Liquid Chromatography-Electrospray Mass Spectrometry Determination of Ibogaine and 12-Hydroxy-Ibogamine in Human Urine

A specific and sensitive liquid chromatography-electrospray ionization mass spectrometry method was developed for the determination of ibogaine and noribogaine in human urine. The work-up procedure involved a solid phase extraction of the compounds and the internal standard (fluorescein) using Oasis HLB columns. The system used a Zorbax eclipse XDB C8 analytical column packed with 5µm diameter particles as the stationary phase. The mobile phase consisted of a 20-min gradient (mobile phase A: 0.02% (v/v) trimethylamine in acetonitrile, mobile phase B: 2 mM ammonium formate buffer (pH 3)). Mass spectrometric data were acquired in single ion monitoring mode at m/z 311.1, 297.2 and 333 for ibogaine, noribogaine and fluorescein, respectively. The drug/internal standard peak area ratios were linked via a quadratic relationship to concentrations (1.78?358 μg L^?1 for ibogaine; 2?400 μg L^?1 for noribogaine). Precision ranged from 5.8 to 14.8% and accuracy was between 93.2 and 112.9%. Mean extraction recoveries of ibogaine, noribogaine and fluorescein were 70.0, 81.7 and 94.8%, respectively. The extraction efficiency was independent of concentration over the range studied. The lower limits of quantitation were 1.78 μg L^?1 for ibogaine and 2 μg L^?1 for noribogaine. In this paper, extensive stability testing was undertaken using a wide range of storage conditions. This method was found suitable for urine analysis of a poisoning involving ingestion of drink made from powdered root of shrub Tabernanthe iboga.     

Fluorescence of tryptophan-containing peptides on paper or silica gel after treatment with formaldehyde, formaldehyde-ozone or formaldehyde-hydrochloric acid.

Sensitive and specific procedures for the chromatographic detection of tryptophan and tryptophan-containing peptides are described. Formaldehyde gas induces strong and characteristic fluorescence from tryptophan and peptides with NH2-terminal tryptophan residues on silica gel. On filter-paper, the detection of small amounts of these compounds requires the additional use of an oxidant, such as ozone. Treatment with formaldehyde-hydrochloric acid was used as a method for inducing fluorescence from tryptophan-containing peptides regardless of the position of the tryptophan residue in the peptide molecule. This reaction is useful for the chromatographic demonstration of small amounts of such peptides on both paper and silica gel. The spectral properties of the fluorophores of such tryptophan-containing peptides are distinctive and serve to distinguish them from all other known biogenic compounds that are capable of giving fluorescence with formaldehyde.     

Estimating measurement uncertainty in an afternoon. A case study in the practical application of measurement uncertainty

The ISO/IEC 17025:1999 standard requires chemical testing laboratories to have an estimate of the uncertainty of their measurements. This is a new requirement for many laboratories and there is confusion as to how to estimate uncertainty. Concerns have been raised about the time and effort required to obtain uncertainty estimates.Uncertainty budgets were prepared for the measurement of benzene, toluene, ethyl benzene and xylenes (BTEX) in water using purge and trap coupled with GC/MS. A time limit of one working afternoon (2 pm–5.30 pm) was imposed for preparing the uncertainty estimate. Details of the uncertainty estimate for toluene are described.The method in question had been in routine use for several years and the laboratory held third party (NATA) accreditation for the test. Consequently a considerable amount of performance data was readily available. Relevant information was extracted from the documented test method, validation data, instrument calibration and from routine quality control. This data was combined according to the principles of the ISO Guide to the Expression of Uncertainty, as promulgated in the Eurachem document "Quantifying Uncertainty in Analytical Measurement."The uncertainty estimates were compared to estimates obtained from generalised empirical models (the Horwitz and Lowthian equations), and from interlaboratory studies of this analysis.A traceability chain from routine measurements to the SI units of metre, kilogram and mole is described.Realistic and useful uncertainty estimates were obtained with a few hours work using data extant in the laboratory.     

Preparation of trout liver microsomes for iron speciation in P-450 enzymes by AE–FPLC with ICP–(ORS)MS detection

Cytochromes P-450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiological and xenobiotic compounds in eukaryotes and prokaryotes. The multiplicity of this group of enzymes has been widely studied by chromatographic techniques, mainly high-performance liquid chromatography (HPLC). Because these enzymes are membrane-bound proteins, sample preparation for chromatographic separation of P-450 enzymes requires a solubilization step. The sample-preparation procedures are critical, because detergents affect not only the efficiency of protein solubilization but also their further chromatographic resolution. Trout liver microsomes have been taken here as a model sample to investigate iron speciation in cytochrome P-450. Trouts were treated intraperitoneally with -naphthoflavone, a potent inducer of some P-450 enzymes, and a microsomal suspension containing 7.4±0.1 nmol mL^–1 P-450 enzymes was obtained by ultracentrifugation. Lubrol PX was selected as detergent for solubilization, resulting in about 90% solubilization recovery. The solubilized cytochromes P-450 were further separated by AE–FPLC, with UV detection, or coupled to ICP–MS with an octapole reaction system, ICP–(ORS)MS (monitoring Fe signals at masses 54, 56, and 57). A sampling procedure and chromatographic conditions are developed and were successfully applied to iron speciation in trout liver P-450 enzymes. ICP–(ORS)MS detection of P-450 enzymes is Fe-specific and so will give accurate information on the prosthetic group of the protein, which can constitute an advantageous alternative to classical methods for detection of these hemoproteins.     

Analysis of organic acids in wines by Fourier-transform infrared spectroscopy

Fourier-transform infrared (FTIR) spectroscopy has been a major point of development in many wine laboratories in recent years. It enables almost instant analysis of several properties of wine, usually with very good precision and accurate results. Nevertheless, validation procedures should not be forgotten and should be fully performed. Recovery experiments were performed by spiking wine samples with different amounts of organic acids (tartaric, malic, lactic, acetic and citric—the most prominent in wines). After FTIR analysis of the total acidity and of each organic acid concentration, recoveries were calculated. For total acidity recovery results were, in general, good and very close to 100% (64–111%). On the other hand, for individual organic acid concentrations, the recovery results were lower than 100% (11–73%) for all spiking additions. These results could be explained by spectroscopic interferences between the organic acids. Because they have similar infrared spectra, it is not easy to distinguish between them and, therefore, to achieve accurate calibration. When total acidity, with a different infrared spectrum from the other abundant compounds in the wine, was taken as a single property the recovery results were acceptable.     

Fluorine containing coordination compounds of Cr(III)

Trans-[Cr(NH₃)₄F₂]I·H₂O (A) has monoclinic P2_l/m (No. 11) space group witha=5.033 (3),b=16.333 (10),c=5.539 (3) Å and =98.47 (3)°,Z=2.Cis-[Cr(NH₃)₄F₂]ClO₄ (B) has tetragonal space group I4_lmd (No. 109) witha=7.417 (1),c=16.610 (2) Å,Z=4. Cr–F and Cr–N bonding distances are 1.894 (3); 2.087 and 2.083 (5) Å for A and 1.887 (6); 2.062 (5) and 2.051 (7) Å for B. Octahedral angles within the cations are close to 90° for both compounds. Cr–N bondtrans to Cr–F bond in thecis compound is shorter. Structures were refined toR ₂ values of 0.072 (A) and 0.058 (B).Trans-[Cr(NH₃)₄F₂]I·H₂O has weak N–H–F hydrogen bonds between the cations. None such interactions were found incis-[Cr(NH₃)₄F₂]ClO₄.

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Fluorhältige Komplexe des Cr(III), 2. Mitt.: Kristall- und Molekülstruktur von trans-[Cr(NH₃)₄F₂]I·H₂O und cis-[Cr(NH₃)₄F₂]ClO₄

Zusammenfassung Trans-[Cr(NH₃)₄F₂]I·H₂O (A) kristallisiert in der Raumgruppe P2_l/m (No. 11) mitZ=2 unda=5,033 (3),b=16,333 (10),c=5,539 (3) Å und =98,47 (3)°.Cis-[Cr(NH₃)₄F₂]ClO₄ (B) kristallisiert in der Raumgruppe I4_lmd (No. 109) mitZ=4,a=7,417 (1) undc=16,610 (2) Å. Die Cr–F- und Cr–N-Abstände sind 1,894 (3); 2,087 (6), 2,083 (5) Å für A und 1,887 (6); 2,062 (5), 2,051 (7) Å für B. Die octaedrischen Bindungswinkel innerhalb der Kationen weichen nicht viel von 90° ab. Der Cr–N-Abstand intrans-Position der Cr–F-Bindung ist kürzer. Die Strukturen wurden bis zu GütefaktorenR ₂ 0,072 (A) und 0,058 (B) verfeinert. Bei der Verbindung A wurden schwache N–H ... F-Wasserstoff-Bindungen zwischen verschiedenen Kationen beobachtet, während bei der Verbindung B keine Wasserstoff-Bindungen vorhanden sind.

     

High accuracy analysis by isotope dilution mass spectrometry using an iterative exact matching technique

The selection of an appropriate isotope dilution mass spectrometry technique for the practical application of this potentially primary method of analysis is highly important. The NARL approach for the application of the exact matching double isotope dilution mass spectrometry technique developed by Henrion is presented. NARL's approach utilises exact matching to minimise the effect of measurement biases within the method but also includes the thorough examination of all other biasing factors. The approach has been successfully tested in international CCQM intercomparisons with other national metrology institutes.     

Determination of pKa values of some hydroxylated benzoic acids in methanol-water binary mixtures by LC methodology and potentiometry

An accurate estimation of pK_a values in methanol-water binary mixtures is very important for several separation techniques such as liquid chromatography and capillary electrophoresis that use these solvent mixtures. In this study, the pK_a values of 11 polyphenolic acids have been determined in methanol-water binary mixtures (10%, 20% and 30% (v/v)) by potentiometry, liquid chromatography (LC) and LC-DAD methodology.The results show a similar trend for the pK_a values of all the studied compounds, as they increase with increasing concentration of organic modifier, which allows a linear relationship between pK_a values and mole fraction of methanol to be obtained. The pK_a values obtained in aqueous medium have been compared with those given in the literature, and also with the values predicted by the SPARC on-line pK_a calculator. The data obtained have been used to test the feasibility of an estimation of dissociation constants in a methanol-water medium from the relationship between pK_a values and the organic cosolvent fraction in the mixtures.     

RNA integrity as a quality indicator during the first steps of RNP purifications : A comparison of yeast lysis methods

Background  

The completion of several genome-sequencing projects has increased our need to assign functions to newly identified genes. The presence of a specific protein domain has been used as the determinant for suggesting a function for these new genes. In the case of proteins that are predicted to interact with mRNA, most RNAs bound by these proteins are still unknown. In yeast, several protocols for the identification of protein-protein interactions in high-throughput analyses have been developed during the last years leading to an increased understanding of cellular proteomics. If any of these protocols or similar approaches shall be used for the identification of mRNA-protein complexes, the integrity of mRNA is a critical factor.     

Distribution of fallout radionuclides through soil surface layer

The distribution of¹³⁷Cs through soil layers has been investigated by field sampling. The investigation deals with¹³⁷Cs concentration found after the Chernobyl accident. The relationship between important soil characteristics, total precipitation and cesium distribution are analyzed. Cesium is strongly sorbed in the top soil layer and its migration downward is limited. Measurements performed over a 3-year period revealed that approximately 20–38 per cent of total¹³⁷Cs is located in the upper soil layer.