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PubMed2006

Trace analysis of fullerenes in biological samples by simplified liquid-liquid extraction and high-performance liquid chromatography.

Fullerene (C60) has several potential biomedical and industrial applications. While pure fullerene is not soluble in water, nanoparticles of the fullerene aggregates (nano-C60) can be prepared in water solutions. The concentration of nano-C(60) in biological media after systemic exposure could be very low and requires trace analytical methods to be developed for the toxicological and pharmacokinetic studies of the nanomaterial. A serious drop in extraction efficiency was observed when the concentration was under 0.5 microg/mL using traditional liquid-liquid extraction (LLE) protocols. The evaporation of the solvent extract to dryness was found to be the main reason for the efficiency drop and an improved evaporation method was proposed to overcome this problem. Optimal proportion of glacial acetic acid (GAA) was used to solublize the proteins and surfactants in the biological samples, so that the emulsion problem was eliminated during LLE. Magnesium perchlorate was used to destabilize the nano-C60 particles in the water solution and promoted the solvent extraction. A simplified LLE method was developed for high throughput while preserved the advantages of the traditional LLE. The developed method was used for trace analysis of fullerenes in protein containing media and tape-stripped skin samples. Under optimal experimental conditions, the detection limit was 0.34 ng/mL and the recovery was in the range of 94-100% (n=5) at a concentration of 10 ng/mL nano-C60 in the biological media.

Chromatography, High Pressure Liquid↗
PubMed2003

Automated solid-phase extraction and liquid chromatographic method for retinoid determination in biological samples.

In this study, a method for partly automated sample preparation and fully automated solid-phase extraction method for plasma, kidney and liver samples for various retinoids like all-trans-4-oxo-retinoic acid, 13-cis-4-oxo-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid, all-trans-retinoic acid, retinol and retinyl palmitate was established. Plasma, embryo-, kidney-and liver-homogenates were automatically mixed and extracted on multiple usage solid-phase (C2) extraction cartridges immediately before HPLC analysis. Automated cleaning, preconditioning and incorporation of the loaded cartridge to fully automated HPLC separation and quantification of the various retinoids in a single HPLC run was established. The recovery of the retinoids was generally between 80 and 90%. Intra-day repeatability was < 11.7%. As little as 1.2 ng/ml could be quantified in lipid-mixture standard samples. This method allows a highly automated sample preparation and a fully automated solid-phase extraction with good selectivity for the study of endogenous retinoids and retinoids after nutritional supplementations and pharmacological applications in several biological samples.

Animals↗
PubMed1989

Determination of beta-19-nortestosterone and its metabolite alpha-19-nortestosterone in biological samples at the sub parts per billion level by high-performance liquid chromatography with on-line immunoaffinity sample pretreatment.

An immunoaffinity precolumn (immuno precolumn) packed with Sepharose-immobilized polyclonal antibodies against the anabolic hormone 17 beta-19-nortestosterone (beta-19-NT) was used for the selective on-line pretreatment of raw extracts of urine, bile and tissue samples by high-performance liquid chromatography. Using UV detection (247 nm), beta-19-NT and its metabolite 17 alpha-19-nortestosterone (alpha-19-NT) can be determined in biological samples with a detection limit of 0.05 microgram/kg. Owing to the high clean-up efficiency of the immuno precolumn and the large sample volumes used, confirmation by gas chromatography-mass spectrometry is possible at this level. In urine samples from a calf treated with 19-nortestosterone 17 beta-laurate, the maximum concentrations of beta-19-NT (1.3 micrograms/l) and alpha-19-NT (3.1 micrograms/l) were found seven days after intramuscular administration. In a bile sample from this calf only alpha-19-NT (55 microgram/l) was detected. In meat samples from three treated calves, the concentration of beta-19-NT varied from 0.1 to 1.6 micrograms/kg and no alpha-19-NT could be detected. In liver samples from these calves, the concentrations of beta-19-NT and alpha-19-NT were less than 0.05-0.1 and 0.5-0.9 micrograms/kg, respectively. In the corresponding kidney samples, the concentrations of beta-19-NT and alpha-19-NT were 0.4-0.5 and 0.5-1.6 micrograms/kg, respectively. The application of the same immuno precolumn to the determination of 17 beta- and 17 alpha-trenbolone, two structurally related steroids, is also demonstrated.

Animals↗
PubMed2000

Determination of malondialdehyde in biological samples by solid-phase extraction and high-performance liquid chromatography.

An analytical procedure for determination of malondialdehyde in tissue homogenates and blood serum was developed. A reaction with 2,4-dinitrophenylhydrazine is used followed by cleaning up of the derivative by solid-phase extraction. The samples were analyzed by isocratic high-performance liquid chromatography (HPLC) using a narrow-bore HPLC-column. A good separation of the 1-pyrazole peak from that of 2,4-dinitrophenylhydrazine was observed. A high linear dependence was established by the concentration of 1-pyrazole in the range of 10-5000 ng/ml. The detection limit of the method applied for tissue homogenates and blood serum was approximately 10 ng/ml or lower, and RSD of the method was 9% (n = 8). The peak of 1-pyrazole for these samples was well separated from the other matrix peaks. Experiments carried out evaluated that the solid-phase extraction might be an effective step of the sample preparation, significantly increasing the selectivity of the analysis and the life-time of the column. The method seems to be applicable for determination of malondialdehyde in different biological samples.

Animals↗
PubMed2006

Selective solid-phase extraction of cholesterol using molecularly imprinted polymers and its application in different biological samples.

Non-covalent molecularly imprinted polymers (MIPs) of cholesterol were prepared by UV initiated polymerization. A polymer that had the highest binding selectivity and capability was used as solid-phase extraction (SPE) sorbents for direct extraction of cholesterol from different biological samples (human serum, cow milk, yolk, shrimp, pork and beef). The extraction conditions of molecularly imprinted SPE (MISPE) were optimized and the optimum protocol was: conditioning MISPE cartridges with n-hexane, loading with n-hexane, washing with n-hexane and n-hexane:toluene=9:1, respectively, then eluting with chloroform:ethanol:acetic acid=3:1:1. Cholesterol MISPE selectively recognized, effectively trapped and pre-concentrated cholesterol over a concentration range of 10-80 microg/mL. Recoveries ranged from 80.6% to 92.7%, with R.S.D. lower than 9.8%. Under the optimal condition, MISPE recoveries of spiked human serum, yolk, cow milk, shrimp, pork and beef were 91.1%, 80.4%, 86.6%, 78.2%, 81.4% and 80.1%, respectively. Compared with C18 SPE, almost all of the matrix interferences were removed after MISPE, and better baselines and higher selectivity were achieved.

Animals↗
PubMed1988

Determination of drug enantiomers in biological samples by coupled column liquid chromatography and liquid chromatography-mass spectrometry.

Liquid chromatography-mass spectrometry (LC-MS) and coupled column chromatography can be used to overcome problems likely to occur in direct separation and determination of drug enantiomers in biological samples. This is exemplified here with the direct separation and determination of terbutaline in human plasma at the nmol/l level. A beta-cyclodextrin column with an aqueous mobile phase was used for chiral separation. For coupled column chromatography, the concentration of each enantiomer was calculated from the enantiomeric area ratio and the racemate concentration. A deuterium-labelled internal standard was used in the LC-MS experiments.

Chromatography, Gas↗
PubMed1995

Diffusion coefficients of ATP and creatine phosphate in isolated muscle: pulsed gradient 31P NMR of small biological samples.

Measurements of the intracellular diffusion coefficients (Di) of ATP and creatine phosphate (PCr) in stable, isolated preparations of skeletal muscle were made by means of pulsed field gradient (PFG) 31P NMR. Experiments used a PFG NMR probe specifically designed for small, superfused biological samples. This provided a magnetic field gradient in the z axis of up to 195 G/cm with minimal eddy currents. DiATP and DiPCr in white (fast, glycolytic) skeletal muscle from goldfish (Carassius auratus) were determined to be 2.48 +/- 0.33 and 3.49 +/- 0.33 x 10(-6) cm2/s, respectively, at 25 degrees C and a diffusion time of approximately 19 ms. For comparison with Di values, diffusion coefficients of ATP and PCr also were measured in solutions of ionic composition similar to that of fish muscle cytosol. The in vitro diffusion coefficients of ATP and PCr were 3.54 +/- 0.11 and 5.28 +/- 0.08 x 10(-6) cm2/s, respectively, at 25 degrees C.

Adenosine Triphosphate↗
PubMed1992

High-performance liquid chromatographic determination of N-epsilon-(2-propenal)lysine in biological samples after derivatization with diethylethoxymethylenemalonate.

A recently reported methodology for amino acid analysis by HPLC has been adapted for quantification of N-epsilon-(2-propenal)lysine (a modified lysine by reaction with malondialdehyde that has been found in enzymatic digests of foods and in urine) in biological samples. We describe its use for investigating the in vitro degradation of N-epsilon-(2-propenal)lysine using rat tissue homogenates. Lysine dipeptide, used as a control in the incubation mixtures, and the lysine released by the hydrolytic action of the homogenates in the in vitro incubations are quantified in the same way. The samples are subjected to a cleanup prederivatization step using PD-10 disposable columns (Pharmacia). This allows precolumn derivatization with diethylethoxymethylenemalonate (50 min, 50 degrees C) and resolution of the derivatives of the compounds of interest by reversed-phase HPLC (binary gradient, 45 min) with quantification based on the uv absorption of the derivatives at 280 nm (detection limits below 1 pmol). The entire analysis takes 110 min. This method can be of general use for the determination of N-epsilon-(2-propenal)lysine in the context of research dealing with protein deterioration by reaction with malondialdehyde in biological systems and in foods. A method for the synthesis of N-epsilon-(2-propenal)lysine, used as external standard for the HPLC analysis, is described.

Animals↗
PubMed2005

Pressurized liquid extraction in the analysis of food and biological samples.

Originally, the use of the pressurized liquid extraction technique (PLE) was mainly focused on the extraction of environmental pollutants present in soil matrices, sediments, and sewage sludge. However, more recently the distinct advantages of this technique are being exploited in diverse areas, including biology, and the pharmaceutical and food industries. The aim of the present review is to explore recent analytical applications of this extraction technique (PLE) in the extraction of contaminant compounds and matrix components in food and biological samples, placing special emphasis on the strategies followed to obtain a rapid, selective, efficient and reliable extraction process.

Chromatography, Liquid↗
PubMed2002

Analysis of six relevant toxaphene congeners in biological samples using ion trap MS/MS.

The quantification of six polychlorinated bornanes (CHBs) was studied using ion trap MS/MS. The significance of the selection of parent ions (Ip) and daughter ions (Id) on the detection of these toxaphene congeners was assessed in standard solution and biological samples. Our results indicate that different Ip and Id, selected at either low or high mass-to-charge (m/z) ratios, influence drastically the response factor of the CHBs and the chemical noise observed. For the octachlorinated toxaphene congeners (Parlar-26 (P-26), Parlar-40/41 (P-40/41), Parlar-44 (P-44)), the detection performance of the ion trap MS/MS is similar whether Ip and Id were chosen at low or high m/z ratios. However, the selection of Ip and Id at high m/z ratios clearly enhances the detection of the nonachlorinated toxaphene congeners (Parlar-50 (P-50), Parlar-62 (P-62)). The improved method, which selects Ip and Id at low m/z ratios for P-26, P-40/41 and P-44 and at high m/z ratios for P-50 and P-62, permitted to obtain low detection limits as well as repeatable and accurate results.

Camphanes↗
PubMed2006

Direct quantitation and detection of salmonellae in biological samples without enrichment, using two-step filtration and real-time PCR.

A new two-step filtration protocol followed by a real-time PCR assay based on SYBR green I detection was developed to directly quantitate salmonellae in two types of biological samples: i.e., chicken rinse and spent irrigation water. Four prefiltration filters, one type of final filter, and six protocols for recovery of salmonellae from the final filter were evaluated to identify an effective filtration protocol. This method was then combined with a real-time PCR assay based on detection of the invA gene. The best results were obtained by subsequent filtration of 100 ml of chicken rinse or 100 ml of spent irrigation water through filters with pore diameters of >40 mum to remove large particles and of 0.22 microm to recover the Salmonella cells. After this, the Salmonella cells were removed from the filter by vortexing in 1 ml of physiological saline, and this sample was then subjected to real-time quantitative PCR. The whole procedure could be completed within 3 h from sampling to quantitation, and cell numbers as low as 7.5 x 10(2) CFU per 100-ml sample could be quantified. Below this limit, qualitative detection of concentrations as low as 2.2 CFU/100 ml sample was possible on occasion. This study has contributed to the development of a simple, rapid, and reliable method for quantitation of salmonellae in food without the need for sample enrichment or DNA extraction.

Animals↗
PubMed2000

Investigation of biological samples for monofluoroacetate and Dichapetalum cymosum poisoning in southern Africa.

A need has existed for the accurate identification of monofluoroacetate (MFA) poisoning in southern Africa. The development of a new method for the determination of MFA has made the analysis of a variety of biological samples (n = 50) feasible. The method has been used in the laboratory over 24 months. Monofluoroacetate was present in 66% of samples from cases of suspected poisoning, reflecting the extent of the problem. Stability of MFA in samples was also determined so as to have a time-bound baseline for the acceptance of samples submitted. It was found that there was a decrease in the level of MFA and, after 14 days at room temperature, only 50% of the spiked dose could be identified. It is suggested that samples be analyzed within 7 days of mortality if they not kept frozen.

Animal Diseases↗
PubMedDate not supplied

Cytotoxicity of EDTA used in biological samples: effect on some human breast-milk studies.

Ethylene diamine tetraacetic acid (EDTA) is a chelator for metal ions that has found widespread application in modern medicine and pharmacy. However, few studies have examined the cytotoxic side-effects of EDTA when added to biological samples upon various laboratory analyses. The present study was aimed at assessing the artefactual side-effects of EDTA on some biochemical assays performed on human milk samples. The secondary effects of addition of EDTA (at a final concentration of 20 mmol l(-1)) to inhibit complement system activation in human breast-milk samples were examined, using different laboratory techniques and methods, compared to milk samples with equivalent volumes of phosphate-buffered saline added. Assays considered included Biuret's test for total protein measurement, CH50 assays of whole complement activity, pH measurements and total cell counts. The toxic effects of EDTA included breast-milk cell loss, disruption of milk fat globule membrane and subsequent release of membrane-bound protein, free fatty acids and reduction in pH. It also caused false-positive results of haemolytic assays.

Animals↗
PubMed1980

Nitrogen estimation in biological samples by use of chemiluminescence.

We describe an apparatus modified for the chemiluminescent estimation of nitrogen in biological and clinical samples. Analytical characteristics have been assessed and results compared with those by the traditional Kjeldahl method. The chemiluminescence method, much faster and more sensitive than the traditional method, is at least as accurate, precise, and reproducible. Costs are low, and the method should find a place in laboratories needing, for example, rapid assessments of nitrogen balance in surgical patients and renal function in patients with renal failure, or estimations of small amounts of specific nitrogen-containing chemical substances in biological samples.

Animals↗
PubMed1988

Automated sequential trace enrichment of dialysates and robotics. A technique for the preparation of biological samples prior to high-performance liquid chromatography.

The development of the sample preparation process, the automated sequential trace enrichment of dialysates, in association with a cartesian robotic sampler is described. The system has been applied to the total automation of the preparation of biological samples and high-performance liquid chromatographic analysis. Concepts of the technique are reported together with an examination of its application to free and total analyte estimation. Examples of chromatographic separations obtained from the preparation of a variety of different analytes and sample materials are given.

Amino Acids↗
PubMed2001

[Direct determination of Pb, Zn and Cd in environmental and biological samples by FAAS].

This paper describes interference and background correction problem of direct determination of Pb, Zn and Cd in soil, sediment, plants, halobios and sea water by FAAS. With the improvement of limit of detection(L.O.D) of instrument, Pb, Zn and Cd in same environmental and biological samples can be analyzed directly by FAAS, but it is worth taking attention that there are quantities of elements which lead to background interference in air-ethyne flame, such as Al, Na, K Ca, Mg and Fe. Compare with no background correction, limit of detection using D2 lamp background correction is improved by 1.05-4.5 times, the recoveries and the relative standard deviation(R.S.D) are better, standard samples were also analyzed with satisfactory results.

Animals↗
PubMed2005

A facile and sensitive chemiluminescence detection of amino acids in biological samples after capillary electrophoretic separation.

It was found that native amino acids enhanced the chemiluminescence (CL) reaction between luminol and BrO(-) in an alkaline aqueous solution. This has led to the development of a facile and highly sensitive CL detection scheme for the determination of amino acids in biological samples after capillary electrophoretic (CE) separation. The CE-CL conditions were optimized. An electrophoretic buffer of 2.5 x 10(-2) M sodium borate (pH 9.4) containing 1 x 10(-4) M luminol was used. The oxidizer solution of 8 x 10(-4) M NaBrO in 0.1 M sodium carbonate buffer solution (pH 12.5) was introduced post-column. Under the optimal conditions, the detection limits were 1.0 x 10(-7) M for glutamic acid (Glu) and 1.3 x 10(-7) M (S/N = 3) for aspartic acid (Asp). The relative standard deviations (RSDs) of peak area and migration time were in the ranges of 3.8-4.3% and 1.4-1.6%, respectively. The present method was applied to the determination of excitatory amino acids (i.e., Asp and Glu) in rat brain tissue and monkey plasma. The levels of these major excitatory amino acids in monkey plasma were quantified for the first time and found to be 1.17 +/- 0.17 x 10(-5) M (mean +/- SD, n = 6) for Glu and 1.64 +/- 0.19 x 10(-6) M for Asp, which were comparable with the levels in human plasma.

Animals↗
PubMed1998

Determination of perfluorinated carboxylic acids in biological samples by high-performance liquid chromatography.

This paper describes a method for the quantitative determination of perfluorinated carboxylic acids (PFCAs), perfluorohexanoic acid (C6-PFCA), perfluoroheptanoic acid (C7-PFCA), perfluorooctanoic acid (C8-PFCA), perfluorononanoic acid (C9-PFCA) and perfluorodecanoic acid (C10-PFCA), in biological samples. PFCA in liver homogenates was extracted as an ion pair with tetrabutylammonium (TBA) ion into organic solvent, then the PFCA was derivatized with 3-bromoacetyl-7-methoxycoumarin (BrAMC) and quantified by HPLC with fluorescence detection. This method is applicable for the studies on tissue accumulation and elimination of PFCAs in animals after the administration.

Animals↗