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Carbonic anhydrase (CA)-immobilized aminopropyl silica precolumn was developed for direct injection determination of certain sulphonamide drugs in biological fluids by column-switching (CS) high-performance liquid chromatography. Under the optimized conditions, only the sulphonamide drugs with an unsubstituted sulphonamide group were retained on the CA precolumn and separated on a reversed-phase analytical column. Of these, the retention of hydrochlorothiazide (HCT), chlorothiazide, acetazolamide, furosemide (FS) and chlorthalidone was almost quantitative. The peak area of HCT was proportional to the concentration in the range of 1-100 nmol/mL with relative standard deviations of 3.7% (5 nmol/mL) and 0.7% (100 nmol/mL). This CS system was applied to urine and plasma samples spiked with HCT and FS. Endogenous components of these were effectively removed, and HCT and FS were selectively retained on the CA precolumn. Almost quantitative recoveries and reproducibility were obtained.

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A method for the derivatization and separation of N-nitroso-N-alkylureas [alkyl = methyl (NMU), ethyl (NEU), and n-butyl (NBU)] has been developed. Fluorescent derivatives were formed with sodium sulphide, taurine and o-phthalaldehyde and separated by reversed-phase high-performance liquid chromatography. The limits of detection of standard NMU, NEU and NBU were 0.25, 0.8 and 1.5 pmol/200 microliters, respectively. The method was applied to the determination of NMU in blood after extraction with acetonitrile in the presence of calcium chloride. NBU was used as the internal standard. The recovery of NMU from blood was ca. 95%, and the limit of detection was 10 pmol/400 microliters blood. NMU levels in rabbit blood following a single oral administration were also measured.

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A column switching high-performance liquid chromatographic system in which peptides retained on an anhydrochymotrypsin (AHC)-immobilized diol-silica precolumn were selectively transferred to and separated on a reversed-phase analytical column was developed. An investigation of the affinity characteristics of 40 peptides to the AHC precolumn showed that among eleven peptides having Tyr or Phe at their C-termini and more than five amino acid residues, ten were retained almost quantitatively on the precolumn, and two peptides having Trp at their C-termini were less retained. Two peptides having C-terminal PheNH2 were also retained, but the peptide having C-terminal D-PheNH2 was not retained. Among eighteen peptides having no aromatic amino acids at their C-termini, two were retained, one slightly and the other moderately. Calibration graphs for rat atrial natriuretic peptide constructed at various sample sizes were nearly identical, indicating that the peptide could be enriched by this system. The AHC precolumn showed no loss of analytical performance over 1 year, during which about 450 samples were analysed.

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A fluorometric method for the assay of rat brain N-methyltransferase (NMT) has been developed using rat brain 9000 x g supernatant fluid as an enzyme preparation. The method is based on the enzyme reaction at pH 8.6 using 4-methylnicotinamide (4-MN) as the methyl acceptor substrate in the presence of cofactor S-adenosyl-L-methionine (AdoMet), and the fluorometric determination of the product 1,4-dimethylnicotinamide by means of its reaction with 4-methoxybenzaldehyde. The apparent Km values for 4-MN and AdoMet were 0.47 mM and 19 microM, respectively. The method was successfully applied to the studies of age- and sex-related differences of brain NMT activity in rats. The enzyme activity decreased gradually between 3 and 14 weeks of age whereas there were no differences between males and females in the levels of NMT activity.

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A method for the determination of cyanide in human red cells has been developed. Cyanide was extracted from red cells by adding water and methanol, and then derivatized with 2,3-naphthalene-dialdehyde and taurine to give a fluorescent product, which was determined by reversed-phase high-performance liquid chromatography with fluorescence detection. The recovery of cyanide from red cells was ca. 83%, and the limit of detection was 100 pmol/ml. The mean concentrations of red cell cyanide from ten smokers and from ten non-smokers were 705 and 466 pmol/ml, respectively. The method was also applicable to whole blood.

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Nicotinamide (NA) and its four isomeric methyl analogs [2-, 4-, 5- and 6-methylnicotinamides (MNs)] were tested as substrates for nicotinamide N-methyltransferase (NNMT) and amine N-methyltransferase (ANMT) using rat liver, kidney, spleen and brain 9000 x g supernatant fluids as model enzyme preparations. The N-methylated products were determined fluorometrically by their reaction with acetophenone or 4-methoxybenzaldehyde to form fluorescent 2,7-naphthyridine derivatives, and the lower limits of the determination were 8-30 pmol/100 microliters. N-Methyltransferase activities were detected in the liver with NA, 4-MN and 5-MN, and in the brain and spleen with 4-MN. On this basis, 5-MN is considered to be a selective substrate for NNMT in addition to NA, which is a known methyl acceptor for this enzyme. Although 4-MN appears to serve as a methyl acceptor for both ANMT and NNMT, it seems to be essentially a selective substrate for brain ANMT because of the absence of NNMT in brain. The fluorometric methods used here are also very useful because of their simplicity, sensitivity and selectivity.

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A column-switching high-performance liquid chromatographic (CS-HPLC) system which consisted of an anhydrotrypsin (AHT)-immobilized diol-silica precolumn and a reversed-phase analytical column was developed for the selective separation of peptides having Arg or Lys at their C-termini. Tuftsin (Thr-Lys-Pro-Arg) could be enriched almost quantitatively on the precolumn when loaded with water as a carrier solvent and the precolumn was washed with 10-30 mM acetate buffer (pH 5.0). An investigation of the affinity characteristics of 55 peptides to the AHT precolumn showed that among twelve peptides having Arg or ArgNH2 at their C-termini and more than four amino acid residues, ten were retained almost quantitatively on the precolumn, and eight out of nine peptides having Lys at their C-termini were less retained. The peptide having D-Arg at its C-termini was not retained. However, twelve out of thirty peptides having no Arg or Lys at their C-termini were also retained, but the retention was greatly decreased, in contrast to the Arg peptides, when the precolumn was washed with 20 mM calcium chloride solution. The results indicate that the CS-HPLC system equipped with an AHT precolumn offers new selectivity in the HPLC selectivity in the HPLC separation of peptides.

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A fluorometric method for the assay of nicotinamide methyltransferase has been established using rat liver 9000 x g supernatant fluid as a model enzyme preparation. 1,4-Dimethylnicotinamide formed enzymatically from a new substrate, 4-methylnicotinamide, is quantified by means of its fluorescence reaction with 4-methoxybenzaldehyde in aqueous alkali. The lower limit of determination of 1,4-dimethylnicotinamide is 100 pmol in the enzymatic reaction mixture. The apparent Km values for 4-methylnicotinamide and for nicotinamide, which is a known substrate for this enzyme, were 0.19 and 0.13 mM, respectively, whereas the relative activity of 4-methylnicotinamide as a methyl acceptor was about 1.5 times the value of nicotinamide.

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A method for the determination of cyanide in human urine has been developed. The method is based on the reaction of cyanide with 2,3-naphthalenedialdehyde and taurine to give a fluorescent product for reversed-phase HPLC separation and fluorometric detection. After centrifugation followed by dilution of urine samples, the specimens could be analysed directly by this method. The recovery of cyanide added to urine at concentration levels of 50-1000 pmol/mL was 85-96%. The detection limit of cyanide was 30 pmol/mL in urine. The method was successfully applied to the analysis of urine from smokers and nonsmokers. The mean concentrations of cyanide were found to be 215 pmol/mL for the former and 84 pmol/mL for the latter.

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A method is described for the determination of trace selenium (impurities in medicinal organic compounds) by ion chromatography (IC) after oxygen flask combustion. All selenium compounds formed by oxygen flask combustion are converted to selenate ion by heating with nitric acid and potassium permanganate. The selenate ion is then determined by IC, using a simple recycle system to eliminate interfering ions. The detection limit of selenium is 0.4 nmol in 50 mg of sample. The recoveries of selenium added to seven drugs are ca. 95-103% with relative standard deviations of 1-6%.

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The method described is based on the reaction of triethylenethiophosphoramide (ThioTEPA) and triethylenephosphoramide (TEPA), through their ethyleneimine groups, with sodium sulphide, taurine and o-phthalaldehyde to give fluorescent products, and separation of the derivatives by reversed-phase high-performance liquid chromatography. The method was successfully applied to the determination of ThioTEPA and TEPA in rabbit plasma samples after clean-up with an Extrelut 3 column. The recoveries of ThioTEPA and TEPA from plasma were 66.1-80.3% and the limits of determination in plasma were ca. 10 and 20 ng/ml, respectively.

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Acetaminophen (paracetamol) showed mutagenicity to Salmonella typhimurium TA100 and TA98 either with or without S9 mix when treated with excess nitrite in acidic solution. The mutagen was isolated and identified as 4-acetylamino-6-diazo-2,4-cyclohexadienone. Mutation tests and product analysis by high-performance liquid chromatography, however, suggested that this type of mutagen was hardly formed in the digestive tract of normal subjects.

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The method described here is based on a separation of deoxynivalenol, nivalenol and fusarenon-X on a C18 column using aqueous acetonitrile, and successive post-column fluorescence derivatization involving an alkaline decomposition to form formaldehyde and modified Hantzsch reaction with methyl acetoacetate and ammonium acetate (lambda ex = 370 nm and lambda em = 460 nm). By this method, 5-10 ng of the standard trichothecenes could be determined. By employing a clean-up procedure with a florisil column and a Sep-Pak CN cartridge, 61.4-96.9% recoveries were obtained for deoxynivalenol and nivalenol added to corn, wheat and barley at concentration levels of 0.05-1 ppm.

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A method is proposed for the fluorometric determination of cyanide based on a fluorogenic reaction with 2,3-naphthalenedialdehyde and taurine at basic pH. As little as 10 pmole of cyanide in 500 mul of sample can be determined.

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TPDMT-4 is a pregnancy-dependent mouse mammary tumor line characterized by progressive growth in pregnant, but dormancy in virgin, hosts and by significant estrogen (ER) and progesterone receptor (PR) levels. Three sublines, T4-OR26, T4-0I96, and T4-0I320, were established from sporadic outgrowths in virgins of TPDMT-4 pieces passaged long in breeding and estradiol (E2)- plus progesterone-treated mice and of enzymatically dissociated TPDMT-4 cells, respectively. T4-OR26 tumors produced ovarian-responsive growth and maintained the parent levels of ER and E2-dependent PR. T4-OI320 and T4-OI96 tumors were ovarian independent or autonomous. The former had ER, but not E2-dependent PR, and the latter had neither. To clarify the mechanism of acquisition of autonomy by hormone-dependent neoplastic cells, comparative ER studies were conducted between TPDMT-4 and T4-OI320 tumors. Although cytosolic, microsomal, and nuclear translocated ER levels were generally lower in T4-OI320 tumors, the dissociation constants, determined by the dextran-charcoal technique, steroid specificity, sedimentation data in sucrose gradients, and activation studies using DNA binding, molecular transformation from 4S to 5S, and [3H]E2 dissociation from [3H] E2-ER complexes as markers, revealed no autonomy-specific changes in cytosolic ER. Small but significant differences in the affinity of [3H]E2 to nuclear ER and the [3H]E2 dissociation rate from nuclear bound [3H]E2-ER complexes were found between both tumors. Hormone-dependent tumors may progress toward autonomy with different receptor statuses in different environments, and lack of E2-dependent PR synthesis in ER-positive tumors may be due to defects at postreceptor or nuclear levels involving subtle changes in interaction between activated ER and nuclei.

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