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.     

Direct, sensitive and selective detection of free fatty acids by high-performance liquid chromatography with post-column ion-pair extraction and absorbance detection

Free fatty acids (C8-C18) are separated by reversed-phase liquid chromatography and detected using a simple post-column dynamic extraction system in which the acids are extracted as ion pairs with chloroform from the aqueous acetonitrile (gradient: 79-99% acetonitrile) mobile phase after the post-column addition of aqueous Methylene Blue solution. The chloroform phase containing the ion pairs is monitored with an absorbance detector at 651 nm. The detection limits ranged from 26 to 83 ng, depending upon the acid, with coefficients of variation of 1.2-14%. Application of the method to butter and margarine samples permitted detection of free fatty acids down to 35 ppm and in orange juice, down to 0.5 ppm using only an organic solvent extraction without further sample clean-up for isolation of the fatty acids.     

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.     

Microanalysis of free fatty acids in plasma of experimental animals and humans by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed for microanalysis of thirteen free fatty acids using 200 microliter of plasma. Fatty acids were derivatized with 9-anthryldiazomethane for HPLC analysis. Use of an ODS minicolumn for pretreatment of plasma gave a more accurate determination of free fatty acids in plasma than by chloroform extraction. Using this method, thirteen free fatty acids in the plasma of normal human, dog, rabbit, guinea pig and rat were determined.     

Analysis of mixtures containing free fatty acids and mono-, di- and triglycerides by high-performance liquid chromatography coupled with evaporative light-scattering detection

Commercial monostearates of glycerol, generally used as antistatic agents for thermoplastic polymers, consist of a complex mixture of mono-, di- and trisubstituted glycerides and the corresponding fatty acids. Thus far, the glycerides and the fatty acids have been analyzed separately by high-performance liquid chromatography (HPLC). In fact, the simultaneous analysis of glycerides and fatty acids requires esterification of the acids in order to avoid overlapping of the chromatographic peaks. This paper describes an HPLC method which allows the direct separation and identification, simultaneously and in a single run, of the variously substituted glycerides, and also the corresponding saturated fatty acids that are found as by-products in commercial glycerol monostearates. The procedure is based on the use of a ternary gradient HPLC instrument equipped with an evaporative light-scattering detector; the stationary phase was a reversed-phase RP-8 end-capped (Merck) column; the mobile phase was two consecutive binary gradients consisting of acetonitile-water plus acetic acid (0.1%, v/v) and acetonitrile-methylene chloride. The method is fast and shows high sensitivity and selectivity, being able to separate also the positional isomers of mono- and digycerides in addition to the mixed di- and triglycerides.     

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.     

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.     

Determination of fatty acids by high-performance liquid chromatography and fluorescence detection using precolumn derivatization with 9-fluorenylmethyl chloroformate

A new high-performance liquid chromatographic method is described for the determination of fatty acids in seed oils. The method was based on precolumn derivatization with 9-fluorenylmethyl chloroformate as a labeling agent and fluorescence detection. Fatty acids were extracted from the samples and subjected to derivatization with the reagent at 60°C for 10?min. The chromatographic separation of 14 fatty acids (C10–C22) was achieved on a combined loading compression octadecyl sulfate (CLC-ODS) column with a run time of 30?min. Three-step gradient elution of a mobile phase consisted of acetonitrile and water was used, and the signal was monitored at excitation and emission wavelengths of 265 and 315?nm, respectively. The method indicated favorable sensitivity and reproducibility for fatty acids’ derivatives. The detection limits, at a signal-to-noise ratio of 3, were 0.01–0.05?µg/ml and relative standard deviations (RSDs) were less than 0.27%. Excellent linear responses were observed with coefficients of 0.9995. This method was applied to quantify fatty acids in white, brown, and black sesame seeds’ oil.     

Routine determination of urinary free catecholamines by high-performance liquid chromatography with electrochemical detection

A simple and reliable high-performance liquid chromatographic method is described for the routine determination of the free catecholamines (norepinephrine, epinephrine and dopamine) in urine. The catecholamines are isolated from urine samples using small affinity chromatography columns prepacked with immobilised m-aminophenylboronic acid, separated by ion-pair reversed-phase liquid chromatography and quantified by electrochemical detection. Total analysis, including sample preparation time, is achieved in less than 30 min with analytical recoveries of 92-96% for all three catecholamines. Long-term stability and reproducibility of the liquid chromatographic system is attained by selection of optimised conditions for chromatographic separation with a formate mobile phase and produces detection limits of 1.4, 1.8 and 2.2 nmol/l for norepinephrine, epinephrine and dopamine, respectively, in urine samples and day-to-day coefficients of variation of less than 6%. Furthermore, the affinity isolation gels can be reused a minimum of ten times providing a rapid and cost-effective means of sample preparation.     

Detection of lyso-platelet-activating factor by high-performance liquid chromatography after derivatisation with fluorescent fatty acids

Lyso-platelet-activating factor (lyso-PAF) was derivatised with 9-anthracenepropionic acid in the presence of dicyclohexylcarbodiimide, p-toluenesulfonic acid and 4-dimethylaminopyridine. The reaction yield exceeded 90% when the fatty acid was present in double molar amounts versus lyso-PAF. The procedure was equally effective in the derivatisation of other lysophospholipids. The derivatized phospholipids are detected by ultraviolet absorption (lambda = 253 nm) or fluorescence detection (using excitation at 254 nm and emission at 450 nm). The technique was applied successfully to the detection of lyso-PAF in complement activated rabbit plasma.     

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.     

Determination of free fatty acids in chocolate by liquid chromatography with tandem mass spectrometry

This paper describes a rapid extraction method, based on a matrix solid-phase dispersion technique using diatomaceous earth as solid support and 50:50 (v/v) chloroform/methanol as extracting solvent, that can determine 11 free fatty acids in chocolate. The extraction procedure is followed by reversed-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) using a normal-bore (4.6 mm i.d.) C-18 column and an electrospray interface operating in the negative ion mode. The tandem mass spectra of selected compounds show that charge-remote fragmentation (CRF) mechanisms are occurring; the intensities of the CRF reactions increase with the carbon number and degree of unsaturation of the fatty acids. Average recoveries, evaluated by the standard addition method, vary between 79-103%, and the estimated quantification limits are less than 153 ng/g. The proposed method has been used to analyse nine chocolate samples from various price ranges, bought from supermarkets.     

Chemiluminescence detection flow cells for flow injection analysis and high-performance liquid chromatography

We have examined a range of new and previously described flow cells for chemiluminescence detection. The reactions of acidic potassium permanganate with morphine and amoxicillin were used as model systems representing the many fast chemiluminescence reactions between oxidising agents and organic analytes, and the preliminary partial reduction of the reagent was exploited to further increase the rates of reaction. The comparison was then extended to high-performance liquid chromatography separations of α- and β-adrenergic agonists, with permanganate chemiluminescence detection. Flow cells constructed by machining novel channel designs into white polymer materials (sealed with transparent films or plates) have enabled improvements in mixing efficiency and overall transmission of light to the photodetector.     

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.     

Specific detection of acetyl-coenzyme A by reversed-phase ion-pair high-performance liquid chromatography with an immobilized enzyme reactor

A selective chromatographic detection system for the determination of acetyl-coenzyme A (CoA) is reported. The short-chain acyl-CoA thioesters were separated by reversed-phase ion-pair high-performance liquid chromatography (HPLC), and then acetyl-CoA was selectively detected on-line with an immobilized enzyme reactor (IMER) as a post-column reactor. Thio-CoA liberated enzymatically from acetyl-CoA was determined spectrophotometrically after reaction with Ellman's reagent in the reagent stream. The IMER with phosphotransacetylase had a substrate specificity sufficient to determine acetyl-CoA and was active and stable in the mobile phase containing methanol and the ion-pair reagent. The calibration graph was linear between 0.2 and 10 nmol, with a detection limit of 0.05 nmol. This HPLC system with detection by IMER allows the selective identification and determination of acetyl-CoA in a mixture of acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA, which are difficult to separate with ion-pair HPLC.