J. De Graeve

Separation, identification and quantitation of ceramides in human cancer cells by liquid chromatography–electrospray ionization tandem mass spectrometry

Ceramides are important intracellular second messengers that play a role in the regulation of cell growth, differentiation and programmed cell death. Qualitative and quantitative analysis of these second messengers requires sensitive and specific analytical methods to detect endogenous levels of individual ceramide species and to differentiate between them. Nine synthetic ceramides were separated by liquid chromatography coupled to tandem mass spectrometry on a C18 bonded silica column. The lipids were eluted in gradient elution mode using a mixture of water, acetonitrile and 2-propanol as mobile phase. They were detected by reaction monitoring performed on positive ion electrospray generated ions. Collision-induced fragmentations conducted on ceramides produced a well characteristic product ion at m/z 264, making multiple reaction monitoring (MRM) well suited for various ceramides quantitative measurements. After optimization of the extraction step, the proposed methodology was able to identify and quantify different ceramide species issued from human cancer cells. The method could be validated for C16, C18 and C20 ceramides, quantified at the nanogram level. The validation exhibits good results with respect to linearity, accuracy and precision.

Metabolism pathway of vinorelbine (Navelbine®) in human: Characterisation of the metabolites by HPLC–MS/MS

The biotransformation of vinorelbine (VRL), an anti-neoplastic vinca-alkaloid derivate already marketed for nonsmall cell lung cancer and advanced breast cancer as an i.v. form and currently registered in several countries as an oral form, was investigated in human. Biological specimen from several human sources constituted the material for the metabolic identification in human. An isocratic liquid chromatographic system composed of 40 mM ammonium acetate (pH 3) and acetonitrile was used for separation of the potential metabolites of VRL. Tandem mass spectrometry with positive electrospray ionisation was used to enable the structural identification of the metabolites. A total of 17 metabolites (12 directly obtained from VRL and 5 involving sequential step pathways) were characterised with proposed structures for most of the metabolites. All metabolites went through phase I reactions by the way of deacetylation, dealkylation, oxidation and hydroxylation. No conjugates were observed. Despite the high number of metabolites quantified, VRL was the major compound observed whatever the matrix. Most of the metabolites rapidly disappeared from blood, except 4-O-deacetyl vinorelbine which was slowly cleared. Most of the enzymatic pathways involved in the metabolites strongly suggested the major role of cytochrome P450 in the biotransformation of vinorelbine.

15- and 16-hydroxylations of androgens and estrogens in the human fetal liver: A critical step in estetrol biosynthesis

To elucidate the main metabolic pathways which lead to the foeto-placental biosynthesis of estetrol (I), we investigated the 15 alpha- and 16 alpha-hydroxylations of potential precursors of this estrogen in the human fetal liver. We determined the 15 alpha- and 16 alpha-hydroxylation capacity of the fetal liver for each precursor by GC-MS. The results suggest that estetrol is derived only from estradiol sulfate (II) and DHEA sulfate (III). 15 alpha-Hydroxy-androstenedione (IV) can no longer be regarded as a good precursor of estetrol. The phenolic pathway appears to be a more likely route than the neutral pathway, even when derived from DHEA sulfate.

Determination par spectrometrie infrarouge de la matiere organique non volatile associee aux particules en suspension dans l'air—II. Facteurs influencant l'indice aliphatique

Abstract The infrared estimation of organic aliphatic material collected on glass fiber filters at commonly used air flow rates shows clearly that the composition of this material, adsorbed on suspended matter, varies greatly with the flow rate and duration of sampling: Most compounds with boiling temperatures lower than 300°C are progressively eliminated from the sample. If sampling lasts 24 h and more, the composition of the sample is thus stabilized and standardization of the spectrophotometer response against a weighed amount of extract is quite satisfactory, the nature of the remaining heavy aliphatic substances showing little variability for different places and periods of the year.

Release of endotoxic lipopolysaccharide by sensitive strains ofEscherichia coli submitted to the bactericidal action of human serum

Free endotoxin was assayed in filtered samples ofE. coli suspensions submitted to the bactericidal and bacteriolytic action of 10% human serum. The Limulus amoebocyte Iysate test, a passive hemolysis inhibition assay based on O antigenic specificity and the determination of 3-OH-myristic acid by mass spectrometry were used as assay methods differing from one another with regard to the part of the endotoxin macromolecule involved in the reaction. The biological activity of endotoxin was assessed in a mouse lethality test.The bactericidal and bacteriolytic action of human serum on sensitive strains ofE. coli released quantities of endotoxic lipopolysaccharide (LPS) amounting to 3,000–12,000 ng/ml, for an inoculum of 1–3×108 colony-forming units. The material thus appearing in the medium was shown to react with the Limulus amoebocyte Iysate, to be lethal for actinomycin D-sensitized mice and to bear O antigen, as well as 3-OH-myristic acid, a marker of lipid A.Samples of serum depleted of lysozyme by adsorption onto bentonite, and displaying a strictly bactericidal effect, released approximately 80% of the quantity of LPS appearing in the medium in a control experiment performed with untreated serum. The LPS release is therefore mainly linked to the bactericidal effect of antibody and complement.The amount of LPS released depended on the concentration of divalent cations in the medium, being reduced by an increase in the concentration of calcium and magnesium beyond the values optimal for complement activity. This effect was already observed for an increase in the concentration of divalent cations too low to alter the bactericidal or bacteriolytic effects.The significance of the release of endotoxin by complement dependent bactericidal reactions occurring in vivo is discussed.

Aromatization of 15α and 16α hydroxylated androgens in the human placenta using [1,2-3H]-substrates

Abstract This in vitro study reports data on the aromatization of [1,2 -3 H]-C 19 steroids in the human term placenta [androstenedione (III), testosterone (IV), 15α-hydroxy-androstenedione (V), 15α-hydroxy-testosterone (VI), 16α-hydroxy-androstenedione (VII)]. The hydroxylated androgens were microbiologically synthesized from commercially radiolabelled [1,2- 3 H]-androstenedione and testosterone. Androstenedione and testosterone were good substrates for the human placental aromatase (low K m values, high V max ; they strongly inhibited the 15 and 16 hydroxylated androgens aromatizations. On the other hand, these hydroxylated compounds acted as poor substrates and were only non-competitive inhibitors of the androstenedione and testosterone aromatizations. However, 15α-hydroxy-androstenedione could not be disregarded as a potential precursor of 15α-hydroxylated estrogens in the human placenta.

Simultaneous determination of methylated barbiturates and other anticonvulsant drugs by high-resolution gas chromatography

Several barbiturates and other anticonvulsant drugs can be analyzed simultaneously as dimethylated derivatives by high-resolution gas chromatography with a solid flash methylation injector. Quantitative determinations were performed on test serum during a quality control scheme. The sensitivity (+/- 0.1 mug/ml) and the accuracy are compatible with the measurement of therapeutic levels of barbiturates. The advantages of the method are discussed.

Determination of L-lysine N-acetylcysteinate and its mono- and dimeric related compounds by liquid chromatography-mass spectrometry

Abstract Methods for the simultaneous determination of N-acetylcysteine, l -lysine, l -cysteine, l -cystine, N,N′-diacetylcystine and N,S-diacetylcysteine in aqueous solutions were developed. The method is based on the on-line coupling of liquid chromatography (LC) to tandem mass spectrometry (MS–MS) with an atmospheric pressure chemical ionisation (APCI) interface. The optimal LC phase system consisted of a C18 stationary phase and an acidic solution of formic acid in acetonitrile as the mobile phase, while d , l -phenylalanine was selected as an internal reference compound. The best transitions for MS–MS detection were selected after optimisation of the APCI ionisation yield in full scan. The optimisation was performed by loop injection. By coupling LC to MS–MS in the APCI+ (positive ion mode) as well as in the multiple reaction monitoring mode, satisfactory results were obtained with a mixture of the seven compounds, each at a concentration level of 1 μg/ml. For a further improvement of the detection limit of the dimeric compounds, electrospray ionisation was then investigated. By use of this interface a quantitation limit down to 10−8 M (3 ng/ml) could be achieved for both dimers. The developed method provides improved selectivity based on the combination of chromatography and mass spectrometry thus facilitating identification.

Preparation and thin-layer chromatography of bromo-derivatives of unsaturated fatty acid esters. A simple and rapid procedure for fatty acid analysis.

Bromo-addition products of unsaturated long-chain fatty acid esters have been prepared and chromatographed on thin layers of unmodified silica gel. The polarity of these derivatives was found to be directly related to the number of double bonds of the parent fatty acid from which they were derived. This has been made the basis of a simple method for assessing the relative proportions of the main fatty acid classes in a mixture.

Determination par spectrometrie infrarouge de la matiere organique non volatile associee aux particules en suspension dans l'air—I. Choix des conditions operatoires

Abstract The paper proposes an i.r. spectrometric method for the determination of the non volatile organic matter associated with the airborne particulates. It describes the operating conditions of the system; data concerning sensitivity and reproducibility are given. Several parameters which must be taken into account for the establishment of the calibration curve are listed.