High-performance liquid chromatographic analysis of bile acids in hamster bile

A high-performance liquid chromatographic procedure has been developed for the determination of pentamidine concentrations in serum samples. A microbore, reversed-phase column was used with a mobile phase consisting of methanol and water with sodium heptanesulfonate and triethylamine as modifiers. Pentamidine could be extracted from serum only by the addition of an ion-pairing agent, di(2-ethylhexyl) phosphoric acid, to the chloroform used for extraction. The method can be used to reliably detect levels as low as 5 ng/ml. The pentamidine concentration in the serum of eleven patients 24 h after their tenth daily dose of pentamidine averaged 60 +/- 34 ng/ml.     

High-performance liquid chromatographic analysis of neurotransmitter amino acids in brain

Rapid and selective determination of neurotransmitter amino acids in rat and human brain is accomplished by pre-column derivatization with o-phthaldehyde-3-mercaptopropionic acid, reversed-phase high-performance liquid chromatographic separation, and fluorimetric detection. Aspartate, taurine, glutamate, and glycine are determined in less than 12 min with intra- and inter-assay precisions of 2.1-9.5% and 4.2-9.2%, respectively. gamma-Aminobutyric acid is determined in less than 8 min with intra- and inter-assay precisions of 1.6 and 11.7%, respectively. Both assays utilize internal standards and require a minimum of sample preparation.     

High-performance liquid chromatographic enantioseparation of monoterpene-based 2-amino carboxylic acids on macrocyclic glycopeptide-based phases

The enantiomers of five monoterpene-based 2-amino carboxylic acids were directly separated on chiral stationary phases containing macrocyclic glycopeptide antibiotics such as teicoplanin (Astec Chirobiotic T and T2) and teicoplanin aglycone (Chirobiotic TAG) as chiral selectors. The effects of pH, the mobile phase composition, the structure of the analyte and temperature on the separations were investigated. Experiments were performed at constant mobile phase compositions in the temperature range 10–40 °C to study the effects of temperature and thermodynamic parameters on separations. Apparent thermodynamic parameters and T_iso values were calculated from plots of ln k or ln α versus 1/T. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes. It was found that the enantioseparations were in most cases enthalpy driven. The sequence of elution of the enantiomers was determined in all cases.     

High-performance liquid chromatographic enantioseparation of beta-amino acids

Direct and indirect high-performance liquid chromatographic methods were developed for the enantioseparation of beta-amino acids (beta-substituted-beta-alanines). Direct separation involved the application of chiral columns: Crownpak CR(+), Chirobiotic T and Chirobiotic R. Indirect separation was based on precolumn derivatization with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate or N-alpha-(2,4-dinitro-5-fluorophenyl)-L-alanineamide (Marfey's reagent), with subsequent separation on an achiral column. The chromatographic conditions were varied to achieve optimum separation.     

High-performance liquid chromatographic analysis of serum short-chain fatty acids by direct derivatization

The application of direct derivatization in conjunction with high-performance liquid chromatography is described for the analysis of short-chain fatty acids in serum. The method is based on the reaction of these acids with acidic 2-nitrophenylhydrazine hydrochloride, without complicated isolation steps, which produces their non-volatile hydrazine derivatives. The hydrazides of fourteen saturated and unsaturated, straight and branched, short-chain fatty acids were separated from other acid hydrazides and interfering components by a simple solvent extraction, and were eluted isocratically on a reversed-phase C8 column within 24 min. UV detection demonstrated that the detection limits for the acids were 200-400 fmol per injection with linearity over the range from 400 fmol to 5 nmol per injection. Analytical recoveries ranged from 96.8% to 103.1% and coefficients of variation ranged from 0.9% to 3.8%. The present method is simple, accurate and adequate for the analysis of short-chain fatty acids in biological fluids and tissues of patients suffering from organic acidemias.     

High-performance liquid chromatographic analysis of free palmitic and stearic acids in cerebrospinal fluid

A relatively simple method for extraction of free fatty acids from cerebrospinal fluid with aminopropyl bonded-phase columns, and the estimation of palmitic acid (C16:0) and stearic acid (C18:0) concentrations by high-performance liquid chromatographic analysis is described. The values of C16:0 and C18:0 in patients with non-neurological disorders lie within a narrow range, with a mean (+/- S.D.) of 4.02 +/- 0.33 micrograms/ml for C16:0 and 2.72 +/- 0.39 micrograms/ml for C18:0.     

High-performance liquid chromatographic analysis of steroid hormones

In the present work, a practical, rapid, reliable and isocratic reversed-phase high-performance liquid chromatographic (HPLC) method is described for the qualitative and quantitative analysis of estriol, estradiol-17 beta, estrone, testosterone, and progesterone. Chromatographic separation is complete in 16 min using a mobile phase of 50% acetonitrile (v/v) in water. The order of elution is estriol, testosterone, estradiol-17 beta, estrone, and progesterone; retention times are 2.5, 5.5, 5.6, 6.9, 16.3 min, respectively. Absorbance maxima of individual steroids is the limiting factor in quantitative determination. The recommended wavelengths for UV monitoring are E3 214, E2 280, T 254, E1 214, and P4 254 nm.     

High-performance liquid chromatographic analysis of dehydroepiandrosterone.

Qualitative and quantitative analysis of dehydroepiandrosterone and its conjugates in biological matrices and establishment of their relationships with physiological functions is a very active field. This review article discusses methods of separation and quantification of dehydroepiandrosterone and its conjugates using high-performance liquid chromatographic techniques.     

High-performance liquid chromatographic analysis of dinitrobenzene isomers

Summary An efficient, reproducible and rapid high-performance liquid chromatographic method, in normal phase mode, for the analysis of the three dinitrobenzene isomers is described. The method affords good linearity for each isomer in the range 10–160 g ml^–1. The total analysis time is only 10 minutes, and the method shows an accuracy of ±1.25% with a coefficient of variation from 0.30% to 2.85% for different levels of the dinitrobenzene isomers.     

High-performance liquid chromatographic analysis of azlocillin in serum

Summary A rapid and sensitive high performance liquid chromatographic method is described for the determination of azlocillin in serum. This method involves a short manual protein precipitation of the sample followed by an injection into a PR 18 column for separation and quantitation. The mobile phase was a 22% (V/V) solution acetonitrile in phosphate buffer pH 4.8 at a flow rate of 2,5 ml/min. The spectrophotometer detector was set at 220 nm with a sensitivity of 0.08 AUFS.     

High-performance liquid chromatographic analysis of serum long-chain fatty acids by direct derivatization method

A new visible-ultraviolet labelling method for the high-performance liquid chromatographic analysis in serum of individual free fatty acids, including polyunsaturated fatty acids, is described. Without commonly used isolation steps, fatty acids in serum were directly derivatized by treatment with acidic 2-nitrophenylhydrazine hydrochloride. The derivatized fatty acids were extracted into n-hexane and separated isocratically on a reversed-phase C8 column within 15 min. The detection limits ranged from 400 fmol to 1 pmol and from 100 to 200 fmol per injection with visible and ultraviolet detection, respectively. Visible detection had better selectivity, and free fatty acid levels were determined in sera obtained from healthy controls and patients with diabetes mellitus. In all the subjects studied, the precise quantitation could be performed with 25 microliters of serum. Analytical recoveries ranged from 98.3 to 103.4%. The intra- and inter-assay coefficients of variation were less than 2.7 and 3.5%, respectively. The present method is superior to the previously published methods for routine analyses: it is cheaper, the procedure is simpler, the analysis time is shorter and both resolution and sensitivity are better.