Muneki Isokawa

Analytical methods involving separation techniques for determination of low-molecular-weight biothiols in human plasma and blood.

Low-molecular-weight biothiols such as homocysteine, cysteine, and glutathione are metabolites of the sulfur cycle and play important roles in biological processes such as the antioxidant defense network, methionine cycle, and protein synthesis. Thiol concentrations in human plasma and blood are related to diseases such as cardiovascular disease, neurodegenerative disease, and cancer. The concentrations of homocysteine, cysteine, and glutathione in plasma samples from healthy human subjects are approximately in the range of 5-15, 200-300, and 1-5 μM, respectively. Glutathione concentration in the whole blood is in the millimolar range. Measurement of biothiol levels in plasma and blood is thought to be important for understanding the physiological roles and biomarkers for certain diseases. This review summarizes the relationship of biothiols with certain disease as well as pre-analytical treatment and analytical methods for determination of biothiols in human plasma and blood by using high-performance liquid chromatography and capillary electrophoresis coupled with ultraviolet, fluorescence, or chemiluminescence detection; or mass spectrometry.

Recent advances in hydrophilic interaction chromatography for quantitative analysis of endogenous and pharmaceutical compounds in plasma samples

There is an increasing need for new analytical methods that can handle a large number of analytes in complex matrices. Hydrophilic interaction chromatography (HILIC) has recently been demonstrated as an important supplement to reversed-phase liquid chromatography for polar analytes, particularly endogenous compounds. With the increasing popularity of HILIC, progressively more polar phases with diverse functional groups have been developed. In addition, the coupling of HILIC to mass spectrometry offers the advantages of improved sensitivity by employing an organic-rich mobile phase. This article reviews recent applications of HILIC for the analysis of endogenous and pharmaceutical compounds in plasma samples. Furthermore, based on recent studies, we provide a discussion of column selection, sample pretreatment for HILIC analysis, and detection sensitivity.

Development of analytical method for catechol compounds in mouse urine using hydrophilic interaction liquid chromatography with fluorescence detection.

An analytical method for catecholamines and related compounds using hydrophilic interaction liquid chromatography (HILIC) with native fluorescence detection has been developed. We found that ZIC-cHILIC with phosphorylcholine was suitable for the separation of catechol compounds with good peak shapes among six different HILIC columns (Inertsil SIL, Inertsil Amide, Inertsil Diol, TSKgel NH2-100, ZIC-HILIC, and ZIC-cHILIC). Using ZIC-cHILIC, eight catechol compounds (dopamine, epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylglycol, 3,4-dihydroxymandelic acid, and internal standard 3,4-dihydroxybenzylamine) were separated within 15min. The limit of detection at a signal to noise ratio of 3 was 3-28nM. An improved sensitivity was obtained as compared to that of reversed-phase liquid chromatography. This was partly attributed to the increase in the fluorescence intensity of the catechol compounds in the acetonitrile-rich mobile phase. Solid phase extraction using a monolithic silica disk-packed spin column with phenylboronate moieties, which have affinity to catechol compounds, was performed for the selective extraction of catechol compounds from mouse urine. Dopamine, epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylglycol were successfully quantified in mouse urine.

Determination and characterization of total thiols in mouse serum samples using hydrophilic interaction liquid chromatography with fluorescence detection and mass spectrometry

Biothiols such as homocysteine, cysteine, and glutathione play many biologically important roles, especially in reduction-oxidation homeostasis and resistance to oxidative stress, and the measurement of their concentrations in model animal fluids is important in clarifying the pathology of thiol-related diseases. In this study, an analytical method for total biothiols in mouse serum using hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection was developed. Mouse serum samples were derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), after reduction by tris(2-carboxyethyl)phosphine. Five biothiols (homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine) in the mouse sera were separated within 16 min on an amide-type HILIC column. The method possessed good linearity, good reproducibility with an intra-day variance of less than 3%, and low detection limits of 0.2-4 nM. Concentrations of homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine in the mouse serum samples were calculated as 6.7 ± 0.3, 227.7 ± 16.9, 1.2 ± 0.4, 77.5 ± 29.2, and 8.2 ± 0.9 μM, respectively (mean ± S.D., n = 4). Furthermore, HILIC-negative electrospray ionization-mass spectrometry (MS) analysis using a high-resolution mass spectrometer was conducted to determine the exact masses of two unknown peaks, which were found in the mouse serum samples with high signal intensity and were not detected in human plasma samples. The exact masses of the unknown compounds were determined as 1184.519 and 800.281 (as SBD-derivatized negative ions), which possessed a product ion common to SBD-thiols (m/z 230.954, as [SBD-SH](-)) upon tandem MS spectrometric analysis.

HPLC-fluorescence determination of thiol compounds in the serum of human male and female subjects using HILIC-mode column

High-performance liquid chromatography-fluorescence detection using a hydrophilic interaction chromatography-mode column (ZIC®-HILIC) was used to determine four kinds of thiol compounds in human serum. Sera were obtained from 34 subjects for this study (17 male subjects aged 22-38 years and 17 female subjects aged 18-38 years). Serum cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine, derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate, were separated on the ZIC®-HILIC column and quantified. The serum concentrations of cysteine, cysteinylglycine, glutathione and γ-glutamylcysteine were 226 ± 4.7, 23.4 ± 1.3, 3.7 ± 0.2 and 3.2 ± 0.1 μm, respectively. In addition, the concentrations of serum thiol compounds from male subjects were significantly higher than those of the female subjects (p < 0.05).

MEMS LC microchip with low dispersion and low pressure drop turn structure using distribution controlled micro pillar array

MEMS liquid chromatography (LC) microchip with low dispersion and a low pressure drop turn structure was fabricated. In the turn structure, we used higher density pillar array at the inner side while lower density pillar array was used at the outer side to control inner and outer speed of a sample flow. An LC chip with an improved turn structure can obtain similar theoretical plate number (1233) compared to the previously proposed tapered turn (1135). The necessary inlet pressure is also reduced from 2.7 to 0.4 MPa under a flow rate of 10 μL/min. These results show the improved turn structure is applicable for fast analysis time under high flow rate conditions.