Molecularly imprinted sol-gel polymers for the analysis of iprodione fungicide in wine: Synthesis in green solvent.

Iprodione is a fungicide widely used in viticulture in most agricultural countries. It was banned recently in the European community because of its carcinogenic and endocrine disrupting characters. In this work, a cheap analytical method able to monitor iprodione in a white wine was developed. Molecularly imprinted sol-gel polymers (MIS) specific to iprodione and using green solvents were synthesized. An experimental design having the following factors (solvent volume and crosslinker quantity) was used to prepare an optimal MIS. In terms of selectivity, the optimal MIS showed the best partition coefficient towards iprodione in a white wine containing four other competing fungicides (procymidone, pyrimethanil, azoxystrobin and iprovalicarb). A solid phase extraction method using the optimal MIS was optimized and applied to analyse iprodione in a white wine. Low detection and quantification limits were reached 11.7 and 39.1 µg/L respectively.

QSAR characterization of new synthesized hydantoins with antiproliferative activity.

Hydantois have been identified as constituents of a number of pharmacologically active molecules. In the present study, we have examined in vitro antiproliferative activity against human colon cancer cell lines HCT-116 of three series of 3-(4-substituted benzyl)-hydantoins with various substituent attached in position 5 of the hydantoin ring. Since the investigated compounds have recently been synthesized and show antiproliferative activity, a good understanding of the properties of the potential drug responsible for their pharmacokinetics is an important goal for their further development. One of the important properties is lipophilicity. Lipophilicity has been assessed by reversed-phase liquid chromatography (high-performance thin-layer chromatography and high-pressure liquid chromatography) by means of direct and indirect (using calibration curve) methods. Chromatographic lipophilicity indices in addition to calculated logP values were compared by hierarchical cluster analysis. The linear solvation energy relationship approach was used to understand and compare the types and relative strength of the molecular interactions that occur in the chromatographic as well as in the n-octanol-water partitioning systems. Finally, correlation between in silico pharmacokinetic predictors and antiproliferative activity was examined. Preliminary quantitative structure-activity relationship modeling indicates that pharmacokinetic predictors capture only one-quarter of all chemical features that are important for antiproliferative activity itself. Among selected descriptors are chromatographic lipophilicity indices.

Simultaneous determination of iprodione, procymidone, and chlorflurenol in lake water and wastewater matrices by GC-MS after multivariate optimization of binary dispersive liquid-liquid microextraction.

This study reports the optimization of a binary dispersive liquid-liquid microextraction method for the determination of iprodione, procymidone, and chlorflurenol by gas chromatography mass spectrometry. The study was aimed at using two extraction solvents to increase the extraction efficiency of all analytes. The binary solvents recorded results higher than the mono-solvents. After examining the effects of main experimental parameters and their interactions by analysis of variance, 200 µL of binary mixture (dichloromethane and 1,2-dichloroethane), 2.5 mL of ethanol, and 15 s vortex were obtained as optimum parameters. The detection and quantification limits calculated for the analytes were found to be between 0.30-1.6 and 1.0-5.3 ng/mL, respectively. Enhancement in detection power calculated as a ratio of the binary extraction detection limit to the detection limit of direct GC-MS analysis was 105-, 214-, and 233-fold for chlorflurenol, iprodione, and procymidone, respectively. In order to check the accuracy of the developed method, recovery study was performed. Water sampled from a lake and two wastewater samples from treatment facilities were spiked at two concentrations, and the percent recovery calculated for the samples ranged between 87 and 116%. These results confirmed the suitability of the method to real samples for accurate determination of the analytes at trace levels.

Design, Synthesis, and Biological Activity of ß-Carboline Analogues Containing Hydantoin, Thiohydantoin, and Urea Moieties.

A series of novel ß-carboline derivatives was designed by combining the anti-tobacco mosaic virus (TMV) lead compound tetrahydro-ß-carboline ester with the hydantoin, thiohydantoin, and urea motifs. These derivatives were synthesized from tetrahydro-ß-carboline ester via a structural diversity-oriented synthesis in one step, and their biological activities were evaluated. Most of the derivatives exhibited anti-TMV activity higher than that of commercial plant virucide ribavirin, such as compounds 2, 4, 5, 7, 9, 15, 16, 19, and 21. Compared with the lead compounds, some of these derivatives showed good insecticidal activity against Plutella xylostella and Culex pipiens pallens. At the same time, these derivatives also showed broad-spectrum fungicidal activity. The systematic study provides strong evidence that the hydantoin, thiohydantoin, and urea motifs of these molecules can improve and modulate the activities of the analogues of natural products.

Iprodione residues and dissipation rates in tobacco leaves and soil.

Field experiments were conducted in two different locations to determine the residue levels and dissipation rates of iprodione in tobacco leaves and soil. Iprodione 50% wettable powder formulation was sprayed once at 12.50 g/ha to study the dissipation behavior and three to four times at 8.33 g/ha (recommended dose) and 12.50 g/ha (1.5 times the recommended field dose) to determine the residue levels of iprodione in tobacco leaves and soil after repeated applications. Iprodione residues in both green tobacco leaves and soil dissipated to about 50% of the initial deposits after 7 days and then further dissipated to more than 90% after 35 days.The dissipation of iprodione followed first order kinetics and the calculated half-life values (T (1/2)) were 5.64-8.80 days in green tobacco leaves and 7.50-9.93 days in soil, respectively. Iprodione residue levels in flue-cured tobacco leaves 21 days after the third and fourth applications ranged from 7.61 to 40.98 mg/kg. Meanwhile, the residues detected in soil decreased to 0.010-0.117 mg/kg 21 days after the last treatment.

A low thermal mass fast gas chromatograph and its implementation in fast gas chromatography mass spectrometry with supersonic molecular beams.

A new type of low thermal mass (LTM) fast gas chromatograph (GC) was designed and operated in combination with gas chromatography mass spectrometry (GC-MS) with supersonic molecular beams (SMB), including GC-MS-MS with SMB, thereby providing a novel combination with unique capabilities. The LTM fast GC is based on a short capillary column inserted inside a stainless steel tube that is resistively heated. It is located and mounted outside the standard GC oven on its available top detector port, while the capillary column is connected as usual to the standard GC injector and supersonic molecular beam interface transfer line. This new type of fast GC-MS with SMB enables less than 1 min full range temperature programming and cooling down analysis cycle time. The operation of the fast GC-MS with SMB was explored and 1 min full analysis cycle time of a mixture of 16 hydrocarbons in the C(10)H(22) up to C(44)H(90) range was achieved. The use of 35 mL/min high column flow rate enabled the elution of C(44)H(90) in less than 45 s while the SMB interface enabled splitless acceptance of this high flow rate and the provision of dominant molecular ions. A novel compound 9-benzylazidanthracene was analyzed for its purity and a synthetic chemistry process was monitored for the optimization of the chemical reaction yield. Biodiesel was analyzed in jet fuel (by both GC-MS and GC-MS-MS) in under 1 min as 5 ppm fatty acid methyl esters. Authentic iprodion and cypermethrin pesticides were analyzed in grapes extract in both full scan mode and fast GC-MS-MS mode in under 1 min cycle time and explosive mixture including TATP, TNT and RDX was analyzed in under 1 min combined with exhibiting dominant molecular ion for TATP. Fast GC-MS with SMB is based on trading GC separation for speed of analysis while enhancing the separation power of the MS via the enhancement of the molecular ion in the electron ionization of cold molecules in the SMB. This paper further discusses several features of fast GC and fast GC-MS and the various trade-offs involved in having powerful and practical fast GC-MS.

Multiresidue method for the determination of 13 pesticides in three environmental matrices: water, sediments and fish muscle.

Pesticides residues in aquatic ecosystems are an environmental concern which requires efficient analytical methods. In this study, we proposed a generic method for the quantification of 13 pesticides (azoxystrobin, clomazone, diflufenican, dimethachlor, carbendazim, iprodion, isoproturon, mesosulfuron-methyl, metazachlor, napropamid, quizalofop and thifensulfuron-methyl) in three environmental matrices. Pesticides from water were extracted using a solid phase extraction system and a single solid-liquid extraction method was optimized for sediment and fish muscle, followed by a unique analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Limits of quantification were below 5 ng L(-1) for water (except for fluroxypyr and iprodion) and ranged between 0.1 ng g(-1) and 57.7 ng g(-1) for sediments and regarding fish, were below 1 ng g(-1) for 8 molecules and were determined between 5 and 49 ng g(-1) for the 5 other compounds. This method was finally used as a new routine practice for environmental research.

Development of a lateral flow immunoassay (LFA) strip for the rapid detection of 1-aminohydantoin in meat samples.

UNLABELLED: Due to the potential toxic effects of the nitrofuran family of antibiotics, their use in animals in the food industry has raised health concerns. This study was aimed to develop a lateral flow assay (LFA) based on competitive format for the detection of 1-aminohydantoin (AHD) in meat samples. The assay could be completed in 1 min and detected AHDs derivates (CPAHD) at 3 ng/mL, equivalent to 1.40 ng/mL of AHD, which was much lower than that reported in the literature by similar method. The antibody showed no cross-reactivity with a panel of more than 10 nitrofuran analogs except for nitrofurantoin at a high concentration. The test strip was stable at room temperature for up to 8 wk or at 37 °C for 4 wk. Parallel analyses of meat samples with LFA and enzyme-linked immunosorbent assay (ELISA) obtained data in good agreement. This developed gold nanoparticle based LFA had a good specificity, sensitivity, stability, and reliability. It was potentially suitable for on-the-spot large-scale screening of meat samples, and even more other applications. PRACTICAL APPLICATION: Nitrofurantoin is one of antibiotics of the nitrofuran family, which has been used not only to prevent and treat diseases, but also to promote growth in animals. However, concerning the carcinogenicity of the metabolite of nitrofurantoin (AHD), a new fast and convenient method for monitoring AHD should be established. We describe the development of a new test assay for rapid screening of meat samples.

Analysis of pesticide residues by fast gas chromatography in combination with negative chemical ionization mass spectrometry.

A combination of fast GC with narrow-bore column and bench top quadrupole mass spectrometer (MS) detector in negative chemical ionization (NCI) mode (with methane as reagent gas) is set up and utilized for the ultratrace analysis of 25 selected pesticides. The observed pesticides, belonging to the endocrine disrupting chemicals (EDCs), were from different chemical classes. A comparative study with electron impact (EI) ionization was also carried out (both techniques in selected ion monitoring (SIM) mode). The programmed temperature vaporizer (PTV) injector in solvent vent mode and narrow-bore column (15mx0.15mm I.D.x0.15microm film of 5% diphenyl 95% dimethylsiloxane stationary phase) were used for effective and fast separation. Heptachlor (HPT) as internal standard (I.S.) was applied for the comparison of results obtained from absolute and normalized peak areas. Non-fatty food matrices were investigated. Fruit (apple - matrix-matched standards; orange, strawberry, plum - real samples) and vegetable (lettuce - real sample) extracts were prepared by a quick and effective QuEChERS sample preparation technique. Very good results were obtained for the characterization of fast GC-NCI-MS method analysing EDCs pesticides. Analyte response was linear from 0.01 to 150microgkg(-1) with the R(2) values in the range from 0.9936 to 1.0000 (calculated from absolute peak areas) and from 0.9956 to 1.0000 (calculated from peak areas normalized to HPT). Instrument limits of detection (LODs) and quantification (LOQs) were found at pgmL(-1) level and for the majority of analytes were up to three orders of magnitude lower for NCI compared to EI mode. In both ionization modes, repeatability of measurements expressed as relative standard deviation (RSDs) was less than 10% which is in very good agreement with the criterion of European Union.

Rapid analysis of multiple pesticide residues in fruit-based baby food using programmed temperature vaporiser injection-low-pressure gas chromatography-high-resolution time-of-flight mass spectrometry.

A rapid method using programmed temperature vaporiser injection-low-pressure gas chromatography-high-resolution time-of-flight mass spectrometry (PTV-LP-GC-HR-TOF-MS) for the analysis of multiple pesticide residues in fruit-based baby food was developed. The fast and inexpensive buffered QuEChERS (quick, easy, cheap, effective, rugged, and safe) extraction method and "conventional" approach that employs ethyl acetate extraction followed by gel permeation chromatography (GPC) cleanup were employed for sample preparation. A PTV injector in solvent venting mode was used to reduce volume of acetonitrile and acetic acid (from the buffered QuEChERS extracts) that caused higher column bleed without their elimination. Otherwise, the time-to-digital converter would become saturated in HR-TOF-MS. For fast GC separation allowing analysis of 100 analytes within a 7 min runtime, both a high temperature programming rate and vacuum conditions in a megabore GC column were employed. The use of HR-TOF-MS allowed the unbiased identification and reliable quantification of target analytes through the application of a narrow mass window (0.02 Da) for extracting analyte ions and the availability of full spectral information even at very low levels. With only a few exceptions, the lowest calibration levels for the pesticides tested were

Stability studies of the metabolites of nitrofuran antibiotics during storage and cooking.

Nitrofuran antibiotics cannot be used in food production within the European Union because of their potential health risks to consumers. The recent discovery of their widespread use in global food industries and the finding of semicarbazide in baby food as a result of packaging contamination have focused attention on the toxicity and stability of these drugs and their metabolites. The stability of the nitrofuran marker residues 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ), 1-aminohydantoin (AHD) and semicarbazide (SEM) were tested. Muscle and liver of nitrofuran treated pigs were cooked by frying, grilling, roasting and microwaving. Between 67 and 100% of the residues remained after cooking, demonstrating that these metabolites are largely resistant to conventional cooking techniques and will continue to pose a health risk. The concentration of metabolites in pig muscle and liver did not drop significantly during 8 months of storage at -20 degrees C. Metabolite stock and working standard solutions in methanol were also stable for 10 months at 4 degrees C. Only a 10 ng ml(-1) solution of SEM showed a small drop in concentration over this extended storage period.

Feasibility of LC/TOFMS and elemental database searching as a spectral library for pesticides in food.

Traditionally, the screening of unknown pesticides in food has been accomplished by GC/MS methods using conventional library-searching routines. However, many of the new polar and thermally labile pesticides are more readily and easily analysed by LC/MS methods and no searchable libraries currently exist (with the exception of some user libraries, which are limited). Therefore, there is a need for LC/MS libraries that can detect pesticides and their degradation products. This paper reports an identification scheme using a combination of LC/MS time-of-flight (accurate mass) and an Access database of 350 pesticides that are amenable to positive ion electrospray. The approach differs from conventional library searching of fragment ions. The concept consists of three parts: (1) initial screening of possible pesticides in actual market-place fruit extracts (apple and orange) using accurate mass and generating an accurate mass via an automatic ion-extraction routine, (2) searching the Access database manually for screening identification of a pesticide, and (3) identification of the suspected compound by accurate mass of at least one fragment ion and comparison of retention time with an actual standard. Imazalil and iprodione were identified in apples and thiabendazole in oranges using this database approach.

Application of comprehensive two-dimensional gas chromatography with nitrogen-selective detection for the analysis of fungicide residues in vegetable samples.

Comprehensive two-dimensional gas chromatography (GCxGC) with nitrogen-phosphorus detection (NPD) has been investigated for the separation and quantitation of fungicides in vegetable samples. The detector gas flows (H(2), N(2) and air) were adjusted to achieve maximum response of signal whilst minimizing peak width. The comparison of different column sets and selection of the temperature program were carried out with a mixture of nine N-containing standard fungicides, eight of which were chlorinated. The results from GCxGC-NPD and GCxGC with micro electron-capture detection (muECD) were compared. External calibrations of fungicides were performed over a concentration range from 1 to 1,000 microgL(-1). The peak area calibration curves generally had regression coefficients of R(2)>0.9980, however for iprodione which was observed to undergo on-column degradation, an R(2) of 0.990 was found. The limit of detection (LOD) and limit of quantitation (LOQ) were less than about 74 and 246 ng L(-1), respectively. The intra-day and inter-day RSD values were measured for solutions of concentration 0.100, 0.500 and 1.50 mg L(-1). For the 0.500 mg L(-1) solution, intra- and inter-day precision of peak area and peak height for most of the pesticides were about 2% and 8%, respectively. Excellent linearity was observed for these standards, from 0.001 to 25.00 mg L(-1). The standard mixture peak positions were identified by using GCxGC with quadrupole mass spectrometry (qMS). To illustrate the potential and the versatility of both GCxGC-NPD and GCxGC-muECD, the method was applied to determination of fungicides in a vegetable extract. Decomposition of one fungicide standard (iprodione) during chromatography elution was readily observed in the two-dimensional (2D) GCxGC plot as a diagonal ridge response in the 2D chromatogram between the degrading compound and the decomposition product.

Development of an SPME-GC-MS/MS method for the determination of pesticides in rainwater: laboratory and field experiments.

A solid-phase microextraction -- coupled to a gas chromatography -- ion trap tandem mass spectrometry (SPME-GC-MS/MS) method was developed for the quantitative determination in rainwater of 8 pesticides amongst the most used in France and 3 triazines metabolites. The main factors affecting the SPME process were studied. Using a 3 mL sample, the method developed showed good linearity for concentrations ranging from 0.05 to 50 microgL(-1) with correlation coefficients between 0.997 and 0.9999 and relative standard deviations (% RSD) below 14%. The study of matrix effects showed that rainwater was too diluted to have any significant influence on the extraction efficiency. To validate the method, a field campaign was carried out on the rain events, which occurred in Strasbourg during a one-year period. The rain concentrations showed patterns of high pesticide concentrations during spring months, which were correlated to the spraying periods of most of these substances.

Complete NMR elucidation of a novel trishomocubane hydantoin and its mono- and bis-t-Boc protected derivatives.

The syntheses of a novel trishomocubane hydantoin and its mono- and bis-protected t-Boc derivatives are described. The less nucleophilic N-3' nitrogen of the hydantoin ring is protected first when treated with di-tert-butyl dicarbonate (t-Boc anhydride), possibly owing to steric hindrance by the bulky trishomocubane cage skeleton. More basic conditions were required to form the bis-protected t-Boc hydantoin with the same reagent. The structures of these novel compounds were elucidated with 2D NMR techniques. The proton spectrum of the trishomocubane skeleton is complex owing to major overlap of proton signals. A high-level DFT calculation was used to determine some of the crucial interatomic positions, which assisted with the elucidation of the structures. The assignment of proton and carbon signals of the three structures is described and it differs significantly from each other and also from the trishomocubanol precursor. The bis-Boc hydantoin is required for a more facile hydrolysis to the corresponding trishomocubane amino acid at room temperature.

Biological monitoring of N, N-dimethylformamide. Reference value for N-methylcarbamoyl adduct at the N-terminal valine of globin as a biomarker of chronic occupational exposure.

OBJECTIVES: The recently identified metabolic product of the industrial solvent N, N-dimethylformamide (DMF), the N-methylcarbamoyl adduct at the N-terminal valine of globin, can be determined after chemical conversion to 3-methyl-5-isopropylhydantoin (MVH). Due to prolonged persistence of the adduct in human erythrocytes (lifetime: 4 months), MVH is a suitable biomarker of integrated exposure to DMF over a period of several months. Here we propose a reference value for MVH, used for biological monitoring of chronic occupational exposure to DMF. METHODS: The reference value for MVH was set equal to its steady-state level in a simulated repeated inhalation exposure to DMF (8 h/day, 5 days/week, >or=20 weeks) at a concentration corresponding to the occupational exposure limits MAK and TLV. The initial data on the toxicokinetic behavior of MVH were obtained after single percutaneous and inhalation exposures to DMF in volunteers. MVH was determined by gas chromatography-mass spectrometry according to Mráz et al. RESULTS: The steady-state level of MVH, attained after repeated inhalation exposure to DMF, 30 mg/m(3), 8 h/day, 5 days/week, >or=20 weeks, was assessed to be approximately 135 nmol MVH/g globin. CONCLUSIONS: We recommend the value of 135 nmol MVH/g globin to be used as a new reference value for biomonitoring of integrated exposure to DMF over a long period. The relationship between the MVH level in globin and the intensity of the exposure to DMF should be further tested in the field studies.

Liquid chromatography-electrospray ionization mass spectrometry of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins.

We report the separation of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins by microbore reverse-phase high-performance liquid chromatography and their detection by on-line electrospray ionization mass spectrometry. These compounds are the products of the chemical stepwise degradation of polypeptides using 4-(3-pyridinylmethylaminocarboxypropyl) phenyl isothiocyanate. We describe chromatographic conditions for on-column concentration of the analytes and for baseline separation of the isobaric amino acid derivatives of leucine and isoleucine. A commercially available protein sequencer was readily interfaced with the described analytical system and used for adsorptive sequence analysis of a panel of synthetic peptides containing collectively all 20 naturally occurring amino acids. On-line mass analysis of derivatives generated by automated sequencing confirmed that the derivatives were of the predicted mass and were detectable at comparable signal strength and sensitivity. Finally, we demonstrate that the additional selectivity in data interpretation provided by mass analysis dramatically improves the signal-to-noise ratio and therefore enhances the ability to conclusively interpret protein and peptide sequence data.

Capillary gas chromatographic-electron-capture assay for the aldose reductase inhibitor imirestat in lens and plasma.

A sensitive and selective gas chromatographic-electron-capture assay was developed for the determination of the aldose reductase inhibitor imirestat in lens and plasma. The method involves solid-phase extraction of drug and internal standard from the plasma specimen or lens sample homogenate using "Baker"-10 SPE extraction columns followed by derivatization with pentafluorobenzyl bromide and further purification. Derivatives of drug and internal standard were separated on a fused-silica capillary column and analyzed using a 63Ni electron-capture detector. The limit of detection was 2.5 ng per lens or ml of plasma. The method was used to evaluate the pharmacokinetics of imirestat in human subjects and to quantitate imirestat in animal lens tissue following topical ocular administration.

A gas chromatographic assay for tocainide carbamoyl-O-beta-D-glucuronide:quantitation of the base hydrolyzed product, 3-(2',6'-xylyl)-5-methylhydantoin.

Quantitation of tocainide carbamoyl-O-B-D-glucuronide, a major metabolite of the antiarrhythmic agent, tocainide, was previously reported using sodium hydroxide hydrolysis (pH greater than 12) of the glucuronide followed by gas chromatographic analysis of the hydrolyzed product, 3-(2',6'-xylyl)-5-methylhydantoin. In this investigation, the hydantoin was found to undergo rapid degradation during base hydrolysis of the tocainide glucuronide. The hydantoin was synthesized and its hydrolysis in sodium hydroxide (pH greater than 12) was found to follow first-order kinetics with an estimated half-life of 24.6 minutes. This spontaneous and rapid degradation of the hydantoin during hydrolysis of the tocainide glucuronide may result in underestimation of the hydantoin. To correct for the degradation of the hydantoin, a gas chromatographic assay with a timed, base-hydrolysis calibration protocol was designed. To demonstrate the applicability of this assay, the elimination kinetics of tocainide and the tocainide carbamoyl glucuronide were studied in three healthy male volunteers after a single 200 mg oral dose of tocainide hydrochloride. From urine data, the elimination half-life of tocainide was approximately 15.5 hours. The urinary excretion half-life of the tocainide carbamoyl glucuronide was approximately 13.8 hours.

Reversed-phase high-performance liquid chromatography separation of dimethylaminoazobenzene sulfonyl- and dimethylaminoazobenzene thiohydantoin-amino acid derivatives for amino acid analysis and microsequencing studies at the picomole level.

A simple and fast reversed-phase high-performance liquid chromatographic method has been developed for the complete separation of 35 dimethylaminoazobenzene sulfonyl (DABS)-amino acids and by-products. This method allows simultaneous determination of primary and secondary amino acids which can be present in protein and peptide hydrolysates and also detects the presence of cysteic acid, S-sulfocysteine, hydroxyproline, taurine, norleucine, cystine, and delta-hydroxylysine. The precolumn derivatization of amino acids with dimethylaminoazobenzene sulfonyl chloride (DABS-Cl) is simple and quick (10 min at 70 degrees C) and allows the complete reaction of primary and secondary amino acids. The separation of the compounds under investigation is achieved in 25 min using a reversed-phase 3-microns Supelcosil LC-18 column at room temperature. The versatility of the proposed method is documented by amino acid determination on protein samples obtained using different hydrolysis techniques (HCl, methane-sulfonic acid, and NaOH), with attention given to the detection of tryptophan in protein samples with high sugar concentration. Furthermore, we have reported the experimental conditions necessary to apply this method to the amino acid analysis of very low amount of proteins (1 to 5 micrograms) electroeluted from a stained band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The stability of DABS-derivatives, the short time of analysis, the high reproducibility and sensitivity of the system, and the complete resolution of all compounds of interest make this method suitable for routine analysis. Furthermore, we have also developed a fast reversed-phase high-performance liquid chromatographic method for the complete separation of dimethylaminoazobenzene thiohydantoin (DABTH)-amino acids. The separation of the compounds under investigation is obtained, at room temperature, in less than 18 min using a reversed-phase Supelcosil LC-18 DB column, 3-micron particles, and also allows the complete separation of DABTH-Ile, DABTH-Leu, and DABTH-Norleu. The short time of analysis, together with the high reproducibility of the system and its sensitivity at picomole levels, make this method very suitable for the identification of DABTH-amino acids released during microsequencing studies of proteins and peptides with the dimethylaminoazobenzene isothiocyanate reagent. In addition, we have shown that it is possible to obtain complete separation of DABTH-amino acids also under isocratic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)