Application of improved iodine-azide procedure for the detection of thiouracils in blood serum and urine with planar chromatography

The application of iodine-azide reaction for the determination of thiouracils in thin-layer chromatography and high-performance thin-layer chromatography is described. The developed plates were sprayed with a freshly prepared mixture of sodium azide, adjusted to a proper pH, and starch solution, and exposed to iodine vapour for 5 s. The detection limits were established at pmol level. The factors depending on the detection limits were described. A comparison of iodine-azide tests reaction with other procedures is presented. The developed method was applied to detection of thiouracils in blood serum and urine. The possibility of detection of a thiouracils mixture was demonstrated.     

Determination of methimazole in urine with the iodine-azide detection system following its separation by reversed-phase high-performance liquid chromatography

The iodine-azide detection system to determine methimazole following its separation by RP-HPLC is described in this paper. The reaction between iodine and azide ions induced by methimazole was applied as a post-column reaction detection system. Neither extraction nor preconcentration of the sample was necessary. The methimazole standards added to normal urine show that the response of the detector, set at 350 nm (corresponding to unreacted iodine in the post-column iodine-azide reaction), was linear within the concentration range 2-10 nmol/mL of urine. The relative standard deviation values for precision and recovery within the calibration range were from 0.3 to 3.2% and from 97 to 102%, respectively. Limits of detection (LOD) and quantitation (LOQ) were 1 and 2 nmol/mL of urine, respectively. The method was applied to the separation and determination of patient urine samples and the analytical results were satisfactory.     

Determination of thiopental in urine sample with high-performance liquid chromatography using iodine-azide reaction as a postcolumn detection system

The reaction between iodine and azide ions induced by thiopental was utilized as a postcolumn reaction for chromatographic determination of thiopental. The method is based on the separation of thiopental on an Nova-Pak CN HP column with an acetonitrile-aqueous solution of sodium azide as a mobile phase, followed by spectrophotometric measurement of the residual iodine (lambda=350 nm) from the postcolumn iodine-azide reaction induced by thiopental after mixing an iodine solution containing iodide ions with the column effluent containing azide ions and thiopental. Chromatograms obtained for thiopental showed negative peaks as a result of the decrease in background absorbance. The detection limit (defined as S/N=3) was 20 nM (0.4 pmol injected amount) for thiopental. Calibration graphs, plotted as peak area versus concentrations, were linear from 40 nM. The elaborated method was applied to determine thiopental in urine samples. The detection limit (defined as S/N=3) was 0.025 nmol/ml urine. Calibration graphs, plotted as peak area versus concentrations, were linear from 0.05 nmol/ml urine. Authentic urine samples were analyzed, thiopental was determined at nmol/ml urine level.     

On the reaction of sulfenyl iodide derivatives with azide

Based on the suggested mechanism of the Raschig catalytic iodine-azide reaction the use of azide for the azotometric estimation of sulfenyl iodide groups is proposed. In the Raschig reaction reduction of iodine to iodide and oxidation of azide to elementary nitrogen is specifically catalyzed by bivalent sulfur compounds; the reaction is usually formulated to proceed via hypothetical sulfenyl iodide derivatives. This has been explored with the use of available, relatively stable sulfenyl iodide derivatives. The -SI group oxidizes azide to nitrogen stoichiometrically: 1 mole of a sulfenyl iodide consumes 2 moles of sodium azide and yields 3 moles of elementary nitrogen. The specificity and limitations of the method are discussed.     

Ion chromatography of azide in pharmaceutical protein samples with high chloride concentration using suppressed conductivity detection

Methods based on reversed-phase liquid chromatography with UV detection of 4-nitrobenzoyl- or 3,5-dinitrobenzoyl azide derivatives lack in accuracy and stability of derivatives to be applied for azide determination in pharmaceutical protein samples with high sodium chloride concentrations. This paper describes a sensitive and selective ion chromatographic method, with simple sample preparation and suppressed conductivity detection, developed for trace determination of azide in protein samples containing sodium chloride in concentrations as high as 11.6 g L(-1). Anion exchange stationary phase with quaternary alkyl amine functional groups and gradient elution with sodium hydroxide enabled good resolution of anions with similar retention times: azide, bromide and nitrate, as well as chloride whose retention time was shorter than azide's. Anions with high affinity to stationary phase (phosphate and citrate) were also eluted within acceptable analysis time of 32 min. The stability of sample solutions and the method selectivity, accuracy, precision and sensitivity satisfied the validation criteria of international organizations competent for pharmaceutical industry. The detection and quantitation limit ranges of sodium azide in protein samples were 0.007-0.02 mg L(-1) and 0.02-0.06 mg L(-1), respectively. Both limits increased with the concentration of sodium chloride.     

Sensitive enzyme-immunostaining and densitometric determination on thin-layer chromatography of N-glycolylneuraminic acid-containing glycosphingolipids, Hanganutziu-Deicher antigens

A sensitive enzyme-immunochemical staining method was developed for detection of N-glycolylneuraminic acid (NeuGc)-containing glycosphingolipids (GSLs) on silica gel thin-layer chromatography. The procedure consists of immune reaction among NeuGc-containing GSLs, affinity-purified chicken anti-NeuGc-LacCer and horseradish peroxidase-conjugated rabbit anti-chicken IgG, and the peroxidase reaction using 4-chloro-1-naphthol as a chromogenic substrate. Quantitative determination was achieved by direct densitometric scanning of the enzyme-immunostained spots on the chromatogram. As little as 0.5 pmol of NeuGc-LacCer, NeuGc-nLcOse4Cer, and NeuGc-nLcOse6Cer (0.64-1.0 ng) could be detected with a good signal-to-noise ratio. A semi-linear detector response was observed up to 50 pmol of each GSL. This procedure can be applied easily to other glycolipid antigen systems.     

Thin-layer chromatography and polyacrylamide gel electrophoresis-based assays for sialyltransferases using tetramethylrhodamine-labeled acceptors

Two novel assay systems for the determination of sialyltransferase activity using a tetramethylrhodamine-labeled disaccharide Galbeta1-4GlcNAc (2) as the acceptor are described. The TMR-labeled disaccharide 2 was synthesized by directly coupling Galbeta1-4GlcNAc-O-(CH(2))(6)NH(2) (1) with 5-tetramethylrhodamine N-hydroxysuccinimide ester. The K(m) value for compound 2 obtained with alpha-2,6-sialyltransferase from rat liver (EC 2.4.99.1) was 160 +/- 20 microM. After incubation of compound 2 with sialyltransferase the product and the unreacted acceptor substrate were separated either by thin-layer chromatography (TLC) on C-18 silica gel plates or by polyacrylamide gel electrophoresis (PAGE). The density of the spots on the TLC plates and the fluorescence of the bands on the gel were quantified. The assay conditions were optimized using crude bovine colostrum extract and also alpha-2, 6-sialyltransferase from rat liver. The detection limits for the TLC and PAGE assays were 1 and 0.4 microU of the rat liver enzyme, respectively. Either assay allows the parallel investigation of several samples at a time and is useful for the testing of fractions during enzyme purification.     

Use and detection of nonradioactive iodine-labeled antibodies for immunoassay

Traditionally radioactively labeled iodine has been used as a reporter group for the detection of antibodies in immunoassay. We have recently developed a microassay system for the detection of very low concentrations of iodide which eliminates the need for the use of radiolabeled iodine (O'Kennedy, R. et al. 1989, Anal. Biochem. 179, 138-144). The successful application of this assay for the detection of mouse IgG is described. The performance of this system compared with enzyme immunoassay was evaluated.     

High-performance liquid chromatography method for determination of N-glucuronidation of 4-aminobiphenyl by mouse, rat, and human liver microsomes

A simple and sensitive method for determination of the N-glucuronidation activity of mouse, rat, and human liver microsomes toward the carcinogenic arylamine 4-aminobiphenyl (4-ABP) using high-performance liquid chromatography with ultraviolet detection has been developed. The method uses chemically synthesized 4-ABP-N-glucuronide (4-ABP-G) as a standard for method validation. Validation was done with respect to specificity, linearity, precision, accuracy, and lower limits of detection. The method was specific since there were no interference peaks from the reaction matrix. The calibration curve for 4-ABP-G was linear from 50 to 5000 pmol/200 microl with R2=0.999. The newly developed method has good precision and accuracy. The intra- and interday precisions were less than 5 and 10%, respectively, and the highest values for intra- and interday accuracies were -4.6 and -12%, respectively. The lower limit of detection was 10 pmol/200 microl. The developed method was used to determine the glucuronidation activity of mouse, rat, and human liver microsomes. Human liver microsomes were the most active in 4-ABP glucuronidation (344.1 pmol/min/mg) followed by rats (30.6 pmol/min/mg) and then mice (12.3 pmol/min/mg). Human UGT1A4 supersomes were much more active than UGT1A9 (184.4 mol/min/mg versus 25.2 mol/min/mg). These results are consistent with those of earlier studies that used the radioactive [C14]UDPGA.     

Determination of arginine-vasotocin and isotocin in fish plasma with solid-phase extraction and fluorescence derivatization followed by high-performance liquid chromatography

A new HPLC assay for plasma arginine-vasotocin (AVT) and isotocin (IT) determination based on fluorescence detection preceded by combination of solid-phase extraction (SPE) and fluorescence derivatization is presented. Plasma samples retained on solid support were purified and then derivatized by the fluorescent compound 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The peptide derivatives were eluted from cartridges, pre-concentrated and analyzed by HPLC system with fluorescent detection. The separation was carried out on a reversed-phase column with solvent gradient system. The assay was linear in the range 15-220 pmol ml(-1) for AVT r2=0.998 and 10-220 pmol ml(-1) for IT r2=0.996. The detection limits for AVT and IT were 0.8 and 0.5 pmol ml(-1) (3:1, signal-to-noise), respectively. The recoveries of derivatized hormones were in the range 89-93%. Both of the inter- and intra-day assay precision were below 5.5 and 9% for AVT and IT, respectively. The assay should be also applicable to plasma and tissue samples from other animals with only minor modification.     

Needle concentrator for gas chromatographic determination of BTEX in aqueous samples

A simple method of solventless extraction of volatile organic compounds (BTEX) from aqueous samples was developed and validated. A new arrangement of the full volume inside needle capillary adsorption trap (INCAT) device with Porapak Q as a sorbent material and wet alumina as a source of desorptive water vapour flow in a closed analytical system is presented. The analytical characteristics of developed device and of compared purge-and-trap (PTI) device for BTEX compounds are similar; the limits of detection as well as quantification are lower than 1 microg l(-1).     

Direct enantioseparation of some β-adrenergic blocking agents using impregnated thin-layer chromatography

The resolution of (±)-atenolol, (±)-propranolol and (±)-metoprolol into their enantiomers was achieved by TLC on silica-gel plates impregnated with optically pure

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-lysine (0.5%) and

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-arginine (0.5%) as the chiral selectors. In all cases, different combinations of acetonitrile–methanol solvent systems were found to be successful in resolving these compounds. Spots were detected using iodine vapour. The detection limit for both (±)-atenolol and (±)-propranolol was 2.6 μg and for (±)-metoprolol, it was 0.26 μg.     

Does impaired mitochondrial function affect insulin signaling and action in cultured human skeletal muscle cells?

Insulin-resistant type 2 diabetic patients have been reported to have impaired skeletal muscle mitochondrial respiratory function. A key question is whether decreased mitochondrial respiration contributes directly to the decreased insulin action. To address this, a model of impaired cellular respiratory function was established by incubating human skeletal muscle cell cultures with the mitochondrial inhibitor sodium azide and examining the effects on insulin action. Incubation of human skeletal muscle cells with 50 and 75 microM azide resulted in 48 +/- 3% and 56 +/- 1% decreases, respectively, in respiration compared with untreated cells mimicking the level of impairment seen in type 2 diabetes. Under conditions of decreased respiratory chain function, insulin-independent (basal) glucose uptake was significantly increased. Basal glucose uptake was 325 +/- 39 pmol/min/mg (mean +/- SE) in untreated cells. This increased to 669 +/- 69 and 823 +/- 83 pmol/min/mg in cells treated with 50 and 75 microM azide, respectively (vs. untreated, both P < 0.0001). Azide treatment was also accompanied by an increase in basal glycogen synthesis and phosphorylation of AMP-activated protein kinase. However, there was no decrease in glucose uptake following insulin exposure, and insulin-stimulated phosphorylation of Akt was normal under these conditions. GLUT1 mRNA expression remained unchanged, whereas GLUT4 mRNA expression increased following azide treatment. In conclusion, under conditions of impaired mitochondrial respiration there was no evidence of impaired insulin signaling or glucose uptake following insulin exposure in this model system.     

A sensitive assay of GTP cyclohydrolase I activity in rat and human tissues using radioimmunoassay of neopterin

A highly sensitive and simple assay for the activity of GTP cyclohydrolase I (EC 3.5.4.16) was established using a newly developed radioimmunoassay. D-erythro-7,8-Dihydroneopterin triphosphate formed from GTP by GTP cyclohydrolase I was oxidized by iodine and dephosphorylated by alkaline phosphatase to D-erythro-neopterin, and quantified by a radioimmunoassay for D-erythro-neopterin. This method was highly sensitive and required only 0.2 mg of rat liver tissues for the measurement of the activity. It was reproducible and can be applied for the simultaneous assay of many samples. The activity of GTP cyclohydrolase I was measured in several rat tissues. For example, the enzyme activity in rat striatum (n = 5) was 13.7 +/- 1.5 pmol/mg protein per hour (mean +/- SE), and agreed well with those obtained by high-performance liquid chromatography with fluorescence detection. The activity in the autopsy human brains (caudate nucleus) was measured by this new method for the first time. The activity in the caudate nucleus from parkinsonian patients (n = 6) was 0.82 +/- 0.56 pmol/mg protein per hour which was significantly lower than the control value, 4.22 +/- 0.43 pmol/mg protein per hour (n = 10).     

Benzanthrone: a new substrate for hepatic microsomal cytochrome P-450

The metabolism of benzanthrone, a commonly used dy intermediate, by rat hepatic microsomes was investigated using thin layer chromatography (TLC) analysis. Incubation of benzanthrone with hepatic microsomes in the presence of NADPH generating system produced at least seven fluorescent metabolites on TLC plates. TLC spots numbered II, III, IV, V and VI were the major metabolites obtained from hepatic microsomes with the Rf values of 0.53, 0.45, 0.38, 0.33 and 0.26, respectively. Metabolites VII and VIII were faint bands with Rf values of 0.08 and 0.04, respectively. Preincubation of hepatic microsomes with either 1-benzyl-imidazole (10(-4)M) or SKF-525 A (10(-4)M) or metyrapone (10(-3)M) or flushing with carbon monoxide substantially inhibited the benzanthrone metabolism. alpha-Naphtho-flavone (10(-4)M) did not cause any change in hepatic microsomal metabolism of benzanthrone. Oral administration of benzanthrone to animals yielded at least six urinary metabolites. TLC spots numbered II, III, IV, V and VI in the urine were same as those of hepatic microsomal metabolites. However, one of the urinary metabolite numbered IX which stays at the origin of TLC plate with the Rf value of 0.05 may be a conjugate. Our results suggest that benzanthrone acts as a substrate for hepatic heme protein, cytochrome P-450 and that some of the metabolites are excreted in urine.     

Validation of HPTLC method for the analysis of taraxerol in Clitoria ternatea

A new, simple, sensitive, selective and precise HPTLC method has been developed for the determination of taraxerol in Clitoria ternatea L. Determination of taraxerol was performed on TLC aluminium plates. Linear ascending development was carried out in twin trough glass chamber saturated with hexane and ethyl acetate (80:20 v/v). The plate was then dried and sprayed with anisaldehyde reagent. A Camag TLC scanner III was used for spectrodensitometric scanning and analysis at 420 nm. The system was found to give compact spots for taraxerol (R(f) 0.53). The calibration plot was linear in the range of 100-1200 ng of taraxerol. The correlation coefficient of 0.9961 was indicative of good linear dependence of peak area on concentration. The concentration of taraxerol was found to be 12.4 mg/g w/w in the hydroalcoholic extract of C. ternatea root. To study the accuracy and precision of the method, recovery studies were performed. Recovery values from 99.65 to 99.74% showed excellent reliability and reproducibility of the method. The limits of detection and quantification were determined to be 31 and 105 ng/spot, respectively. The proposed HPTLC method for quantitative monitoring of taraxerol in C. ternatea can be used for routine quality testing of C. ternatea extract used in Ayurvedic formulations.     

Phospholipase A2 activities with a plasmalogen substrate in brain and in neural tumor cells: a sensitive and specific assay using pyrenesulfonyl-labeled plasmenylethanolamine

We have developed a new assay method for phospholipase A2 (EC 3.1.1.4.), towards ethanolamine plasmalogen using pyrenesulfonyl-labeled plasmenylethanolamine as the substrate. This procedure is sensitive to about 3 pmol/ml per min and is absolutely specific for plasmalogen. In this method, the product of phospholipase A2, pyrenesulfonyl-labeled lysoplasmalogen, is hydrolyzed to aldehyde and labeled glycerophosphoethanolamine with hydrochloric acid exposure, and after TLC separation, the pyrenesulfonyl-glycerophosphoethanolamine is quantitated spectrofluorometrically. The excitation and emission wave lengths were 340 and 376 nm, respectively. The activity of bovine brain homogenate was 44.1 +/- 6.47 pmol/min per mg protein (n = 3). Among bovine brain subcellular fractions, the distribution and specific activity of the enzymes were highest in cytosol (38.7 +/- 1.58% and 102.6 +/- 16.2 pmol/min per mg protein, n = 3). The activities of neural tumor cells, PC12 pheochromocytoma, Neuro2A and SKNSH neuroblastoma and U1242MG glioblastoma, were 34.4 +/- 6.83 (n = 5), 7.05 +/- 0.97 (n = 4), 5.25 +/- 1.69 (n = 5), and 9.68 +/- 1.35 (n = 4), pmol/min per mg protein (M +/- S.E.M.), respectively.     

Specific and rapid analysis of ubiquinones using Craven's reaction and HPLC with postcolumn derivatization

A new method for the analysis of ubiquinones in various samples was developed using an HPLC system with postcolumn derivatization. Craven's reaction, a specific color reaction for the analysis of ubiquinones, was used in the system. Because the reaction progressed in organic solvents that contained ubiquinones and ethylcyanoacetate under an alkaline condition, the selectivity for ubiquinone detection was higher than that for ubiquinone detection using the nonderivatized ultraviolet detection system at 275 nm, a system widely used for the analysis of ubiquinones. The new detection system can avoid the adverse effects of impurities. Furthermore, it can confirm specificity by stopping the color reaction under a neutral condition. The detection limit for ubiquinone-10 was 1 ng (1.2 pmol). A good linearity for the calibration curve was observed in the range of 11.7 pmol to 11.7 nmol. To investigate the possible application of this method, various samples, such as soybean capsules used as a dietary supplement and biological materials (rice as well as bovine plasma and liver samples), were applied to the system and their ubiquinone contents were quantified. This method is thought to be widely and conveniently applicable for determining the level of ubiquinones because of its high selectivity for ubiquinone detection.     

Detection of gangliosides by direct binding ofLimax flavus agglutinin to thin layer chromotograms

A simple and sensitive method for detecting gangliosides on TLC plates is described. Gangliosides are extracted by phase partition in chloroform/methanol, developed on TLC plates in chloroform/methanol/0.25% aqueous KCl (5/4/1 by vol) and identified by binding of¹²⁵I-labelled, sialic acid-specificLimax flavus agglutinin (LFA) autoradiography and scanning densitometry. The detection limit of the method is below 1 ng (0.5 pmol) for GM3, GM1 and GT1b, and below 0.3 ng (0.2 pmol) for GM2 and GD1a. Binding of¹²⁵I-LFA is not inhibited by 10⁶-fold molar excess concentrations ofN-acetylneuraminic acid or lactose but is decreased in a dose-dependent manner by eitherN-acetylneuraminyllactose or unlabelled lectin. Gangliosides were not detected after their treatment byClostridium perfringens sialidase in the presence of taurocholic acid. Ten gangliosides were detected in human brain and seven in normal human serum. Extracts from 0.2 mg of brain and 20 l of serum were sufficient for the detection of major gangliosides.Abbreviations LFA Limax flavus agglutinin - ELLA Enzyme Linked Lectin Assay - PIM Poly(isobutyl methacrylate) - PVP Polyvinylpyrrolidone mol.wt. 40,000 - PBS Phosphate buffered saline - BSA Bovine serum albumin     

TLC-densitometric analysis of artemisinin for the rapid screening of high-producing plantlets of Artemisia annua L

A simple TLC-densitometric technique has been developed for the rapid and accurate analysis of artemisinin in a large number of Artemisia annua plantlets cultured in vitro. This new analytical method is based on the structural conversion of artemisinin on a silica gel layer by ammonia vapour to form 10-azadesoxyartemisinin, a chromophore-containing compound (lambdamax 320 nm) that can be detected by UV-based TLC densitometry. The TLC system was evaluated quantitatively in terms of product stability, precision, accuracy and calibration. Good linearity was obtained in the range of 0.01-0.12 microg artemisinin. The technique appeared to be accurate and sensitive as compared with the complicated pre-column reaction-HPLC technique. Among 90 samples of A. annua plantlets, the artemisinin content in the leaves appeared to be highly variable, ranging from 0.02 to 0.67% w/w dry weight. These results demonstrate that densitometric TLC can be a cheap and simple technique for the accurate screening of high-artemisinin-producing plants.