Masako Maeda

Chemiluminescence high-performance liquid chromatography using N-(4-aminobutyl)-N-ethylisoluminol as a precolumn labelling reagent

Abstract N-(4-Aminobutyl)-N-ethylisoluminol (ABEI) was used as a pre-labelling reagent for primary and secondary amines and carboxylic acids. Chemiluminescence generated by the reaction of ABEI—hydrogen peroxide—potassium hexacyanoferrate(III) was applied to a detection system for high-performance liquid chromatography. The combination of 2.2 ml/min of 0.3 M hydrogen peroxid, 3.9 ml/min of 1.25 · 10 −2 M potassium hexacyanoferrate(III) in 2.6 M sodium hydroxide and 1.5 ml/min of eluent (methanol—water) using a reversed-phase column was optimum for the detection of femtomole amounts of ABEI derivatives. The detection limit for the ABEI derivative of cholic acid was 20 fmol.

Chemiluminescence enzyme immunoassay of cortisol using peroxidase as label

A new and highly sensitive enzyme immunoassay of cortisol was established using horseradish peroxidase as the label. Separation of free and bound cortisol was effected by insolubilized anti-cortisol antibody which was prepared by coupling the purified immunoglobulin G of antiserum with Sepharose 4B. The enzyme activity was measured by the chemiluminescence reaction using luminol and hydrogen peroxide as substrate. The faint chemiluminescence was measured by a photon counter. Comparison of assay results obtained by radioimmunoassay and this enzyme immunoassay showed excellent agreement of results in all cases (r = 0.913). The detection limit of cortisol was about 10 pg per assay tube. This enzyme immunoassay is applicable to the routine determination of plasma cortisol.

Separation and determination of bile acids by high-performance liquid chromatography using immobilized 3α-hydroxysteroid dehydrogenase and an electrochemical detector

A high-performance liquid chromatographic method using an immobilized 3 alpha-hydroxysteroid dehydrogenase column and an electrochemical detector was developed for the determination of individual bile acids in serum and bile. Bile acids in the eluate from a Radial-Pak A column reacted with NAD in the enzyme column to generate NADH, which was monitored by a voltammetric detector after mixing with phenazine methosulphate solution. Each bile acid was measurable at the 20 pmole level at the highest sensitivity of the detector. The mean recoveries and reproducibilities of bile acids were 86.7-104.6% [coefficient of variation (C.V.) = 0.3-8.7%] within-assay and 83.8-103.4% (C.V. = 1.6-7.0%] between-assay.

Fluorescence high-performance liquid chromatographic determination of free and conjugated bile acids in serum and bile using 1-bromoacetylpyrene as a pre-labeling reagent

A fluorescence high-performance liquid chromatographic method is described for the determination of free and conjugated bile acids in serum and bile. Free and conjugated bile acids are extracted from serum or bile using a Sep-Pak C18 cartridge and then fractionated on a piperidinohydroxypropyl Sephadex LH-20 column. Free and glycine-conjugated bile acids are labeled with 1-bromoacetylpyrene in acetonitrile using dicyclohexyl-18-crown-6-ether as catalyst. Taurine-conjugated bile acids are hydrolyzed by cholylglycine hydrolase and then derivatized by the same reagent. Derivatized bile acids are separated stepwise on a reversed-phase column (Radial Pak A) using acetonitrile-methanol-water (A) (100 : 50 : 40) and (B) (100 : 50 : 20) as mobile phase. The eluate is monitored by a fluorophotometer at 370 nm (excitation) and 440 nm (emission). Linearities of fluorescence intensities (peak heights) with the amounts of free and conjugated bile acids were obtained between 50 pmol and 200 pmol for free bile acids and between 25 pmol and 100 pmol for glycine-conjugated bile acids, respectively. Recoveries from serum and bile samples are not less than 90%. This method is sensitive, reliable and useful for the simultaneous determination of free and conjugated bile acids in serum and bile.

Fluorescene high-performance liquid chromatography of reducing using Dns-hydrazie as a pre-labelling reagent

Abstract The fluorescence high-performance liquid chromatography of reducing sugars is describe. Dns-hydrazine was used as a fluorescent pre-labelling reagent. Various reducing sugars, such as fucose, glucose, galactose and maltose, were labelled with Dns-hydrazine in trichloroacetic acid—ethanol solution and then chromatographed on LiChrosorb SI 100 column using ethanol—water—chloroform as eluent. Good separations of thirteen reducing sugars involving pentoses, hexoses and disaccharides were obtained within 25 min by stepwise elutin system. Calibration graphs are linear in the range from 0.05 to 1.0 nmol for each sugar. The detection limits were about 3– 20 pmol of reducing sugar.

Analysis of single-strand DNA conformation polymorphism by capillary electrophoresis

Analysis of single-strand conformation polymorphism (SSCP) by capillary electrophoresis (CE) was developed. The conformational change of single-strand DNA is caused by a mutation in a DNA fragment. The change is detected as mobility shift in CE. The effects of acrylamide gel concentration, running temperature and fragment size amplified by the polymerase chain reaction (PCR) were studied to develop the separation of SSCP. The model DNA used was the divE 42 gene carrying wild- and mutant-type (G-->A point mutation at the 141 site). The results show that two single-strand DNA fragments that differ in one nucleotide can be separated by CE within minutes. This method was also applied to the separation of SSCP for N-ras gene including four kinds of mutations. All mutations tested in this study could be distinguished. CE is well suited for clinical analysis of SSCP because it is rapid and reproducible, allows on-line detection and is easy.

Chemiluminescence enzyme immunoassay of dehydroepiandrosterone and its sulfate using peroxidase as label

We report a highly sensitive enzyme immunoassay for dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) using horseradish peroxidase as the label enzyme. Separation of free and bound DHEA-peroxidase conjugate was by insolubilized antibody, prepared by coupling purified IgG of goat anti-rabbit IgG serum with Sepharose 4B or a polystyrene tube. The enzyme activity was measured by the chemiluminescence reaction using luminol and hydrogen peroxide as substrate. The faint chemiluminescence was measured by a photon counter. The sensitivity was 25 pg/assay tube for DHEA and 100 pg/assay tube for DHEA-S. Upon comparison, results obtained by radioimmunoassay and this method showed good agreement ; r = 0.86 for free DHEA, r = 0.92 for acid-hydrolyzed DHEA-S and r = 0.91 for solvolyzed DHEA-S. The present method is applicable in the routine determination of DHEA and DHEA-S in biological fluid.

Simultaneous determination of alpha-fetoprotein, human chorionic gonadotropin and estriol in serum of pregnant women by time-resolved fluoroimmunoassay

We have developed a simple and rapid time-resolved fluoroimmunoassay (TR-FIA) for simultaneous determination of alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG) and estriol (E3) using europium and samarium ion chelate. In the proposed method, we used a combination of a 96-well microtiter plate for the AFP and hCG assay and transferable solid phase plate for the E3 assay. Therefore, these analytes could be measured simultaneously. The measurable ranges for AFP, hCG and E3 by the proposed method were 3.91-1000 ng ml(-1), 877-250000 IU l(-1) and 0.39 100 ng ml(-1), respectively. The proposed method which utilized characteristics of a rare earth ion chelate, was convenient (unnecessary diluting samples), quick (96 assays for 2 h), and required only a small quantity sample (50 microl). The principle of this proposed method is applicable to other antigens.

Analysis of polymerase chain reaction-product by capillary electrophoresis with laser-induced fluorescence detection and its application to the diagnosis of medium-chain acyl-coenzyme A dehydrogenase deficiency

Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) has been developed to detect polymerase chain reaction (PCR) amplified samples. LIF detection was performed using Thiazole Orange as the fluorescent intercalating dye. This method was ca. 100x as sensitive as that with UV detection. The highly sensitive CGE-LIF was applied to the detection of the most prevalent mutation (lysine329-to-glutamic acid substitution) in medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency. The disorder, which shows an autosomal recessive inheritance, is known to be highly prevalent among Caucasian population and often mimics as Reye-like syndrome or sudden infant death. A DNA fragment containing the mutation site was PCR-amplified with two sets of allele specific oligonucleotide primers, followed by CGE-LIF. The mutant allele produced a 175-base pairs DNA fragment, which the normal allele generated a 202-base pairs DNA fragment. CGE-LIF clearly distinguished these PCR products, facilitating rapid diagnosis of MCAD deficiency.

Chemiluminscence with lucigenin as post-column reagent in high-performance liqusd chromatography of corticosteroids and p-nitrophenacyl esters

Abstract Compounds having an α-hydroxycarbonyl group give an intense chemiluminescence with lucigenin in alkalilne solution. Based on this reaction, the chemiluminescence detection of corticosteroids and p -nitrophenacycl esters of car☐ylic acids has been developed. These compounds were separated by high0performance liquid chromatography on a reversed-phase column (Zorbax ODS) with a methanol—water (7:3) mixture as eluent, and detected by the chemiluminescence reaction with lucigenin—potassium hydroxide solution as the post-column reagent. The detection limits of corticosteroids and p -nitrophenacyl eters were ca . 0.5 pmol per injection.