Osamu Suzuki

Determination of phenothiazines in human body fluids by solid-phase microextraction and liquid chromatography/tandem mass spectrometry

Eleven phenothiazine derivatives with heavy side-chains were found to be extractable from human whole blood and urine samples by solid-phase microextraction (SPME) with a polyacrylate-coated fiber. The fiber was then injected into the desorption chamber of an SPME-liquid chromatography (LC) interface for LC/tandem mass spectrometry (MS/MS) with positive ion electrospray (ES) ionization. All compounds formed base peaks due to [M + 1](+) ions by LC/ES-MS/MS. By use of LC/ES-MS/MS, the product ions produced from each [M + 1](+) ion showed base peaks due to side-chain liberation. Selected reaction monitoring (SRM) and selected ion monitoring (SIM) were compared for the detection of the 11 phenothiazine derivatives from human whole blood and urine. SRM showed much higher sensitivity than SIM for both types of sample. Therefore, a detailed procedure for the detection of drugs by SRM with SPME-LC/MS/MS was established and carefully validated. The extraction efficiencies of the 11 phenothiazine derivatives spiked into whole blood and urine were 0. 0002-0.12 and 2.6-39.8%, respectively. The regression equations for the 11 phenothiazine derivatives showed excellent linearity with detection limits of 0.2-200 ng ml(-1) for whole blood and 4-22 pg ml(-1) for urine. The intra- and inter-day precisions for whole blood and urine samples were not greater than 15.1%. The data obtained after oral administration of perazine or flupentixol to a male subject are presented.

Analysis of methanol or formic acid in body fluids by headspace solid-phase microextraction and capillary gas chromatography

Methanol and its metabolite formic acid have been found extractable from human whole blood and urine by headspace solid-phase microextraction (SPME) with a Carboxen/polydimethylsiloxane fiber. The headspace SPME for formic acid was carried out after derivatization to methyl formate under acidic conditions. The determinations of both compounds were made by using acetonitrile as internal standard (IS) and capillary gas chromatography (GC) with flame ionization detection. The headspace SPME-GC gave sharp peaks for methanol, methyl formate and I.S.; and low background noises for whole blood and urine samples. Extraction efficiencies were 0.25-1.05% of methanol and 0.38-0.84% formic acid for whole blood and urine. The calibration curves for methanol and formic acid showed excellent linearity in the range of 1.56 to 800 and 1.56 to 500 microg/0.5 ml of whole blood or urine, respectively. The detection limits were 0.1-0.5 microg/0.5 ml for methanol and 0.6 microg/0.5 ml for formic acid for both body fluids. The within-day relative standard deviations in terms of extraction efficiency for both compounds in whole blood and urine samples were not greater than 9.8%. By using the established SPME method, methanol and formic acid were successfully separated and determined in rat blood after oral administration of methanol.

Improved extraction of ethanol from human body fluids by headspace solid-phase microextraction with a carboxen-polydimethylsiloxane-coated fiber

SummaryEthanol can be extracted from human whole blood and urine by headspace solid-phase microextraction (SPME) with a Carboxen-polydimenhylsiloxane-coated fiber. Whole-blood containing ethanol, with isobutanol as internal standard (IS), is heated at 60 °C in the presence of (NH4)2SO4 and sodium dithionite. A Carboxen-polydimethylsiloxane-coated SPME fiber is then exposed to the headspace in the vial for 15 min and then injected into a mdium-bore capillary GC injection port. For urine the SPME procedure is essentially the same as that for whole blood except that the sodium dithionite is omitted. Recoveries of ethanol and IS were, respectively, 1.98 and 28.1% for whole blood, and 2.91 and 35.1% for urine. The calibration curves for ethanol were linear in the range 2.5–400 mg L−1 for whole blood and 0.5–400 mg L−1 for urine; the detection limits for whole blood and urine were 0.5 mg L−1 and 0.2 mg L−1, respectively. Data obtained for determination of ethanol after the drinking of beer are presented for two subjects. This SPME method with the new fiber for ethanol and IS resulted in sensitivity 40–1080 times greater than that obtained with the previous SPME method employing a Carbowax-divinylbenzene-coated fiber.

Ultra-sensitive method for determination of ethanol in whole blood by headspace capillary gas chromatography with cryogenic oven trapping

We have established an ultra-sensitive method for determination of ethanol in whole blood by headspace capillary gas chromatography (GC) with cryogenic oven trapping. After heating a blood sample containing ethanol and isobutyl alcohol (internal standard, IS) in a 7.0-ml vial at 55 degrees C for 15 min, 5 ml of the headspace vapor was drawn into a glass syringe and injected into a GC port. All vapor was introduced into an Rtx-BAC2 wide-bore capillary column in the splitless mode at -60 degrees C oven temperature to trap entire analytes, and then the oven temperature was programmed up to 240 degrees C for GC measurements with flame ionization detection. The present method gave sharp peaks of ethanol and IS, and low background noise for whole blood samples. The mean partition into the gaseous phase for ethanol and IS was 3.06+/-0.733 and 8.33+/-2.19%, respectively. The calibration curves showed linearity in the range 0.02-5.0 microg/ml whole blood. The detection limit was estimated to be 0.01 microg/ml. The coefficients of intra-day and inter-day variation for spiked ethanol were 8.72 and 9.47%, respectively. Because of the extremely high sensitivity, we could measure low levels of endogenous ethanol in whole blood of subjects without drinking. The concentration of endogenous ethanol measured for 10 subjects under uncontrolled conditions varied from 0 to 0.377 microg/ml (mean, 0.180 microg/ml). Data on the diurnal changes of endogenous ethanol in whole blood of five subjects under strict food control are also presented; they are in accordance with the idea that endogenous blood ethanol is of enteric bacterial origin.

High performance liquid chromatography/electrospray tandem mass spectrometry for phenothiazines with heavy side chains in whole blood.

Eleven phenothiazine derivatives with heavy side chains contained in human whole blood have been analyzed by high-performance liquid chromatography (HPLC)/electrospray (ES) tandem mass spectrometry (MS). All compounds gave the base peaks due to [M + 1](+) by HPLC/ES single MS. The product ions formed from each quasi-molecular ion by HPLC/ES tandem MS showed the base peaks due to side chains liberated. The mass chromatography of HPLC/ES tandem MS showed much higher sensitivity than that of HPLC/ES single MS for phenothiazines spiked to whole blood. Therefore, regression equations, detection limits, recovery rates and reproducibility were studied for thiethylperazine, clospirazine and flupentixol spiked to human whole blood by means of mass chromatography of HPLC/ES tandem MS. The three compounds showed good linearity in the range of 2-40 ng/mL with a detection limit of about 0.5 ng/mL. Recoveries of the three compounds spiked to whole blood (2 and 8 ng added to 1 mL whole blood) were 43.4-72.5 %; the coefficients of intraday and interday variations were 3.7-9.3 and 12.6-17.9 %, respectively. Thiethylperazine, clospirazine and flupentixol in whole blood could actually be determined with sufficient sensitivity 3 and 6 h after oral administration of 5-10 mg of each compound in a volunteer.

Determination of triazine herbicides in human body fluids by solid-phase microextraction and capillary gas chromatography

SummaryEight triazine herbicides, prometon, propazine, atrazine, simazine, prometryn, ametryn, metribuzin, and cyanazine, have been extracted from human whole blood and urine samples by headspace solid-phase microextraction (SPME) with a polydimethylsiloxane-coated fiber and quantified by capillary gas chromatography with nitrogen-phosphorus detection.Extraction efficiencies for all compounds were 0.21–0.99% for whole blood, except for cyanazine (0.06%). For urine, the extraction efficiencies for prometon, propazine, atrazine, prometryn and ametryn were 13.6–38.1%, and those of simazine, metribuzin and cyanazine were 1.35–8.73%.The regression equations for the compounds extracted from whole blood were linear within the concentration ranged 0.01–1 μg (0.5 mL)−1 for prometon, propazine, atrazine, prometryn, and ametryn, and 0.02–1 μg (0.5 mL)−1 for simazine, metribuzin, and cyanazine. For urine, regression equations for all compounds were linear within the concentration range 0.005–0.25 μg mL−1. Compound detection limits were 2.8–9.0 ng (0.5 mL)−1 and 0.4–2.0 ng mL−1 for whole blood and urine, respectively. The coefficients of within-day and day-to-day variation were satisfactory for all the compounds, and not greater than 10.3 and 14.2%, respectively.Data obtained from determination of atrazine in rat whole blood after oral administration of the compound are also presented.

MONOAMINE OXIDASE ACTIVITIES IN CATFISH (PARASILURUS ASOTUS) TISSUES

The substrate- and inhibitor-related characteristics of monoamine oxidase (MAO) were studied for catfish brain and liver. The kinetic constants for MAO in both tissues were determined using 5-hydroxytryptamine (5-HT), tyramine and beta-phenylethylamine (PEA) as substrates. For both tissues, the Vmax values were highest with 5-HT and lowest with PEA. The Km value for the brain was highest with 5-HT, followed by tyramine and PEA; but for the liver its value was highest with PEA, followed by 5-HT and tyramine, although all values were in the same order of magnitude. The inhibition of MAO by clorgyline and deprenyl by use of 5-HT, tyramine and PEA as substrates showed that the MAO-A inhibitor clorgyline was more effective than the MAO-B inhibitor deprenyl for both catfish tissues; a single form was present since inhibition by clorgyline or deprenyl with 1000 microM PEA showed single phase sigmoid curves. It is concluded that catfish brain and liver contain a single form of MAO, relatively similar to mammalian MAO-A.

Sensitive determination of xylenes in whole blood by capillary gas chromatography with cryogenic trapping

A new and sensitive method for measurement of o-, m- and p-xylenes in human whole blood by capillary gas chromatography (GC) with cryogenic trapping is presented. After heating 0.5 ml of whole blood and 0.5 ml of distilled water containing the xylenes and aniline (internal standard, I.S.) in a 4.0-ml vial at 100 degrees C for 30 min, 2 ml of the headspace vapor was drawn into a glass syringe. All vapor was introduced through the GC port into an AT-Wax middle-bore capillary column in the splitless mode at an oven temperature of 5 degrees C to trap the entire analytes, and the oven temperature was then programmed up to 180 degrees C. The present conditions gave sharp peaks for xylenes and aniline (I.S.), and low background noises for whole blood samples; the peaks of p- and m-xylenes showed about 90% separation with the AT-Wax column. As much as 41.0-46.3% of xylenes, which had been spiked to whole blood could be recovered. The calibration curves showed linearity in the range of 0.1-0.5 microg/0.5 ml of whole blood. The detection limit was estimated to be about 10 ng/0.5 ml. The coefficients of intra-day and inter-day variations for xylenes were not greater than 9.38%. The data for actual detection of xylenes in post-mortem blood of self-ignition suicide cases by the present method were also presented.

Analyses of butyrophenones and their analogues in whole blood by high-performance liquid chromatography–electrospray tandem mass spectrometry

Five butyrophenones and two analogues contained in human whole blood have been analyzed by high-performance liquid chromatography (HPLC)-electrospray (ES)-tandem mass spectrometry (MS). All compounds gave the base peaks due to [M+1]+ by HPLC-ES-single MS. The product ions formed from each quasi-molecular ion by HPLC-ES-tandem MS showed the base peaks at m/z 165 for four compounds. The mass chromatography of HPLC-ES-tandem MS showed much higher sensitivity than that of HPLC-ES-single MS for all drugs spiked to whole blood. Therefore, regression equations, detection limits, recovery rates and precision were studied for haloperidol, bromperidol and fluoropipamide spiked to human whole blood by means of mass chromatography of HPLC-ES-tandem MS. The three compounds showed good linearity in the range of 0.2-0.8 ng/ml with a detection limit of about 0.1 ng/ml. Recoveries of the three compounds spiked to whole blood (0.2 and 0.8 ng added to 1 ml whole blood) were 23.6-81.2%; the coefficients of intra- and inter-day variations were 8.4-10.4 and 14.5-17.5%, respectively. The three compounds in whole blood could be actually determined 3 and 6 h after oral administration of 1 mg each of haloperidol and bromperidol, and 10 mg of floropipamide in a volunteer.

Determination of solvent thinner components in human body fluids by capillary gas chromatography with trapping at low oven temperature for headspace samples

A simple and sensitive method is presented for determination of solvent thinner components in human body fluids by capillary gas chromatography (GC) with a low oven temperature for trapping headspace vapor components. After heating a blood or urine sample containing ethyl acetate, benzene, butan-1-ol, toluene, butyl acetate, isoamyl acetate and ethylbenzene (internal standard) in a 7.5 ml vial at 90 degrees C for 30 min, 5 ml of headspace vapor were drawn into a glass syringe. All vapor was introduced through an injection port in the splitless mode into a DB-624 medium-bore capillary column at a 5 degrees C oven temperature for trapping the volatile compounds, and the oven temperature was programmed up to 110 degrees C for their detection by GC. These conditions gave sharp peaks, a good separation of each peak and low background noise for both whole blood and urine samples. As much as 3.58-55.1 and 3.52-57.9% of the six compounds, which had been added to vials, could be introduced to the GC instrument for whole blood and urine, respectively. The intra-day RSD values in terms of the introduction rate (net recovery) of the six compounds in whole blood and urine samples were < or = 8.1%. The calibration curves showed linearity in the range 0.78-400 ng per 0.5 ml whole blood or urine. The detection limits were 0.5-5 ng per 0.5 ml. The data on toluene in post mortem blood in an actual case are also presented.