Homogeneous fluorescent derivatization of large proteins

A method of homogeneously derivatizing large proteins for highly sensitive analysis is described. Homogeneity of the derivative was realized by tagging all the free amino groups of proteins. With this method, alpha-chymotrypsinogen A, ovalbumin and bovine serum albumin were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Prior to the derivatization, all the proteins were reduced and alkylated. After reacting the resulting unfolded proteins with excessive amounts of AQC, the samples were analyzed with matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to determine the derivatization degree. The results indicated that all three proteins had been, or had almost been, fully derivatized. HPLC and CE were used for characterizing these protein derivatives. Under the optimized fluorescence detection conditions, the detectability of the tagged proteins was 2400-6200 times better than that detected at UV 280 nm, 170-300 times better than detected at UV 214 nm, and 150-420 times better than measured with their native fluorescence.     

RP-HPLC assay for 1,2-5,6-dianhydro-3,4-disuccinyl-galactitol and its metabolites in blood plasma and liver

Summary A method involving pre-column derivatization and HPLC assay is described for measuring submicrogram quantities of 1,2-5,6-dianhydro-3,4-disuccinyl-galactitol (1,2-5,6-dianhydro-3,4-bis(carboxypropionyl)-galactitol), an effective cytostatic drug and its metabolites in blood plasma and liver homogenate. The substance and its metabolites were derivatized with sodium pentamethylene-dithiocarbamate to form different bis(dithiocarbamoyl) esters, which can be detected by UV absorbance at 254 and 280 nm. The directly derivatized products were then extracted into CHCl₃, and after sample preparation resolved by RP-HPLC on SAS-Hypersil column.     

Capillary electrophoresis analysis of gentamicin sulphate with UV detection after pre-capillary derivatization with 1,2-phthalic dicarboxaldehyde and mercaptoacetic acid

A selective, sensitive, and rapid pre-capillary derivatization method for determination of the multicomponent aminoglycoside antibiotic gentamicin is described. The derivatization reagents 1,2-phthalic dicarboxaldehyde and mercaptoacetic acid were used and the thioisoindole derivative was UV detected at 330 nm. A central composite experimental design was performed to optimize selectivity and derivatization conditions. Baseline separation of gentamicin C1, C1a, C2, C2a, C2b, sisomicin and several minor components was achieved with a background electrolyte containing 30 mM sodium tetraborate, 7.5 mM beta-cyclodextrin and 12.5% (v/v) methanol at pH 10. Quantitative analysis was performed and illustrated the potential use of capillary electrophoresis for the identification and quantitation of gentamicin as an alternative to methods prescribed in the United States Pharmacopeia and European Pharmacopoeia.     

Derivatization of peptides and small proteins for improved identification and detection in capillary zone electrophoresis (CZE)

Peptides and small proteins, of limited molecular weight (MW) can be derivatized with a 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (6-AQC) reagent, leading to a single capillary zone electrophoresis (CZE) peak, suggestive of a completely tagged product. The number of tags per molecule was demonstrated by matrix assisted, laser desorption, time-of-flight mass spectrometry (MALDI-TOFMS) studies. In CZE, these species have greatly improved plate count and peak shape, improved (lowered) detectabilities, and in general, improved identification properties in the CZE mode in high performance capillary electrophoresis (HPCE). The formation of what appears to be a single, homogeneously tagged product is a function of how the derivatizations are performed. Once these conditions are optimized, virtually all peptides and small proteins tested (limited MW) can form single, fully tagged products, with the desirable CZE properties. These derivatization approaches thus lead to products that perform and are detected much better in CZE than their precursors (native, untagged peptides). The determined plate counts for these tagged peptides were as high as 6 million plates/m, which was very reproducible, and 59–12,000 times higher than the untagged (native) molecules. The peak symmetry was also improved greatly. The limit of detection (LOD) of some tested 6-AQC tagged peptides were nine to 209 times improved (lower) with ultraviolet (UV) absorption detection, again as compared with that for the native species. The LOD could be further lowered via laser induced fluorescence (LIF) detection in CZE, especially when acetonitrile (ACN) containing buffers were used.     

Degradation of complexons derived from succinic acid under UV radiation

The destruction of complexons derived from succinic acid under the action of UV radiation was studied. IR spectroscopy, thin-layer paper chromatography, and complexometric titration were used to determine the destruction products of these complexons. It was found that the complexons decompose under UV irradiation substantially more easily than ethylenediaminetetraacetic acid does, and the products of their decomposition can undergo a biological utilization under natural conditions. The data obtained in the study make it possible to choose, instead of ethylenediaminetetraacetic acid, ligands that will be nearly fully destructible in the light without deteriorating the ecology.     

Short-end injection capillary electrophoresis for quantification of plasma chondroitin sulfate isomer disaccharides

We describe a new ultra-rapid capillary electrophoresis method with UV detection for analysis of the disaccharides obtained after enzymatic depolymerization of plasma chondroitin sulfates. The free reducing groups of the released carbohydrate molecules are derivatized with 2-aminoacridone by reductive amination in the presence of cyanoborohydride. The fluorotagged products can be separated by short-end injection capillary electrophoresis in a capillary with an effective length of 10.2 cm. The migration times of Δdi-0S and Δdi-4S were 0.95 and 1.81 min, respectively. We compared the proposed method with UV detection to a reference CE-LIF assay by measuring plasma chondroitin sulfate in 94 subjects. The described assay for total plasma CS measurement may, owing to the high throughput and the fast analytical times, be a good tool for routine studies both in research and in clinical applications.     

Confirmation of okadaic acid, dinophysistoxin-1 and dinophysistoxin-2 in shellfish as their anthrylmethyl derivatives using UV radiation

A rapid and simple method for confirmation of the diarrhetic shellfish poisons (DSP): okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) using fluorescence detection following derivatization with 9-chloromethylanthracene, has been established as an alternate to LC/MS. Exposure of the anthrylmethyl derivatives of OA, DTX-1 and DTX-2 to near UV light (300-400 nm) resulted in the loss of these compounds to below detection limits within 30 min, with a concurrent appearance of two additional compounds. Based on the mass spectral evidence, we propose that these newly formed compounds are the decarboxylation products of the derivatized diarrhetic shellfish poisons. UV radiation is, therefore, proposed as a rapid and simple confirmation technique for these DSP in mussel samples.     

Fluorescence derivatization of single-walled carbon nanotubes for analysis by means of conventional CE-LIF

Single-walled carbon nanotubes (SWNTs) are now widely used in many fields, and while many analytical methods for SWNTs have been reported, there are few practical analytical methods that combine the necessary levels of selectivity and sensitivity. We have developed a highly sensitive separation method for fluorescence-derivatized SWNTs by means of conventional CE with laser-induced fluorescence. First, SWNTs were dispersed using a triphenylene derivative into the water, and the excess dispersant was removed by nitric acid treatment. The dispersed SWNTs were then derivatized with a fluorescence reagent, 4-aminofluorescein. Finally, the derivatized SWNTs were analyzed using a conventional apparatus CE-LIF detection. The SWNTs migrated within 20 min. The detection sensitivity of SWNTs was improved by about 170 times with LIF detection as compared with UV detection. We anticipate that the derivatized SWNTs can also be detected with high sensitivity using LC.     

Capillary electrophoretic separations of peptides using micelle-forming compounds and cyclodextrins as additives

The value of electrokinetic capillary chromatography for separating structurally similar model peptides and tryptic digests is demonstrated. The behavior of model peptides in buffer systems containing dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, sodium dodecyl sulfate and two cyclodextrins as additives is described. These additives, under different analytical circumstances, exhibit certain beneficial effects for peptides with similar net charges but different hydrophobicities. Separations of underivatized peptides, utilizing UV detection, are presented. In addition, separation of fluorescent products of peptides derivatized with o-phthalaldehyde, fluorescamine, and a new reagent, 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde, are demonstrated and discussed. Beneficial spectroscopic detection effects with cyclodextrin are also noted.     

Photocontrolled Release of Chemicals from Nano‐ and Microparticle Containers

A benzoin‐derived diol linker was synthesized and used to generate biocompatible polyesters that can be fully decomposed on demand upon UV irradiation. Extensive structural optimization of the linker unit was performed to enable the defined encapsulation of diverse organic compounds in the polymeric structures and allow for a well‐controllable polymer cleavage process. Selective tracking of the release kinetics of encapsulated model compounds from the polymeric nano‐ and microparticle containers was performed by confocal laser scanning microscopy in a proof‐of‐principle study. The physicochemical properties of the incorporated and released model compounds ranged from fully hydrophilic to fully hydrophobic. The demonstrated biocompatibility of the utilized polyesters and degradation products enables their use in advanced applications, for example, for the smart packaging of UV‐sensitive pharmaceuticals, nutritional components, or even in the area of spatially selective self‐healing processes.     

Capillary isoelectric focusing with UV-induced fluorescence detection

Low-molecular-mass fluorescent compounds excitable in the near UV region with suitable acidobasic and electrophoretic properties are suggested as isoelectric point (pI) markers for isoelectric focusing (IEF) with UV photometric and UV excited fluorometric detection. The experimental set-up of capillary IEF with UV excited fluorometric detection and properties of new UV-induced fluorescent pI markers are given. The pI values of 18 new pI markers determined independently of IEF methods range from 2.1 to 10.3. The examples of separation of new pI markers together with derivatized proteins by capillary IEF with photometric or fluorometric detection are presented.     

New biochemical separations using precolumn derivatization and microcolumn liquid chromatography

Microcolumn liquid chromatography with slurry-packed capillary columns has been used to resolve mixtures of biologically important steroids and prostaglandins. In order to enhance detection sensitivity, the samples are first derivatized with a suitable chromophore-yielding agent. Hydroxy steroids were derivatized with either benzoyl chloride or 7-(chlorocarbonylmethoxy)-4-methylcoumarin, while Dns hydrazine was employed to react with the ketonic groups of certain steroidal conjugates. Bile acids can also be derivatized with bromomethylcoumarin to yield fluorescent products. The use of microcolumns permits both a high degree of component resolution and enhanced detection sensitivity.     

Determination of Amikacin Isomers by High Pressure Liquid Chromatography

Abstract

A high-pressure liquid chromatographic (HPLC) method for quantitative monitoring of amikacin isomers is described. Four isomers, BB-K8, BB-K29, BB-K6 and BB-K11 were applied to a silica gel column. While adsorbed, the isomers were derivatized with o-phthalaldehyde and the derivatized products eluted with ethanol. A decrease in the fluorescence of the derivatized products with time was observed. Heating at 50°C for 5 min produced products with stable fluorescence for at least three hours. Using the fluorescent properties of the amikacin derivative for detection, the four isomers of amikacin were separated by reverse phase (HPLC). A linear relationship from 1 to 10 μg/mL was obtained for all four isomers.     

The structure of granaticin and of granaticin B. 1. Spectroscopic charcteristics and chemical analysis

UV.-, IR.- and NMR.-spectra of the antibiotic granaticin and several of its derivatives and degradation products are discussed. The spectroscopic properties agree with the formula XX for the antibiotic, which was fully elucidated by an X-ray structural analysis (see following communication).     

High-performance liquid chromatographic analysis of dianhydrogalactitol in plasma by derivatization with sodium diethyldithiocarbamate.

A high-performance liquid chromatographic method is described for measuring submicrogram quantities of dianhydrogalactitol, a promising anti-neoplastic agent, in plasma. The drug is derivatized directly in plasma with sodium diethyldithiocarbamate to form a bis(dithiocarbamoyl) ester which absorbs UV light at 254 nm (am 17,000). The derivatized product is then extracted quantitatively into chloroform and separated by normal phase chromatography (muBondpak CN column). Dianhydrogalactitol concentration below 50 ng/ml of plasma can be detected in the eluent.     

气相色谱/质谱法鉴定黑芝麻中黑色素的结构类型

 为获得天然高分子化合物黑芝麻中黑色素的分子结构信息 ,对其进行碱熔降解 ,然后对降解产物进行硅烷化衍生 ,再利用气相色谱 /质谱联用分析 ,结果发现产物中含有儿茶酚、对苯二酚和儿茶酸 ,从而证明黑芝麻中的黑色素具有儿茶酚型黑色素的结构特征。     

Photoflocculation of TiO2 microgel mixed suspensions

Photocatalytic TiO 2 was derivatized with an amino silane to give a positively charged colloidal suspension in water. UV irradiation of the cationic titania oxidized the organo silane coating converting the titania to a negatively charged colloid. Irradiation of mixed suspensions of cationic titania and cationic polyvinylamine microgel induced catastrophic flocculation. Electrophoresis and XPS evidence suggests that the UV removed the amine groups from the titania resulting in heteroflocculation of anionic titania with cationic microgel particles.     

Identification of oligomeric domains within dermatan sulfate chains using differential enzymic treatments, derivatization with 2-aminoacridone and capillary electrophoresis

Galactosaminoglycans, i.e. dermatan sulfate (DS) and chondroitin sulfate, are linear heteropolysaccharides consisting of repeating disaccharide units of L-iduronic acid (L-IdoA) or D-glucuronic acid (D-GlcA) residues linked to N-acetyl-galactosamine. High-performance capillary electrophoresis (HPCE or CE) has been successfully used for determining the disaccharide composition of glycosaminoglycans. However, only limited information is available on how to identify oligomeric domains rich in D-GlcA or L-IdoA. The aim of this study was therefore to develop a rapid and accurate CE procedure by which such oligosaccharides can be determined together with the variously sulfated disaccharides. Isolated dermatan sulfates of human origin were separately digested with chondroitinases ABC, AC and B and the enzymic products were derivatized with 2-aminoacridone. CE analysis of these products was performed using a phosphate buffer, pH 3.0, and reversed polarity at 30 kV. The derivatization enabled their detection with laser-induced fluorescence (LIF) and UV at 260 nm at much higher sensitivity than the detection of nonderivatized delta-saccharides at 232 nm and therefore components undetectable at 232 nm were nicely detected after derivatization. Except for delta-disaccharides, altogether five distinct oligosaccharides with differences in charge density were identified. Depending on the lyase that produced these oligomers, information on the presence of L-IdoA- or D-GlcA-containing domains within the DS chain and the sulfation pattern of these oligomeric domains was obtained. This CE method could also be useful in studying the functional oligomeric domains in galactosaminoglycan chains.     

Square wave anodic stripping voltammetric determination of Pb2+ using acetylene black paste electrode based on the inducing adsorption ability of I-

In the study, we developed a simple, rapid and sensitive method for the determination of tiopronin (TP) in human plasma, which was based on derivatization with p-bromophenacyl bromide (p-BPB) followed by liquid-liquid extraction and reverse-phase HPLC-UV detection. For the first time, the p-BPB was introduced into the derivatization of TP. The thiol group of TP was trapped with p-BPB to form a TP-p-BPB adduct, which can be very suitable for UV detection. From acidified plasma samples, the derivatized TP was extracted with 5 mL dichloromethane. Effective chromatographic separation was achieved using a C18 column (DIAMONSIL 150 mm × 4 mm i.d., 5 μm) based on an acetonitrile-water-trifluoroacetic acid (40:59.88:0.12, v/v/v) elution at a flow-rate of 1 mL/min. The IS and the derivatized TP were detected at 263 nm. No endogenous substances were found to interfere. The limit of quantification for derivatized TP (TP-p-BPB) in plasma was 40 ng/mL. The calibration curve for the derivatized TP showed linearity in the range 0.04-4 μg/mL with a regression coefficient corresponding to 0.9991 and the coefficient of the variation of the points of the calibration curve being lower than 10%. Extraction recoveries of the derivatized TP in plasma were greater than 72%. The method was suitably validated and successfully applied to determination of TP in human plasma samples.     

Hydrazine determination in sludge samples by high-performance liquid chromatography

A relatively simple and cost-effective method utilizing HPLC with UV detection was developed to detect and quantify hydrazine in sludge samples. The method was developed primarily for sludge samples, but it can also be applicable to soil and other environmental samples. The hydrazines in the matrices were derivatized to hydrazones with benzaldehyde. The hydrazones were separated using HPLC with an RP C-18 column in an isocratic mode with methanol-water (95:5 v/v) and detected with UV detection at 313 nm. The detection limit (25 microL injection) for the method is 0.02 microg/mL of hydrazine.