We developed several, acoustic-wave sensors for clinical tests. Piezoelectric quartz crystals (AT-cut, 9 MHz) were used as a sensor device. (1) A piezoelectric immunosensor for microalbumin with an anti-human albumin-immobilized crystal was sufficiently sensitive to monitor the levels of microalbumin in urine. This sensor does not require any radioisotope- or enzyme-labeled antibody, a second antibody, which is essential for ordinary immunoassay, like an immunoradiometric assay or an enzyme-linked immunosorbent assay. (2) Latex piezoelectric immunoassay (LPEIA) is a new immunoassay that requires no immobilization of an antigen or antibody on the crystals. LPEIA was used for the detection of the C-reactive protein (CRP), antistreptolysin O (ASO) and rheumatoid factor (RF), and is sufficiently sensitive for clinical applications. (3) Human collagen Type I-immobilized quartz crystals responded successfully to the adhesion of human platelets onto the crystals. The platelet adhesion sensor can monitor the adhesion of platelets without radioisotope labelling or a dye-staining method. These sensors are simple in operation and are cost-effective.

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The simultaneous determination of cinchona alkaloids (qunine, quinidine, cinchonine and cinchonidine) in Cinchona bark water extracts were investigated by capillary electrophoresis. Micellar electrokinetic chromatography (MEKC) in 5 mM phosphate-borate buffer containing 15% tert-butanol at pH 8.5 with 50 mM SDS as surfactant gave complete separation of four alkaloids within 20 min. A sample solution was introduced by hydrostatic method (10 s) and on-column detection was performed at 214 nm. The calibration curves of alkaloid gave good linearities over the concentration range at 840 μg ml^-1. Qunine, quinidine, cinchonine and cinchonidine in Cinchona Bark water extracts were determined with good reproducibility.

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2- (5-Nitro-2-pyridylazo) -5- (N-propyl-N-sulfopropylamino) phenol (nitro-PAPS) reacts with iron(II), copper and zinc, but not with iron(III). The iron(II) complex is formed at pH 3.49 and has an absorption maximun at 582 nm. Copper forms a 1: 1 complex at pH 2.54.5 and a 1: 2 complex at pH 89. Zinc reacts at pH 89. The absorption maxima of copper and zinc complexes are at 565 nm. When sodium periodate was added to a sample solution containing copper, iron and zinc at pH 3.5, only the copper complex was formed. Accordingly, only copper could be determied. On the other hand, the sum content of iron and copper was determined in the presence of ascorbic acid at pH 3.5. Also, the total contents of iron, copper and zinc were determined at pH 8.5. Three metal ions in the range of 0.020.5μg ml^-1 could be determined individually and the proposed method is useful for determinations in sera.

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A specific and sensitive method for the determination of three retinoic acid isomers (13-cis-retinoic acid, all-trans-retinoic acid and 9-cis-retinoic acid) and retinol using column-switching, HPLC was developed. The three acids and retinol were completely separated within 25 min. The detection limit was ca. 20 pg for the three acids, respectively. Linearity was obtained up to 200 ng for, all three acids. The relative standard deviation (n=10, 0.260.37 μg/ml) of a within run was 0.51.9% for all three acids. This method was applied to the determination of three acids and retinol in human plasma following oral administration with 13-cis-retinoic acid.

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Acrinol, cetylpyridinium chloride and benzalkonium chloride react with tetrabromophe-nolphthalein ethyl ester (TBPE) to form, blue ion associates. Also, dl-methylephedrine hydrochloride forms a red ion associate. Both associates can be quickly extracted into l, 2-dichloroethane. When sodium tetraphenylborate in a burette is dropped as a titrant, the onium compound remaining in the aqueous phase forms a colorless ion associate (R₄N⁺ ·TPB^-or R₃NH⁺ ·TPB^-) which is extracted quantitatively. At the end point, the tetraphenylborate anion takes the onium compound away from the colored ion associate in the organic phase and TBPEH, having a maximum absorption near to 400 nm, is dissociated. Consequently, the color of the organic phase turns to yellow. The color change in the organic phase provides a clear end point.

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The Cu and Zn levels in human bladder carcinoma and non-carcinoma tissues obtained by a biopsy were successfully analyzed by a total-reflection X-ray fluorescence (TXRF) analysis using monochromatic X-rays (11 keV) of synchrotron radiation. The analysis was made by a calibration-curve method combined with a standard addition technique. An analysis of the biopsy samples (minimum 1 mg) from 6 carcinoma patients indicates that the Cu level falls in 210 μg/g and that the Zn level is in the range of 3270 μg/g. It is found that the Cu level in carcinoma tissues was significantly smaller than that of the non-carcinoma one, whereas no clear tendency was observed for the Zn level. The present study has demonstrated that a TXRF analysis of biopsy samples allows us to reveal the dynamical behavior of trace elements in carcinoma tissues, such as the time-dependent level of trace elements at different progress stages of carcinoma and the correlation among various trace elements.

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The effect of neutrophils on polystyrene irradiated by gamma-ray or Ar-plasma was investigated. The physical and chemical changes in the irradiated polystyrene surface were evaluated by FT-IR and measuring the contact angle. The activation of neutrophil-like HL-60 cells was estimated by measuring the luminol-dependent chemiluminescence derived from the interaction between the cells and the polystyrene surface. Although the surface hydrophilicity was increased by a further irradiation of gamma-ray or Ar-plasma, no variation is a polystyrene surface treated by gamma-ray or Ar-plasma irradiation was observed by FT-IR analysis. Further, the chemiluminescence measurement for reactive oxygen species, generated from neutrophil-like HL-60 cells in contact with a polystyrene surface, could discriminate the treated polystyrene. Based on these fundamental studies, a biological assay with the activation of differentiated HL-60 cells could be applied for a modified or denatured polystyrene surface.

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A simple system for the potentiometric measurement of cyanide in blood samples has been developed for forensic poisoning in fire accidents based on a HCN gas sensor with a gaspermeable PTFE diaphragm. HCN gas produced from a sample (1 ml) containing cyanide was measured by adding 1 ml of 50% phosphoric acid in a 25 ml sealed Teflon vessel. Although the presence of sulfide in the sample interfered significantly with the measurements, no influence was observed by CO in the present system. The analytical results for blood samples obtained by the present measuring system were in excellent agreement (r=0.998) with those obtained by gas chromatography, which has been the conventional method used so far. The proposed method is very much advantageous and useful for rapid field analyses of cyanide in blood samples, for example to clarify the cause of death at the location of a fire due to the following reasons. No contamination by the blood sample takes place, because the sensor is placed in the gas phase and the blood sample does not touch the sensor at all; thus, many repetitive measurements are possible with high reliability and reproducibility; the system is simple enough to be portable to any place; it is safe in operation for measurement, it provides a rapid analysis etc.

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It is well known that dextran for clinical use should have narrow molecular-weight(M_W) distributions, because any material with a M_W, that is too small is rapidly lost from circulation, and is therefore therapeutically ineffective; also, any material with a M_W that is too high can interfere with the normal coagulation process of the blood. Therefore, accurate, and rapid methods are necessary for measuring the M_W the distribution of dextran. In this study, the molecular weight of dextran for injection and dextran preparations were estimated by a method adopted in the European Pharmacopoeia(EP) using the polydisperse standard of dextran and also by the usual method using the pullulan standards. From the results, it has been clarified that pullulan is a useful standard for the injection of dextran, such as dextran 70 and dextran 40. It was also found that the molecular weights of almost all clinical dextran in Japan seem to be smaller than the specifications described in EP, whereas their limiting viscosities are in the range of the specifications in the Japanese Pharmacopoeia.

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The magnitude of the current increase for five min due to the oxidation of p-nitrophenol (PNP) released from p-nitrophenyl-β-glucuronide (PNPGD) by β-glucuronidase (βGlu) from a snail was proportional to the concentration of the enzyme ranging from 0.3 to 3 Ul^-1. The steady state current by PNP was measured at 1 V in acetate buffer of pH 5 stirred at 500 rpm using a dialysis membrane-covered glassy carbon electrode, and, was proportional to the concentration of PNP from 1 to 500 μM. Since the, GβGlu activities of serum were very small, they were determined by measuring the difference in current at 0 and 120 min after a reaction of acetate buffer solution containing 10 mM PNPGD and serum diluted by 1/5. The βGlu activities of twelve serum samples obtained by the present method and a colorimetric method are in good agreement with the correlation coefficient of 0.96 over the range 0.83.6 Ul^-1. This method, which is free from a process for color development, is simple compared with a colorimetric method. The K_M value of serum β glu for PNPGD was determined to be 2 mM.

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A method is described for the determination of methamphetamine (MP) by a high-performance liquid-chromatographic method using a peroxyoxalate chemiluminescence reaction. After MP was derivatized with 4-(N, N-dimethylaminosulphony1)-7-fluoro-2, 1, 3 benzoxadiazole (DBD-F), it was detected by a chemiluminescence method. Bis(2, 4, 6 trichlorophenyl) oxalate (TCPO) and hydrogen peroxide were used as postcolumn chemiluminescence reagents. The separation of the DBD-F derivative of MP was carried out using a reversed-phase column (GL Science, Lichrospher RP 18-5, 4.6 i.d. × 250 mm) and an acetonitrile aqueous solution as the mobile phase(flow rate, 1.0 ml/min). The optimum pH, temperature and reaction time for fluorogenic reactions of DBD-F and MP were 8, 70°C, 30 min, respectively. The linear calibration graph was observed over the range of 5 × 10^-95 ×10^-6 M. The correlation coefficient for this line was 0.999. The detection limit (3σ) was 2× 10^-9 M (40 fmol) and the relative standard deviation was 1.4% (n=6) for a concentration of 5 × 10^-9 M. The effect of urine on the MP determination was investigated. The urine sample solutions were prepared by adding a 1 × 10^-7 M or 1 × 10^-8 M MP standard solution to a normal human urine. The relative chemiluminescence intensities were 98102% toward the chemiluminescence intensity of the MP standard solution.

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5, 10, 15, 20-tetrakis(4-sulfonatophenyl)porphine (TPPS), a water-soluble porphyrin as an ultra-high-sensitive chromogenic reagent, was used for the determination of small amounts of albumin. In the association reaction of TPPS and albumin under a weak acidic condition (pH 3.0), their aggregates were quantitatively formed within one minute at room temperature, and the absorption spectra in the Soret band changed from the absorption maxima of TPPS (434 nm, ε=5.0× 10⁵ M^-1 cm^-1) to that of an aggregate (420 nm, ε= 2.8 × 10⁵ M^-1 cm^-1) along with an increase in the albumin concentration. Based. on these results, a spectrophotometric detection-FIA system was developed. The proposed FIA is a double-line manifold system: A line for an acetic acid-sodium chloride solution (0.05 M), and B line for a TPPS solution (5 ×10^-7M). The mixing coil uses a 1 mmφ × 1 m teflon tube. The liquid flow rate is 2.0 ml/min and the detection wavelength is 434 nm. A one hundred μl sample solution was injected into the FIA. The calibration curve gave a straight line over the concentration range of [albumin]_T=0 20 μg/ml, and the detection limit (S/N=3) was 0.15 μg/ml. At least one hundred and twenty samples could be determined within one hour {the RSD: 1.51 %, 5 determinations for [albumin]_T=10 μg/ml}. On the other hand, although the coexistence of γ-globulin seriously interfered with the determination of albumin, this influence was removed by the addition of sodium chloride ([NaCl]_T=0.05 M). This method was applied to a dog's spinal fluid sample with satisfactory results.

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It was found that the mutagenicity of 1-methyl-1, 2, 3, 4-tetrahydro-β-carboline-3-carboxylic acid (MTCCA) treated with 50 mM nitrite at pH 3, 37°C for 60 min in the presence of 7.5 % ethanol was strongly reduced upon adding the decoctions of green, black and oolong teas in reaction mixture with the Ames Salmonella mutagenicity test. The antimutagenicity and reducing power, which were determined of tea decoctions, gave a positive correlation, suggesting that reducing compounds, such as catechins contained in the teas, may play a significant role in the mutagenicity-reducing activity.

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A spectrophotometric method for the determination of lysozyme chloride was established by using the ο-sulfophenylfluorone (SPF)-titanium(IV) complex. This method can be used to determine about 10200 μg/10 ml lysozyme chloride, and is about 2-times more sensitive than the Japanese Standards of Pharmaceutical Ingredients method. This was applied to assays of lysozyme chloride in pharmaceutical preparations (tablet, granule, syrup, drysyrup, capsule and cold medicine). The analytical results and recoveries were satisfactory. The proposed method should be useful for the determination of various enzymes, such as α-amylase, cytochrome ο and hyaluronidase.

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Many patients suffer from allergic contact dermatitis due to textile products. For the safety of textile products, a systematic analysis using spectrophotometry and gas chromatography/mass spectrometry (GC/MS) for substances which have previously caused dermatitis was proposed. The textile products were extracted with water to detect formaldehyde by an acetylacetone method. Alternatively, they were extracted with organic solvents, and the extracts were separated by silica-gel column chromatography. The fractions were then analyzed by spectrophotometry and GC/MS to detect naphthol AS and phosgene(2, 5-dichlorophenyl)hydrazone. These detected substances were identified as being allergens by closed patch tests for the patients. Consequently, this systematic analysis can be applied for research on the cause of allergic contact dermatitis due to textile products.

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We have designed a portable cocaine-selective electrode which can be used at the location of a crime. The cocaine electrode is based on a poly (vinyl chloride) membrane containing sodium tetrakis [3, 5-bis (trifluoromethyl) phenyl] borate as an ion-exchanger and tetrakis (2-ethylhexyl) pyromellitate as a membrane solvent. The following two points were considered in miniaturizing an instrument. First, we used a digital multimeter having a small size (16×7.5×3 cm) for a potential measurement. Second, the structure of a reference electrode was changed from a double to a single junction in order to reduce the size (10 cm in length and 4 mm in diameter). Calibration plots between the electric potentials and cocaine concentrations were measured in the presence of 0.5 M MgCl₂ in order to adjust the ionic strength of a solution. The detection limit was 4×10^-7 M and the slope of the electrode in the linear region was 56 mV per concentration decade. Interference by inorganic ions (Mg²⁺, Ca²⁺, H⁺, Na⁺, K⁺ and NH₄⁺), drugs (morphine and codeine) and stimulants (methamphetamine and amphetamine) was almost negligible. However, lipophilic medicines, such as diphenhydramine, chlorpromazine and imipramine, interfered significantly.

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