Kenji Matsuura

Determination of Aconitum alkaloids in blood and urine samples. II. Capillary liquid chromatographic-frit fast atom bombardment mass spectrometric analysis.

Determination of fourteen alkaloids, toxic Aconitum alkaloids, aconitine, mesaconitine, jesaconitine, hypaconitine and deoxyaconitine, and their hydrolysis products, benzoylaconines and aconines, have been established using capillary liquid chromatography (LC) fast atom bombardment mass spectrometry (FAB-MS) with a frit interface. Protonated molecular ions were observed as base peaks in the FAB-MS for these fourteen alkaloids. All the alkaloids were simultaneously quantified with linear gradient LC elution by solvent mixture of acetonitrile and 0.3% trifluoroacetic acid using selected ion monitoring of the protonated molecular ions. The calibration curves of these alkaloids were linear in injection amounts ranging from 5 to 500 pg, and their detection limits were 1 pg per injection (S/N=3). Solid-phase extraction using Sep-Pak Plus PS-1 was also investigated to clean-up and concentrate alkaloids in blood and urine samples, and showed satisfactory recoveries. This capillary LC-frit-FAB-MS method enables determination of low levels of Aconitum alkaloids in blood and urine samples, coupled with solid-phase extraction.

Altered metabolism of bile acids in cholestasis: Determination of 1β- and 6α-hydroxylated metabolites

Trihydroxy and tetrahydroxy bile acid metabolites substituted at the C-1 or C-6 position were studied using the urine, serum and liver tissue from sixteen patients with cholestatic liver diseases. Following extraction, isolation and hydrolysis, bile acids were converted into the dimethylethylsilyl derivatives and assayed by capillary gas chromatography—mass spectrometry. Five 1β-hydroxylated bile acids, viz. 1β,3α,12α-trihydroxy-, 1β,3α,7β-trihydroxy-1, 1β,3α,7α,12α-tetrahydroxy-5β-cholanoic acids and an epimer of the first compound, and two 6α-hydroxylated bile acids, viz. 3α,6α,7α-trihydroxy-, 3α,6α,7α,12α-tetrahydroxy-5β-cholanoic acids, were completely or partially identified. Large amounts of 1β-hydroxylated and 6α-hydroxylated bile acids were found in the urine, whereas only trace amounts were detected in the serum and liver tissue. These findings indicate that altered metabolism, such as 1β- or 6α-hydroxylation of bile acids, is enhanced in cholestasis, and that the resulting hydroxylated metabolites are eliminated in the urine.

Determination of pilocarpine in aqueous humour by liquid chromatography—atmospheric pressure chemical ionization mass spectrometry

A new method has been developed for rapid analysis and determination of pilocarpine in aqueous humour using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. The chromatography was carried out on a reversed-phase phenyl column with 0.1% acetic acid-acetonitrile (95:5, v/v). Pilocarpine and its analogues, isopilocarpine, pilocarpic acid and isopilocarpic acid, were separated. An aqueous humour sample was deproteinized with methanol. After evaporation, the residue was dissolved in the mobile phase. The method was applied to the analysis of the metabolite in aqueous humour after the topical application of 2% pilocarpine (w/v) eye-drops. The main metabolite, pilocarpic acid, was easily identified. The protonated molecular ion of pilocarpine was used for the determination. The calibration curve had a good linearity within the concentration range investigated (2 ng to 10 micrograms/ml). The limit of determination was estimated to be an aqueous humour concentration of ca. 2 ng/ml. The method was applied to the determination of unchanged pilocarpine after the topical application of 2% pilocarpine (w/v) eye-drops.

Determination of Isoprostanes as Marker Oxidant Stress by LC-ESI/MS.

The isoprostane, 8-iso-PGF2α is formed from arachidonic acid in vivo by a mechanism independent of cyclooxygenase pathway. We developed a new assay method for 8-iso-PGF2α using [2H4]-8-iso-PGF2α as the internal standard (I.S.) by LC-ESI/MS. For this assay, we established a very simple and rapidly pretreatment method using a membrane filter-type solid phase extraction column (Empore™ disk cartridge) for human plasma and urine. LC-ESI/MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 353.4757 (8-iso-PGF2α) and m/z 357.5073 (I.S.) with a resolution of 3,000. The imprecision for this method was below 14%. Mean inaccuracy was 9% for added levels of 8-iso-PGF2α up to 5,000 pg/mL of urine and 500 pg/mL of plasma. The study of human urinary and plasma 8-iso-PGF2α concentrations may be a convenient diagnostic tool to be able to assess the extent of oxidative stress in vivo not only by smoking but also in other disease states.

Determination of 8-methyl ether of xanthurenic acid in human urine by high-performance liquid chromatography

We developed a simple and sensitive assay for the urinary 8-methyl ether of xanthurenic acid (XA-OMe) by high-performance liquid chromatography with fluorescence detection (excitation at 340 nm; emission at 450 nm). Urine samples were diluted with 0.03 M potassium phosphate buffer (pH 6.0) and applied to an octadecylsilane-bonded column (Nucleosil 5C18, 150 x 4 mm I.D.). The mobile phase used was a mixture of this same buffer and acetonitrile (1000:140, v/v). Both direct injection of urine and solvent extraction prior to HPLC were tested and showed a good correlation and sensitivity, although the peak of XA-OMe was occasionally less distinguishable from close peaks in urine from normal controls by the direct injection method. The quantification limit was 5 x 10(-14) mol which was sensitive enough to detect XA-OMe in urine from normal subjects. The method was applied to samples from patients with a deficiency in tryptophan catabolism, xanthurenic acid/3-hydroxykynurenine-uria and showed a striking elevation in urinary XA-OMe excretion.