Dansylation Procedure Research Articles

Liquid chromatography-tandem mass spectrometric analysis of ten estrogen metabolites at sub-picogram levels in breast cancer women.

The measurement of estrogens at sub-picogram levels is essential for research on breast cancer and postmenopausal plasma. Heretofore, these concentration levels have rarely been achieved. However, it is possible through derivatization but still represent problems for monitoring catechol estrogens and 16α-hydroxyestrone (16α-OH-E1). Estrogens possess poor ionization efficiency in MS/MS, which results in insufficient sensitivity for analyzing samples at trace concentrations. The method presented here was used to extract ten estrogen metabolites (EMs) with a derivatization step involving a new adduct. The electrospray ionization (ESI) MS/MS sensitivity for the EMs was enhanced by derivatization with 3-bromomethyl-propyphenazone (BMP). The lower limits of quantification (LLOQ) of the EMs were 12-100 femtogram on-column, equivalent to 0.3-3.6pg/mL plasma, and the limits of detection (LOD) were 0.1-0.8pg/mL plasma. The percentage coefficient of variation (CV%) at the LLOQ was <20 for all investigated EMs. Ionization suppression was minimized by reacting the excess reagent, BMP, with methanol. The method was successfully applied for the determination of ten EMs in the plasma of fifty healthy postmenopausal and fifty ductal breast cancer women aged 47-65 years old. 16α-OH-E1 and three catechol estrogen metabolites, 4-OH-E1, 2-OH-E2 and 4-OH-E2, were successfully measured in the plasma of healthy and breast cancer women. The methyl-propyphenazone-EM derivatives exhibited better sensitivity in ESI-MS (7.5-fold) compared to the commonly used dansylation procedure.

Study on Derivatising Condition of Cadaverine with Dansyl Chloride as Pre-Column Derivatising Agent

A rapid ultrasonic wave-assisted dansylation procedure was developed for the derivatization of cadaverine prior to HPLC determination. Cadaverine was quantitatively dansylated in 10 min using a ultrasonic power of 200 W and a operating frequency of 40 KHz .In this case, the excellent derivatising condition are determined as follows: pH of buffer, 9.5; concentration of dansyl chloride, 5 mg/mL; time of reaction, 10 min. This method is fast, reproducible and practical for the analysis of cadaverine.

Fast microwave-assisted dansylation of N-nitrosamines : Analysis by high-performance liquid chromatography with fluorescence detection

A fast microwave-assisted dansylation procedure has been developed for the derivatization of N-nitrosamines prior to high-performance liquid chromatography determination. N-Nitrosomorpholine, N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosopyrrolidine and N-nitrosopiperidine are first denitrosated by hydrobromic acid–acetic acid to produce secondary amines, which are then quantitatively dansylated in 5 min using radiation power of 378 W and a maximum pressure of 1.4 bar inside the reactor. The reaction mixture is separated on a C 18 column with acetonitrile–water (55:45, v/v) as mobile phase with fluorimetric detection at 531 nm (excitation at 339 nm). The detection limits range from 8 to 75 pg for N-nitrosomorpholine and N-nitrosodiethylamine, respectively. The method was applied to study the recoveries of N-nitrosamines in beer and their determination in cigarette smoke.

Improvement of the analysis of dansylated derivatives of polyamines and their conjugates by high-performance liquid chromatography

The paper described a method for improving the hydrolysis of conjugated polyamines in PH fraction, isolated from the lichen Evernia prunastri, as well as the optimization of dansylation procedure of these polyamines on the basis of the pH value to which derivatization is achieved. Dansylated polyamines have been later separated by high-performance liquid chromatography (HPLC) using a gradient elution. Hydrolysis of conjugates requires acid treatment at room temperature rather than at 110°C, as usually described. Dansylation is improved at high pH values, whereas removal of phenolics (mainly evernic acid), from the conjugates requires low pH values.

High-performance liquid chromatographic determination and standardization of agmatine

In order to standardize the reversed-phase high-performance liquid chromatographic (RP-HPLC) determination of natural polyamines, particularly of agmatine, which is important in plant physiology, a simple dansylation and thin-layer chromatography (TLC) procedure was developed for the separation and purification of dansyl-agmatine. The monodansyl structure of the agmatine derivative was verified by mass spectrometry. Dansyl-agmatine was used as a reliable reference compound for the validation of an improved RP-HPLC method and to the quantitation of plant polyamines. In two varieties of wheat that had been stressed by cold treatment, significant changes were demonstrated in the agmatine content of plant seedlings.

Rapid microwave-assisted dansylation of biogenic amines: Analysis by high-performance liquid chromatography

A fast microwave-assisted dansylation procedure has been developed for the derivatization of biogenic amines prior to HPLC determination. Putrescine, cadaverine, spermidine and spermine are quantitatively dansylated in 5 min using a radiation power of 252 W and a maximum pressure of 3.4 bar inside the reactor. As compared to classical overnight dansylation, the microwave-assisted procedure is 150-times faster. Data concerning repeatability and recovery of method are reported. The method was successfully applied to the determination of the above amines in plant tissues.

Quantification of polyamine losses during manipulation and assay procedures

AbstractLosses in polyamine recovery during manipulation and assay procedures have been detected. They are partly due to the binding of polyamines at low concentration (around 5 μM for putrescine and 100 μM for spermine) to the containers and depend on the chemical nature of the latter, on the pH of the solution and on the length of time for which they remain in contact with the container. The recovery of labelled polyamines after dansylation was about 45% of the initial radioactivity. Remarkably high losses occurred when bound polyamines, obtained by in vitro incubation with cell‐free extracts, were estimated after acidic hydrolysis. Standard free polyamines, subjected to acidic hydrolysis conditions, yielded a 50–60% recovery, both measured as radioactivity and fluorescence, compared to standard polyamines not subjected to hydrolysis. If carbohydrates are present, the recovery after hydrolysis can be even lower, depending on the sequence in which the reagents of the dansylation procedure are added. Ion exchange chromatography to remove carbohydrates improved this recovery.

Purification and structural comparisons of the cytosolic and mitochondrial isoenzymes of fumarase from pig liver

A method has been developed for the purification of cytosolic and mitochondrial isoenzymes of fumarase from total homogenates of pig liver. Separation of the isoenzymes from one another was achieved using chromatofocusing. The isoenzymes were pure as judged by production of single bands on electrophoresis in the presence of sodium dodecyl sulphate; they appeared to have identical or very similar subunit molecular weights. The isoenzymes differed in electrophoretic properties under nondenaturing conditions. One-dimensional peptide maps of fragments produced from the two isoenzymes by chemical cleavage at cysteine residues were identical; maps obtained after digestion with the V 8 proteinase from Staphylococcus aureus showed small differences at short times of digestion which could have reflected variations in rates of hydrolysis rather than structural differences. However, two-dimensional peptide maps of digests obtained by treatment of the isoenzymes with trypsin followed by chymotrypsin had 58 peptides in common, but showed two peptides unique to the mitochondrial isoenzyme and five peptides unique to the cytosolic form. Using the dansylation procedure, the mitochondrial isoenzyme was shown to have N-terminal alanine and the cytosolic form to have N-terminal glutamic acid or glutamine. We conclude that the isoenyzmes of fumarase are identical over nearly all of their amino acid sequences but differ at their N-termini; the extent of these differences is yet to be established. These results are consistent with the claim (Edwards, Y.H. and Hopkinson, D.A. (1979) Ann. Human Genet. Lond. 42, 303–313) that the isoenzymes are determined at the same genetic locus, but they raise interesting questions about the biosynthesis of the isoenzymes.

Lack of detectable polyamines in an extremely halophilic bacterium

Polyamines (putrescine, spermidine, spermine and other analogs) were not detectable by the dansylation procedure coupled with HPLC analysis in an extremely halophilic bacterium, Halobacterium halobium . Based on the detection limit of this analytical method, we estimated that the polyamine content in H. halobium , if present, was less than 0.06% of that of E. coli . Putrescine uptake and the metabolic conversion of ornithine or arginine to polyamines were negligible in this bacterium. In a H. halobium cell-free extract, a saturated amount of KCl was needed for poly(U) directed polyphenylalanine synthesis; neither putrescine nor spermidine could replace KCl. These results suggest that polyamines may play an insignificant role in the growth of this halophilic bacterium.

Improved dansylation procedure for the determination of ketosteroids by liquid chromatography

A new procedure for derivatizing ketosteroids with dansyl hydrazine, a fluorogenic tagging agent, is reported. Termed “evaporative-derivatization”, this technique employs simultaneous heating and evaporation for the derivatization step. Presumably, as the evaporation approaches completion, the analyte is exposed to an infinite concentration of dansyl reagent. The impact of this work provides for a short reaction time (10min), low temperature (60°C), and almost complete derivatization. The short derivatization time enables labile steroids to be tragged with little decomposition. While the prior dansylation techniques tag only the 3-position, this new procedure also tags the sterically hindered 20-position forming a bis-dansyl derivative. The bis-derivative exhibits significantly greater chromatographic retention, which means that chromatographic interferences are less likely when complex matrices, such as extracts of blood plasma are analyzed.

Determination of pipecolic acid in rat brain areas by high-performance liquid chromatography of dansyl derivatives with fluorimetric detection

A method for the determination of pipecolic acid in the rat brain is reported. The identification and quantification of pipecolic acid is accomplished with reverse-phase high-performance liquid chromatography including precolumn dansylation procedure by using nipecotic acid, a regioisomer of pipecolic acid, as an internal standard. The lower limit of quantification for the method is in the picomole range. Higher concentration of pipecolic acid in the rat brain regions were found in cerebellum, medulla oblongata, hypothalamus, and midbrain than the other regions. The method establishes the usefulness of dansyl chloride for the simple and sensitive detection of pipecolic acid, and is easily adapted to routine analysis of pipecolic acid in the rat brain regions.

Determination of catecholamines in rat tissue by precolumn dansylation using micro high-performance liquid chromatography with fluorescence detection

A micro high-performance liquid chromatography (MHPLC)-precolumn dansylation procedure for determination of norepinephrine (NE), epinephrine (E), and dopamine (DO) in various kinds of rat tissue has been developed. After dansylation of the extracted catecholamines to get fluorescent derivatives, they were separated and determined using MHPLC equipment with a fluorimeter. The standard deviation of the method was ± 8% for NE, ± 4% for E, and ± 8% for DO. This relatively simple and rapid procedure for determination of catecholamines provides an efficient and reproducible method. Furthermore, the procedure is readily adaptable to routine pharmacological study of catecholamine metabolism in various kinds of rat tissue.

Amino acid sequence of the tryptic peptide containing the catalytic-site thiol group of bovine liver uridine diphosphate glucose dehydrogenase.

The catalytic-site thiol groups of UDP-glucose dehydrogenase from bovine liver were carboxymethylated with iodo[2-14C]acetate or with iodoacetamidofluorescein. After the residual thiol groups were carboxymethylated with iodoacetate, the proteins were digested with trypsin. The 14C-labelled peptide from the carboxymethylated enzyme was purified to homogeneity by successive thick-layer chromatography on silica gel, paper electrophoresis and chromatography, and column chromatography on Bio-Gel P-6. Homogeneous fluoresceincarboxamidomethylated peptide was prepared from a tryptic digest of fluoresceincarboxamidomethylated enzyme by specific adsorption--desorption from Sephadex G-25. The sequences of either peptide determined by the manual Edman dansyl procedure is: Ala-Ser-Val-Gly-Phe-Gly-Gly-Ser-Cys-Phe-Glx-Glx-Gly-Lys.

Dansylation of amino acids for high-performance liquid chromatography analysis

An amino acid dansylation procedure compatible with reversed-phase high-performance liquid chromatography is presented. The method provides high yield derivatization while the yield appears to be independent of the ratio of dansyl chloride to amino acid over a 1000-fold range. Studies of derivatization yields as a function of reaction time at ambient temperature and at elevated temperatures permit the proper choice of reaction conditions. A comparative study of the dansyl-liquid chromatography, gas chromatography, and amino acid analyzer methods of analysis for amino acids in complex bacteria broths demonstrates the advantageous use of the dansyl-liquid chromatography approach.

The estimation of lysine by a simplified dansylation procedure

AbstractA method is described for the estimation of free lysine which depends on its reaction with excess dimethylaminonaphthalene sulphonyl (dans) chloride to form dans‐5‐aminovaleraldehyde. This product is extracted into toluene, separated by thin layer chromatography and estimated by fluorimetry. The mean recovery of lysine added to casein hydrolysate using this method was 99% (s.e. 4.3%).

Purification and characterization of α1-proteinase inhibitor from microsomal fraction of normal human liver

A plasma α 1-proteinase inhibitor was purified, for the first time, from the microsomal fraction of normal human liver by combined procedures of deoxycholate extraction, (NH 4) 2SO 4 precipitation, gel filtration and immunoadsorbent column chromatographies. The final yield of the material was approx. 15 mg/300 g liver. The preparation was shown to be homogeneous according to polyacrylamide gel electrophoretic and immunological criteria. The estimated molecular weight of the glycoprotein as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was 53 000, a value comparable to that reported for plasma α 1-proteinase inhibitor. The liver material was, however, devoid of trypsin-, chymotrypsin- or elastase-inhibitory activity. The loss of activity could be due to deoxycholate treatment during the extraction procedure. SDS-polyacrylamide gel electrophoresis of reduced (β-mercaptoethanol) and alkylated (iodoacetamide) preparation revealed no change in mobility suggesting the presence of only polypeptide chain. The amino acid and carbohydrate compositions of the microsomal material were in close agreement with values reported in the literatures for the corresponding glycoprotein purified from the plasma. The liver microsomal α 1-proteinase inhibitor has a single amino terminal residue, namely, glutamic acid, as determined by the dansyl procedure, and a single carboxyl terminal residue, lysine as shown by the carboxypeptidase digestion method. These terminal amino acids are the same as those found in α 1-proteinase inhibitor purified from normal human plasma.

Electrically induced release of amino acids formed from (U14C) glucose in rat brain cortex slices, studied by a simplified dansylation procedure.

The nonessential amino acids glutamate, aspartate, glutamine, gamma-aminobutyrate (GABA), alanine, glycine, and proline present in rat thin brain cortex slices were labeled by in vitro incubation of these with [U-14C]glucose, and the efflux of such endogenous radioactive amino acids and of lactate was studied in a superfused system, under control conditions or when the slices were depolarized by varous procedures. When electrical stimuli known to induce selective neurotransmitter release (1 or 1.5 volt, sine wave 60 Hz) were applied for 10 sec to the slices, no significant increase in amino acid efflux was found. When more intense stimuli (4 volt, 60 Hz) were applied for 60 sec, or extracellular potassium was raised to 56 mM, both conditions being known to induce nonselective substance release, the efflux of essentially all amino acids and of lactate was markedly increased. Increases in efflux were proportionately larger for glutamate, aspartate, and gamma-aminobutyrate, and this could be accounted for by their greater intracellular chemical (or electrochemical) potentials, but not because of a selective release mechanism for them. Amino acids were analyzed as their 1-dimethylaminoaphthalene-5-sulfonyl (dansyl) derivatives, by a modification of existing procedures in which the dansyl (DNS) derivatives were efficiently extracted from acidified incubation fluid into an organic phase. This rapidly desalted the derivatives and allowed their concentration and chromatographic separation on thin-layer silica gel sheets with little loss.

Synthesis and content of polyamines in bloodstream Trypanosma brucei.

The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [3H]ornithine, [14C]arginine and [14C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [4C]methionine. Putrescine and sperimidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous 14C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.

The composition, structure and origin of proteose-peptone component 5 of bovine milk.

Proteose-peptone component 5 has been isolated from bovine milk. Molecular weight values within the range 12000--13500 were obtained by sedimentation equilibrium, dodecylsulphate/polyacrylamide gel electrophoresis and gel filtration in urea-containing buffers. A dansylation procedure showed that the sequence Arg-Glu occupied the N-terminal position while hydrazinolysis revealed C-terminal lysine. The latter was confirmed by experiments with carboxypeptidases B and C which indicated that a mixture of molecules was present, about 80% of which had a C-terminal sequence -(Ala-Met)-Ala-Pro-Lys while about 20% had an additional -His-Lys in the terminal position. These results, together with data on the overall composition, showed that this component of the proteose-peptone fraction of milk corresponded to a mixture of molecules representing residues 1--105 and 1--107 of the beta-casein molecule, a finding that was confirmed by peptide mapping. This demonstration that proteose-peptone components correspond to the N-terminal portions of the beta-casein molecule while the gamma-caseins represent the matching C-terminal portions provides strong evidence in favour of a proteolytic mechanism for the formation of these substances in vivo and in vitro.

Canine pulmonary angiotensin-converting enzyme. Physicochemical, catalytic and immunological properties

Antiontensin-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) has been solubilized from canine pulmonary particles and purified to apparent homogeneity. A value of approx. 140000 was estimated for the molecular weight of the native and the reduced, denatured forms of the enzyme. No free NH 2-terminal residue was detected by the dansylation procedure. Carbohydrate accounted for 17% of the weight of the enzyme, and the major residues were galactose, mannose and N- acetylglucosamine with smaller amounts of sialic acid and fucose. Removal of sialic acid residues with neuraminidase did not alter enzymatic activity. The enzyme contained one molar equivalent of zinc. Addition of this metal reversed stimulation and inhibition of activity observed in the presence of Co 2+ and Mn 2+, respectively. Immunologic homology of pure dog and rabbit enzymes was demonstrable with goat antisera. Fab fragments and intact IgG antibodies displayed similar inhibition of dose vs. response curves with homologous enzyme, whereas the fragments were poor inhibitors of heterologous activity compared to the holoantibodies. The canine glycoprotein was much less active than the rabbit preparation in catalyzing hydrolysis of Hip-His-Leu. In contrast, the two enzymes exhibited comparable kinetic parameters with angiotensin I as substrate.