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In this study, we examined the affinities of many (21) compounds such as hormones, pharmaceuticals, industrial chemicals, and phytoestrogens to the estrogen receptor (ER) by ER binding assay using fluorescence polarization (FP). This method is based on the competitive binding assay using fluorescein-labeled estradiol (F–E2), in which the polarization values decreased with the addition of the compounds (competitors). The obtained sigmoidal inhibition curves were transformed into the pseudo-Hill plots, and the concentrations at 50% inhibition (IC₅₀) and Hill coefficients were obtained from the regression equations. We examined the relationship between the chemical structures and estrogenic activities, and finally classified the tested compounds into three categories, agonists, partial agonists and antagonists based on their Hill coefficients.

A reversed-phase high-performance liquid chromatographic (HPLC) method for the femtomole determination of nine saturated carboxylic acids, n-butyric (C₄), n-hexanoic (C₆), n-caprylic (C₈), n-decanoic (C₁₀), lauric (C₁₂), n-tetradecanoic (C₁₄), palmitic (C₁₆), stearic (C₁₈) and arachidic (C₂₀), based on the condensation reaction of these acids with a newly synthesized water-soluble benzofurazan fluorescent reagent, 7-(N,N-dimethylaminosulfonyl)-4-N-(4-N-aminoethyl)piperazino-2,1,3-benzoxadiazole (DBD-PZ-NH₂), was developed. The derivatization reaction proceeds with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) in the presence of the catalyst 4-(dimethylamino)pyridine (DMAP). A model derivative of the reagent with n-caprylic acid (C₈) was synthesized for fluorescence excitation/emission characterization. Depending on the solvents, including water, methanol, acetonitrile, 1,4-dioxane or N,N-dimethyformamide (DMF), the C₈ derivative has a fluorescence emission with a fluorescence quantum yield (ϕ) ranging from 0.01 to 0.20 in the region from 545 to 580 nm. An exponential increase in ϕ was observed with increasing acetonitrile content. The calculated detection limits (signal-to-noise ratio = 3∶1) of the proposed method for the above nine carboxylic acids were 9.1, 4.0, 2.5, 2.2, 2.0, 1.8, 1.2, 1.0 and 1.3 fmol, respectively. Biological samples including Intralipos 20% and rat plasma were analysed satisfactorily.

A column-switching HPLC method employing both octadecylsilica (ODS) and chiral columns with fluorescence detection was developed for the determination of enantiomer of fluoxetine (FLX), an antidepressant drug, in rat plasma. Racemic FLX was derivatized with a fluorescent reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) or 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and the enantiomeric separation of the resultant derivatives was examined on an amylose-based chiral column (CHIRALPAK AD-RH) in the reversed-phase mode. The derivative with NBD-COCl (NBD-FLX) showed a sufficient separation factor (α) and resolution (R_s) compared with that with DBD-COCl. Thus, FLX was derivatized with NBD-COCl and the resultant NBD-FLX was first quantified on the ODS column and then introduced to the CHIRALPAK AD-RH column via a six-port switching valve to examine the enantiomeric ratio. The intra- and inter-day accuracy (97.6–112.7%) and precision (1.47–10.60%) were satisfactory in the range 10–1000 nM FLX and the limit of quantification was approximately 10 nM. The absolute recoveries of FLX with hexane from rat plasma were in the range 87.5–92.2% (n = 3). The method was applied to determine FLX enantiomers in the plasma of rats administered FLX orally, and it was shown that the R-isomer was eliminated faster than the S-isomer.

A fully automated analyzer for methylated l-arginine metabolites [N,N-dimethyl-l-arginine (ADMA), N-methylarginine (NMMA) and N,N'-dimethyl-l-arginine (SDMA)] by high-performance liquid chromatography with post-column fluorescence derivatization was developed. This system consists of an on-line extraction, a separation on a reversed phase ion-pair chromatograph, a post-column derivatization by o-phthaladehyde (OPA) and thiol reaction, and fluorescence detection. NMMA, ADMA and SDMA were separated in 40 min with isocratic elution by a combination of octanoate and cyclohexane carboxylate as ion-pair reagents. The eluate was monitored at 450 nm with excitation at 337 nm. The calibration curves for NMMA, ADMA and SDMA showed linearity over the range from 0.05 μmol l−1 (0.5 pmol on column) to 5.0 μmol l−1 (50 pmol on column). This method does not require any time-consuming pre-treatment and requires only 10 μl of plasma sample for assay.

We propose a highly sensitive method for the measurement of catechol-O-methyltransferase (COMT) activity with norepinephrine (NE), an endogenous native substrate. The product, normetanephrine, was determined by high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence reaction detection or, if required, less sensitive fluorescence detection. For the measurement of membrane-bound (MB)-COMT activity in the rat erythrocyte, the HPLC-peroxyoxalate chemiluminescence reaction detection was employed. Soluble (S)- and MB-COMT activities in the rat erythrocyte were 22.9 ± 2.5 and 4.62 ± 1.23 pmol min⁻¹ (mg protein)⁻¹, respectively (n = 5). The K_m values obtained for S- and MB-COMT were 366 ± 31 μmol l⁻¹ and 12.0 ± 1.1 μmol l⁻¹, respectively (n = 5), suggesting that the use of NE as a substrate would give more precise information on the role of both isoenzymes. However, with dihydroxybenzoic acid as an artificial substrate, the K_m values for S- and MB-COMT were similar, with values of 69.2 ± 11.4 μmol l⁻¹ and 72.2 ± 9.2 μmol l⁻¹, respectively. The proposed method is thought to be useful for the measurement of both S-COMT and MB-COMT activities, and would give us critical information on the role of metabolism of catecholamines in rat tissues.

In order to elucidate the fluorescence ‘on–off’ switching mechanism of benzofurazan compounds bearing an aromatic substituent group, we first studied the intermolecular electron transfer between fluorescent benzofurazan compounds and other aromatic compounds. The fluorescence was quenched by the added aromatic compounds without changing the absorption and excitation wavelengths, indicating that quenching was caused by intermolecular electron transfer. The quenching abilities of the added aromatic compounds reflected the K values of the Stern–Volmer plot. The K values were also co-related with the molecular orbital energies of the benzofurazan and the added aromatic compounds calculated with the semi-empirical PM3/COSMO method. Next, 11 benzofurazan compounds bearing an aromatic substituent group were synthesized and the effects of the intramolecular electron transfer between the benzofurazan skeleton and the aromatic substituent group were studied. A benzofurazan compound bearing an aromatic substituent group which has a high quenching ability (large K value) in the study of intermolecular electron transfer did not fluoresce (fluorescence ‘off’) because of the intramolecular electron transfer, whereas a compound bearing an aromatic substituent group which has a low quenching ability (small K value) fluoresced (fluorescence ‘on’). From the results, the fluorescence ‘on–off’ properties of benzofurazan compounds bearing an aromatic substituent group were predictable from the estimation of the quenching ability of the aromatic substituent group using the Stern–Volmer plot. Finally, the prediction of the fluorescence ‘on–off’ properties of benzofurazan compounds was applied to the development of a new fluorescent ‘on–off’ reagent for oxidation. The results are useful in the context of the development of new fluorescent ‘on–off’ reagents for use in analytical chemistry.

A fully automated and highly sensitive method with a semi-microcolumn liquid chromatography system for the determination of rat plasma catecholamines (CAs) was developed. Automated on-line extraction of CAs in diluted plasma using a precolumn packed with strong acidic cation exchange resin was coupled with separation of CAs on a semi-microcolumn (250 × 1.5 mm id), fluorogenic derivatization with ethylenediamine and finally postcolumn peroxyoxalate chemiluminescence detection utilizing bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl]oxalate (TDPO) and hydrogen peroxide. The detection limits were 0.91, 0.36 and 1.1 fmol for norepinephrine (noradrenaline), epinephrine (adrenaline) and dopamine, respectively, at a signal-to-noise ratio of 3. A good linearity of the calibration curve for each CA was observed in the range of 5.0 to 500 fmol for each CA using N-methyldopamine (N-MeDA) as an internal standard. The RSD for the proposed method (n = 5) were 3.7–9.5% for the intra-day assay and 6.6–10.0% for the inter-day assay. The volume of rat plasma required for the determination of CAs was 10 μl.

In our previous studies, we found a relationship between the fluorescence characteristics (the fluorescence intensity, the maximum excitation and emission wavelengths) and Hammett substituent constants (ςp) of the substituent groups at the 4- and 7-positions of benzofurazan compounds, which enabled us to predict the fluorescence characteristics of the benzofurazan compounds. This prompted us to design a new fluorogenic derivatization reagent having a benzofurazan skeleton for carboxylic acids along this line of thought. Accordingly, the fluorogenic reagent, 7-acetylamino-4-mercapto-2,1,3-benzoxadiazole (AABD-SH) was synthesized. The reagent itself fluoresced very weakly but fluoresced strongly after the reaction with carboxylic acids. AABD-SH reacted with carboxylic acids within 5 min at room temperature in the presence of triphenylphosphine and 2,2′-dipyridyl disulfide. The derivatives of five carboxylic acids were separated on a reversed phase column and detected fluorimetrically at 524 nm with excitation at 368 nm. The detection limits were 10–20 fmol (signal-to-noise ratio of 3).

Two newly synthesized fluorescence Edman reagents with the benzofurazan structure, 7-phenylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (PSBD-NCS) and 7-methylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MSBD-NCS), were compared with 7-aminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (ABD-NCS) and 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) for peptide and protein sequence analysis by the generation of fluorescent 2,1,3-benzoxadiazolylthiazolinone (TZ)-amino acids. The effects of the substituent group at the para position to the isothiocyanate moiety of these reagents on the rate of the cyclization/cleavage reaction, the repetitive yield and the fluorescence quantum yield and stability of TZ amino acids were investigated. MSBD-TZ-amino acids were most sensitively detected and the detection limit for MSBD-TZ-Pro was 7 fmol (S/N = 3). ABD-NCS afforded the highest repetitive yield in the sequencing analysis. Fewer interfering peaks were observed in the chromatogram with DBD-NCS.

In order to improve the high-performance liquid chromatographic separation of α-amino acids derivatized with the fluorogenic reagent 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) on commercially available chiral stationary phases (CSPs) such as SUMICHIRAL OA-2500(S) (CSP 1) and OA-4700 (CSP 3), the preparation of two new CSPs (CSP 2 and CSP 4) having 11-aminoundecanoic acid between the aminopropyl silica gel support and the chiral moiety in CSP 1 and CSP 3 is described. CSP 2 and CSP 4 improved both the mutual and enantiomeric separation of NBD-amino acids compared with CSP 1 and CSP 3. Thus, 17 pairs of NBD-amino acid enantiomers and NBD-glycine were separated on CSP 2 except for six NBD-amino acids (D-Asn, D-Ser, D-Gln, L-Pro, L-Ser and Gly). CSP 2 and CSP 4 also showed better enantiomeric separation of NBD-amino acid esters and amides than CSP 1 and CSP 3. It was considered that the achiral long alkyl chains in the CSPs might form a hydrophobic space which assisted the stereoselective interaction of analytes with the chiral moiety by changing the environment around the chiral moiety. On CSP 1 and CSP 2, NBD-β-amino acid was also enantiomerically separated.

A semi-automatic method for the simultaneous determination of the sequence and the D/L-configuration of amino acids in peptides is reported. The automated sequencing program was set up based both on the yield in the cyclization/cleavage reaction with 40 mmol dm–3 boron trifluoride (BF3) etherate at 48 °C for 5 min and on the recovery of anilinothiazolinone- (ATZ-) amino acids extracted with ethyl acetate and 1-chlorobutane. The D/L-configuration of phenylthiocarbamoylamino acids was determined manually after hydrolysis of ATZ-amino acids by HPLC using a combination of a reversed phase and a chiral stationary phase of β-cyclodextrin. The hydrolysis conditions for ATZ-amino acids were examined with HCl to suppress the amino acid racemization, and 0.1 mol dm–3 HCl at room temperature for 5 min was selected for hydrolysis. The sequence and D/L-configuration determination of a synthetic β-amyloid 1–40 peptide was carried out and up to 10 residues could be successfully analyzed by the semi-automated sequencing procedure without racemization.

It is shown that an electron-withdrawing or -donating group at the para-position of aromatic isothiocyanate significantly affects the racemization of 2,1,3-benzoxadiazolylthiazolinone (TZ) derivatives of amino acids, derivatized with newly synthesized benzoxadiazolyl isothiocyanates in Edman sequence analysis. A linear relationship between the logarithms of the TZ-amino acid enantiomer ratio and the para-substituent constants (ς_p) for the isothiocyanate moiety was obtained, and the D/L configuration of the amino acid residue was retained with an isothiocyanate containing an electron-donating group at the para-position. The para-substitution effect on the racemization of phenylthiohydantoin (PTH) amino acids was also confirmed by several para-substituted phenylisothiocyanate (PITC) reagents, including nitro-PITC, chloro-PITC, PITC, methyl-PITC and methoxy-PITC. The relationship between the fluorescence intensity of the 2,1,3-benzoxadiazolyl TZ amino acid and ς_p was also demonstrated. When the isothiocyanate containing an electron-donating group was used, the fluorescence intensity of the TZ-amino acid decreased while retaining the D/L configuration of the amino acid residues.