Samples Of Biological Origin Research Articles

The analysis and crystallographic characterization of 20-hydroxyecdysone.

20-Hydroxyecdysone (20E) is an insect molting hormone that is also widely spread in various plants. Many chromatographic methods can be used to identify and/or determine 20E content in samples of biological origin and various spectroscopic methods serve to identify its structural elements. We have utilized X-ray crystallography to reveal the stereostructures of 20E. Our data demonstrates that 20E exists in two different crystalline forms that are both orthorhombic modifications. One form is homo-molecular, with a limited freedom of internal rotation of the side chain around the C23-C24 bond and the other, which is a clathrate formed with methanol and water, which minimize the conformational freedom of the side chain.

An updated look at the analysis of unsaturated C27 sterols by gas chromatography and mass spectrometry

Gas chromatography-mass spectrometry (GC-MS) and GC are commonly used methods for the identification and quantitation of sterols from samples of biological origin. To investigate the utility and limitations of these methods, we have determined gas chromatographic mobilities and mass spectral properties of 5alpha-cholestan-3beta-ol and 26 unsaturated C27 sterols as their acetate and trimethylsilyl (TMS) ether derivatives by GC and GC-MS. The GC retention data showed that numerous sterols were essentially coeluted on capillary GC columns coated with either 5% phenyl-95% methyl polysiloxane or polyethylene glycol, although the peaks were more widely dispersed on the latter column. Mass spectra of many groups of sterol isomers were also quite similar. Sterol mixtures of any complexity are likely to contain coeluting components, and attempts to establish structures based on mass spectra that may represent a mixture of sterol isomers could easily lead to errors. Our results demonstrate that GC and GC-MS alone cannot generally be used for rigorous structure determinations of individual components in mixtures of unsaturated sterols. However, all but a few of the 26 sterols could be distinguished by their combined chromatographic mobilities on the two GC columns coupled with critical examination of their mass spectra. GC-MS analysis of appropriate sterol subclasses or preferably individual sterol components obtained by prior purification by other methods may provide valuable supporting evidence for the identification of sterol structures. Reliability of identification is dependent upon careful attention to GC and MS conditions, calibration of GC and MS data with authentic sterol standards, and consideration of possible decomposition under GC conditions and of the effect of overloading on GC retention times.

An intercomparison of γ-spectrometry on two samples of biological origin by eight laboratories in four countries

This report gives details of the first inter-laboratory comparison of γ-spectrometry to be run within SPERA, the South Pacific Environmental Radioactivity Association since its inauguration in 1991. Laboratories in Australia, Chile, French Polynesia and New Zealand participated in the exercise. Two ‘unknown’ samples of biological origin were analysed. The first was a sample of milk powder derived from IAEA reference material. This sample provided an assessment of overall accuracy of 134Cs, 137Cs and 40K determinations. The second sample consisted of dried fish flesh including natural 40K and spiked with a mixed nuclide solution containing 210Pb, 109Cd, 54Mn, 60Co and trace 133Ba. Together the samples gave information on analytical precision over a range of energies and activities. When the results were compared with the recommended values and confidence intervals of the IAEA reference material, the overall accuracy of the γ-spectrometry analytical procedures was found to be good. The average mean values for combined laboratory data fell within the recommended value ranges for each isotope. Ninety percent of the individual laboratory isotope mean values were within two standard errors of the 95% confidence interval of the standard, 75% were within 1 s.e., and 33% of the analyses fell within the confidence interval. Technical precision was also adequate with the overall errors being of the same magnitude as that of the reference material values for each isotope with relative standard deviations of 5–10%. There was a tendency for standard deviations of the combined results to be larger than those reported or derived from individual laboratory results by a factor between 1.2–5.6. This result suggested an under-estimate of systematic errors within individual laboratories. The largest sources of error were derived from reporting and calculation of results which gave a 16% gross error rate.

Antimony speciation in freshwater plant extracts by using hydride generation–gas chromatography–mass spectrometry

An analytical method based on a semi-continuous hydride generator coupled on-line with a gas chromatograph and mass spectrometric detectro was developed and used for the identification of hydride-forming antimony compounds. This method allows for the characterization of compounds based on their retention times and mass spectral data. Organoantimony compounds were identified to be present in extracts of a freshwater plant, namely pondweed (Potamogetan pectinatus). This is the first time that such compounds have been detected in samples of biological origin. The problem of organostibine molecular rearrangements which have been reported to occur during the hydride generation process was also investigated. An experimental procedure is described that allows for the elimination of such rearrangements.

Simple quantitative transesterification of lipids 2. Applications

AbstractThe following samples of biological origin were submitted to transesterification by means of methanolic hydrogen chloride, generated from methanol and acetyl chloride: triglycerides from black currant oil, butter, pig lard tissue, fat tissue of inner part of goose, lyophilized human blood plasma, larvae of fleshfly of the genus Sarcophaga, lipid A from Shigella dysenteriae, esters from beeswax and esters from the cuticular lipids of pea aphid Acyrthosiphon pisum. Attention was also paid to sterol esters and to free fatty acids. Conditions of transesterifications are discussed, as well as the conditions of gas chromatographic analysis of components after transesterification.

Rapid and Selective Sample Preparation for the Chromatographic Determination of Brassinosteroids from Plant Material Using Solid-Phase Extraction Method

This report describes a rapid and selective method of sample preparation for the determination of brassinosteroids in samples of biological origin, based on solid-phase extraction. The steroids, brassinolide and castasterone, which possess a C-22,23-diol group, were retained as their cyclic boronates formed on immobilized phenylboronic acid gel in a mixed solvent of pyridine and acetonitrile. After removal of impurities in the sample, parent brassinosteroids could easily be released from their boronates by a mixed solution of hydrogen peroxide and acetonitrile. The phenylboronic acid phase was shown to have a high affinity for brassinosteroids containing a C-22,23-diol. The present method is a first example for the selective extraction of brassinosteroids in a biological sample using immobilized phenylboronic acid gel.

Immunoassay for native enzyme quantification in biological samples

In order to detect low levels of enzyme activity, specifically glucose oxidase, in biological samples, an immunoenzymatic assay was developed since currently available methods could not be used because of either their lack of sensitivity or the conditions prevailing in our samples: turbidity of the medium, presence of redox systems other than glucose oxidase, and high concentration of proteins. The principle of the method is to coat a polystyrene surface with a fragment Fc-specific anti-IgG, then with an antibody directed against the looked-for enzyme, which is simultaneously the antigen and the enzyme activity required for immunoenzymatic detection. We applied this concept to biological samples after glucose oxidase administration to mice. This method achieves specificity and sensitivity (20 ng/mL or 1 ng) with samples of biological origin. No marker is needed since the antigen itself possesses an enzyme activity. This method, which requires a small sample volume (50 microL, 20 microL, if necessary), can be extended easily to the many enzymes currently used as markers. It could also be applied to the native enzymes of medical interest for which antibodies and a colorimetric reaction are available.

Elimination of interferences in the determination of arsenic and selenium in biological samples by inductively coupled plasma mass spectrometry

The determination of As and Se in biological samples by inductively coupled plasma mass spectrometry is well known to be degraded by spectral interferences. The resolution of quadrupole mass analysers is insufficient to resolve As + and Se + ions from polyatomic species such as ArCl +, ArAr + and So + 3. A study of this problem in human serum also revealed substantial non-spectral interferences on these elements occuring in the presence of organic compounds. It is shown that both problems can easily be overcome by a combination of chemical modification (addition of 4% ethanol) with nebulizer flow-rate gas adjustment. Under these conditions the use of standard additions for calibration allowed As and Se to be determined accurately in samples of biological origin. The method developed was applied to human serum and urine and for both As and Se excellent agreement with certified values was obtained.

Cryo-Scanning tunneling microscopy of DNA

Scanning tunneling microscopy has found increasing interest in biological sciences. Several problems face the investigator imaging samples of biological origin. These include low conductivity, poor attachment of the sample to substrate and thermal/mechanical drift of the sample while scanning. Several approaches have tried to overcome these problems. These include coating with metal, freeze drying and/or freeze fracturing and tunneling in water. Another useful method is to freeze the sample on the STM holder and image frozen. The biological structure should stay rigid under the scanning tip, should not be swept away from the substrate and should provide highly reproducible images.Calf thymus DNA (Sigma #D-1501) was mixed in deionized water-tween 20 (0.1%). DNA concentrations varied between 10 μg/ml and 1 μg/ml. The sample was heated slightly, a small drop placed on highly oriented pyrolytic graphite (HOPG) and air dried. The sample holder was immediatel) placed in an Atomis variable temperature cryo-STM with a 6 micron scan range head (Surface Interface, Inc., Mountain View, CA), frozen and scanned.

Analysis of volatile fatty acids in biological specimens by capillary gas chromatography

AbstractA method was developed to analyze and quantitate volatile fatty acids such as acetic, propionic, butyric, iso‐butyric, valeric, and iso‐valeric acid from samples of biological origin. A capillary column system including an automatic on‐column injection device as well as a precolumn of larger internal diameter than the analytical column was elaborated for this purpose.In order to obtain well resolved and correctly quantifiable chromatographic peaks it turned out to be essential to work under acidic/aqueous conditions. To achieve a better sample transfer into the chromatographic system an organic solvent had to be used together with the aqueous milieu, thus improving wetting properties of the liquid sample plug introduced into the column. Cold on‐column injection was applied in order to avoid discrimination of the various acids due to sample splitting and the automatic technique was chosen in view of the large number of samples from biological extractions which had to be analyzed.

An amperometric detector for use with small-bore liquid chromatographic columns

A miniaturized electrochemical flow cell for amperometric detection in liquid chromatography has been designed to meet the requirement of low volumetric dispersion when using small-bore columns. The volume of the thin-layer cell is in the range 10–50 nl, depending on the spacer dimensions, with the electrode area being about 1 mm 2. The cell is constructed from glass and designed in such a way that the working electroded can be mounted without alterations to its surface state. The flow-rate dependence, contribution to peak dispersion and linear dynamic range were investigated. After separation on a 250 × 1 mm I.D. column, a detection limit for dopamine of about 0.15 was ahieved with a linear dynamic range of 10 6. The variance of the extra-column dispersion was determined to be less than 0.3 μl 2, including injector and connecting capillaries. The applicability of the detector is illustrated with the detection of dopamine and its major metabolites, dihydroxyphenylacetic acid and homovanillic acid, and of Met- and Leu-enkephalin in samples of biological origin.

Electrophoresis of opioid peptides on columns with agarose suspension

Opioid peptides have been fractionated by column electrophoresis in agarose suspension. The procedure provides a high resolution efficiency and compares favorably with high-performance liquid chromatography. Peptides were recovered from the column in 70% or higher yields when run in the femtomole to the nanomole range. By choosing different pHs it is possible to get complete resolution of a number of different endorphins. The electrophoreses were performed in volatile buffers, which are easily evaporated, and the residues are suitable for direct screening by radioreceptor- or radioimmunoassays. In combination with these assays the separation procedure makes it possible to detect and identify very low concentrations of endorphins in samples of biological origin.

Biochemical and biomedical applications of capillary column GLC using SIM.

This report focuses on recent applications of capillary column GLC to the analysis of drugs and metabolites, other xenobiotics, natural products, and environmental contaminants in samples of biological origin. The increasing use of selected ion monitoring, combined with stable isotope methods as a means of GLC detection and quantification, is emphasized. Specific topics covered include the use of capillary column GLC for 1) determining the pharmacokinetics of timolol (BLOCADREN) in human volunteers by simultaneous oral and intravenous administration of this beta-blocker and its 13C3-labeled analog; 2) measuring residue levels of the forced molting agent, xylonidine, in yolk and albumen of chicken eggs; 3) monitoring animal plasma and tissues for the depletion of 5-hydroxy-1,3-dioxane and 4-hydroxymethyl-1,3-dioxolane (isomeric components of glycerol formal, an animal drug formulation component); 4) detecting 13C, 15N-methyltryptamine (a biosynthesis product from 13C, 15N-tryptamine) in human urine; and 5) measuring estradiol levels in biological samples.

Analysis of drugs and related compounds by capillary column GLC

AbstractThis article describes the development of the application of capillary column GLC methods to steroids, prostaglandins and especially drugs and their metabolites in samples of biological origin. The wide variety – in terms of structures and pharmacological properties – of drugs now analyzed by capillary column GLC is made evident. Reasons for use of capillary rather than packed columns are given. The frequent use of and need for derivatization procedures prior to chromatography is emphasized. A bibliography of 83 references is included in the manuscript.

Experience with Quantitative Thin-Layer Chromatography in the Analysis of Drugs in Plasma

Abstract A survey is presented concerning the possible techniques of quantitative thin-layer chromatography. Most of the possibilities mentioned have been used successfully in our laboratory for the determination of drugs and metabolites in plasma. With respect to our own experience, the advantages and disadvantages of quantitative thin-layer chromatography are discussed. Although gas chromatography and high-performance liquid chromatography are more popular, quantitative thin-layer chromatography has a high potential in the analysis of drugs in samples of biological origin and should not be neglected in the development of an assay.

Contact Spotting : A new approach to sample application in thin-layer chromatography

The introduction of high-performance adsorbent layers to thin-layer chromatography has necessitated improved methods of sample application in order to realize fully the resolution, sensitivity and speed achievable with this quantitative analytical technique. Systems utilizing microcapillary pipettes are capable of maintaining minimal sample spot diameters; but when the solution viscosity is high or the concentration of analyte is small, this method of delivery may be impractical or may require a large number of serial applications to the same spot. The process described in this paper enables rapid concentration of sample solutions of up to 50 μl with transfer of the residue directly to the plate resulting in spot diameters as low as 0.1 mm located with excellent precision. Multiple samples are applied simultaneously, and the densitometric measurement of separated components is highly reproducible. The method is particularly valuable in dealing with samples of biological origin where concentrated extracts are too viscous to permit delivery by micropipettes.

Liquid column chromatography of nucleoside monophosphates on deae-spheron 1000

The ion exchange capacity, radiation stability and particle distribution have been estimated for two fractions of the weak anion exchanger DEAE-Spheron 1000. Capacity factors are reported for eight nucleotides measured under various conditions in citrate and formate buffers. This type of sorbent may be used for the separation of deoxy- or ribonucleotides under mild conditons in ammonium formate buffer. No interferences from other substances which are usually found in samples of biological origin have been observed.

Automatic isotope gas analysis of tritium labelled organic materials

An isotope analytical procedure and an automatic instrument developed for the determination of tritium in organic compounds and biological materials by internal gas counting are described. The sample is burnt in a stream of oxygen and the combustion products including water vapour carrying the tritium are led onto a column of molecular sieve-5A heated to 550 °C. Tritium is retained temporarily on the column, then transferred into a stream of hydrogen by isotope exchange. After addition of butane, the tritiated hydrogen is led into an internal detector and enclosed there for radioactivity measurement. The procedure, providing quantitative recovery, is completed in five minutes. It is free of memory effect and suitable for the determination of tritium in a wide range of organic compounds and samples of biological origin.

The Use of Activated Carbon for the Recovery and Concentration of Nucleic Acid Derivatives and other Compounds from Biological Samples

Activated carbon was used to recover nucleic acid derivatives from samples of biological origin, such as wort (grain extract) and beer (fermented liquor). The sorption-desorption phenomena of different types of activated carbon were investigated using various experimental conditions. Simple and mixed solutions of nucleic acid derivatives, as well as wort and beer samples were treated with activated carbon. The physical structure and other peculiar characteristics of the adsorbant were studied. It was found that the pH, possibly the visconsity, and the concentration of the compounds in the samples are important factors affecting the recovery of nucleic acids. The composition of the desorbates and the distribution of nucleic acid derivatives in these desorbates were analysed.Maximum recovery was obtained in the first desorbate using the ammonia ethanol solution. The ratios of total UV absorption in the desorbates vs. nucleotides and other compounds are variable in each fraction. Not only nucleotides but also several other types of compounds such as carbohydrates, amino acids, tanninogens, colored compounds and mineral salts could be detected in various amounts in the desorbates.

Micro-Determination of Nitrogen

The Kjeldahl method remains the method which will be used for the analysis of most samples of biological origin, the advantage being the “wet ashing” technique. The advantages of automation are app...