Pre-column Derivatization Reaction Research Articles

Separation of Alkyne Enantiomers by Chiral Column HPLC Analysis of Their Cobalt-Complexes.

Separation of the enantiomers of new chiral alkynes in strategic syntheses and bioorthogonal studies is always problematic. The chiral column high-performance liquid chromatography (HPLC) method in general could not be directly used to resolve such substrates, since the differentiation of the alkyne segment with the other alkane/alkene segment is not significant in the stationary phase, and the alkyne group is not a good UV chromophore. Usually, a pre-column derivatization reaction with a tedious workup procedure is needed. Making use of easily-prepared stable alkyne-cobalt-complexes, we developed a simple and general method by analyzing the in situ generated cobalt-complex of chiral alkynes using chiral column HPLC. This new method is especially suitable for the alkynes without chromophores and other derivable groups.

Analysis of Amikacin in Human Serum By UHPLC With Fluorescence Detector Using Chloro-Formate Reagent With Glycine

Background: Amikacin belongs to aminoglycosides family, commonly administered in the treatment of systemic infections due to gram negative bacteria. Its narrow therapeutic index results in adverse effects like nephrotoxicity and ototoxicity. Objective: Optimize an ultra-high performance liquid chromatography (UHPLC) based analytical method for the determination of amikacin sulfate in human serum using derivatizaon with FMOCCl and glycine. Methods: Pre-column derivatization reaction of amikacin performed using fluorescence reagent 9-fluorenylmethyl chloroformate (FMOC-Cl) at ambient temperature in the presence of borate buffer (0.2 M). Stabilizing reagent glycine (0.1 M) added into the reaction mixture solution after completion of the derivatization reaction for stabilization of fluorescent complex product. Fluorimetric detection of amikacin was performed at excitation and emission wavelength of 265 nm and 315 nm respectively, using C18 UHPLC column. The reported method was validated by performing linearity, precision, recovery and ruggedness. Results: The optimum mobile phase composition was found to be Acetonitrile:water in the ratio of 70:30 (v/v) at flow rate of 0.4 ml/min. A linear response of amikacin in serum samples ranging from 0.5-10 μg/ml was obtained, with correlation co-efficient of 1.00. The limit of detection (LOD) was found to be 50 ng/ml. Both inter- and intra-day analysis co-efficient values were found to be less than 10%. Conclusion: The developed UHPLC method will be useful for pre-clinical and pharmacokinetic study of amikacin in human serum. Key words: Amikacin Sulphate, Ultra High Performance Liquid Chromatography (UHPLC), 9-Fluorenylmethyl chloroformate (FMOC-Cl) reagent, Borate buffer, Glycine, Fluorescence detector.

Pre-Column Derivatization HPLC Procedure for the Quantitation of Aluminium Chlorohydrate in Antiperspirant Creams Using Quercetin as Chromogenic Reagent.

This article describes the development and validation of a selective high-performance liquid chromatography method that allows, after liquid–liquid extraction and pre-column derivatization reaction with quercetin, the quantification of aluminium chlorohydrate in antiperspirant creams. Chromatographic separation was achieved on an XTerra MS C18 analytical column (150 × 3.0 mm i.d., particle size 5 μm) using a mobile phase of acetonitrile:water (15:85, v/v) containing 0.08 % trifluoroacetic acid at a flow rate of 0.30 mL min−1. Ultraviolet spectrophotometric detection at 415 nm was used. The assay was linear over a concentration range of 3.7–30.6 μg mL−1 for aluminium with a limit of quantitation of 3.74 μg mL−1. Quality control samples (4.4, 17.1 and 30.6 μg mL−1) in five replicates from five different runs of analysis demonstrated intra-assay precision (% coefficient of variation <3.8 %), inter-assay precision (% coefficient of variation <5.4 %) and an overall accuracy (% recovery) between 96 and 101 %. The method was used to quantify aluminium in antiperspirant creams containing 11.0, 13.0 and 16.0 % (w/w) aluminium chlorohydrate, respectively.

Liquid Chromatographic Determination of Amikacin Sulphate after Pre-Column Derivatization

A novel high performance liquid chromatographic (HPLC) method with a pre-column derivatization reaction has been developed and validated. The method was used for the determination of the aminoglycoside antibiotic amikacin sulphate (AMK) in the presence of its synthetic precursor kanamycin sulphate in pure form and in different pharmaceutical preparations. The pre-column derivatization was based on Hantzsch condensation reaction and the obtained coloured products were separated using an isocratic reversed-phase high performance liquid chromatographic method. The separation was achieved on a Spherisorb C18 ODS2 (250 × 4.6 mm, 5 μm) column using a mobile phase composed of acetonitrile-0.1 M sodium acetate buffer (pH 5.0; 25:75, v/v). The column temperature was adjusted at 35 °C and the flow rate at 2 mL min(-1). The detection was carried out at 330 nm by using photo-diode array detector. Different conditions for the optimization of the derivatization reaction as well as for the HPLC measurement were studied. Moreover, AMK was subjected to forced degradation by oxidation, hydrolysis, photolysis and dry heat. Degradation products did not interfere with the assay, which can thus be considered selective and specific. The proposed method was validated for linearity, precision, accuracy, specificity and robustness. Also, it was used to check the purity of AMK in the presence of KAN (related impurity) at the pharmacopoeial limit (0.5%).

Highly sensitive liquid chromatographic analysis of artemisinin and its derivatives after pre-column derivatization with 4-carboxyl-2,6-dinitrobenzene diazonium ion

Summary A highly sensitive and reproducible isocratic liquid chromatographic method has been developed for the analysis of artemisinin and its three commonly used derivatives (artesunate, dihydroartemisinin, and artemether). The method involves a precolumn derivatization reaction with 4-carboxyl-2,6-dinitrobenzene diazonium ion to produce azo adducts that are UV-active. The critical parameters for the derivatization such as temperature, reaction time, and reagent concentrations were studied and optimized. The chromatographic separations were carried out on a C-18 column with mobile phase consisting of acetonitrile-0.1% acetic acid (60:40) at a flow rate of 1 mL min−1. UV detection was set at 254 nm. Dynamic linear calibration range was obtained at concentrations of artemisinins ranging from 0.26 to 1.44 μg mL−1. The low limits of detections of artemisinin, artesunate, dihydroartemisinin, and artemether were found to be 0.091, 0.0125, 0.0489, and 0.0128 ng μL−1, respectively. The developed methods were precise (RSD <3%) and accurate (% error < 5%). The developed methods may find application in dosage form analysis and pharmacokinetic studies.

Automated pre‐column derivatization of thiolic fruit‐antibrowning agents by sequential injection coupled to high‐performance liquid chromatography using a monolithic stationary phase and an in‐loop stopped‐flow approach

The present study reports the very first application of ethyl propiolate (EP) as an advantageous pre-column derivatization reagent for the determination of thiols by liquid chromatography (LC). Cysteine (CYS), glutathione (GSH) and N-acetylcysteine (NAC) were derivatized online under stopped-flow conditions in a sequential injection (SI) system coupled to HPLC. The formed derivatives were separated isocratically with a monolithic stationary phase (100×4.6 mm id) and UV detected at 285 nm. Critical parameters that affected the online pre-column derivatization reaction (e.g. the reaction time and the amount concentration of EP) and the separation (e.g. pH and the composition of the mobile phase) were investigated. The developed analytical scheme offers a total analysis time of less than 10 min, limits of detection in the range of 0.24-0.35 μmol/L and satisfactory linearity up to 200 μmol/L for all analytes. The proposed method was applied to the analysis of the selected thiols--that are often employed as antibrowning agents--in fresh fruit samples.

Highly sensitive and selective pre-column derivatization high-performance liquid chromatography approach for rapid determination of triterpenes oleanolic and ursolic acids and application to Swertia species: Optimization of triterpenic acids extraction and pre-column derivatization using response surface methodology

Oleanolic acid (OA) and ursolic acid (UA) are the ubiquitous triterpenic acids in plant kingdom and have multiple biological effects. In the present study, a highly sensitive and selective method using a dual-sensitive probe 2-(5-benzoacridine)ethyl-p-toluenesulfonate (BAETS) as pre-column labeling reagent has been developed for rapid determination of the triterpenes OA and UA by HPLC with fluorescence detection (FLD) and online mass spectrometry identification. Response surface methodology as an efficient tool was employed to optimize the ultrasonic-assisted extraction of triterpenic acids from Swertia plants and the pre-column derivatization reaction, respectively, which ensured the highest triterpenic acids recoveries within the shortest extraction time and the sufficient labeling of the analyzed components. Fast separation of the isomers OA and UA could be achieved on a Hypersil BDS C8 column within 7 min. Both of OA and UA gave the good correlation coefficients of 0.9999. This developed method offered the satisfactory detection limits of 1.10 and 1.30 ng mL −1 for UA and OA, respectively. When applied to Swertia species, it showed good reproducibility.

A SIMPLE AND VALIDATED LC METHOD FOR THE SIMULTANEOUS ANALYSIS OF GLUCOSAMINE AND CHONDROITIN SULFATE EQUIVALENT IN DIETARY PRODUCTS

A simple and reliable reversed-phase liquid chromatographic (RP-LC) method was developed and validated to determine simultaneously a glucosamine and chondroitin sulfate equivalent in dietary products. The procedure is based upon the reaction of o-phthaldialdehyde with glucosamine and galactosamine coming from the galactosaminoglycan hydrolysis. The hydrolysis reaction was carried out with hydrochloric acid (7.5 N) at 80°C for 8 hr, whereas, the pre-column derivatization reaction was carried out in alkaline media for 1 min at ambient temperature. The chromatographic separations were performed on a Phenomenex Synergi 4 μ fusion-RP 80 A (250 mm × 3.0 mm i.d.) using a mobile phase consisting of a mixture of sodium acetate buffer (pH 5.9; 0.05 M) and methanol (85:15, v/v). UV-DAD detection at λ = 340 nm was used. Linear responses were observed and the limit of quantitation for both aminosaccharides was about 60 pmol. The intra-day precision (RSD) was ≤1.8% and there was no significant difference between intra- and inter-day data. Recovery studies showed good results (99.3–101.0%) with RSD ranging from 1.1 to 2.1%.

Quantitative determination of free glycerol and myo-inositol from plasma and tissue by high-performance liquid chromatography

A high-performance liquid chromatographic method that accurately measures glycerol and myo-inositol from plasma and tissue is described. The method incorporates a pre-column derivatization reaction using aqueous extracts with benzoyl chloride as a modifying agent. The benzoylated derivatives are isolated by HPLC using reversed-phase gradient chromatography and quantified via absorbance detection at 231 nm. The benzoylated derivatives of glycerol and myo-inositol are well resolved from other known carbohydrates, internal standard and other contaminants encountered within samples and during incubation. The benzoylation of these analytes reach a maximum between 3.5 and 6 h of incubation and are stable for at least 24 days at 4 °C. The limit of quantization (LOQ) of glycerol was equal to 2.5 nmol/ml plasma and 6.4 nmol/g tissue and the LOQ of myo-inositol was 1.8 nmol/ml plasma and 3.6 nmol/g tissue. Incubation of known standards and samples with benzoyl chloride at 40 °C for 4 h showed fully benzoylated products as determined by mass spectral analysis. Calibration curves were linear between 2.7 and 174 nmol for glycerol and 1.4–89 nmol for myo-inositol. Comparison of tissue and plasma concentrations of glycerol and myo-inositol found using this method are in good agreement with other reported values using other techniques.

A new selective pre-column ninhydrin-based derivatization for a RP-HPLC determination of plasma asymmetric dimethyl-l-arginine (ADMA) by fluorescence detection

We report a new selective and direct pre-column ninhydrin-based derivatization reaction for determination of plasma ADMA levels. This original derivatization procedure matched to a validated and rapid RP-HPLC method can be a useful alternative to other assays in which time consuming and expensive extraction and/or purification steps are required.

Determination of nateglinide in human plasma by high-performance liquid chromatography with pre-column derivatization using a coumarin-type fluorescent reagent

A sensitive and selective high-performance liquid chromatographic method has been developed and validated for the determination of nateglinide in human plasma. Nateglinide and the internal standard, undecylenic acid, were extracted from plasma by liquid–liquid extraction using a mixture of ethyl acetate–diethyl ether, 50:50 (v/v). Pre-column derivatization reaction was performed using a coumarin-type fluorescent reagent, N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide. The derivatization proceeded in acetone in the presence of potassium carbonate and catalyzed by 18-crown-6 ether. The fluorescent derivatives were separated under isocratic conditions on a Hypersil BDS-C8 analytical column (250.0 mm × 2.1 mm i.d., particle size 5 μm) with a mobile phase that consisted of 65% acetonitrile in water and pumped at a flow rate of 0.50 mL min −1. The excitation and emission wavelengths were set at 345 and 435 nm, respectively. The assay was linear over a concentration range of 0.05–16.00 μg mL −1 for nateglinide with a limit of quantitation of 0.05 μg mL −1. Quality control samples (0.05, 4.50 and 16.00 μg mL −1) in five replicates from five different runs of analysis demonstrated intra-assay precision (%coefficient of variation <6.8%), inter-assay precision (%coefficient of variation <1.6%) and an overall accuracy (%relative error) less than −3.4%. The method can be used to quantify nateglinide in human plasma covering a variety of pharmacokinetic or bioequivalence studies.

A novel pre-column derivatization reaction for the determination of dithiocarbamates in plasma by high-performance liquid chromatography

A new, fully automated pre-column derivatization reaction was developed for the quantitative determination of dithiocarbamates in plasma samples. For complexation, an aliquot of the sample was mixed with an aliquot of a 5 mM Co(II) solution on the autosampler tray. Optimum conditions for the reaction were achieved at a tray temperature of 20 °C and a reaction time of 11 min. Liquid chromatography (LC) separation was performed on a C18 ODS column using a binary gradient consisting of phosphate buffer and methanol. The colored complex of dithiocarbamate and Co(III) was quantified by UV detection at 330 nm. The assay was applied for the determination of pyrrolidine dithiocarbamate, a potent antiviral compound for the inhibition of human rhinoviruses and influenza viruses, in plasma samples. Limits of quantification were 1.4–2.9 μg ml −1.

Capillary zone electrophoresis with pre-column NDA derivatization and amperometric detection for the analysis of four aliphatic diamines

A method was developed for the analysis of four aliphatic diamines by capillary zone electrophoresis using pre-column derivatization with naphthalene-2,3-dicarboxaldehyde (NDA)/CN − and amperometric detection. The pre-column derivatization reaction conditions including the molar ratio of NDA to amines, the cyanide concentration, the pH value of derivatization buffer, and the reaction time, were investigated. The separation of four derivatives of aliphatic diamines has been optimized by capillary zone electrophoresis (CZE) using end-column amperometric detection with a carbon fiber microelectrode, at a constant potential of 0.7 V versus SCE. The optimum conditions for the separation were 10 mM Tris–H 3PO 4 (pH 4.0) for the running buffer solution, 15 kV for the separation voltage. The detection limits for diaminopropane, putrescine, cadaverine, diaminohexane were 6.7×10 −8, 5.1×10 −8, 1.9×10 −7 and 3.8×10 −7 M, respectively (S/N=3). The proposed method was applied to the determination of aliphatic diamines in a lake water sample by the standard addition method. The recovery of these amines in water was 89.9–107%.

Validation of a LC-fluorescence method for determination of free captopril in human plasma, using a pre-column derivatization reaction with monobromobimane

Both derivatization of free captopril in human plasma samples using monobromobimane as fluorescent label and the corresponding HPLC-fluorescence detection (FLD) method were validated. Calibration curve for the fluorescent captopril derivative in plasma samples is linear in the ppb–ppm range with a detection limit of 4 ppb and an identification limit of 10 ppb ( P%: 90; ν≥5). These methods were successfully applied on bioequivalence studies carried out on some marketed pharmaceutical formulations.

Optimization in the Formaldehyde Determination at Sub-PPM Level from Acetals by HPLC-DAD

ABSTRACT Determination of formaldehyde at sub-ppm level as impurity in acetals using HPLC-DAD is described. Automated on-line precolumn derivatization reaction with 2,4 dinitrophenylhydrazine has been used. Breakdown rates of some industrial scale used acetals (Methylal, Ethylal) to formaldehyde by hydrolysis in aqueous media, according to pH, are described.

PRE-COLUMN DERIVATIZATION REACTION COUPLED WITH ULTRASONIC WAVE EXTRACTION FOR THE TRACE ANALYSIS OF ATRAZINE AND SIMAZINE IN SOIL AND CROPS BY LIQUID CHROMATOGRAPHY, ULTRAVIOLET DETECTION

Combined with ultrasonic wave extraction, a pre-column derivatization reaction high performance liquid chromatographic ultraviolet detection procedure was developed for the determination of atrazine and simazine in soil and crop. The 4-(2-phthalimidyl) benzoyl chloride (PIB-Cl) was used as a pre-column derivatization reagent for high performance liquid chromatography (HPLC). The ultrasonic wave technique was applied to the extraction for trace atrazine and simazine in soil and crop. The clean-up and second-time concentration procedures, which was indispensable in the conventional analytical methods for soil and crop analysis because of the complexity of the samples, were replaced by derivatization reaction between PIB-Cl and analytes. The optimum absorption wavelength of 345 nm and the molar absorption coefficients of 105 level for the derivatives were found. The derivatization reaction and chromatographic separation conditions were optimized systematically. The detection limits of 1.1 ng/g for atrazine and 1.0 ng/g for simazine were obtained with the recoveries of 90–95% for soil and crop samples. On the basis of the practical application to 5 soil and 3 crop samples, this method was compared with the conventional GC-MS method. The reliability of this procedure was verified.

Analysis of trace atrazine and simazine in environmental samples by liquid chromatography–fluorescence detection with pre-column derivatization reaction

Coupled with off-line extraction, a pre-column derivatization liquid chromatographic fluorescence detection (LC–FL) procedure was developed for the determination of atrazine and simazine in soil, crop and water samples. Concentrations in real samples were expected to be at or below the low ng/g level, which requires pre-concentration of analytes and improved detection. 4-(2-Phthalimidyl) benzoyl chloride (PIB-Cl) was used as a pre-column derivatization reagent for high-performance liquid chromatography. The clean-up and second-time concentration procedures, which were indispensable in the conventional analytical methods for soil and crop analysis because of the complexity of the samples, were replaced by a derivatization reaction between PIB-Cl and the analytes. The fluorescent and ultraviolet characteristics of the derivatives were investigated. The derivatization reaction and chromatographic separation conditions were optimized systematically. Detection limits of 1.2 ng/g for atrazine and 1.1 ng/g for simazine were obtained with recoveries of 84–95% for environmental samples. On the basis of practical application to five soil and five crop samples, the LC–FL method was compared with the conventional GC–MS method.

Capillary Electrophoresis of Herbicides. 1. Precolumn Derivatization of Chiral and Achiral Phenoxy Acid Herbicides with a Fluorescent Tag for Electrophoretic Separation in the Presence of Cyclodextrins and Micellar Phases

A novel, selective precolumn derivatization reaction was introduced and evaluated in the fluorescence labeling of phenoxy acid herbicides with 7-aminonaphthalene-1,3-disulfonic acid (ANDSA). The ANDSA-phenoxy acid derivatives were readily detected by capillary electrophoresis/laser-induced fluorescence (CE-LIF) at 0.2 ppb, a concentration at which environmental herbicide samples are expected to occur. The precolumn derivatization was very quantitative (99.7% yield) and produced stable derivatives and no side products. Furthermore, the ANDSA-phenoxy acid herbicide enantiomers exhibited higher chiral resolution than their underivatized counterparts in the presence of cyclodextrin (CD) in the running electrolyte. Several native and modified CDs were investigated in the enantiomeric separation of chiral phenoxy acid herbicides. The best enantioselectivity was achieved when 2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CD) was used as the chiral selector. To effect the enantiomeric resolution of a large number of AN...

Capillary electrophoresis of carboxylated carbohydrates I. Selective precolumn derivatization of gangliosides with UV absorbing and fluorescent tags

We demonstrate that the precolumn derivatization reaction, recently introduced by our laboratory for the selective labeling of carboxylated monosaccharides, cab be readily transposed to other glycoconjugates containing carboxylated sugar residues, namely sialogangliosides. The selective derivatization reaction described here involved the attachment of sulfanilic acid (a UV-absorbing tag) or 7-aminonaphthalene-1,3-disulfonic acid (a UV-absorbing and also fluorescing tag) to the sialic acid moiety of the gangliosides via the carboxylic group in the presence of water-soluble carbodiimide. This labeling of the sialic acid moiety of the gangliosides with a chromophore and/or fluorophore leads to the formation of an amide bond between the carboxylic group of the sugar residue and the amino group of the derivatizing agent, thus replacing the weak carboxylic acid group of the carbohydrate species by the stronger sulfonic acid group which is ionized over the entire pH range. Furthermore, novel electrolyte systems were introduced and evaluated for the separation of the derivatized and underivatized gangliosides. The addition of acetonitrile or α-cyclodextrin (α-CD) to the running electrolyte was necessary to break-up the aggregation of amphiphilic gangliosides and allowed for their efficient separation as monomers in aqueous media using capillary electrophoresis. Several operating parameters were investigated with these electrolyte systems including the additive concentration as well as the ionic strength, pH and nature of the running electrolyte. Acetonitrile at 50% (v/v) in 5 m M sodium phosphate at high and low pH or 15 m M α-CD in 100 m M sodium borate, pH 10.0, proved ideal, in terms of resolution and separation efficiency, for the group separation of mono-, di- and trisialogangliosides. On the other hand, the complete resolution of disialoganglioside isomers (e.g., G D1a and G D1b) necessitated the superimposition of a chromatographic component on the electrophoretic process. This was achieved by adding either a hydrophobic (e.g., decanoyl-N-methylglucamide-borate surfactant complex) or hydrophilic [e.g., poly(vinyl alcohol) or hydroxypropyl cellulose] selectors to the running electrolyte.

A PC‐controlled module system for automatic sample preparation and analysis

A simple automatic analytical system, consisting of separate modules, for liquid chromatography has been constructed. The different parts of the automatic machine are an auto sampler, an auto dispenser, a selector valve with eight channels, a heater/cooler, a mixing chamber and a pressure air driven injector valve. The process was controlled by a PC from an easily changeable run protocol. The system was applied to analysis of primary amines. The analysis was performed as a pre-column derivatization reaction of the amines and separation by isocratic reversed-phase HPLC with fluorescent detection. Reproducibility and analytical precision have been studied. Comparison between automatically and manually made derivatization reaction and injection was also made. The automatic system was easy to handle, cost-effective and gave good reproducibility.