Reversed-phase high-performance liquid chromatographic method for the determination of peptidoglycan monomers and structurally related peptides and adamantyltripeptides

The reversed-phase HPLC method using UV detection was developed for the determination of (a) immunostimulating peptidoglycan monomers represented by the basic structure GlcNAc-MurNAc-L-Ala-D-isoGln-meso-DAP(omegaNH(2))-D-Ala-D-Ala (PGM) and two more lipophilic derivatives, Boc-Tyr-PGM and (Ada-1-yl)-CH(2)-CO-PGM, (b) two diastereomeric immunostimulating adamantyltripeptides L- and D-(adamant-2-yl)-Gly-L-Ala-D-isoGln and (c) peptides obtained by the enzyme hydrolyses of peptidoglycans and related peptides. The enzymes used, N-acetylmuramyl-L-alanine amidase and an L,D-aminopeptidase are present in mammalian sera and are involved in the metabolism of peptidoglycans and related peptides. Appropriate solvent systems were chosen with regard to structure and lipophilicity of each compound. As well, different gradient systems within the same solvent system had to be applied in order to achieve satisfactory separation and retention time. HPLC separation was developed with the aim to use this method for the study of the stability of the tested compounds, the purity during preparation and isolation and for following the enzyme hydrolyses.     

Reversed-phase liquid chromatographic method for the determination of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labelled lipid analogues

This paper reports the development of a dual column system for the simultaneous separation of fluorescent short-chain ceramide, 6-[(7-nitrobenz-2-oxa-1,3,-diazol-4-yl[NBD])amino]hexanoyl-sphingosine and its metabolites, C6-NBD-sphingomyelin and C6-NBD-glucosylceramide, as well as the fluorescent derivatives of choline and serine phosphatides. The method enables the separation of these lipids in a single run on the basis of the polarity of their headgroups and hydrophobicity of their acyl backbone. The fluorescent properties of the NBD-label make it possible to quantitate small amounts of NBD-lipid analogues. The sensitivity of the presented method thus permits the use of small sample volumes and the determination of NBD-lipid analogues secreted into mouse bile directly, without prior extraction or concentration steps.     

Rapid and specific high-performance liquid chromatographic method for the determination of iodide in urine

A rapid and specific method for the determination of iodide in urine by high-performance liquid chromatography on an anion-exchange column with electrochemical detection is described. The assay is reproducible as judged by the coefficient of variation of less than 4% at all concentrations used. The limit of detection was 0.1 μ mol, and the calibration graph was linear for concentrations between 0.1 and 200 μmol. Using this method, healthy volunteers were found to excrete 69±39 μmol of iodide per mole of creatinine.     

Reversed-phase liquid chromatographic determination of plasma levels of adriamycin and adriamycinol

A method is given for the determination of adriamycin and its main metabolite, adriamycinol in plasma from cancer patients after administration of adriamycin as the free drug or as a complex with DNA.Adriamycin and adriamycinol are extracted in a column from 1 ml of plasma (pH 8.6) using a mixture of chloroform—1-heptanol (8:2). After re-extraction into phosphate buffer pH 2.2, the separation is performed as reversed-phase liquid chromatography on a LiChrosorb RP-2 (5 μm) column with a mobile phase of acetonitrile—water, acidified with phosphoric acid.The precision by quantitation with photometric detection was better than 5% within the range 50–300 ng/ml. Plasma levels of adriamycin and adriamycinol in a cancer patient are presented in this paper.     

Reversed-phase high-performance liquid chromatographic method for the determination of the 11-hydroxythromboxane B2 anomers equilibrium

An improved reversed-phase HPLC method for the separation and detection of both hemiacetalic 11-hydroxy anomers of thromboxane B₂ (TXB₂) is described. Water-acetonitrile mixtures have served as mobile phases. By diminishing stepwise the temperature of the chromatographic system from 40 to 0°C, the UV-absorbance profile of TXB₂ changed from one broad peak to two clearly separated narrow peaks corresponding to the two anomers existing in equilibrium. Modification of the mobile phase pH from 1.6 to 6.9 (0°C) resulted in different concentration ratios of the anomers. The equilibrium constant and the Gibbs free energy were calculated. The intermediate open aldehyde form of TXB₂ is unstable and, therefore, cannot be observed either by HPLC or by ¹H NMR measurements.     

Evidence for glutathione peroxidase activities in cultured plant cells

Extracts from cultured plant cells of spinach, maize and sycamore and from Lemna plants contain detectable glutathione peroxidase activity, using either hydrogen peroxide or t-butyl hydroperoxide as substrates. Using extracts from cultured maize cells, two peaks of glutathione peroxidase activity could be resolved by a combination of gel filtration and ion exchange chromatography. One peak was eluted along with glutathione transferase activity; the second was distinct from both glutathione transferase and ascorbic acid peroxidase, and was active with both hydrogen peroxide and organic hydroperoxides. It seems likely that at least two enzymes with glutathione peroxidase activity exist in higher plant cells.     

Reversed-phase liquid chromatographic determination of idarubicin and its 13-hydroxy metabolite in human plasma

A method is given for the determination of idarubicin and its main metabolite, idarubicinol, in plasma from cancer patients. Idarubicin and idarubicinol are extracted from 2-ml samples of buffered plasma (pH 8.1) using chloroform-1-heptanol (9:1). After reextraction into phosphoric acid (0.1 M), separation is performed by reversed-phase liquid chromatography on a LiChrosorb RP-2 column (5 μm) with a mobile phase of acetonitrile-water, acidified with phosphoric acid. The absolute recovery in the range 5–100 ng/ml was greater than 83% with a precision better than 8% (relative standard deviation), using photometric detection at 484 nm. Proper handling of whole blood samples containing idarubidin is essential to avoid metabolic conversion into idarubicinol. Prolonged storage of the drug and its main metabolite under alkaline conditions should be avoided to prevent chemical degradation.     

Geraniol-inducible glutathione S-transferase in cultured soybean cells

When the cultured cells of Glycine max (soybean) were treated with 5 mM geraniol as a chemical stress, an mRNA level was elevated in a rapid but transient increase. The mRNA was cloned and sequenced, and found to correspond to the mRNA encoding glutathione S-transferase (GST). The GST mRNA level and GST activity were elevated to maxima at 4-6 h and 8 h, respectively, after treatment of the cultures with geraniol. These indicate that GST is one of the geraniol-responsive factors in soybean cells.     

A method for phospholipid-mediated delivery of specific antibodies into adherent cultured cells.

A simple and rapid procedure for the intracellular delivery of macromolecules into adherent cultured cells is described. Cells are incubated with cold glycerol, then transiently made permeable with L-alpha-lysophosphatidylcholine (LPC) in the presence of test compound to be loaded into cells. LPC induces temporary permeability of the plasma membrane, as evidenced by the loss and recovery of the cells' ability to exclude trypan blue. Molecules at least as large as antibodies are internalized during this transient permeability. Antibodies delivered intracellularly in this manner are able to complex with their specific antigen and exert functional consequences on normal cell metabolism, suggesting that this procedure is useful for determining protein function. As one example of this, we present data on the ability of specific antibodies, delivered intracellularly in this manner, to inhibit morphological differentiation (i.e., neurite outgrowth) in a neuroblastoma cell line.     

Reliable and specific high-performance liquid chromatographic method for simultaneous determination of loratadine and its metabolite in human plasma

A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the simultaneous determination of loratadine (L) and its metabolite, descarboethoxyloratadine (DCL), in human plasma. Following a two-step liquid-liquid extraction with toluene, the analytes were separated using a gradient mobile phase consisting of methanol-acetonitrile-phosphate buffer. The linearity for L and DCL was within the concentration range of 0.5-16 ng/ml. The coefficient of variation of intra- and inter-day assay was <8.3%, with accuracy ranging from 98.3 to 105.7%. The lower limit of quantification was 0.5 ng/ml for both L and DCL. This method has been demonstrated to be reliable, and is an improvement over existing methods due to its capability for determining L and DCL simultaneously in a single chromatographic run.     

Reversed-phase liquid chromatographic method with fluorescence detection for the simultaneous determination of albendazole sulphoxide, albendazole sulphone and albendazole 2-aminosulphone in sheep plasma

A rapid and sensitive HPLC method for the simultaneous quantification of albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO2) and albendazole 2-aminosulphone (ABZ-SO2NH2) in sheep blood plasma has been developed. Plasma samples were extracted with ethyl acetate under alkaline conditions. Separation was achieved on a C18 reversed-phase analytical column, in the presence of positively- (tetra-n-butylammonium hydrogen sulphate) and negatively-charged (octanesulphonate sodium) pairing ions, while detection was performed fluorometrically. Excitation and emission wavelengths were 290 and 320 nm, respectively. Limits of quantification were defined at 39 ng/ml for ABZ-SO, 4.95 ng/ml for ABZ-SO2 and 4 ng/ml for ABZ-SO2NH2. Accuracy data, in terms of recovery efficiency showed overall values (+/- S.E.M.) of 85.6 +/- 1.0% for ABZ-SO, 100.0 +/- 1.0% for ABZ-SO2 and 89.1 +/- 0.6% for ABZ-SO2NH2. The method was successfully applied to quantitatively determine the three albendazole metabolites in plasma samples collected from sheep that had been orally administered albendazole.     

Reversed-phase high-performance liquid chromatographic screening method for serum steroids using retention index and diode-array detection

A multisteroid screening method has been developed based on the use of 1-[4-(2,3-dihydroxypropoxy)phenyl]-1-alkanones as retention index standards and UV absorbance spectra recorded on-line with a diode-array detector using reversed-phase high-performance liquid chromatographic gradient elution with acetonitrile and water. The effect of chromatographic conditions on retention indices of steroids were studied. The method was tentatively applied to profiling of steroids in serum samples.     

Enzymic method for the quantitative determination of reduced glutathione

A new specific and sensitive assay method for reduced glutathione has been developed. It is based on the reaction HCHO + NAD⁺ + H₂O → HCOOH + NADH + H⁺, catalyzed by formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC.1.2.1.1) in the presence of reduced glutathione. Oxidized glutathione and other thiols do not interfere in this reaction. A purification procedure for formaldehyde dehydrogenase from beef liver is presented.The influence of cysteine and some other thiols, leucine, ascorbate, lactate, pyruvate and four acids generally used for deproteinization is reported. The results obtained by this method from human blood and rat tissues are compared with those obtained by Ellman's method.