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Improved preparation of small biological samples for mercury analysis using cold vapor atomic absorption spectroscopy.

Concentrations of mercury in biological samples collected for environmental studies are often less than 0.1 microgram/g. Low mercury concentrations and small organ sizes in many wildlife species (approximately 0.1 g) increase the difficulty of mercury determination at environmentally relevant concentrations. We have developed a digestion technique to extract mercury from small (0.1 g), biological samples at these relevant concentrations. Mean recoveries (+/- standard error) from validation trials of mercury fortified tissue samples using cold vapor atomic absorption spectroscopy for analysis ranged from 102 +/- 4.3% (2.5 micrograms/L, n = 15) to 108 +/- 1.4% (25 micrograms/L, n = 15). Recoveries of inorganic mercury were 99 +/- 5 (n = 19) for quality assurance samples analyzed during environmental evaluations conducted during a 24 month period. This technique can be used to determine total mercury concentrations of 60 ng Hg/g sample. Samples can be analyzed in standard laboratories in a short time, at minimal cost. The technique is versatile and can be used to determine mercury concentrations in several different matrices, limiting the time and expense of method development and validation.

Animals↗

An improved wet digestion procedure for the analysis of total arsenic in biological samples by direct hydride atomic absorption spectrophotometry.

The presence of dimethylarsinic acid (DMAA) in biological samples can cause an underestimation of total arsenic content when analyzed relative to an inorganic arsenic standard by direct hydride flame atomic absorption spectrophotometry. An acid digestion procedure is described that quantitatively recovers DMAA as well as monomethylarsonic acid, inorganic arsenic(III), and arsenic(V) from aqueous and biological samples. Methylated arsenicals are converted to inorganic arsenic by wet digestion with HNO3, H2SO4, and K2Cr2O7 and subsequently reduced to arsenic(III) with Nal. Arsine is generated with NaBH4 and converted to atomic arsenic following immediate introduction into a nitrogen-entrained air-hydrogen flame. This method produces a linear relationship to absorbance within a mass range of 50 to 300 ng arsenic/arsine reaction. A sensitivity of 2 ng arsenic and a detection limit of 7 ng arsenic/arsine reaction were also obtained. Recovery of DMAA from water, urine, feces, or whole blood ranged from 92 to 105% with a coefficient of variation of 5 to 10%.

Animals↗

Direct measurement of lipid-soluble arsenic species in biological samples with HPLC-ICPMS.

Lipid-soluble arsenicals (arsenolipids) occur in a wide range of biological samples where they may play a key role in the biosynthesis of organoarsenic compounds from inorganic arsenic. The study of these compounds has been hindered, however, by the lack of a suitable analytical technique able to separate and measure the various lipid species. As a source of arsenolipids, we used 10 crude fish oils from various regions of the world. Total arsenic analyses on the fish oils, performed with ICPMS following acid digestion with microwave-assisted heating, gave concentrations from 4.3 to 10.5 mg As kg(-1). All of the arsenic was soluble in non-polar solvents such as hexane. Analysis of the fish oils for arsenolipids was performed by normal phase HPLC-ICPMS with various mixtures of organic solvents as mobile phases. Inherent problems of instability associated with the introduction of organic solvents to the plasma were overcome by the use of reduced column flow, a chilled spray chamber, and the addition of oxygen directly to the plasma. All ten fish oils appeared to contain the same 4-6 major arsenolipids, but in varying amounts depending on the origin of the fish. Further chromatography with both normal phase and reversed-phase conditions on some of the oils indicated the presence of many more minor arsenolipids. Quantification was achieved by external calibration against triphenylarsine oxide or triphenylarsine sulfide, and the sum of species following HPLC of the oils matched well the total arsenic results (92-107%). The method was applied to samples of food supplements (fish oil capsules) and a packaged food product (cod liver) whereby arsenolipids were measured and found to be significant arsenic constituents. This study represents the first attempt to directly measure intact arsenolipids and, with appropriate sample preparation, may be suitable for quantitative measurement of these arsenicals in a range of biological samples, including foodstuffs.

Animals↗

A high performance liquid chromatographic method of analysis of 4'-O-tetrahydropyranyladriamycin and their metabolites in biological samples.

A method for measuring 4'-O-tetrahydropyranyladriamycin (THP) and its metabolites in biological samples are described. By reversed-phase high performance liquid chromatography using fluorescence detection, THP and its metabolites were all separated on a single chromatogram within 18 minutes. A linear calibration curve was obtained up to 2,000 ng/ml of THP in plasma. The recovery of THP in the analysis was more than 95% above 5 ng/ml and 87.1% even at 1.25 ng/ml. Thus the lower limit was 1.25 ng/ml in biological samples. Blood levels and urinary excretion in mice and dogs were satisfactory measured by this analytical method.

Animals↗

Electrophoretic separation of tryptophan enantiomers in biological samples.

A method for the determination of D- and L-tryptophan (Trp) in biological samples is described. The amino acid enantiomers were precolumn-derivatized with a fluorescence tagging reagent, naphthalene-2,3-dialdehyde (NDA). In the presence of hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) as the chiral selector, NDA-tagged Trp enantiomers were well resolved by micellar electrokinetic chromatography (MEKC). Using laser induced fluorescence (LIF) detection, a detection limit of 3.3 x 10(-8) M Trp was obtained. The method was applied to the determination of Trp enantiomers in biological samples including human urine and cerebrospinal fluid (CSF), rat brain tissue, and Aplysia ganglia. No interference from other amino acids or the endogenous compounds in the sample matrices was observed. D-Trp was found at the sub-microM level in human urine samples collected from several healthy subjects. Further, the determination of DL-Trp residues in small quantities (10 microg) of peptides after acid hydrolysis is demonstrated.

Chromatography, Micellar Electrokinetic Capillary↗

Reversed-phase and ion-pair separations of nucleotides, nucleosides and nucleobases: analysis of biological samples in health and disease.

Methods for the assay of nucleotides, nucleosides and nucleobases in biological samples in health and disease are reviewed, with emphasis on reversed-phase and ion-pair reversed-phase techniques for their determination. Modes of extraction from biological samples are discussed with respect of the determination of in vivo concentrations. Advantages and limitations of ion-pair reversed-phase chromatography are discussed with examples from biochemistry and clinical chemistry. The capacity of the high-performance capillary electrophoresis is compared with that of ion-pair reversed-phase chromatography.

Animals↗

A chemiluminescent assay for hydroperoxide level of phosphatidylcholine hydroperoxide fraction purified by two Sep-Pak cartridges in biological samples.

A chemiluminescent assay for hydroperoxide level of phosphatidylcholine hydroperoxide (PCOOH) fraction purified from biological samples was presented. This method utilized of two Sep-Pak cartridges. A lipid soluble fraction was isolated from each homogenized tissue or blood by Folch's method. The mixture of phosphatidylcholine (PC) and PCOOH was separated from the lipid soluble fraction by a Sep-Pak silica cartridge. A Sep-Pak tC18 cartridge made complete separation of both PCOOH and PC possible. The hydroperoxide level of PCOOH fraction was quantified by the reaction with ferrous ion using 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin++ +-3-one as a chemiluminescent dye. The mixture of positional isomers, 1-hexadecanoyl-2-[9, or 10-hydroperoxyl octadecanoyl]-sn-glycero-3-phosphocholine was used as an authentic standard. The good recovery rate for authentic PCOOH of 87.1 +/- 11.6% (mean +/- S.E., n = 4) was obtained by using two Sep-Pak cartridges. Linear calibration curve was obtained in the range from 2.5 to 20 nmol, and the detection limit of the standard was 10 pmol (signal-to-noise ratio > 3). This method was applied to the investigation of the lipid peroxidation induced by reperfusion of the liver with cold preservation, mimicking liver transplantation in rats. The effect of liposome-encapsulated dichloromethylene diphosphonate (LEDD), which eliminate of Kupffer cells to prevent the generation of oxygen radicals on the lipid peroxidation, was compared with the untreated group as a control. After 1 h reperfusion at 37 degrees C the hydroperoxide level obtained the liver without preservation in the untreated group was 12.4 +/- 2.4 nmol/100 mg lipid (n = 4) and levels increased significantly by prolongation of the preservation time. On the other hand, the hydroperoxide level in the LEDD treated group did not change up to 24 h preservation. These results suggest that this improved assay for hydroperoxide level of PCOOH fraction in biological samples can be applied to investigations involving lipid peroxidation because of its simplicity and accuracy.

Animals↗

Mutagenicity testing of protein-containing and biological samples using the Ames/Salmonella plate incorporation test and the fluctuation test.

Mutagenicity testing of biological samples and proteins is complicated by the presence of histidine and histidine-related growth factors which may produce a false positive result in the Ames/Salmonella plate incorporation test. A bioassay method, utilizing an automated dispenser-photometer and Salmonella typhimurium strain TA1535 as the indicator bacteria, was used to estimate the presence of histidine-related growth factors in three enzyme solutions submitted for mutagenicity testing. One of the solutions was clearly positive in the Ames/Salmonella test and also contained the highest amount of L-histidine-HCl-equivalents. The two other solutions, with low or undetectable amounts of L-histidine-HCl-equivalents, gave equivocal and negative results, respectively, in the Ames/Salmonella test. Studies were also performed with strains TA98, TA100 and TA1535 to determine the amount of added L-histidine-HCl that would result in a 'positive' result in the Ames/Salmonella test. Because the minimum amount of L-histidine-HCl required to double the number of revertant colonies was 150 nmol/plate, and the maximum amount of L-histidine-HCl-equivalents supplied by the enzyme preparations was 40 nmol/plate at the highest tested dose, the mutagenicity test results of the enzyme solutions cannot be explained solely by histidine or related compounds. Smokers' and non-smokers' urines, concentrated with liquid extraction (CHCl3) and adsorbent (XAD-2 and XAD-2/Sep-Pak C18) techniques, were studied to reveal differences in efficiencies to extract histidine and histidine-related compounds in the urines. Amounts of 'histidine' in concentrates of urine were measured using the bioassay method and a chemical method employing derivatization with fluorescamine. The fluorescamine method also efficiently detected 3-methyl-L-histidine, a product of muscle metabolism excreted in urine, which was found to be unable to support auxotrophic growth in TA1535, leading to exaggerated estimations of the auxotrophic growth enhancing properties of urine extracts. The urine extracts, and pure L-histidine-HCl, were tested using a two-step fluctuation test to estimate auxotrophic growth factor effects in this type of test. Because of a strong dilution effect when adding the histidine-free selection medium, the fluctuation test employed in this study was not found to be particularly sensitive to growth factors. The results of this study indicate that use of a bioassay, employing the same indicator bacteria as the mutagenicity test themselves, is a reliable way to measure histidine-related growth factors in biological samples.(ABSTRACT TRUNCATED AT 400 WORDS)

Enzymes↗

Analysis of low-angle x-ray scattering peaks from lyophilized biological samples.

Low-angle x-ray scattering (LAXS) from lyophilized blood and its constituents is characterized by the presence of two peaks in the forward direction of scattering. These peaks are found to be sensitive to the variations in the molecular structure of a given sample. The present work aims to explore the nature of LAXS from a variety of lyophilized biological samples. It also aims to investigate the possibility that a certain biological macromolecule is responsible of the production of LAXS peaks. This is carried out through measurements of LAXS from complex biological samples and their basic constituents. Among the measured samples are haemoglobin (Hb), globin, haem, packed red blood cells, bovine albumin, egg albumin, milk, casein, glutamine, alanine, fat, muscle and DNA. A table containing some characteristic parameters of the LAXS profiles of these samples is also presented. Analysis of measured profiles shows that all lyophilized samples produce at least one relatively broad peak at a scattering angle around 10.35 degrees. The full width at half maximum (FWHM) of this peak varies considerably among the measured samples. Except for milk and casein. one additional peak at a scattering angle around 4.65 degrees is observed only in the LAXS profiles of proteins or protein-rich samples. This fact strongly suggests protein to be the biological macromolecule from which this characteristic peak originates. The same idea is further strengthened through discussion of some previous observations.

Blood Chemical Analysis↗

Isotope dilution technique for the estimation of l-alpha-[2-3H]-acetylmethadol and its metabolites in biological samples.

An isotope dilution technique for the determination of l-alpha-(2-3H)-acetylmethadol and its metabolites from biological samples is described. The parent drug and metabolites were extracted from biological samples with chlorobutane after the addition of unlabeled internal standards. The extracts were purified by two-dimensional thin-layer chromatography for quantification by gas-liquid chromatography and scintillation counting. Dinoracetylmethadol (NNAM) was found to be converted to 6-acetamido-4,4-diphenyl-3-heptanol (AMIDE) during the extraction procedure. The conversion of NNAM to AMIDE was confirmed by gas chromatography-mass spectrometry.

Animals↗

Liquid chromatography analysis of N-(2-mercaptopropionyl)-glycine in biological samples by ThioGlo 3 derivatization.

N-(2-Mercaptopropionyl)-glycine (MPG) is a synthetic aminothiol antioxidant that is used in the treatment of cystinuria, rheumatoid arthritis, liver and skin disorders. Recent studies have shown that MPG can function as a chelating, cardioprotecting and a radioprotecting agent. Several other studies have shown that it may also act as a free radical scavenger because of its thiol group. Thiol-containing compounds have been detected in biological samples by various analytical methods such as spectrophotometric and colorimetric methods. However, these methods require several milliliters of a sample, time-consuming procedures and complicated derivatization steps, as well as having high detection limits. The present study describes a rapid, sensitive and relatively simple method for detecting MPG in biological tissues by using reverse-phase HPLC. With ThioGlo 3 [3H-Naphto[2,1-b] pyran, 9-acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl) phenyl-3-oxo-)] as the reagent, highly fluorescent derivatives of thiols can be obtained that are suitable for HPLC. MPG is derivatized with ThioGlo 3 and is then detected flourimetrically by reverse phase HPLC using a C18 column as the stationary phase. Acetonitrile: Water (75:25) with acetic acid and phosphoric acid (1 mL/L) is used as the mobile phase (excitation wavelength, 365 nm; emission wavelength, 445 nm). The calibration curve for MPG is linear over a range of 10-2500 nM (r=0.999) and the coefficients of the variation of within-run and between-run precision were found to be 0.3 and 2.1%, respectively. The detection limit was 5.07 nM per 20 microL injection volume. Quantitative relative recovery of MPG in the biological samples (plasma, lung, liver, kidney and brain) ranged from 90+/-5.3 to 106.7+/-9.3 %. Based on these results, we have concluded that this method is suitable for determining MPG in biological samples.

Animals↗

Observation of significant signal voids in images of large biological samples at 11.1 T.

Proton MRI of large biological samples were obtained on an 11.1 T / 40 cm instrument. Images were obtained of a fixed human brain and a large piece of fresh beef. The proton MR images demonstrate severe distortions within these conductive samples, indicative of shortened electrical wavelengths and wave behavior within the sample. These observations have significant implications with respect to the continuing evolution of MR to higher magnetic field strengths on large samples, particularly on humans.

Animals↗

A fluorescence plate reader assay for monitoring the susceptibility of biological samples to lipid peroxidation.

The susceptibility of biological samples to lipid peroxidation can be determined by exposing samples to a lipid peroxidation initiator and measuring the length of time prior to the onset of lipid peroxidation. Previous studies have shown that aldehydes generated by lipid peroxidation can react with amines to produce fluorescent products. We have utilized this principle to develop a fluorescence plate reader assay for measuring susceptibility to lipid peroxidation. In this assay, samples are placed in glycine/phosphate buffer and loaded into a 96-well plate. Lipid peroxidation initiators are added, and fluorescence is monitored over time. Samples were assayed for susceptibility to lipid peroxidation by both the thiobarbituric acid reactive substances assay and the fluorescence plate reader assay. We found good agreement between these two methods in assessing relative susceptibility to lipid peroxidation in liver microsomes and mitochondria. The fluorescence assay was also used to monitor lipid peroxidation in liposomes and rat liver homogenates. Fluorescence was stable over an extended time period and could be induced by a variety of lipid peroxidation initiators. The fluorescence plate reader assay offers a rapid method for monitoring lipid peroxidation in a large number of samples.

Animals↗

Mass spectrometry of trace elements in biological samples.

Mass spectrometry is a powerful analytical tool for determining the isotope ratios and concentrations of trace elements in various samples at levels ranging from major constituents to subparts per billion. Because isotope dilution is free from matrix effects, it has the potential of being incorporated into a definitive analytical approach that can provide reference values for concentrations in physiological and pathological conditions. In addition, isotope dilution mass spectrometry results are free from the constraints of quantitative recovery of the analyte, an essential requirement in other analytical techniques that is difficult to achieve with complex biological samples. A variety of mass spectrometric approaches have been used for determining the concentration of trace elements in biological samples. The more commonly used are thermal ionization mass spectrometry, inductively coupled plasma mass spectrometry, fast atom bombardment mass spectrometry, and gas chromatography mass spectrometry. This article reviews the work on trace element determination in biological samples using different mass spectrometric techniques and highlights the experiments performed by the authors in establishing gas chromatography mass spectrometry.

Animals↗

Preparation of biological samples in space: the experiment preparation unit.

The Experiment Preparation Unit (EPU) is a facility that prepares biological samples for their upcoming experiment within Biolab (Columbus). For logistical reasons and to avoid the influence of the heavy launch loads and the microgravity environment before the actual initiation of the experiment, biological samples will be sent cryogenically frozen to the International Space Station. The Experiment Preparation Unit will prepare those samples by thawing and extracting the cryogenic fluid automatically after minimal crew intervention for set-up of the system. The EPU can be tele-operated directly from ground and consists of three major subsystems to guarantee its functionality: Functional Platform, Base and Control Panel.

Journal Article↗

High resolution electron paramagnetic resonance imaging of biological samples with a single line paramagnetic label.

The application of electron paramagnetic resonance imaging (EPRI) to obtain information from biological samples has been limited by the lack of ideal single line radical labels. The commonly used nitroxides exhibit multiple lines causing either hyperfine-based limitations in the maximum obtainable image resolution or hyperfine-based artifacts in the reconstructed image. The use of a novel single-line triarylmethyl paramagnetic label that enables marked enhancement in image quality and resolution is reported. This label exhibits a single line EPR spectrum that is sharp (linewidth approximately 60 mG in the absence of oxygen) and relatively stable in tissues. The potential of this label in enabling high resolution EPR imaging of biological samples was demonstrated in a series of phantoms and isolated biological organs such as the rat kidney. The images demonstrate that resolutions better than 100 microns could be obtained at L-band on samples of up to 20 mm in size.

Animals↗

Simultaneous determination of nitrate and nitrite in biological samples by multichannel flow injection analysis.

An automated method for the simultaneous determination of nitrite and nitrate in biological samples by using a multichannel flow injection analyzer has been developed. The method was based on the reaction of nitrite with Greiss reagent. The sample solution was injected and equally divided into two channels; channel one (1) represented total nitrite obtained by cadmium reduction of nitrate to nitrite while channel two (2) represented only nitrite. The absorbance of the color product was measured by photometric detectors with 540-nm filters. This method combines high reproducibility of sample introduction via flow injection and sensitivity of spectrophotometric detection. The detection limit is 25 nM for both nitrite and nitrate. The chemistry manifolds are constructed of Teflon tubing which, along with a low-pressure Flowfit connector system, provides for low maintenance, ease of use, and high sample throughput. We demonstrated that the system can be used for the determination of both nitrate and nitrite in a variety of biological samples as well as a comparison of the results from this system and the HPLC system.

Animals↗

AISP position statement: Standardising biological sample collection and handling for advanced diagnostics and multi-omic analyses in pancreatic cancer.

The quality of biological samples is a major determinant of analytical reliability and translational relevance in patients with pancreatic ductal adenocarcinoma (PDAC). However, variability in specimen procurement, handling, transport, processing, and storage can substantially affect tissue integrity and the robustness of downstream analyses. This paper, promoted by the Pathology and Basic Science Task Force of the Italian Association for the Study of the Pancreas (AISP), brings together experts in pathology, molecular biology, translational research, medical oncology, and gastroenterology to provide practical recommendations for the collection, handling, and pre-analytical management of biological samples. Draft recommendations were discussed during dedicated working group meetings and approved by consensus among all authors, supported by key literature. The document identifies the biological specimen as the critical link between patient care, pathology, and research, and provides guidance for clinicians and professionals involved in sample procurement and processing. By addressing the requirements of different analytical platforms, including genomics, organoid generation, immunophenotyping, pharmacogenomics, and multiplex/spatial analyses, this paper aims to reduce pre-analytical variability, improve diagnostic accuracy, and enhance the clinical and translational value of molecular investigations in pancreatic cancer. Standardised procedures across centres may facilitate comparable data collection, support multicentre studies, and strengthen collaboration between clinicians, pathologists, and research laboratories.

Biobanking↗