Pre-column Derivatization Reagent Research Articles

Marfey′s Reagent: Past, Present, and Future Uses of 1‐Fluoro‐2,4‐dinitrophenyl‐5‐L‐alanine Amide

AbstractFor Abstract see ChemInform Abstract in Full Text.

Residue analysis of spectinomycin in tissues of chicken and swine by HPLC

A reversed-phase HPLC method with ultraviolet detection using p-nitrophenyl hydrazine as a pre-column derivatizing reagent was investigated for the determination of the antibiotic spectinomycin (SPCM) in muscle, liver, kidney and fat of chicken and swine. SPCM was extracted from samples with 10% trichloroacetic acid saturated with EDTA-2Na, and then cleaned up with coupled Sep-Pak Plus PS-2 cartridges. The detection limit of SPCM was 0.02 microgram (potency)/g. Recoveries of SPCM ranged from 77.4 to 97.4% for chicken tissues and from 74.5 to 91.8% for swine tissues. The present method was used for the analysis of chicken tissues after the 11th day of withdrawal (SPCM-medicated drinking water: 500 mg (potency)/L, for 7 days), and swine tissues after the 14th day of withdrawal (SPCM-medicated feed: 100 mg (potency)/kg, for 7 days). Results showed that SPCM concentrations were lower than the MRLs in all tissues.

Study on determination of iron, cobalt, nickel, copper, zinc and manganese in drinking water by solid-phase extraction and RP-HPLC with 2-(2-quinolinylazo)-5-diethylaminophenol as precolumn derivatizing reagent.

A new method for the determination of iron, cobalt, nickel, copper, zinc and manganese in drinking water by the reversed-phase high-performance liquid chromatography (RP-HPLC) with 2-(2-quinolinylazo)-5-diethylaminophenol (QADEAP) as precolumn derivatizing reagent was studied in this paper. The iron, cobalt, nickel, copper, zinc, and manganese ions react with QADEAP to form color chelates in the presence of cetyl trimethylammonium bromide (CTMAB) and acetic acid-sodium acetic buffer solution medium of pH 4.0. These chelates were enriched by solid-phase extraction with a Waters Nova-Pak C18 cartridge and eluted the retained chelates from the cartridge with tetrahydrofuran (THF). The enrichment factor of 100 was achieved. Then the chelates were separated on a Waters Nova-Pak C18 column (3.9 x 150 mm, 5 microm) by gradient elution with methanol (containing 0.2% of acetic acid and 0.1% of CTMAB) and 0.05 mol L(-1) acetic acid-sodium acetic buffer solution (containing 0.1% of CTMAB) (pH 4.0) as mobile phase at a flow rate of 0.5 ml min(-1), and monitored with a photodiode array detector from 450 approximately 700 nm. The detection limits (S/N = 3) of iron, cobalt, nickel, copper, zinc and manganese are 0.8, 1.1, 0.9, 1.1, 1.5 and 2.0 ng L(-1), respectively, in the original sample. This method can be applied to determination at the microg L(-1) level of iron, cobalt, nickel, copper, zinc and manganese in drinking water with good results.

A NEW FLUOROGENIC REAGENT FOR LABELLING CARBOXYLIC ACIDS IN HPLC

A new fluorescent reagent for carboxylic acids, N-(4-bromomethyl-7-hydroxy-2-oxo-2H-6-chromenyl) bromoacetamide (Br-MAMC) was synthesized. The fluorescence properties of Br-MAMC were monitored for a series of polar and non-polar solvents. Its applicability as a precolumn derivatization reagent in high-performance liquid chromatography was examined using undecylenic acid as a model compound. The derivatization reaction was performed in acetone in the presence of potassium carbonate and the crown ether, 18-crown-6 at 30°C for 20 min. The reaction solution was subjected to a high-performance liquid chromatographic procedure on a reversed-phase ODS C-18 column (150×4.6 mm i.d., 5 μm) with a mobile phase of acetonitrile and water (75 : 25, v/v) at a flow rate of 1.0 mL/min. The derivative of undecylenic acid was detected fluorometrically; excitation and emission wavelengths were set at 345 and 435 nm, respectively. The detection limit of the method was determined to be 12.5 pg (signal to noise ratio = 3) of the derivatized undecylenic acid on column. The derivatives of butyric, hexanoic, octanoic, decanoic, and dodecanoic acid with Br-MAMC were also well resolved with a mobile phase of acetonitrile and water (60 : 40, v/v).

A NEW FLUOROGENIC REAGENT FOR LABELLING CARBOXYLIC ACIDS IN HPLC

A new fluorescent reagent for carboxylic acids, N-(4-bromomethyl-7-hydroxy-2-oxo-2H-6-chromenyl) bromoacetamide (Br-MAMC) was synthesized. The fluorescence properties of Br-MAMC were monitored for a series of polar and non-polar solvents. Its applicability as a precolumn derivatization reagent in high-performance liquid chromatography was examined using undecylenic acid as a model compound. The derivatization reaction was performed in acetone in the presence of potassium carbonate and the crown ether, 18-crown-6 at 30°C for 20 min. The reaction solution was subjected to a high-performance liquid chromatographic procedure on a reversed-phase ODS C-18 column (150×4.6 mm i.d., 5 μm) with a mobile phase of acetonitrile and water (75 : 25, v/v) at a flow rate of 1.0 mL/min. The derivative of undecylenic acid was detected fluorometrically; excitation and emission wavelengths were set at 345 and 435 nm, respectively. The detection limit of the method was determined to be 12.5 pg (signal to noise ratio = 3) of the derivatized undecylenic acid on column. The derivatives of butyric, hexanoic, octanoic, decanoic, and dodecanoic acid with Br-MAMC were also well resolved with a mobile phase of acetonitrile and water (60 : 40, v/v).

N-HYDROXYSUCCINIMIDYL-α-NAPHTHYLACETATE AS A DERIVATIZING REAGENT FOR AMINO ACIDS AND OLIGOPEPTIDES IN RP-HPLC

A new derivatizing reagent, N-hydroxysuccinimidyl-α-naphthylacetate (SINA), has been used as a precolumn derivatizing reagent for the separation and determination of amino acids (AAs) and oligopeptides with HPLC. The spectral properties of SINA derivatives with AAs and oligopeptides have been studied. The derivatization and separation conditions of SINA derivatives with most amino acids and some oligopeptides have been investigated. With spectrophotometric detection at 280 nm, the detection limits were in the range of 0.49–2.76 pmol; with fluorescence detection at λex/λem = 299/338 nm, the detection limits were in the range of 19–376 fmol when the ratio of signal to noise (S/N = 3) was 3.

Improved method for the measurement of large neutral amino acids in biological matrices

A high-performance liquid chromatographic method for measuring neutral amino acids in rat sera, brain tissues, and perfusates was developed by using o-phthalaldehyde sulfite as a pre-column derivatization reagent. With the present method, it was possible to separate the neutral amino acids within a single run in 25 min, while the acidic amino acids were eluted near or at the solvent front. The recovery was above 88.8% with a relative standard deviation (RSD) below 4.2%. The within- and between-day assay reproducibility for the determination of rat serum amino acids showed RSDs below 1.35 and 7.61%, respectively. In the present study, the neutral amino acids were assayed with high sensitivity, accuracy and good reproducibility in a relatively short time and on a small sample size.

N-hydroxysuccinimidyl fluorescein- O-acetate as a highly fluorescent derivatizing reagent for aliphatic amines in liquid chromatography

A sensitive fluorescent reagent, N-hydroxysuccinimidyl fluorescein- O-acetate (SIFA), has been developed as a precolumn derivatizing reagent for aliphatic amines in liquid chromatography (LC). SIFA was shown to be a successful reagent with advantages including high fluorescent quantum yield in the visible region, specificity to aliphatic amines, mild derivatization conditions and few by-products. Methylamine, ethylamine, ethylene diamine, isopropylamine, isobutylamine, n-butylamine, 1,4-butylene diamine and 1,5-amylene diamine were used to investigate the separation and derivatization conditions. In a pH 8.5 H 3BO 3–Na 2BO 7 buffer solution, SIFA reacted with amines at 45°C for 30 min to form stable derivatives. The derivatives could be separated on a C 18 column in 18 min with methanol–water (46/54, v/v) containing 10 mmol l −1 pH 5.40 citric acid–Na 2HPO 4 buffer as a mobile phase. The detection limits of these amines were in the range 85–520 amol (signal-to-noise ratio=3), which were better than those using the other derivatizing reagents for amine analysis by LC.

Chemiluminescence determination of catecholamines in human blood plasma using 1,2-bis(3-chlorophenyl)ethylenediamine as pre-column derivatizing reagent for liquid chromatography

A highly sensitive method for the determination of the human blood plasma catecholamines (norepinephrine, epinephrine and dopamine) is described which employs liquid chromatography (LC) with chemiluminescence detection. Solid-phase extraction using cation exchange cartridges was used for selective and quantitative isolation of the catecholamines and isoproterenol (an internal standard) from 20 μl of human plasma containing 0.1 pmol ml −1 isoproterenol. The amines and isoproterenol, derivatized with 1,2-bis(3-chlorophenyl)ethylene diamine, were separated on a reversed-phase LC column with isocratic elution using a mixture of imidazole buffer (120 mM, pH 5.8), methanol, acetonitrile (13 : 4 : 18, v/v/v). The eluate was detected by a post-column chemiluminescence reaction system using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate and hydrogen peroxide. The detection limits (a signal-to-noise ratio = 3) for each catecholamine are 40–120 amol in a 100 μl injection volume.

Determination of domoic acid in phytoplankton by high-performance liquid chromatography of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivative.

Domoic acid is the toxin that can cause amnesic shellfish poisoning. In this work, a new precolumn derivatization reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, was used to react with domoic acid to form a stable unsymmetrical urea derivative, which was readily analyzed by reversed-phase HPLC with fluorescence detection. The one-step derivatizing method is rapid and straightforward. The method was applied to detect domoic acid in phytoplankton using a 3.9 x 150 mm Nova-Pak, 4 microm, C(18) column.

Studies on 2-(2-thiazolylazo)-5-diethylaminophenol as a precolumn derivatizing reagent in the separation of platinum group metals by high performance liquid chromatography

Five platinum group metals, Pt(II), Ir(IV), Ru(III), Rh(III) and Os(IV) have been separated by high performance liquid chromatography (HPLC) using 2-(2-thiazolylazo)-5-diethylaminophenol (TADAP) as a precolumn derivatizing reagent. The whole analysis was completed on a C 18 column in 23 min at 574 nm, with the mobile phase of methanol–water (69.5:30.5, v:v) containing 4 mmol l −1 tetrabutylammonium bromide (TBA Br) and 10 mmol l −1 pH6.0 acetate buffer. The detection limits (S/N=3) of Pt(II), Ir(IV), Ru(III), Rh(III) and Os(IV) were 0.39, 9.74, 1.64, 0.29 and 1.29 ng ml −1, respectively. This method was rapid, sensitive and simple.

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.

N-(4-nitro-2,1,3-benzoxadiazoyl-7-yl)- N-methyl-2-aminoacetohydrazide (NBD-CO-Hz) as a precolumn fluorescent derivatization reagent for carboxylic acids in high-performance liquid chromatography

A new fluorescent reagent for carboxylic acids, N-(4-nitro-2,1,3-benzoxadiazoyl-7-yl)- N-methyl-2-aminoacetohydrazide (NBD-CO-Hz) was synthesized and its applicability as a precolumn derivatization reagent in high-performance liquid chromatography was examined. NBD-CO-Hz reacted with 2-arylpropionic acids (2-APAs), a group of non-steroidal antiinflammatory drugs (NSAIDs) in the presence of a condensing agent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and pyridine at room temperature for 2 h to give fluorescent adducts. The reaction solution was subjected to a reversed phase or a chiral stationary phase HPLC and the derivatives were detected fluorometrically at a wavelength of 530 nm with an exitation of 475 nm. The detection limits were in the fmol range on column.

Chemiluminescence Detection of Thiols by High-Performance Liquid Chromatography Using o-Phthalaldehyde and N-(4-Aminobutyl)-N-ethylisoluminol as Precolumn Derivatization Reagents

A method for the determination of thiols by high-performance liquid chromatography (HPLC) with chemiluminescence detection has been developed. The method is based on the precolumn derivatization of thiols with o-phthalaldehyde and N-(4-aminobutyl)-N-ethylisoluminol to form isoindole derivatives and the reversed-phase HPLC separation of the derivatives, followed by successive postcolumn chemiluminescence reactions with hydrogen peroxide and hematin. The limits of the detection of glutathione (GSH) and cysteine were 1.5 nM (15 fmol/injection) and 3.5 nM (35 fmol/injection), respectively. The calibration curve for GSH was linear in the range 5 - 1000 nM (1 - 200 pmol/200 μl). The method was applied to the determination of GSH in human blood. The recovery of GSH added to the blood at the concentration level of 500 μM was about 70%. The GSH concentrations in the blood of three healthy male persons were 502 - 770 μM.

N-Hydroxysuccinimidyl-3-indolylacetate As Pre-column Derivatizing Reagent to Separate and Determine Peptides and Their Hydrolysates by Reversed-Phase High Performance Liquid Chromatography

N-Hydroxysuccinimidyl-3-indolylacetate (SIIA) was firstly synthesized in a very simple route and used as a pre-column derivatizing reagent, which can react with amino compounds selectively under mild conditions. Its derivatives of peptides and their hydrolysates were separated and determined by reversed-phase high performance liquid chromatography (HPLC). In a mobile phase of methanol-water (17:83, v:v) containing 10 mmol/L citric acid-sodium phosphate buffer (pH 3.8), the derivatives of glutathione (GSSG), glycyl glycine (Gly-Gly), glutamic acid (Glu), cystine ((Cys)

Separation and simultaneous determination of cobalt, nickel and copper with 2-(4-methyl-2-quinolylazo)-5-diethylaminophenol by reversed-phase high-performance liquid chromatography

Performance of six quinolylazo compounds was studied as a pre-column derivatizing reagent for the separation of metal ions in the reversed-phase high-performance liquid chromatography. 2-(4-Methyl-2-quinolylazo)-5-diethylaminophenol (QADP) was suited for the determination of Co, Ni and Cu in the presence of a large amount of Fe. The detection limits under the optimized conditions [stationary phase: L-column ODS; mobile phase: acetone-water (62:38, v/v) containing 0.01 M NH 4SCN and 0.01 M 2-morpholinoethanesulfonic acid-NaOH (pH 6); injection volume: 10 μl] were 0.24 ng for Co, 0.22 for Ni and 0.53 for Cu. This method was successfully applied to the determination of these metal ions in a standard steel sample.

Determination of free amino acids in pig plasma by precolumn derivatization with 6- N-aminoquinolyl- N-hydroxysuccinimidyl carbamate and high-performance liquid chromatography

A high-performance liquid chromatographic method for measuring amino acids in pig plasma has been developed by using 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate as a precolumn derivatization reagent. With the method presented here it was possible to separate 24 amino acids in pig plasma within one single run in 45 min, while only 18 amino acids were properly separated with the original AccQ.Tag conditions used for analysis of hydrolysate samples.The recovery was above 90% for most amino acids, and the mean coefficient of variation of the retention times below 0.30%. The within- and between-assay reproducibility for the determination of plasma amino acids showed C.V.s below 2.2% and 4.5%, respectively, for most amino acids analyzed. In the present work, most of the plasma amino acids were assayed with high sensitivity, accuracy and good reproducibility in a relatively short time and on very small amounts of sample.

Visible diode laser-induced fluorescence detection of phenylacetic acid in plasma derivatized with Nile blue and using precolumn phase transfer catalysis.

This study reports the application of Nile blue (NB), a farred oxazine label, as a precolumn derivatization reagent for the measurement of free levels of phenylacetic acid (PAA) in plasma. The measurement of PAA in psychiatric populations is important because it provides a marker for 2-phenylethylamine (PEA), which has been implicated in the pathogenesis of schizophrenia and major depression. PAA was derivatized with NB through an amide linkage in the presence of 2-chloro-1-methylpyridinium iodide (carboxylic acid activator, CMP) and triethylamine (base catalyst, TEA), respectively. The formation of the NB-PAA derivative was confirmed using normal phase and reversed phase thin-layer chromatography, reversed phase liquid chromatography, and electrospray mass spectrometry. The formation of the NB-PAA derivative was optimized using a sequential single factor approach. The optimal conditions for the formation and chromatographic separation of the derivative were determined to be 8.0 nmol/mL NB, 390 nmol/mL CMP, 2 mumol/mL TEA, a reaction time of 45 min, and a reaction temperature of 25 degrees C. This derivatization scheme was performed in a phase transfer catalysis mode that enabled the simultaneous extraction, preconcentration, and derivatization of the analyte in a single step. The limit of derivatization of PAA was determined to be 1.0 x 10(-9) M in phosphate-buffered saline, a PAA-free matrix. This derivatization was limited not by the kinetics of the reaction but by the chromatographic separation of the derivative from a side reaction product. The method was used to estimate endogenous free levels of PAA in human plasma samples. The levels of PAA in four sources of plasma were determined to be within 30-70 ng/mL using the method of standard addition and reflected levels that have been reported in the literature. The limit of detection of the derivative was determined to be 7.33 x 10(-11) M using a laboratory-constructed HPLC-VDLIF detector.

HPLC determination of biologically active thiols using pre-column derivatisation with 5,5′-dithio-(bis-2-nitrobenzoic acid)

5,5′-Dithio-(bis-2-nitrobenzoic acid), Ellmans reagent (ESSE), is used as a pre-column derivatisation reagent for the determination of biologically active thiols by HPLC. d-penicillamine, N-acetyl- d-penicillamine, N-acetylcysteine, cysteine, captopril and thiomalic acid all give well resolved derivatives. The calibration graph and reproducibility (%R.S.D.±1.3%) for the analysis of glutathione indicates that the method could be used for quantitative analysis. ESSE is widely used as a reagent in thiol determinations by electronic spectroscopy via the detection of the Ellmans anion (ES −) generated without any prior separation procedures. However, there are considerable reservations over its use for the spectrophotometric determination of thiols because of the possibility of side reactions which generate another Ellmans based species (ESO 2 −). The assay described determines the thiol as a derivatised mixed disulphide (ESSR) and since speciation between the anion ES − and the oxidation product ESO 2 − occurs it enables the process of oxidation to be monitored simultaneously.