A highly sensitive method for quantitative determination of abscisic Acid

An abscisic acid derivative was formed by reaction with pentafluorobenzyl bromide which allowed highly sensitive detection by gas-liquid chromatography with electron capture detection. In comparison to the methyl ester derivative, the pentafluorobenzyl derivative of abscisic acid was four times more sensitive to electron capture detection and was stable at room temperature in the presence of ultraviolet light. Derivatization was rapid and the molecular weight of the new compound was confirmed by gas-liquid chromatography-mass spectrometry.     

Spore dipicolinic acid contents used for estimating the number of endospores in sediments

Endospores are heat-resistant bacterial resting stages that can remain viable for long periods of time and may thus accumulate in sediments as a function of sediment age. The number of spores in sediments has only rarely been quantified, because of methodological problems, and consequently little is known about the quantitative contribution of endospores to the total number of prokaryotic cells. We here report on a protocol to determine the number of endospores in sediments and cultures. The method is based on the fluorimetric determination of dipicolinic acid (DPA), a spore core-specific compound, after reaction with terbium chloride. The concentration of DPA in natural samples is converted into endospore numbers using endospore-forming pure cultures as standards. Quenching of the fluorescence by sediment constituents and background fluorescence due to humic substances hampered direct determination of DPA in sediments. To overcome those interferences, DPA was extracted using ethyl acetate prior to fluorimetric measurements of DPA concentrations. The first results indicated that endospore numbers obtained with this method are orders of magnitude higher than numbers obtained by cultivation after pasteurization. In one of the explored sediment cores, endospores accounted for 3% of all stainable prokaryotic cells.     

A rapid and sensitive method for HPLC cholesterol determination in bile.

A relatively little time consuming simple method based on the treatment of bile with cholesterol oxidase and subsequent high performance liquid chromatography measurement of the 3-ketocholesterol produced in order to determine the level of the cholesterol concentration is described. The method avoids bilirubin interferences, has high reproducibility and recovery assays give 100% values. It is highly sensitive and suitable for use in the determination of cholesterol concentrations in bile and other bilirubin containing biological fluids.     

A simple and highly sensitive method for sequence determination of 32P-labeled oligonucleotides

A new procedure is described for the sequence determination of oligonucleotides produced by digestion of RNA with pancreatic RNase A. The oligonucleotide is treated with spleen exonuclease and all intermediates are resolved by thin-layer chromatography on polyethyleneimine plates. On the basis of the increase in mobility it can be decided for each successive step whether a Gp- or an Ap-residue has been removed by reference to a calibration grid. The method is very simple and can easily be applied to a large number of samples. An amount of ³²P-radioactivity corresponding to 40 dpm/nucleotide is sufficient for analysis.     

A simple and sensitive HPLC method for determination of gliclazide in human serum

A simple, rapid and specific method for analysis of gliclazide in serum by a sensitive high-performance liquid chromatographic method is described. Only 100 microl of serum and a little sample work-up is required. A simple procedure of extraction by toluene followed by evaporation to dryness under a gentle stream of air and dissolving the dried residue in mobile was used. The gliclazide peak was separated from endogenous peaks on a C(8) column by a mobile phase of acetonitrile-water (45:55, v/v), pH 3. Gliclazide and internal standard (phenytoin) were eluted at 6.8 and 3.8 min, respectively. The limit of quantitation (LOQ) for gliclazide in serum was 75 ng/ml at 230 nm. The method was linear over the range of 75-10,000 ng/ml with r(2) of 0.999. Mean recovery for gliclazide and internal standard was 84.5 and 87%, respectively.     

A cell-based method for the detection of nanomolar concentrations of bioactive amyloid

A cell-based method for the detection of nanomolar concentrations of bioactive amyloid peptide is described. The method is based upon the observation that fibrillogenic amyloid peptides specifically and dramatically enhance the exocytosis of the intracellular vesicles that are involved in transporting the reduced tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT formazan), with the formation of unique formazan crystals on the cell surface. It is found that the ability of amyloid peptides to induce MTT formazan exocytosis is closely associated with both their neurotoxicity and their ability to activate glia cells, two biological activities of amyloid peptides that are believed to cause neurodegeneration. This simple assay for bioactive amyloid species can be of great value in the screening of anti-amyloid drugs and in the study of amyloid fibrillogenesis with a cell-based model.     

A simple and sensitive HPLC fluorescence method for determination of tadalafil in mouse plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method utilizing fluorescence detection was developed for the determination of the phosphodiesterase type 5 inhibitor tadalafil in mouse plasma. This method utilizes a simple sample preparation (protein precipitation) with high recovery of tadalafil (∼98%), which eliminates the need for an internal standard. For constituent separation, the method utilized a monolithic C₁₈ column and a flow rate of 1.0 mL/min with a mobile phase gradient consisting of aqueous trifluoroacetic acid (0.1% TFA in deionized water pH 2.2, v/v) and acetonitrile. The method calibration was linear for tadalafil in mouse plasma from 100 to 2000 ng/mL (r > 0.999) with a detection limit of approximately 40 ng/mL. Component fluorescence detection was achieved using an excitation wavelength of 275 nm with monitoring of the emission wavelength at 335 nm. The intra-day and inter-day precision (relative standard deviation, RSD) values for tadalafil in mouse plasma were less than 14%, and the accuracy (percent error) was within −14% of the nominal concentration. The method was utilized on mouse plasma samples from research evaluating the potential cardioprotective effects of tadalafil on mouse heart tissue exposed to doxorubicin, a chemotherapeutic drug with reported cardiotoxic effects.     

A sensitive liquid chromatographic method for the spectrophotometric determination of urinary trans,trans-muconic acid

Benzene is a human carcinogen and its metabolite, urinary trans,trans-muconic acid (ttMA), is a biomarker for risk assessment. However, most of the existing methods were not sensitive enough for monitoring of low level exposure. This paper describes a HPLC-UV method for ttMA determination with enhanced selectivity and sensitivity. A 30 mg OasisMAX cartridge was used to clean-up 50 microl of urine sample and gradient elution was performed on a Zorbax SB-C(18) column (30 degrees C). ttMA was detected at wavelength 263 nm using a UV diode array detector (DAD). The two mobile phases used were (A) 150 mM ortho-phosphoric acid containing of 9% (v/v) methanol; and (B) 125 mM ortho-phosphoric acid containing 30% (v/v) acetonitrile. The method was validated with 61 urine samples collected from non-occupationally benzene exposed individuals and 14 quality control specimens from an international quality assessment scheme. The urinary ttMA concentrations (mean+/-S.D.microg/g creatinine) were 90+/-34 for smokers (n=26), 49+/-39 for non-smokers (n=21) and 23+/-18 for non-smoking hospital staff (n=14). A correlation coefficient, r=0.99 was found with 14 external quality specimens for ttMA ranged from 0.4 to 6.8 mg/l. The recovery and reproducibility were generally over 90% and the detection limit was 5 microg/l.     

A sensitive and specific HPLC method for the determination of total pentosidine concentration in plasma

Pentosidine is an advanced glycation end-product (AGE) appearing when arginine and lysine residues in proteins are cross-linked with carbonyl derivatives. This paper presents an improved method for the synthesis of pentosidine and reversed-phase chromatography of this substance with fluorometric detection that enables sensitive (0.01 pmol/mg protein) and specific determination of pentosidine in plasma. Separation is done twice on the same C(18) Vydac 218TP54 column, first with trifluoroacetic acid and next with heptafluorobutyric acid as ion pair. The inter-day coefficient of variation is 6.4% at pentosidine concentration in plasma of 25 pmol/mg protein and 8% at 1.7 pmol/mg protein. Spectral properties of pentosidine exploited during identification of the substance with UV absorption and fluorescence detectors are described. Maximum of absorbance was observed at 325 nm, maximum fluorescence at lambda(ex)/lambda(em)=330/373 nm. The method may prove useful for the study of processes associated with generation and accumulation of pentosidine in the body as a marker of AGE production in healthy subjects and patients with chronic renal failure.     

A sensitive spectrophotometric method for determination of Tris

The paper describes a sensitive spectrophotometric method for qualitative and quantitative determination of Tris, in the 0.1–1.0 μmole range. The procedure is almost identical with that used for histidine. The green-colored reaction product has two absorption peaks with maxima at 422 nm and 640 nm, in contrast to the single peak (540 nm) in the case of histidine. Absorbance and Tris concentration are mutually linear at both wavelengths, but reading conditions are more favorable at 640 nm.     

A specific, sensitive method for the determination of hyaluronate.

A sensitive and specific assay for hyaluronate was devised. Hyaluronate contained in biological mixtures was digested with a commercially available microbial hyaluronate lyase. The β-Δ4,5-eneglucopyranuronic acid residues contained at the nonreducing termini of the resulting oligosaccharides were oxidized with periodic acid to yield, among other products, formyl pyruvic acid. The latter compound reacted with thiobarbituric acid to yield a chromophore with an absorption maximum at 549 nm. Optimal conditions for quantitative assay of hyaluronate are described.     

A highly specific and sensitive determination of gamma-aminobutyric acid by gas chromatography mass spectrometry

A procedure for the identification and quantification of picomole quantities of gamma-aminobutyric acid in tissue samples is given. This procedure combines the chemical specificity of dinitrophenylation with that of gas chromatography mass spectrometry to eliminate the interferences encountered with other direct derivatization procedures. Only a limited number of dinitrophenyl amino acid ethyl esters and some fatty ethyl esters are detected in the solution used for analysis. Identification is based on retention time and on the relative abundances of the three major ion fragments of the gamma-aminobutyric acid derivative. Quantitation is accomplished using isotope dilution techniques with [2H2]gamma-aminobutyric acid as an internal standard. The procedure has been successfully applied to samples of human cerebrospinal fluid and to extracts of ganglia from the mollusc, Aplysia californica.