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.     

Analysis of carbohydrates in glycoproteins by high-performance liquid chromatography and high-performance capillary electrophoresis

We describe two methods for the analysis of oligosaccharide chains in glycoproteins by high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE).O-andN-glycosidically linked oligosaccharides released from glycoproteins can be identified as their borohydride-reduced forms by anion-exchange HPLC with pulsed amperometric detection.N-Glycosidically linked oligosaccharides can also be analyzed as 2-aminopyridine derivatives by HPCE in direct zone electrophoresis mode in an acidic phosphate buffer and zone electrophoresis mode as borate complexes in an alkaline buffer. We also present a convenient procedure for the analysis of the constituent monosaccharides of these oligosaccharides chains by HPLC based on reversed-phase partition mode as 1-phenyl-3-methyl-5-pyrazolone derivatives.     

Pyrimidine nucleoside phosphorylase assay by automated high-performance liquid chromatography

A new assay for pyrimidine nucleoside phosphorylase is reported. This method utilizes an isocratic reversed-phase high-performance liquid chromatographic system for separation of nucleosides and bases. Product detection is accompanied by ultraviolet monitoring and radioactive flow detection. Use of an automated sample injector allows for the analysis of a series of samples, with data recorded onto a microprocessor-based cassette recorder. Data can then be downloaded into computer memory. The velocity of uridine phosphorylase (E.C. 2.4.2.3) was a linear function of enzyme concentration. The Michaelis constant for uridine at pH 8.0 was found to be in close agreement with the value obtained by a thin-layer chromatographic assay method.     

Separation of pyrimidine derivatives by reversed-phase high-performance liquid chromatography

Chromatographic behavior and separation conditions of pyrimidine derivatives were studied by high-performance liquid chromatography using a reversed-phase column and a multiwave UV detector.     

Measurement of acid-catalyzed isomerization of unsaturated aldehyde-2,4-dinitrophenylhydrazone derivatives by high-performance liquid chromatography analysis

Aldehyde-2,4-dinitrophenylhydrazones exist as (E)- and (Z)-geometrical isomers, and adventitious isomerization during sample preparation can cause analytical errors. Purified alkenal-2,4-dinitrophenylhydrazone derivatives comprise only the (E)-isomer. However, partial isomerization to the (Z)-isomer occurs upon addition of acid to attain an equilibrium isomer ratio. The UV-visible spectral properties of the isomers differ; the (Z)-isomer exhibiting a 6-10 nm lower absorption maximum compared to the (E)-isomer. Alkenal-2,4-dinitrophenylhydrazones having a CC double bond at the 2- or 3-position of the alkenal exhibited similar absorption maxima with an equilibrium isomer ratio (0.035) that was much lower than those of other alkenals. The CC double bond at the 3-position migrates to a position of conjugation with the CN double bond during hydrazone synthesis to form a stabilized molecular structure. Alkenal-2,4-dinitrophenylhydrazones having a double bond at the 4-position or greater exhibited a similar absorption maxima equilibrium isomer ratio (0.14) to alkanal-2,4-dinitrophenylhydrazones. The quantitative analysis of carbonyl compounds in air or water using DNPH is usually conducted in the presence of an acid catalyst. Consequently, the solution of the direct extract prepared for HPLC or GC analysis contains both (E)- and (Z)-isomers.     

Separation of pantothenic acid derivatives by reversed-phase high-performance liquid chromatography

A variant of the use of reversed-phase high-performance liquid chromatography is described which permits the separation of pantothenic acid derivatives. The stationary phase used was a μBondapak-C₁₈ (4.1 × 250 mm column; 4.6 × 50 nm precolumn). Elution was performed in the isocratic regime using as mobile phase 20 M potassium phosphate buffer (pH 5.0)-methanol (91.5:8.5). The rate of elution was 1 ml/min. Retention times in the column for phosphopantothenate, pantothenate, phosphopantetheine, CoA, and dephosphoCoA were about 3.5, 6, 10.5, 16, and 42 min, respectively. This method, with radioactive detection, can be used for the analysis of pantothenic acid derivatives in liver extracts. One hour after white rats had been injected with [¹⁴C]pantothenic acid, the abovementioned components (with the exception of dephosphoCoA) contained the label in a ratio of 4:18:54:24. Institute of Biochemistry, Academy of Sciences of the Belorussian SSR, Grodno. Translated from Khimiya Prirodynkh Soedinenii, No. 6, pp. 855–858, November–December, 1988.     

Separation of pantothenic acid derivatives by reversed-phase high-performance liquid chromatography

A variant of the use of reversed-phase high-performance liquid chromatography is described which permits the separation of pantothenic acid derivatives. The stationary phase used was a Bondapak-C₁₈ (4.1 × 250 mm column; 4.6 × 50 nm precolumn). Elution was performed in the isocratic regime using as mobile phase 20 M potassium phosphate buffer (pH 5.0)-methanol (91.5:8.5). The rate of elution was 1 ml/min. Retention times in the column for phosphopantothenate, pantothenate, phosphopantetheine, CoA, and dephosphoCoA were about 3.5, 6, 10.5, 16, and 42 min, respectively. This method, with radioactive detection, can be used for the analysis of pantothenic acid derivatives in liver extracts. One hour after white rats had been injected with [¹⁴C]pantothenic acid, the abovementioned components (with the exception of dephosphoCoA) contained the label in a ratio of 4:18:54:24.Institute of Biochemistry, Academy of Sciences of the Belorussian SSR, Grodno. Translated from Khimiya Prirodynkh Soedinenii, No. 6, pp. 855–858, November–December, 1988.     

Measurement of epidoxorubicin and its metabolites by high-performance liquid chromatography using an advanced automated sample processor.

A sensitive and rapid method for measuring epidoxorubicin and its six metabolites by high-performance liquid chromatography using an advanced automated sample processor is described. Plasma samples (1 ml) were extracted using C2 cassettes, and reversed-phase chromatography was performed with an Apex II ODS column. The isocratic mobile phase of acetonitrile-0.019 M NaH2PO4 (pH 4.0) had a flow-rate of 1 ml/min and the fluorescence detector an excitation wavelength of 480 nm with an emission at 580 nm. Linear calibration curves were obtained which were reproducible both within-day and day-to-day (coefficients of variation less than 10%). The extraction efficacy of epidoxorubicin was 88% and ranged from 51 to 88% for the metabolites. This method has been successfully applied to measure the plasma levels of these compounds in patients receiving epidoxorubicin over a wide dose range (12-120 mg/m2) and in patients with disturbed liver biochemistry.     

Regioselectively modified polysaccharide derivatives as chiral stationary phases in high-performance liquid chromatography

This review summarizes several regioselectively modified polysaccharide derivatives which have been prepared in order to be used for chiral separations chromatography. The goal was to combine the effects of the known tris-arylcarbamate and tris-arylesters of polysaccharides. The use of new materials as chiral stationary phases (CSPs) in liquid chromatography for enantiomeric discrimination was investigated and compared to the homogeneously substituted polysaccharide derivatives. The different works describing the preparations, the performances and the applications of regioselectively modified polysaccharide derivatives have been resumed.     

Covalently bonded polysaccharide derivatives as chiral stationary phases in high-performance liquid chromatography

Polysaccharide derivatives have been extensively used as chromatographic chiral selectors in chiral stationary phases (CSPs) for the separation of enantiomers by HPLC. When coated onto a silica matrix, they represent nowadays one of the most popular type of CSPs. However, they are only compatible with a limited choice of solvents. The main drawback of these CSPs is related to the solubility of the chiral selector in a number of solvents, which limits their applicability. The different attempts which have been described up to now to overcome this problem by covalently fixing the chiral selector to a matrix are reviewed in this paper.     

Determination of biogenic amines as dansyl derivatives in alcoholic beverages by high-performance liquid chromatography with fluorimetric detection and characterization of the dansylated amines by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A sensitive high-performance liquid chromatographic method for the simultaneous determination of 11 biogenic amines has been developed. The method involves addition of an internal standard (1,7-diaminoheptane), pre-column dansylation of the amines and subsequent solid-phase extraction of the derivatives through C18 cartridges. The dansylamides were separated on an Inertsil ODS-3 column (250 x 4 mm I.D., 5 microm) using a 35-min gradient elution with a binary system of acetonitrile-water, a flow-rate of 1 ml min(-1) and fluorescence detection at excitation and emission wavelengths of 320 and 523 nm, respectively. The identity of the dansyl derivatives was confirmed by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. Linearity of derivatization was obtained for concentrations ranging from 0.008 to 40.0 mg l(-1). The within- and between-day relative standard deviations ranged from 0.2 to 7.6% and 0.3 to 8.6%, respectively. The overall process was successfully applied to identify and quantify biogenic amines in white-, red- and Retsina Greek wines and Greek beers, after treatment with polyvinylpyrrolidone.     

Analysis of OPA-derivatized amino sugars in tobacco by high-performance liquid chromatography with fluorimetric detection

An analytical method is developed to quantitatively determine glucosamine, galactosamine, and mannosamine in dried-and-ground burley and flue-cured tobaccos. Extraction is shown to be quantitative in the range of 0.01 to 2.0% (w/w). The extraction procedure consists of shaking one g of sample with 50 mL of deionized water adjusted to pH 7 for 30 min. This extract is filtered directly into an autosampler vial. An autosampler is programmed to withdraw two different aliquots: one with o-phthalaldehyde (OPA) derivatizing solution and the second one from the tobacco extract. The derivatization reaction occurs in the tubing connecting the autosampler and the chromatographic column. The OPA derivatives of these aminosugars are then detected with a fluorimetric detector, and their simultaneous analysis is performed with an external standard. This method is shown to be selective, accurate, and precise.     

Measurement of beta-carbolines by high-performance liquid chromatography with fluorescence detection

A method using high-performance liquid chromatography with fluorescence detection was developed for the determination of beta-carboline compounds norharman, harman, norharmol, and harmol in lung. Aqueous derivatization with acetic anhydride was used to facilitate the isolation and separation of the phenolic compounds and to reduce the fluorescence background of the biological samples. Harman was identified and quantitated in rat lung (1.88 +/- 0.55 ng/g) using this method and its identity confirmed by means of gas chromatography-negative-ion chemical ionization mass spectrometry.     

Determination of plasma and urinary cortisol by high-performance liquid chromatography using fluorescence derivatization with dansyl hydrazine

A method is described for the determination of cortisol in human plasma and urine by high-performance liquid chromatography using fluorophotometric detection. After extraction with methylene chloride, cortisol is labelled with dansyl hydrazine, and then separated by high-performance chromatography. The eluate is monitored by a fluorophotometer at 350 nm (excitation) and 505 nm (emission). The optimum conditions for the determination, such as HCl and dansyl hydrazine concentrations, reaction time and reaction temperature, and for the eluent of high-performance liquid chromatography, are discussed. Linearity of the fluorescence intensity (peak height) with the amount of cortisol was obtained between 0.5 and 60 ng. The recoveries for 50 and 100 ng of added cortisol were 98.7 and 95.4% for plasma, and 96.4 and 90.6% for urine, respectively. Comparison with a radioimmunoassay gave a correlation coefficient of 0.978. The proposed method is suitable for the routine analysis of cortisol in plasma and urine.     

Chloromethylphenylcarbamate derivatives of cellulose as chiral stationary phases for high-performance liquid chromatography

A new class of eight chloromethylphenylcarbamate derivatives of cellulose was prepared by introducing both an electron-donating methyl group and an electron-withdrawing chloro group on to the phenyl moieties and their chiral recognition abilities were evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography. The superiority of these derivatives over dichloro- and dimethylphenylcarbamates of cellulose as CSPs was demonstrated for some racemic compounds. The elution order and enantioselectivity were greatly dependent on the positions of the substituents. Meta- and para-disubstituted derivatives showed higher chiral recognition than ortho- and meta- or para- disubstituted derivatives. The correlation between the chemical shifts of the N---H protons of the carbamate moieties and the enantiomer-resolving abilities of the derivatives is discussed. Some of the derivatives were effective CSPs in both normal- and reversed- phase conditions and could efficiently separate some chiral drug enantiomers.