Kazuaki Akasaka

Study on aromatic phosphines for novel fluorometry of hydroperoxides (I): synthesis and spectral properties of diphenyl aryl phosphines and their oxides

Abstract 1-Naphthyldiphenylphosphine (NDPP), 9-anthryldiphenylphosphine (ADPP) and diphenyl-1-pyrenylphosphine (DPPP) and their oxides were prepared from triphenyl-phosphine (TPP) and the corresponding aryl bromides. These phosphines had no fluorescence, but their oxides showed blue fluorescence. The order of fluorescence intensities of these oxides were DPPP oxide > ADPP oxide > NDPP oxide. Hydroperoxides oxidized these phosphines to the corresponding oxides quantitatively. The relative reactivities were TPP > NDPP > DPPP > ADPP. The phosphines were stable in the dark, however, unstable in the light in chloroform. The reaction is proposed as novel type of fluorometry for hydroperoxides.

Determination of triacylglycerol and cholesterol ester hydroperoxides in human plasma by high-performance liquid chromatography with fluorometric postcolumn detection

Cholesterol ester (ChE) and triacylglycerol (TG) hydroperoxides in human plasma were determined by high-performance liquid chromatography with postcolumn detection with diphenyl-1-pyrenylphosphine. Human plasma was extracted once with n-hexane. 2,6-Di-tert.-butyl-4-methylphenol and N-stearylcinnamide were added to human plasma before extraction as an antioxidation agent and an internal standard, respectively. The detection limits of both ChE and TG hydroperoxides were 1 pmol. The sample size was minimized to 250 microliters for each run. The recoveries of ChE and TG hydroperoxides from fresh plasma were ca. 90 and 80%, respectively. The relative standard deviations (n = 8) of their values in frozen human plasma were 5.4% (ChE hydroperoxides, 298 nM) and 5.7% (TG hydroperoxides, 267 nM). No TG hydroperoxides and 24.5 +/- 9.6 nM (n = 15) ChE hydroperoxides were detected in fresh human plasma. The relative standard deviation (n = 8) of ChE hydroperoxides values in fresh plasma was 5.8% (27.1 nM).

Study on Aromatic Phosphines for Novel Fluorometry of Hydroperoxides (II) -The Determination of Lipid Hydroperoxides with Diphenyl-1-Pyrenylphosphine -

Abstract A new fluorometric determination of hydroperoxides was proposed. Non-fluorescent diphenyl-1-pyrenyl-phosphine (DPPP) was oxidized quantitatively by hydroperoxides to a strong fluorescent DPPP oxide. A sensitive assay for lipid hydroperoxides was developed based on the reaction. The reaction of lipid hydroperoxides with DPPP was conducted in the dark in the mixture of chloroform and methanol at 60°C within 60 min. The contents of hydroperoxides showed linear relation to the fluorescence intensities in a wide concentration range. The sensitivity was 10,000 times higher than conventional iodometry. The correlation constant between peroxide values obtained by iodometry and by this method was 0.9993 (n=10). The sample size was minimized to less than 3 mg per tube.

Automatic determination of hydroperoxides of phosphatidylcholine and phosphatidylethanolamine in human plasma.

An automatic method for the determination of hydroperoxides of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is reported. Sample plasma was deproteinized with a fourfold volume of methanol. After centrifugation, the supernatant was injected directly into an HPLC system without further treatment. The hydroperoxides of PC and PE were concentrated and washed on an ODS column followed by introduction into two analytical columns, a silica gel and an aminopropylsilica gel column, which were connected in series, by column switching. After the separation, they were detected by postcolumn detection with diphenyl-1-pyrenylphosphine. The compounds were determined at picomole levels within 30 min with good reproducibilities. By using only a silica gel column as an analytical column, PC hydroperoxides were determined within 20 min, and samples could be injected into it at 15-min intervals. Those methods made it possible to inject a sample of up to 2 ml at one time and up to 8 ml by repeated injections and to determine phospholipid hydroperoxides in human plasma at picomole levels.

Determination of carboxylic acids by high-performance liquid chromatography with 2-(2,3-anthracenedicarboximido)ethyl trifluoromethanesulfonate as a highly sensitive fluorescent labelling reagent

2-(2,3-Anthracenedicarboximido)ethyl trifluoromethanesulfonate (AE-OTf) is a highly sensitive fluorescent labelling reagent for carboxylic acids for use in liquid chromatography. The labelling reaction of carboxylic acids with AE-OTf was completed within 10 min at room temperature in acetonitrile in the presence of tetraethylammonium carbonate as a base. The 2-(2,3-anthracenedicarboximido)ethyl esters of 18 kinds of fatty acids including polyunsaturated fatty acids were separated from each other on an ODS column with an isocratic solvent system within 30 min. The relative standard deviations of their peak areas were within 2%(n= 8, 1.4–3.3 pmol on-column). Their relative peak areas were almost 1.0 and their detection limits were 0.8–2.7 fmol (signal-to-noise ratio = 3). These fatty acids were successfully determined at least over the range 20 fmol-6 pmol (correlation coefficient r >0.999 for all fatty acids tested).

Simultaneous determination of hydroperoxides of phosphatidylcholine, cholesterol esters and triacylglycerols by column-switching high-performance liquid chromatography with a post-column detection system

A method for the simultaneous determination of hydroperoxides of phosphatidylcholines (PC), triacylglycerols (TG) and cholesterol esters (CE) has been developed. A sample was separated into a combined TG and CE hydroperoxides fraction and a PC hydroperoxides fraction on a short silica column. The fractions were introduced into an ODS column and another silica column by a valve-switching device. The PC hydroperoxides were monitored by a post-column detection system with diphenyl-1-pyrenylphosphine, and the TG and CE hydroperoxides were monitored by another switching device. With this system, the hydroperoxides were determined at the picomole level within 32 min. Their detection limits were 2-4 pmol at a signal-to-noise ratio of 3, and the relative standard deviations of the peak areas were 1.6-3.1%. This method was successfully applied to determine lipid hydroperoxides in human plasma.

Development of phosphine reagents for the high-performance liquid chromatographic-fluorometric determination of lipid hydroperoxides

Phosphine reagents were designed and synthesized as a new type of fluorescent reagents for the determination of lipid hydroperoxides in foodstuff and biological materials. All phosphine reagents prepared had no fluorescence but their oxides, which were produced by the reaction of the phosphines with hydroperoxides, had strong fluorescence. Among the phosphine reagents prepared, diphenyl-1-pyrenylphosphine had the most suitable properties as a fluorescent reagent and was successfully applied to the determination of hydroperoxides by batch, flow injection and HPLC post-column methods.

An aromatic phosphine reagent for the HPLC-fluorescence determination of hydroperoxides ― Determination of phosphatidylcholine hydroperoxides in human plasma

Abstract A new method is developed to determine picomole levels of phosphatidylcholine hydroperoxides (PC-HPO) in human plasma. The PC-HPO was extracted from 500 μ1 of human plasma with a mixture of chloroform and methanol and separated on a HPLC with a silica gel column using the chloroform - methanol - water system. Detection was carried out by fluorometry after post column derivatization with diphenyl-1-pyrenylphosphine CDPPP). The determination of PC-HPO was performed by the method of standard addition. The sampling error was corrected by using the UV peak of inherent unoxidized PC as an internal standard. By this method, the PC-HPO levels in healthy adult human plasma were 20–40 pmol/ml which compares favorably to the normal expected values.

Normal-phase high-performance liquid chromatography with a fluorimetric postcolumn detection system for lipid hydroperoxides

Abstract Hydroperoxides (HPO) of triacylglycerols (TG) and cholesterol esters (ChE) were selectively determined at picomole levels with a fluorescence detector by postcolumn reaction with diphenyl-1-pyrenylphosphine. Hydroperoxides were separated on a normal-phase silica gel column with gradient elution with n-hexane-1-butanol. With this system, TG-HPO and ChE-HPO were separated according to their class and determined in the range 5–1000 pmol. Detection limits of hydroperoxides of cholesterol linolate and trilinolein were about 2 pmol (signal-to-noise ratio = 3) and the relative standard deviations of their peak areas were 3.4% (39.1 pmol, n = 7) and 1.8% (32.4 pmol, 4 = 7), respectively.

Enantiomeric separation of carboxylic acids having chiral centers remote from the carboxyl group by labelling with a chiral fluorescent derivatization reagent

Abstract Enantiomeric separations of 2-, 3-, 4-, 5- and 6-methyl fatty acids and 3-, 4- and 5-hydroxy fatty acids derivatized with ( S )-(+)-2-(anthracene-2,3-dicarboximido)-1-propyl trifluoromethanesulfonate are described. Although there are 4–8 bond distances between the chiral centers of these diastereomeric derivatives, they are separated on HPLC and detected at fmol levels.