Hisakuni Sato

Separation and determination of amino acids, creatinine, bioactive amines and nucleic acid bases by dual-mode gradient ion-pair chromatography

A simple and fast high-performance liquid chromatography method for the analysis of amino acids and biological bases such as creatinine was developed using a technique termed dual-mode gradient ion-pair chromatography. A butyl-silica reversed-phase column and water-acetonitrile eluent containing sodium dodecyl sulfate (SDS) and perchloric acid were used for the separation. A concentration gradient of acetonitrile from 15 to 35% provided a good separation of such organic cations. Since change in concentration of acetonitrile causes change in distribution equilibrium of SDS between mobile and stationary phases, a complete regeneration of the column to the initial state is required for the reproducible separations. Completion of the reequilibrium was indicated by a system peak appearing in the UV chromatogram and by conductivity measurements. The formation mechanism of the system peak was revealed. A flow-rate gradient from 1 to 2 ml/min was introduced in addition to the concentration gradient to shorten the cycle time of the chromatography. More than twenty kinds of amino acids, creatine and creatinine were simultaneously separated within 50 min and the cycle time was 80 min including the reequilibration time. A post-column derivatization fluorescence detection system was usable as well as UV detection. This elution system was also useful for the separation of bioactive amines and nucleic acid bases. The developed method was applied to the simultaneous determination of urinary creatinine and diagnostic amino acids due to inherited metabolic disorders.

Low-capacity cation-exchange chromatography of ultraviolet-absorbing urinary basic metabolites using a reversed-phase column coated with hexadecylsulfonate

A low-capacity cation-exchange HPLC method for the determination of UV-absorbing organic cations such as amino acids, histidine dipeptides, and creatinine was developed. A commercially available reversed-phase column was dynamically coated with hexadecylsulfonate, and was successfully used for the cation-exchange separation with ethylenediammonium eluting ion at pH 2.5. The coated column was enough stable for the specific use with a completely aqueous mobile phase at low and constant pH; and the day-to-day reproducibility for retention time was 0.9-1.7% of RSD (relative standard deviation). The linear relation between concentrations and detector responses (area) by using a photodiode-array UV detection at 210 nm ranged from 0.2 to 1000 microM (sample size 50 microl) for 1-methylhistidine, 3-methylhistidine, histidine, creatinine, anserine, carnosine, and homocarnosine, and from 0.5 to 2000 microM for creatine, tyrosine, and phenylalanine, with less than 5% of RSD. The UV spectrum (190-300 nm) obtained during chromatography was very indicative for each analyte. Overall recoveries were 97-104%. The developed HPLC method in conjunction with preliminary fractionation technique could be applied to the analysis of urine of patient with metabolic disorder such as phenylketonuria.

Simultaneous determination of silicate and phosphate in environmental waters using pre-column derivatization ion-pair liquid chromatography

A highly sensitive HPLC method for the simultaneous determination of soluble silicate and phosphate in environmental waters was developed, using ion-pair liquid chromatography preceded by the formation of their yellow alpha-heteropolymolybdates. The moderate-pH mobile phase enabled to use a highly efficient reversed-phase silica column. The pre-column coloring reactions at moderate-pH were reproducible for both silicate and phosphate in all quantification ranges with R.S.D.s less than 2% and 5%, respectively. The linear calibration lines between concentrations (mg-SiO(2)/L and mg-PO(4)/L) and peak area intensities were obtained for silicate and phosphate both with acceptable determination coefficients (r(2)) of 0.9999. The limits of determination for both analytes were 0.007 mg-SiO(2)/L and 0.003 mg-PO(4)/L, which were calculated theoretically using 10sigma/slope. The four-digit dynamic ranges were obtained for 0.007-10mg-SiO(2)/L and 0.003-20mg-PO(4)/L. The developed method was applied for the analysis of tap water, river water, coastal seawater, well water, hot-spring water, commercial mineral water, and laboratory water. The results were very reasonable and acceptable from the environmental viewpoints, which were well correlated with those confirmed by the molybdenum-blue spectrophotometry.

Reversed-phase high-performance liquid chromatographic determination of linear alkylbenzenesulphonates in river water at ppb levels by precolumn concentration

A high-performance liquid chromatography method for the determination of linear alkylbenzenesulphonates (LASs) in river waters has been developed. The ppb levels of LASs can be determined by reversed-phase high-performance liquid chromatography with ultraviolet detection after on-line anion-exchange concentration and successive injection. LASs were quantitatively concentrated on the anion-exchange precolumn and easily cleaned up from river water matrix, because of its specific affinity, for the anion-exchange resin. A weak non-polar reversed-phase column was useful for the determination of LASs. The relationships between concentration and summation of peak areas were linear from 10 to 200 ppb for total LAS concentrated from 5 ml of standard solutions. Overall recovery for total LAS was found to be 99%. Total LAS in the Tama River waters was determined to be around 100 ppb by the proposed method.

Determination of alkylbenzenesulphonates in environmental water by anion-exchange chromatography

Abstract A simple and selective method for the determination of μg/1 levels of alkylbenzenesulphonates (ABSs) in environmental waters is presented. Selectivity for ABSs was obtained by using anion-exchange pre-column concentration followed by anion-exchange high-performance liquid chromatography separation and ultraviolet detection. The method is quantitative and easily applicable to the analysis of real samples. The concentrations of ABSs in tap water and river water were determined to be ca . 0.1 and 100 μg/1, respectively.

Determination of sweat constituents by liquid ionization mass spectrometry

Liquid ionization (LI) mass spectrometry was applied to the determination of organic compounds in sweat. LI mass spectra of ordinary sweat exhibited characteristic peaks of lactic acid, urea and several kinds of amino acids. The relative peak intensities of these organic compounds changed according to the sweating rate. An unknown compound giving an intense peak at m/z 107 was found in sweat produced during walking excercise and was identified as diethylene glycol (DEG) by comparison with standard compounds. This identification was confirmed by liquid chromatography (LC). The concentrations of DEG in the sweat samples changed similarly to those of inorganic cations, depending on the change in momentum of the subject. The complementary use of LI mass spectrometry and LC is one of the best methods for the analysis of complicated organic mixtures such as biological fluids.

Chelate formation of zirconium with xylenol orange and semi-xylenol orange

Abstract The composition, formation constants, and molar absorptivities of the chelates of zirconium ion wtih xylenol orange and semi-xylenol orange are investigated spectrophotometrically in strong acid medium at ionic strength 3.0 (NaClO4 and HClO4). The data obtained were processed with a newly-constructed computer program and with LETAGROP/SPEFO. In the zirconium—xylenol orange system, Zr · H3 L, Zr· H4L, and Zr2 · L are present with logarithmic overall formation constants of 37.80, 38.68, 43.47, and molar absorptivities of 3.10 × 104 (485 nm), 5.98 × 104 (528 nm), 9.50 × 104 (551 nm) I mol-1 cm-1, respectively. The chelates Zr · L and Zr · HL were found in the zirconium—semi-xylenol orange system with logarithmic overall formation constants of 26.25 and 27.56, and molar absorptivities of 5.70 × 104 (532 nm) and 8.30 × 104 (535 nm) 1 mol-1 cm-1, respectively. Semi-xylenol orange is more sensitive and reliable than xylenol orange as a spectrophotometric reagent for zirconium.

Determination of creatinine and ultraviolet-absorbing amino acids and organic acids in urine by reversed-phase high-performance liquid chromatography

A simple and reliable method for the determination of urinary creatinine, amino acids and organic acids was developed. A urine sample was preliminarily separated into an organic acid fraction (including neutral species) and an amino acid fraction by cation-exchange chromatography. Both fractions were analysed by reversed-phase high-performance liquid chromatography, with a phosphoric acid-methanol gradient elution system and ultraviolet detection at 210 nm. Relationships between concentrations and peak heights were linear from 2 to 500 microM for the analytes. Overall recoveries were ca. 100%. The concentrations of creatinine for 37 urine samples, from 20 healthy newborns and from 17 patients with several inherited metabolic disorders, were 2.35 +/- 2.29 mM (ranging between 0.27 and 10.15 mM). The method was applied to the determination of several diagnostically useful metabolites in urine. The concentrations of phenylalanine and phenylacetic acid for five urine samples from patients with phenylketonuria were 347 +/- 177 and 282 +/- 224 microM/mM creatinine, respectively. The concentrations of tyrosine and 4-hydroxyphenyllactic acid in the urine of a patient with tyrosinemia were 112 and 1871 microM/mM creatinine, respectively.

Isocratic elution of sodium, ammonium, potassium, magnesium and calcium ions by ion-exchange chromatography

Abstract Isocratic elution conditions for five cationic species were investigated using conductivity and UV absorption detectors. Using several kinds of eluents and silica and styrene—divinylbenzene separating columsn, the detection sensitivity, separation efficiency, separation time, system peak interference, etc., were examined. As a result, two types of eluents were concluded to be useful: a solution of benzylamine, citric acid and N-hydroxyethylenediamine-N,Nt,Nt-triacetic acid (EDTA · OH) and a solution of 1,1′-di-n-heptyl-4,5′-bipyridinium (DHBP) ion and citric acid. When the former eluent is used, monovalent cations are separated clearly and divalent cations can be eluted without being caputred in the column. With a slightly lower precision, divalent cations at the parts per million level can also be determined. The latter eluent is suitable mainly for determining Mg2+ and Ca2+ ions with no interference from heavy metal ions. The separation of monovalent ions was poor with a commercially available separation column, but the detection sensitivity with a UV absorption detector was higher than that with the benzylamine eluent. Several examples of application are shown

High-sensitivity conductivity detection in non-suppressed ion chromatography using sulfoisophthalic acid as eluent

A non-suppressed ion chromatographic (IC) system for the high-sensitivity detection of common anions was developed using sulfoisophthalic acid as the eluent. The detection sensitivity was ten times higher than that using conventional non-suppressed IC with sodium phthalate as eluent, and was almost the same as that using conventional suppressed IC with a carbonate-hydrogencarbonate eluent under the same electrical conditions with a conductivity detector. Temperature regulation was very important in minimizing the baseline drift. A commercial incubator, in which a separation column and a sample injector were placed, was useful. The developed non-suppressed system facilitated the determination of low concentrations of phosphate, chloride, bromide, nitrate and sulfate at micromolar levels.