Simultaneous separation and sensitive determination of free fatty acids in blood plasma by high-performance liquid chromatography

The quantitative determination of saturated and unsaturated fatty acids (ranging from acetic acid to lignoceric acid) in biological samples is presented. The secondary amine group of 5-(dimethylamino)-1-naphthalenesulponyl-semipiperazide (dansyl-semipiperazide) reacts with the carboxyl group of the fatty acids to form an amide linkage in order to obtain fluorescent derivatives of the acids. The fluorescent derivatives are analysed by high-performance liquid chromatography (HPLC) using an internal standard.     

Simultaneous separation and sensitive determination of free fatty acids in blood plasma by high-performance liquid chromatography

Fatty acids are separated by reversed-phase high-performance liquid chromatography after derivatization with a fluorescence reagent, 4-bromomethyl-7-acetoxycoumarin. Each derivative eluted from a column is successively hydrolysed by mixing it with an alkaline solution, and the produced fluorescence is detected. The derivatives of series of both saturated and unsaturated fatty acids (C6:0--C20:4) are simultaneously separated by a continuous gradient elution method using a methanol-based solvent containing acetonitrile. The quantitative detection of fatty acids is over a range of 5-1000 pmol per derivatization mixture. This method is applicable to the quantitative analysis of free fatty acids in normal human blood samples and blood samples from diabetic patients. Ten microliters of blood plasma are sufficient to carry out the determination. The analytical results show good recovery and good reproducibility. This sensitive method is very useful for the analysis of fatty acids in very low concentrations.     

Determination of fluorescent diamidines in plasma of experimental animals by high-performance liquid chromatography

The fluorescent diamidines (E)-2,2'-vinylenedi-1-benzo [b] furane-5-carboxamidine dihydrochloride (I) and 2-[2-(6-amidinoindole-2-yl)-(E)-vinyl]-1-benzofurane-5-ca rbo xamidine dihydrochloride (II) were determined in the plasma of experimental animals by high-performance liquid chromatography with a mobile phase of methanol-water (60:40, v/v) containing 0.005 M octanesulphonic acid and 0.003 M dimethyloctylamine. Samples were prepared by precipitation of plasma proteins with methanol-perchloric acid. Quantitation was performed by measuring the peak heights after monitoring the native fluorescence. The assay was linear over the range 5-750 ng/ml for I and 5-500 ng/ml for II, with limits of determination of 2.5 ng/ml for I and 1.5 ng/ml for II. Coefficients of variation were below 10% at all concentrations studied.     

Separation and quantitation of long-chain free fatty acids in human serum by high-performance liquid chromatography

A rapid, simple and highly sensitive reversed-phase high-performance liquid chromatographic method is described for the separation and quantitation of fatty acids in human serum using a very reactive fluorescent labeling reagent, 9-anthryldiazomethane. Quantitative esterification proceeds at room temperature without heat or catalysis. Baseline separation of nineteen select fatty acids from a standard mixture was achieved on two C18-bonded silica columns connected in tandem using stepwise gradient elution of an acetonitrile-methanol-water mobile phase. The eluent was monitored by a fluorescence detector (maximum excitation wavelength, 365 nm; maximum emission wavelength, 412 nm). The procedure was applied to the analysis of both saturated and unsaturated long-chain free fatty acids (C8 to C22) extracted from human serum. Sera from fasting and non-fasting subjects were analyzed to show the applicability of this assay to biological samples. Detection limit and recovery of free fatty acids in serum were less than 10 pmol/microliter and greater than 92%, respectively.     

Separation and quantitation of fatty acids by high-performance liquid chromatography

To facilitate the determination of the fatty acid composition of tissues and the investigation of fatty acid metabolism, we developed a method for the rapid separation by high-performance liquid chromatography and quantitation (by ultraviolet light absorption) of p-bromophenyl esters of fatty acids which vary in chain length from 10 to 22 carbon atoms. The utility of the method was demonstrated by evaluating the fatty acid composition of human uterine decidua vera tissue and human endometrial stromal cells that are maintained in monolayer culture.     

Determination of diagnostically important free and conjugated bile acids in blood plasma by high-performance liquid chromatography

A procedure was proposed for the determination of free bile acids and their conjugates in blood plasma by the reversed-phase HPLC using the column Lichrospher 100 RP-18 (250 + 4.6 mm) with gradient elution and UV-detection at 206 nm. The procedure allowed the simultaneous determination of diagnostically important cholic acids, tauro-and glyco-cholates in blood plasma of patients with no preliminary separation of the analytes into subtypes. The bile acids and their conjugates were isolated from the sample matrix by solid phase extraction in a Sep-Pack C18 cartridge. The limits of detection were 0.11–0.15 mM for free acids and 0.015–0.025 mM for conjugates.     

Determination of free phenytoin in plasma by ultrafiltration and high-performance liquid chromatography

A process for determining the free concentration of the highly protein-bound anti-convulsant drug phenytoin (5,5-diphenylhydantoin) is described. The procedure involves rapid isolation of the unbound drug from the drug/protein complex by ultrafiltration followed by high-performance liquid chromatography. Total time for a free phenytoin determination is about 15 min. The procedure is used to examine the protein-binding of phenytoin, as well as the effects of a displacer, salicylic acid. Commonly prescribed anticonvulsant drugs are resolved under the selected chromatographic conditions.     

Highly sensitive determination of free fatty acids in human serum by high-performance liquid chromatography with fluorescence detection

Endotoxins from four bacterial species extracted by three different procedures were acid-methanolyzed and the methyl esters of the fatty acids were analyzed by packed-column gas chromatography. There were qualitative and quantitative differences in the fatty acid profiles of the lipopolysaccharides isolated from four Gram-negative bacteria. Our data show considerable lot-to-lot variations in amounts of four methyl esters from the same bacterial serotype extracted by the same procedure and in the same bacterial serotype extracted by different procedures. These results indicate that extraction and perhaps culture conditions, as well as bacterial species, affect the fatty acid composition of endotoxins, hydrolyzed and derivatized by these procedures.     

Sensitive determination of free and plasma protein-bound dipyridamole by high-performance liquid chromatography

For many years dipyridamole (DP) has been used in the treatment of hypertension as a vasodilator, but recently it has been recognised as an anti-platelet aggregation agent and to potentiate anti-metabolite activity. A rapid and sensitive (20 nM) procedure for the determination of free and protein-bound DP in plasma, using reversed-phase high-performance liquid chromatography on an Ultrasphere XL ODS (3 microns) column (70 mm x 4.6 mm I.D.) with ultraviolet detection (280 nm), is reported. Free and bound DP were separated using ultrafiltration. Concentrations of DP between 0.1 and 10 microM were measured in plasma with a relative standard deviation of less than 9.6%. The subsequent determination of DP levels in patients orally administered 450 mg per day showed that DP binding to plasma protein is higher than 90%.     

Analysis of free amino acids in fermented shrimp waste by high-performance liquid chromatography

This work presents an HPLC method for the quantification of free amino acids in lyophilized protein fraction from shrimp waste hydrolysate which is obtained by acid lactic fermentation and analyzed using pre-column derivatization with 9-fluorenylmethyl-chloroformate. The amino acids were separated in a Hypersil ODS 5 microm column (250 mm x 4.6 mm) at 38 degrees C. The mobile phase was a mixture of phase A: 30 mM ammonium phosphate (pH 6.5) in 15:85 (v/v) methanol/water; phase B: 15:85 (v/v) methanol/water; and phase C: 90:10 (v/v) acetonitrile/water, with flow rate 1.2 ml/min. Fluorescence detection was used at an excitation wavelength of 270 nm and an emission wavelength of 316 nm. Method precisions for the different amino acids were between 4.4 and 7.1% (relative standard deviation, RSD); detection limits were between 23 and 72 ng/ml; and the recoveries were between 89.0 and 95.0%. The amino acid present at the highest concentration was tyrosine.     

Microanalysis of methotrexate by high-performance liquid chromatography using a fluorescence detector

Methotrexate (N¹⁰-methyl-4-aminofolic acid) has been determined by HPLC after oxidizing it with permanganate to a fluorogenic derivative. The detection limit has been 0.01 g/ml. The method has been applied for the determination of methotrexate in blood serum of rats.     

Quantitation of phenacyl esters of retinal fatty acids by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for the separation and quantitation of retinal fatty acids containing long-chain polyunsaturated fatty acids is described. Fatty acids from frog retinal lipids were converted to the corresponding phenacyl derivatives which were separated on a C18 reversed-phase column and detected at 242 nm. Molar absorptivities (peak area units/nmol) of up to seventeen fatty acid phenacyl derivatives were determined and used for quantitation of fatty acids separated by HPLC. Compared with gas chromatography, the HPLC method gave a similar molar percent distribution of the fatty acids and was twenty to fifty times more sensitive. This HPLC method provides a useful means for the study of chemistry and metabolism of long-chain polyunsaturated fatty acids in retina and other tissues where amounts of material may be limited or recovery of individual components desirable.     

Analysis of fatty acids in mouse cells using reversed-phase high-performance liquid chromatography

Summary A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed to analyze various fatty acids in recombinant mouse L cells. These fatty acids were the metabolites of oleic acid. A process was developed to extract fatty acids from the cell samples before RP-HPLC analysis. The samples were first saponified with 0.5 M NaOH in 96% ethanol then extracted with acidified ethyl acetate. After extraction, the sample was dried and dissolved in HPLC-grade methanol. After centrifugation to remove insoluble impurities, the sample was applied to a C₁₈RP-HPLC column using a gradient of acetonitrile (ACN)-H₂O. The eluted fatty acids were monitored by ultraviolet (UV) absorption at 195 nm and identified by retention time and adsorption spectrum comparison. This method successfully resolved various fatty acids and provided a tool for the elucidation of the fatty acid metabolic pathway in the cells.     

Resolution of nucleic acids by high-performance liquid chromatography

High-performance liquid chromatography is a very powerful technique for the separation and isolation of nucleic acids. Nucleic acids can be generally characterized as long, negatively charged polymers with hydrophobic nucleobases. Chromatographic processes which use electrostatic interactions (anion-exchange), hydrophobic interactions (reversed-phase or salting-out) or both types of interactions (mixed-mode) are most effective for resolution of these materials.