Tadashi Hayama

Determination of polar organophosphorus pesticides in water samples by hydrophilic interaction liquid chromatography with tandem mass spectrometry.

A method combining hydrophilic interaction liquid chromatography (HILIC) with tandem mass spectrometry (MS/MS) was developed for the determination of polar organophosphorus pesticides (OPPs; acephate, methamidophos, monocrotophos, omethoate, oxydemeton-methyl, and vamidothion) in water samples. To extract the polar OPPs and minimize matrix effects from the water sample, an activated carbon cartridge was used for pretreatment. After pretreatment of the water sample, the eluate from the activated carbon cartridge was directly injected into the HILIC/MS/MS system. The OPPs were separated on an Atlantis HILIC silica column by isocratic elution with a mixture of acetonitrile, isopropanol, and ammonium formate buffer as a mobile phase, and they were detected by positive electrospray ionization MS/MS in the selected reaction monitoring mode. The method was validated at 0.05, 0.5, and 5 microg/L levels in water samples, and the recoveries of polar OPPs were between 76.4 and 98.6%. The limits of detection were between 0.13 and 1.0 pg on-column, and the limits of quantification were between 0.43 and 3.4 pg on-column. The method can be applied to the determination of trace amounts of OPPs in environmental water samples.

Highly sensitive and selective derivatization-LC method for biomolecules based on fluorescence interactions and fluorous separations

A fluorescence derivatization LC method is a powerful tool for the analysis with high sensitivity and selectivity of biological compounds. In this review, we introduce new types of fluorescence derivatization LC analysis methods. These are (1) detection-selective derivatization methods based on fluorescence interactions generated from fluorescently labeled analytes: excimer fluorescence derivatization and fluorescence resonance energy transfer (FRET) derivatization; (2) separation-selective derivatization methods using the fluorous separation technique: fluorous derivatization, F-trap fluorescence derivatization, and fluorous scavenging derivatization (FSD).

Binary fluorous alkylation of biogenic primary amines with perfluorinated aldehyde followed by fluorous liquid chromatography-tandem mass spectrometry analysis.

We have developed a novel method for the determination of biogenic amines (dopamine, norepinephrine, 3-methoxytyramine, normetanephrine, serotonin, tyramine, tryptamine, 5-methoxytryptamine, and histamine) utilizing liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) combined with a separation-oriented derivatization technique. Using this approach, primary amino groups in the target amines were selectively dialkylated with a perfluorinated aldehyde reagent (2H,2H,3H,3H-perfluoroundecan-1-al) through reductive amination. The derivatives were directly injected onto an LC column containing perfluoroalkyl-modified stationary phase and were separated via gradient elution using a water/methanol/trifluoroacetic acid mixture and trifluoroethanol with formic acid as mobile phases. Matrix-induced signal suppression effects were eliminated because the binary fluorous-labeled amines were strongly retained on the fluorous-phase LC column, whereas the nonfluorous derivatives, including matrix components and monofluorous-labeled compounds such as the derivatization reagent, were poorly retained under the separation conditions. The linear dynamic ranges of the target amines were established over a concentration range of 0.01-1 nM (r > 0.9978), and the limits of detection were found to be 7.8-26 amol on column. The feasibility of this method was further evaluated by applying it to human plasma samples.

Fluorous derivatization and fluorous-phase separation for fluorometric determination of naproxen and felbinac in human plasma

Fluorous derivatization followed by fluorous-phase liquid chromatographic (LC) separation exploits the affinity between perfluoroalkyl compounds for highly selective and quantitative isolation of various analytes. However, the applicability of this technique as a simple pretreatment for fluorometric determination in clinical settings has not been fully explored. Here we show the applicability of this technique to the clinical determination of non-steroidal anti-inflammatory drugs (NSAIDs) in human plasma. Naproxen and felbinac, widely used native-fluorescent NSAIDs with a carboxyl group, can have toxic effects at acute doses, and were therefore chosen as representative NSAIDs. Samples were precolumn derivatized with a non-fluorescent fluorous amine, which allowed highly selective retention of only derivatized substances in the fluorous LC column. Thus, subsequently, only the retained fluorous-labeled and fluorescent analytes were detected fluorometrically at appropriate retention times. The detection limits for these two drugs were less than 11fmol on column. Correlation curves were liner over the range of 0.04-10 and 5-250nmol/mL plasma for both two drugs (r>0.999) with good repeatability. Thus, this method offers a simple, sensitive, and selective solution for determination of NSAIDs in clinical settings.

Concerted derivatization and concentration method with dispersive liquid–liquid microextraction for liquid chromatographic analysis of 5-hydroxyindoles in human serum

We developed a concerted derivatization and concentration method based on dispersive liquid-liquid microextraction (DLLME) for the liquid chromatography (LC) determination of 5-hydroxyindoles (5-HIs; serotonin, 5-hydroxyindole-3-acetic acid, N-acetylserotonin, and 5-hydroxytryptohol). Concerted derivatization and concentration could be affected by adding a mixture of an ionic liquid (1-hexyl-3-methylimidazolium hexafluorophosphate, extraction solvent), methanol (disperser), and water containing fluorescence derivatization reagents [benzylamine and potassium hexacyanoferrate(III)] into the sample. The resulting sedimented phase was injected into a reversed-phase LC column using a mixture of acetonitrile and 250 mM acetate buffer (pH 4.3) as the mobile phase for gradient elution, and the derivatives obtained were fluorometrically detected at excitation and emission wavelengths of 345 nm and 452 nm, respectively. The derivatization (reagent concentrations and pH) and extraction (extraction and disperser solvent type) conditions were optimized simultaneously. The limits of detection of the 5-HIs were in the range of 0.08-0.33 nM. The method was validated for 10 and 50 pmol/mL human serum levels, and the recovery of 5-HIs was between 66% and 98%, within a relative standard deviation of 9.5%. The proposed method is well suited for the highly sensitive analysis of trace amounts of 5-HIs in human serum samples.

Fully automated reagent peak-free liquid chromatography fluorescence analysis of highly polar carboxylic acids using a column-switching system and fluorous scavenging derivatization.

In this study, we combined a column-switching system with a fluorous scavenging derivatization method to develop a fully automated reagent peak-free LC fluorescence detection protocol for the analysis of highly polar carboxylic acids. In this method, highly polar carboxylic acids were derivatized with fluorescent 1-pyrenemethylamine in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-hydroxy-1H-benzotriazole. Residual excess of the unreacted reagent was tagged with 2-(perfluorooctyl)ethyl isocyanate and then removed selectively using a fluorous column-switching system placed in front of an analytical reversed-phase column. The signal of the fluorous-tagged unreacted reagent was completely absent in the resulting chromatograms; therefore, it did not interfere with the quantification of each acid especially those eluted before 20 min. The detection limits (S/N = 3) for the examined acids were in the range from 4.0 to 22 fmol per injection. We have applied this method to comparative analysis of highly polar carboxylic acids in urine samples obtained from diabetes mellitus type-II model mice and their control.

Liquid chromatographic determination of microcystins in water samples following pre-column excimer fluorescence derivatization with 4-(1-pyrene)butanoic acid hydrazide

A method to measure the concentrations of microcystins (MCs) in water samples has been developed by incorporating pre-column fluorescence derivatization and liquid chromatography (LC). A solid-phase extraction for pretreatment was used to extract the MCs in water samples. The MCs were derivatized with excimer-forming 4-(1-pyrene)butanoic acid hydrazide (PBH). The MCs could then be detected by fluorescence after separation with a pentafluorophenyl (PFP)-modified superficially porous (core shell) particle LC column. The derivatization reactions of MCs with PBH proceeded easily in the presence of 4,6-dimethoxy-1,3,5-triazin-2-yl-4-methylmorpholinium (DMT-MM) as a condensation reagent, and the resulting derivatives could be easily separated on the PFP column. The derivatives were selectively detected at excimer fluorescence wavelengths (440-540 nm). The instrument detection limit and the instrument quantification limit of the MCs standards were 0.4-1.2 μg L(-1) and 1.4-3.9 μg L(-1), respectively. The method was validated at 0.1 and 1.0 μg L(-1) levels in tap and pond water samples, and the recovery of MCs was between 67 and 101% with a relative standard deviation of 11%. The proposed method can be used to quantify trace amounts of MCs in water samples.

Direct tandem mass spectrometric analysis of amino acids in plasma using fluorous derivatization and monolithic solid-phase purification.

In this study, we developed a novel direct tandem mass spectrometric method for rapid and accurate analysis of amino acids utilizing a fluorous derivatization and purification technique. Amino acids were perfluoroalkylated with 2H,2H,3H,3H-perfluoroundecan-1-al in the presence of 2-picoline borane via reductive amination. The derivatives were purified by perfluoroalkyl-modified silica-based monolithic solid-phase extraction (monolithic F-SPE), and directly analyzed by tandem mass spectrometry using electrospray ionization without liquid chromatographic separation. The perfluoroalkyl derivatives could be sufficiently distinguished from non-fluorous compounds, i.e. the biological matrix, due to their fluorous interaction. Thus, rapid and accurate determination of amino acids was accomplished. The method was validated with human plasma samples and applied to the analysis of amino acids in the plasma of mice with maple syrup urine disease or phenylketonuria.

Fluorous-assisted metal chelate affinity extraction for nucleotides followed by HILIC-MS/MS analysis

We herein developed a selective method for the determination of nucleotides by fluorous-assisted metal chelate affinity extraction followed by hydrophilic interaction liquid chromatography (HILIC) combined with tandem mass spectrometric (MS/MS) analysis. In this study, the nucleotides were selectively chelated by Fe(III)-immobilized perfluoroalkyliminodiacetic acid, and the resulting chelates were subsequently extracted into a fluorous solvent. The nucleotides present in the fluorous solvent were then back-extracted into a non-fluorous solution, such as a solution of ammonia in aqueous acetonitrile. The resulting non-fluorous solution containing the nucleotides was then directly injected into an amide-type HILIC column using a mixture of acetonitrile and aqueous ammonium bicarbonate as the mobile phase for gradient elution, and the nucleotides were detected using the negative electrospray ionization MS/MS mode. In this method, the extraction recoveries of the nucleotides ranged from 43.2 to 94.7% within a relative standard deviation of 17%. This method enabled the determination of intracellular concentrations of nucleotides.

Fluorous-assisted metal chelate affinity extraction technique for analysis of protein kinase activity.

We have developed a fluorous affinity-based extraction method for measurement of protein kinase activity. In this method, a fluorescent peptide substrate was phosphorylated by a protein kinase, and the obtained phosphopeptide was selectively captured with Fe(III)-immobilized perfluoroalkyliminodiacetic acid reagent via a metal chelate affinity technique. Next, the captured phosphopeptide was selectively extracted into a fluorous solvent mixture, tetradecafluorohexane and 1H,1H,2H,2H-tridecafluoro-1-n-octanol (3:1, v/v), using the specificity of fluorous affinity (fluorophilicity). In contrast, the remained substrate peptide in the aqueous (non-fluorous) phase was easily measured fluorimetrically. Finally, the enzyme activity could be assayed by measuring the decrease in fluorescence. The feasibility of this method was demonstrated by applying the method for measurement of the activity of cAMP-dependent protein kinase (PKA) using its substrate peptide (kemptide) pre-labeled with carboxytetramethylrhodamine (TAMRA).