4-Iodobenzonitrile as a fluorogenic derivatization reagent for chromatographic analysis of L-p-boronophenylalanine in whole blood samples using Suzuki coupling reaction.

BACKGROUND: L-p-Boronophehylalanine (BPA) is used in boron neutron capture therapy (BNCT), which is a novel selective cancer radiotherapy technique. It is important to measure BPA levels in human blood for effective radiotherapy; a prompt gamma-ray spectrometer, ICP-AES, and ICP-MS have been used for this purpose. However, these methods require sophisticated and expensive apparatuses as well as experienced analysts. Herein, we propose an HPLC-FL method for the determination of BPA after precolumn derivatization. A new fluorogenic reagent for aryl boronic acid derivatives, namely, 4-iodobenzonitrile, was employed for the fluorogenic derivatization of BPA based on the Suzuki coupling reaction. RESULTS: After the fluorogenic derivatization, a fluorescent cyanobiphenyl derivative is formed with maximum fluorescence at 335 nm after excitation at 290 nm. The developed method showed good linearity (r2=0.997) over the concentration range of 0.5-1000 nmol/L, and the detection limit (S/N = 3) was 0.26 nmol/L. The proposed method is more sensitive than previously reported methods for the determination of BPA, including the ICP-MS. Finally, the proposed method was successively applied to the measurement of BPA in human whole blood samples with a good recovery rate (≥95.7 %) using only 10 µL of blood sample. The proposed method offers a simple and efficient solution for monitoring BPA levels in BNCT-treated patients. SIGNIFICANCE: 4-Iodobenzonitrile was investigated as a new fluorogenic reagent for BPA based on Suzuki coupling. A new HPLC-FL method for BPA in whole blood samples with ultrasensitivity was developed. The developed method is superior in sensitivity to all previously reported methods for BPA. The method requires only a very small sample volume, making it suitable for micro-blood analysis of BPA via fingerstick sampling.

Extremely Simple and Rapid HPLC Analysis of Tocilizumab in Human Serum with Selective Precipitation Using Alkylamine.

An assay using HPLC with fluorescence (FL) detection method for monitoring native FL of tocilizumab (TCZ) in human serum combined with extremely simple and rapid pretreatment without any antigen-antibody reaction was developed. Good separation of TCZ was achieved within 13 min on a Presto FF-C18 column (100 × 4.6 mm i.d., 2 µm). Simple pretreatment with acetonitrile containing primary and secondary alkylamines having longer than C3 in the alkyl chain removed immunoglobulin G subclass 1 and TCZ could be recovered selectively. The spiked calibration curve of TCZ in human serum showed good linearity in the range of 40-1000 µg/mL (r > 0.997). The lower limit of quantitation (S/N = 10) of the TCZ was 19.7 µg/mL. The accuracy was within 103.5-114.9%, and the intra- and inter-day precisions as relative standard deviations were less than 5.3 and 7.8% (n = 5), respectively. The recovery of TCZ was 42.2 ± 3.4% (n = 3). The TCZ in pretreated sample was confirmed to be stable for 6 h (>95%) at room temperature and 24 h (>95%) at 4 °C. The proposed method is considered extremely superior to the previous methods in terms of time requirement for analysis. Therefore, the developed method may be more useful than conventional methods in urgent situations, such as confirming therapeutic efficacy of cytokine-release syndrome by 2019 coronavirus disease.

Different properties of two types of red fluorescent proteins in octocoral, Scleronephthya spp. as Akane families.

We report the different properties of two types of red fluorescent proteins (RFP), undescribed species, extracted from two octocorals, Scleronephthya sp. 1 (S. sp. 1) and S. sp, 2 (Alcyonacea, Nephtheidae). S. sp. 1, named Alc-Orange, emits strong green emission at 492 nm and weak red emission at 590 and 630 nm when excited at 449 and 574 nm, respectively. S. sp. 2, LS-Red, emits strong deep red at 642 nm and weak green at 480 and 510 nm when excited at 574 nm and 434 nm, respectively. LS-Red has a very large Stokes shift of about 208 nm emitting at 642 nm when excited at 434 nm. Interestingly, LS-Red shows some emissions at 480 (blue emission), 514 (green emission), 563 (orange emission), and 642 nm (deep red emission) continuously at pH 7.5, which means multicolored fluorescence protein by one excitation at 434 nm. In pH dependence of fluorescence of Alc-Orange (pH 13 to 3.5), no relation between 'green and red FPs' was observed, whereas LS-Red showed the interconversion between 'green and red forms' depending on pH (11.5 to 4.5).

Simple and rapid analysis of tocilizumab using HPLC-fluorescence detection method.

We developed a novel assay using high-performance liquid chromatography (HPLC) with fluorescence detection for the determination of tocilizumab (TCZ), after it has undergone a facile and rapid pretreatment. TCZ belongs to the same subclass as IgG1 (Immunoglobulin G subclass 1), and we could separate TCZ from IgG1 without antigen-antibody reactions, with the novel detection method. The separation of these antibodies was achieved by pretreatment with an organic solvent containing a base, such as trimethylamine and triethylamine. The effect of these bases on the separation of TCZ is related to the hydrophobicity of the base rather than the electrostatic charge. The results indicated that the surface charge of antibodies changed because of the structural change, even though the difference in the amino acid sequences of the antibodies was very low. Our method is available for the separation of the antibody subclasses, and it would be useful to assay TCZ in blood.

Evaluation of chemiluminescence reagents for selective detection of reactive oxygen species.

In order to evaluate the chemiluminescence (CL) reagents for selective detection of reactive oxygen species (ROS), we comprehensively measured the CL responses of 20 CL reagents (three luminol derivatives, two imidazopyrazinone derivatives, eight lophine derivatives, six acridinium ester derivatives and lucigenin) against six types of ROS (superoxide anion: O(2)(*-), hydroxyl radical: *OH, hydrogen peroxide: H(2)O(2), hypochlorite anion: ClO(-), singlet oxygen: (1)O(2), and nitric oxide: NO). As a result of the screening, it was found that nine CL reagents selectively detected O(2)(*-) while one CL reagent selectively detected *OH. However, no CL reagent had selectivity on the detection of H(2)O(2), ClO(-), (1)O(2) and NO. Our screening results could help to select the most suitable CL reagent for selective determination of different ROS. As an application study, 4-methoxyphenyl-10-methylacridinium-9-carboxylate (MMAC), one of the acridinium ester derivatives, showed high selectivity on the detection of O(2)(*-), and thus was applied to the assay of superoxide dismutase (SOD) activity. The dynamic range and detection limit of the developed CL assay were 0.1-10 and 0.06 U mL(-1), respectively. Significant correlation (r=0.997) was observed between the results by the CL assay using MMAC and the spectrophotometric assay using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt.

A validated HPLC-fluorescence method with a semi-micro column for routine determination of homocysteine, cysteine and cysteamine, and the relation between the thiol derivatives in normal human plasma.

A semi-micro column HPLC-fluorescence method for routine determination of thiol derivatives such as homocysteine (Hcy), cysteine (Cys) and cysteamine (CA) is described. The thiol derivatives labeled with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) were isocratically separated within 12 min on a semi-micro ODS column (Daisopak-SP-120-5-ODS-BP) with a mixture of 25 mm acetate buffer (pH 2.00) and CH(3)CN as a mobile phase. The purity and similarity of SBD-thiols by a multi-wavelength fluorescence detector were more than 92.3 and 96.7%. The detection limits of Hcy, Cys and CA at a signal-to-noise ratio of 3 were 0.16, 0.47 and 0.03 microm, respectively. Furthermore validation parameters such as accuracy, precision and robustness of the proposed method showed satisfactory results. Almost 850 plasma sample injections (range 572-1076, n = 3) for a column could be performed without differences in retention time and peak heights of labels. As an application of the proposed method, the determination of thiol derivatives in normal human plasma (n = 103) was demonstrated. The correlation coefficients between Hcy vs Cys and Hcy vs CA were 0.38 and -0.35, respectively.

Preparation and characterization of poly(l-phenylalanine) chiral stationary phases with varying peptide length.

Three new chiral stationary phases with different lengths of l-phenylalanine peptide were prepared by solid-phase synthesis with tert-butoxycarbonyl (Boc)-l-phenylalanine on silica. The effect of phenylalanine peptide length on enantioselectivity was studied. The best separation of R/S-warfarin was achieved by the chiral stationary phase with intermediate peptide length. These stationary phases were found to exist mainly in alpha-helical conformation by using FT-IR spectra. The end-capping reagents for the N-terminus of the peptide were also evaluated.

Determination of aromatic compounds by high-performance liquid chromatography with on-line photoreactor and peroxyoxalate chemiluminescence detection.

This paper describes a novel high-performance liquid chromatographic (HPLC) method for the determination of aromatic compounds with peroxyoxalate chemiluminescence (PO-CL ) detection following on-line UV irradiation. Aromatic compounds were UV irradiated (254 nm, 15 W) to generate hydrogen peroxide, which was determined via PO-CL detection using a mixture of bis(2,4,6-trichlorophenyl)oxalate (aryloxalate) and 2,4,6,8-tetrathiomorpholinopyrimido[5,4-d]pyrimidine (fluorophore) as a post-column CL reagent. Generation of hydrogen peroxide from aromatic compounds was confirmed using a flow injection analysis (FIA) system incorporating an enzyme column reactor immobilized with catalase. The conditions for UV irradiation were optimized using benzene and monosubstituted benzenes (phenol, benzaldehyde, nitrobenzene and N,N-dimethylaniline) by an HPLC system to evaluate the analytical performance of the proposed system. The detection limits for benzene and monosubstituted benzenes were in the range 2.1-124 pmol/injection at signal:noise (S:N) ratio = 3. Monocyclic and polycyclic hydrocarbons were also employed to investigate their CL properties. The possibility of PO-CL detection for a wide variety of aromatic compounds was shown for the first time.

Quasi-simultaneous determination of antioxidative activities against superoxide anion and nitric oxide by a combination of sequential injection analysis and flow injection analysis with chemiluminescence detection.

A method that combines sequential injection analysis (SIA), flow injection analysis and chemiluminescence (CL) detection was developed for the quasi-simultaneous determination of antioxidative activities against superoxide anion (O2-) and nitric oxide (NO). The antioxidative activity was expressed as the decrease in luminol CL intensity caused by the quenching of O2- or NO by an antioxidant. The SIA system consisted of two syringe pumps, two selection valves, two holding coils, an HPLC pump to deliver luminol solution, and a CL detector. Operation of the syringe pumps and multiport valves was controlled automatically using a personal computer with appropriate software. A hypoxanthine (HX)-xanthine oxidase (XOD) system was used for the generation of O2-, and (+/-)-(E)-4-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide (NOR1) was employed as NO donor agent. The repeatability of the method was evaluated with 35.2 microg ml(-1) L-ascorbic acid, and the relative standard deviations (RSD) of the antioxidative activities were less than 3.8%. The quasi-simultaneous determination of the antioxidative activities in one sample was completed within 2.0 min. The antioxidative activities of some antioxidants and commercially available supplements containing certain antioxidants were successfully determined using this system. The proposed system is rapid and reproducible, and thus may be useful for the screening of functional foods, supplements and pharmaceutical formulations that exhibit antioxidative activity.

Determination of haloperidol and reduced haloperidol in human serum by liquid chromatography after fluorescence labeling based on the Suzuki coupling reaction.

A simultaneous method for the determination of haloperidol (HP) and its metabolite, reduced haloperidol (RHP), in human serum was developed by means of high-performance liquid chromatography (HPLC) with fluorescence detection. Suzuki coupling reaction with a fluorescent arylboronic acid, 4-(4,5-diphenyl-1H-imidazol-2-yl)phenylboronic acid (DPA), was employed to convert HP and RHP into highly fluorescent compounds. HP and RHP were extracted from human serum by liquid-liquid extraction with a mixture of n-hexane and isoamyl alcohol (99:1, v/v) and subsequently labeled by reaction with DPA. Separation of DPA derivatives of HP and RHP was performed on a silica column with a mixture of acetonitrile and H(2)O (90:10, v/v) containing triethylamine and acetic acid as a mobile phase. The proposed method allowed sensitive detection of HP and RHP in human serum with a detection limit (at a signal to noise ratio of 3) of 0.22 and 0.20 ng/mL, respectively. The applicability of the method for therapeutic drug monitoring (TDM) was demonstrated by analyzing human serum samples from schizophrenic patients receiving HP.

Selective determination of quinones by high-performance liquid chromatography with on-line post column ultraviolet irradiation and peroxyoxalate chemiluminescence detection.

A new HPLC method was developed for the simultaneous determination of quinones with peroxyoxalate chemiluminescence (PO-CL) detection following on-line UV irradiation. Quinones [i.e., 1,2-naphthoquinone, 1,4-naphthoquinone, 9,10-anthraquinone, 9,10-phenanthrenequinone] were UV irradiated (254 nm, 15 W) to generate hydrogen peroxide and a fluorescent product that were determined via PO-CL detection. Generation of hydrogen peroxide from quinones with on-line UV irradiation was confirmed using flow injection analysis (FIA) system whereby incorporating an enzyme column reactor immobilized with catalase. Moreover, the structure of the produced fluorophore was confirmed using LC-MS, IR, and (1)H NMR. Afterwards, the conditions for UV irradiation and PO-CL detection were optimized. The separation of four quinones by HPLC was accomplished isocratically on an ODS column within 25 min. The detection limits (signal-to-noise ratio=3) were 6.0 pmol/injection for 1,2-naphthoquinone, 4.4 pmol/injection for 1,4-naphthoquinone, 0.2 pmol/injection for 9,10-anthraquinone, and 0.45 pmol/injection for 9,10-phenanthrenequinone.

Determination of artemisinin in human serum by high-performance liquid chromatography with on-line UV irradiation and peroxyoxalate chemiluminescence detection.

Artemisinin is an antimalarial drug containing an internal endoperoxide linkage in its structure. A simple, selective and sensitive high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence (PO-CL) method for the determination of artemisinin was developed. This method is based on the fact that endoperoxide in artemisinin structure can be converted to hydrogen peroxide (H(2)O(2)) under ultraviolet (UV) irradiation and the generated hydrogen peroxide can be measured using PO-CL detection. The HPLC-PO-CL system was optimized on a mobile phase, post column chemiluminescence reagent, UV source and irradiation time. In addition, the system was combined with simple liquid-liquid extraction using n-hexane that allowed selective and sensitive determination of artemisinin in serum. The limit of detection using 0.5 mL of blood was 0.062 micromol/L (17.5 ng/mL) at a signal-to-noise ratio of 3. Calibration curve obtained for artemisinin in human serum 4-80 micromol/L (1.1-22.6 microg/mL) showed a good linearity (r = 0.999).

Sequential injection analysis with chemiluminescence detection for the antioxidative activity against singlet oxygen.

A sequential injection analysis (SIA) with chemiluminescence (CL) detection was developed for the measurement of antioxidative activity against singlet oxygen ((1)O2). Lactoperoxidase-hydrogen peroxide-bromide ion system was used for the generation of (1)O2. When a 100 mM sodium acetate buffer (pH 4.5) was used as a carrier solution, the SIA-CL system could be optimized with respect to the flow-rate of the carrier, concentration of reagents and their aspiration order. The antioxidative activity was expressed as an attenuation of luminol CL due to the quenching of (1)O2 by an antioxidant. The relative standard deviations of antioxidative activity (n=3) against (1)O2 for within- and between-day analyses were < or = 1.6% (20 microM Trolox). The system was successfully applied to the assay of antioxidative activities of various antioxidants including vitamin supplements at a rate of 10 samples within 15 min. The proposed SIA-CL system was rapid and reproducible with minimum consumption of the sample and of reagents, and thus was useful for the screening of compounds possessing antioxidative activity against (1)O2.

Study on immunocapture-chemiluminescence assay of lipase activity in a biological sample.

A new approach for the determination of lipase (triacylglycerol lipase, EC.3.1.1.3) activity in a biological sample was investigated by combining an immunocapture technique with a chemiluminescence (CL) assay method in order to eliminate interference with CL detection. The proposed method consists of an immunocapture step to trap lipase and a subsequent step for CL detection of the activity of the captured lipase. The CL detection is based on the luminol-hydrogen peroxide (H(2)O(2))-horseradish peroxidase (HRP) reaction and utilizes a proenhancer substrate [a lauric acid ester of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI)] which liberates an active enhancer, HDI, by enzymatic hydrolysis. A polyclonal antibody prepared with porcine pancreas lipase was used for the immunocapture. The proposed immunocapture-CL method effectively eliminated the interference with the CL reaction from biological components and enabled the determination of spiked porcine pancreas lipase activity in serum samples in the range 0.41-1.1 U(HDI) (1 U(HDI) corresponds to the amount which liberates 1 pmol HDI/min at 37 degrees C from the substrate). The method was further applied to the assay of the activity for human pancreas lipase in serum and the results showed good correlation (r = 0.871) with those by the conventional colorimetric method.

Concentration and trend of 9,10-phenanthrenequinone in airborne particulates collected in Nagasaki city, Japan.

9,10-Phenanthrenequinone (PQ), one of the components of atmospheric pollutants, has potent harmful effects on human health. PQ in airborne particulates collected in Nagasaki city was determined by HPLC with fluorescence derivatization. PQ extracted from airborne particulates using methanol was derivatized with benzaldehyde in the presence of ammonium acetate to give a fluorescent compound. The average concentration (mean+/-SD, n=52) of PQ found in airborne particulates collected from July 1997 to June 1998 was 0.287+/-0.128 ng m-3. Concentrations of PQ in winter were higher than those in summer. In a weekly variation study, PQ concentrations were higher during weekdays and lower at weekend. The levels of PQ were obviously correlated with those of phenanthrene (PH) that is considered as a parent compound of PQ. This observation suggested that PQ was emitted into the atmosphere from the same source as PH, or PQ was converted from PH in the atmosphere.

Retrospective analyses of atmospheric polycyclic and nitropolycyclic aromatic hydrocarbons in an industrial area of a western site of Japan.

Thirteen polycyclic aromatic hydrocarbons (PAHs) and four nitropolycyclic aromatic hydrocarbons (NPAHs) on the surfaces of airborne particulates, which were collected at an industrial area of a western site of Japan during periods from 1976 to 1998, were retrospectively analyzed. PAHs and NPAHs were extracted from airborne particulates using hexane with ultrasonication, and then analyzed by HPLC systems with fluorescence detection and chemiluminescence detection, respectively. The total concentrations (mean +/- SD, n = 34) were 15.54 +/- 21.24 ng/m3 for PAHs and 5.85 +/- 8.16 pg/m3 for NPAHs. The concentrations of PAHs and NPAHs were found to be highest during the period between 1979 and 1982, and then reduced. The annual concentrations of PAHs and NPAHs were highly correlated with those of air pollutants from motor vehicle origin, such as carbon monoxide, suspended particulates and non-methane hydrocarbons. The results suggested that motor vehicle emissions were one of the predominant sources of atmospheric PAHs and NPAHs.

Investigation of a novel mixed-mode stationary phase for capillary electrochromatography. Part III: Separation of nucleosides and nucleic acid bases on sulfonated naphthalimido-modified silyl silica gel.

Capillary electrochromatography (CEC) with a novel stationary phase, 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP), proved useful for the separation of nucleosides and nucleic acid bases. The application scope of SNAIP, which is a relatively polar reversed-phase (RP)-type stationary phase, was successfully expanded to include the CEC separation of polar compounds although the combination of non-polar RP phase with highly aqueous mobile phase is often inadequate. Due to the permanently charged sulfonic acid groups and the naphthalimidopropyl moiety, the retention of charged and relatively polar nucleosides as well as bases on the SNAIP stationary phase was effected by electrostatic and hydrophobic interactions. This yielded a unique selectivity on SNAIP toward nucleosides and bases. The characteristic EOF on SNAIP, which was stronger at higher aqueous content in the mobile phase, proved suitable for the separation of polar compounds in reversed-phase mode with highly aqueous mobile phase. In addition, when a double stepwise gradient was employed to accelerate the latest peak (adenine), the elution time was shortened to less than half its original duration.

Fluorescence labeling method for aryl halides with 4-(4,5-diphenyl-1H-imidazol-2-yl)phenylboronic acid based on Suzuki coupling reaction.

For the first time, fluorescence labeling methods for aryl halides with a fluorescent arylboronic acid was developed on the basis of a Suzuki coupling reaction. 4-(4,5-diphenyl-lH-imidazol-2-yl)phenylboronic acid (DPA) was used as a fluorescence labeling reagent. In order to explore its analytical performance, the reaction conditions were optimized using simple bromobenzene derivatives. The reactivity was then investigated with chloro- and iodobenzene derivatives, and also bromobenzene derivatives with different position of substituents. The order of reactivity with DPA: iodobenzene > bromobenzene more more than chlorobenzene derivatives, and p- > m- > o-substituted bromobenzenes. The detection limits of bromobenzene, 4-bromotoluene, and 4-bromoanisole ranged from 0.2 to 1.4 pmol/injection at a signal-to-noise ratio, (S/N) of 3. The applicability of the method to biological samples was also evaluated using clofibrate as the analyte. The reaction was found not only to proceed well but also to be selective for clofibrate even in the presence of plasma components. The method allowed the sensitive detection of clofibrate in human plasma with the detection limit of 170 pmol/mL (260 fmol/injection) at a S/N = 3. The proposed method is highly selective and sensitive and thus would be useful for labeling of aryl halides that do not have other functional groups that could be labeled by currently available fluorescent labeling reagents.

Stepwise gradient of buffer concentration for capillary electrochromatography of peptides on sulfonated naphthalimido-modified silyl silica gel.

The advantage of using a stepwise gradient of buffer concentration in CEC was demonstrated with the mixed-mode stationary phase, 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP). Before the application of a stepwise gradient, the effect of buffer concentration on the separations of six peptides and tryptic digests was investigated. Bubble formation caused by Joule heating at currents up to 95 microA was successfully suppressed by using SNAIP column even without pressurization, which contributed to a stepwise gradient of buffer concentration. Utilizing the stepwise gradient improved and shortened the separation of six peptides as compared to the separation under an isocratic elution.

Capillary electrochromatography of caffeine and its metabolites in rat brain microdialysate.

A capillary electrochromatography (CEC) method has been developed for the separation of caffeine and its two metabolites 1-methylxanthine (1-MX) and 1,7-dimethylxanthine (1,7-DX). The stationary phase was 3-(1,8-naphthalimido) propyl-modified silyl silica gel (NAIP) and the best separations were achieved with 4.0 mM citrate buffer (pH 5.0) containing 80% methanol at an applied voltage of 25 kV. The compounds were completely separated in less than 3.5 min with good repeatability, which was approximately 3-times less than that in high-performance liquid chromatography (HPLC) with NAIP. The proposed method coupled with microdialysis was successfully applied to the monitoring of caffeine concentration in rat brain with detection limits of 1.11 microg/mL.