Determination of Homocystine as Dabsyl Derivative by Hplc Using Precolumn Derivatization

ABSTRACT

An HPLC method has been developed for the determination of homocystine. Homocystine was converted to its chromophoric dabsyl derivative and was chromatographed by a reversed phase chromatographic column (μ-Bondapak C) with pH = 3 phosphate buffer-acetonitrile (60:40) as isocratic mobile phase. The derivative was detected by a variable wavelength detector operating at 436 ran. A linear relationship was obtained between peak areas and homocystine concentrations over the range of 0.54-2.68 μg/20 μl.     

Dabsyl chloride derivatisation of melamine followed by high-performance liquid chromatography determination in water samples

A new and sensitive precolumn derivatisation with dabsyl chloride was developed for the analysis of melamine in water samples by high-performance liquid chromatography (HPLC) with visible detection. Derivatisation with dabsyl chloride leads to improving sensitivity and hydrophobicity of melamine. Under optimum conditions of derivatisation and microextraction, the method yielded a linear calibration curve ranging from 10 to 2000 µg L^?1 with a determination coefficient (R²) of 0.9952. Limit of detection (LOD) and limit of quantification (LOQ) were 2.0 and 6.0 µg L^?1, respectively. The relative standard deviation per cent (RSD%) for intraday and inter-day extraction and determination at 20 and 200 µg L^?1 levels of melamine was less than 8.2% (n = 6). Finally, the proposed method was successfully applied for the determination of melamine in different water samples and satisfactory results were obtained (relative recovery ≥91%).     

Micro high performance liquid chromatography of aliphatic amines by means of resonance raman detection

A micro high performance liquid chromatography coupled with a resonance Raman detection system is described. For highly sensitive Raman detection, aliphatic amines were derivatized with dabsyl chloride (4-dimethylaminoazobenzene-4′-sulfonyl chloride). The derivatives were separated on an ODS micro column (0.5 mm i.d. × 145 mm PTFE tube). Chromatograms were obtained by measuring the intensity of Raman scattering at 1136 cm^?1 with the 488.0 nm line of an Arion laser. The lower detection limit was 1.5 ng and the RSD of relative peak height (n = 9) was 5.9% at 11 ng of methylamine derivative. Moreover, by stopping the flow of the micro HPLC system at the retention time of the individual derivatives, it was possible to measure their resonance Raman spectra.     

聚苯乙烯基N,O-二酰基磺酰羟胺树脂: 一种双酰基转移试剂在合成酰胺库中的应用

用聚苯乙烯基磺酰羟胺树脂1与酰氯2反应合成了聚苯乙烯基N,O-二酰基磺酰羟胺树脂3. 树脂3作为一种新的双酰基转移试剂可与胺4发生酰基转移反应, 合成了含有24个结构类似的酰胺化合物库. 改变酰氯的种类, 结果发现双对硝基苯甲酰树脂3a的活性较高. 双酰基树脂3胺解结果表明, 由脂肪族胺得到的酰胺收率较芳香族胺高. 当解脱试剂同时含有羟基和氨基时, 双酰基树脂3能选择性地在氨基端发生酰基转移, 而羟基端不受影响.     

Synthesis of 4′-alkyl-8-azaspiro[bicyclo[3.2.1]octane-3,2′-morpholin]-5′-ones

N-Boc-protected 8-azaspiro[bicyclo[3.2.1]octane-3,2′-oxirane] reacted with primary aliphatic amines through opening of the epoxide ring with formation of the corresponding amino alcohols which were converted into N-chloroacetyl derivatives. The latter underwent cyclization to N-Boc-protected 4′-alkyl-8-azaspiro[bicyclo[3.2.1]octane-3,2′-morpholin]-5′-ones by the action of sodium hydride in DMF, and subsequent treatment with hydrogen chloride in ethyl acetate afforded 8-azaspiro[bicyclo[3.2.1]octane-3,2′-morpholin]-5′-one hydrochlorides.     

Direct amidation of non-activated carboxylic acid and amine derivatives catalyzed by TiCp2Cl2

This paper described a mild and efficient direct amidation of non-activated carboxylic acid and amine derivatives catalyzed by TiCp₂Cl₂. Arylacetic acid derivatives reacted with different amines to afford the corresponding amides in good to excellent yield except of aniline. Aryl formic acids failed to react with aniline but smoothly reacted with aliphatic amines and benzylamine in moderate to good yield, fatty acids reacting with benzyl and aliphatic amines give amides in good to excellent yield. Chiral amino acids derivatives were transformed into amides without racemization in moderate yield. The possible mechanism of direct amidation catalyzed by TiCp₂Cl₂ was discussed. This catalytic method is very suitable for the amidation of low sterically hindered arylacetic acid, fatty acids with different low sterically hindered amines except aniline, as well as the amidation of aryl formic acid with benzyl and aliphatic amines.     

A fluorous tag-bound fluorescence derivatization reagent, F-trap pyrene, for reagent peak-free HPLC analysis of aliphatic amines

We have developed a novel pre-column fluorescence derivatization reagent for amines, F-trap pyrene. This reagent comprises a fluorescent pyrene moiety, an amine-reactive Marshall linker, and a fluorophilic perfluoroalkyl group known as fluorous tag. When the reagent reacts with aliphatic amines and amino acids to give fluorescent derivatives, the fluorous tag in the reagent is eliminated simultaneously. Therefore, excess unreacted reagents in the derivatization reaction solution still have the fluorous tag and could be removed by fluorous solid-phase extraction selectively before high-performance liquid chromatography (HPLC) analysis. By using this reagent, 13 kinds of aliphatic amine (C₂–C₁₆) derivatives can be separated within 40 min by reversed-phase HPLC with gradient elution. In this chromatogram, unreacted reagents peak at around 28 min, greatly decrease after fluorous solid-phase extraction, and do not interfere with the quantification of each amine. The detection limits (S/N = 3) for examined aliphatic amines are 3.6–25 fmol per 20 μL injection. We have also applied this reagent successfully to the amino acid analysis.

Characterization and application of a new ultraviolet derivatization reagent for amino acids analysis in capillary electrophoresis

A new ultraviolet (UV) labeling reagent, p-acetamidobenzenesulfonyl fluoride (PAABS-F), was designed and synthesized to label and determine the amino acids by capillary electrophoresis (CE) with diode-array detector (DAD). PAABS-F is very stable and easy to synthesize. It reacted with primary or secondary amino acids very quickly under facile conditions to give corresponding derivatives in high yield with excellent sensitivity and stability. No by-products were observed in amino acid derivatives when stored at room temperature under natural daylight for at least 7 days. Both amino acids standard solution and real samples reacted with this new UV labeling reagent smoothly to form high UV-absorption derivatives. The labeled 20 standard amino acids were efficiently separated by CE and the mass detection limits (S/N = 3) were ranged from 59.3 fmol for l-tryptophan to 1.70 pmol for l-histidine.     

(2-Naphthoxy)acetyl chloride,a simple fluorescent reagent

In continuing the search for fluorescent reagents for analytical derivatization in chromatography, we found a simple chemical, (2-naphthoxy)acetyl chloride, with potential fluorophore/chromophore characteristics for the highly sensitive detection of analytes with an amino function. The reagent has an auxochrome (a substituted alkoxy moiety) attached to the fluorophoric/chromophoric naphthalene system, resulting in favorable spectrophotometric properties. The reagent can be easily prepared from (2-naphthoxy)acetic acid and has been used in organic synthesis; it is initially introduced as a fluorescent reagent to derivatise amantadine and memantine (amino pharmaceuticals) as model analytes. The resulting naphthoxy derivatives of the drugs can be analyzed at sub-microM levels by HPLC with fluorimetric detection (excitation wavelength 227 nm, emission wavelength 348 nm). Application of the reagent to the fluorimetric derivatization of important biological amines for sensitive detection can be expected.     

Anthraquinone-2-sulfonyl chloride: a new versatile derivatization reagent-synthesis mechanism and application for analysis of amines

A new sulfonating agent, anthraquinone-2-sulfonyl chloride, has been synthesized. The reagent consists of three important moieties: a cyclic conjugation system (with 18 pi-electrons), a p-quinone system and a group of sulfonyl chloride and is thereby a versatile derivatization reagent for analytical chemistry. The mechanism about the synthetic reaction was first elucidated in aid of mass spectrometry. Several primary and secondary amines were selected to evaluate the new reagent and their standard derivatives were prepared via a facile pathway. Analytical derivatization carried on through a one-step procedure at room temperature within 3 min. The new reagent reacts quantitatively with amines to form stable sulfonamides, which are readily amenable to analysis by normal-phase and reversed-phase HPLC. Compared with standard derivatives, excellent response linearity is demonstrated over the concentration range 0.4-400 muM at 320 nm for normal-phase HPLC and 4 nM to 4 muM at 256 nm for reversed-phase HPLC. Detection limits are 0.8 nmol and 8 pmol, respectively.     

Solid-phase reagent containing the 3,5-dinitrophenyl tag for the improved derivatization of chiral and achiral amines, amino alcohols and amino acids in high-performance liquid chromatography with ultraviolet detection

A solid-phase reaction technique is described for improved derivatization of aliphatic amines, amino alcohols and amino acids. A polymeric activated ester is used for the immobilization of the 3,5-dinitrobenzoyl group, which can then be used for derivatizations of strong or weak nucleophiles, while avoiding solution-phase derivatization conditions. The reagent is easily prepared and can be regenerated after use to attain its original reactivity. The resulting chromatograms are free of system peaks due to excess derivatizing reagent, and sample handling is kept to a minimum. The reagent can be used in conjunction with both reversed- and normal-phase chromatography and can be used for off-line gas chromatographic or high-performance liquid chromatographic (HPLC) derivatizations. In addition, the reagent can be used on-line for derivatization in HPLC. Since the labelling reagent is a strong pi-acid, chiral substrates can be derivatized and separated on a Pirkle-type pi-donor column. The confirmation and quantitation of amphetamine in urine was accomplished using a polymer containing two labelling moieties, p-nitrobenzoyl and 3,5-dinitrobenzoyl. The derivatization and separation of chiral and achiral amines, amino alcohols and amino acids is described.     

Sensitive determination of aliphatic amines by high-performance liquid chromatography with a new fluorogenic probe 3-(4-fluorinebenzoyl)-2-quinoline carboxaldehyde

A new fluorogenic reagent 3-(4-fluorinebenzoyl)-2-quinoline carboxaldehyde (FBQCA) has been synthesized and used as a derivatizing reagent for the determination of aliphatic amines with HPLC. The reagent is nonfluorescent, but forms highly fluorescent isoindole upon the reaction with primary amines in alkaline medium. Eleven amine derivatives were baseline separated in 8 min using a gradient elution on a C(8) column and detected with fluorescence detection at lambda(ex)/lambda(em) = 480/546 nm. The detection limits were in the range of 0.5-2 nM (S/N = 3). The proposed method has been successfully applied to the analysis of aliphatic amines in food and environmental samples, including white wine, soybean oil, soil, and tap water with satisfactory recoveries in the range of 94-106%.     

Optimization of experimental variables in the dabsyl chloride derivatization of biogenic amines for their determination by RP-HPLC

Summary In the last few years special attention has been paid to the pre-column derivatization of biogenic amines with dabsyl chloride because proper experimental conditions for this reaction are very important. In this study, an experimental design (Doehlert design) was used to optimize the variables involved in the dabsylation of the following amines: histamine, tyramine, phenylethylamine, tryptamine, cadaverine, putrescine, spermidine, and spermine. The optimum experimental conditions for forming the dabsyl derivatives are: reagent concentration, 1.75.10⁻³ M; pH, 8.2; temperature, 70°C; heating time (t _h ), 21 min. Under these conditions good chromatographic repeatability is obtained.     

NEW APPLICATIONS OF SOLID SILICA CHLORIDE (─SiO2Cl): EFFICIENT OXIDATION OF CYCLIC THIOACETALS,TMS, TBDMS,AND THP ETHERS TO THEIR CARBONYL COMPOUNDS BY SOLID SILICA CHLORIDE/KMnO4 SYSTEM

Solid silica chloride/KMnO₄ system oxidizes effectively aromatic and aliphatic cyclic thioacetals (1,3-dithiolanes, 1,3-dithianes) and silyl and pyranyl ethers into their corresponding carbonyl compounds in dry CH₃CN at room temperature in high yields. Over-oxidation of aldehydes to carboxylic acids has not been observed by this system. In the absence of silica chloride, KMnO₄ in dry CH³CN is an ineffective reagent for these oxidations.     

BrMOZPhC, a novel coumarin type reagent for the fluorescent derivatisation of carboxylic acids

A new fluorescent reagent was synthesized in order to be used as a fluorescent probe in analytical HPLC derivatisation reactions. Briefly, 2,4-dimethoxyaniline was acylated and demethylated and the resulting diphenol reacted with α-bromoacetoacetate in the presence of conc. sulfuric acid (the Von Pechmann reaction). The resulting acetamido coumarin was hydrolyzed and the aminohydroxy derivative cycled to form the corresponding bromo-coumarinoxazine. The analytical reaction was performed with butyric acid as model compound and catalyzed by 18-crown-6 ether. The calibration curve constructed was characterized by good linearity whereas the precision of the reaction gave an R.S.D. values below 3%. The reaction applied to the separation of five aliphatic acids.     

Preparation of polymeric amines from poly(schiff bases)

A study was made of the preparation of aromatic polymeric amines in order to test their thermal stability. The most useful method was the hydrogenation of polymeric Schiff bases by the dimethylamine—borane reagent or the borane—tetrahydrofuran reagent. The Schiff bases were prepared by the solution polymerization of terephthalaldehyde with various aromatic diamines, including 4,4′-methylenedianiline, benzidine, and p-phenylenediamine, and for comparison, 1,6-hexanediamine. The Schiff bases and the polyamines from the aromatic diamines were found to be dimers or trimers, not high polymers: the polymers from the aliphatic diamine had a degree of polymerization of about 14. Thermogravimetric analyses of the aromatic polyamines under nitrogen showed that the initial temperatures of marked degradation were 350–400°C.     

Zirconium(IV) Chloride–Mediated Chemoselective Conjugate Addition of Aliphatic Amines to α,β‐Ethylenic Compounds

Zirconium chloride efficiently catalyzes the conjugate addition of a variety of aliphatic amines to α,β‐unsaturated ester, nitriles, and ketones to give the corresponding β‐amino derivatives in excellent yields under mild reaction conditions. Aromatic amines do not participate in this transformation.     

HPLC of 9-fluorenylmethylchloroformate-polyamine derivatives with fluorescence detection

Summary There are a number of reagents available for fluorescent labelling of primary amines. These include dansyl chloride, o-phthalaldehyde, fluorescamine, and a new reagent, 9-fluorenylmethylchloroformate (FMOC), reported recently. This paper describes a reversed-phase HPLC procedure for the separation and fluorescence detection of polyamines following pre-column derivatization with FMOC. The polyamines studied by this method include putrescine, cadaverine, spermidine, and spermine. Experiments were carried out to determine maximum fluorescence excitation and emission wavelengths, optimum reaction pH, linear ranges, and minimum detection limits for each of the polyamines. The HPLC method includes a gradient program which provides complete separation from serum hydrolysate components and specificity for the four polyamines with detection limits ranging from 2 to 9 pg. This procedure was applied to hydrolyzed serum samples.     

Synthesis of new substituted acetamide derivatives of 6′,7′-dimethoxy-2′,3′-dihydro-1′<Emphasis Type="Italic">H</Emphasis>-spiro[isoquinoline-1,4′-cyclopentane]-1′-carboxamide

Reaction of ethyl 6′,7′-dimethoxy-3′Н-spiro[isoquinoline-1,4′-cyclopentane]-1′-carboxylate with ammonia and isopropylamine afforded dihydroisoquinoline carboxamides that were reduced with NaBH₄ in amides of 6,7-dimethoxy-4-spirocyclopentane-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid. The reaction of unsubstituted carboxamide with chloroacetyl chloride led to chloroamide that reacted with versatile secondary amines and heterylthiols providing the corresponding substituted amino- and sulfanylamides of tetrahydroisoquinoline series.     

Fluorescence Probe of 10-Phenyl-acridone-2-sulfonyl Chloride and Its Application for Determination of Free Aliphatic Amines in Environmental Samples by HPLC with Fluorescence Detection and APCI-MS

A simple and sensitive method for the determination of free aliphatic amines using 10-phenyl-acridone-2-sulfonyl chloride (PASC) as a labeling reagent by high-performance liquid chromatography with fluorescence detection and online mass spectrometry identification (HPLC-FLD-MS) has been developed. Derivatization conditions including reagent concentration, buffer pH, reaction time and temperature were optimized. PASC reacted with aliphatic amines at 50 °C for 4 min in aqueous acetonitrile (ACN) in the presence of sodiumtetraborate–NaOH buffer (0.10 mol L⁻¹, pH 9.0) to give high yields of PASC-amine derivatives. Derivatives exhibited intense fluorescence with an excitation maximum at λ_ex 265 nm and an emission maximum at λ_em 418 nm. The separation of derivatives was performed by a reversed-phase Hypersil BDS C8 column in combination with a gradient elution. The identification of derivatives was carried out by online post-column mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive-ion detection mode. Excellent linear responses were observed with the correlation coefficients of larger than 0.9997, and detection limits (at a signal-to-noise of 3:1) were from 3.0 to 24.3 fmol. Comparing with 10-ethyl-acridine-2-sulfonyl chloride (EASC), PASC exhibited more intense fluorescence and ultraviolet absorbance. The proposed method is sensitive and reproducible for the determination of aliphatic amines from water and soil samples.