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 urinary trypsin inhibitors by reversed-phase high-performance liquid chromatography

Summary Human urines contain a family of trypsin inhibitors (UTIs) in small quantities, which seem to be involved in important biological processes. A procedure for separation and quantitative determination of such endogenous inhibitors in human urine has been developed. The urine sample is adjusted to pH 8.3 and percolated through a trypsin-Sepharose 4B column: the inhibitors are eluted with acid solution. The eluate (1000 l) is analysed by RP-HPLC with programmed elution and ultraviolet detection (200 nm). Three principal peaks have been evidenced: they are due to the elution of urinary trypsin inhibitors (UTIs) having apparent m.w. of ca. 6000, 72000, 18000 daltons, respectively. Characteristics of the procedure are: limited sample volume (ca. 200 ml) and recovery of the global inhibition activity (95%). For each UTI determination reproducibility and linearity ranges are reported.

---

Trennung und Bestimmung von Trypsin-Inhibitoren im Harn durch Umkehrphasen-HPLC

     

Separation of carotenol fatty acid esters by high-performance liquid chromatography

Employing isocratic and gradient-elution high-performance liquid chromatography (HPLC) a number of straight-chain fatty acid esters (decanoate, laurate, myristate, palmitate) of violaxanthin, auroxanthin, lutein, zeaxanthin, isozeaxanthin, and beta-cryptoxanthin, prepared by partial synthesis, have been separated on a C18 reversed-phase column. Several chromatographic conditions were developed that separated a mixture of di-fatty acid esters (dimyristate, myristate palmitate mixed ester, dipalmitate) of violaxanthin, auroxanthin, lutein, and zeaxanthin in a single chromatographic run. Hydroxycarotenoids such as lutein, zeaxanthin, and isozeaxanthin that are not easily separated by HPLC on C18 reversed-phase columns, can be readily separated after derivatization with fatty acids and chromatography of their esters. Chromatographic conditions for optimum separation of carotenoids from various classes are discussed.     

Separation of urinary aromatic acids and measurement of vanilmandelic acid by high-performance liquid chromatography

Summary The rapid separation of urinary aromatic acids by reversed-phase chromatography on 5-m octadecyl-silica columns and with gradient elution is described. The use of a tandem UV- and fluorescence detector system is demonstrated. The technique can be employed either to obtain chromatographic profiles of over 100 components in less than 30 minutes, or for the quantitative determination of some of the clinically significant aromatic acids. The latter approach is illustrated by the measurement of vanilmandelic acid (VMA) in human urine.     

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.     

Separation of porphyrin isomers by high-performance liquid chromatography

A high-speed reversed-phase high-performance liquid chromatographic method using an octadecylsilyl 3 cm long (3 microns particle size) column to separate the free acids of uroporphyrins I and III and coproporphyrins I and III from each other, and from the type I isomers of several other porphyrin carboxylic acids, is described. Separation of the porphyrins was achieved in less than 8 min, and injections were possible every 12 min. The detection limits of uroporphyrin, coproporphyrin, and mesoporphyrin were 75, 45, and 35 fmol (at a signal-to-noise ratio of 2), respectively. Application of the method to the determination of urinary and liver porphyrin patterns is shown.     

Separation and quantification of basic hydroxycinnamic amides and hydroxycinnamic acids by reversed-phase high-performance liquid chromatography

A method for the separation of basic hydroxycinnamic amides and hydroxycinnamic acids by high-performance liquid chromatography is described. N-p-Coumaryl-, N-caffeyl- and N-ferulylpetrescine, N-p-coumryl-, N-caffeyl- and N-ferulylspermidine and p-coumaric, caffeic, ferulic and sinapic acids were chromatographed on a μBondapak C₁₈ reversed-phase column (particle size 9 μm) with different methanol—water gradients as the mobile phase. It is possible with this high-resolution and reproducible method to assay biological samples containing mre that 10⁻⁵ M of hydroxycinnamic amides, using either p-coumaric or ferulic acid as the internal standard: this is demonstrated for tobacco extracts.     

Volatile fatty acids analysis from pig slurry using high-performance liquid chromatography

A simple and sensitive liquid chromatography method designed to determine the volatile fatty acids (VFA) content in pig slurry sample is proposed. The acids are isolated with a cation exchange column for carbohydrates using an isocratic phosphate eluant coupled with an ultraviolet detector in series. Centrifuged and filtered samples can be injected directly into the liquid chromatographic system. The accuracy of this new liquid-chromatographic chain using spiked solutions ranging between 20 and 5000?mg/L of VFA standard solution varied from 87 to 124%. The precision of the new procedure, expressed as variability, was between 9.2 and 0.6% for a spike solution concentration of 20 and 5000?mg/L, respectively.     

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.     

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.     

Separation and quantitation of nicergoline and related substances by high-performance liquid chromatography/atmospheric pressure ionization mass spectrometry

Summary This study demonstrated the utility of high-performance liquid chromatography/atmospheric pressure ionization mass spectrometry (HPLC/API-MS) in the investigation of 10-methoxy-1,6-dimethylergoline-8-methanol 5-bromonicotinic acid ester (Nicergoline) and its related substances. The analysis was performed by using an ODS column with ammonium acetate and methanol mixture as the mobile phase. Nicergoline and its related compounds could be characterized by HPLC/API-MS in terms of their molecular weight. The use of multiple ion detection techniques for the quantitation of these compounds was also investigated. The detection limits of nicergoline and its related substances were 5 to 10 ng each at a signal-to-noise ratio of 4. The method was also applied to the study of the decomposition products of nicergoline in simulated gastric and intestinal fluids.     

Separation of cytochromes c by reversed-phase high-performance liquid chromatography

Six kinds of cytochrome c of different origin, i.e., bovine, chicken, dog, horse, rabbit and tuna, were subjected to separation by reversed-phase high-performance liquid chromatography on three commercial packing materials; octadecyl-, octyl- and cyanoalkyl-silicas. The effects of reversed-phase material, mobile phase and temperature on the separation of cytochromes c were examined. The parameters of the mobile phase were the organic modifier, the pH, the salt concentration and additives. Under optimal conditions, five of the six cytochromes c were resolved in 10 min. The relative retention values cannot be explained in terms of the relative lipophilicities of the side-chains of the amino acid residues.     

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.     

Separation of polypeptide antibiotics by reversed-phase high-performance liquid chromatography

Summary The influence of different reversed-phase packings and the addition of acidic modifiers to the mobile phase was observed on the separation of basic and neutral polypeptide antibiotics by gradient elution. A dependence of pore size, coverage, reaction type and endcapping of the packings was not observed. Nevertheless, not all reversed-phase packings were suitable for the separation of polypeptides, especially of basic molecules. The addition of phosphoric or perchloric acid to the mobile phase prevented adsorption of the basic polypeptide antibiotics on the stationary phase.