Construction of dual-chiral covalent organic frameworks for enantioselective separation.

Developing novel chiral stationary phases (CSPs) with versatility is of great importance in enantiomer separation. This study fabricated a dual-chiral covalent organic framework (PA-CA COF) via successive post-synthetic modifications. The chiral trans-1,2-cyclohexanediamine (CA) and (D)-penicillamine (PA) groups were periodically aligned within nanochannels of the COF, allowing selective recognition of enantiomers through intermolecular interactions. It can be a versatile high-performance liquid chromatography (HPLC) CSP for separating a wide range of enantiomers, including chiral pharmaceutical intermediates and chiral drugs. With separation performance comparable to commercial chiral columns and even greater versatility, the PA-CA COF@SiO2 column held promise for practical applications. Chiral separation results combined with molecular simulation indicated that the mixed mode of PA and CA resulted in the broad separation capability of PA-CA COF. The introduction of the dual-chiral COFs concept opens up a new avenue for chiral recognition and separation, holding great potential for practical enantiomer separation.

A new BODIPY-based long-wavelength fluorescent probe for chromatographic analysis of low-molecular-weight thiols.

A new long-wavelength fluorescent probe, 1,7-dimethyl-3,5-distyryl-8-phenyl-(4'-iodoacetamido)difluoroboradiaza-s-indacene (DMDSPAB-I), was designed and synthesized for thiol labeling in high-performance liquid chromatography (HPLC). The excitation and emission wavelengths of DMDSPAB-I are 620 and 630 nm, respectively, with a high fluorescence quantum yield of 0.557, which is advantageous in preventing interference of intrinsic fluorescence from complex biological matrices and enabling high sensitivity HPLC. Based on DMDSPAB-I, a reversed-phase HPLC method was developed for measuring low-molecular-weight thiols including glutathione, cysteine, homocysteine, N-acetylcysteine, cysteinylglycine, and penicillamine. After the specific reaction of DMDSPAB-I with thiols, baseline separation of all six stable derivatives was achieved through isocratic elution on a C18 column within 25 min, with the limits of detection (signal-to-noise ratio = 3) from 0.24 nmol L(-1) for glutathione to 0.72 nmol L(-1) for penicillamine. The proposed method was validated in part by measuring thiols in blood samples from mice, with recoveries of 95.3-104.3%.

Separation and determination of chiral composition in penicillamine tablets by capillary electrophoresis in a broad pH range.

A chiral capillary electrophoretic method with nearly full pH window was explored for the separation and determination of dl-penicillamine. A facile one-pot labeling technique was coupled in the method for introduction of chromophore and charge groups onto the analytes to facilitate the electromigration and sensitive detection. By using simply a cost-effective neutral ß-cyclodextrin as chiral selector, baseline separation of the dl-penicillamine was achieved from pH 2.0 to over pH 10. Quantification of standard d- and l-penicillamines was demonstrated by taking pH 4.5, 7.4, and 9.7 as the representatives of acidic, neutral, and basic conditions. The working curves were constructed between peak area and concentration, having linear ranges of 8.56-8.56 × 10(2) µg/mL for pH 4.5 and 8.56-1.71 × 10(3) µg/mL for pH 7.4 and 9.7, with correlation coefficients all better than 0.999. The limit of detection (S/N = 3) was 2.58 µg/mL in acidic and neutral conditions or 1.41 µg/mL in basic condition. The method was further validated by assaying the commercial penicillamine tablets, applicable to quantification of the effective enantiomer and the trace impurity of l-penicillamine at a content of down to 0.2, 0.6, and 2.0% for pH 9.7, 4.5, and 7.4, respectively. The recovery determined by spiking technique was in a range from 93.1 to 105 %. The method is easily extendable to the analysis of other chiral amines or amino acids.

Application of an unusual ninhydrin-based reaction for the indirect chiral resolution of D,L-penicillamine.

An unusual reaction involving ninhydrin and aminothiols was exploited to set an indirect method for the chiral recognition of stereoisomers of penicillamine. Separation of diastereoisomers was achieved on a C18 column in isocratic mode by using a mixture of propionic acid (pH 3.0)/acetonitrile/water (10:10:80, v/v/v) as a mobile phase. Diastereoisomers were detected by a fluorescence detector in fairly short times (about 7 min) and with a good resolution. The lowest detectable amount of toxic isomer of penicillamine (l-enantiomer) in samples of the d-enantiomer, was around 0.01%. The method was also suitable for the indirect chiral recognition of other aminothiols such as cysteine and cysteinylglycine.

A facile light-emitting-diode induced fluorescence detector coupled to an integrated microfluidic device for microchip electrophoresis.

In this paper, a compact and inexpensive light emitting diode induced fluorescence (LED-IF) detector with simplified optical configuration was developed and assembled in an integrated microfluidic device for microscale electrophoresis. The facile detector mainly consisted of an LED, a focusing pinhole, an emission filter and a photodiode, and was encapsulated in the upper layer of an aluminum alloy device with two layers. At the bottom layer, integrated circuit (IC) was assembled to manipulate the voltage for sample injection and separation, LED emission and signal amplifying. A high-power LED with fan-shaped heat sink was used as excitation source. The excitation light was focused by a 1.1mm diameter pinhole fabricated in a thin piece of silver foil, and the obtained sensitivity was about 3 times as high as that using electrode plate. Other important parameters including LED driven current, fluorescence collection angle and detection distance have also been investigated. Under optimal conditions, considerable high-response of 0.09 fmol and 0.18 fmol mass detection limits at 0.37 nL injection volume for sodium fluorescein (SF) and FITC was achieved, respectively. This device has been successfully employed to separate penicillamine (PA) enantiomers. Due to such significant features as low-cost, integration, miniaturization, and ease of commercialization, the presented microfluidic device may hold great promise for clinical diagnostics and bioanalytical applications.

Synthesis of (S)-naproxen-benzotriazole and its application as chiral derivatizing reagent for microwave-assisted synthesis and indirect high performance liquid chromatographic separation of diastereomers of penicillamine, cysteine and homocysteine.

(S)-Naproxen-benzotriazole was synthesized by the reaction of (S)-naproxen with 1H-benzotriazole using coupling reagent dicyclohexyl carbodiimide and 4-dimethylamino pyridine (DCC/DMAP). It was used as chiral derivatizing reagent for microwave irradiated synthesis of diastereomers of penicillamine, cysteine and homocysteine. The diastereomers were separated by reversed phase high performance liquid chromatography using gradient elution of triethylammonium phosphate (pH 3.5)-acetonitrile (30-65% within 30 min). The method was validated for accuracy, precision, and limit of detection.

Application of (S)-N-(4-Nitrophenoxycarbonyl) phenylalanine methoxyethyl ester as a chiral derivatizing reagent for reversed-phase high-performance liquid chromatographic separation of diastereomers of amino alcohols, non-protein amino acids, and PenA.

Indirect enantioresolution of 15 primary and secondary amino group containing compounds (amino alcohols, non-protein amino acids, PenA) was done using the reagent (S)-N-(4-Nitrophenoxycarbonyl) phenylalanine methoxyethyl ester [(S)-NIFE] by reversed-phase high-performance liquid chromatography. The diastereomeric derivatives were analyzed under reversed-phase conditions using linear gradient. The detection was at 205 nm and sharp peaks were obtained. The reagent used is comparatively economic than the other derivatizing reagents. Method validation was also done.

Analytical and preparative enantioseparation of DL-penicillamine and DL-cysteine by high-performance liquid chromatography on alpha-acid glycoprotein and beta-cyclodextrin columns using ninhydrin as a reversible tagging reagent.

Two sulfur-containing amino acids, DL-cysteine (Cys) and DL-penicillamine (PenA), were condensed with ninhydrin to form their spirothiazolidine derivatives. These were separated by HPLC using alpha-acid glycoprotein (AGP) and beta-cyclodextrin (beta-CD) columns. The resolution conditions were optimized and the results were compared. Since the method provided resolution greater than 2 it was also applied to preparative separation. After separation, each of them was detagged using Zn dust and 10% aqueous trifluoroacetic acid. For analytical purposes dinitrophenyl (DNP) derivatives of DL-Cys and DL-PenA were also prepared and were resolved on both the columns. The detection was carried out using photodiode array detection system at 231 nm. The limits of detection were found to be 0.01% and 0.004% for spirothiazolidine carboxylic acid and DNP derivatives, respectively.

Synthesis of succinimidyl-(S)-naproxen ester and its application for indirect enantioresolution of penicillamine by reversed-phase high-performance liquid chromatography.

Synthesis of N-succinimidyl-(S)-2-(6-methoxynaphth-2-yl) propionate was carried out by the reaction of (S)-naproxen with N-hydroxysuccinimide in the presence of dicyclohexyl carbodiimide. It was characterized and was used as a chiral derivatizing reagent, under mild conditions, to form diastereomers of dl-penicillamine which were resolved by reversed-phase high-performance liquid chromatography using triethyl ammonium phosphate buffer (pH 4.0, 5mM)-acetonitrile (linear gradient (30min) of acetonitrile from 30 to 70%). Excellent separation was achieved with gradient mobile phase. The detection limit was at pmol level.

Enantiomeric reversed-phase high-performance liquid chromatography resolution of D-/L-penicillamine after spirocyclization with ninhydrin and by using copper(II)-L-proline complex as a chiral selector in the mobile phase.

A new HPLC method by fluorescence or UV/vis absorbance detection has been developed for the separation and quantification of penicillamine stereoisomers after their spirocyclization with ninhydrin. The separation was performed on an achiral C18 column by isocratic elution with a copper(II)-l-proline complex as a chiral selector in the mobile phase. The method was able to detect traces of l-penicillamine in samples of d-penicillamine below 0.1% in fairly short times (about 16 min) with a good resolution (R(s)=1.31). On the whole, the method was found to be stable and useful in the quality control of the bulk material and formulations.

Direct enantiomeric TLC resolution of dl-penicillamine using (R)-mandelic acid and l-tartaric acid as chiral impregnating reagents and as chiral mobile phase additive.

DL-Penicillamine has been resolved into its enantiomers by normal-phase TLC using L-tartaric acid as chiral impregnating reagent as well as chiral mobile phase additive, while (R)-mandelic acid has been found to be successful as a chiral impregnating reagent. The solvent system acetonitrile-methanol-water (5:1:1, v/v) was found to be successful when L-tartaric acid was used as impregnating agent while the solvent combination acetonitrile-methanol-(0.5% l-tartaric acid in water, pH 5)-glacial acetic acid (7:1:1.1:0.7, v/v) was successful as mobile phase as it contained L-tartaric acid as the chiral additive. (R)-mandelic acid was successful as chiral impregnating reagent with ethyl acetate-methanol-water (3:1:1, v/v), as the mobile phase. The effects of concentration of chiral selectors, temperature and pH were examined on enantiomeric resolution. The spots were detected with iodine vapors and the detection limits were found to be 0.12 microg for each enantiomer of penicillamine with L-tartaric acid, under both the conditions, and 0.11 microg with (R)-mandelic acid.

Spectrophotometric determination of penicillamine and carbocisteine based on formation of metal complexes.

A simple spectrophotometric method was developed for the determination of penicillamine and carbocisteine. The method depends on complexation of penicillamine with Ni, Co and Pb ions in acetate buffer pH of 6.3, 6.5 and 5.3, respectively, and carbocisteine with Cu and Ni ions in borate buffer pH of 6.7; 1-70 microg/ml of these drugs could be determined by measuring the absorbance of each complex at its specific lambdamax. The results obtained are in good agreement with those obtained using the official methods. The proposed method was successfully applied for the determination of these compounds in their dosage forms. Also, the molar ratio and stability constant of the metal complexes were calculated and a proposal of the reaction pathway was postulated.

High-performance liquid chromatographic separation of penicillamine enantiomers labelled with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl] maleimide on a chiral stationary phase.

Penicillamine enantiomers derivatized with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM) were separated and determined by high-performance liquid chromatography. A fluorogenic reagent, DBPM easily reacted with D- or L-penicillamine to give each two kinds of strong fluorescent derivatives (D1-, D2-, L1- and L2-DBPM), which could be separated on a Pirkle-type chiral stationary phase using an eluent of 75% aqueous methanol solution containing 0.15 M CH3COONH4 and 0.05 M tetra-n-butylammonium bromide. Two of the peaks (D1- or L1-DBPM), having a shorter retention time than the others, had almost the same retention times (25 min for D1-DBPM and 25.7 min for L1-DBPM). The retention times of the peaks eluted later were 28 min and 31.6 min for D2- and L2-DBPM respectively. Linear calibration curves over the range of 2-50 pmol per injection were obtained for D- and L-penicillamines with a detection limit of 290 and 350 fmol at respectively at a signal-to-noise ratio of 3. Using the proposed method, the absence of contamination of L-penicillamine in a commercially available D-penicillamine preparation (capsule) was confirmed.

Application of chromatographic chiral stationary phases to pharmaceutical analysis. Enantiomeric purity of D-penicillamine.

The enantiomeric purity of the pharmaceutical D-penicillamine (I) has been determined by a novel high-performance liquid chromatographic technique involving the formation of 5,5-dimethylthiazolidine-4-carboxylic acid (III) by reaction of I with formaldehyde and separation of the optical antipodes by means of ligand exchange chromatography using the copper(II) complex of (2S,4R,2'RS)-4-hydroxy-1-(2'-hydroxydodecyl)proline, which is coated on a reversed-phase column. The limit of determination for the L-antipode is ca. 0.1%. The validation of the method is accomplished by comparison with an independent gas chromatographic procedure. Thirteen commercially available lots of I are shown to be of equally high enantiomeric purity (ca. 99.9%).

The discovery of the therapeutic use of D-penicillamine.

An account is given of the identification of penicillamine in human urine by chromatographic and analytical techniques. At that time this observation appeared to be of esoteric interest only. Some years later, working at the Thorndike Memorial Laboratory at the Boston City Hospital, it occurred to me that the formula of this compound was ideally suited for use as a copper chelating agent for the treatment of Wilson's disease. The subsequent work leading to the acceptance of penicillamine as an important new therapy and also as to its mode of action is given with illustrations of some key experiments and with reference to the first patient ever treated with this drug.