Mumio Ishibashi

Fluorescent chiral derivatization reagents for carboxylic acid enantiomers in high-performance liquid chromatography

Six chiral derivatization reagents, viz., (+)-4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (D-DBD-APy), (–)-4-(N,N-dimethylaminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (L-DBD-APy), (+)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (D-NBD-APy), (–)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (L-NBD-APy), (+)-4-(aminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (D-ABD-APy) and (–)-4-(aminosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (L-ABD-APy), have been synthesized to permit separation of carboxylic acid enantiomers by high-performance liquid chromatography (HPLC). The reagents react with carboxylic acids at room temperature in the presence of activation agents (2,2′-dipyridyl disulfide and triphenylphosphine). The maximum emission wavelengths of the diastereomeric amide derivatives formed from D-naproxen (Nap) with D-DBD-APy, D-ABD-APy and D-NBD-APy were approximately 580 nm (excitation at 470 nm), 585 nm (excitation at 470 nm) and 540 nm (excitation at 470 nm), respectively. The emission wavelengths of the derivatives shifted towards the blue, and the fluorescence intensities increased with increasing acetonitrile concentration in the medium. The fluorescence intensities of three derivatives at pH 2–11 were higher in neutral than in acidic and alkaline solutions. The diastereomers derived from anti-inflammatory drugs and N-acetylamino acids were efficiently resolved by a reversed-phase column (5 µ Inertsil ODS-2) with a water-acetonitrile mixture as mobile phase. The detection limits (S/N = 2) of DBD-APy-Nap, NBD-APy-Nap and ABD-APy-Nap on HPLC chromatograms were 10, 15 and 30 fmol, respectively.

4-(Aminosulphonyl)-2,1,3-benzoxadiazole derivatives as pre-column fluorogenic tagging reagents for carboxylic acids in high-performance liquid chromatography

Three 4-(aminosulphonyl)-2,1,3-benzoxadiazole amine derivatives (ABD-amines), 4-(aminosulphonyl)-7-(1-piperazinyl)-2,1,3-benzoxadiazole (ABD-PZ), 4-(aminosulphonyl)-7-(5-aminopentylamino)-2,1,3-benzoxadiazole (ABD-AP) and 4-(aminosulphonyl)-7-N-(2-aminoethylamino)-2,1,3-benzoxadiazole (ABD-AE), have been synthesized as fluorogenic reagents for carboxylic acids. All three reacted with fatty acids at room temperature (20–30 °C) in the presence of diethyl phosphorocyanidate (DEPC) to produce fluorescent adducts. The maximum wavelengths of arachidic acid labelled with ABD-PZ, ABD-AP and ABD-AE were 580 nm (excitation, 440 nm), 570 nm (excitation, 438 nm) and 573 nm (excitation, 429 nm), respectively. The rate of derivatization of ABD-PZ with arachidic acid was the slowest compared with those of ABD-AP and ABD-AE. However, the fluorescence intensity with ABD-PZ after a reaction time of 75 min was the highest among the three benzoxadiazoles. The ABD-amine reagents are applicable to the determination of carboxylic acids. Eight saturated free fatty acids derivatized with ABD-amines were completely separated with a linear elution gradient by use of a reversed-phase high-performance liquid chromatography column. The detection limits (signal-to-noise ratio = 3) that could be obtained for eight fatty acids were in the 10–50 fmol range.

Resolution of carboxylic acid enantiomers by high-performance liquid chromatography with peroxyoxalate chemiluminescence detection

Abstract The peroxyoxalate chemiluminescence (CL) detection of carboxylic acid enantiomers, combined with high-performance liquid chromatography (HPLC), is described. The CL reaction is influenced by various factors (e.g., cluent pH, type of aryl oxalate, relative concentrations of aryl oxalate and hydrogen peroxide and reaction time). Good linearity between CL intensity and injected amounts (5 fmol-5 pmol) of authentic-derivatives, DBD-Apy-Nap and ABD-APy-Nap, which were synthesized by the reaction with naproxen (Nap) with (+)-4-(N,N-dimethylaminosulphonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole [(+)-DBD-APy] and (+)-4-(aminosulphonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole [(+)-ABD-APy)], were obtained with the proposed procedures. The reproducibility of the CL intensity during 6 h was also excellent, and no peak decrements were observed. The detection limits (signal-to-noise ratio = 2) of authentic DBD-APy-Nap, ABD-APy-Nap and NBD-APy-Nap {synthesized from naproxen and (+)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole [(+)-NBD-APyl]} with the bis[4-nitro-2-(3,6,9-trioxadecyloxy)phenyl] oxalate (TDPO)-hydrogen peroxide (H2O2) system after separation by HPLC were 0.49, 1.9, and 15 fmol, respectively, whereas those with bis(2,4,6-trichlorophenyl_ oxalate (TCPO)-H2O2 were 0.74, 2.8 and 29 fmol, respectively. Some carboxylic acid enantiomers were converted on reaction with (+)-DBD-APy into the corresponding fluorescent diastereomers after 2 h at room temperature in the presence of 2,2′-dipyridyl disulphide and triphenylphosphine, activating agents for carboxylic acids. The diastereomers derived from each pair of enantiomers of anti-inflammatory drugs and N-acetylamino acids were efficiently resolved by reversed-phase chromatography with on ODS column and a 0.1 M imidazole-NO3 (pH 7.0)-acetonitrile mixture as the mobile phase. The applicability of the proposed procedure was also evaluated for the detection of racemic ibuprofen (anti-inflammatory drug) added to rat plasma and human urine.

Resolution of carboxylic acid enantiomers by high-performance liquid chromatography with highly sensitive laser-induced fluorescence detection

Abstract Optical resolution of carboxylic acid enantiomers by high-performance liquid chromatography with laser-induced fluorescence detection after chiral derivatization was investigated. Both laser power and time constants influence the detection limit of the fluorophore. The minimum detectable levels of three derivatives of naproxen [4-(aminosulphonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzodioxazole, 4-(N,N-dimethylaminosulphonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole and 4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzodiazole derivatives] were in the femtomole-attomole ranges, and the lowest detection limit was obtained with the last derivative.

4-(N,N-dimethylaminosulfonyl)-7-(2-chloroformylpyrrolidin-1-yl)-2,1,3-benzoxadiazole: novel fluorescent chiral derivatization reagents for the resolution of alcohol enantiomers by high-performance liquid chromatography

Optically active derivatization reagents, 4-(N,N-dimethylaminosulfonyl)-7-(2-chloroformylpyrrolidin-1-yl)-2,1,3-benzoxadiazole [(R)-(+)-DBD-Pro-COCI and (S)-(–)-DBD-Pro-COCI], were synthesized to permit the separation of alcohol enantiomers by high-performance liquid chromatography. The reagents react with hydroxyl groups in the presence of pyridine, which functions as a catalyst and reacts with hydrogen chloride. The maximum excitation and emission wavelengths of the diastereomers derived from the alcohols and the reagents were approximately 450 and 560 nm, respectively. The emission wavelengths of the derivatives shifted slightly towards the blue with increasing acetonitrile concentration in the medium; however, the excitation wavelengths remained constant. The diastereomers derived from some aliphatic alcohols were efficiently resolved by normal-phase chromatography with hexane-ethyl acetate as the eluent. Incomplete separation was realized with a reversed-phase column with water-acetonitrile as the eluent. When (R)-(+)-DBD-Pro-COCI was used as the derivatization reagent, alcohols corresponding to the R-configuration were eluted faster than those corresponding to the S-configuration. As expected, the elution order of the alcohols was reversed when the diastereomers were prepared with (S)-(-)-DBD-Pro-COCI. The Rs values of diastereomers derived from hydrophobic alcohols are larger than those from hydrophilic alcohols.

Further studies on the resolution of carboxylic acid enantiomers by liquid chromatography with fluorescence and laser-induced fluorescence detection

Abstract The optical purities of chiral derivatization reagents 4-( N,N -dimethylaminosulphonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (DBD-APys), 4-(aminosulphonyl)-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (ABD-APys) and 4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (NBD-APys) were determined by chromatographic separation with an Ultron ES-PHCD (β-cyclodextrin phenylcarbamate derivative) chiral stationary phase column. With the exception of (+)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole ( d -NBD-APy), which was found to have a purity of 99.5%, the enantiomers were determined to be more than 99.8% pure. The rate of the derivatization reaction for each enantiomer of DBD-APy at room temperature in the presence of the carboxylic acid reaction promoters 2,2′-dipyridyl disulphide (DPDS) and triphenylphosphine (TPP), was essentially the same for each enantiomer of naproxen. The reaction rates of 50°C were comparable at room temperature. When the derivatizations with DBD-APy and NBD-APy were carried out in the presence of diethyl phosphorocyanidate (DEPC) instead of DPDS and TPP quantitative derivatizations were not obtained even after a prolonged reaction time. The diastereomers derived from anti-inflammatory drups and N -acetylamino acid enantiomers were efficiently resolved by both normal-phase ( R s = 2.58–7.60) and reversed-phase ( R s = 1.62–6.96) chromatography. The resolution of carboxylic acid enantiomers by liquid chromatography with laser-induced fluorescence (LIF) detection was also investigated. The minimum detectable levels by reversed-phase chromatography with an argon ion laser at 488 nm were 29 fmol of ABD-APy-Nap, 11 fmol of DBD-APy and 2.9 fmol of NBD-APy. Ibuprofen enantiomers added to rat plasma and human urine were completely separated by an ODS column with water-acetonitrile as eluent without interference from endogenous compounds. Sensitive detection was achieved with a commercially available LIF detector. Negligible mutagenicity of the reagents was observed by the Ames test using Salmonella typhimurium strains TA100 and TA98

An information theory of chromatography : II. Application of FUMI to the optimization of overlapped chromatograms

Abstract The fu nction of m utual i nformation ( FUMI ) was used as a quality criterion in the optimization of the injection interval in overlapped chromatograms. FUMI , which represents the amount of Shannons mutual information involved in the chromatograms, was calculated for overlapped chromatograms with various injection intervals. The most efficient peak separation was selected with respect to the amount of the mutual information and the observation time. Overlapped chromatograms containing negative peaks were also optimized successfully.

An information theory of chromatography : I. Evaluation of analytical systems by means of FUMI

Abstract The application of the function of mutual information (FUMI) to the logical evaluation of methods for chromatographic quantitation is described. FUMI provides the Shannon mutual information of overlapped peaks with various resolutions. Hence an analytical method can be evaluated logically based on both the amount of information transmitted by overlapped peaks and the observation period of chromatography. As an example of the evaluation, a previously proposed chromatographic analysis consisting in a rapid but incomplete separation of naphthalene and diphenyl, and peak-deconvolution based on the Kalman filter, is considered. The “best” chromatogram that can transmit the maximal mutual information in unit time is given.

Use of a function of mutual information for optimization of mobile-phase composition in reversed-phase liquid chromatography

Abstract Optimization of the chromatographic separation of antipyretic mixtures is described. A function of mutual information, FUMI, based on information theory and the Kalman filter, is used as the criterion. The separation patterns are predicted on the basis of the model that relates the logarithm of the capacity factor, log (k′), of a solute to the volume fraction of the organic modifier in the mobile phase by a second-order equation. Overlapped chromatograms and single chromatograms were simulated and evaluated by the separation time and the FUMI. Optimal conditions were selected two-dimensionally with respect to mobile-phase composition and injection interval and then confirmed by experiments.

Totally automated robotic system for liquid chromatographic analysis of solid dosage formulations with the aid of a reduced Kalman filter

Abstract An automated system for the analysis of solid dosage formulations is described, comprised of a commercially available robotic system, an high-performance liquid chromatograph and microcomputers. By means of the robotic system, several sample solutions are simultaneously prepared from the solid formulations and then injected successively into the chromatograph at regular, excessively short intervals. The overlapped peaks in the resulting complex chromatogram are resolved and quantitatively analyzed by a reduced Kalman filter. A content uniformity test was performed for twenty commercial diazepam tablets; the total analysis time involving the tablet disintegration and chromatographic determination was significantly reduced. The use of this robotic system has been shown to result in a great improvement in the throughput of the total analytical procedures without adverse effects on the accuracy and precision of the chromatographic system.