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ABSTRACT: In this report, we present a simple and rapid method for analysis of 21 kinds of bile acids and the conjugates in rat serum and liver samples by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) in the negative ionization mode, using cholic-2, 2, 4, 4-d4 acid as internal standard. After liquid-liguid extraction from serum and liver samples, specimens were analyzed by UPLC equipped with an Acquity TQD tandem quadrupole mass spectrometer. All of the 21 bile acids were sufficiently separated within 5 min. For most bile acids, calibration curves showed good linearities in the range of 0.25 to 5000 ng/mL for serum samples, 2.5 ng/g to 50 microg/g for liver samples. The limits of detection (LOD) were estimated to be less than 0.25 to 7.5 ng/mL in serum, less than 2.5 to 10 ng/g in liver samples. The present method was validated with respect to repeatability; the coefficient of variation (CV) values were less than 26.7% in the serum and 25.9% in the liver. In the animal study, we compared 21 bile acids in the serum and liver samples of the stroke-prone spontaneously hypertensive (SHRSP) rats fed with control (SP) diet or high-fat and high-cholesterol-containing (HFC) diet. By feeding with HFC diet, the glycine conjugates of some bile acids significantly increased and the taurine conjugate of ulsodeoxicolate (TUDC) decreased in serum and liver samples. Our results suggest that the change of bile acid profiles could be applied for the diagnosis of non-alcoholic fatty liver disease (NAFLD).
Nagoya journal of medical science 02/2013; 75(1-2):57-71.
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ABSTRACT: Blonanserin is a novel antipsychotic agent having dopamine D2 and serotonin 5-HT2A receptor antagonist properties. In this communication, a simple method for analysis of blonanserin in human plasma by gas
chromatography-mass spectrometry (GC-MS) using hexobarbital as internal standard (IS) is presented. One milliliter of plasma
containing blonanserin and 50 ng of IS was mixed well with 0.5 ml of 0.1 M hydrochloric acid and 2.5 ml of distilled water;
the mixture was applied to an Oasis HLB cartridge. The cartridge was washed with 5 ml of distilled water, and the target compound
and IS were eluted with 2 ml of chloroform. The eluate was evaporated to dryness under a stream of nitrogen. The residue was
reconstituted in 50 μl of methanol for use in GC-MS analysis. The calibration curve for blonanserin spiked into human plasma
showed good linearity in the range of 0.5–20 ng/ml. The detection limit (signal-to-noise ratio = 3) was about 0.25 ng/ml.
The recovery rate was 81.0% at 2.5 ng/ml and 97.5% at 15 ng/ml. The accuracy and precision were also satisfactory. A 60-year-old
male volunteer took a 10-mg oral dose of blonanserin; the plasma concentrations of the drug were 0.61 and 1.20 ng/ml at 1.5
and 4 h after ingestion, respectively.
KeywordsBlonanserin-Antipsychotic-Second generation-GC-MS-Hexobarbital-Oasis HLB
Forensic Toxicology 05/2012; 28(2):105-109. · 3.00 Impact Factor
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ABSTRACT: A number of reports are available in the literature that describe liquid chromatography-mass spectrometry (LC-MS) and LC-tandem
mass spectrometry (LC-MS-MS) analysis of amanitins, very toxic mushroom toxins, in biological samples. However, the extractive
pretreatment methods and LC separation column materials vary remarkably according to the different reports. This communication
presents a very simple and suffi ciently sensitive method for LC-MS analysis of amanitins. A plasma sample was diluted with
distilled water and buffer solution, and applied to a Discovery DSC 18 column (500 mg packing material), followed by washing
with distilled water and elution with methanol. The extract, after evaporation and reconstitution in mobile phase solution,
was subjected to LC-MS analysis with a conventional octadecyl LC separation column. The selected ion monitoring of α-amanitin
and β-amanitin at m/z 919–921 and m/z 920–922, respectively, gave symmetrical peaks and good separation of both amanitin peaks. Using an external calibration method,
linearity, detection limits, recovery rates, and precision were tested; they were all satisfactory. To our knowledge, the
present method gives the simplest LC-MS analysis for amanitins among those so far reported. We recommend the method for use
in actual forensic and clinical toxicological analysis of amanitins in biological samples.
Keywordsα-Amanitin-β-Amanitin-LC-MS (-MS)-Solid-phase extraction-Discovery DSC 18 column-Plasma
Forensic Toxicology 04/2012; 28(2):110-114. · 3.00 Impact Factor
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ABSTRACT: A Japanese female in her thirties was found dead in her apartment; the postmortem interval was estimated to be approximately 3 days. Several postmortem changes were evident. Acute gastroenteritis had been diagnosed 3 days earlier. On autopsy, no specific findings other than fatty liver were observed. On hematoxylin eosin staining, mild fibrosis, and invasion of neutrophils in the pancreatic parenchyma, severe fatty liver, and extensive vacuolation of renal tubular cells were observed. Biochemical analyses revealed extremely high β-hydroxybutyrate concentrations in body fluids with moderate elevation of hemoglobin A1c. Toxicological analyses of organ and urine samples were negative. We concluded that severe ketoacidosis had occurred in the deceased. Subsequently, selective destruction of pancreatic β-cells was demonstrated. Considered together, results indicated that the cause of death was fulminant type 1 diabetes mellitus. This report illustrates the fact that a combination of biochemical and immunohistochemical investigations can be useful for diagnosing this condition in cases with evident postmortem changes.
Legal Medicine 08/2011; 13(5):250-3.
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ABSTRACT: In this report, a high-throughput and sensitive method for analysis of eight central-acting muscle relaxants in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in the positive and negative ionization modes using tolbutamide as internal standard is presented. After pretreatment of a plasma sample by solid-phase extraction with an Oasis HLB cartridge, muscle relaxants were analyzed by UPLC with Acquity UPLC BEH C(18) column and Acquity TQD tandem quadrupole mass spectrometer equipped with an electrospray ionization interface. The calibration curves for muscle relaxants spiked into human plasma equally showed good linearities in the nanogram per milliliter order range. The detection limits (signal-to-noise ratio = 3) was as low as 0.1-2 ng/mL. The method gave satisfactory recovery rates, accuracy, and precision for quality control samples spiked with muscle relaxants. To further validate the present method, 250 mg of chlorphenesin carbamate was orally administered to a healthy male volunteer, and the concentrations of chlorphenesin carbamate in plasma were measured 0.5, 1, 2, 4, 6, and 8 h after dosing; their concentrations in human plasma were between 0.62 and 2.44 μg/mL. To our knowledge, this is the first report describing simultaneous analysis of over more than two central-acting muscle relaxants by liquid chromatography-tandem mass spectrometry. This has been realized by the capability of our instrument for simultaneous multiple reaction monitoring of the target compounds in both positive and negative ionization modes. Therefore, the present method seems very useful in forensic and clinical toxicology and pharmacokinetic studies.
Analytical and Bioanalytical Chemistry 03/2011; 400(7):1959-65. · 3.78 Impact Factor
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ABSTRACT: A rapid and sensitive method for analysis of blonanserin in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry is presented. After pretreatment of a plasma sample by solid-phase extraction, blonanserin was analyzed by the system with a C(18) column. This method gave satisfactory recovery rates, reproducibility, and good linearity of calibration curve in the range of 0.01-10.0 ng/mL for quality control samples spiked with blonanserin. The detection limit was as low as 1 pg/mL. This method seems very useful in forensic and clinical toxicology and pharmacokinetic studies.
Analytical Sciences 01/2010; 26(10):1099-102. · 1.25 Impact Factor
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Tetsuya Arinobu,
Hideki Hattori,
Takeshi Kumazawa,
Xiao-Pen Lee,
Yoko Mizutani,
Takao Katase,
Sadao Kojima,
Takayuki Omori,
Rina Kaneko, Akira Ishii,
Hiroshi Seno
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ABSTRACT: Automated high-performance liquid chromatography/mass spectrometry (HPLC-MS) with backflush column-switching was established
for ultra-fast determination of theophylline and caffeine. A 400-μl portion of serum sample diluted with ultrapure water was
injected and transferred to an Oasis HLB cartridge used as a precolumn for extraction. After switching the valves, the analytes
trapped in the precolumn were eluted in the backflush mode and separated with a Chromolith Performance RP-18e column (C18-bonded monolithic silica); the compounds in column effluents were then detected by atmospheric pressure chemical ionization
(APCI)-MS. The present method successfully provided high-throughput determination of theophylline and caffeine within 2 min.
Satisfactory linearity, reproducibility, and sensitivity could be obtained for analysis of therapeutic and toxic levels of
both compounds. Because of the very simple procedure and high throughput using the conventional HPLC system, the present method
seems to have high potential in the fields of forensic toxicology and emergency medicine.
Forensic Toxicology 01/2009; 27(1):1-6. · 3.00 Impact Factor
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ABSTRACT: A detailed procedure has been established for simultaneous analysis of aconitine, mesaconitine, hypaconitine, and jesaconitine
in whole blood using liquid chromatography-tandem mass spectrometry (LC-MS-MS). The method uses a new unique polymer column
(Shodex ODP2 HP-4B) for separation, which enabled the injection of relatively crude organic extracts without complicated pretreatments.
Quantitation was made by mass chromatography with each product ion referenced against dextromethorphan as internal standard.
Aconitine and its three analogues showed good linearity over the range of 1.25–40 ng/ml; the detection limits were 0.3–0.5
ng/ml. Validation data including accuracy, precision, and recovery rates are presented. To further validate the present method,
the mixture of aconitine, mesaconitine, and hypaconitine (0.2 mg each) was orally administered to rats, and the concentrations
of the three compounds in whole blood specimens were measured 15, 30, and 45 min after dosing; their concentrations in rat
whole blood were 0.74–18.3 ng/ml. The present LC-MS-MS method is very effective for simple simultaneous analysis of aconitine
and its analogues in forensic and clinical toxicology.
Forensic Toxicology 01/2009; 27(1):7-11. · 3.00 Impact Factor
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ABSTRACT: Three selective serotonin reuptake inhibitors (sertraline, fluvoxamine, and paroxetine) in human serum specimens were analyzed
by liquid chromatography-tandem mass spectrometry using a new polymer column (Shim-pack MAYI-ODS), which enabled direct injection
of crude biological samples without complicated pretreatments. Quantitation was made by mass chromatography for each product
ion using dextromethorphan as internal standard. The recoveries of the three drugs from human serum were 29.2%–45.7% at 20
ng/ml and 52.0%–53.7% at 80 ng/ml. The regression equations showed good linearity for the three drugs in the range of 5–80
ng/ml. Each drug had a detection limit of 1–3 ng/ml. Thus, the present method of using a new polymer column is effective for
rapid and sensitive analysis of both therapeutic and toxic levels of sertraline, fluvoxamine, and paroxetine in biological
specimens.
Forensic Toxicology 01/2007; 25(2):100-103. · 3.00 Impact Factor
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ABSTRACT: Mequitazine has been found to be extractable from human plasma samples using MonoTip C(18) tips, inside which C(18)-bonded monolithic silica gel was fixed. Human plasma (0.1mL) containing mequitazine and cyproheptadine as an internal standard (IS) was mixed with 0.4mL of distilled water and 25muL of 1M potassium phosphate buffer (pH 8.0). After centrifugation of the mixture, the supernatant fraction was extracted to the C(18) phase of the tip by 25 repeated aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C(18) phase were then eluted with methanol by five repeated aspirating/dispensing cycles. Without evaporation and reconstitution, the eluate was injected into a gas chromatograph injector and detected by a mass spectrometer with selected ion monitoring in the positive-ion electron impact mode. The separation of mequitazine and the IS from each other and from impurities was generally satisfactory using a DB-1MS capillary column (30mx0.32mm i.d., film thickness 0.25mum). The recoveries of mequitazine and the IS spiked into plasma were more than 90.0%. The regression equation for mequitazine showed excellent linearity in the range of 0.2-200ng0.1mL(-1), and the detection limit was 0.05ng0.1mL(-1)of plasma. The intra-day and inter-day coefficients of variation for mequitazine in human plasma were not greater than 8.16 and 9.24%, respectively. Accuracy for the drug was in the range of 90.0-97.4%. The data obtained from determination of mequitazine in human plasma after oral administration of the drug are also presented.
Talanta 10/2006; 70(2):474-8. · 3.79 Impact Factor
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ABSTRACT: The Aconitum species (Ranunculaceae) are widely distributed in northern Asia and North America. Their roots are popularly used in herbal medicines in China and Japan. Many cases of accidental, suicidal and homicidal intoxication with this plant have been reported; some of these were fatal because the toxicity of Aconitum is very high. It is thus important to detect and quantify Aconitum alkaloids in body fluids, with high sensitivity. We have developed a simple and sensitive method for measuring four kinds of Aconitum alkaloids (aconitine, hypaconitine, jesaconitine and mesaconitine) by LC/electrospray (ESI)-time-of-flight (TOF)-MS. For all of them, only molecular ions were observed at an orifice voltage of 75 V; at 135 V, base peaks corresponding to [M - 60 + H]+ ions were observed. These four compounds and methyllycaconitine (internal standard) in human plasma samples were purified by solid-phase extraction. The four extracted compounds were completely separated in mass chromatograms; the calibration curves showed good linearity in the range 10-300 ng/ml, and the detection limits were estimated to be 0.2-0.5 ng/ml. Using our method, we also determined the amounts of these compounds in tuber samples. The present method is applicable in clinical and forensic toxicology.
Journal of Mass Spectrometry 07/2006; 41(6):810-4. · 3.27 Impact Factor
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ABSTRACT: Breath alcohol tests are widely used to control DUI (driving under the influence) in Japan. However, this test is not applied to injured drivers transported to emergency hospitals. In such cases, BAC (blood alcohol concentration) testing should be done to prove DUI. In this paper, we tried to clarify two important issues on the BAC testing in Japan using a questionnaire survey and experiments about contamination of antiseptic ethanol. First, we have described the doctor's dilemma with DUI cases; our present questionnaire survey showed that the police often request the doctor to volunteer blood samples of the suspected drunk drivers brought to emergency hospitals since they have not been granted the right to order blood sampling in Japan. Then, doctors face a serious dilemma whether comply with the police request or not, resulting in widely different responses. Secondly, we have estimated the effects of antiseptic ethanol routinely used as a dermal antiseptic on the BAC tests. Our present experiments showed that uptake of ethanol can occur under certain conditions. Given the actual conditions outlined in the questionnaire, there seem to be a definite risk of ethanol contamination in BAC testing. Obviously, the time has come to discuss problems in BAC testing of injured drivers brought to emergency hospitals in Japan.
Legal Medicine 11/2005; 7(5):299-305.
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ABSTRACT: Paraquat (PQ) and diquat (DQ) in human whole blood and urine were analyzed by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) with positive ion electrospray ionization (ESI). The compounds were extracted with Sep-Pak C18 cartridges from whole blood and urine samples containing ethyl paraquat as an internal standard. The separation of PQ and DQ was carried out using ion-pair chromatography with heptafluorobutyric acid in 20 mM ammonium acetate and acetonitrile gradient elution for successful coupling with MS. Both compounds formed base peaks due to [M-H]+ ions by HPLC/ESI-MS and the product ions produced from each [M-H]+ ion by HPLC/MS/MS. Selective reaction monitoring (SRM) showed much higher sensitivity for both body fluids. Therefore, a detailed procedure for the detection of compounds by SRM with HPLC/MS/MS was established and carefully validated. The recoveries of PQ and DQ were 80.8-95.4% for whole blood and 84.2-96.7% for urine. The calibration curves for PQ and DQ showed excellent linearity in the range of 25-400 ng ml(-1) of whole blood and urine. The detection limits were 10 ng ml(-1) for PQ and 5 ng ml(-1) for DQ in both body fluids. The intra- and inter-day precision for both compounds in whole blood and urine samples were not greater than 13.0%. The data obtained from the determination of PQ and DQ in rat blood after oral administration of the compounds are also presented.
Journal of Mass Spectrometry 11/2004; 39(10):1147-52. · 3.27 Impact Factor
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ABSTRACT: Simultaneous determination of seven barbiturates in human whole blood and urine by combining direct immersion solid-phase microextraction (DI-SPME) with gas chromatography-mass spectrometry (GC-MS) is presented. The main parameters affecting the DI-SPME process, such as SPME fibers, salt additives, pHs, extraction temperatures and immersion times were optimized for simultaneous determination of the drugs. The extraction efficiencies were 0.0180-0.988 and 0.0156-2.76% for whole blood and urine, respectively. The regression equations of the drugs showed excellent linearity for both samples; the correlation coefficients (r(2)) were 0.994-0.999. The detection limits for whole blood were 0.05-1 microg x ml(-1), and those for urine 0.01-0.6 microg x ml(-1). Actual quantitation could be made for pentobarbital in whole blood and urine obtained from volunteers, who had been orally administered a therapeutic dose of the drug. The DI-SPME/GC-MS procedure for barbiturates established in this study is simple and sensitive enough to be adopted in the fields of clinical and forensic toxicology.
Journal of Chromatography B 07/2004; 806(1):65-73. · 2.89 Impact Factor
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ABSTRACT: We have presented a simple and sensitive method for determining pethidine, a narcotic analgesic drug in body fluids by gas chromatography-tandem mass spectrometry (GC-MS/MS). Pethidine and 4'-piperidinoacetophenone (internal standard) were extracted from body fluids with Bond Elut C(18) columns; the recoveries were above 85% for both compounds. The calibration curves for blood and urine showed good linearities in the range of 1.25-40 ng/ml. Its detection limits (signal-to-noise ratio=3) were estimated to be approximately 0.5 ng/ml of whole blood and urine.
Journal of Chromatography B 08/2003; 792(1):117-21. · 2.89 Impact Factor
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ABSTRACT: A 50-year-old male was hit by a high-speed golf ball on the left lateral side of his neck. He collapsed immediately and was sent to hospital, where he was pronounced dead. The autopsy showed an extensive basal subarachnoid haemorrhage. Careful gross and histological examinations disclosed a rupture of the right vertebral artery at a site very close to the bifurcation. It was estimated that the impact of the golf ball on the left side of his neck resulted in the rupture of the contralateral vertebral artery, owing to its hyperextension. Although there are many reports on traumatic basal subarachnoid haemorrhage, the present type of trauma seems rare to our knowledge.
Medicine, science, and the law 05/2003; 43(2):174-8. · 0.45 Impact Factor
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ABSTRACT: The abuse of alkyl nitrites is becoming a serious social problem worldwide. In this report, a simple and sensitive method is presented for the determination of n-butyl alcohol, isobutyl alcohol, and isoamyl alcohol as decomposition products of alkyl nitrites in human whole blood and urine samples using capillary gas chromatography (GC) with cryogenic oven trapping. After heating a whole blood or urine sample containing each alkyl alcohol and t-butyl alcohol [the internal standard (IS)] in a 7-mL vial at 55 degrees C for 15 min, 5 mL of the headspace vapor is drawn into a gas-tight syringe and injected into a GC inlet port. The vapor is introduced into an Rtx-BAC2 medium-bore capillary column in the splitless mode at 0 degrees C oven temperature in order to trap the entire analytes, and then the oven temperature is programmed up to 240 degrees C for the GC measurements by flame ionization detection. These conditions give sharp peaks for each compound and the IS and low background noise for whole blood or urine samples. The detection limits of the analytes are 10 ng/mL for whole blood and 5 ng/mL for urine. Linearity and precision are also tested to confirm the reliability of this method. Isobutyl alcohol and methemoglobin could be determined from the whole blood samples of three male volunteers who had sniffed isobutyl nitrite.
Journal of chromatographic science 03/2003; 41(2):63-6. · 0.88 Impact Factor
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ABSTRACT: Phenothiazines are widely prescribed as neuroleptics; some are used as antihistaminics. These compounds are important in clinical and forensic toxicology. Seventeen phenothiazine compounds with heavy side chain structures have been found to be detectable by high-performance liquid chromatography/fast atom bombardment-mass spectrometry (HPLC/FAB-MS) method. Authentic samples of the compounds were subjected to our HPLC/FAB-MS system; their mass spectra were obtained by positive and negative modes. Four typical phenothiazines, in the serum samples of two patients, were also analyzed. All 17 phenothiazines were sufficiently separated on the chromatogram. In the positive mode, all the base peaks were quasimolecular ions; their main fragment ions observed were [M-R(1)+CH(2)](+), [R(1)](+), [M-R(1)](+) and [M+H+Gly](+). In the negative mode, the base peaks were [Cl](-) for chlorpromazine, prochlorperazine and perphenazine, three compounds containing chloride. For the other compounds, they were [M-R(1)-CH(3)](-), [M-R(1)-CH(2)CH(3)](-) or [M-R(1)-(CH(3))(2)](-) ions. We observed [M+H](-) ions in all the compounds, however, the ir intensities were variable (3-74%). The spectra and mass chromatograms of four compounds and their metabolites in the extracted serum samples from two patients, were also obtained. The approximate detection limits for phenothiazines were less than 1 ng on-column in the positive mode, and about 1 microg on-column in the negative mode. We have succeeded in the identification and characterization of 17 phenothiazine compounds at therapeutic concentrations in body fluids using our HPLC/FAB-MS system. The present method would be useful in forensic toxicological practice.
Legal Medicine 01/2003; 4(4):207-16.
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ABSTRACT: We report a rare case of the death of a stunt man who jumped into the sea driving a motorcycle on location for a TV drama. A special ramp had been constructed for the jump. The stunt man and a passenger drove up the ramp at a speed of 60-70 km/h and jumped into the sea from a height of about five metres. Both men swam up, but the driver fell unconscious at the surface. He was sent to hospital, but did not recover. The autopsy revealed a deep laceration of the right axilla with complete rupture of the axillary artery, bone fractures of the right ribs and the right pelvis, and a small laceration of the upper lobe of the right lung. Other visceral organs were not injured. We finally judged that his cause of death was exsanguination due to rupture of the right axillary artery on the basis of the presence of a small amount of blood inside the heart and ischemic organs. All injuries were probably inflicted by a collision with the motorcycle upon reaching the water surface; the axillary injury might be due to the left handlebar piercing into the part.
Medicine, science, and the law 11/2002; 42(4):355-7. · 0.45 Impact Factor
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ABSTRACT: Surface ionization (SI), which consists in the formation of positive and negative ions along the course of thermal desorption of particles from a solid surface, was first applied as a detector for gas chromatography (GC), GC-surface ionization detection (SID); we developed many new sensitive methods for the determination of abused and other drugs by GC-SID. Recently, Fujii has devised a combination of SI and a quadrupole mass spectrometer and named this system a surface ionization organic mass spectrometer (SIOMS), which is highly selective and sensitive for organic compounds containing tertiary amino groups. We have tried to apply this mass spectrometer to forensic toxicological study; so far we have succeeded in determining important drugs-of-abuse and toxic compounds, such as phencyclidine (PCP), pethidine, pentazocine, MPTP and its derivatives from human body fluids with high sensitivity and selectivity. In this review, we describe our recent studies on the application of GC-SIOMS to forensic toxicology.
Journal of Chromatography B 09/2002; 776(1):3-14. · 2.89 Impact Factor
