Hiroshi Seno

Trace levels of pyrroloquinoline quinone in human and rat samples detected by gas chromatography/mass spectrometry

A detailed procedure for the assay of free pyrroloquinoline quinone (PQQ) in human and rat samples by gas chromatography/mass spectrometry (GC/MS) has been established with stable-isotopic PQQ as internal standard. PQQ was extracted from the samples, after addition of the internal standard, with butanol under acid conditions and with Sep-Pak C18 cartridges. After derivatization of PQQ with phenyltrimethylammonium hydroxide, molecular peaks at m/z 448 and 462 were used for detection of PQQ and [U-13C]PQQ by selected ion monitoring, respectively. Trace amounts of free PQQ were detected in eight organs, plasma and urine of the human, and in three organs of the rat. The PQQ level was highest in the human spleen (5.9 +/- 3.4 ng/g tissue, followed by the pancreas and lung, and it was below detection limits for human brain and heart. Trace levels of PQQ were also found in rat small intestine, liver and testis. Our data are far below those measured by the redox cycling method of Gallops group for human plasma, adrenal and urine.

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.

Headspace solid-phase microextraction and gas chromatographic determination of dinitroaniline herbicides in human blood, urine and environmental water

Solid-phase microextraction (SPME) is a unique extraction and sampling technique, and it has been used for separation of volatile organics from water or other simple matrices. In this study, we have used SPME to separate dinitroaniline herbicides from complicated matrices of human urine and blood in order to broaden its application to biomedical analysis. The SPME conditions were optimized for water, urine and blood samples, in terms of pH, salt additives, extraction temperature, and fiber exposure time. Urine or water (1.0 ml) spiked with herbicides and 0.28 g of anhydrous sodium sulfate was preheated at 70 degrees C for 10 min, and a polydimethylsiloxane-coated fiber for SPME was exposed to the headspace at 70 degrees C for another 30 min; while spiked blood (0.5 ml) diluted with water (0.5 ml) was treated at 90 degrees C in the same way. The herbicides were extractable under these conditions, and could be determined by gas chromatography-electron capture detector (GC-ECD). The recoveries of the herbicides, measured at the concentrations of 0.50 and 1.0 ng/ml urine or water, or 6.0 and 20 ng/0.5 ml blood, ranged from 35 to 64% for different herbicides from water or urine, and from 3.2 to 7.2% from blood. The headspace SPME yielded clean extracts of dinitroaniline herbicides from urine, blood or water, which could be directly analyzed by GC-ECD without further purification. The peak areas of the extracted herbicides were proportional to their concentrations in the range 0.1-10 ng/ml in water or urine, or 1-60 ng/0.5 ml in blood. The lowest detectable concentration of the herbicides lay in 0.1 ng/ml water or urine, or in 0.5 ng/0.5 ml blood. The intra- and inter-day coefficients of variation were within 14% for most of the analytes. Although the recoveries of the herbicides were rather low, the linearity of calibration curve and the precision were good. The developed method is more sensitive and much simpler in sample preparation than previously reported ones. With the established SPME method, a dosed herbicide was successfully separated and determined in rats blood.

Detection of ethanol in human body fluids by headspace solid-phase micro extraction (SPME)/capillary gas chromatography

SummaryEthanol has been found extractable from human whole blood and urine samples by headspace solid-phase micro extraction (SPME) with a Carbowax/divinylbenzene-coated fiber. After heating a vial containing the body fluid sample with ethanol, and isobutanol as internal standard (IS) at 70°C in the presence of (NH4)2SO4, a Carbowax/divinylbenzene-coated SPME fiber was exposed in the headspace of the vial to allow adsorption of the compounds. The fiber needle was then injected into a middle-bore capillary gas chromatography (GC) port. The headspace SPME-GC gave intense peaks for both compounds; a small amount of background noises appeared, but did not interfere with the detection of the compounds. Recoveries of ethanol and IS were 0.049 and 0.026% for whole blood, respectively, and 0.054 and 0.085% for urine, respectively. The calibration curves for ethanol showed excellent linearity in the range of 80–5000 mg L−1 for whole blood and 40–5000 mg L−1 for urine; the detection limits for both samples were 20 and 10 mg L−1, respectively. The data on actual determination of ethanol after the drinking of beer are also presented for two subjects.

Extraction of local anaesthetics from human blood by direct immersion-solid phase micro extraction (SPME)

SummaryLocal anaesthetics have been shown to be extractable from human whole blood samples by direct immersion (DI)-solid phase micro extraction (SPME). After deproteinization with perchloric acid, the pH of the clear supernatants of human whole blood samples containing the drugs were adjusted to about 7 with 10 M NaOH in the presence of NaCl; a polydimethylsiloxanecoated SPME fiber was then immersed directly into the sample solution to allow adsorption of the drugs before capillary gas chromatography (GC) with flame ionization detection. The DI-SPME for 1-mL whole blood gave peaks for all the drugs; only a small amount of background noise appeared and this gave no problems in the detection of the drugs. Recoveries of the ten drugs from human whole blood was 0.74–19.7 %. The calibration curves for seven drugs showed linearity in the range of 0.25–12 μg mL−1 whole blood, with detection limits of 54–158 ng mL−1.

Extraction of methylxanthines from human body fluids by solid-phase microextraction

Abstract Four methylxanthines, caffeine, theobromine, paraxanthine and theophylline, have been found extractable from human whole blood and urine samples by solid-phase microextraction (SPME) with a Carbowax/divinylbenzene-coated fiber. Their determination was made by using capillary gas chromatography with nitrogen phosphorus detection. Extraction efficiency of the methylxanthines was 0.01–0.047% for whole blood, and 0.05–0.229% for urine, respectively. The regression equations for the compounds showed excellent linearity in the range 1.88–60xa0μgxa0ml −1 for whole blood and urine samples, except for caffeine (0.31—-20xa0μgxa0ml −1 for whole blood and 0.16–20xa0μgxa0ml −1 for urine). The detection limits for each of the compounds were 0.2–0.9 and 0.06–0.7xa0μgxa0ml −1 for whole blood and urine, respectively. The coefficients of within-day variation in terms of extraction efficiency for all compounds in whole blood and urine samples were 3.2–9.3% and 2.6–8.4%, respectively. The data obtained for the actual determination of four methylxanthines in a male subject after ingestion of coffee or cocoa were also presented.

Rapid isolation with Sep-Pak C18 cartridges and wide-bore capillary gas chromatography of some barbiturates.

A simple and rapid method for isolation of nine barbiturates with Sep-Pak C18 cartridges from human urine, plasma and whole blood, is presented; the detection of drugs was made by wide-bore capillary gas chromatography (GC) with flame ionization. The drug-containing samples, after mixing with dilute acid solution, were directly applied to the cartridges and eluted with either chloroform/methanol (9:1) or acetonitrile. Separation of the nine drugs was satisfactory with use of an intermediately polar HP-17 capillary column. Recoveries of most compounds were excellent for both chloroform/methanol and acetonitrile as elution solvents. However, backgrounds were cleaner and evaporation time was much shorter for the chloroform/methanol system. The present isolation method with use of Sep-Pak C18 cartridges and wide-bore capillary GC seem very useful in the fields of forensic chemistry, clinical toxicology and clinical pharmacology.

Rapid isolation with Sep-Pak C18 cartridges and wide-bore capillary gas chromatography of organophosphate pesticides

A simple and rapid method for isolation of eleven organophosphate pesticides with Sep-Pak C18 cartridges from human urine and plasma, is presented. The detection of the pesticides was made by wide-bore capillary gas chromatography (GC) with flame ionization detection. The pesticide-containing samples, after mixing with water, were directly applied to the cartridges and eluted with chloroform/isopropanol (9:1). The recoveries with use of the cartridges were excellent for most pesticides. Separation of each pesticide peak from each other and from impurities on the gas chromatograms was also satisfactory with use of non-polar and slightly polar capillary columns. The isolation method with use of the cartridges, together with the wide-bore capillary GC, seems very useful in forensic and environmental chemistry and clinical toxicology.

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.

Simple extraction of phencyclidine from human body fluids by headspace solid-phase microextraction (SPME)

SummaryPhencyclidine (PCP) was found to be extractable by headspace solid-phase microextraction (SPME) from human whole blood and urine. Sample solutions were heated at 90°C in the presence of NaOH and K2CO3, and an SPME fiber was exposed in the headspace of a vial for 30 min. Immediately after withdrawal of the fiber, it was analyzed by gas chromatography with surface ionization detection (GC-SID). Recoveries of PCP were approximately 9.3–10.8% and 39.8–47.8% for whole blood and urine samples, respectively. The calibration curve for PCP showed good linearity in the range 2.5–100 ng mL−1 whole blood and 0.5–100 ng mL−1 urine. The detection limits were approximately 1.0 ng mL−1 for whole blood and 0.25 ng mL−1 for urine.