Yukie Tsukamoto

Determination of the fluorescent drugs dipyridamole and benzydamine in rat plasma by liquid chromatography with peroxyoxalate chemiluminescence detection

Abstract A facile and sensitive method for the determination of the fluorescent drugs dipyridamole (coronary vasodilator) and benzydamine hydrochloride (anti-inflammatory) in rat plasma is presented. The method consists of the addition of an internal standard (DNS- l -phenylalanine for dipyridamole and dipyridamole for benzydamine hydrochloride), deproteinization of plasma (1 μl for dipyridamole and 10 μl for benzydamine hydrochloride) with 13 volumes of acetonitrile, direct injection of the supernatant into an ODS column, separation by liquid chromatography and peroxyoxalate chemiluminescence detection using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate and hydrogen peroxide. Calibration graphs were linear over the ranges 2.5–200 nM and 2.5–100 μM for dipyridamole and benzydamine hydrochloride in plasma, respectively. The detection limits (signal-to-noise ratio = 2) were 345 pM for dipyridamole and 147 nM for benzydamine hydrochloride in plasma. The relative standard deviation for four determinations of dipyridamole at 2.5 nM and benzydamine hydrochloride at 2.5 μM in plasma were 3.7% and 2.6%, respectively. The method was applied to real samples and the time courses of the plasma concentration after oral administration of the two drugs [0.50 mg kg −1 (0.99 μ mol kg −1 ) and 10 mg kg −1 (29 μ mol kg −1 ), respectively] were obtained.

Application of bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)-phenyl] oxalate to post-column chemiluminescence detection in high-performance liquid chromatography

Bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate (TDPO) was used to examine the peroxyoxalate chemiluminescence (CL) reaction for the detection of fluorescent compounds. Some fluorescent compounds (perylene, eosine, rhodamine, Rose Bengal, fluorescein and umbelliferone) gave higher CL intensities as the proportion of water in the reaction medium increased to ca. 40%, whereas dansylalanine, 8-anilinonaphthalene-1-sulphonic acid, 7-nitrobenzo-2-oxa-1,3-diazol-4-ylproline and dihydronicotinamide adenine dinucleotide gave opposite results. The effects of temperature and time on the post-column reaction in reversed-phase high-performance liquid chromatography (HPLC) were investigated. Under the optimal conditions, the detection limit for dansylamino acids was at the sub-femtomole level. The advantage of using TDPO in HPLC is its stability in the presence of hydrogen peroxide [ca. 10% loss of activity per 8 h vs. 60% per 8 h for bis(2,4,6-trichlorophenyl) oxalate].

Sensitive detection of oxo-steroids and oxo-bile acids by liquid chromatography with peroxyoxalate chemiluminescence detection

Abstract Oxo-steroids and oxo-bile acid ethyl esters were derivatized with 5- N,N -dimethylamino-naphthalene-1-sulphonohydrazide (DNS-hydrazine) to DNS-hydrazone in the presence of hydrochloric acid in ethanol or trifluoroacetic acid in benzene, purified by high-performance gel-permeation chromatography, separated on an ODS column with an eluent containing tetrahydrofuran and detected via a peroxyoxalate chemiluminescence reaction using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate (TDPO). Sensitive detection (femtomole level) of each oxo-steroid which appeared as a single peak was achieved. The procedure for the isolation of oxo-bile acids developed for GC-MS allowed the detection by this system of an unusual oxo-bile acid, 7α-hydroxy-3-oxo-5β-cholanic acid, at the nanomole l −1 level in urine from a patient with cholestatic liver disease.

Prediction of the detection ranges for HPLC-peroxyoxalate chemiluminescence detection system of fluorescent compounds

SummaryA manual method for predicting the detection ranges of fluorescent compounds for the HPLC-peroxyoxalate chemiluminescence detection system is presented utilizing bis(4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) as a chemilumigenic reagent. The generated chemiluminescence decay curves were measured on a photomultiplier and extrapolated to zero time based on the first part of the decay curve. Dipyridamole, perylene, DNS-Ser, Rose Bengal, DNS-Asp, Trp-P-1, pyrencarboxylic acid methyl ester, perfenazine, alimemazine, oxypertine, Glu-P-2, benzydamine and doxorubicin gave chemiluminescence intensity (Icl) values of 3.2 × 106, 2.8 × 106, 2.3 × 105, 2.1 × 105, 1.9 × 105, 1.4 × 105, 9.4 × 103, 6.2 × 103, 4.2 × 103, 3.3 × 103, 2.3 × 103, 9.7 × 102 and 3.1 × 102, respectively. The reaction conditions for the HPLC-CL detection system were investigated and optimum conditions obtained.

Synthesis and evaluation of aryl oxalates as peroxyoxalate chemiluminescence reagents

Eight aryl oxalates having an alkyoxy moiety were synthesised to improve the solubility in acetonitrile for the peroxyoxalate chemiluminescence reaction system. Of these, bis[2-(3,6-dioxaheptyloxycarbonyl)-4-fluorophenyl] oxalate showed the highest solubility (1685 mM). To evaluate these esters together with some known aryl oxalates as chemiluminescence reagents, a simple and rapid method using a flow injection system was developed. The chemiluminescence reaction system consisted of an oxalate, hydrogen peroxide, triethylamine and 9,10-diphenylanthracene in acetonitrile. Of the oxalates synthesised, bis[2-(3,6-dioxaheptyloxycarbonyl)-4-bromophenyl] oxalate gave the highest intensity, which was about 100 times lower than that of bis(2,4-dinitrophenyl) oxalate. However, the chemiluminescence intensities of the oxalates synthesised continued for a far longer period of time than those of commercially available oxalates.

Synthesis of pyrimido[5,4-d]pyrimidine derivatives and their ultraviolet absorption and fluorescence spectral properties

Abstract This paper describes the synthesis of some tri- and tetra-substituted pyrimido[5,4-d]pyrimidine derivatives and their absorption and fluorescence spectral properties, in the expectation of such compounds having application as fluorescence reagents in the peroxyoxalate-chemiluminescence reaction system. Of the 18 derivatives investigated, the tetra-substituted m-methoxyanilino compound showed the strongest relative fluorescence intensity.

Effect of tetrahydrofuran on reversed-phase liquid chromatographic separation of dansylhydrazones of oxo-steroids

The oxo-steroids corticosterone, testosterone and progresterone were converted with 5-N,N-dimethylaminonaphthalene- 1 -sulphonohydrazide (DNS-hydrazine) into DNS-hydrazones, which were separated by reversed-phase liquid chromatography and detected fluorimetrically. The anti and syn conformers of the hydrazones were recognized on a C18 column with eluents consisting of imidazole buffer (nitrate, pH 6.0) containing acetonitrile, methanol, acetone or dioxane, and thus two peaks were observed for each oxo-steroid derivative. On the other hand, a single peak was obtained when an eluent containing tetrahydrofuran was used, affording highly sensitive detection of oxo-steroids. A further improvement in the detection of oxo-steroids down to subpicomole level was achieved by adding a gel permeation chromatographic clean-up procedure to remove the excess reagent before the analysis by reversed-phase liquid chromatography.

Dynamic analytical chemistry: a trial study of the interaction of fluorogenic reagents with living chinese hamster ovary cells

Abstract The concept of dynamic analytical chemistry, which deals with the qualitative localization or the quantitative transportation of a known or unknown component or its conversion into a product within living cells, is introduced. The concept was tested using cultured Chinese hamster ovary cells in a phosphate-buffered saline medium in the presence of fluorogenic reagents for amines and thiols. The phenomenon was observed and recorded with a fluorescence microscope equipped with a SIT camera. Among the reagents, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) added to the culture medium was first trapped at the surface of the cells and then passed inside the cells to react with the mitochondria to give yellow fluorescence, o -Phthalaldehyde and 4-aminosulphonyl-7-fluoro-2, 1,3-benzoxadiazole reacted with the cells to give a blue florescence at certain sites inside the cells. One of the two major components labelled with NBD-F appeared to be phosphatidylethanolamine, a component of the plasma membrane. Its identification and quantitation were effected by conventional analytical techniques, such as thin-layer and liquid chromatography, following enzymatic hydrolysis. The fate of the N -NBD-phosphatidylethanolamine is discussed.

A Newly Designed Simple Method for the Measurement of Percutaneous Absorption of Drugs into Human Skin from Ointments. Its Application to 2% Aspirin Ointment.

A simple method was newly designed and compared with a conventional method for the measurement of percutaneous absorption of drugs into human skin from ointments.The ointment was put on the gauze (8×17mm) of a sticky plaster, the rear side of which was covered with a selophan tape, and applied to the human skins.At intervals, the residual drug in the ointment was extracted and measured.The percutaneous absorption of the drug was calculated on the residual drug concentration.The absorption of aspirin from 2% aspirin ointment, which was effective for the cure of postherpetic neuralgia, was investigated by the proposed methods using isopropanol extraction and HPLC determination of the residual aspirin in the ointment.There was no great difference between the absorption of aspirin into the arm and that into the backskin of a normal subject.About 35% of the aspirin was absorbed for 7 hours.A similar absorption pattern was observed in the two patients of postherpetic neuralgia. Thus, the proposed method might be useful for measuring absorption of many other drugs and investigating the effect of bases and accelerators on the absorption into the human skins.