Selectivity enhancement for trans-2-(2,3-anthracenedicarboximido)-cyclohexane-derived diastereomers in HPLC by using an ordered organic stationary phase.

2-(2,3-anthracenedicarboximido)cyclohexane derivatives (AC) have been known as the evolutionary diastereomerizing reagents for enantiomer discrimination in HPLC with ODS. However, a substantial separation of diastereomers can be observed only at lower temperatures, such as -40 degrees C. Therefore, in this work, poly(octadecyl acrylate)-grafted silica, ODAn was applied as an alternative stationary phase to ODS for the separation of AC-derived diastereomers. As a result, complete separation was achieved even under the conventional condition: for example, methanol as the mobile phase and 0 degrees C as the column temperature. An investigation on the temperature dependency of the selectivity demonstrated that ODAn shows a remarkable increase in selectivity at temperatures below 30 degrees C, which almost agreed with the peak-top temperature of the endothermic peak in a DSC thermogram for ODA35 immersed in a mobile phase. The better separation would be derived from a highly ordered structure of ODAn and a carbonyl-pi interaction with AC-derived diastereomers.     

Reversed-phase HPLC separation of the enantiomers of denopamine after derivatization with GITC chiral reagent

Summary A reversed-phase high-performance liquid chromatographic (HPLC) method was developed to determine the optical purity of denopamine, which is a new cardiotonic agent having an asymmetric carbon in a molecule. The enantiomers were converted to diastereomeric thiourea derivatives using 2,3,4,6-Tetra-O-acetyl--D-glucopyranosyl isothiocyanate (GITC) reagent. Separation of the enantiomers of denopamine as diastereomers was successfully achieved by reversed-phase HPLC within 10 min using an ODS column and UV detection. Derivatization of denopamine proceeded rapidly under the alkaline conditions and the ambient temperature. This method was applied to the determination of the optical purity of denopamine drug substances and those in tablets. The favorable UV absorption of the derivatives enabled the optical antipode to be determined down to the 0.2% level.     

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.

Determination of biogenic amines in food by automated pre-column derivatization with 2-naphthyloxycarbonyl chloride (NOC-CI)

Summary The fluorogenic reagent 2-naphthyloxycarbonyl chloride (NOC-Cl) has been used for the automated precolumn derivatization of biogenic amines (BAs) at ambient followed by liquid-chromatographic separation of the derivatives formed. For optimized derivatization samples in 0.5 M borate buffer (pH 9.0) were derivatized with 5 mM NOC-Cl in acetonitrile (MeCN) for 3 minutes. Excess of reagent was scavenged by addition of 20 mM glycine in water. For HPLC a Superspher^? RP-18e column and gradient elution using 0.1 M sodium acetate buffer (pH 4.4) and MeCN were used. The NOC-derivatives were detected by fluorescence at an emission wavelength of 335 nm at an excitation wavelength of 274 nm. This method allows the detection of BAs (2-phenylethylamine, putrescine, histamine, cadaverine, tyramine, spermidine, spermine) found in food and beverages (fruit juices, wines, various vinegars, fermented cabbage juice, and salmon). Detection limit of BAs are approximately 49–113 μg kg⁻¹ with the exception of histamine (747 μg kg⁻¹) (injected amounts: 18–41 pg histamine 267 pg), at a signalto-noise ratio of 3:1. The limits of determination are approximately 82–189 μg kg⁻¹ (histamine 1245 μg kg⁻¹) at a signal-to-noise ratio of 5:1. The correlation coefficients of linearity are 0.9910–0.9976. Recoveries from different matrices range from 65 to 109%, depending on the sample investigated. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996.     

对甲氧基苯磺酰氯柱前衍生生物胺的高效液相色谱分离

采用对甲氧基苯磺酰氯作为柱前衍生试剂,RP-HPLC为分析模式,建立了一种新的生物胺衍生化方法,并对葡萄酒中生物胺含量进行检测。通过液质联用对产物进行定性,研究并确定了最适衍生化条件:衍生温度50℃,缓冲液pH 9.0,衍生时间15 min。实验建立了7种生物胺的HPLC分离方法:Beckman ODS柱;流动相A为10 mmol/L的NH4Ac溶液(pH 6.37),B相为乙腈;采用梯度洗脱;流速1 mL/min;检测波长240 nm,室温。     

Determination of biogenic amines by RPHPLC with fluorescent detection after derivatization with 2-(9-carbazole)ethyl chloroformate (CEOC)

Summary The use of 2-(9-carbazole)ethyl chloroformate (CEOC) for pre-column derivatization of biogenic amines (BA) has been tested for the first time. The reagent reacts completely with BA within 3 min at ambient temperature in acetonitrile solution to form stable derivatives that are readily analyzed by reversed-phase HPLC. Study of the derivatization conditions revealed derivatization yields to be excellent in borate buffer over the pH range 9.0–10.0. Maximum yields were obtained by use of a three- to fourfold molar excess of reagent. The reaction is extremely tolerant of common buffer salts, no decrease in reaction yield is discernible in well-buffered samples. The emission maximum for the CEOC-derivatives is 360 nm (λ _ex = 293 nm). All the derivatives fluoresced strongly and direct injection of the reaction mixture was possible, with no significant disturbance from the major fluorescent reagent degradation by-products, 2-(9-carbazole)ethanol (CEOC-OH) and bis-(2-(9-carbazole)ethyl) carbonate (CEOC)₂. Separation of the derivatized BA by high-performance liquid chromatography with gradient elution was tested on a Hypersil BDS C18 column. Excellent response linearity was observed over the concentration range from 0.25 to 94.6 μmol L⁻¹ for the labeled BA. Detection limits were 117–840 fmol at a signal-to-noise ratio of 3∶1. Analysis of BA in a shrimp sauce extract was conducted to demonstrate the applicability of the technique to real sample matrixes; results were satisfactory.     

HPLC with fluorescence detection of urinary phenol, cresols and xylenols using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a fluorescence labeling reagent.

A simple and sensitive HPLC method for the determination of phenolic compounds, i.e., phenol (Phe), cresols (Cres) and xylenols (Xyls), was developed. After a pre-column fluorescence derivatization of these compounds with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) at 60 degrees C for 30 min, 11 DIB derivatives were successfully separated within 50 min with an ODS column using CH3CN-H2O-CH3OH (25 + 22 + 53, v/v) as the eluent. The detection limits of DIB derivatives at a signal-to-noise ratio of 3 ranged from 0.15 to 1.09 microM (0.2-1.6 pmol per 20 microliters). The precision of the proposed method for both within- and between-day assays of free and total phenol related compounds was satisfactory (RSD < 9.5%). By the proposed method, Phe and p-Cre could be detected in normal urine samples, and the calculated concentrations of free Phe and p-Cre in unhydrolysed urine samples were 1.5 +/- 1.3 and 23.9 +/- 24.3 microM and those of total Phe and p-Cre in hydrolysed urine samples were 87.3 +/- 81.2 and 200.7 +/- 195.4 microM (n = 21), respectively.     

R(-)-4-(3-Isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole, a fluorescent chiral tagging reagent: sensitive resolution of chiral amines and amino acids by reversed-phase liquid chromatography

The usefulness of R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS], a fluorescent chiral tagging reagent, for the determination of racemic amines and amino acids, was studied. The reagent reacted with beta-blockers selected as representative secondary amines to produce corresponding fluorescent diastereomers (excitation at 460 nm and emission at 550 nm). The yields of the derivatization reaction were dependent on the stereostructure arround the NH group in beta-blockers. The resulting diastereomers were completely separated with single chromatographic run using linear gradient elutions by reversed-phase chromatography. R(-)-DBD-PyNCS was also applied to the determination of DL-amino acid, considered to be one of the primary amines, in human urine and foodstuffs. DL-amino acids tested equally reacted with the reagent, and the thiocarbamoyl derivatives were separated with an ODS column. The epimerization during the derivatization reaction was negligible judging from the resolution of opposite diastereomers on the chromatogram. The occurence of D-amino acids (D-Ala, D-Ser, D-Asp and/or D-Glu) was identified in the samples tested. The structures and the purities were elucidated with on-line HPLC-MS. The chiral reagent possessing an isothiocyanate group (-NCS) in the structure seems to be applicable to continuous sequential analysis of peptides containing D-amino acids. The thiocarbamoyl derivatives obtained from the reaction with DL-amino acids were converted to thiohydantoins via thiazolinones in acidic medium. The thiohydantoins produced from acidic, basic, neutral, hydroxyl and aromatic amino acids were completely separated with isocratic elutions using acidic mobile phase containing 0.1% TFA. The separations were sufficient for the identification of DL-amino acid in peptide sequences. Although the epimerization during the conversion reaction to thiohydantoins was not avoidable, the descrimination of D- and L-configuration was demonstrated with some commercially available peptides such as beta-lipotropin and [D-Ala2]-deltorphin II. The Edman degaradation method using R(-)-DBD-PyNCS was also adopted to autoanlaysis by gas-phase sequencer. The separation and the detection (UV 254 nm) conditions of the derivatives were used without any change from those for the Edman degradation method using PITC as the tagging reagent. The three DL-amino acid residues (Tyr, Ala and Gly) in [L-Ala2]-leucine-enkephalin and [D-Ala2]-leucine-enkephalin were perfectly identidied with the autoanalysis.     

Determination of aliphatic amines by high-performance liquid chromatography-amperometric detection after derivatization with naphthalene-2,3-dicarboxaldehyde

A simple and sensitive liquid chromatographic method has been developed for the determination of low molecular weight aliphatic amines after their pre-column derivatization with naphthalene-2,3-dicarboxaldehyde (NDA). Derivatization conditions, including the NDA concentration, reaction pH and reaction time have been investigated for method optimization. The chromatographic separation of five amines was performed on ABZ PLUS column using mobile phase of methanol-water (80:20, v/v) at a flow rate of 0.2 mL min⁻¹. The detection was carried out with a 6 mm glassy carbon electrode at the applied potential of 0.7 V versus Ag/AgCl reference electrode. The detection limits were between 23.3 and 34.4 nmol L⁻¹ of amines with a sample injection volume of 2 μL. The present method was applied for the determination of aliphatic amines in lake water. The recovery ranged 52.2-127.9%. The RSD in analytes retention time was less than 0.3% and 2.4% for intra- and inter-day analyses, respectively. The RSD in peak area was below 5.8% for both intra-day and inter-day analyses. The total analysis was completed within 20 min.     

Determination of amines using 2-(11H-benzo[a]carbazol-11-yl) ethyl chloroformate (BCEC-Cl) as labeling reagent by HPLC with fluorescence detection and identification with APCI/MS

A pre-column derivatization method for the sensitive determination of amines using a labeling reagent 2-(11H-benzo[a]-carbazol-11-yl) ethyl chloroformate (BCEC-Cl) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by LC/APCI/MS in positive-ion mode. The chromophore of 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC-Cl) reagent was replaced by 2-(11H-benzo[a]-carbazol-11-yl) ethyl functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEC-Cl. BCEC-Cl could easily and quickly label amines. Derivatives were stable enough to be efficiently analyzed by HPLC and showed an intense protonated molecular ion corresponding m/z [M + H]⁺ under APCI/MS in positive-ion mode. The collision-induced dissociation of the protonated molecular ion formed characteristic fragment ions at m/z 261.8 and m/z 243.8 corresponding to the cleavages of CH₂O-CO and CH₂-OCO bonds. Studies on derivatization demonstrated excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% were observed with three- to four-fold molar reagent excess. In addition, the detection responses for BCEC-derivatives were compared to those obtained using 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC-Cl) and 9-fluorenyl methylchloroformate (FMOC-Cl) as labeling reagents. The ratios I_BCEC/I_BCEOC = 1.94-2.17 and I_BCEC/I_FMOC = 1.04-2.19 for fluorescent (FL) responses (here, I was relative fluorescence intensity). Separation of the derivatized amines had been optimized on reversed-phase Eclipse XDB-C₈ column. Detection limits calculated from 0.50 pmol injection, at a signal-to-noise ratio of 3, were 1.77-14.4 fmol. The relative standard deviations for within-day determination (n = 11) were 1.84-2.89% for the tested amines. The mean intra- and inter-assay precision for all amines levels were <3.64% and 2.52%, respectively. The mean recoveries ranged from 96.6% to 107.1% with their standard deviations in the range of 0.8-2.7. Excellent linear responses were observed with coefficients of >0.9996.     

New chiral derivatization reagent for the resolution of amino acids as diastereomers by TLC and HPLC

Summary The applicability of a new chiral reagent to the resolution of amino acid enantiomers has been investigated. The new reagent, S(-)-N-1-(2-naphthylsulphonyl)-2-pyrrolidinecarbonyl chloride (NSP-C1), was synthesized by the chlorination of S(-)-N-1-(2-naphthylsulphonyl)-2-pyrrolidinecarboxylic acid which was prepared by the reaction of 2-naphthalene sulphonyl chloride with L-proline. Derivatization of the amino acids proceeds rapidly at ambient temperature and no racemization takes place during the reaction. The resolution of the diastereomeric amides was performed by TLC and normal phase HPLC. Complete resolutions were observed for the enantiomers of all amino acids examined except cysteine, cystine and histidine. The favourable UV absorption of the derivatives enabled the optical antipode to be determined down to the 0.1% level.     

Determination of primary biogenic amines in various food products using isocratic HPLC with 3-(4-chlorobenzoyl)-quinoline-2-carboxaldehyde as a new precolumn fluorogenic reagent

A simple HPLC approach has been successfully established for the sensitive determination of six biogenic amines (BAs) in food samples. The method involves derivatization with 3-(4-chlorobenzoyl)-quinoline-2-carboxaldehyde newly synthesized as a new fluorogenic reagent followed by LC isocratic elution mode. The optimization of both derivatization and separation conditions is carefully studied. Related analyses of the eluted compounds, in the presence of MeOH/THF/H(2)O (78:2.5:19.5 v/v/v) as a mobile phase containing 8 mM, pH 6.0 phosphate buffer solution, have been carried out on a C(18) column. The LOD (S/N = 3) of 2.5 nM, RSD value from 1.0 to 5.1% in peak areas, and good response linearity (R(2) >0.9936) are provided with fluorescence detection at lambda(ex)/lambda(em) = 480/545 nm. Obviously, recovery ranging from 95 to 107% in this method demonstrates its accuracy for determination of histamine, tyramine, 2-phenylethylamine, putrescine, cadaverine, and spermidine in the storage fish sample. Thus, the present method could be developed to monitor BAs in fish, cucumber, and spinach samples.     

Non-aqueous capillary electrophoretic separation of enantiomeric amines with (-)-2,3:4,6-di-O-isopropylidene-2-keto-L-gulonic acid as chiral counter ion

(-)-2,3:4,6-Di-O-isopropylidene-2-keto-L-gulonic acid [(-)-DIKGA] has been introduced as a chiral counter ion in non-aqueous capillary electrophoresis. High enantioresolutions (R(s)> or =3) were obtained for amines, e.g., pronethalol, labetalol and bambuterol. Methanol containing NaOH and (-)-DIKGA was used as the background electrolyte. The counter ion concentration and the nature of the injection medium were found to affect the chiral separation. Covalent coating of the fused-silica capillary reduced the electro-osmotic flow resulting in improved enantioresolutions.     

Chiral NMR discrimination of secondary amines using (18-crown-6)-2,3,11,12-tetracarboxylic acid

[reaction: see text] Enantiomeric discrimination is observed in the (1)H NMR spectra of chiral secondary amines in the presence of (R)-(+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid. Secondary amines are protonated by one of the carboxylic acid groups of the crown ether to produce the corresponding ammonium and carboxylate ions. The secondary ammonium ion likely forms two hydrogen bonds to crown ether oxygen atoms and an ion pair with the carboxylate anion.     

(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.     

Quantitative determination of memantine in human plasma by GC using negative ion chemical ionization MS detection after derivatization with a new reagent, o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride

A method is presented for the quantitative determination of memantine in plasma by use of the derivatization reagent o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride. Memantine can be quantitatively analyzed down to 49?pg per mL of plasma using a 250?μL sample and negative ion chemical ionisation mass spectrometry (GC-NICI-MS). Plasma samples were made alkaline with carbonate buffer and extracted with n-hexane. The extracts were treated with reagent solution for 20?min, concentrated, and submitted to GC-NICI-MS. The method is rapid because extraction and derivatization occur in one single step. Amantadine is used as an internal standard. The utility and robustness of the assay is demonstrated by giving data on specificity, linearity, accuracy and precision, benchtop stability, freeze-thaw stability, autosampler stability, aliquot analysis, and prospective analytical batch size accuracy.

Determination of (2,3-epoxypropyl)trimethylammonium chloride, (3-chloro-2-hydroxypropyl)trimethylammonium chloride, and (2,3-dihydroxypropyl)trimethylammonium chloride by capillary electrophoresis

Capillary electrophoresis (CE) is a powerful technique to determine (2,3-epoxypropyl)trimethylammonium chloride, (3-chloro-2-hydroxypropyl)trimethylammonium chloride and their hydrolysis product (2,3-dihydroxypropyl)trimethylammonium chloride in complex matrices. The separation was performed in a 180 mM copper(II) sulphate-4 mM formic acid buffer at pH 3. The UV inactive target compounds were detected by indirect UV detection at 215 nm with a reference wavelength of 300 nm--copper(II) being the chromophor. The results were compared to data acquired by ion-pair HPLC with perchlorate as ion pair former in a 1 M aqueous sodium perchlorate solution in 0.2 mM phosphoric acid (pH 3.4) on a RP 18 column and refractive index detection as reference method. Results of both methods are in good agreement.     

Sensitive determination of biogenic amines by capillary electrophoresis with a new fluorogenic reagent 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde

An effective approach was proposed to the derivatization of seven biogenic amines using 3-(4-fluorobenzoyl)-2-quinolinecarboxaldehyde (FBQCA) as a fluorogenic reagent. The sensitive determinations of these derivatives were achieved by micellar electrokinetic capillary chromatography (MEKC) with laser-induced fluorescence (LIF) detection. The derivatization and electrophoretic conditions have been optimized. A running buffer was composed of mixtures of 25 mM pH 9.5 boric acid, 25 mM SDS, and 27% ACN. At 25 °C and 22.5 kV, the baseline separation of the derivatives was accomplished in 13 min. The detection limit (S/N = 3) was found as low as 0.4 nM. The proposed method was validated by the linearity of two orders magnitude and correlation coefficient in the range 0.9969–0.9998. Also, the procedure was successfully applied to the determination of biogenic amines in soy sauce, fish and wine samples.     

Enantiomeric discrimination : II. Incorporation of chiral amines platinum complexes. Crystal and molecular structure of (chloro)-(methyl)((+)-2S,3S-O-isopropylidene-2,3-dihydroxy-1,4-bis-(diphenylphosphino)platinum(II)

Diastereomeric platinum complexes of formula [Pt(chelate)CH₃L]ClO₄ have been prepared, where (chelate) is disphosphineligand, either 2S ,3S-bis(diphenylphosphino)butane, or (+)-2S, 3S-O-isopropylidene-2,3-dihydroxy-1,4-bis-(diphenylphosphino)butane)platinum(II) has shown that the compound crystallises in space group P2₁2₁2₁, with cell dimensions a 16.282(4), b 16.995(4) and c 10.973(2) Å. Full-matrix least-squares refinement on aF, using 2504 observations and 170 variables, converged at R  0.0605. The coordination at the Pt atom is square planar, with Pt—P distances of 2.322(4) and 2.233(4) Å. Pt—Cl and Pt—C were 2.323(5) and 2.17(1) Å respectively. The seven-membered chelate ring adopts a twisted chair conformation. Enantiomeric discrimation in these Pt-amine systems is discussed with reference to the solid state structure of the complex.