Determination of amine-containing phosphonic acids and amino acids as dansyl derivatives

Conditions are selected for the analytical separation of (N-phosphonomethyl glycine), products of its microbiological conversion, glutamic acid, and alanine as dansyl derivatives using reversed-phase HPLC: column (250 × 4.6 mm) ReproSil-PAH EPA; mobile phase, methanol + 20 mM CH₃COONa (pH 5.1) (20: 80); rate of mobile phase, 1 mL/min; working detector wavelength, 330 nm. The duration of separation is 35 min. The lower limits of the analytical range (in ng) for dansyl derivatives are as follows: glyphosate, 8.2; aminomethyl phosphonic acid, 24.2; glutamic acid, 9.4; alanine, 12.6: glycine, 17.7; and sarcosine, 19.3. The TLC study of dansyl derivatives of amino acids was performed on sorbfil plates PTSH-P-V using two-dimensional chromatography in the systems ethyl acetate-isopropanol-24% aqueous ammonia (45: 35: 20) in the first direction and chloroform-methanol-acetic acid (25: 5: 1) in the second one. For determining phosphonic acids (glyphosate and aminomethyl phosphonic acid), a version of one-dimensional chromatography with the sequential use of two systems, chloroform-methanol-acetic acid (25: 5: 0.2) and ethanol-24% aqueous ammonia (7: 3), was proposed.     

Determination of biogenic amines as dansyl derivatives in intestinal digesta and feces by reversed phase HPLC

Summary A new method was developed for the determination of fifteen biogenic amines in the intestinal digesta and feces of animal or human origin. The method involves the addition of an internal standard (heptylamine), extraction of the amines, and precipitation of the proteins with perchloric acid. The amines are derivatised with dansyl chloride, separated on a C18 column using gradient elution with 0.2M ammonium acetate at pH 5, water and acetonitrile, and detected with a fluorescence detector. The separation was achieved in a 40 min run. Recoveries ranged from 67 to 110%, the relative standard deviation for intra-assay precision being <5% and the limit of determination 1–5 mg kg⁻¹. The method is specific for biogenic amines in intestinal and fecal samples.     

High-speed analysis of dansyl derivatives of polyamines

Summary Analytical conditions for the high-speed, reversed-phase, liquid chromatographic of a diamine (putrescine) and polyamines (spermine and spermidine) were determined. Various elution modes were employed using the same mobile phase constituents: 20mM sodium heptane sulfonate and 20mM acetic acid as solvent A and, pure acetonitrile as solvent B. Samples were derivatized with dansyl chloride before injection.Under isocratic conditions, the separation of the three polyamines was achieved in 7 min. The use of a linear elution gradient led to the same analytical time but with a better resolution of the putrescine peak from non-polyamine frontal peaks. For these measurements, the sample size was 5l. This volume was increased to 20l and the use of a steep gradient combined with the peak-compression technique allowed a fast analysis in 2–3 minutes, which may be compared with a 30 min run time necessary when a conventional column is used.     

HPLC determination of catabolic amines in silage using their dansyl derivatives and an electrochemical detector

Summary The use of the electrochemical detector (EC) in the high performance liquid chromatography (HPLC) of dansyl derivatives of biogenic amines is reported. Isocratic and gradient elution patterns of synthetic mixtures of putrescine, cadaverine, 1,6-diaminohexane, tryptamine, histamine, tyramine, spermine and spermidine on a reversed phase column are shown. Hydrodynamic voltammograms of standard compounds are presented. The detection limits of the EC are compared with those of ultraviolet and spectrofluorimetric detectors. A chromatographic profile of amines from the proteolytic catabolism of maize silage byClostridia is shown.Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.     

Determination of 27 dansyl amino acid derivatives in biological fluids by reversed-phase high-performance liquid chromatography

The concentrations of free amino acids in plasma and in ascitic liquid of mice with Ehrlich ascitic tumours were determined by reversed-phase high-performance liquid chromatography using pre-column derivatization with Dns chloride and UV detection at 254 nm. Sample preparation is simple, and the Dns derivatives are stable. Complete separation of 27 amino acids, including proline and cysteine, was achieved in 70 min with detection limits of less than 25 pmol. There was no interference from Dns-Cl, Dns-OH and Dns-NH2. Retention time reproducibility was better than 1%. The described method enables a rapid, economical and reproducible quantification of free amino acids in biological fluids.     

Thin layer chromatography of dansyl and dinitrophenyl derivatives of amino acids. A review

The dinitrophenyl (DNP) derivatives of amino acids have found continual application in protein sequencing since Sanger used them for the first time for the sequencing of insulin. Dansyl derivatives of amino acids have been widely used in protein sequencing because of their fluorescent nature. The success of protein sequencing largely depends upon correct identification of such derivatives. The choice for the method of identification is related to cost, the availability of instrumentation and to the sensitivity needed for the analysis. Thin layer chromatography (TLC) is simple and has several advantages over other chromatographic methods. Therefore the literature after 1972 is reviewed for TLC analysis of dansyl- and DNP-amino acids, the two important amino acid derivatives required for identifying protein sequences. Additionally, the literature on the TLC resolution of enantiomeric mixtures of dansyl amino acids is reviewed. Application of various adsorbents, composition of solvent systems and other experimental conditions together with successful resolution data have been discussed. TLC provides a direct and inexpensive method for the resolution of enantiomers, and is fast becoming a sensitive instrumentalized quantitative analytical technique.     

Measurement of neutral sugars in glycoproteins as dansyl derivatives by automated high-performance liquid chromatography

Automated high-performance liquid chromatography was used to analyse dansylhydrazine derivatives of neutral sugars in unfractionated acid hydrolysates of four well-characterized glycoproteins: fetuin, ovalbumin, alpha-1-acid glycoprotein and bovine submaxillary mucin. After a simple single-step derivatization at 65 degrees C the sugar derivatives in protein hydrolysates chromatographed as single peaks on reversed-phase C18 columns. The isocratic solvent consisted of 20% (v/v) aqueous acetonitrile containing 0.01 M formic acid, 0.04 M acetic acid and 0.001 M triethylamine. The triethylamine significantly increases the sugar peak height at 254 nm. Repeated automatic sample injection without deterioration of column performance or interference from dansyl hydrazine is not possible with published methods, but was achieved by cleaning the column between each analysis with a solvent of 20% (v/v) acetonitrile and 80% (v/v) methanol. Hydrolysis with 2 M trifluoroacetic acid is superior to 2 M hydrochloric acid for both sugar recovery and convenience but must continue for 6-8 h at 105 degrees C to ensure complete sugar release. We confirmed that mannose is present in most preparations of human high-molecular-weight salivary glycoproteins, and also examined purified bovine skin proteodermatan sulphate. p-Nitrophenylhydrazine derivatives of neutral sugars are readily produced, but do not chromatograph as successfully as the dansyl derivatives while phenylhydrazine derivatives are not easily produced at 65 degrees C. Further development of the method should be possible by producing other hydrazine derivatives of neutral sugars.     

High-pressure liquid chromatographic analysis of carbofuran and two non-conjugated metabolites in crops as fluorescent dansyl derivatives

Carbofuran, and non-conjugated 3-hydroxycarbofuran and 3-ketocarbofuran were extracted from carrots, corn and potatoes with acetone and partitioned into hexane-methylene chloride. The organic extract was evaporated to a small volume for clean-up on a 2% deactivated Florisil column. All three carbamates were eluted with 15% acetone in hexane. The pesticide residues were hydrolysed to their corresponding phenols with 0.1 M sodium carbonate followed by derivatization with dansyl chloride in acetone. The derivatives were extracted and analysed by high-pressure liquid chromatography with fluorescence detection (excitation, 360 nm; emission, greater than 400 nm). Absolute recoveries for all three compounds were between 50 and 65% for spiked samples by the extraction method used. Detection limits approached 0.01 ppm in the foods studied.     

Determination of the structure of glycolaldehyde as their trimethylsilyl derivatives by gas chromatography-mass spectrometry

Summary The structure of glycolaldehyde and glyceraldehyde was determined using gas chromatography — mass spectrometry. Glycolaldehyde and glyceraldehyde, mixed with lactic acid, 2-furoic acid, furoin, xylose and ascorbic acid were separated on a capillary column coated with SE-52 as trimethylsilyl derivatives. The mass spectra show two dimeric forms for TMS-glycolaldehyde and one dimeric form for TMS-glyceraldehyde.     

Determination of the insecticide promecarb by fluorogenic labelling with dansyl chloride.

The use of spectrofluorimetry to determine the fluorescent derivative of the insecticide promecarb, following hydrolysis to the corresponding phenol in basic media and subsequent coupling with the labelling agent dansyl chloride, is described and discussed. A study of media of different basicity and of different temperatures for both reactions gave optimum conditions of 20 min for the hydrolysis reaction and 10 min for the labelling reaction at 55 degrees C in 0.05 mol dm-3 sodium hydrogen carbonate solution with a reagent to insecticide ratio of 12:1. The effect of the solvent on the formation of the dansyl derivative and on the extraction process was studied using nine and seven solvents, respectively. The use of a mixture of acetone and water (50 + 50, v/v) and an extraction into cyclohexane gave the best results. The minimum detectable concentration of promecarb in the experimental assays was 100 ng ml-1. The error and relative standard deviation at a concentration level of 0.6 micrograms ml-1 were 9.7 and 10.9%, respectively. Air samples containing promecarb at different concentration levels were analysed.     

Determination of bromophos residues by fluorogenic labelling with dansyl chloride and thin-layer. Chromatography

The use of a highly sensitive and inexpensive method for determination of residues of bromophos (an organophosphate pesticide) in peanut crops, by fluorogenic labelling and thin-layer chromatography (TLC) is described. The bromophos is hydrolysed and the product 4-bromo-2,5-dichlorophenol (BDCP) dansylated with dansyl chloride. The fluorescent dansylated BDCP is separated by TLC and detected fluorimetrically. Linear fluorimetric analytical curves are obtained for bromophos weights ranging between 0.5 and 50 ng. The minimum detectable quantity is estimated to be 0.5 ng. The method can be used to recover as little as 400 ng of bromophos residue from 25 g of peanut seeds.     

Quantitative analysis of 15N-labeled positional isomers of glutamine and citrulline via electrospray ionization tandem mass spectrometry of their dansyl derivatives

The enteral metabolisms of glutamine and citrulline are intertwined because, while glutamine is one of the main fuel sources for the enterocyte, citrulline is one of its products. It has been shown that the administration of ¹⁵N‐labeled glutamine results in the incorporation of the ¹⁵N label into citrulline, but it is not clear which of the three nitrogen groups of citrulline is actually labeled. To determine the ¹⁵N‐enrichment of the positional isomers of glutamine and citrulline, a rapid liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed. The amino acids were analyzed as their dansyl derivatives. The product ion resulting from the loss of NH₃ from the omega carbon allows for the determination of the enrichment of the ureido (citrulline) or amido groups (glutamine). The protonated pyrrolidine (citrulline) or 5‐oxopyrrolidine (glutamine) product ion contains the 2‐N (amino group) and is used to determine its enrichment. The method described showed no ion suppression and a wide dynamic range ranging from 1.3 picomoles to 2 nanomoles for citrulline. Background samples and standards resulted in enrichments not different from those theoretically expected. The enrichment curves for the different glutamine and citrulline isotopomers were linear (R² > 0.998) over the range of enrichments studied. The method developed provides an additional insight into the metabolism of glutamine and citrulline tracing the precursor‐product relationship between these two amino acids. Copyright © 2011 John Wiley & Sons, Ltd.     

Gas chromatography of bile acids as their hexafluoroisopropyl ester-trifluoroacetyl derivatives.

Bile acids, such as cholic, chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids, were allowed to react with hexafluoroisopropanol and tri-fluoracetic anhydride at 37 for 30 min. The resulting derivatives were gas chromatographed on QF-1, with flame ionization detection, and were identified by gas chromatography-mass spectrometry. Separation was good. By using this method, these acids were detected in samples of human duodenal fluid; the ratios of each were 24.4, 41.5, 24.9, 2.3 and 6.9%, respectively.