This review surveys various chromatographic methods for the extraction and the determination of drugs and toxic compounds in biological and environmental materials reported during 1980-1985. The column liquid chromatographic methods by use of XAD-2, XAD-4, Extrelut, Clin Elut (Chem Elut), Sep Pak C₁₈, Bond Elut, Bio Beads SX-2 and SX-3 gel were applied to simple and rapid extraction and clean up of drugs and toxic compounds in biological and environmental materials. These chromatographic methods include high performance thin-layer chromatography, capillary gas chromatography, capillary gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC) with photodiode array detection, high performance liquid chromatography-mass spectrometry, and isotachophoresis. Trace drugs and toxic compounds in biological and environmental materials were rapidly, sensitively and selectively detectable by HPLC with photodiode array detection, and capillary GC-MS.

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Simplified methods with SEP-PAK cartridges (Waters Associates) for the determination of diphenyl ether herbicides (CNP, NIP, X-52) in various samples, i. e. water, fish and river sediment, were developed. 1) A water sample was aspirated through SEP-PAK C₁₈ cartridge. Diphenyl ether herbicides were eluted with n-hexane and subjected to GC analysis. The recoveries of CNP, NIP and X-52 were 88.0-98.5%. 2) In the analysis of fish sample, SEP-PAK AgNO₃-coated Florisil, prepared originally, was used for cleaning up instead of glass column packed with AgNO₃-coated Florisil. Overall recoveries of diphenyl ether herbicides, CNP, NIP and X-52, in fortified samples were 88.0, 81.2 and 93.4%, respectively. Correlation coefficient between the levels of CNP in actual samples by the proposed method with SEP-PAK cartridge and by the other method by using glass column reported previously was 0.992. 3) In the case of river sediment, SEP-PAK Florisil was used to clean up instead of glass column packed with Florisil. Diphenyl ether herbicides in fortified materials were quantitatively detected. And all over recoveries with this cartridge were 94.5-98.4%. As a result, SEP-PAK cartridges were found to be very useful for their simplicity, rapidity, efficiency and reproducibility, as a pretreatment for the analysis of diphenyl ether herbicides in various samples.

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Beryllium in atmospheric air was determined. At the same time, the concentration of strontium in the same genus element was determined. The sampler used was operated continuously for 24-48 h. For the determination of two metals in atmospheric air, flameless atomic absorption spectrophotometry (AAS) for beryllium and flame AAS for strontium were used. Beryllium and strontium in 76 samples of atmospheric air were detected at the average levels of 0.042 ng/m³ and 6.68 ng/m³, respectively. Beryllium in these samples has a narrow range of values, while strontium has wider values. On the other hand, atmospheric air samples in areas of seven cities in Japan were collected with quartz fiber filters according to particulate size ranges by use of an Andersen high volume sampler. Two metals (Be, Sr) in these particulates were measured. Distributions of particulates size of each area were divided into two groups with their distribution pattern. At the same time, concentration ratios of strontium to beryllium by particulate size ranges were divided into three groups with their distribution characteristics.

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A simple and rapid method for the determination of paraquat in the serum was developed by a modification of the method originally reported by Knepil. Serum samples were treated with a mixture of chloroform-ethanol and ammonium sulfate and centrifuged to obtain a clear aqueous paraquat solution. Alkaline dithionite solution was added to the supernatant and the absorbance at 396 nm was recorded as A. A small amount of acetic acid was added to the same cuvette to distinguish the coloured paraquat radical and the absorbance at 396 nm resulted from the serum constituents was again measured as B. From the difference between A and B, the concentration of paraquat was determined in the range of 0.1-4 μg/ml serum, with the recovery of about 90%. This method posesses the advantages of 1) no need of special instrument and 2) short time for the determination. By using this method, paraquat concentration in the serum was monitored succesively during 7 d in a poisoning case.

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When bactericidal antiseptics are discharged into the environment, they are degraded but their behavior has not been cleaned. In this paper, carcinogenicity of chlorhexidine digluconate and its seven kinds of metabolites (p-chloroaniline, p-chlorophenol, p-chloroacetoanilide, aniline, phenol, pyrocatechol and pyrogallol) were screened with Ames test and liquid rec-assay based on Ashby's report. Ames test was performed with Salmonella typhimurium TA98 and TA100, and liquid rec-assay was performed with Bacillus subtilis H-17 (Rec⁺) and M-45 (Rec^-). Pg was positive on Ames test and liquid rec-assay, and recognized as positive carcinogenic compound. CPh was positive on Ames test with S-9 mix (+). Pc was found to have positive DNA injury by liquid rec-assay with S-9 mix (+). These results suggest the necessity of discarding counterplan of drugs.

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For the biological identification of fish species by electrophoresis including isoelectric focusing (Y. Ukishima, et al., Eisei kagaku, 30, 189 (1984)) of the sarcoplasmic proteins, it is generally required to electrophoresize the standard samples together with the test samples. However, each standard sample is not always kept in the laboratories. We, therefore, attempted to identify fish species, without standard samples, by applying numerical values which indicate isoelectric focusing patterns of sarcoplasmic proteins. To determine the pH gradient of polyacrylamide gel, a method with pI markers was more satisfactory in the reproducibility and operability than the methods with pH micro electrode. The stained protein bands on isoelectro-focused polyacrylamide gels were scanned by a densitometer, and pI values and relative peak heights of the five to ten bands were numerically expressed. Relative standard deviations of pI values of the protein bands were below 1%. Isoelectro-focused sarcoplasmic protein bands of 45 fish species were also numerically expressed. The numerical values were species-specific except bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares), indicating that fish species are successfully identified by using the tables of numerical values of isoelectro-focused sarcoplasmic proteins. Furthermore, isoelectro-focused sarcoplasmic proteins of seasoned fish slices were numerically expressed. Although a few protein bands were disappeared or weakened by seasoning process, the present identification method might also be applicable to the seasoned slice samples.

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In order to obtain mono-, di- and triisopropyl citrate as standard of food additives, their syntheses were studied. On refluxing anhydrous citric acid (I) and isopropanol with a small amount of sulfuric acid, five kinds of esters, monoesters (IIa and IIb), diesters (IIIa and IIIb) and triester (IV) were obtained, and IIb, IIIb and IV were isolated. On the other hand, alkaline hydrolysis of IV gave I, IIa and IIIa, and IIa and IIIa were isolated. These compounds II, III and IV were distinguished by gas chromatography and mass spectrometry, but chemical structures of isomers of monoesters (IIa and IIb) and diesters (IIIa and IIIb) could not be revealed. The study on ¹H- and ¹³C-NMR spectra made it clear that IIa, IIb, IIIa and IIIb were 2-monoisopropyl citrate, 1-monoisopropyl citrate, 1, 2-diisopropyl citrate and 1, 3-diisopropyl citrate, respectively. Recovery of 100mg/l of our authentic each isopropyl citrate (IIa, IIb, IIIa, IIIb and IV) from edible oil by our established method was more than 96%.

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Lethal toxicity of endotoxin was observed on killifish (Oryzias latipes) at relatively high temperature (30°C). Effect of endotoxin on the lethal toxicity of various pollutants was examined for killifish at 30°C. Permissible volumes of various dissolvents with and without endotoxin were determined at 30°C. Ten percent diethylether-dimethysulfoxide solution was chosen as a suitable dissolvent for water-insoluble pollutants with and without endotoxin. Reared concentration of about 90 kinds of pollutants were decided previously to the acute toxicity test in the presence of endotoxin. Endotoxin increased the lethal toxicities of HgCl₂, NaSeO₄, MnCl₂, methanol, ethanol, propanol, butanol, pentanol, Span 60, Tween 60, ethylene glycol, propylene glycol and methyl cellosolve for killifish, and decreased the lethal toxicities of CuCl₂, CuSO₄, AgNO₃ and Cu (CH₃COO)₂.

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A method for the determination of dithiocarbamates (DTCs), rubber accelerators, was studied by reversed-phase high performance liquid chromatography (HPLC) with Nucleosil 5C18 column by using methanol-water (95 : 5, 90 : 10, 85 : 15) as mobile phases. After extraction of rubber samples by shaking with a mixture of acetone-chloroform (1 : 1) at room temperature, zinc (II) complexes (Zn complexes) of DTCs were converted into their cobalt (III) complexes (Co complexes) and analyzed by HPLC with a ultraviolet (UV) detector at 320 nm. Accelerators in 37 different brands of commercial rubber gloves mainly for domestic use, which were used as samples for the analysis of antioxidants in previous paper [Eisei Kagaku, 30, 126 (1984)], were analyzed by the proposed method. However, when more than two Zn complexes were present in the rubber sample, "mixed complexes" with different ligands in a Co complex molecule were formed and HPLC chromatograms obtained were found to be too complicated to determine DTCs directly. The following procedure was chosen to analyze Zn complexes : 1) analyze amines by nitrogen-phosphorus detection gas chromatography, 2) prepare standard mixtures of Zn complexes corresponding to amines detected, 3) convert the mixtures in to Co complexes and analyze by HPLC, 4) choose the standard mixture which gives the most similar HPLC chromatogram to that of the sample, 5) calculate the contents of Zn complexes by the comparison of sum of all peak heights between the chosen standard mixture and sample. Zinc dimethyldithiocarbamate (ZDMC), zinc diethyldithiocarbamate (ZDEC), zinc dibutyldithiocarbamate (ZDBC), zinc ethylphenyldithiocarbamate (ZEPC) and zinc pentamethylenedithiocarbamate (ZPC) were detected. Their frequencies of detection and range of content were as follows : ZDMC, 10/37, 9-156 μg/g ; ZDEC, 18/37, 27-16500 μg/g ; ZDBC, 19/37, 11-755 μg/g ; ZEPC, 6/37, 105-2940 μg/g ; ZPC, 10/37, 19-3290 μg/g. ZEPC was detected only in the gloves manufactured in Japan. Thiuram disulfides (Thiurams), accelerators, were also analyzed by HPLC. But, none of them was detected in the gloves. Patch testing to DTCs and Thiurams was also studied in 40 patients with allergic contact dermatitis from rubber or polyvinyl chloride materials (rubber group or PVC group), who were subjects for patch testing to antioxidants reported in the previous paper. Generally speaking, rubber group reacted to DTCs and Thiurams in higher incidence than PVC group. Thiurams reacted in higher incidence than DTCs. The order of the incidence of positive reactions among DTCs and Thiurams with 5 kinds of N-substituents was as follows : dimethyl » diethyl, ethylphenyl>dibutyl, pentamethylene.

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Gonyautoxin-1, 4 (GTX_1-4), -2, 3 (GTX_2-3) and saxitoxin (STX) group (neoSTX, STX) were isolated from the toxic plankton Protogonyaulax tamarensis in Funka Bay, Hokkaido. Some chemical properties of the toxins were attempted to be examined. The cells of P. tamarensis cultured in BSW-4 medium were extracted with acidic ethanol. The extract was applied successively to three types of column chromatography on Bio-Gel P-2, Amberlite CG-50 and Bio-Rex 70. The toxins obtained were finally purified by cellulose acetate membrane electrophoresis. GTX_1-4, GTX_2-3 and STX group thus obtained were treated, respectively, by two methods as described below. One was treatment by heating at 60°C for 1 h with Raney nickel, and the other was treatment by heating at 90°C for 20 min with hydroiodic acid and red phosphorus. The composition of reaction products obtained was examined by fluorodensitometry. The results were as follows. By the treatment with Raney nickel, GTX_1-4 was partially reduced to GTX_2-3. Namely, GTX_1-4 (GTX₁ : 93%, GTX₄ : 7%) was treated with Raney nickel, to give a mixture of GTX₁ (62%), GTX₄ (11%), GTX₂ (18%) and GTX₃ (9%). However, GTX_2-3 were not reduced to other PSP components, and there was not a remarkable change in the composition of STX group. By the treatment with hydroiodic acid and red phosphorus, GTX_1-4, GTX_2-3 and STX groups were converted to the components whose electrophoretic behaviors differed from PSP standards (GTX_1-4, neoSTX, STX) used at this time.

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A fluorometric determination of H₂O₂ based on the formation of amprochrome was developed. Amprochrome is an oxidation product of amprolium, an agent used for treating coccidiosis of chickens, and has a strong fluorescence. Though potassium ferricyanide has been used for oxidizing amprolium, H₂O₂ is also considered to be useful for the reaction. The analytical procedure was as follows. To a sample solution containing a suitable quantity of H₂O₂, 1 ml of amprolium (500 μg/ml), NaOH (10%) and peroxidase solution (20 units/ml) were added. The mixture was shaken for 45 min at room temperature and amprochrome produced was extracted with 10 ml of isoBuOH after saturating the solution with sodium sulfate. Relative fluorescence intensity of the separated isoBuOH layer was measured (excitation wavelength 400 nm and emission wavelength 460 nm) against that of 10^-6M quinine sulfate in 0.1 N H₂SO₄. The calibration plot was linear up to H₂O₂ 0.2 μg/tube. On the basis of the proposed method, remaining H₂O₂ in Japanese noodles (udon) could be determined with a recovery of 82.4-108%.

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The calculation of most probable number which has been widely used for enumeration of bacteria, especially coli-forms, was carried out by Newton-Raphson method and its micro-computer program was presented.

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