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PubMed2006

Recent developments in methods and technology for analysis of biological samples by MALDI-TOF-MS.

Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) is widely used in a variety of fields because it has the characteristics of speed, ease of use, high sensitivity, and wide detectable mass range for obtaining molecular weights and for structural characterization of macromolecules. In this article we summarize recent developments in matrix additives, new matrices, and sample-pretreatment methods using off-probe or on-probe techniques or nanomaterials for MALDI-TOF-MS analysis of biological samples.

Analytic Sample Preparation Methods↗
PubMed2002

Methylmercury determination in biological samples by derivatization, solid-phase microextraction and gas chromatography with microwave-induced plasma atomic emission spectrometry.

A method for the extraction and gas chromatographic determination of methylmercury in biological matrices is presented. By combining the advantages of two extraction techniques-microwave-assisted extraction (MAE) and solid-phase microextraction (SPME)--the separation of methylmercury from biological samples is possible. Specifically, the procedure involves microwave extraction with 3 M hydrochloric acid, followed by aqueous-phase derivatization with sodium tetraphenylborate and headspace SPME with a silica fibre coated with polydimethylsiloxane (PDMS). For optimization of the derivatization-SPME procedure, a central composite experimental design with alpha = 1.682 and two central points was used to model gas-chromatographic peak areas as functions of pH, extraction temperature and sorption time. A desirability function was then used for the simultaneous optimization for methylmercury and Hg(II). The optimal derivatization-SPME conditions identified were close to pH 5, temperature 100 degrees C, and sorption time 15 min. The identification and quantification of the extracted methylmercury is carried out by gas chromatography with microwave-induced plasma atomic emission spectrometry detection. The validity of the new procedure is shown by the results of analyses of certified reference materials.

Calibration↗
PubMed2005

Shotgun lipidomics of phosphoethanolamine-containing lipids in biological samples after one-step in situ derivatization.

This article presents a novel methodology for the analysis of ethanolamine glycerophospholipid (PE) and lysoPE molecular species directly from lipid extracts of biological samples. Through brief treatment of lipid extracts with fluorenylmethoxylcarbonyl (Fmoc) chloride, PE and lysoPE species were selectively derivatized to their corresponding carbamates. The reaction solution was infused directly into the ion source of an electrospray ionization mass spectrometer after appropriate dilution. The facile loss of the Fmoc moiety dramatically enhanced the analytic sensitivity and allowed the identification and quantitation of low-abundance molecular species. A detection limitation of attomoles (amoles) per microliter for PE and lysoPE analysis was readily achieved using this technique (at least a 100-fold improvement from our previous method) with a >15,000-fold dynamic range. Through intrasource separation and multidimensional mass spectrometry array analysis of derivatized species, marked improvements in signal-to-noise ratio, molecular species identification, and quantitation can be realized. The procedure is both simple and effective and can be extended to analyze many other lipid classes or other cellular metabolites by adjustments in specific derivatization conditions. Thus, through judicious derivatization, a new dimension exploiting specific functional reactivities in each lipid class can be used in conjunction with shotgun lipidomics to penetrate farther into the low-abundance regime of cellular lipidomes.

Animals↗
PubMedDate not supplied

[The most frequent bacterial isolates in biological samples from HIV-infected patients].

A descriptive and retrospective study was conducted aimed at knowing the behavior and the frequency of the bacterial isolations most frequently found in different biological samples (sputum, blood and urine) from patients with HIV/AIDS diagnosis that were seen at the Outpatient Department or that were admitted at the Hospital of "Pedro Kouri" Institute of Tropical Medicine from 1997 to 2001. It was observed that most of the isolations were obtained from sputum samples and that Pseudomonas aeuroginosa was the most isolated microorganism in all samples. Pseudomonas aeuroginosa proved to be the most isolated microorganism in sputum with 28.5%. The bacteria most commonly isolated in blood was Staphylococcus epidermidis with 48.5%. The uropathogenic microorganism most found in urine was Escherichia coli with 43.5%.

HIV Infections↗
PubMedDate not supplied

Selective determination of methyl mercury in biological samples by means of programmed temperature gas chromatography.

A programmed temperature gas chromatographic method is presented by which it is possible to carry out routine analysis of methyl mercury in biological samples prepared according to the AOAC official first action recommendations without the need for preliminary treatment of the columns. This method greatly extends the life of the columns as well as the useful time for analysis; it has good linearity and repeatability. With the proposed method a total of 36 samples can be analyzed daily.

Animals↗
PubMed1991

Sensitized immunochemical method for the specific detection of human haemoglobin in dried biological samples.

The authors describe a modified version of counterimmunoelectrophoresis of easy performance and high sensitivity. With this method human haemoglobin can easily and specifically be determined either in fresh or dried biological samples. Glutaraldehyde pretreatment of the samples results in the development of complexes with highly favourable electrophoretic mobility and precipitating capacity. By this chemical modification and use of a double set of samples with varying antigen-antibody proportion, the sensitivity of haemoglobin detection in erythrocyte-containing haemolysate and native blood was 300 ng/ml. The examination is easily performed, and without considerable outlay, even with conventional laboratory facilities. It is equally suitable for the detection of occult colorectal bleeding and for species-specific study of blood stains of unknown origin. The authors succeeded in identifying human haemoglobin even in a 42-month dried blood stain.

Blood Stains↗
PubMed2004

Determination of organotin compounds in biological samples using accelerated solvent extraction, sodium tetraethylborate ethylation, and multicapillary gas chromatography-flame photometric detection.

A method has been developed for species-selective analysis of organotin compounds in solid, biological samples. The procedure is based on accelerated solvent extraction (ASE) of analytes and includes extraction of the tin species with a methanol-water (90% methanol) solution of acetic acid/sodium acetate containing tropolone (0.03% w/ v), their ethylation with NaBEt(4), and separation and detection by GC-FPD. The analytical procedure was optimized with an unspiked sample of harbor porpoise ( Phocoena phocoena) liver. Effects of ASE operational variables (extraction temperature and pressure, solvent composition, number of static extraction steps) are discussed. Method detection limits (MDL) were in the range 6-10 ng(Sn) g(-1) dry weight and 7-17 ng(Sn) g(-1) dry weight for butyl- and phenyltin compounds, respectively. Recoveries were comparable with or better than those obtained by use of other procedures reported in the literature. The analytical procedure was validated by analysis of NIES No. 11 (fish tissue) certified reference material.

Alkylation↗
PubMed2005

Optical zymography for specific detection of urokinase plasminogen activator activity in biological samples.

Zymography techniques are routinely used to quantify proteolytic activity. In the current study, we describe an optical zymographic procedure that specifically detects urokinase-type plasminogen activator (uPA) activity in biological samples. The method employs a synthetic polymeric uPA fluorescent probe, which is copolymerized in sodium dodecyl sulfate (SDS)-polyacrylamide gel. Following electrophoresis and renaturation, enzymatic digestions of the substrate in 50 mM of Tris buffer at pH 7.4 generates fluorescence emission at 695 nm. The enzymatic activities can be analyzed directly by conventional gel imaging systems with a detection limit of 40 pg. This protocol is fast (hours) and does not require staining and destaining steps. The procedure is independent of plasminogen and, therefore, can efficiently distinguish the active two-chain uPA from its proenzyme. Densitometry analysis demonstrated a highly correlative relationship (r2=0.999) between the amount of uPA (over the range of 0.1-8.0 ng) and the average intensity of the fluorescent band. We were able to directly measure uPA activities in different cancer cell lines. This newly developed technique could be expanded to nearly all proteases, including the ones that cannot be analyzed by traditional zymography.

Caseins↗
PubMed2002

The determination of homocysteine-thiolactone in biological samples.

Homocysteine-thiolactone, a cyclic thioester of homocysteine, is synthesized by methionyl-tRNA synthetase in all cell types. A new assay for the determination of homocysteine-thiolactone in biological samples is described. The assay involves separation of homocysteine-thiolactone from macromolecules by ultrafiltration. Homocysteine-thiolactone is further purified and quantified by high-pressure liquid chromatography either on a reverse phase or a cation exchange micro-bore column. The detection and quantitation are obtained by monitoring the absorbance at 240 nm, a maximum in a UV spectrum of homocysteine-thiolactone. The sensitivity of detection is 5 pmol. This assay has been applied to bacteria (Escherichia coli and Mycobacterium smegmatis), the yeast Saccharomyces cerevisiae, cultured human vascular endothelial cells, and human plasma. The data support the conclusion that homocysteine-thiolactone is a ubiquitous metabolite whose levels are directly related to homocysteine levels.

Calibration↗
PubMed2002

Separation methods for taurine analysis in biological samples.

Taurine plays an important role in a variety of physiological functions, pharmacological actions and pathological conditions. Many methods for taurine analysis, therefore, have been reported to monitor its levels in biological samples. This review discusses the following techniques: sample preparation; separation and determination methods including high-performance liquid chromatography, gas chromatography, ion chromatography, capillary electrophoresis and hyphenation procedures. It covers articles published between 1990 and 2001.

Chromatography↗
PubMed2004

Screening and identification of glycosides in biological samples using energy-gradient neutral loss scan and liquid chromatography tandem mass spectrometry.

A rapid, selective, and reliable strategy has been developed for the screening and identification of glycosides in biological samples: a crude extract was directly infused to a triple-quadrupole MS/MS, and major glycosides were screened out with high confidence by an energy-gradient neutral loss scan (EGNLS) for the loss of sugar(s); then these glycosides were further identified with LC/MS/MS. The proposed EGNLS method was established and optimized with 16 representative glycosides (including ginsenosides and the glycosides of flavones, anthraquinones, and terpenoids). The EGNLS method has two major advantages over the conventional fixed-energy neutral loss scan: (1) The latter is liable to '"omit" some target compounds due to the usual mismatch between the preset collision energy and interested compounds' optimal collision energy (OCE), while EGNLS solves this problem by scanning over an energy range. (2) The EGNLS simultaneously measures the screened compounds' OCE, which not only are essential parameters for further LC/MS/MS analysis but also carry some structural information, as proved by this study. This strategy has been successfully demonstrated with the analysis of glycosides in Scutellaria viscidula Bge and transformed Panaxhairy roots (the glycoside constitutions of both had not been studied before): without laborious separation processes; comprehensive glycoside information on those two plants was obtained by a rapid and simple procedure. This strategy is valuable for the study of glycosides in complex samples.

Chromatography, High Pressure Liquid↗
PubMed2005

Determination of microcystins in biological samples by matrix solid-phase dispersion and liquid chromatography-mass spectrometry.

A method for the detection and quantification of the microcystins (MCs)-MC-LR, MC-RR and MC-YR-in biological samples by matrix solid-phase dispersion (MSPD) has been developed. The optimum extraction conditions were 500 mg of liver or kidney, C18 bonded silica as dispersant, and a mixture methanol-water (70:30) as eluent. The MCs were determined by liquid chromatography electrospray mass spectrometry (LC/ES/MS). Recoveries of biological extracts at three different spiked levels (1-10 mg kg(-1)) ranged from 40.5 to 87.0% in liver, and from 52.5 to 74.5 in kidney. R.S.D.s were < 15.6% and < 10.6%, respectively. The detection and quantification limits were 0.05 and 0.5 mg kg(-1), for all MCs. The method was applied to MCs detection in liver and kidney of rat previously injected i.p. with MC-LR. Results showed the presence of MC-LR in the liver of the animals injected with the highest dose.

Animals↗
PubMed2003

Oxidative stress monitoring in biological samples by bioluminescent method.

The integral bioluminescent biotest with lyophilized fluorescent bacteria was used for monitoring of LPO processes in tissue extracts and serum of rats exposed to stress. A relationship between the content of MDA (LPO indicator) and fluorescence of bacteria was observed in all biological samples.

Animals↗
PubMed1999

Analysis of the amino acid indospicine in biological samples by high performance liquid chromatography.

Indospicine is a hepatotoxic amino acid that accumulates in the meat of horses that consume the legume Indigofera linnaei. A method to determine indospicine concentration in biological samples using an amino acid analyser has been reported, but the analysis time is long and therefore not suited to the analysis of large numbers of samples. A rapid and reliable method was developed for the analysis of indospicine in horsemeat and serum using High Performance Liquid Chromatography. Horsemeat and serum were extracted with either water or 0.01 N hydrochloric acid, respectively, and deproteinized by ultrafiltration. Precolumn derivatization of samples with phenylisothiocyanate was followed by separation of indospicine from other amino acids on a Pico-Tag C 18 column and UV detection at 254 nm. The calibration curves for indospicine in horsemeat extract were linear over the concentration range 0.4 microg ml(-1) to 20 microg ml(-1), while for indospicine in serum, the linear range was from 0.17 microg ml(-1) to 16.67 microg ml(-1). The mean recovery of indospicine in horsemeat extract was 87.2 +/- 6.8% and in serum was 97.3 +/- 9.9%. Analysis time for indospicine in horsemeat samples was 31 min and in serum samples was 36 min.

Animals↗
PubMed1985

Determination of pentacaine, trans-2-(1-pyrrolidinyl)cyclohexyl-3-pentyloxycarbanilate hydrochloride, in biological samples by gas chromatography/mass spectrometry.

A quantitative and selective method has been developed for the determination of a novel local anaesthetic compound pentacaine, trans-2-(1-pyrrolidinyl)cyclohexyl-3-pentyloxycarbanilate hydrochloride, in biological samples. After ion pair extraction from 1 M HCl into 1,2-dichloroethane, pentacaine and a structurally related internal standard were derivatized to prevent thermal decomposition in the gas chromatograph. An on-column methylation technique with trimethylanilinium hydroxide was used. Determination was performed by gas chromatography/mass spectrometry (GC/MS) with selected ion monitoring. Interferences by endogenous lipophilic constituents were avoided by including an n-hexane wash before the ion pair extraction. This wash step did not reduce the drug recoveries. The method gave linear results over a concentration range of 5-100 ng ml-1 with a coefficient of variation less than 10% at 5 ng pentacaine ml-1. Specimens of plasma, whole blood, urine as well as in vitro preparations such as hepatic microsomes were successfully analysed.

Anesthetics, Local↗
PubMed2006

Use of egg yolk antibody (IgY) as an immunoanalytical tool in the detection of Indian cobra (Naja naja naja) venom in biological samples of forensic origin.

An immunoglobulin Y (IgY) based indirect double antibody sandwich enzyme linked immunosorbent assay (ELISA) was developed for the detection of Indian cobra (Naja naja naja) venom in the biological samples of forensic origin. Polyclonal antibodies were raised and purified from chick egg yolk and rabbit serum. The cobra venom was sandwiched between immobilized affinity purified IgY and the rabbit IgG. The detection concentration of cobra venom was in the range of 0.1 to 300ng. The calibration plot was based on linear regression analysis (y=0.2581x+0.4375, r(2)=0.9886). The limit of detection of the assay was found to be 0.1ng. The coefficient of variation (CV) of different concentrations of working range in inter (n=6) and intra-assay (n=6) was observed to be less than 10%. The recovery of venom was found to be in the range of 80-99%, when different concentrations (0.002, 0.1, 0.2, 1, and 2microg) of cobra venom were spiked to pooled normal human serum (ml(-1)). No cross reactivity was observed with krait and viper venom in the immunoassay system in the concentration range of 0.1-1000ng. The method was initially, validated by analyzing specimens (autopsy) of experimental rats injected with cobra venom (1.2mgkg(-1) body mass). Further, human specimens (autopsy and biopsy) of snake bite victims of forensic origin were also analyzed. The methodology developed may find diagnostic application in forensic laboratories.

Animals↗
PubMed1994

Determination of aluminium by instrumental neutron activation analysis in biological samples with special reference to NBS SRM 1577 bovine liver.

It was found that a well thermalized heavy water-moderated research reactor can be used to determine the Al content of biological samples without separation of P, Na and Cl. The contribution of the 31P(n,alpha)28Al reaction was corrected from the known concentration of P and the measured effective value. It was found that the contribution is only about 10%. A method is suggested for the determination of Al using instrumental neutron activation analysis.

Aluminum↗
PubMed2001

Specific blocking to improve biopanning in biological samples such as serum and hybridoma supernatants.

Screening phage-displayed peptide libraries (biopanning) is an important technique for acquiring peptide ligands and for mapping peptide epitopes recognized by antibodies (Ab). In biological samples, other materials, not only contaminants but also natural constituents, often interfere with biopanning. Capture methods use anchoring Abs that reduce the need for purification of the intended panning target from a crude sample. This capture method is analogous to sandwich ELISA. However, when the target molecule concentration is low in the initial mixture, the panning of a captured target often yields epitopes that bind to the capture antibody rather than the target of interest. We have developed a methodology that utilizes specific blocking reagents of the capture Ab to extend the sensitivity and applicability of the capture approach to phage panning. A flowchart is presented to enable the worker to begin panning with the simplest approach and then to employ sandwich capture and specific blocking reagents as necessary.

Animals↗