Kenichiro Nakashima

Simultaneous determination of homocysteine, methionine and cysteine in maternal plasma after delivery by HPLC-fluorescence detection with DBD-F as a label

An HPLC-fluorescence (FL) method for determination of sulfur-containing amino acids such as homocysteine (Hcy), methionine (Met) and cysteine (Cys) in human plasma was developed. The sulfur-containing amino acids were labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F). Calibration curves in the range of 1-100 µm (Hcy and Met) and 5-500 µm (Cys) indicated good linearities (r ≥ 0.998). The limits of detection at a signal-to-noise ratio of 3 were 0.13 (Hcy), 0.02 (Met) and 0.11 µm (Cys), respectively. Acceptable results for accuracy and precision of intra- and inter-day measurements were obtained. The results of Hcy and Cys obtained by the proposed method indicated good correlations with the conventional method (r > 0.911, n = 20). Furthermore, the method was applied to determination of the sulfur-containing amino acids in maternal plasma (n = 200) after delivery. The concentrations of Hcy, Met and Cys as a median (inter quartile range, Q1 and Q3 ) were 5.37 (3.32-7.79) μm, 25.20 (20.10-31.06) μm and 147.25 (102.81-189.31) μm, respectively.

Simultaneous determination of amphetamine-type stimulants in abusers’ hair: clinical usefulness of hair analysis in prehospitalization for abusers

Simultaneous determination of amphetamine-type stimulants (ATSs), such as methamphetamine (MA), amphetamine (AP), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), and p-methoxymethamphetamine (PMMA), has been made for hair of 32 female abusers from different ethnic backgrounds. One milligram of abusers hair spiked with 1-methyl-3-phenylpropylamine as internal standard was used for each determination of the ATSs. After digestion with 1xa0M NaOH, the samples were extracted with n-heptane; the organic layer was evaporated to dryness, dissolved in 75xa0mM of borate buffer solution (pH 8.5), and labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. The determination of the ATSs in hair was performed by the semi-micro high-performance liquid chromatography-chemiluminescence method. Thirty-two subjects who had self-report histories of ATS use for more than 1xa0week prior to hair sample collection participated in this study. Concentration ranges of ATSs in abusers’ hair were 0.07–6.73xa0ng/mg for MA (nxa0=xa024), 0.18–2.47xa0ng/mg for AP (nxa0=xa013), 0.05–0.86xa0ng/mg for MDMA (nxa0=xa012), 0.52–1.38xa0ng/mg for MDA (nxa0=xa03), and 0.09–1.88xa0ng/mg for PMMA (nxa0=xa06). Several discrepancies between detected drugs and information obtained from the interviewed abusers were found. Most of the drugs determined in this study could not be detected by the urine test performed at hospitalization. Correlation between ATS concentration and psychiatric symptoms on the basis of Brief Psychiatric Rating Scale (BPRS) was also studied. Simultaneous determination of ATSs in hair of outpatients and inpatients at psychiatric clinics and hospitals should be effective in providing proper diagnosis and treatment. This is the first report to demonstrate the presence of PMMA in hair of human subjects.

Evaluation of lophine derivatives as L-012 (luminol analog)-dependent chemiluminescence enhancers for measuring horseradish peroxidase and H2O2.

8-Amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione (L-012) was recently synthesized as a new chemiluminescence (CL) probe; the light intensity and the sensitivity of L-012 are higher than those of other CL probes such as luminol. Previously, our group developed four lophine-based CL enhancers of the horseradish peroxidase (HRP)-catalyzed CL oxidation of luminol, namely 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI), 2-(4-hydroxyphenyl)-4,5-di(2-pyridyl)imidazole (HPI), 4-(4,5-diphenyl-1H-imidazol-2-yl)phenylboronic acid (DPA), and 4-[4,5-di(2-pyridyl)-1H-imidazol-2-yl]phenylboronic acid (DPPA), and showed that DPPA was suitable for the photographic detection of HRP. In this study, we replaced luminol with L-012 and evaluated these as L-012-dependent CL enhancers. In addition, to detect HRP and/or H2O2 with higher sensitivity, each detection condition for the L-012-HRP-H2O2 enhanced CL was optimized. All the derivatives enhanced the L-012-dependent CL as well as luminol CL; HPI generated the highest enhanced luminescence. Under optimized conditions for HRP detection, the detection limit of HRP was 0.08 fmol. By contrast, the detection limit of HRP with the enhanced L-012-dependent CL using 4-iodophenol, which is a common enhancer of luminol CL, was 1.1 fmol. With regard to H2O2 detection, the detection limits for enhanced CL with HPI and 4-iodophenol were 0.29 and 1.5 pmol, respectively. Therefore, it is demonstrated that HPI is the most superior L-012-dependent CL enhancer.

A novel lophine-based fluorescence probe and its binding to human serum albumin

The binding of a lophine-based fluorescence probe, 4-[4-(4-dimethylaminophenyl)-5-phenyl-1H-imidazol-2-yl]benzoic acid methyl ester (DAPIM) with human serum albumin (HSA) was investigated by fluorescence spectroscopy under physiological conditions. While DAPIM shows extreme low fluorescence in aqueous solution, DAPIM binding with HSA emits strong fluorescence at 510nm. The binding constant and binding number determined by Scatchard plot was 3.65×10(6)M(-1) and 1.07, respectively. Competitive binding between DAPIM and other ligands such as warfarin, valproic acid, diazepam and oleic acid, were also studied fluorometrically. The results indicated that the primary binding site of DAPIM to HSA is site II at subdomain IIIA. DAPIM can be a useful fluorescence probe for the characterization of drug-binding sites. In addition to the interaction study, because the fluorescence intensity of DAPIM increased in proportion to HSA concentration, its potential in HSA assay for serum sample was also evaluated.

Quantitation of sulfur-containing amino acids, homocysteine, methionine and cysteine in dried blood spot from newborn baby by HPLC-fluorescence detection.

Sulfur-containing amino acids (SAAs), homocysteine (Hcy), methionine (Met) and cysteine (Cys) in blood are related to homocystinuria, an inborn error of metabolism. In this study, an assay method with HPLC-fluorescence detection to quantify the SAAs in a dried blood spot was established and applied to samples from newborn babies (n=200). Sample pretreatment involving reduction, derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole, and liquid-liquid extraction with ethyl acetate gave the separation of the derivatives with retention times within 12 min. The method was enough sensitive to determine the SAAs in a dried blood spot with 0.04-0.14 µm as the limit of detection at a signal-to-noise ratio of 3. However, the absolute recoveries were very low (5.7% for Hcy, 4.6% for Cys) except for Met (105.4%) owing to inefficient recovery of Hcy and Cys from the blood matrix. Other validation parameters such as accuracy (93.5-106.2%) and intra- (≤ 9.0%) and inter-day precisions (≤ 8.7%) were acceptable. The reliability of a dried blood spot as an analytical sample was estimated. Furthermore, the proposed method was successfully applied to dried blood spots prepared from newborn babies.

Simultaneous determination of propofol and remifentanil in rat plasma by liquid chromatography–tandem mass spectrometry: application to preclinical pharmacokinetic drug–drug interaction analysis

Propofol (Pro) is an ultra-short-acting hypnotic agent used for general anesthesia that has no analgesic properties. Remifentanil (Rem) is an ultra-short-acting opioid administered concomitantly as an analgesic with Pro. To evaluate the pharmacokinetic interactions between Pro and Rem, we developed and validated a method combining high-performance liquid chromatography with tandem mass spectrometry for simultaneous determination of Pro and Rem. The proposed method was successfully used to study the pharmacokinetic interactions of Pro and Rem coadministered to rats.

Evaluation of the neurochemical effects of methoxetamine using brain microdialysis in mice

The ketamine analogue, 2-(3-methoxyphenyl)-2-(ethylamino)cyclohexanone (methoxetamine) has emerged as a drug of abuse. Both methoxetamine and ketamine are antagonists of glutamate N-methyl-d-asparate receptors, and several case reports show that methoxetamine produces similar schizophrenia-like symptoms and hallucinations to ketamine. Although methoxetamine is believed to change levels of dopamine, glutamate, and serotonin in the brain, few studies thus far have examined these effects. We investigated the influence of methoxetamine on dopamine and serotonin concentrations using microdialysis and high performance liquid chromatography with electrochemical detection. To reveal the effects of methoxetamine, we monitored dopamine and serotonin concentrations in several brain areas [striatum, nucleus accumbens, and prefrontal cortex (mPFC)] after an administration of 20xa0mg/kg of methoxetamine. We compared the effects of methoxetamine with those of ketamine using two ketamine doses. Methoxetamine increased dopamine and serotonin concentrations most robustly in the mPFC. In addition, its effects were stronger than those of ketamine at the same molar dose, suggesting that methoxetamine causes schizophrenia-like symptoms and hallucinations by increasing the dopamine and serotonin concentrations. We conclude that consumption of methoxetamine may be more dangerous than consumption of ketamine.

Validation of an LC-MS/MS Method for the Determination of Propofol, Midazolam, and Carbamazepine in Rat Plasma: Application to Monitor Their Concentrations Following Co-administration

Propofol (PRO) is a hypnotic used to induce and maintain general anesthesia. A risk of drug-drug interactions exists in cases of clinical co-administration of PRO and midazolam (MDZ) or carbamazepine (CBZ). Therefore a sensitive and rapid assay is needed to monitor these drugs. In this study, a sensitive and selective liquid chromatography-tandem mass spectrometry technique was developed for simultaneous determination of PRO, MDZ, and CBZ in plasma. Simultaneous selected reaction monitoring in the positive and negative ionization modes was used for mass detection. Analytes were isolated from plasma samples by a simple, economic, and rapid solid-phase extraction method. Chromatographic separations were achieved using a Chromolith Performance RP-18e analytical column (100×4.6u2009mm i.d.) with a mixture of acetonitrile-ammonium acetate buffer (10u2009mM, pH 3.5) (90u2009:u200910, v/v) as the mobile phase. The method was fully validated for PRO, MDZ, and CBZ over concentrations ranging at 1-100, 2-100, and 7-1000u2009ng/mL, respectively, with acceptable validation parameters. Furthermore, the method was applied to monitor PRO and MDZ or CBZ following co-administration in rats.

The physicochemical interactive mechanism between nanoparticles and raffinose during freeze-drying

New methods of preparing nanoparticles and in vivo studies of their behavior have been the subject of much study. However, there exist few studies on maintaining the nanoparticle size. In this work, we report on the interaction mechanism between raffinose and nanoparticles during freeze-drying. The mean particle size of the rehydrated freeze-dried raffinose-containing nanoparticles (170.5 nm) was similar to the initial particle size before freeze-drying (156.1 nm), indicating that the particle size was maintained. The powder X-ray diffraction of the freeze-dried raffinose-containing nanoparticles shows a halo pattern, while that of the normal-dried raffinose shows a crystalline pattern. No endothermic peak of the freeze-dried raffinose appeared, while the normal-dried raffinose had an endothermic peak at 84.0 °C. These results suggest that there exists a relationship between the nanoparticles and the raffinose, and that the relationship depends on whether the mixture is freeze-dried or normal-dried. In the case of normal drying, the raffinose molecules have space and time to arrange themselves into regular arrangement because the nanoparticles and raffinose molecules can move around freely in water. In contrast, in the case of freeze-drying, the moisture was sublimed while the raffinose molecules and nanoparticles were immobilized in the ice, thereby preventing aggregation.

A simple and rapid chemiluminescence assay for on-site analysis of paraquat using a portable luminometer

Paraquat (N,N′-dimethyl-4,4′-bipyridinium dichloride) is one of the most widely used herbicides owing to its high efficacy and low environmental persistence. However, because paraquat has significant acute toxicity, fatalities are often caused by accidental or voluntary ingestion of paraquat. In consideration of the strong toxicity and fast-acting property of paraquat, on-site analysis at accident scenes should be effective in facilitating immediate medical treatment. In this study, a simple and rapid chemiluminescence assay using a portable luminometer was developed for on-site analysis of paraquat. The proposed assay is based on luminol chemiluminescence detection of superoxide anion radical resulting from the redox reaction between paraquat and dithiothreitol. Intense chemiluminescence was observed after mixing of paraquat and dithiothreitol in the presence of luminol. Because the chemiluminescence intensity was proportional to the concentration of paraquat, a quantitative measurement of paraquat was possible. The calibration curve for standard paraquat solution was linear from 0.025 to 2.5xa0μM with the correlation coefficient of 0.992; the detection limit (blankxa0+xa03SD) was 22xa0nM. The proposed assay was applied to determine paraquat in beverage samples after a cation exchange clean-up procedure. Given that the portable luminometer used in this study is small and lightweight, the proposed assay should be useful for on-site analysis of paraquat.