A. fumigatus-specific retrotransposon-like sequence. Patients with no symptom of aspergillosis may carry several strains, whereas patients with pulmonary aspergillosis may carry one or two strains; nocosomial transmission of aspergillosis was proven in 39% of the patients studied; any given environmental strain can be infectious; the environmental population of A. fumigatus is extremely diverse and no specific niche was found in the hospital. A PCR assay was designed to target conserved 18S-ribosomal DNA (rDNA) sequences shared by most fungi and a 687 bp product was amplified from 25 medically important fungal species. Studies with blood, cerebrospinal fluid and sputum specimens from patients with mycoses indicated that the PCR assay is more sensitive in diagnosing invasive fungal infections than blood culture methods. More specific identification is obtainable with genus/species-specif c probes designed from within the PCR-amplified sequences for C. albicans, C. krusei, C. lusitaniae, Pneumocystis carinii, Cryptococcus neoformans, Aspergillus/Penicillium spp. and C. glabrata/Saccharomyces cerevisiae. A. fumigatus and A. niger were differentiated by denaturing gradient gel electrophoresis. In situ hybridization (ISH) detected a 648 bp fragment of the 18S rDNA of C. neoformans and a 568 bp fragment of the alkaline proteinase gene of A. fumigatus in tissues from experimentally infected animals. In ISH, the entire process can be automated, making this procedure rapid and easy. The difficulty in establishing a diagnosis of invasive candidiasis has prompted the quest for a clinically useful PCR test for candidaemia. The universal fungal oligonucleotide primer pair, ITS3 and ITS4, amplifies portions of the 5.8S ad 28S rDNA subunits, and the ITS2 region. Although rRNA genes are highly conserved, the ITS regions are distinctive. DNA probes were designed from ITS2 that were specific for 16 different Candida species. Simple, rapid sample preparation was suitable for PCR analysis of BacT/Alert blood culture bottles. Sample preparation, PCR, and EIA detection of the amplicon from five different Candida species was accomplished in 7 h, 2.5 days sooner than by conventional culture methods. As well as saving time, minor yeast species among a major species, or among bacteria, were simultaneously detected. PCR-EIA using a microtitration plate format had sensitivity 10-times greater than that obtained with ethidium bromide-stained agarose gels. Taqman combines in one step PCR, probe hybridization, and fluorescent signal generation. Taqman PCR had sensitivity equivalent to PCR-EIA and required only 5 h, including sample preparation.

Aspergillosis↗

[Establishment of two-dimensional gel electrophoretic protein map of human spermatozoa].

OBJECTIVE: To establish the method of 2-dimensional electrophoresis(2-DE) for proteins of human spermatozoa and to construct a protein map of human spermatozoa. METHODS: The sperm pellet was prepared with simple Percoll layer protocol. We studied the effects of various sample preparation methods, loading quantities and isoelectric-focusing protocols on the quality of silver-stained 2-DE map, and constructed a primary protein map of human spermatozoa. RESULT: Up to 703 protein spots were acquired with sample preparation Method I while only 194-210 spots with Method II. With immobilized pH gradients and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(IPG-DALT) we could acquire over 700 spots while only 280-300 with isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(ISO-DALT). CONCLUSION: It is satisfactory to lyse sperm with sample preparation Method I and to separate sperm proteins by IPG-DALT for establishing 2-D map of human sperm.

Adult↗

Fast liquid chromatographic-tandem mass spectrometric (LC-MS-MS) determination of metformin in plasma samples.

Liquid chromatographic-tandem mass spectrometric (LC-MS-MS) methods for the determination of metformin in plasma from different species are presented. The first method employed a YMC cyano 2mm x 50 mm, 3 microm analytical column. For minimum sample preparation direct injection of samples after protein precipitation was performed. The polar column used with highly organic mobile phases provided a normal phase retention mechanism. The elution conditions were optimized to obtain reproducible peak areas and good peak shape. A step gradient from 100% acetonitrile to acetonitrile-water 80:20 (v/v) containing 10mM ammonium acetate and 1% acetic acid was applied, leading to a sample-to-sample cycle time of 2 min. In a second method, a column-switching LC-MS-MS assay for on-line trapping was developed. The analyte and internal standard were trapped on a YMC cyano 2 mm x 10 mm, 5 microm column using acetonitrile-methanol 95:5 (v/v). Elution was performed isocratically in back-flush mode on to the analytical column (YMC cyano 2 mm x 50 mm, 3 microm) using 10 mM ammonium acetate in acetonitrile-water 80:20 (v/v) with 1% formic acid. With this approach, the signal-to-noise ratio was improved and the run time could be shortened to 1 min. Calibration samples were prepared in the matrix to be assayed in the range of 10-10,000 ng/ml. Quality control (QC) samples were prepared at 40, 400 and 4000 ng/ml and interspersed with the unknown study samples in the assays. Deviations for precision and accuracy were less than 20% for the lower limit of quantification (LLOQ) and low QC sample and less than 15% for other calibrators and QCs.

Animals↗

Quantitation and stability of piperacillin and tazobactam in plasma and ultrafiltrate from patients undergoing continuous venovenous hemofiltration by HPLC.

Simple and reproducible HPLC methods for the determination of piperacillin and tazobactam have been developed and a complete stability study carried out. The method for piperacillin plasma samples consisted of protein precipitation with methanol using penicillin G as internal standard. No sample preparation was needed for ultrafiltrate samples. Tazobactam sample preparation involved protein precipitation with acetonitrile and the removal of lipids with dichloromethane. Piperacillin separation was performed on a microBondapack C(18) column (300 x 3.9, 10 microm) and tazobactam on a Novapack C(18) column (150 x 3.9, 4 microm) with UV detection set at 229 and 225 nm, respectively. The mobile phase consisted of phosphate buffer-acetonitrile, delivered at 1.5 mL[sol ]min. Calibration curves determination coefficients were >or=0.999 and response factors CV% < 5%. Intra- and inter-assay precision and accuracy of the quality control and limit of quantification were satisfactory. Plasma and ultrafiltrate samples were stable at -20 and -80 degrees C for 2 months and after three freeze-thaw cycles. In the chromatographic rack, tazobactam ultrafiltrate samples were stable for 24 h and plasma samples for 12 h, piperacillin ultrafiltrate samples for 8 h, but plasma samples for only 4 h. Storage of piperacillin samples at 4 degrees C until analysis is recommended. Piperacillin was stable in the presence of tazobactam.

Anti-Bacterial Agents↗

Automated on-line solid-phase extraction-gas chromatography with nitrogen-phosphorus detection: determination of benzodiazepines in human plasma.

An automated sample preparation module, the ASPEC (automated sample preparation with extraction columns) was interfaced with a capillary gas chromatograph (GC) by means of a loop-type interface. The system was optimized for the determination of four benzodiazepines in plasma. Extraction from the untreated plasma was carried out on disposable C18 cartridges and involved several washing steps. The analytes were desorbed with 2 ml of ethyl acetate and a 110-microliter aliquot of the eluate was injected into the gas chromatograph via the loop-type interface using fully concurrent solvent evaporation conditions. Detection of the benzodiazepines was carried out with a nitrogen-phosphorus detector (NPD). The ASPEC-GC-NPD was fully automated and could run unattended overnight. With a sample volume of 1 ml the procedure showed good linearity and repeatability in the range 5-50 ng/ml using a sample volume of 1 ml. The limits of detection in plasma were 0.5-2 ng/ml.

Acetates↗