Masataka Ishibashi

Biosynthesis of α-ecdysone by prothoracic glands in vitro

An in vitro study in which isolated prothoracic glands of the Bombyx silkworm were cultured has provided definite evidence that the prothoracic gland is the site where molting hormone is synthesized. The hormone behaved very similarly to free ecdysone on thin-layer chromatography. Analysis by liquid chromatography and mass fragmentography revealed that the hormone is identical with α-ecdysone.

Microanalysis of bile acid in human liver tissue by selected ion monitoring

A method of microquantitative determination of bile acid in 5–30 mg of human liver tissue was developed. Bile acids were converted to their ethyl ester dimethylethylsilyl ether derivatives and analyzed by capillary gas chromatography-selected ion monitoring, using [2H4]-lithocholic (LCA), [2H5]deoxycholic (DCA), [2H4]chenodeoxycholic (CDCA), [2H4]-ursodeoxycholic (UDCA), and [2H3]cholic (CA) acids as internal standards. Precision and reproducibility of the present method were tested using surgically obtained liver specimens. The results were statistically analyzed according to one-way layout and the orthogonal polynomial equation. Bile acids except LCA were determined with 2.3 to 11.4% of the coefficient of variation. Recoveries of conjugated bile acids ranged from 72.2 to 96.0% with a mean of 84.3%. The amount of bile acids present in histologically normal liver specimens (n = 10) was found to be 29.56 ± 8.62 μg/g liver. The relative compositions (%) of CDCA and CA were 38.8 ± 8.9 and 41.1 ± 11.0, respectively.

Changes in the levels of prostaglandins and thromboxane and their roles in the accumulation of exudate in rat carrageenin-induced pleurisy — a profile analysis using gas chromatography-mass spectrometry —

Injection of lambda-carrageenin into the pleural cavity of rats caused the accumulation of the pleural exudate. When levels of prostaglandins (PGs) and thromboxane (TX) B2 were quantified by gas chromatography-mass spectrometry as their methyl ester (ME)-dimethylisopropylsilyl (DMiPS) ether or ME-methoxime-DMiPS ether derivatives, 6-keto-PGF1 alpha reached the maximum at 1 hr after carrageenin, then PGE2 and TXB2 showed peaks at 3 hr and waned off before 9 hr. The PGF/ alpha level was kept low, but PGD2, PGE1 and PGF1 alpha were not detected. Aspirin (100 mg/kg, i.p.) significantly decreased the PG and TXB2 levels and suppressed the rate of plasma exudation until 5 hr, but did not at 7 hr, when it was measured by the amount of exuded pontamine sky blue injected intravenously. OKY-025 (300 mg/kg, i.p.), a selective TXA synthetase inhibitor, and tranylcypromine (20 mg/kg, i.p.), a PGI synthetase inhibitor, could not extensively inhibit the accumulation of the exudate. These results suggest that the cyclooxygenase products of arachidonic acid, particularly PGE2, definitely play an important role in the exudation during the first 5 hr.

Simultaneous qualitative and quantitative analyses of bile acids by mass chromatography

Abstract For the profile analysis of bile acids by mass chromatography, the extraction and clean-up procedure, hydrolysis and derivatization have been studied. A new method of mass chromatography for qualitative and quantitative determinations involving the multi-deuterium-labeled conjugated bile acids as internal standards and the reciprocal of the relative intensity of the peaks on the mass chromatogram was developed. This method can be applied to the separation of isomers which have almost identical retention times and which undergo the same fragmentation with different intensities.

Gas chromatography of dimethylalkylsilyl ether derivatives of bile acid methyl esters

The gas chromatographic separation of the DMAS ether derivatives of ten bile acid methyl esters, namely DMES, DMnPS and DMiPS ethers, have been studied by use of an open tubular glass capillary column, coated with SE-30. The DMAS ether derivatives were eluted in sequence according to the number of hydroxyl groups except of 12-KCDCA, and separated. This contrasts with the poorly resolved gas chromatographic peaks produced by the TMS ether derivatives. The DMES ether derivatives were resolved and baseline separation in 30 min, but those of HCA and 12-KCDCA had similar methylene unit values.

Quantitative analysis of berberine in urine samples by chemical ionization mass fragmentography

A highly specific and sensitive method has been developed for the quantitative determination of berberine in human urine. In order to carry out the microdetermination of berberine by chemical ionization mass fragmentography, berberine was reduced with sodium borohydride in methanol to tetrahydroberberine and subjected to gas chromatography-mass spectrometry. Berberine concentrations as low as 1 ng/ml urine can be measured by this method, with [2H3]berberine chloride as an internal standard.

Simultaneous determination of glyceryl trinitrate and its principal metabolites, 1,2- and 1,3-glyceryl dinitrate, in plasma by gas chromatography-negative ion chemical ionization-selected ion monitoring

A specific and sensitive method for the quantitation of glyceryl trinitrate (GTN) and its principal metabolites, 1,2- and 1,3-glyceryl dinitrate (GDN) in dog plasma by capillary gas chromatography-negative ion chemical ionization-selected ion monitoring using dichloromethane as a reagent gas and the corresponding compounds labelled with stable isotopes as internal standards. The quantitation limits of the method for GTN and the GDNs were 0.1 and 1.0 ng/ml in plasma, respectively. When GTN was administered intravenously to four anaesthetized beagle dogs at a dose of 6 micrograms/kg . min for 30 min, the plasma levels of GTN and 1,2- and 1,3-GDN reached a maximum at the end-point of infusion and decreased with bi-exponential decay. The half-lives of the alpha- and beta-phases were 0.50 and 4.95 for GTN, 8.10 and 40.6 for 1,2-GDN and 8.50 and 48.5 for 1,3-GDN, respectively.

Use of new silylating agents for separation and identification of prostablandins by gas chromatography and gas chromatography—mass spectrometry

Abstract A method for the complete separation and selective ion monitoring of five kinds of primary prostaglandins was developed involving gas chromatography (GC) and gas chromatography—mass spectrometry (GC—MS) using dimethylethylsilyl and dimethyl- n -propylsilyl imidazoles as silylating agents. Of these derivatives of prostaglandins, the dimethylethylsilyl ether derivative provided the most suitable properties in GC and GC—MS. The method described will be useful for the profile analysis of biologically important prostaglandins.

Determination of glyceryl trinitrate in human plasma by gas chromatography—negative ion chemical ionization—selected ion monitoring

A specific, sensitive and accurate quantitation method for glyceryl trinitrate was developed using gas chromatography--negative ion chemical ionization--selected ion monitoring with dichloromethane as a reagent gas. [15N3] and [2H5, 15N3] variants were synthesized from non-labelled or [2H8]glycerol and [15N]nitric acid. The former variant was used for preventing adsorption of glyceryl trinitrate onto active sites on column materials and the latter was used as an internal standard for quantitation of glyceryl trinitrate in biological fluids by selected ion monitoring. The quantitation limit of this method is 0.1 ng/ml of human plasma. When glyceryl trinitrate was administered intravenously in the dose of 4 micrograms/kg to patients receiving hypotensive anesthesia for surgical operation, the plasma levels exhibited a biexponential decay. The mean and standard deviation of half-lives of the alpha and beta phases were found to be about 0.41 +/- 0.13 and 5.34 +/- 1.60 min, respectively.

Microdetermination of prostaglandins and thromboxane B2 by capillary gas chromatography and negative ion chemical ionization mass spectrometry

Application of dimethyl-n- propylsilyl ( DMnPS ) ether derivatives of prostaglandins (PGs) and thromboxane (TX) B2 pentafluorobenzul ( PFB ) esters of negative ion chemical ionization mass spectrometry ( NICIMS ) was investigated. These derivatives were separated completely within 10 min by the use of a fused-silica capillary column coated with methyl silicone. In NICIMS , all of the DMnPS ether derivatives of PGs and TXB2 PFB esters yielded the characteristic negative ion [M - 181]- which was produced by elimination of PFB from the molecule. The detection limit of the DMnPS ether derivative of PGF2 alpha PFB ester was found to be 200 fg with a signal-to-noise ratio of 5 when monitoring the ion of m/z 653 ([M - 181]-) in the high-resolution mode (R = 2500) using ammonia as a reagent gas. The method was applied to the quantitation of PGE2 and PGF2 alpha in an extract obtained from the plasma of a lung-heart preparation from a dog.