Kazuo Iwaki

Amino acid analysis by reversed-phase high-performance liquid chromatography : Automatic pre-column derivatization with activated carbamate reagent

Succinimido alpha-naphthylcarbamate, an activated carbamate reagent, facilitated the simple and rapid pre-column derivatization of amino acids for fluorimetric detection. The method is based on the formation of naphthylcarbamyl derivatives of amino acids. The carbamylation of amino acids is completed within 1 min at room temperature, and the reaction mixture can be injected directly into a liquid chromatograph equipped with an octadecylsilyl reversed-phase column. The effluent stream is monitored fluorimetrically at 370 nm excited at 290 nm at the sub-picomole level. Naphthylcarbamyl derivatives of common protein amino acids were separated within 30 min by gradient elution with aqueous sodium acetate and acetonitrile. Excess of the reagent does not interfere with the analysis of amino acids, because it is hydrolysed in 2-3 min to give naphthylamine, which is more strongly retained than any amino acid derivatives.

Determination of tetracycline antibiotics by reversed-phase high-performance liquid chromatography with fluorescence detection

A highly sensitive method for the determination of tetracycline antibiotics (TCs) using reversed-phase high-performance liquid chromatography with fluorescence detection is presented. This method was based on the use of disodium ethylenediaminetetraacetate (EDTA) and calcium chloride as fluorescence-increasing reagents in the mobile phase. The concentrations of each reagent in the mobile phase greatly influenced the fluorescence intensity of TCs. When the concentration of EDTA and calcium chloride were 25 and 35 mM, respectively, and the pH of the mobile phase was 6.5, the maximum fluorescence intensity was obtained. The column temperature hardly influenced the fluorescence intensity. At 3.75 ng of TCs injected, the precision (relative standard deviation) ranged from 1.12 to 2.20%. In the range 0.075-37.5 ng for tetracycline and oxytetracycline and 0.225-37.5 ng for chlortetracycline, a linear response was observed. The detection limits of this method were 49-190 pg for three different TCs. The proposed method was applied to the determination of one of the TCs in pharmaceuticals by the internal standard method using other TCs as internal standards and was also applied to determination of TCs added to fish tissue.

Precolumn derivatization technique for high-performance liquid chromatographic determination of penicillins with fluorescence detection

A precolumn derivatization method was developed for the high-performance liquid chromatographic (HPLC) determination of penicillins using fluorescence detection. Penicillins were derivatized by a two-step reaction, the beta-lactam ring being opened by hydrolysis in aqueous sodium carbonate solution in the first step to give a secondary amine functionality, and the secondary amino group being reacted with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole in the second step to give a fluorescent derivative. The resulting reaction mixture was injected directly onto a reversed-phase column and analysed by HPLC. At a penicillin concentration of 10 micrograms/ml, the precision (relative standard deviation) ranged from 1.49 to 2.20%. In the concentration range 0.2-100 micrograms/ml, a linear response was observed. The detection limits of this method were 30-85 ng/ml for five different penicillins at a signal-to-noise ratio of 3:1. The proposed method was applied to the determination of penicillins added to serum following pretreatment by deproteinization and removal of compounds containing amino functionalities with a cation-exchange resin.

Rapid determination of tetracycline antibiotics in serum by reversed-phase high-performance liquid chromatography with fluorescence detection

A rapid and accurate determination of tetracycline antibiotics in human serum by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed, based on protein precipitation in serum. Various reagents for precipitation were investigated, and 24% trichloroacetic acid in methanolic solution gave the maximum recovery (at least 94.3%) and interference-free chromatograms of different three tetracyclines. At a concentration of 0.5 micrograms/ml, the precision (relative standard deviation) ranged from 1.12 to 1.94%. In the range 0.04-10.0 micrograms/ml for oxytetracycline and chlorotetracycline and 0.01-10.0 micrograms/ml for tetracycline, linear responses were observed. The detection limits of this method were 10-35 ng/ml for all three antibiotics. The proposed method was applied to the determination of serum concentrations in subjects receiving tetracycline antibiotics.

Determination of sodium 5,6-benzylidene-l-ascorbate and related compounds by high-performance liquid chromatography

Abstract A simple high-performance liquid chromatographic process with ultraviolet detection was established to determine ascorbic acid, benzoic acid, benzaldeh

Routine check of baseline noise in ion chromatography

The long-term check of baselines in an ion chromatograph for more than two years reveals that the suppressor breakdown and solvent contamination can be detected by the anomalous patterns of the power spectra of the baseline noise. The signal is not referred to in this study. The early detection of the troubles is possible and the quality of a new suppressor can also be examined by the noise pattern. Coupled with the signal information such as shape and elution time, the above methodology will provide a rapid and reliable routine check for ion chromatography.

Statistical and probabilistic approaches to confidence intervals of linear calibration in liquid chromatography.

The aim of this paper is to compare the reliability of two approaches to estimate the 95% confidence intervals of linear calibration in real situations. One is the statistical approach, which is well known in statistics, and the other is the probabilistic approach, which is based on a theory to predict the precision of instrumental analyses mainly from signal and noise, called FUMI (Function of Mutual Information) theory. The high-performance liquid chromatographic determination of quisalofop and maltose is taken as an example. Calibration lines obtained under the same experimental conditions are superimposed on the 95% confidence intervals to investigate whether the resulting confidence intervals can include all the calibration lines reasonably. A pair of 95% confidence intervals (upper and lower limits) can be calculated from each calibration line, but varies from calibration line to calibration line, although obtained under the same experimental conditions. The variability and reliability of the 95% intervals are also examined.

Optimization of naphthylethylurea multiple-bonded chiral stationary phases for optical resolution of enantiomeric amino acid derivatives

Abstract The polyethyleneamine spacer length of naphthylethylurea multiple-bonded chiral stationary phases (CSPs) was optimized for optical resolution of p-bormophenylcarbamyl (Br-PC) amino acid enantiomers. Four multiple-bonded CSPs having different lengths of polyethyleneamine spacer, diethylenetriamine, triethylenetetramine, tetraethylenepentamine and pentaethylenehexamine, were prepared via an activated carbamate intemediate from aminopropylsilyl silica gel. The resolution data of Br-PC anmino acids on these CSPs by elution with an aqueous mobile phase were compared. The CSP with the triethylenetetramine spacer showed the best resolution of the enantiomers in the four CSPs. The other conditions, pH, concentration and temperature, etc., were also investigated using this CSP. They influenced the retention time of Br-PC amino acids, but they hardly affected the enantio recognition of the CSP.

Co-operative study for precision and limit of detection in HPLC analysis.

The limit of detection (LOD) was determined for three p-hydroxybenzoic acid esters at nine laboratories. The result of each laboratory was analyzed by computer software (called TOCO) which predicts the precision and LOD based on the baseline fluctuation without repeated measurements. In every laboratory, the shorter was the retention time of the analytes, the lower was the LOD. In each analyte, the peak-height measurement was more precise than the entire area measurement. The LOD obtained by the laboratories ranged from 0.16 to 30 μg/l. The validity of the TOCO, when applied in practical situations, has been proved.

An information theory-based evaluation of stationary phases developed for optical resolution of amino acids

SummaryNaphthylethylurea multiple-bonded chiral stationary phases prepared for the optimal resolution of p-bromophenylcarbamyl derivatives of enantiomeric amino acids are examined from the viewpoint of information theory. The criterion used is the function of mutual information (FUMI) which describes the precision of analysis as a function of peak shape (area and width), overlap and noise level. The length of the polyethyleneamine spacer in the chiral stationary phases is evaluated by the precision (FUMI) calculated from the peak shape and overlap. The best column is defined as the one which gives the highest precision. The temperature effect on the precision is also examined.