enantioresolution of dl-penicillamine

Copyright 2009 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2010; 24: 6682

Special Issue: Review

Received 13 September 2009, Accepted 16 September 2009 Published online in Wiley Interscience: 10 November 2009

(www.interscience.wiley.com) DOI 10.1002/bmc.1355

Enantioresolution of DL-penicillamineRavi Bhushan* and Rajender Kumar

ABSTRACT: Penicillamine (PenA) is a nonproteinogenic amino acid containing a thiol group. The three functional groups in penicillamine undergo characteristic chemical reactions and diff er in their ability to participate in various chemical and bio-chemical reactions. D-penicillamine is more active pharmacologically, while the L-isomer occurs naturally. This review deals with the enantioresolution of PenA both by direct and indirect methods using liquid chromatography. HPLC separation of its diastereomers prepared with diff erent chiral derivatizing reagents (on reversed-phase columns) and separation of the deriva-tives prepared with achiral reagents (on chiral columns or via ligand exchange mode) has been discussed. Separation of enantiomers tagged with achiral reagent (to add a chromophore for ehanced detection) when there is no diastereomer forma-tion has been considered separately. In addition, application of PenA and its derivatives as chiral selector for enentioresolution of certain other compounds has also been discussed. Copyright 2009 John Wiley & Sons, Ltd.

Keywords: penicillamine; direct resolution; indirect resolution; TLC; HPLC; chiral derivatizing reagents; chiral selector; tagging

* Correspondence to: R. Bhushan, Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247 667, India. E-mail: [email protected]

Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247 667, India

Abbreviations used: AGP, a-acid glycoprotein; BOC-L-Leu-SU, t-butyloxy-L-leucine-N-hydroxysuccinimide ester. ; CE, capillary electro-phoresis; b-CD, b-cyclodextrin; Cys, cysteine; CSP, chiral stationary phase; CMPA, chiral mobile phase additive; CIMS, chiral ion mobility spectrometry; DBPM, N-[4-(6-dimethylamino-2-benzofuranyl) phenyl] maleimide; DNCB, dinitrochlorobenzene; DNP-, dinitrophenyl-; DBD-PyNCS, 4-(3-isothiocyanato-pyrrolidin-1-yl)-7-(N,N-dimethyl-aminosulfonyl)-2,1,3 benzoxadiazole; ESB; 1,1-[ethenylidenebis-(sulfonyl)] bis-benzene; DCC, dicyclohexylcarbodiimide; FDLA, 1-fl uoro-2,4-dinitrophenyl-5-l-alanine amide; FDPA, 1-fl uoro-2,4-dinitrophenyl-5-L-phenylalanine amide; FDVA, 1-fl uoro-2,4-dinitrophenyl-5-L-valine amide; GITC, 2, 3, 4, 6-tetra-O-acetyl-b-D-glucopyranosyl isothiocyanate; MEKC, micellar electrokinetic chromatography; NAP, N-acetyl-D-PenA; NPM, N-(1-pyrenyl) maleimide; OPA, o-phthalaldehyde; PenA, penicillamine; SINP, N-succinimidyl-(S)-2-(6-methoxynaphth-2-yl) propionate; SDS, sodium dodecyl sulfate; STC, spirothiazolidinecarboxylic acid.

IntroductionPenicillamine

Penicillamine (PenA) is a structural analog (3,3-dimethylcysteine) of cysteine as it has two methyl groups in place of the two hydro-gen atoms attached to the second carbon atom of cysteine. It shows chemical properties similar to cysteine (Cys) and is consid-ered as a nonproteinogenic amino acid containing thiol group. It diff ers from valine in having a sulfydryl group. The -SH group in PenA is therefore much more sterically hindered than the corre-sponding group in cysteine.

Penicillamine is a trifunctional amino acid in which an amino group and a carboxyl group are attached to one carbon atom and a sulfydryl and two methyl groups to a second. The three func-tional groups in penicillamine undergo characteristic chemical reactions and diff er enormously in their ability to participate in acidbase equilibria, nucleophilic addition and displacement, combination with various metals, oxidation and free radical transformations (induced by ultraviolet or gamma radiation or by free radicals). The pH of the reaction medium, ionization (as spec-ifi ed by pKa values) and the respective nucleophilic and electro-philic reactivities, which are infl uenced in turn by steric and electronic factors, as well as the hydrogen-bonding and hydro-phobic interactions are the factors responsible for such diff erences.

PenA is no exception to the well-recognized fact that stereo-mers can have diff erent activities, toxicities, pharmacokinetics and pharmacodynamics because a biological system has inher-ent chiral selectivity to enzyme and receptor. D-Penicillamine is usually, or always, more active pharmacologically, while the L-iso-mer occurs naturally. D-PenA is used in treatment of Wilsons disease (Walshe, 1956), cystinuria (Stephens, 1989) and heavy metal poisoning (Gooneratne and Christensen, 1997). It has been shown to be eff ective against rheumatoid arthritis (Jaff e, 1983) and scleroderma (LeRoy et al., 1991). PenA exists in D- (or S) and L- (or R) enantiomer forms and, similarly to others drugs, the toxi-cological eff ects of the two stereoisomers diff er considerably.

Thus, if D-PenA (eutomer) is employed for therapeutic purposes, even with caution and under careful medical supervision, the high toxicity of L-PenA (distomer) and of the racemate mixtures restricts its use (Friedman, 1977). It has been used in the treat-ment of rheumatoid arthritis and hepatitis and for the prevention of infants retina disease (Phelps et al., 2001). L-Penicillamine has been found to have considerable toxicity (Kean et al., 1991). Copper chelation of D-PenA has been shown to generate reactive oxygen species that are cytotoxic to human leukemia and breast cancer cells (Gupte and Mumper, 2007). The evaluation of enan-tiomeric purity of PenA, especially when it is intended for phar-maceutical preparations, is therefore of the utmost importance.

In spite of the presence of thiol group, and like other amino acids without aromatic chains, PenA (Fig. 1) is relatively a poor absorber in the UVvis region; therefore, functionalization of the stereoisomers is generally needed for enantioseparation.

66

Enantioresolution of DL-penicillamine

Biomed. Chromatogr. 2010; 24: 6682 Copyright 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/bmc

Enantiomeric Resolution

Chirality and stereochemistry have become very important topics in pharmacology and analytical chemistry. In drug devel-opment, analytical methods are required to evaluate the enan-tiomeric purity of starting materials, reagents and catalysts, because the quality of these compounds limits the enantiomeric purity of the resulting products. Therefore, the goal of the analyti-cal laboratory in the pharmaceutical industry is to develop as many separations as possible in a minimum of time.

There have been two basic approaches for the chromato-graphic resolution of enantiomers: a direct and an indirect method. The resolution of a pair of enantiomers by reacting them with an optically pure chiral reagent, i.e. the formation of diaste-reomers followed by their separation by chromatography in an achiral environment, is considered as an indirect approach and has been the most common means of achieving the resolution. The following basic considerations are to be taken into account for indirect enantioseparation:

(1) The chiral derivatizing agent should be readily available in high and known optical purity and should not racemize during storage.

(2) The chiral derivatizing agent should react quantitatively or at least the reaction rates with the compounds to be resolved must be kinetically equal; in fact the rates are normally diff erent.

(3) The racemization at the stereogenic center is negligible under the reaction conditions.

As diastereomers have diff erent physico-chemical properties, there is a diff erential retention in a chromatographic system. Introduction of a chromophore enhances detection for HPLC resolution. Resolution via diastereomer formation is usually improved when bulky groups are attached to the chiral center and when the chiral centers of both the reagent and the analyte are in close proximity in the resulting diastereomer.

The direct approach requires no chemical derivatization prior to separation process. Resolution is possible through reversible diastereomeric association between the chromatographic chiral environment and the solute enantiomers. The enantiomers may interact during the course of chromatographic process with a chiral stationary phase (CSP) or a chiral selector added to the mobile phase (CMPA) or a chiral selector immobilized (especially in TLC) on the stationary phase by mixing it with the slurry at the time of plate making. Some important binding types present in the enantioselective sorption process include coordination to transition metals (ligand exchange), charge transfer interaction, ion exchange and inclusion phenomena (hostguest complex).

Direct methods have certain critical disadvantages, particu-larly in HPLC, e.g. many of the stationary phases are not durable

over time and pH and also have low sample capacity. In addition, the correct elution order is diffi cult to predict because of the complexity of interactions with the CSP. Stationary phases involv-ing hostguest type complexation often result in poor band shape and have slow kinetics on a chromatographic time scale. In addition, the CSPs are very expensive.

Enantioresolution by TLCThere are only a few reports on enantiomeric resolution of PenA by TLC using both direct and indirect approaches. Ion pair forma-tion, in situ, by diff erent methods of impregnation of the chiral selector such as the use of L-tartaric acid and (R)-mandelic acid as chiral impregnating reagent or chiral mobile phase additive has been successful for direct TLC resolution. Although ligand-exchange TLC provides direct resolution of enantiomers of a variety of compounds, it has been used for indirect separation of DL-PenA.

Ligand Exchange

One of the earliest TLC resolutions of enantiomers of DL-PenA includes application of ChiralPlate (marketed by Macherey-Nagel, Germany) which involves (2S, 4R, 2RS)-N-(2-hydroxy dodecyl)-4-hydroxy proline as the chiral selector (Martens et al., 1986). DL-PenA was treated with paraformaldehyde (50 mg each) to form enantiomeric 5,5-dimethyl-4-thiazolidinecarboxylic acids. The reaction mixture was applied directly onto Chiralplate, the chromatogram was developed with methanolwateraceto-nitrile (1 : 1 : 4, v/v) for 30 min and after drying the spots were visualized using ninhydrin reagent, showing RF values of 0.48 and 0.62 for the D- and L-derivative, respectively. The respective antip-odes could be determined with the lowest level of detection >0.5% with the conventional TLC technique (Martens et al., 1986).

Ligand-exchange TLC is a typical case of complexation chro-matography. It takes advantage of stereoselectivity of the Cu2+ complex of the hydroxy proline derivative, and has a relatively high degree of selectivity, i.e. the separated enantiomers were those capable of forming diastereomeric complexes of diff erent stabilities with the metal ion (Cu2+) and the chiral selector. Enantiomeric molecules (ligands) are able to donate a lone pair of electrons. The formation of fi ve-membered ring is considered responsible to obtain a ternary complex of suffi cient stability.

Chiral selector as the reagent for impregnating the silica gel or as mobile phase additive

Bhushan and Agarwal (2008) have reported a method for direct TLC resolution and isolation of enantiomers of DL-PenA using L-tartaric acid as chiral impregnating reagent as well as chiral mobile phase additive (CMPA) and (R)-mandelic acid as chiral impregnating reagent. The spots were detected with iodine vapors and the detection limits were found to be 0.12 g for each enantiomer of penicillamine with L-tartaric acid, under both con-ditions, and 0.11 g with (R)-mandelic acid. Enantiomeric resolu-tions were observed at 16 2C and at pH 5 and it has been suggested that only an optimum temperature and pH provide the desired mobility to the diastereomeric ion pair formed in situ and any change in these parameters adversely aff ects the resolu-tion. Recovery of pure L-PenA from the samples of pure D-PenA, prepared by spiking L-PenA with fi xed amounts of pure D-PenA

Figure 1. Structure of D-penicillamine.

67

R. Bhushan and R. Kumar

www.interscience.wiley.com/journal/bmc Copyright 2009 John Wiley & Sons, Ltd. Biomed. Chromatogr. 2010; 24: 6682

in the range 0.15%, was found to be 98.599% and average RSD was less than 1%. Thus the method can be applied for the detec-tion of L-PenA in D-PenA up to 0.1%.

Preparation of impregnated plates and development of chro-matogram. Solutions of L-tartaric acid (0.5%) were prepared in distilled water (50 mL), a few drops of NH3 were added to bring the pH to 5 and the slurry of silica gel G (25 g) was prepared in this solution. TLC plates of (10 5 cm 0.5 mm) were made by spreading the slurry with a manual applicator; the plates were activated overnight at 60 2C. Similarly, plates were prepared using (R)-mandelic acid as a chiral selector. Solutions of racemic and pure isomers of penicillamine were prepared in methanol (102 M) and applied side by side on the plates. Cleaned, dried and paper-lined rectangular glass chambers were used for devel-oping the chromatograms. These were pre equilibrated with mobile phases (Table 1) at 16 2C for 1015 min. Chromatograms were dried at 40C in oven for 1015 min and cooled to room temperature; spots were located in an iodine chamber. L-Tartaric acid was dissolved in double-distilled water (0.5%) and then the pH was adjusted by adding a few drops of NH3; this solution was used as a component of the mobile phase for TLC on plain plates.

The experimental conditions were controlled and the chamber was pre-equilibrated for nearly 15 min for each increase or decrease in the temperature made systematically and tested for chromatographic separation. Each temperature was maintained inside an incubator and the chromatographic chambers were placed inside and allowed to attain the specifi c temperature before development.

Resolution of enantiomers. Of the various solvent systems tried, the solvent system acetonitrilemethanolwater (5 : 1 : 1, v/v) was found to be successful when L-tartaric acid was used as impregnating agent while the solvent combination acetonitrilemethanol(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 mobile phase additive. (R)-mandelic

acid was successful as chiral impregnating reagent with ethyl acetatemethanolwater (3 : 1 : 1, v/v), as the mobile phase. (R)-mandelic acid was not successful as the chiral mobile phase additive under identical conditions for resolving the enantio-mers. The successful solvent combinations along with hRF (RF 100) values are reported in Table 1. The results are the averages of at least fi ve runs under identical conditions. It was observed that the (+)-isomer eluted before the ()-isomer in all cases. The resolution was calculated by dividing the distance between two spots by the sum of two spot radii; a value of 1.50 was taken as an indication of complete resolution whereas a value of 1.00 or below indicated incomplete resolution. Actual photographs of chromatograms are shown in Figs 24.

Isolation of enantiomers. The spots representing the two enantiomers of PenA, as located by exposure to iodine vapors, on the plate impregnated with L-tartaric acid were marked and iodine was allowed to evaporate off . The spots were scraped (from nearly 40 chromatograms) and extracted with methanol. The combined extracts, pertaining to each of the enantiomers, were fi ltered and concentrated in vaccuo. It was expected that only (+)- or ()-penicillamine, from the two cut spots, went into the solution since tartaric acid is insoluble in methanol. For each extract, optical density was measured at the lmax (209 nm) and the concentration was estimated using standard plots. These solutions were examined by polarimeter, and their specifi c rota-tions were calculated using the concentration determined from the calibration plots, e.g. [a]D = +63.5 (c = 0.5, 1 M NaOH); the specifi c rotation value for each of the enantiomers was found to be in agreement with literature. These measurements also showed that the two isomers were in the ratio of 1 : 1. Similarly, isolation of enantiomers was achieved from the plates impreg-nated with (R)-mandelic acid and the plates developed with the mobile phase containing L-tartaric acid as the chiral additive.

Resolution mechanism. The mobile phase with L-tartaric acid and the plates impregnated with either L-tartaric acid or

Table 1. hRF (RF 100) values of enantiomers of DL-PenA using (R)-mandelic acid and L-tartaric acid

Sample no. TLC resolution of DL-PenA using

hRFSolvent Pure L Racemic

d l

1. mandelic acid as impregnating agent

I 52 39 52

2. tartaric acid as impregnating agent

II 61 30 61

3. tartaric acid as CMPA III 71 61 71

Specifi c rotation; +63.5 (c = 0.5, 1 M NaOH), in agreement with literature, Sigma-Aldrich Catalogue, 2007). I, ethyl acetatemethanolwater, 3 : 1 : 1, v/v); II, acetonitrilemethanolwater, 5 : 1 : 1, v/v); III, acetonitrilemethanol(0.5% L-tartaric acid in water, pH 5)glacial acetic acid (7 : 1 : 1.1 : 0.7, v/v).Time, 10 min; solvent front, 8 cm; detection, iodine vapor; temperature, 16 1C; pH 5 for both the chiral selectors (under all conditions).Adapted from: Bhushan R and Agarwal C. 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. Biomedical Chromatography 2008; 22: 12371242.68



Figure 2. Photograph of an actual chromatogram showing resolution of ()-penicillamine using ()-mandelic acid as a chiral selector: left to rightspot 1, lower spot is of D-enantiomer and upper spot is of (+)-enantiomer; spot 2 for pure L- enantiomer. Development time 10 min; temperature 16 2C; detection, iodine vapors. Adapted from: Bhushan R and Agarwal C. 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. Biomedical Chromatography 2008; 22: 12371242. This fi gure is available in colour online at www.interscience.wiley.com/journal/bmc.

Figure 3. Photograph of an actual chromatogram showing resolution of ()-penicillamine on plate impregnated with L-tartaric acid. From left to rightspot 1, lower spot for ()-isomer and the upper spot for L-isomer resolved from the mixture; spot 2, pure D-isomer; spot 3, pure L-isomer. Development time, 10 min; temperature, 16 2C; detection, iodine vapors. Adapted from: Bhushan R and Agarwal C. 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. Biomedical Chromatography 2008; 22: 12371242. This fi gure is available in colour online at www.interscience.wiley.com/journal/bmc.

Figure 4. Photograph of an actual chromatogram showing resolution of ()-penicillamine, using L-tartaric acid as CMPA. From left to rightspot 1, lower spot for D-isomer and the upper spot for L-isomer resolved from the mixture; spot 2, pure D-isomer; development time, 10 min; tem-perature, 16 2C; detection, iodine vapors. Adapted from: Bhushan R and Agarwal C. 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. Biomedical Chromatography 2008; 22: 12371242. This fi gure is available in colour online at www.interscience.wiley.com/journal/bmc.

(R)-mandelic acid had a pH of about 5; both L-tartaric acid and (R)-mandelic acid existed in anionic form under the experimental conditions while the enantiomers in the DL-PenA existed as pro-tonated cations. Thus, Coulombic/chargecharge interaction, hydrogen bonding and steric interactions occurred that favored formation of diastereomers in situ and hence enantioresolution. This is evidenced by the presence of chiral selector(s) in both the spots on the TLC plate and isolation of individual enantiomer(s), as described above. The resolution can be considered as based on ion exchange mechanism when diastereomers of the type (+)-PenA-L-tartaric and ()-PenA-L-tartaric acid were formed in situ and were separated. The overall resolution can thus be attrib-uted to the diff erence in the physical properties of the diastereomers.

There appeared a single spot only when TLC was performed on plates not impregnated with either of the chiral selectors but spotted with the racemic analyte and developed under identical experimental conditions; this confi rmed that the impregnation with L-tartaric acid or (R)-mandelic acid played a role as a neces-sary requirement for enantiomeric resolution.

Factors infl uencing resolution. Experiments carried out at dif-ferent concentrations of each of the chiral selectors showed the best resolution at 0.5% of the impregnating reagent. There was no resolution at 0.1 and 0.9% of the chiral selectors while elon-gated spots were observed at 0.3 and 0.7%. This behavior reveals that the low concentration lacks the suffi cient level of chiral selector to make an ion pair while the higher concentration blocks the capillary action and thus resolution too. Since enan-tiomeric resolution was observed at 16C, and at other tempera-tures either no resolution or poor resolution was observed, it can 69



be considered that only an optimum temperature provides the desired mobility to the diastereomeric ion pair and therefore any change in temperature adversely aff ects the resolution.

Resolution studies carried out on plates prepared in solutions of pH 4, 4.5, 5, 5.5 and 6 showed resolution at pH 5 while there was poor resolution with tailing of spots at pH 4 and 6. Lowering the pH might result in neutral tartaric and mandelic acid mole-cules while at higher pH the analytes might exist as neutral mol-ecules, providing no sites for coulombic interactions and no enantioresolution under these changed pH conditions. Thus, a change in pH changes the ionic states of the chiral selector(s) and the isomers of PenA, resulting in noninteraction among the ionizable groups and a failure of enantiomer separation. The same interpretation is applicable for enantiomeric resolution using a mobile phase having pH 5 and L-tartaric acid as chiral additive.

Indirect Resolution using Marfeys Reagent and its Variants

A TLC method was developed (Bhushan et al., 2007) for indirect chiral separation of penicillamine enantiomers after derivatiza-tion with Marfeys reagent (MR, FDNPAlaNH2) and two of its structural variants, FDNPPheNH2 and FDNPValNH2. The binary mobile phase of phenolwater (3 : 1 v/v) and solvent com-binations of acetonitrile and triethylamine phosphate (TEAP) buff er were found to give the best separation in normal and reversed-phase TLC, respectively. The recovery of the L enantio-mer from the samples of pure D enantiomer prepared by spiking L-PenA with fi xed amounts of D-PenA in the range 0.505.0% was found to be between 98.9 and 99.1%, and the average RSD was less than 1.5%. The results indicate that this method can be applied for the detection of L-PenA in D-PenA up to 0.05% by TLC.

Derivatization. The penicillamine samples (both enantiomeri-cally pure and the racemic) were derivatized with MR and two of its chiral variants according to the method used for derivatization of amino acids and as described in previous chapters. Solutions of DL-PenA (50 L and 2.5 mol) and 1 M NaHCO3 (20 L and 20 mol) were mixed with 1% acetone solution (100 L) of FDNPAlaNH2 (1 mg) in a plastic tube. The contents were incu-bated at 40C for 1 h with stirring over a hot plate. After cooling to room temperature, the reaction was ended by adding HCl (10 L and 2 M). After mixing, the contents were dried in a vacuum desiccator over NaOH pellets. Acetonitrile (200 L) was then added to dissolve the diastereomeric derivatives. Similarly, samples were derivatized with FDNPPheNH2 and FDNPValNH2 (Fig. 5).

The derivatives were yellow colored and visible. Since these derivatives are light-sensitive, all procedures were protected from light exposure and the derivatives were kept in the dark at 4C. During derivatization, maintaining the reaction tempera-ture at 40C, or a little below, was generally required for success-ful coupling with NH2 group of PenA. The diastereomers so formed can be referred to as DNPA-, DNPP- and DNPV-penicillamine.

Derivatization with Marfeys reagent and its structural variants, namely FDNPPheNH2 and FDNPValNH2 provided easier loca-tion on TLC plates due to their self-visibility and greater sensitiv-ity in comparison to literature reports (Busker et al., 1985; Lodemann et al., 1980) for control of enantiomeric purity both by TLC and HPLC, as described below.

Resolution of diastereomers of DL-PenA prepared with MR and its variants. TLC was performed on pre-coated normal-phase plates Alugram SIL G/UV254 and RP- plates RP Alugram RP-18W/UV254 (both 20 20 cm 0.15 mm) received from Macherey-Nagel, Germany, gratis. Solutions (2 L, 25 nmol) of each of the diastereomers of DL-PenA prepared with each of the CDRs, and those of pure D- and L- PenA, were spotted on pre-coated normal and reverse-phase TLC plates. The chromatograms were developed in a pre-equilibrated rectangular glass chamber at 25C. After development, the plates were removed from the chamber and dried with a hair-dryer.

Phenolwater (3 : 1, v/v) at 25C in 40 min on normal-phase plates provided the best resolution of the diastereomeric pairs, prepared from all the three CDRs. In RP-TLC, the best resolution was obtained with combination of TEAP buff er (50 mM, pH 5.5)MeCN (50 : 50, v/v) at 25C. The hRF values on both normal- and reversed-phase TLC are given in Table 2. A photograph of a rep-resentative chromatogram is shown in Fig. 6A for normal-phase and Fig. 6B for reversed-phase conditions. The D-derivative was eluted earlier than the L-isomer for all three cases under normal-phase conditions as expected according to the separation mech-anism (Brckner and Carmen, 1991). The elution order of diastereomers in reversed phase was found to be the reverse of normal phase. Among all the three chiral reagents, the best reso-lution was of the diastereomers prepared with FDNPValNH2 under both normal and reversed phase conditions, as indicated by the hRF values given in Table 2.

Enantioresolution by HPLCIndirect Approach

Lodemann et al. (1980) desulfurized D-PenA with Raney nickel to give D-valine. The L-valine content of the sample was determined by a combination of enzymatic and calorimetric methods. Konig et al. (1984) obtained thiazolidin-2-ones by esterifi cation of peni-cillamine and cyclization with phosgene and the enantiomers were separated by gas chromatography (GC).

Figure 5. Pair of diastereomers obtained by the reaction of Marfeys reagent with DL-Penicillamine.

70



Some of the chiral derivatizing agents used for indirect enan-tiomeric resolution of DL-PenA by HPLC include t-butyloxy-L-leu-cine-N-hydroxy succinimide ester (BOC-L-Leu-SU) (Natchtman, 1980) and 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl isothiocya-nate (GITC) (Ito et al., 1992). Chiral separation and determination of fl uorescent derivatives of PenA enantiomers prepared with N-[4-(6-dimethylamino-2-benzofuranyl) phenyl] maleimide (DBPM) on Pirkle-type CSP has been reported (Nakashima et al., 1995). A fl uorescent chiral reagent, 4-(3-isothiocyanato-pyrro-lidin-1-yl)-7-(N,N-dimethyl-aminosulfonyl)-2,1,3 benzoxadiazole (DBD-PyNCS), has been used for indirect HPLC resolution on an ODS column (Jin and Toyooka, 1998) while the fl uorescent tagging reagent, N-(1-pyrenyl) maleimide (NPM), has been used for direct resolution on Teicoplanin and b-CD columns (Kullman et al., 2000). Enantiomers of PenA derivatized with OPA and 2-mercaptoethanol were separated on a b-CD column (Merino et al., 1992), but sensitivity was low in spite of fl uorometric detection.

Indirect resolution using Marfeys reagent and its variants as the CDRs. An HPLC method was developed (Bhushan et al., 2007) for indirect chiral separation of penicillamine enantiomers after derivatization with Marfeys reagent (FDNPAlaNH2) and two of its structural variants FDNPPheNH2 and FDNPValNH2 using a reversed-phase C18 HPLC column with gradient elution of acetonitrile and 0.01 M trifl uoroacetic acid (TFA). The method was successful for checking the enantiomeric impurity of L-penicillamine (L-PenA) in D-penicillamine and the enantiomeric purity of pharmaceutical formulations of D-penicillamine. The recovery of L enantiomer from the samples of pure D enantiomer prepared by spiking L-PenA with fi xed amounts of D-PenA in the range 0.505.0% was found to be between 98.9 and 99.1%, and average RSD was less than 1.5%. The results indicate that this method can be applied for the detection of L-PenA in D-PenA up to 0.005% by HPLC.

Resolution of diastereomers. Separations were carried out on an Agilent C18 column (150 4.6 mm, 5 m) using acetoni-trile0.01 M TFA as mobile phase under a linear gradient elution mode (acetonitrile, 2565%, 45 min) at a fl ow rate of 1 mL/min with UV detection at 340 nm. Mobile phases for HPLC were fi l-tered through a 0.45 m fi lter and degassed before use.

Retention factors were calculated from the equation K = tR-tO/tO, where tR is the retention time of resolved diastereomers and tO is the retention time of the marker acetone. The separation factors (a) were calculated from the equation a = KD/KL, where KD and KL are the retention factors of diastereomers of D- and L-pen-icillamine, respectively. The HPLC data for resolution of deriva-tives of DL-PenA with MR and its variants is given in Table 3. The sections of chromatograms showing enantioseparation of deriv-atives of PenA prepared with FDNPAlaNH2, FDNPPheNH2 and FDNPValNH2 are shown in Fig. 7. Among the three sets of diastereomers, the order of diff erence between the retention times of resolved diastereomers was as follows FDNPValNH2 > FDNPPheNH2 > FDNPAlaNH2. Very large retention times were obtained in the case of FDNPPheNH2. In each case, the diastereomer of L-PenA eluted earlier than D-PenA. The elution order was determined by derivatizing individual enantiomers of penicillamine with all the three reagents followed by HPLC analy-sis of the diastereomers so formed under identical conditions.

Table 2. hRF values for normal and reversed-phase TLC resolution of diastereomers of DL-PenA prepared with FDAA and its PheNH2 and ValNH2 variants

Diasteromeric mixture of DL-PenA prepared with

hRFNormal phase Reversed phase

From DL-mixture Pure L From DL-mixture Pure L

FDNPAlaNH2 (MR) D 76.9 40.0 L 61.5 61.5 43.1 43.1

FDNPPheNH2 D 67.7 24.6 L 46.2 46.2 30.7 30.7

FDNPValNH2 D 89.2 35.4 L 58.5 58.5 44.6 44.6

Mobile phase: for the diastereomeric pairs, prepared from all the three chiral derivatiz-ing reagents. Normal phase: phenolwater (3 : 1 v/v) at 25C in 40 min. RP-TLC: a com-bination of TEAP buff er (50 mM, pH 5.5)MeCN (50 : 50, v/v). Temperature, 25C.Adapted from: Bhushan R, Brckner H and Kumar V. Indirect resolution of enantiomers of penicillamine by TLC and HPLC using Marfeys reagent and its variants. Biomedical Chromatography 2007; 21: 10641068.

Figure 6. TLC separation of DL- penicillamine derivatized with FDNPPheNH2. (A) Normal phase; (B) reversed phase. Adapted from: Bhushan R, Brckner H and Kumar V. Indirect resolution of enantiomers of penicil-lamine by TLC and HPLC using Marfeys reagent and its variants. Biomedical Chromatography 2007; 21: 10641068. This fi gure is available in colour online at www.interscience.wiley.com/journal/bmc.

71



The RSD was 1.12% for D-, and 1.09% for L-penicillamine, as the mean values for precision.

These results showed a better sensitivity of 0.05% by TLC and of 0.005% by HPLC of derivatives of PenA prepared with MR and its structural variants, which is superior to earlier reports where 0.05% of L-PenA was detected by HPLC as its dimethyl-thiazoli-dine-4-carboxylic acid derivative separated via ligand exchange (Busker et al., 1985), and 0.1% L-PenA was detected in commercial samples in the desulfurized (with raney nickel) form by GC (Lodemann et al., 1980).

Indirect resolution using succinimidyl-(S)-naproxen ester as the CDR. Bhushan and Tanwar (Bhushan and Tanwar, 2008) have

reported indirect enantiomeric resolution of DL-PenA by RP-HPLC using N-succinimidyl-(S)-2-(6-methoxynaphth-2-yl) propionate (SINP), as the chiral derivatizing reagent which was synthesized for this purpose (Fig. 8) by the reaction of (S)-naproxen with N-hydroxysuccinimide in the presence of dicyclohexylcarbodi-imide (DCC). The CDR reacted with PenA readily at room tem-perature in one step and the diastereomeric derivatives were detected by UV. The detection limits were in the 0.52.5 pmol range. The reagent was shown to be useful with the advantage of mild derivatization conditions, shorter reaction time and sen-sitivity to amino group. The method is briefl y described below.

Synthesis of the CDR, SINP. The CDR was synthesized by drop-wise addition of 226 mg (1.1 mmol) of DCC in 3 mL of dry tetra-hydrofuran (THF) to a stirred solution of 230 mg (1 mmol) of (S)-naproxen and 115 mg (1 mmol) of N-hydroxysuccinimide in 2 mL THF under nitrogen atmosphere at room temperature. The solution was then stirred for 3 h, over the course of which a pre-cipitate (dicyclohexyl urea) appeared. After fi ltration, the THF solution was dried in vacuo using a rotatory evaporator and the residue redissolved in 15 mL ethyl acetate. The extract was washed fi ve times with H2O (5 mL), fi ve times with brine (5 mL) and twice quickly with ice-cold saturated NaHCO3 (5 mL). The washed EtOAc extract was then dried in vacuo using a rotatory evaporator; the residue was fi nally recrystallized from hot ethanol to give the reagent as white solid, 212 mg (92.5% yield); m.p. = 117118C; [a]D25 = +42.8 (c = 0.035, MeOH), UV (nm, in MeOH): 231 (lmax), 272, 331; IR(KBr): 3487, 2925, 2357, 1785, 1629, 1539,

Table 3. HPLC resolution of diastereomers of DL-PenA pre-pared with FDAA and its PheNH2 and ValNH2 variants

Diastereomeric mixture of DL-PenA prepared with

Resolution dataKL KD a RS

FDNPAlaNH2 5.081 5.637 1.109 0.595FDNPPheNH2 9.596 10.090 1.090 0.813FDNPValNH2 7.685 8.989 1.170 0.815

Mobile phase: acetonitrile0.01 M TFA under a linear gradient (acetonitrile, 2565%, 45 min) at a fl ow rate of 1 mL/min with UV detection at 340 nm.Adapted from: Bhushan R, Brckner H and Kumar V. Indirect resolution of enantiomers of penicillamine by TLC and HPLC using Marfeys reagent and its variants. Biomedical Chromatography 2007; 21: 10641068.

Figure 7. The sections of the chromatograms showing HPLC resolution of diastereomers of DL-penicillamine prepared with FDNPAlaNH2, FDNPPheNH2 and FDNPValNH2 (left to right). Adapted from: Bhushan R, Brckner H and Kumar V. Indirect resolution of enantiomers of penicil-lamine by TLC and HPLC using Marfeys reagent and its variants. Biomedical Chromatography 2007; 21: 10641068.

Figure 8. Pair of diastereomers obtained by the reaction of a CDR [pre-pared from (S)-naproxen] with DL-penicillamine.

72



1450, 1364, 1268, 1205, 1066, 1031, 996, 859, 820, 780, 648 cm1; 1H NMR (500 MHz, DMSO-d6): d 1.59 [d, 3H, CH(CH3)], 2.7 (s,4H, CH2-CH2), 3.8 (s, 3H, OCH3), 4.3[q, 1H, CH(CH3)], 7.08.0 (m, 6H, C10H6); anal. calcd for C18H17NO5: C, 66.03%; H, 5.24%; N, 4.28%. Found: C, 66.10%; H, 5.35%; N, 4.34%.

The reaction takes place at room temperature and can be com-pleted in 3 h. The base washes should be accomplished quickly and at reduced temperature to avoid cleavage of the ester. The formation of ester is also successful when the reaction mixture is sonicated for 20 min (Mitsunobu reaction condition) when the dicyclohexyl urea is completely precipitated and the yield of the desired product is found to be the same as obtained under the conditions of stirring for 3 h.

Synthesis of diastereomers of PenA with SINP. PenA (20 L, 60 nmol) and 0.2 M borate buff er (pH 9.5, 50 L) were transferred to a 1.0 mL derivatization vial. The solution of derivatizing reagent in acetonitrile (30 L, 150 nmol) was added to the vial and the vial was capped. Thus, PenA and SINP were in the mole ratio of 1 : 2.5. The maximum yield was obtained when the reaction mixture was vortexed at 700 rpm for 3 min and then kept at room temp for 1518 min. Because of high pKa value of PenA, the basic medium was required, e.g. the borate buff er (H3BO3Na2B4O7) was used to facilitate the derivatization. Optimization experiments revealed that 50 L of 0.2 M borate buff er (pH 9.5) was required and the derivatization was found to increase with increase in pH from 7.5 to 9.5 (as shown in Fig. 9). Under these conditions of temperature and time a 2.5-fold molar excess (PenA, 60 nmol and SINP 150 nmol) of the CDR was successful for complete derivatization. A very slight kinetic resolution (due to diff erent reaction rates of enantiomers) was observed at 2.2-fold molar excess.

Separation of the diastereomers of PenA obtained with SINP. The mixture of diastereomeric derivatives was injected onto the column after a 10-fold dilution. Separation of the said diastereomers was achieved on a C18 column (250 4.6 mm, i.d.

5 m) with a fl ow rate of 1.0 mL/min with UV detection at 231 nm; the mobile phase was degassed with nitrogen prior to use. The eff ect of diff erent HPLC conditions on the separation of diaste-reomeric pairs and the data obtained for their separation is shown in Table 4. A representative chromatogram showing base-line separation is given in Fig. 10. The derivatization yield can be calculated from the peak area. Diastereomeric peaks were well separated with RS > 20. The L-isomer eluted after the D. Separation of diastereomers of DL-PenA prepared with SINP was found to be better in terms of resolution than that reported for BOC-L-Leu-SU (Nachtmann, 1980). Table 4 shows little diff erence in resolution when buff er concentration is in the range 550 mM. Therefore the 5 mM buff er concentration is the optimized concentration because high concentration could be harmful for the column. Among the various mixtures of MeCN and TEAP buff er used for isocratic and gradient elution of diastereomers, sharp peaks were obtained under gradient elution. MeCN was found to be a better organic solvent in comparison to methanol as broader peaks and larger retention times were obtained with the latter. At a fl ow rate lower than 1 mL/min both resolution and retention increased

Figure 9. Eff ect of buff er pH on synthesis of diastereomer of PenA with SINP reagent. Adapted from: Bhushan R and Tanwar S. Synthesis of suc-cinimidyl-(S)-naproxen ester and its application for indirect enantioreso-lution of penicillamine by reversed phase high-performance liquid chromatography. Journal of Chromatography A 2008; 1209: 174178.

Table 4. HPLC separation data of diastereomers of PenA prepared with SINP

k1 a RSFlow rate (mL/min)

0.8 9.33 1.49 29.661.0 9.26 1.24 23.161.5 5.91 1.43 22.67

Isocratic systema 3.28 1.16 12.15b 1.58 2.19 13.10c 1.74 2.19 15.95Gradient systemd 9.26 1.24 23.19e 3.00 1.78 17.64pH of TEAP buff er

3 6.18 1.33 20.114 9.26 1.24 23.195 NR NR NR

Buff er concentration (mM)5 9.26 1.24 23.19

10 6.77 1.34 22.8920 6.42 1.35 21.9430 6.97 1.33 22.2940 6.18 1.68 22.8150 8.00 1.28 22.59

Column C18 Eurospher (250 4.6 mm i.d., 5 mm); fl ow rate, 1 mL/min; detection, 231 nm; k1 represents the retention factor of D-diastereomer; a, separation factor; RS, resolution; NR, not resolved.Mobile phase: TEAP buff er (pH 4.0, 5 mM); a = TEAPCH3CN (50 : 50); b = TEAPCH3CN (30 : 70); c = TEAPCH3CN (40 : 60); d = TEAPCH3CN (70 : 30) to TEAPCH3CN (30 : 70) in 30 min; e = TEAPCH3CN (50 : 50) to TEAPCH3CN (25 : 75) in 30 min.Adapted from: Bhushan R and Tanwar S. Synthesis of succin-imidyl-(S)-naproxen ester and its application for indirect enantioresolution of penicillamine by reversed phase high-performance liquid chromatography. Journal of Chromatography A 2008; 1209: 174178. 73



along with the separation factor (Table 4). After separation, the percentage of the peak area of diastereomer of L-PenA was cal-culated relative to the peak area of the diastereomer of D-PenA. When the presence of the L-form and racemization was not detectable (



most of the cases derivatizing reagent is irreversibly attached to both the enantiomers. A brief comparison of the two approaches has also been discussed. In general, for a large number of com-pounds, the sensitivity achieved by the direct separation of underivatized enatiomers on chiral stationary phases (CSPs) or by using chiral additives in the mobile phases (CMPAs) may be unsatisfactory for most real applications.

Sometimes, HPLC separation on chiral stationary phases requires tagging of the analyte with a suitable chromophore or a fl uorophoric reagent. Tagging in most of the cases is irrevers-ible, resulting in a permanent change in the structure of enantio-mers. Most of the time, the experimental conditions applied in an attempt to remove the tagging reagent, after chromato-graphic separation, lead to risk of racemization or some nones-sential decomposition product, thereby reducing the importance and sensitivity of the direct method that makes use of tagging. Thus the term tagging should be used, and is being used in this paper, in a diff erent context, i.e. when the derivatization is carried out with achiral reagent and there is no diastereomer formation, and it is the separation of tagged enantiomers rather than the diastereomeric pair.

Condensation of PenA with formaldehyde. In a diff erent approach, Busker et al. (1985) determined enantiomeric purity of the pharmaceutical D-penicillamine by RP-HPLC involving the formation of 5,5-dimethylthiazolidine-4-carboxylic acid by the reaction of D-PenA with formaldehyde; penicillamine (150 mg) was treated with 100 L of 37% (w/w) aqueous formaldehyde solution and 4 mL of water. After stirring for 2 h at 50C the reac-tion mixture was diluted with water to give a total volume of 25 mL. Then 10 L of the resulting solution, containing the optical antipodes, was injected for chromatographic analysis. Separation was achieved by means of ligand exchange chroma-tography using copper (II) complex of (2S,4R,2RS) 4-hydroxy-1-(2 hydroxydodecyl) proline coated on an RP8 column and using the mobile phase of 12% methanol and 88% water contain-ing 1 104 mol/L copper sulfate (pH adjusted to 4.5 by addition of small amounts of orthophosphoric acid) at a fl ow rate of 3 mL/min and UV detection at 235 nm. The column was maintained at

50C using a water bath. The limit of determination for the L-antipode was ca 0.1%. The validation of the method was accom-plished by comparison with an independent gas chromatographic procedure. Thirteen commercially available lots of D-PenA were shown to be of equally high enantiomeric purity (ca 99.9%).

The approach can be considered as a diff erent one in the sense that derivatization of PenA was carried out but there were no diastereomers as the derivatizing reagent was achiral. It is the condensation of penicillamine with formaldehyde to form the enantiomeric dimethylthiazolidinecarboxylic acids followed by resolution of the isomers by HPLC.

Spirocyclization of DL-PenA with ninhydrin. The classical reac-tion of aminothiols with ninhydrin to form spirothiazolidine adduct is a valid approach for chiral chromatographic recogni-tion of stereoisomers of penicillamine. Since 1910, ninhydrin (triketohydrindene hydrate) has been extensively used as a reagent for qualitative and quantitative determination of a-amino acids (Hutzler and Dancis, 1983). The product of the reaction is a purple colored complex named Ruhemanns purple or, formally, diketohydrindylidenediketohydrindamine, which forms the basis of identifi cation or location of the spots in planar chromatography. The color intensity of this complex can be mon-itored for the majority of amino acids at 570 nm (Friedman, 2004) and for proline and hydroxyproline, which have an a-imino group instead of an a -amino group, at 440 nm. The colored complex is formed via an initial formation of a Schiff s base obtained by the condensation of the amine function of the amino acid with the central carbonyl of the anhydrous keto form of the ninhydrin. Once formed, it undergoes decarboxylation and the resulting intermediate reacts with another molecule of nin-hydrin (Fig. 11) to yield the purple chromophore (Friedman, 2004; Hansen and Joullie, 2005).

The proximity of the a-amine and b-thiol nucleophiles, in peni-cillamine and other aminothiols such as cysteine, in fact, confers a peculiar chemical reactivity to these amino acids toward ninhy-drin. In particular, b-thiol adds to the Schiff s base intermediate (Fig. 12), trapping both molecules as a spirothiazolidine (Pool et al., 2004; Prota and Ponsiglione, 1973). Thus penicillamine and

Table 6. Accuracy and precision of HPLC method (n = 6) for the determination of D- and L-PenA via derivatization with SINP reagent

Actual concentration (ng/mL)

D-PenA L-PenAMean SD measured

concentration (ng/mL)Recovery (%) RSD (%) Mean SD measured

concentration (ng/mL)Recovery (%) RSD (%)

Intra-day 5 4.78 0.10 95.6 2.0 4.88 0.05 97.6 1.115 14.97 0.01 99.8 0.1 15.03 0.01 100.2 0.130 31.02 0.46 103.4 1.5 30.05 0.02 100.1 0.1Inter-day 5 4.57 0.15 93.4 3.2 4.91 0.13 98.2 2.615 14.67 0.11 98.4 0.7 15.09 0.04 100.6 0.330 29.83 0.15 99.4 0.5 19.69 0.15 105.3 1.5

SD, standard deviation; RSD, relative standard deviation; column, C18 Eurospher (250 4.6 mm i.d., 5 mm). Mobile phase: TEAP buff er (pH 4.0, 5 mM); fl ow rate, 1 mL/min; detection, 231 nm.Adapted from: Bhushan R and Tanwar S. Synthesis of succinimidyl-(S)-naproxen ester and its application for indirect enantioresolu-tion of penicillamine by reversed phase high-performance liquid chromatography. Journal of Chromatography A 2008; 1209: 174178.

75



other aminothiols do not give the expected color reaction with ninhydrin (Friedman, 2004). This atypical behavior is of consider-able advantage for PenA, cysteine and other b-thiols.

In fact, during ninhydrin derivatization the amino acid is usually destroyed and the same adduct (the purple complex) is formed with all amino acids. As a result, ninhydrin has been used only as a post-column derivatizing agent. On the other hand, the spirocyclization between PenA and ninhydrin prevents the irre-versible transformation of the amino acid into Ruhemanns purple and thus the spirothiazolidine adducts thus formed are unique for each of the stereoisomers of PenA (Fig. 12) and can be resolved, being the enantiomeric derivatives (say, by HPLC), taking advantage of the acquired chromatographic and optical properties.

Achiral derivatization of DL-PenA with ninhydrin and separa-tion of the spirothiazolidine carboxylic acid derivatives on achiral C18 column. Taking into account the atypical behavior of PenA in its reaction with ninhydrin, Sotgia et al (2008) reported an HPLC method for the separation and quantifi cation of stereo-isomers of PenA after their spirocyclization with ninhydrin; copper (II)-L-proline complex as a chiral selector in the mobile phase was used which provided resolution via ligand exchange but, due to copper in the mobile phase, there was a very high noise background that limited the sensitivity of the detection

system, particularly with the fl uorescence detector. The method was able to detect traces of L-penicillamine in samples of D-pen-icillamine below 0.1% in fairly short times (about 16 min) with a good resolution (RS = 1.31).

Derivatization of DL-PenA with ninhydrin. A 200 L volume of aqueous penicillamine standard solution or pharmaceutical for-mulation was mixed with 20 L of an ethanolic 3% (w/v) ninhy-drin solution. After vortex-mixing, 20 L of an aqueous H2SO4 5 mol/L solution was added, then the reaction mixture was left for 5 min at 100C in a thermoblock heater. These conditions provided maximum reaction rate, short reaction time, sensitivity and best peak shape. Spirocyclization may also take place at room temperature, but the time for maximum reaction rate increases (Sotgia et al., 2008).

HPLC separation of the spirothiazolidine carboxylic acid derivatives of DL-PenA on achiral C18 column. The separation was achieved (Sotgia et al., 2008) using an achiral C18 column (15 cm 4.6 mm, 5 m, with a 4.6 20 mm guard column car-tridge) at room temperature (about 2324C) and applying a fi ve-fold dilution of derivatized solutions with injection amount of 20 L. The chiral mobile phase consisted of an aqueous solution of L-proline (22 mmol/L) and of copper chloride dihydrate (2 mmol/L) containing 30% MeCN (v/v). The eluent was delivered isocratically at a fl ow-rate of 0.5 mL min1 and prior to use it was fi ltered through a 0.22 m disposable fi lter to remove any par-ticulate matter. Detection was made by both the UVvis dual-wavelength absorbance detector set at 231 and 246 nm and the fl uorescent detector with the gain set at 1000 scale expansion and excitation and emission wavelengths set at 390 and 497 nm, respectively. The evaluation of eff ectiveness of metal complexes of Cu2+ with some amino acids such as L- Pro, L-Arg, L-Phe, L-His, L-Asp and L-Val as chiral selectors showed that the degree of reso-lution between the two spirothiazolidine enantiomers of penicil-lamine was poor by using L-Arg, L-Asp and L-Val, incomplete with L-Phe and L-His while it was good with L-Pro. Mobile phase showed a blue color and very high noise background that limited the sensitivity of the detection system. The loss of sensitivity was more pronounced when a fl uorescence detector was used instead of the UVvis absorbance detector. However, attempts to overcome the high detector background signal by replacing Cu2+ with Zn2+, which forms colorless complexes, gave unsatisfactory results in terms of resolution. The recoveries for the added amount of L-PenA were all found to be between 98.56 and 99.11%, and the limit of detection was 0.06 and 0.09% for UV and fl uorescence detection, respectively.

Achiral derivatization of DL-PenA with ninhydrin and dinitro-fl uorobenzene and separation on a-acid glycoprotein and b-cyclodextrin columns along with recovery of native enan-tiomers. In a similar approach, Bhushan and Kumar (2009) carried out achiral derivatization (spirocyclization) of DL-PenA and DL-Cys with ninhydrin and obtained the enantiomeric spiro-thiazolidine carboxylic acid (STC) derivatives; these were resolved by HPLC with UV detection on chiral a-acid glycoprotein (AGP) and b-CD columns. The enantiomers so separated as STC deriva-tives of DL-PenA were subjected to detagging to obtain the native enantiomers. The method was optimized and was scaled up for preparative separation of enantiomers. The chiral purity of the enantiomers obtained after detagging was further confi rmed using MR (Bhushan and Kumar, 2008). Additionally, DNP-

Figure 11. General reaction showing condensation of ninhydrin with an a-amino acid to form the Ruhemanns purple complex via the forma-tion of a Schiff s base.

Figure 12. General reaction showing formation of spirothiazolidines by condensation of ninhydrin with DL-penicillamine.

76



derivatives of DL-PenA and DL-Cys were also prepared and sepa-rated on both of these stationary phases.

Derivatization of DL-PenA with ninhydrin was carried out in a vial by adding an ethanolic solution of ninhdrin (30 L, 3 mmol) to an aqueous solution of DL-PenA (200 L, 3 mmol). After vortex mixing at 5000 rpm for 5 min, H2SO4 (20 L of 0.2 M) was added. The resultant mixture was heated at 100C for 5 min and then cooled to room temperature. The structures and synthesis of the two spirothiazolidine derivatives are shown in Fig. 12. Presence or absence of any peak corresponding to ninhydrin or other side products in the density plot from PDA detector is an indication of the success, as well percentage completion, of the derivatiza-tion reaction.

Similarly, the DNP derivatives were prepared by taking a stan-dard solution of DL-PenA (50 L, in 1 M NaHCO3) in a derivatiza-tion vial and adding a solution of 2,4-dinitrofl uorobenzene (100 L, 60 mM in acetone). The solution mixture was vortex mixed and was heated at 45C for 45 min in incubator. To this was added 20 L of 2 M HCl. The structure and general scheme for synthesis of DNP-DL-PenA and DNP-DL-Cys is shown in Fig. 13. Synthesis of DL-DNP derivatives can also be carried out by using dinitrochlorobenzene (DNCB), although it is relatively less reac-

tive in comparison to dinitrofl uorobenzene. Further, high basic condition, higher temperature and higher reaction time should be avoided to prevent formation of disubstituted derivatives.

HPLC of STC-, and DNP-derivatives on AGP and b-CD columns. The enantiomeric mixture of STC-derivatives pertain-ing to DL-PenA or DL-Cys was diluted 10-fold with mobile phase and injected into the 20 L HPLC loop. HPLC was performed, for analytical separation, on an AGP column (100 4 mm i.d., 5 m) and b-CD-bonded chiral phase column (250 4.0 mm i.d., 5 m) from Chromtech Merck (Darmstadt, Germany). The separation was carried out at a fl ow rate of 1 mL/min with UV detection at 231 nm and at ambient temperature of 24C. Mobile phases used for AGP column were (I) 0.5% 2-propanol in sodium phosphate buff er (10 mM), (II) 1% 2-propanol in sodium phosphate buff er (10 mM), (III) 2% MeCN in sodium phosphate buff er (10 mM) and (IV) 3% MeCN in sodium phosphate buff er (10 mM). For b-CD column, mobile phase, (V) MeCN20 mM sodium phosphate buff er, 40 : 60 (v/v) was used. The buff er in all the mobile phases had pH 4 for DL-STC derivatives and pH 7 for DL-DNP derivatives.

Preparative separation was performed using mobile phases I and IV on an AGP column and the mobile phase (V) on a b-CD column. A fl ow rate of 1 mL /min was applied as a higher fl ow rate with higher sample concentration and load volume could lead to column disintegration. The HPLC run was performed using 200 L injection loops with a sample concentration of 2mg/mL and the injections were repeated several times.

Resolution of enantiomeric STC- and DNP-derivatives. The data for resolution of enantiomeric STC- and DNP- derivatives is shown in Table 7. On the AGP column, DNP-DL-derivatives had higher resolution (RS values) compared with the STC-DL-derivatives and DNP-DL-PenA was better resolved, while on the b-CD column STC-DL-derivatives were better resolved compared with DNP-DL-derivatives.

The chiral AGP column was shown to have better enantiose-lectivity compared with b-CD as the AGP column, in general, showed a better enantioresolution compared with the b-CD

Figure 13. Synthesis and structure of DNP derivatives of DL-penicillamine.

Table 7. Chromatographic data for resolution of enantiomers of STC -, and DNP- derivatives of DL-PenA on AGP and b-CD columns

Analyte AGP column b-CDMobile phase I Mobile phase II Mobile phase III Mobile phase IV Mobile phase V

pH KL RS a KL RS a KL RS a KL RS a KL RS a

STC derivative 4 10.08 6.92 1.44 6.00 3.82 1.34 14.52 3.22 1.30 14.16 4.38 1.35 7.83 2.38 1.10DNP-DL- PenA 7 5.90 7.60 1.46 3.52 2.05 1.34 6.45 1.80 1.22 3.42 1.18 1.07 3.11 1.75 1.19

Mobile phase I: 0.5% 2-propanolsodium phosphate buff er (10 mM, pH 4) for resolving DL- STC derivatives and the same mobile phase with buff er of pH 7 for resolving DL-DNP derivatives. Mobile phase II: 1% 2-propanol and the rest of the composition and application is the same as for mobile phase I. Mobile phase III: 2% MeCNsodium phosphate buff er (10 mM, pH 4) for resolving DL- STC derivatives and the same mobile phase with buff er of pH 7 for resolving DL-DNP derivatives. Mobile phase IV: 3% MeCN and the rest of the composition and application is the same as for mobile phase III. Mobile phase V: MeCNTEAP buff er (20 mM, pH 4), 40 : 60 (v/v) for resolution of DL-STC derivatives and the same mobile phase with buff er of pH 7 for DL-DNP derivatives.STC derivatives: DL mixture of spirothiazolidine carboxylic acid derivatives with respect to confi guration of PenANote: (i) There was no resolution of enantiomers of STC derivatives using mobile phases IV with buff er of pH 7. (ii) There was no resolution of DNP-DL-Cys and DNP-DL-PenA using mobile phases IV with buff er of pH 4.Adapted from: Bhushan R and Kumar R. Analytical and preparative enantioseparation of dl-penicillamine and dl-cysteine by high-performance liquid chromatography on a-acid glycoprotein and b-cyclodextrin columns using ninhydrin as a reversible tagging reagent. Journal of Chromatography A 2009; 1216: 34133417. 77



column for both types of derivatives. On both the columns, the STC derivatives of D-enantiomer eluted earlier than the L-isomer while the DNP derivatives of L-enantiomer eluted earlier than that of the D-enantiomer. The elution order of the D-, and L-iso-mers of STC- and DNP-derivatives was confi rmed by elution of corresponding single enantiomeric derivatives prepared for this purpose. The HPLC chromatograms showing resolution of DL-STC and DL-DNP derivatives using mobile phase I on AGP and mobile phase V on b-CD columns are shown in Figs 14 and 15.

The recovery for STC-derivatives of D- and L- PenA varied from 98.9 to 99.7% and from 98.5 to 99.5% for intraday assay and from 98.5 to 99.4% and from 97.6 to 99.4% for inter-day assay, respec-tively. The recovery for DNP-derivatives of L- and D-PenA deriva-tives varied from 97.5 to 98.3% and from 96.9 to 97.8% for intraday assay and from 97.1 to 98.1% and from 97.1 to 97.7% for inter-day assay, respectively. It detected up to 0.01% of STC-derivative of D- PenA in STC-derivative of L- PenA and 0.004% of DNP- D-PenA in DNP- L-PenA on the AGP column.

Resolution mechanism. AGP is known to have 181 amino acid residues and has an isoelectric point of 2.7. The separation is based on ion pair, hydrogen bonding and hydrophobic interac-tions. b-CD consists of seven glucose units with a shape of cone having hydrophobic interior and hydrophilic hydroxyl groups on the rim. The separation on this stationary phase is principally based on the inclusion phenomenon, hydrophobic interactions and hydrogen bonding. Based on experimental data and the literature on mechanism of enantioseparation on chiral AGP and b-CD stationary phases, it is suggested that inclusion phenom-ena and ionic interactions along with hydrogen bonding (that are highly pH sensitive) are the two main forces responsible for chiral separation of these compounds on b-CD and AGP columns, respectively. Thus it may be possible that STC- DL-derivatives having an almost naphthalene type of structure have a better fi t

in chiral cavities of stationary phase, particularly for b-CD column compared with DNP- DL-derivatives.

The observation that DL-DNP derivatives were separated only at pH 7 while the STC- DL-derivatives were separated at pH 4 indicates that the functional groups (such as carboxylic, second-ary amino-, and SH groups) of STC- and DNP- derivatives infl u-ence the ionic interactions (with AGP) and play a role in the formation and stability of the inclusion complex with b-CD. The dinitrofl uoro benzene moieties also serve as suitable substrates for the inclusion phenomenon with the chiral material in the column for enantiomeric resolution.

Detagging of spirothiazolidine moiety from enantiomeric STC-derivatives

The solutions of individual fractions of STC derivatives, corre-sponding to D-, and L- isomers of PenA obtained from HPLC columns, were concentrated under vacuum and dried under nitrogen. The residue obtained was dissolved in 10% TFA in puri-fi ed water and then 200 mg Zn dust was added to it. The reaction was required to be continuously ventilated since hydrogen gas was produced. The reaction mixture was extracted fi rst with dichloromethane to remove any undesired organic impurities and then the aqueous fraction was extracted with ethyl acetate three times. Combined ethyl acetate extract was dried under stream of nitrogen.

The enantiomeric purity of individual enantiomers, viz., D-PenA and L-PenA, obtained after detagging and purifi cation workup was verifi ed. The enantiomeric purity of the recovered native enantiomers was further confi rmed using MR and as reported earlier for Cys (Bhushan and Kumar, 2008) and PenA (Bhushan et al., 2007). The yield and data on enantiomeric purity is shown in Table 8.

Preparative separation of enantiomeric STC- and DNP-derivatives. The analytical method optimized for separation of enantiomers of DL- STC and DL-DNP derivatives was scaled up for

Figure 14. Section of chromatograms showing resolution of enantio-mers of STC derivative and DNP-DL-PenA on AGP column using mobile phase (0.5% 2-propanol in sodium phosphate buff er, 10 mM, pH 4) for STC derivatives and using the same mobile phase with buff er of pH 7 for DNP-DL-PenA. (Retention times are in minutes.) Adapted from: Bhushan R and Kumar R. Analytical and preparative enantioseparation of DL-peni-cillamine and DL-cysteine by high-performance liquid chromatography on a-acid glycoprotein and b-cyclodextrin columns using ninhydrin as a reversible tagging reagent. Journal of Chromatography A 2009; 1216: 34133417.

Figure 15. Section of chromatograms showing resolution of STC deriv-ative and DNP-DL-PenA on b-CD column using mobile phase (MeCNTEAP buff er, 20 mM, pH 4) for STC derivative and using the same mobile phase with buff er of pH 7 for DNP-DL-PenA (Bhushan and Kumar, unpub-lished, 2009). (Retention times are in minutes.)

78



Table 8. Yield and purity of the enantiomers obtained by detagging of STC-derivatives along with recovery of enantiomeric STC-, and DNP- derivatives from AGP and b-CD columns

Analyte Resolved compound

Percentage recovery from DetaggedAGP column using b-CD column using

mobile phase VEnantiomer Yield (%) Purity (%)

Mobile phase I Mobile phase IV

DL- STC derivatives

L- STC derivative 96.08 93.04 95.44 L-PenA 71 98.7D- STC derivative 98.32 96.72 96.72 D-PenA 77 99.3

DNP-DL-PenA DNP-L-PenA 99.44 99.12 98.48 Detagging of DNP-derivatives was not performedDNP-D-PenA 98.48 97.36 97.52

Mobile phases are as per Table 7.Percentage purity of the detagged enantiomers was established using Marfeys reagent.Adapted from: Bhushan R and Kumar R. Analytical and preparative enantioseparation of DL-penicillamine and DL-cysteine by high-performance liquid chromatography on a-acid glycoprotein and b-cyclodextrin columns using ninhydrin as a reversible tagging reagent. Journal of Chromatography A 2009; 1216: 34133417.

preparative separation using both the AGP and b-CD analytical columns. The chromatograms obtained for preparative separa-tion on chiral AGP stationary phases for DL-STC derivatives and DL-DNP-PenA are shown in Fig. 7. For preparative separation, the chiral AGP column was found to be better in terms of resolution (Table 7), yield and enantiomeric excess (Table 8) compared with the b-CD column. With the RS > 2, a > 1.2 and K < 10, the scaling of analytical method to preparative one proved to be good and suitable.

The transposed method was found to have no appreciable disposition from analytical chromatographic data in terms of resolution. Pure enantiomers were obtained from individual frac-tions after detagging.

Direct Resolution after Achiral Derivatization

The enantioresolution on CSPs may not be fully exceptional to derivatization, particularly when there is a need to increase detection sensitivity; this involves achiral derivatization, which is practically free from kinetic resolution and associated with diff er-ent spectral properties. Application of chiral stationary phases for enantioresolution minimizes the chances of racemization and kinetic resolution, but the CSPs are extremely expensive. In general, for a large number of compounds, the sensitivity achieved by direct separation of underivatized enantiomers on chiral stationary phases (CSPs) or by using chiral additives in the mobile phases (CMPAs) may be unsatisfactory for most real applications.

Enantioseparation of dansyl-DL-PenA was reported along with several other dansyl amino acids on the stationary phase to which proline and BOC-proline were bonded (Engelhardt et al., 1986). These phases were used with both non-polar eluents and aqueous systems in the presence of Cu2+ ions in the ligand-exchange mode. Isoindole adduct derivatives prepared with OPA, 2-mercaptoethanol (Merino et al., 1992) were separated using HPLC on b-CD column. The method was also applied to determine the enantiomeric purity of D-PenA in pharmaceutical tablet forms. It was successful for determination of L-PenA impu-rity above 0.5%; however, the sensitivity was lower compared with those reported with OPA by Lindroth and Mopper (1979). This lower sensitivity was assumed to be due to incomplete derivatization of PenA possessing both thiol and amine groups. Nakashima et al. (1995) reported resolution of enantiomers of

PenA on Pirkle type stationary phase as their DBPM derivatives with a detection limit of 290 and 350 fmol for D-, and L-PenA, respectively.

Capillary electrophoresis with b-CD as chiral selector in running buff er was employed for the enantioseparation of PenA (Gotti et al., 1999) derivatized with 1,1-[ethenylidenebis-(sulfonyl)] bis-benzene (ESB), which is a thiol specifi c derivatization reagent and results in enhanced UV detection; baseline resolution was obtained only when L-camphor-10-sulfonic acid was added to the mobile phase, which acted as an ion pair reagent. Dwivedi et al., (2006) reported chiral separation of a number of analytes including DL-PenA by chiral ion mobility spectrometry (CIMS). The separation was based on size and shape and on the stereo-specifi c interaction with a chiral gas. The drift gas was doped with the volatile chiral reagent, which was (S)-2-butanol in this study.

Application of PenA as chiral selectorAs CDR

PenA and its derivatives, particularly N-protected ones, have been employed as chiral derivatizing reagents for enantioresolu-tion of a few amino acids and amino group-containing drugs using RP-HPLC. PenA has also been used as chiral derivatizing reagent for CE or micellar electrokinetic chromatographic sepa-ration. There is no report on PenA being used as a chiral selector in TLC.

N-acetyl-D-penicillamine was used as CDR for formation of diastereomers of several racemic amine drugs such as (a-methylbenzene ethanamine, 6-amino-2-methyl-2-heptanol and 1-aminoethylbenzenmethanol) and thiol drugs [N-(2-mercaptopropionyl) glycine, 2-mercaptopropionic acid, and N-acetyl-3-mercaptovaline] in conjunction with OPA and naph-thalene-2,3-dicarboxaldehyde. The resulting diastereomers were resolved using RP-HPLC and CE (Leroy et al., 1995). The diastereo-mers of racemic amino drugs were resolved by either micellar electrokinetic chromatography (MEKC) or b-CD-MEKC using sodium dodecyl sulfate (SDS) as surfactant. Tivesten et al. (1996) reported chiral separation of DL-amino acids after on-column derivatization with OPA and N-acetyl-D-PenA, as CDR. The result-ing diastereomers were then successfully resolved by micellar electrokinetc chromatography, enabling the derivatization of samples in the picoliter range. 79



As chiral selector in stationary or mobile phase

D-PenA, N-acetyl-D-PenA (NAP) and L-Val were employed as chiral ligand exchange additives in running buff er for enantioseparation of dansyl amino acids (Zheng et al., 2003) by micellar electrokinetic chromatography. PenA and N-acetyl PenA act as tridentate ligands while L-Val acts as a bidentate ligand. It was found that SDS was crucial for enantioresolution. There was no resolution with NAP while all the studied amino acids were well resolved using D-PenA and L-Val as chiral selectors. The resolution obtained with L-Val was better compared with that obtained with D-PenA, which sug-gested that the SH group did not contribute towards enantiodis-crimination. Under optimum conditions, chiral separation of eight pairs of enantiomers of dansyl amino acids was achieved with resolution (RS) in the range of 1.1 to 5.9. Use of L-PenA instead of D-PenA resulted in reversal of elution order. N-acetyl-D-PenA and D-PenA were applied as chiral selectors (along with chiral alcohols, which were used as chiral co-surfactant) by incorporation in micelles of sodium dodecyl sulfate with microemulsion electroki-netic chromatography for enantioseparation of ephedrine, nor-ephedrine, N-methylephedrine, nadolol and propranolol (Zheng et al., 2004).

Low-molecular-weight strong chiral cation exchanger, based on penicillamine sulfonic acid, was immobilized on thiol-modi-fi ed silica particles for enantioseparation (Bicker et al., 2003) and determination of enantiomeric excess of ephedrine using a non-aqueous capillary electrochromatographic method. Resolutions greater then 4 were obtained and the limit of detection for the L-enantiomer in a D-ephedrine solution was found to be 0.035%.

Two chiral ligand exchange stationary phases were synthe-sized, namely, N,S-dioctyl-D-penicillamine from D-penicillamine by the reaction of 1-bromooctane and triethylamine in a chloro-formmethanol mixture at 7080C, and N,S-dioctyl-N-methyl-D-penicillamine prepared by reductive N-methylation of N,S-dioctyl-D-penicillamine (Oi et al., 1992). These stationary phases were successful for enantioresolution of underivatized amino acids, amino alcohols and hydroxy acids by HPLC. The stationary phase containing Cu (II) complex of N,S-dioctyl-D-penicillamine was found to be more successful for the resolution of most of the amino acids with very good selectivity except for glutamic acid and tryptophan, which were better resolved on N,S-dioctyl-N-methyl-(D)-penicillamine stationary phase. The elution order for all the amino acids with the exception of histi-dine was found to be L followed by D on the former while on the latter a reversal in elution order was obtained. The reversal in elution order for histidine was suggested to be due to the imid-azole group of histidine. Diff erent patterns of chiral recognition obtained on two chiral stationary phases was speculated to be due to the hydrogen bonding eff ect of NH group in N,S-dioctyl-D-penicillamine stationary phase compared with the other one where chiral discrimination was believed to be aff ected by the N-CH3 group. The stationary phase containing N,S-dioctyl-N-methyl-D-penicillamine was found to be successful in resolving a multicomponent mixture of enantiomeric amino acids.

A chiral ligand exchange column packed with octadecyl silanized silica coated with N,S-dioctyl-D-penicillamine was found to be successful for the resolution of some nonprotein amino acids (Miyazawa et.al, 1997, 2004) by varying the con-centration of organic modifi er (2-propanol) and Cu (II) in the mobile phase. The study revealed good resolution and selectiv-ity for a number of a-amino acids with aromatic, aliphatic or cyclic side chains including cyclic imino acids. The same CSP

was employed (Miyazawa et al., 2000) for enantioresolution of some underivatized a-Me-a-amino acids. It provided good resolution and selectivity for amino acids having both aliphatic and aromatic side chains in comparison to cellulose tris (3, 5-dimethylphenylcarbamate). Bisel et al. (2001) reported syn-thesis of four stereomers of a,a-quaternary-a-amino acids which were successfully separated on D-a penicillamine-based chiral stationary phase via ligand exchange. The method proved to be successful in determination of enantiomeric excess in the range 92.9 to >98%.

Chiral ligand exchange stationary phase prepared by embed-ding Cu (II)N,S-dioctyl-D-penicillamine in a reversed-phase C18 silica gel as reported by Oi et al. (1995) was employed for resolu-tion of enantiomers and diastereomers of cyclic b-substituted a-quaternary a-amino acids (Schlauch et al., 2000). The devel-oped method proved to be successful for resolution of all the stereoisomers under study except for the two diastereomers of 1-amino-2-methylcyclopentanecarboxylic acid. The elution order of the chiral amino acids was reported to be dependent upon the concentration of acetonitrile in the mobile phase. Penicillamine was found to give more stable complexes due to it being a tri-dentate ligand compared with proline, which acts as bidentate one. Schlaulch and Frahm (2001) carried out thermodynamic studies related to retention and separation of a-amino acid enan-tiomers on chiral stationary phase derived from D-penicillamine (i.e. N,S-dioctyl-D-penicillamine) with the help of the vant Hoff principle. The study revealed that the concentration of Cu (II) ions in mobile phase had little eff ect on enantioselectivity; it was the organic modifi er (acetonitrile, in this case) and pH of mobile phase that were the real forces that contributed to enantioselec-tivity. Thus, the role of hydrophobic interaction with chiral sta-tionary phase was justifi ed for enantiodescrimination.Schlaulch et al. (2001) employed D-penicillamine bonded stationary phase for the separation of diastereomers of cycloaliphatic b-substituted-a-quaternary-a-amino acids based on chiral ligand exchange, and was found to be economical and better in terms of sensitivity and the resolution of polar substituted compounds.

Okamato et al. (1995) resolved all four enantiomers of a plate-let-activating factor antagonist SM-10661 using ligand exchange chiral stationary phases with copper (II) complex of N,S-dioctyl-D-PenA. The mechanism of resolution involved the hydrophobic interactions between SM-10661 and D-penicillamine-Cu(II) complex. The D-penicillamineCu(II) complex was used as chiral selector for ligand exchange HPLC resolution of enantiomers of unusual secondary amino acids such as pyrrolidine-2-carboxylic acid, piperidine-2-carboxylic acid, piperazine-2-carboxylic acid, morpholine-3-carboxylic acid and thiomorpholine-3-carboxylic acid analogs (Ilisz et al., 2006).

ConclusionThe literature review is suggestive that there is a scope for devel-opment of methods for chiral analysis of PenA in biological speci-mens. TLC provides a simple and less expensive method for direct enantioseparation for analytical studies. Native enantiomers at a semi-preparative scale were obtained in the laboratory by tagging with ninhydrin followed by detagging. There are fewer CDRs providing indirect resolution of PenA and the CSPs are extremely expensive for direct resolution; therefore, there may be developed other reagents that could derivatize the thiol or 80



amino functional group selectively, leading to the formation of diastereomers having enhanced sensitivity and detection.

ReferencesBhushan R and Agarwal C. Direct enantiomeric TLC resolution of DL-pen-

icillamine using (R)-mandelic acid and L-tartaric acid as chiral impreg-nating reagents and as chiral mobile phase additive. Biomedical Chromatography 2008; 22: 12371242.

Bhushan R and Kumar V. Synthesis and application of new structural vari-ants of Marfeys reagent for liquid chromatographic enantiosepara-tion of -amino acids. Acta Chromatographica 2008; 20: 329347.

Bhushan R and Kumar R. Analytical and preparative enantioseparation of DL-PenA and DL-cysteine by high performance liquid chromatography on a-acid glycoprotein and b-cyclodextrin columns using ninhydrin as a reversible tagging reagent. Journal of Chromatography A 2009; 1216: 34133417.

Bhushan R and Tanwar S. Synthesis of succinimidyl-(S)-naproxen ester and its application for indirect enantioresolution of penicillamine by reversed phase high-performance liquid chromatography. Journal of Chromatography A 2008; 1209: 174178.

Bhushan R, Brckner H and Kumar V. Indirect resolution of enantiomers of penicillamine by TLC and HPLC using Marfeys reagent and its vari-ants. Biomedical Chromatography 2007; 21: 10641068.

Bicker W, Hebenstreit D, Laemmerhofer M and Lindner W. Enantiomeric impurity profi ling in ephedrine samples by enantioselective capillary electrochromatography. Electrophoresis 2003; 24: 25322542.

Bisel P, Breitling E, Schlauch M, Volk, FJ and Frahm A W. Asymmetric syn-thesis and chromatographic resolution of all four stereomers of the a,b-propanoleucine. Pharmazie 2001; 56: 770772.

Brckner H and Gah C. High-performance liquid chromatographic sepa-ration of DL-amino acids derivatized with chiral variants of Sangers reagent. Journal of Chromatography 1991; 555: 8195.

Busker E, Gnther K and Martens J. Application of chromatographic chiral stationary phases to pharmaceutical analysis. Enantiomeric purity of D-penicillamine. Journal of Chromatography 1985; 350: 17985.

Dwivedi P, Wu C, Matz LM, Clowers BH, Siems WF and Hill HH Jr. Gas-phase chiral separations by ion mobility spectrometry. Analytical Chemistry 2006; 78: 82008206.

Engelhardt H, Konig Th and Kromidas St. Optimization of enantiomeric separations with chiral bonded phases. Chromatographia 1986; 21: 205213.

Friedman M. Chemical basis for pharmacological and therapeutic actions of penicillamine. Proceedings of the Royal Society of Medicine 1977; 70(suppl. 3): 5060.

Friedman M. Application of ninhydrin reaction for analysis of amino acids, peptides and proteins to agriculture and biomedical sciences. Journal of Agricultural and Food Chemistry 2004; 52: 385406.

Gooneratne SR and Christensen DA. Eff ect of chelating agents on the excretion of copper, zinc and iron in the bile and urine of sheep. Veterinary Journal 1997; 153: 171178.

Gotti R, Pomponio R, Andrisano V and Cavrini V. Analytical study of peni-cillamine in pharmaceuticals by capillary zone electrophoresis. Journal of Chromatography A 1999; 844: 361 369.

Gupte A and Mumper RJ. Copper chelation by D-penicillamine generates reactive oxygen species that are cytotoxic to human leukemia and breast cancer cells. Free Radical Biology and Medicine 2007; 43:12711278.

Hansen DB and Joullie MM. The development of novel ninhydrin ana-logues. Chemical Society Review 2005; 34: 408417.

Hutzler J and Dancis J. The determination of pipecolic acid: method and results of hospital survey. Clinica Chimica Acta 1983; 128: 7582.

Ilisz I, Tourwe D, Armstrong DW and Peter A. High-performance liquid chromatographic enantioseparation of unusual secondary amino acids on a D-penicillamine-based chiral ligand exchange column. Chirality 2006; 18: 539543.

Ito S, Ota A, Yamamoto K and Kawashima Y. Resolution of the enantiomers of thiol compounds by reversed-phase liquid chromatography using chiral derivatization with 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl isothiocyanate. Journal of Chromatography 1992; 626: 187196.

Jaff e IA. Penicillamine: an anti-rheumatoid drug. American Journal of Medicine 1983; 75: 6368.

Jin D and Toyooka T. Indirect resolution of thiol enantiomers by high-performance liquid chromatography with a fl uorescent chiral tagging reagent. Analyst 1998; 123: 12711277.

Kean WF, Lock CJ and Howard-Lock HE. Chirality in antirheumatic drugs. Lancet 1991; 338: 15651568.

Konig WA, Steinbach E and Ernst K. Phosgene a versatile reagent enan-tiomer separation by capillary gas chromatography. Journal of Chro-matography 1984; 301: 129135.

Kullman JP, Yu T, Chen X, Neal R, Ercal N and Armstrong DW. Resolution of chiral thiol compounds derivatized with N-(1-pyrenyl)-maleimide and ThioGlo3. Journal of Liquid Chromatography and Related Technology 2000; 23: 19411952.

Leroy P, Bellucci L and Nicolas A. Chiral derivatization for separation of racemic amino and thiol drugs by liquid chromatography and capil-lary electrophoresis. Chirality 1995; 7: 235242.

LeRoy EC, Trojanowska M and Smith EA. The pathogenesis of scleroderma (systemic sclerosis, SSc). Clinical and Experimental Rheumatology 1991; 9: 173177.

Lindroth P and Mopper K. High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fl uorescence derivatization with o-phthaldialdehyde. Analytical Chemistry 1979; 51: 16671674.

Lodemann E. El-Kirdassy ZHM and Wacker A. Quantitative determination of small amounts of L-penicillamine in D-penicillamine after desulfur-ization with Raney nickel. Arzneimittel-Forschung 1980; 30: 395397.

Martens J, Gnther, K and Schickedanz M. Resolution of optical isomers by thin-layer chromatography. Enantiomeric purity of D-penicillamine. Archiv der Pharmazie (Wienheim) 1986; 319: 461465.

Merino MI, Blanco GE and Sanz-Medel A. Liquid chromatographic separa-tion of penicillamine enantiomers derivatized with OPA/2-ME on a b-cyclodextrin bonded phase. Mikrochimica Acta 1992; 107: 7380.

Miyazawa T, Minowa H, Imagawa K and Yamada T. Enantiomeric separa-tion of non-protein amino acids by chiral ligand-exchange high-performance liquid chromatography. Analytical Letters 1997; 30: 867882.

Miyazawa T, Minowa H, Shindo Y and Yamada T. Chiral high performance liquid chromatographic separation of enantiomers of a-methyl-a-amino acids. Journal of Liquid Chromatography and Related Technolo-gies 2000; 23: 10611068.

Miyazawa T, Minowa H, Imagawa K and Yamada T. Separation of enantio-mers of non-protein amino acids by high-performance liquid chro-matography on a chiral ligand-exchange column. Chromatographia 2004; 60: 4550.

Nakashima K, Tomoko I, Naotaka K and Akiyama S. High-performance liquid chromatographic separation of penicillamine enantiomers labeled with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide on a chiral stationary phase. Biomedical Chromatography 1995; 9: 9093.

Natchtman F. Determination of the L-isomer in D-penicillamine by derivatization liquid chromatography. International Journal of Pharmaceutics 1980; 4: 337345.

Oi N, Hajimu K and Reiko K. Direct separation of enantiomers by high-performance liquid chromatography on a new chiral ligand-exchange phase. Journal of Chromatography 1992; 592: 291296.

Oi N, Kitahara H and Aoki F. Direct separation of carboxylic acid and amine enantiomers by high-performance liquid chromatography on reversed-phase silica gels coated with chiral copper (II) complexes. Journal of Chromatography A 1995; 707: 380383.

Okamoto M, Ueda Y, Ken-ichi T, Hiroshi N and Tadashi D. Direct enantio-meric separation of platelet-activating factor receptor antagonist SM-10661 by ligand-exchange high-performance liquid chromatog-raphy with copper (II) N,S-dioctyl-D-penicillamine complex. Biosci-ence, Biotechnology, and Biochemistry 1995; 59: 17401741.

Phelps DL, Lakatos L and Watts JL. Penicillamine for preventing retinopa-thy of prematurity in preterm infants. Cochrane Database of System-atic Reviews 2001; 1: CD001073.

Pool CT, Tam JP and Boyd JG. Ninhydrin as a reversible protecting group of amino-terminal cysteine. Journal of Peptide Research 2004; 63: 223234.

Prota G and Ponsiglione E. On the reaction of ninhydrin with cysteine and its analogues: a revision. Tetrahedron 1973; 29: 42714274.

Schlauch M and Frahm AW. A thermodynamic study of the temperature-dependent elution order of cyclic a-amino acid enantiomers on 81



a copper (II)-D-penicillamine chiral stationary phase. Analytical Chemistry 2001; 73: 262266

Schlauch M, Volk FJ, Fondekar KP, Wede J and Frahm AW. Enantiomeric and diastereomeric high-performance liquid chromatographic separation of cyclic b-substituted a-amino acids on a copper (II)-D-penicillamine chiral stationary phase. Journal of Chromatography A 2000; 897: 145152.

Schlauch M, Kos O and Frahm AW. Comparison of three chiral stationary phases with respect to their enantio- and diastereoselectivity for cyclic b-substituted a-amino acids. Journal of Pharmaceutical and Bio-medical Analysis 2001; 27: 409419.

Sotgia S, Zinellu A, Pisanu E, Pinna GA, Deiana L and Carru C. 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. Journal of Chromatography A 2008; 1205: 9093.

Stephens AD. Cystinuria and its treatment: 25 years experience at St. Bartholomews hospital. Journal of Inherited Metabolic Disease 1989; 12: 197209.

Tivesten A, Oernskov E and Folestad S. On-column chiral derivatization of D- and L-amino acids in micellar electrokinetic chromatography by zone mixing. Journal of High Resolution Chromatography 1996; 19: 229233.

Walshe JM. Penicillamine, A new oral therapy for Wilsons disease. Ameri-can Journal of Medicine 1956; 21: 487495.

Zheng Z, Lin, J and Huie CW. Chiral separations by ligand-exchange micellar electrokinetic chromatography and microemulsion electro-kinetic chromatography. Sepu 2004; 22: 309312.

Zheng ZX, Lin JM, Qu F and Hobo T. Chiral separation with ligand-exchange micellar electrokinetic chromatography using a D-penicil-laminecopper(II) ternary complex as chiral selector. Electrophoresis 2003; 24: 42214226.

82

enantioresolution of dl-penicillamine

Documents