Resolution of chiral drugs by liquid chromatography based upon diastereomer formation with chiral derivatization reagents

J. Biochem. Biophys. Methods 54 (2002) 25 – 56 www.elsevier.com/locate/jbbm

Review article

Resolution of chiral drugs by liquid chromatography based upon diastereomer formation with chiral derivatization reagents Toshimasa Toyo’oka * School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan

Abstract Chiral derivatization reagents for resolution of biologically important compounds, such as chiral drugs by high-performance liquid chromatography (HPLC), based upon pre-column derivatization and diastereomer formation, are reviewed. The derivatization reagents for various functional groups, i.e., amine, carboxyl, carbonyl, hydroxyl and thiol, are evaluated in terms of reactivity, stability, wavelength, handling, versatility, sensitivity, and selectivity. The applicability of the reagents to the analyses of drugs and bioactive compounds are included in the text. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Resolution of chiral drugs; UV and FL chiral derivatization reagents; Diastereomer formation; HPLC

1. Introduction Macro molecules, such as proteins, maintain their chirality in living organisms. Large differences in the activities of drug enantiomers are usually observed in biological system. The chiral protein environment is responsible for the different reactivities of drug enantiomers. The difference derived from chirality applies equally to all bioactive substances, such as drugs and agrochemicals. Enantioselectivity plays an important role not only in pharmacodynamics involving the interaction of bioactive agents with macromolecules (enzymes and receptors) in the target organs, but also in pharmacokinetics, involving the absorption, distribution, metabolic conversion, and excretion (ADME) of the drug. The different pharmacodynamics and pharmacokinetics of eutomers (isomers with higher affinity) and distomers (isomers with lower affinity) in racemates lead to variety of * Tel.: +81-54-264-5656; fax: +81-54-264-5593. E-mail address: [email protected] (T. Toyo’oka). 0165-022X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X ( 0 2 ) 0 0 1 2 7 - 6

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effects. For instance, the distomer sometimes exhibits undesirable side-effects, such as the hallucinogenic effect of (R)-ketamine, the mutagenicity of (R)-penicillamine, and the teratogenicity of (S)-thalidomide [1]. The distomers in some cases, such as h-blockers, may not display any serious side-effects. The a-arylpropionic acids, like ibuprofen, are all chiral molecules, and (S)-enantiomers are responsible for the desired therapeutic effect (treatment of rheumatoid arthritis). Since the (R)-isomers undergo metabolic inversion of configuration by racemase to form the active (S)-isomers, most enantiomeric drugs, except naproxen, have been administrated as racemates. The majority of naturally occurring drugs are chiral molecules and are marketed as the single enantiomer. In spite of inherent differences between pairs of enantiomers, a number of synthetic chiral drugs are sold as racemates, instead of a single enantiomer [2,3]. The Food and Drug Administrations in the USA (FDA) still accepts racemates as new chemical entities [4]. According to the guidelines of the FDA, pharmaceutical companies continue to develop chiral drugs as racemates. When the racemates are developed as drugs, however, risk and benefit of candidate chemicals must be examined in detail [5]. Thus, chiral separation is equally important in chiral synthesis as in the investigations of differences in biological effects, such as pharmacological and toxicological properties.

2. Resolution of enantiomers The principles of the chiral resolution by high-performance liquid chromatography (HPLC) are divided into two categories; one is direct resolution using a chiral stationary phase (CSP), and the other relies on diastereomer formation with a suitable chiral derivatization reagent. A third technique is the method of chiral ligand-exchange chromatography, which utilizes mobile-phase additives, such as amino acid enantiomers and metal ions, to form chelates formation between analytes and mobile-phase additives. Although a limited number of racemates, such as amino acids, are separated by the method, applications of the method to chiral drug analysis are rare. A number of enantioseparations have been achieved with the direct methods that employ CSP columns containing immobilized chiral selectors. Commercially available CSP columns are classified into cavity-phase (cyclodextrin a, h, g, etc.), helical-phase (cellulose and amylose esters), affinity-phase (a1-acid glycoprotein, ovomucoid, etc.), kdonor (semichiral columns), k-acceptor (Pirkle-type columns), and ligand-exchange columns. The separation is due to the stability difference of the diastereomeric complexes formed between the stationary phase and each enantiomer in the chromatographic system. Since there is no derivatization step in direct resolution, no racemization occurs during the reaction with a chiral tagging reagent. Consequently, the direct method, based upon CSP columns seems to be preferable for the analysis of traces of antipode enantiomers in main components, e.g., the determination of optical purity in bulk drugs. However, the resolving power of the columns and the detection sensitivity are not always adequate for sample analysis. The choice of the best column for the separation of each racemate is difficult, because the separation highly depends on the interaction between CSP and enantiomer. Furthermore, the elution order of enantiomers is also dependent upon the CSP column used, and cannot be changed easily.

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The indirect resolution, involving a derivatization step with a chiral tagging reagent, is an efficient technique for the separation of many enantiomers. The separation is based upon diastereomer formation by the reaction with a chiral derivatization reagent. This indirect derivatization method is suitable for trace analysis of enantiomers in biological samples, such as blood and urine, because of the option of coupling with highly sensitive reagents which have high molar absorptivity (e) or high fluorescence quantum yield (u). There are various reagents for HPLC that provide ultra violet (UV) or visible (VIS) absorption, fluorescence (FL), and chemiluminescence (CL) [6– 9]. In indirect methods involving a derivatization step, the choice of tagging reagent is of great importance for the resolution of chiral molecules. A pair of enantiomers is labelled with a chiral derivatization reagent to generate the two corresponding diastereomers. The separation is based on the difference of physicochemical properties (e.g., stereochemistry and stability) on an achiral stationary phase. The elution order and degree of separation of the diastereomer derived from each reagent is not easily predictable for conventional achiral stationary phases, such as octadesylsilane (ODS). As the separation is influenced by the distance between the two asymmetric centers in the substrate and the reagent, the distance should be minimize for best separation. The conformational rigidity around the chiral centers is another important factor for the separation. A resolving reagent is recommended in which free rotation near the asymmetric center of the substrate is hindered by the formation of the diastereomer. Since there is no obvious rule concerning the separation of both diastereomers, the structural differences must also be considered. Gas chromatography (GC) on capillary columns is one of the effective tools for the separation of volatile substances, owing to its excellent separating and detecting. Chiral substances analysed mainly by GC are hydroxyl (alcohols and phenols) and carbonyl (aldehydes and ketones) compounds. Since only volatile compounds are suitable for GC, a reagent that yields highly volatile derivatives is essential for the determination of nonvolatile analytes. As the separation by GC is usually carried out at high temperature, the stability of the derivatives is one of the important considerations in the choice of the reagent. HPLC is an important technique in separation science, as are GC and high-performance capillary electrophoresis (HPCE). HPLC, carried out at around room temperature, is suitable for chiral resolution, because the possible racemization during separation on the column is negligible. The separation occurs on the column, but sensitivity and selectivity must be improved in the detection step. Thus, highly sensitive and selective detection techniques, such as FL and CL, have been adopted for the determination of trace substances. This review focuses on indirect HPLC resolution by means of a chiral derivatization reagent. Enantiospecific separations of racemic compounds such as drugs and biologically active compounds, via chiral derivatization reagents are described in the text. The structures of important chiral derivatization reagents are depicted in Figs. 1– 7.

3. Reaction and detection of chiral molecules A number of optically active reagents, having various functional groups, have been developed for HPLC analysis of chiral molecules. Chiral primary and secondary amines

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are easily labeled with carboxyl, chloroformate, isocyanate, and isothiocyanate groups to yield the corresponding amides, carbamates, ureas and thioureas, respectively. Racemic carboxylic acids are usually labeled with a chiral primary amine in the presence of activation reagents (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2,2V-dipyridyl disulfide (DPDS)/triphenylphosphine (TPP)). The reaction proceeds under mild conditions at room temperature. Ester formation with a chiral alcohol is also frequently used for the derivatization of carboxylic acids. Since the reaction conditions are generally drastic, the possibility of racemization should be monitored during the reaction. The labelling of alcoholic OH groups is most difficult, because of competitive reaction with water in the reaction medium. Hence, reactions with acid chloride type reagents are mainly performed in anhydrous solvents, such as chloroform and benzene. A variety of organic reactions is suitable for the carbonyl compounds (aldehydes and ketones). However, few labels have been reported for carbonyl enantiomers in liquid chromatography (LC). They are amine- and hydrazine-type reagents that produce the corresponding nitriles and hydrazones. Since the resulting CjN structure is unstable, the derivatives are converted to CUN with reducing agents, such as sodium borohydride. Tagging of analytes with reagents that afford structures absorbing in the UV or VIS regions is the most popular means of derivatization, because most laboratories possess a UV – VIS detector and the analysts are experienced in their manipulation. It is desirable that the reagents have large molar absorptivity. There are many substances in samples that also absorb in the UV –VIS regions. Since interfere by impurities absorbing at the detection wavelength must be considered in real samples, especially in complex matrices, such as biological specimens, reagents absorbing in the visible band are preferable in terms of selectivity. However, the selectivity problem may be solved with the use of efficient columns having a high number of theoretical plates, such as capillary columns. Although a number of UV labels have been applied to the tagging of various functional groups, the sensitivity of the derivatives is not good enough for some real samples. To overcome this disadvantage, various types of FL labels have been developed. As fluorometry is both sensitive and selective, a great number of papers concerning fluorescence tagging have been published. The fluorescence properties of the substances tend to be greatly affected by temperature, viscosity of the solvent, pH of the medium, and contamination with halide ions, such as Cl and Br . It should be also noted that undesirable FL materials contaminating test samples, especially biological specimens, interfere with the determination. However, the FL label is the most effective for determinations in biological specimens, in terms of sensitivity and/or selectivity. Thus, different types of FL labelling reagents have been developed for the enantioseparation of drugs and biologically important materials. The selection of the reagent dominates the accuracy, precision, and repeatability of quantitative analysis. Several important points worthy of consideration for the choice of chiral derivatization reagent are: (i) The optical purity of the reagent should be as great as same as the chemical purity. Since the opposite enantiomer contaminating the reagent also produces a corresponding diastereomer, it is obvious that erroneous results will be obtained with the use of impure reagents. (ii) The degree of racemization during labelling reaction and storage of the reagent itself is an another important issue for quantitative determinations. Further-

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more, the chemical stability of the resulting diastereomers also influences the results. Good stability (at least 1 day) is required for many analyses, because autoanalysis overnight is usually planed. (iii) The reactivity of the reagent for each enantiomer and the fluorescence properties (wavelength and intensity) of the resulting derivatives are essentially the same. (iv) The reagent possess specificity for the target functional group and quantitatively labels the analyte under mild conditions. The resulting diastereomers exhibit an adequate detector response for sample analysis. (v) Another important point is the solubility of the reagent—whether it is freely soluble in water or misible with aqueous solvents, such as alcohol and acetonitrile—because many bioactive chiral molecules are in aqueous solution. Items (iv) and (v) are considerations similar to those for achiral quantitative analysis with achiral reagent. Item (vi) presupposes that both enantiomers of the reagent are commercially available or easily obtained by simple synthesis, because the elution order can be controlled by the selection of the reagent enantiomer. This is necessary when the determination of a trace enantiomers is required in the presence of a large amount of the antipode enantiomer. Items (i) – (vi) are of general importance for all chiral tagging reagents, not only for FL but also UV – VIS. The FL detection in HPLC provides excellent sensitivity and selectivity. However, the sensitivity is often insufficient for trace determination in real samples. Laser-induced fluorescence (LIF) detection is used in such cases. Various laser sources, such as Ar-ion, He – Cd ion, and semiconductor laser are commercially available. The minimum detectable concentrations in LIF are typically one to five orders of magnitude lower than those in FL and UV detection. That the excitation wavelengths available for use are limited with each laser source selected is an important disadvantage of LIF detection. However, it may be overcome by development of various laser sources and tagging reagents matched to the wavelengths. Some FL compounds emit light upon chemical reaction without the need of optical excitation with lamps such as the xenon arc. Since the flicker noise based on the lamp is negligible, extremely high sensitivity is theoretically obtained in this method. Indeed, trace analysis at attomole levels has been achieved with this technique in the reaction with CL reagents, such as luminol, lucigenin, and the combination of oxalates and hydrogen peroxide. In HPLC/CL detection systems, the CL reagent is mixed with the eluate after column separation and made to reacted just before the detector, because the CL reaction is very fast, and the light generated disappears in a few seconds. The CL method can be applied to the detection of diastereomers derived from FL chiral tagging reagents.

4. Tagging of various functional groups Labelling with chiral derivatization reagent is carried our by reaction with a functional group in the analyte, e.g., amine (primary and secondary), carboxyl, carbonyl, hydroxyl, and thiol. Many organic reactions are adopted for the labelling of various functional groups in analytes [6,7,10,11]. The characteristics of individual reagents (e.g., reactivity, stability, wavelength, handling, and versatility) are described in the following section.

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4.1. Amines Many biochemically important compounds, such as biogenic amines, amino acids, and drugs, have at least one amino functional group in their structure. Among various functional groups, the tagging reactions of primary and secondary amines have been extensively investigated. The major types of reactions for chiral amines, involving amino acids and amino alcohols, are based on the formation of amides, carbamates, ureas, and thioureas. The formation reactions of diastereomeric amides are widely used for the resolution of various amines. The reactions with acid chloride and chloroformate reagents rapidly proceed to produce the corresponding amides and carbamates. The acid halides are good labels, because the reaction proceeds under mild conditions at room temperature or in an ice bath. However, since hydrolysis of the reagents easily proceeds with water in the sample solution, contamination with water in the medium should be avoided. Chiral drugs possessing a – COOH group, such as naproxen and benzoxaprofen, are converted to corresponding – COCl compounds and used as the resolving reagents for chiral amines. Another amide formation is the reaction with Nsuccinimidyl ester. This type of reagent is fairly stable and can be used in aqueous media. Therefore, the main application is as a reagent for staining of biological specimens, such as cells and organs. Reagents for HPLC analysis include 1-amethoxy-a-methylnaphthaleneacetic acid N-succinimidyl ester [10] and (S)-2-methoxy2-phenylacetic acid N-succinimidyl ester [12]. 4.1.1. UV labels Although a number of UV labels for amino functional groups have been developed and applied to real sample analysis, chiral reagents that permit the enantioseparation are not so numerous. Important chiral UV-labels are shown in Fig. 1. 2,3,4,6-Tetra-O-acetyl-h-Dglucopyranosyl isothiocyanate (GITC) [13 –18], which has an isothiocyanate group as a reactive site, is one of the important labels for amines; it produces thiourea derivatives in the presence of base catalyst. The thiourea diastereomers are readily separated on a reversed-phase (RP) ODS column in spite of the great distance between the two chiral centers. The GITC method has been applied to various drug analyses: h-methylamino acids and peptide epimers containing h-methylamino acids [19], propranolol and its metabolite (4-hydroxypropranolol) in human plasma [20], salbutamol in human urine [21], bevantolol [22], terbutaline [23], and ephedrine and pseudoephedrine [24]. Methyl chloroformate ((S)( )-MCF), a-methylbenzyl isocyanate ((S)( )-MBIC), and GITC which can react with the secondary amino group of bevantolol were evaluated as chiral derivatization reagents [22]. Among them, GITC gave the best separation. The derivatization reaction with GITC generally proceeds under mild condition at room temperature within a few hours. Racemization during the reaction is negligible. However, GITC possesses no effective chromophore to provide high sensitivity. Thus, the limits of detection and quantitation are dependent upon the strength of absorbance of analytes. Similar reagents, namely the GITC isomer 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl isothiocyanate (TAGIT) [25], 2,3,4-tri-O-acetyl-a-D-arabinopyranosyl isothiocyanate (AITC) [26 –29] and 2,3,4,6-tetra-O-benzoyl-h-D-glucopyranosyl isothiocyanate (BGIT) [30] were reported and applied to drug analysis.

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Fig. 1. Structures of UV labels for the derivatization of chiral amines.

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Kleidernigg et al. [31] have synthesized three isothiocyanate-based chiral trans-1,2diaminocyclohexanes, i.e., N-3,5-dinitrobenzoyl-trans-1,2-diaminocyclohexane isothiocyanate ((SS)- and (RR)-DDITC), N-[(2-isothiocyanato)-cyclohexyl]-pivalinoyl amide (PDITC) [32], and N-[(2-isothiocyanato)-cyclohexyl]-6-methoxy-4-quinolinyl-carboxamide (CDITC) [33,34]. The derivatives obtained with DDITC, PDITC, and CDITC are well separated by RP-HPLC. The separation factors and the resolutions of the diastereomeric thioureas were higher than those of GITC derivatives, which are well established as the labels for primary and secondary amines. A number of h-blockers, such as metoprolol, normetoprolol, and carvedilol were successfully separated by the DDITC method [31]. However, the derivatization of secondary amines (e.g., bupranol, penputolol, and timolol), containing a tertiary butyl group at the a-position of the amino functional group, was not quantitative with DDITC. This may be due to steric hindrance of the bulky tert-butyl group. Although DDITC and PDITC have been developed as the UV-labels, the derivative obtained from CDITC fluoresces highly at 430 nm (excitation at 333 nm). The excitation wavelength almost matched the nitrogen laser emission line of 335 nm and one line of the He –Cd laser. The fluorescence properties make it possible to use laser-induced fluorescence detection. Nineteen proteinogenic amino acids were labelled with CDITC and separated by reversed-phase HPLC and CE, employing polyvinyl pyrolidone (PVP) as a pseudo-stationary phase [33]. Separation and reproducibility by HPLC were superior to CE. However, CE may be of interest for further application, because of the possibility of laser use. The above three chiral derivatization reagents, which show higher separation efficiency than GITC, are good labels for primary and secondary amines. However, in terms of derivatization conditions, GITC is milder than these reagents (60 jC and 2 h in the presence of sodium carbonate). The other isothiocyanate labels include 1-phenylethyl isothiocyanate ((S)( )- and (R)(+)-PEIT) [35]. Isocyanate ( – NCO)-bearing reagents, such as phenylethylisocyanate ((R)(+)- and (S)( )-PEIC) are used for primary and secondary amines [36 – 41]. (SS)- and (RR)dorzolamide (a carbonic anhydrase inhibitor) were labeled with (S)-PEIC and separated on a normal-phase silica gel column [42]. The opposite enantiomer, (R)-PEIC, was used for the determination of 5,6-dihydroxy-2-methyl-aminotetralin and catecholamines (e.g., epinephrine and isoproterenol) in biological fluids, such as plasma and serum [43]. Salbutamol enantiomers in human urine were determined with (S)( )-MBIC [44]. The derivatization reaction conditions with isocyanate reagents are more severe than those with isothiocyanate reagents and generally require more time and a higher temperature for completion of the derivatization. Chloroformates (e.g., ( )-MCF [45 –50] and (S)-tert-butyl-3-(chloroformoxy)butyrate [51]) and carboxylic acid chlorides (e.g., N-trifluoroacetyl-1-prolylchloride (TFAP-Cl) [52], 1-[(4-nitrophenyl)sulfonyl]prolyl chloride (NSP-Cl) [53,54], a-methoxyl-a-(trifluoromethyl)phenylacetyl chloride (MTPA; Mosher’s acid chloride), ( )-camphanic acid chloride [55], and benzoxaprofen chloride ((S)(+)-BZOP-Cl) [56 –58]), are important labels for amines because of their excellent reactivity. ( )-MCF is a good chiral label for primary and secondary amines. ( )-MCF was applied for the determination of metoprolol (h1-adrenergic antagonist) [59]. Prakash et al. [45] demonstrated that the reagent also reacts with tertiary amino functional group to produce carbamate diastereomer. The resulting derivatives of encainide (an anti-arrhythmic drug) enantiomers [45] are effi-

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ciently separated by normal-phase chromatography on a silica gel column. 4-(6-Methoxy2-naphtyl)-2-butyl chloroformate ((+)- and ( )-NAB-C), prepared from the prochiral non-steroidal anti-inflammatory drug, nabumetone, were compared with a couple of chiral derivatization reagents of naproxen analogs: the naproxen chloride (NAP-Cl), 1-(6methoxy-2-naphthyl)ethyl isothiocyanate (NAP-IT), and 2-(6-methoxy-2-naphtyl)-1propyl chloroformate (NAP-C) [60]. (S)( )-TFAP-Cl was used for the determination of the enantiomers of fluoxetine and norfluoxetine (anti-depressants) [61]. In this case, GCMS was adopted, owing to the high volatility of the resulting derivatives. Marfey’s reagent, 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDNP-Ala-NH2), which is a chiral derivatization reagent having an aromatic fluorine atom in the molecule, was tested for the labeling of primary and secondary amines [62]. This reagent belongs to the family of Sanger-type reagents. Fluoride is easily substituted with an amino functional group because of the strong electron-withdrawing activity of the dinitro group at the Positions 2 and 4 in the aromatic ring. Therefore, derivatization generally proceeds under mild reaction conditions. Marfey’s reagent was used for the tagging of amino acids and peptides [63 –65]. The separation and quantification of DL-phosphoserine in rat brain were performed with this reagent [66]. The detection sensitivity is 11 pmol with the ODS column and UV detection at 340 nm. The derivatized samples are stable at room temperature for at least 2 weeks. Unusual aromatic amino acids, having tetrahydroisoquinoline (e.g., DL-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) and tetraline (e.g., DL-6hydroxy-2-aminotetraline-2-carboxylic acid) ring structures were separated after derivatization with Marfey’s reagent [67]. In order to evaluate the effect of substituents in Marfey’s reagent, three Marfey’s reagents, in which alanine is replaced with other chiral aamino acids (1-fluoro-2,4-dinitrophenyl-5-L-valine amide (FDNP-Val-NH2), 1-fluoro-2,4dinitrophenyl-5-L-phenylalanine amide (FDNP-Phe-NH2), and 1-fluoro-2,4-dinitrophenyl5-L-proline amide (FDNP-Pro-NH2)) were synthesized and applied to the resolution of amino acids along with FDNP-Ala-NH2 [68]. In most cases, the separation efficiency of the diastereomers obtained from FDNP-Val-NH2 was higher than that from the other three reagents. Bru¨ckner and Gah [62] also synthesized analogs of Marfey’s reagents. The reagents representatives of the general structures FDNP-Val-NHR (R=H, tert-butyl, chiral aralkyl, phenyl, p-nitrophenyl), FDNP-Val-OR (R=H, CH3, tert-butyl), FDNP-(Ala)n-NH2 (n = 1, 2), made it possible to separate D- from L-amino acids as diastereomers. The difference in retention times in HPLC are explained from considerations of Corey – Pauling– Koltun (CPK) space-filling molecular models. h-Blockers are labelled with tertbutoxycarbonyl-L-leucine anhydride [69] in basic medium. An advantage of the chiral reagent is that the separation of the enantiomers is possible with subsequent remove of the tert-butoxycarbonyl group by treatment with trifluoroacetic acid (TFA). The derivatives of tartaric acid anhydrides, such as O,O-diacetyl tartaric acid anhydride ((R,R)-DATAAN) [70], were used for the determination of alkanolamines, such as hblockers, in biological fluids [71 – 73]. The reaction of anhydrides with h-blockers are carried out in aprotic medium, and the resulting tartaric acid monoester derivatives are easily separated by reversed-phase chromatography, owing to intramolecular hydrogenbond formation. O,O-Dibenzoyl tartaric acid anhydride ((R,R)-DBTAAN) and O,O-di-ptoluoyl tartaric acid anhydride ((R,R)-DTTAAN) were also synthesized by Lindner et al. [70]. The effect of the substituent groups of these reagents on the enantioseparations were

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compared. The separation factors increased about twice with change from the acetyl (DATAAN) to the p-toluoyl group (DTTAAN). Furthermore, the a-values of the diastereomers of all six h-blockers with DBTAAN are much larger than those with DATAAN. The results suggest that the bulkiness of the substituents in the chiral reagent molecule is one of the predominant factors in the separation [71]. 4.1.2. FL labels o-Phtalaldehyde (OPA) has been widely used for the fluorometric determination of nonchiral amines in the presence of thiols such as 2-mercaptoethanol (2-ME). The derivatization reaction essentially requires thiol compounds to produce the corresponding isoindoles. The reaction is usually completed within 5 min at room temperature. Since the resulting isoindole derivatives are unstable, labelling after separation on an analytical column is usually practiced in the determination of primary amines. The OPA method has been extended to the enantioseparation of chiral primary amines and amino acids. When a chiral mercaptan, instead of the achiral thiol, is used together with OPA for the labelling of racemic amines, a pair of enantiomers is converted to the corresponding diastereomers and is easily separated on an achiral-phase column, such as ODS. N-Acetyl-L-cysteine (NAC) [74 – 85], N-tert-butyloxycarbonyl-L-cysteine (BOC-C) [78,79,86], N-acetyl-D-penicillamine (NAP) [76 – 79], 1-thio-h-D-glucose (TG) [77, 87], D-h-mercapto-2-methylpropionic acid (MMPA) [88], 2,3,4,6-tetra-O-acetyl-1-thio-h-D-glucopyranoside (TATG) [77,87], N-isobutylyl-L-cysteine (IBLC), and N-isobutylyl-D-cysteine (IBDC) [89] have been used as the chiral mercaptans of OPA for the resolution of primary amine enantiomers (Fig. 2). A series of chiral thiols, e.g., N-propanoyl-L-cysteine (Pr-L-Cys), N-n-butanoyl-L-cysteine (n-But-L-Cys), N-tert-valeroyl-L-cysteine (t-But-L-Cys), Nvaleroyl-L-cysteine (Va-L-Cys), N-isovaleroyl-L-cysteine (iso-Va-L-Cys), and N-trimethylacetyl-L-cysteine (Tma-L-Cys), with varying chain lengths of the N-acyl group in NAC, were also synthesized [90]. Naturally occurring compounds, like L-cysteine derivatives are expected to be ideal as the chiral mercaptan, because they are available in optically pure

Fig. 2. Structures of chiral thiols for the derivatization of chiral amines by the OPA method.

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form. Actually, the purity of BOC-C was calculated to be 99.8%, judging from the apparent D-amino acid diastereomer obtained from the reaction with L-amino acids. The diastereomers of 21 pairs of amino acids, derived from OPA/BOC-C, can be resolved by reversed-phase chromatography with a linear elution gradient. However, proline does not react with OPA/BOC-C owing to the lack of a primary amino group, and cysteine forms only a weakly fluorescent derivative. The L-enantiomers all of the amino acids tested were eluted before the corresponding D-antipode. The detection wavelengths of the derivatives are around ex. 345 nm and em. 445 nm, respectively, and the minimum detectable amount is at the sub-pmol level. Nimura and Kinoshita [80] obtained adequate separation of the common protein amino acid enantiomers within 70 min in a single chromatographic run by gradient elution. Numerous amines, e.g., biogenic amines (norepinephirine and norephedrine), amino acids, amino alcohols (h-blockers), and primary-amine drugs, have been resolved with a combination of OPA and a chiral mercaptan. The N-N-oxalyl diamino acids [79], lombricine [78], tranylcypromine [81], and baclofen [82] were analyzed by precolumn labeling with OPA/chiral thiols. Desai and Gal [77] reported the enantioseparation of various primary amines and drugs: ( F )-p-chloroamphetamine (PCA), ( )-amphetamine (AMP), 3-amino-1-(4-hydroxyphenyl)butane (AHB), ( F )-1-methyl-3-phenylpropylamine (APB), ( F )-p-hydroxyamphetamine (HAM), rimantadine (RIM), tocainide (TOC), and mexiletine (MEX). OPA/NAC was used for the determination of MEX and its metabolites, hydroxymethylmexiletine (HMX), in Chagasic women with ventricular arrhythmias [91] and N-hydroxymexiletine glucuronide in female patients with arrhythmic form of chronic Chagas’ heart disease [92]. The OPA/NAC method was also applied to the determination of LL-, DD- and meso-diastereomers of diaminopimelic acid (DAP) in the peptideglycan of Gram-positive bacteria [93]. The resolution values depend upon the kind of chiral thiols and the primary amines. Relatively good separations were obtained with use of TG or TATG. Bru¨ckner et al. [94] systematically investigated the chromatographic resolvability of DL-amino acid diastereomers, derived from OPA, together with chiral Nacetylated cysteines. As a result, IBLC and IBDC were selected as chiral thiols for OPA and 41-component standards containing 17 protein amino acids, were satisfactorily resolved with a fully automated pre-column derivatization technique. Replacement of IBLC with IBDC led to a reversal in the elution order of the derivatives of DL-amino acids. The method has been applied to the determination of D-amino acids in various biological samples, such as bacteria, fungi, plants, and vertebrates. Amino acids, including nonprotein amino acids, allo-isoleucine, a-amino-n-butyric acid, g-aminobutyric acid and Dallo-threonine, were labeled with IBDC or IBLC and separated by reversed-phase HPLC with a ternary gradient system [95]. The most important FL-label-possessing chloroformate as a reactive functional group is chiral 1-(9-fluorenyl)ethylchloroformate (FLEC), developed by Einarsson et al. [96]. FLEC was initially used for the determination of amino acids by HPLC. The reaction conditions at room temperature in basic solution are mild, and the resulting FL diastereomers are stable. The rates of reaction with aromatic amino acids are faster than those with acidic amino acids, i.e., aspartic and glutamic acids. Good separation is obtained with increased hydrophobicity of the derivatives. The bulky and planar fluorene moiety in the reagent structure seems to play an important role in the separation of the diastereomers. The D-enantiomers of all of the amino acids were consistently eluted before

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the L-enantiomers. This elution order is a distinct advantage, because the L-form is the dominant component in most amino acid samples. Another advantage is the thermal stability of the carbamates derived from (+)-FLEC. Although FLEC equally labels primary and secondary amino acids, for a selective derivatization of secondary amino acids, such as proline and hydroxyproline, the labeling of the amino acids, having a primary amino group with OPA/thiol is carried out before derivatization of secondary amino acids with FLEC [96]. The enantioseparation of pharmaceuticals in synthetic mixtures and biological fluids is equally important for the resolution of amino acids and biogenic amines. Enantioseparations of chiral amines by means of FLEC were carried out with the (+)enantiomer in most of the reports. In the case of separations of biogenic amines and drugs, however, the elution of the main component may be faster than that of trace amounts of the antipode. Since ( )-FLEC shows similar optical purity and reactivity, it can be used as readily as (+)-FLEC for the chiral separation of amine enantiomers with a reversal of the elution order [97]. Therefore, trace quantities of the D- or L-enantiomers may be determined with a switch in the elution order by selecting the appropriate enantiomer of FLEC reagent. The cardiovascular drugs atenolol [98], propranolol, metoprolol, ahydroxymethoprolol, tocainide, (RS)-2-[(RS)-a-(2-ethoxyphenoxy)benzyl]morpholine methanesulfonate (reboxetine) [99], and (RS)-1-methyl-8-[(morpholin-2-yl)methoxy]1,2,3,4-tetrahydroquinoline [100], were separated by reversed-phase HPLC. The sensitivity of the methamphetamine derivative with FL detection is ca. 200 times higher than with UV detection at 254 nm [101]. Simultaneous assays of the diastereomers of D- and Lcarnitines, derived from (+)-FLEC were performed not only by HPLC but also by CZE [97,102]. As the fluorene-bearing reagents, Herraez-Hernandez et al. [103] used 9fluorenylmethyl chloroformate-L-phenylalanine (FMOC-L-Phe) and 9-fluorenylmethyl chloroformate-L-proline (FMOC-L-Pro) for the resolution of chiral amines. Although a pair of FMOC-L-Pro diastereomers was successfully separated by HPLC, the separation of FMOC-L-Phe diastereomers was a failure [104]. The difference in separatability between the diastereomers of FMOC-L-Pro and FMOC-L-Phe may be due to the rigidity of the amino acids, i.e., L-Pro is much more rigid at around its chiral center than L-Phe. Fluorogenic reactions can be classified into FL generation, and FL labelling. The reagents, FLEC and OPA/thiol, are categorized as FL generators that yield highly fluorescent products. Chiral derivatization with a fluorophore reagent is also used for indirect resolution of chiral molecules. FL chiral derivatization reagents for amines are shown in Fig. 3. The reagents having the naphthalene structure as the fluorophore, e.g., 1(1-naphthyl)ethyl isocyanate ((R)( )- or (S)(+)-NEIC) [105 –113], a-methoxy-a-methyl1-naphthaleneacetic acids (( )-MM1NA), and a-methoxy-a-methyl-2-naphthaleneacetic acids (( )-MM2NA) [114], have been used for the chiral separation of the serotonin reuptake inhibitor (fluoxetine) and its desmethyl metabolite (norfluoxetine) [109], the calcium antagonist prenylamine [N-(3,3-diphenylpropyl)-N-(a-methylphenethylamine)] [115], and h-adrenoceptor blocking agents, such as propranolol [110], betaxolol [107], mefloquine (MFQ) [113], and nadolol [106,108]. Although these chiral compounds are determined after tagging with (R)( )-NEIC, the opposite enantiomer (S)(+)-NEIC was also used for the separation of both enantiomers of acebutolol [105]. The tagging is based upon the reaction of a primary or secondary amine with an isocyanate moiety in the reagent.

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Fig. 3. Structures of FL labels for the derivatization of chiral amines.

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Toyo’oka et al. developed fluorescent chiral tagging reagents with the benzofurazan (2,1,3-benzoxadiazole) structure to permit enantioseparation of various types of racemates involving amino, carboxyl, carbonyl and hydroxyl functional groups (Fig. 4). While working on the development of chiral tagging reagents, optically active fluorescent ‘‘Edman-type’’ reagents having a NCS functional group, i.e., 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole ((S)(+)- and (R)( )DBD-PyNCS), and 4-(3-isothiocyanatopyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole ((S)(+)- and (R)( )-NBD-PyNCS), were synthesized [116]. Although the reaction of

Fig. 4. Structures of chiral tagging reagents having a benzofurazan moiety as the fluorophore.

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amino acids with these reagents proceeded at 55 jC for 20 min in the presence of a base catalyst, TEA, the labeling of h-blockers required further drastic conditions (i.e., 60 jC and 90 min) because of steric hindrance around the NH group. The thiocarbamoyl derivatives obtained from aromatic and basic amino acid enantiomers were well separated by reversed-phase chromatography [117 – 119]. The FL properties of the derivatives obtained from the DBD and NBD moieties are preferred for the analysis of real sample because of their long wavelengths (ex. 460– 470 nm, em. 530– 570 nm) and high FL intensities. These chiral reagents were applied to the determination of the stereochemical purity of synthetic peptides [120]. The enantiomers of DBD-PyNCS were used to determine DL-amino acids in foodstuffs, such as milk, yogurt and alcoholic beverages [121]. The usefulness to the resolution of various racemic amines including, h-blockers, was demonstrated for these reagents [122]. Many bioactive peptides consisting of L-amino acids have been separated from reptiles and insects. It is firmly believed that only L-amino acids participate in the function of higher animals. However, some peptides (e.g., achatin I and N-agatoxin-TK), containing D-amino acid(s) in their sequences, have been discovered in the ganglia of snails and in the venom of spiders. Thus, chiral sequence analysis of peptides and proteins, in which Lamino acids in the sequence are converted to D-amino acids, is another conceivable application for these reagents as part of a chiral Edman-degradation technique. The reactions of labelling, cleavage, cyclization, and conversion seems to proceed according to the Edman degradation with PITC. The proposed method with chiral DBD-PyNCS was applied to the discrimination of DL-amino acids in the sequences of various peptides, including D-amino acids such as [D-Ala2]-leucine-enkephalin and deltorphin II [123 –126]. As chiral reagents having benzofurazan structure, Al-Kindy et al. [127] synthesized 4-(N1-carboxyethyl-N-methyl)amino-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD-N-Me-Ala) and 4-(N-1-carboxyethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole (NBD-N-Me-Ala). These reagents react with amino functional groups at room temperature in the presence of such activation agents as DPDS and TPP. The separation efficiency of the resulting amides was compared with 4-(2-carboxypyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD-Pro) and 4-(2-carboxypyrrolidin-1-yl)-7-nitro2,1,3-benzoxadiazole (NBD-Pro). The fluorescent prolines gave better separation than the corresponding N-Me-Ala derivatives. Similary, Kondo et al. [128] synthesized N-[4-(6methoxy-2-benzoxazolyl)]benzoyl-L-phenylalanine (BOX-L-Phe) and N-[4-(6-methoxy-2benzoxazolyl)]benzoyl-L-proline (BOX-L-Pro), which possess a COOH group of an Lamino acid as the reactive site. Chiral amines were readily derivatized with these reagents in the presence of DPDS and TPP. The resulting BOX-L-Pro amides were separated by both normal-phase and reversed-phase chromatography and detected fluorometrically at 432 nm (excitation at 325 nm). The separability of the BOX-L-Pro diastereomers was much better than that of the BOX-L-Phe diastereomers. Chiral non-steroidal anti-inflammatory drugs, such as naproxen and ibuprofen, which have a 2-arylpropionic acid moiety, can be used as derivatization reagents for amines. Acid chlorides, chloroformates, isocyanates, and isothiocyanates, derived from 2-arylpropionic acid drugs have been synthesized as chiral coupling reagents for amines. These reagents include: NAP-IT [129], NAP-C [129], (S)(+)-NAP-Cl [130], flunoxaprofen chloride ((S)(+)-FLOP-Cl) [131,132], (S)(+)-BZOP-Cl [130], and flunoxaprofen isocyanate

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((S)( )-FLOPIC) [133] (Fig. 3). The separations of the diastereomers of h-adrenoceptor antagonist and anti-arrhythmic agents obtained with NAP-IT were compared with those with NAP-C. The thioureas derived from NAP-IT were readily separated by reversedphase chromatography while the resolution of the carbamates obtained by derivatization with NAP-C is insufficient. A possible explanation for the decreased resolvability is the increased distance between the two chiral centers [129]. (S)(+)-FLOP-Cl and (S)( )FLOPIC were applied to the determination of (RS)-propranolol in plasma and urine [132], while baclofen and its fluoro analogue in biological samples, i.e., urine, plasma and cerebrospinal fluid (CSF), were assayed with (S)(+)-BZOP-Cl and (S)(+)-NAP-Cl [130]. Nishida et al. [134] synthesized various FL reagents possessing 1,3-benzodiazole-4and 5-carboxylic acids (Fig. 3). The reagents with a COOH group at the C-4 position exhibited higher separation ability toward the original enantiomers than the C-5 isomers. The separation efficiency was dependent upon the chain length of the C-2 substituent; greater separation was achieved in the following order, iso-pentyl>iso-butyl>iso-propyl. Introduction of a phenyl group at the C-2 position was not effective in increasing the separation, but a h-naphtyl group significantly increased the separation. However, the reagents having an aromatic substituent showed the weakest separation for phenylalanine. Among the FL reagents, 2-tert-butyl-2-methyl-1,3-benzodioxole-4-carboxylic acid ((S)(+)-TBMB-COOH) and 2-methyl-2-h-naphthyl-1,3-benzodioxole-4-carboxylic acid ((S)(+)-MNB-COOH) were applied to the enantioseparation of amino acids by reversedphase HPLC [134 –138]. Although the derivatization conditions for amino acids were relatively mild, these reagents required activation to the acid halogenide, such as – COCl and – COF before coupling. The resulting derivatives provided a high fluorescence intensity (ca. 50 fmol) at 370 –380 nm (excitation at 310 nm). The activation of (S)TBMB-COOH to (S)-TBMB-COCl was also used for the analysis of enantiomeric diamines and diols [139], glycosyl diacylglycerols [140], and amino deoxy sugars [141]. Per-O-methylated monosaccharides (pentopyranoses and hexopyranoses) were converted to their glycosyl chlorides and coupled with the cesium salt of (S)(+)-MNBCOOH. The resulting 1-O-(S)(+)-MNB carboxylates were separated by normal-phase HPLC and determined at the pmol level [142]. 4.2. Carboxyls Carboxyl compounds are analytes as important as amines, because a wide range of drugs and biologically imporatant substances possess a carboxylic acid in their structures. The tagging is usually carried out by ester formation. The esterification with a chiral alcohols requires extremely drastic condition, like high temperature and a long reaction period in strong acid solution. The reaction proceeds smoothly with activation of the carboxylic acid to the acid chloride. However, racemization during the reaction should be considered in the case of trace determination of antipode enantiomer in excess amounts of enantiomer. Aliphatic primary and secondary amines are used as the resolving reagent for carboxylic acid labelling to yield diastereomeric amides. The amide formation reaction with chiral amine reagents in the presence of activation reagents, such as N,N-dialkylcarbodiimides, usually proceeds under relative mild conditions at room temperature. In general, the amide diastereomers are more readily separated by HPLC than the corre-

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sponding esters. The separation seems to depend upon the ease of hydrogen bonding with the stationary phase. Hence, a chiral amine reagent is favorable for the tagging of carboxylic acids, in terms of reactivity and resolution. Fig. 5 shows chiral derivatization reagents for carboxylic acids. 4.2.1. UV labels A wide variety of amines, possessing – NH2 and – NH, are essentially suitable for use in the tagging of carboxylic acids. However, amide formation by direct tagging is fairly difficult under mild condition. Thus, derivatization is usually carried out after activation of carboxylic acids. Chiral 1-phenylethylamine (PEA) [143 – 153], 1-(1-naphtyl)ethylamine (NEA) [154 – 159], and their analogs, e.g., a-methyl-4-nitrobenzylamine [160,161] and 1(dimethylamino-1-naphtyl)ethylamine (DANE) [162,163], are the most popular reagents for derivatization. (S)( )- and (R)(+)-NEA have been used for the determination of compounds of pharmacological interest, such as a leukotriene D4 antagonist in human plasma [164] and non-steroidal anti-inflammatory drugs [165,166]. The diastereomers derived from NEA may be determined not only by UV but also FL detection, due to the naphtalene skeleton (ex. ca. 285 nm; em. ca. 330 nm). However, the fluorescence properties are not always used for real-sample analysis because of the relatively short wavelengths of excitation and emission. (R)(+)-NEA was used for the analysis of free and total ibuprofen in serum and urine [159]. The amide formation with this type of reagents is performed in the presence of activation reagents, such as 1,3-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBT). 2-[4-(2-Oxocyclohexylidenemethyl)phenyl]propionic acid (CS-670), a nonsteroidal anti-inflammatory drug, and three metabolites in plasma were determined with NEA [158]. Enantiomeric ratios of the propionic acid moiety of the metabolites, i.e., M-B (unsaturated-alcohol), M-C (cis-alcohol) and M-D (transalcohol), were estimated by HPLC after conversion to the amides with (R)(+)-NEA. (S)(+)-NEIC was used for the determination of the stereoselective metabolite (MDL 16.455) of the H1-antihistamic drug terfenadine in human serum and urine [167]. (1R, 2R)- or (1S, 2S)-2-amino-1-(4-nitrophenyl)-1,3-propanediol was used for the indirect chiral resolution of chiral carboxylic acids [168]. The carboxylic acid analytes were activated with DCC before coupling to the reagents. N-Protected amino acids (Z-, BOC- and FMOC-) were labeled with these reagents and separated by reversed-phase HPLC. Recently, N-substituted perylene-3,4-dicarboximide reagents, i.e., N-(2-hydroxy-1phenylethyl)perylene-3,4-dicarboximide ((R)-HPD) and N-(a-carboxyphenethy)perylene3,4-dicarboximide ((R)-CPD), were synthesized as optically pure forms by Nakaya et al. [169]. The reagents fluoresce in the long-wavelength region at ex. 500 nm and em. 550 nm. HPD was coupled with the carboxylic acid functional group in ibuprofen to yield the ester in the presence of 4-pyrrolidinopyridine. Alternatively, CPD reacted with the amino functional group in alanine methyl ester to produce the corresponding amide. In spite of perylene structures, the detection sensitivity was not so good (ibuprofen, 1 pmol; alanine methyl ester, 13 pmol). As other chiral amine reagents, L-alanine-h-naphtylamide (L-Ala-h-NA), L-phenylalanine-h-naphtylamide (L-Phe-h-NA) [170], and L-leucinamide [171 – 180] are reported. Acetyl-DL-carnitines were labeled with L-Ala-h-NA, separated by reversed-phase chromatography, and detected at 254 nm. The reaction requires the activation of the carboxyl

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Fig. 5. Structures of chiral tagging reagents for carboxylic acids.

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group of carnitine with ethyl chloroformate. The derivatives of DL-carnitines were simultaneously eluted with acetyl-DL-carnitines within 25 min. The diastereomers of DLcarnitine esters derived from (+)- or ( )-FLEC were also successfully separated by reversed-phase HPLC and capillary zone electrophoresis (CZE) [97]. 4.2.2. FL labels The reaction of chiral amines with DANE is carried out in the presence of activation reagents, such as EDC and pyridine [181]. The resulting amide diastereomers of naproxen were separated more efficiently by normal-phase rather than by reversed-phase chromatography. In the case of N-acetylamino acids, the resolution values increased with the carbon number of the alkyl residue at the a-position [182]. Aromatic amino acids (e.g., phenylalanine) and anti-inflammatory drugs (e.g., naproxen, ibuprofen, and indoprofen) provided good resolution (Rs>3.0) [182]. Even with the naphthalene fluorophore, the maximal excitation and emission wavelengths of DANE shifted to the relatively long wavelength region (ex. 320 nm; em. 395 – 420 nm), owing to the N,N-dimethylamino group at the Position 4 [181 – 184]. The sensitivity depends upon the naphthalene moiety, and detection of 0.1 ng is achieved with naproxen. The DANE method was applied to the determination of loxoprofen and its alcohol metabolites in urine [184] and rat plasma [183] and of naproxen in human serum [181]. Highly sensitive chiral reagents, 1-(1-anthryl)ethylamine (L- and D-AEA), were developed for the derivatization of carboxylic acids by Goto et al. [185]. Although the reaction conditions are similar to those of DANE and NEA, the sensitivity (ca. 100 fmol) is much greater than that of DANE and NEA. The excellent sensitivity is due to the large fluorescence quantum yield (u) of the anthracene moiety in the reagent. No racemization during the tagging reaction with AEA occurred, even after prolonged reaction. Chiral benzofurazan-bearing reagents for carboxylic acid labelling, 4-(3-aminopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole ((S)(+)- and (R)( )DBD-APy) and 4-(3-aminopyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole ((S)(+)- and (R)( )-NBD-APy), were synthesized by Toyo’oka et al. [186 – 188] (Fig. 4). Carboxylic acids, such as N-acetylamino acids, and anti-inflammatory drugs (e.g., naproxen and ibuprofen) react with DBD-APy and NBD-APy at room temperature in the presence of DPDS and TPP as the activation agents. The resulting diastereomers are completely separated by both reversed-phase (Rs = 1.62 –6.96) and normal-phase (Rs = 2.58 –7.60) chromatography [188]. The detection limits were 20 –50 fmol with a conventional FL detector. The diastereomers, derived from NBD-APy, provide sensitive detection not only with conventional FL detection but also with argon ion (488 nm) LIF detection [187,188]. The minimum detectable levels of DBD-APy, ABD-APy and NBD-APy with the LIF were 11, 29 and 3 fmol, respectively [187,188]. The enantiomeric separation and the detection of 2-arylpropionic acid drugs, i.e., ketoprofen, ibuprofen, and flurbiprofen, after derivatization with (S)-DBD-APy and (R)-NBD-APy were carried out by reversed-phase chromatography with ESI-MS [189]. The detection limits of (S)-ketoprofen, labeled with (S)-DBD-APy and (S)-NBD-APy were 125 fmol and 131 fmol, respectively. The sensitivity was 5– 8 times higher than with the conventional FL detection. The direct resolution of the drugs labeled with achiral benzofurazan reagents, such as 4-(N,Ndimethylminosulfonyl)-7-(1-piperazinyl)-2,1,3-benzoxadiazole (DBD-PZ) and 4-(N-

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hydrazinoformyl-N-methyl)-amino-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD-COHz), was also investigated by the HPLC-electrospray ionization mass spectrometry (ESI-MS). In this case, the separation was performed on a Pirkle-type chiral stationary phase, Sumichiral OA-2500(S). Alternatively, the DBD-APy derivatives are also amenable to peroxyoxalate CL [186,188]. The detection limits are at the sub-fmol levels with the CL method. The fluorescent chiral amine derivatives, ( p-fluorophenyl)-a-methyl-5-benzoxazoleethylamine (FLOPA), ( p-chlorophenyl)-a-methyl-5-benzoxazoleethylamine (BOPA) and 6methoxy-a-methyl-2-naphthaleneethylamine (NAPA) were synthesized from the corresponding enantiomer of 2-arylpropionic acid (flunoxaprofen, benoxaprofen and naproxen) (Fig. 5) [190]. Racemic anti-inflammatory drugs (e.g., flunoxaprofen, benoxaprofen, flubiprofen, and ibuprofen) were successfully separated by normal-phase and reversedphase chromatography after diastereomer formation with (S)(+)-FLOPA. (S)(+)-FLOPA was applied to the determination of ( F )-a-phenylcyclopentylacetic acid [( F )-PCA] in human plasma and urine [191]. Kondo et al. [192,193] synthesized a series of 2-phenylbenzoxazole derivatives for carboxylic acid labeling, i.e., 2-[4-(1-aminoethyl)phenyl]-6methoxybenzoxazole (( )-APMB), 2-[4-(L-leucyl)aminophenyl]-6-methoxybenzoxazole (L-LeuBOX), 2-[4-(L-phenylalanyl)aminophenyl]-6-methoxybenzoxazole (L-PheBOX) and 2-[4-(D-phenylglycyl)aminophenyl]-6-methoxybenzoxazole (D-PgBOX) (Fig. 5). (RS)-Ibuprofens in rat plasma were determined with ( )-APMB, a derivative of 2-phenylpropionic acid [194]. Since these reagents possess an amino functional group as the reactive site, the tagging conditions and detection methods are essentially the same as for amine-type reagents. The detection limit of ( )-APMB is 10 fmol at a signal-to-noise ratio of 3 [192]. The high sensitivity with fluorometry is almost same with DBD-APy derivative [195]. It is well known that dansylated amino acids give excellent sensitivity because of high fluorescence quantum yield. Exploiting this important property, Iwaki et al. [196] synthesized the dansyl-amine derivative, 1-(4-dansylaminophenyl)ethylamine (D- and LDAPEA) for carboxylic acid enantiomers. Similarly, a chiral tagging reagent having the dansyl structure, 1-(5-dimethylamino-1-naphthalenesulfonyl)-(S)-3-aminopyrrolidine ((S)DNS-APy), was synthesized and used for the enantioseparation of carboxylic acids, such as anti-inflammetry drugs [197]. DNS-APy involves a 3-aminopyrrolidine moiety as a reactive site and a fluorophor different from DBD-APy and NBD-APy. The reactivity of DNS-APy toward 2-arylpropionic acid drugs and the separatability of the resulting derivatives were compared with those from DBD-APy and NBD-APy. Yasaka et al. [198] developed a chiral reagent involving powerful alkylating ability, (S)(+)-1-methyl-2-(2,3-naphthalimido)ethyl trifluoromethanesulfonate ((S)(+)-MNE-OTf) (Fig. 6). The reaction of the reagent with carboxylic acids requires anhydrous K2CO3 and 18-crown-6 in acetonitrile. As a more sensitive chiral fluorescent conversion reagent, Akasaka et al. [199,200] synthesized (S)(+)-2-(anthracene-2,3-dicarboximido)-1-propyl trifluoromethanesulfonate ((S)(+)-AP-OTf), which has made it possible to separate the enantiomers of chiral fatty acids with a hydroxy or methyl group at the Positions 2, 3, 4, 5, or 6 by HPLC. Four stereoisomers of beraprost sodium, which is an analog of prostaglandin I2 (PGI2) are also separated, after derivatization with the reagent, by normal-phase chromatography and detected at the fmol level [200]. As reagents possessing anthracenedicarboximido structure, (S)(+)-2-(anthracene-2,3-dicarboximido)-2-propanol ((S)(+)-

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Fig. 6. Structures of (S)(+)-1-methyl-2-(2,3-naphthalimido)ethyl trifluoromethanesulfonate, MNE-OTf, (S)(+)-2(anthracene-2,3-dicarboximido)-1-propyl trifluoromethanesulfonate, and trans-2-(2,3-anthracenedicarboximido) cyclohexanol.

AP-OH) [201] and trans-2-(2,3-anthracenedicarboximido)cyclohexanol (trans-AC-OH) [202] have also been tested for the enantiomeric separation of branched-chain fatty acids. The maximal wavelengths of FL for the derivatives obtained with the anthracenedicarboximido-bearing reagents were: excitation at 298 nm and emission at 462 nm. The diastereomeric trans-AC-OH derivatives of chiral, branched-chain fatty acids that had methyl/ethyl chirality at the Positions 2 –12 are resolved into two peaks by reversed-phase chromatography and detected at the 10 15 mol level by fluorometry [202]. The absolute configuration of a ceramide with a novel branched-chain fatty acid, isolated from the epiphytic dinoflagellate, Coolia monotis, was decided on the basis of the procedure with (RR)-AC-OH [203]. The ceramide was cleaved to 12-methylpentadecanoic acid before conversion with the reagent. The elution order of all branched-chain fatty acids tested was dependent on the position of the branched-chain methyl group and independent of the chain length. Therefore, the proposed procedure seems to be useful for the enantiomeric discrimination of methyl-branched fatty acids. There is a review concerning the development and the use of anthracenedicarboximido reagents [204]. The benzotriazole derivative (S)( )-2-[4-(1-aminoethyl)naphthyl]-6-methoxy-2H-benzotriazolyl-5-amine ((S)( )ANBT) was also reported as a tagging reagent for carboxylic acid enantiomers [205]. 4.3. Hydroxyls Hydroxyl compounds are classified into two types, alcohols and phenols. The phenolic OH is more easier to label because of ionization to form Ph –O in slightly alkaline medium. Alcoholic compounds are difficult to label because of the limited reactivity and the relatively poor stability of the reagents used [112,206,207]. Esterification with acids and acid chlorides, carbonation with chloroformates, and carbamation with isocyanates have mainly been adopted as labelling reactions for alcohols. The esterification with acid chloride reagents and the carbonation with chloroformate reagents seem to be suitable for labelling because of the good reactivity of the – COCl group in the reagent. Since the

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isocyanate reacts with alcohols to produce carbamate diastereomers at elevated temperatures, care should be taken to avoid racemization during the derivatization reaction. The ester formation with acids is also performed under severe reaction conditions, such as a strong basic medium. Therefore, the esters are normally prepared after the activation of carboxylic acid to the chloride or anhydride. Since the labelling for alcohols competes with the hydrolysis of the reagent, the reaction should be performed in anhydrous solvents under protection from moisture. Consequently, the reaction conditions should be optimized in detail for each sample. UV and FL derivatization reagents for hydroxyls are shown in Fig. 7. 4.3.1. UV labels Isocyanate-type reagents, such as NEIC, can react with hydroxyls and thiols. However, the reaction with alcohols requires more drastic conditions than that with amines. Although thiol compounds react easily with the reagent, the resulting diastereomer is not stable in many samples. Although the fluorometric detection based on the naphthyl moiety of NEIC is more sensitive, the derivatives obtained with NEIC are sometimes determined by UV detection, like those with PEIC [208]. Mosher’s acid chloride (MTPA) is a good label for the analysis of hydroxyl compounds by GC, because the derivatives are highly volatile and have excellent electron-capturing properties. The derivatives of PAH dihydrodiols are resolved not only by GC, but also by normal-phase LC [209 –212]. Although MTPA is widely used for the resolution of alcohol enantiomers, the method sometimes fails, either because of low efficiency of derivatization due to steric hindrance or due to insufficient separation of the esters. To overcome this disadvantage, chlorofluoroacetic acid ((S)- and (R)-CFA) were applied to the resolution of chiral alcohols [213]. The reaction of CFA with alcohols readily affords the corresponding diastereomers, even when other derivatization methods fail. Furthermore, the chromatographic behavior of CFA esters is superior to that of esters obtained by Mosher’s procedure. The esters derived from the reaction of various chiral alcohols and racemic 2-methoxy-2-(1naphthyl)propionic acid ((R)( )- and (S)(+)-MaNP acids) were separated by normal-phase liquid chromatography [214]. The derivatization reaction with these acid reagents usually requires various catalysts, e.g., 4-dimethylaminopyridine (DMAP) and DCC. The separatability of ( )-menthol derivatives was similar to that of Trost’s chiral acid (a-methoxyphenylacetic acid; MPA acid) derivatives [215], but higher than that of Mosher’s acid derivatives [216]. Asymmetric alcohols, formed from anthracene 1,2-oides, were also labelled with a chiral reagent, tetrahydro-5-oxo-2-furancarboxylic acid ((S)-TOF) [217] or menthenyloxyacetic acid (( )-MOA) [218 – 220]. Doolittle and Health [217] compared the separation factor of the enantiomeric esters derived from MTPA, TOF and acetoxypropionic acid ((S)-AP) by GC and HPLC. The results showed that both are similar; TOF is most suitable for acetylenic, olefinic, and aromatic alcohols, whereas AP and MTPA are recommended for aliphatic alcohols and alcoholic lactones, respectively. CarbobenzoylL-proline is used for the formation of diastereomeric esters [221]. The determination of warfarin and its metabolites in plasma and urine was carried out by the procedure with carbobenzoyl-L-proline. Although the determination of the diastereomeric ester of warfarin with UV detection at 313 nm is feasible, the sensitivity is insufficient to measure the minute amounts in biological samples. In this paper, therefore, detection was performed by FL after

T. Toyo’oka / J. Biochem. Biophys. Methods 54 (2002) 25–56

Fig. 7. Structures of chiral tagging reagents for alcohols.

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post-column aminolysis with n-butylamine. The limit of detection for the enantiomers was improved to 50– 100 ng. Shimizu et al. [222,223] synthesized two chiral tagging reagents for alcohol enantiomers, N-(1-naphthylsulfonyl)-2-pyrrolidinecarbonyl chloride ((S)( )-NSPC) and 2-(2naphtyl)propionyl chloride (D-NPC), containing the – COCl group as a reactive group. The reaction of alcohols with these reagents proceeds in an aprotic solvents, such as chloroform, in the presence of a base catalyst (e.g., TEA) under mild conditions at room temperature. Since – COCl-type reagents decompose readily in aqueous reaction media, considerable care should be exercised to avoid moisture. Four isomers (DL-cis and DLtrans) of deacetyl diltiazem labelled with NSPC were completely separated by normalphase chromatography. The resulting derivatives were monitored at 254 nm. However, FL detection based on the naphtalene moiety is probably effective. 2-Methoxy-2-(1-naphthyl)propionic acid (a-MNPA) [224], a-cyano-a-fluoro(2-naphtyl)acetic acid (2-CFNA) [225] and fluoro-(1-naphtyl)acetic acid (1-FNA) [226] were reported as chiral derivatization reagents having a naphthalene moiety. a-MNPA was used for the enantiomeric resolution of monoterpene alcohols by HPLC with monitoring by 1HNMR [224]. 2-CFNA was superior for the determination of enantiomeric excess (ee) of primary alcohol, when compared to MTPA and a-cyano-a-fluoro( p-tolyl)acetic acid (CFTA) [225]. The esters derived from the reaction of 1,2-diglycerides, such as 1,2diolein, with 1-FNA were well separated by normal-phase HPLC [226]. Comparison of several chiral naphthylacetic acids showed that florinated acids provided good overall separation of chiral alcohols, especially 1,2-diglycerides [226]. The ester derivatives of aMNPA, 2-CFNA and 1-FNA were detected at UV 300, 254, and 280 nm, respectively. Harada et al. [227] developed a chromatographic method for the resolution of acyclic secondary alcohols differing in absolute configuration by using 1,5-difluoro-2,4-dinitrobenzene (FFDNB) and leucinamide. The method is based on the formation of a fixed favorable conformation of a secondary alcohol-2,4-dinitrophenyl-5-leucinamide (DLA) derivative and its recognition by ODS. Secondary amino acids were allowed to react with FFDNB under basic condition and then L-leucinamide or DL-leucinamide was introduced into the FDNB derivative (Fig. 7). The conformation of the resulting alcohol-DLA derivatives was rigidly fixed by the dinitrobenzene plane. The proposed method was successfully applied to various chiral acyclic secondary alcohols, including chloramphenicol and 4-hydroxyphenyllactic acid. 4.3.2. FL labels Use of the chiral reagent FLEC, introduced for the resolution of amines, has been extended to the enantiomeric separation of a-hydroxy acids, such as lactic, mandelic, and malic acids [228]. In this case, FLEC forms esters with hydroxy functional group in ahydroxy acids. A unique chiral axis reagent, 2-methyl-1,1V-binaphthalene-2V-carbonyl cyanide (( )and (+)-Methyl-BNCC), with a binaphthalene moiety as the fluorophore and carbonyl cyanide as a group reactive toward the hydroxyl function has been developed by Goto et al. [229]. The tagging reaction progresses favorably with heating at 60 jC in the presence of 0.01% quinuclidine as a base. The diastereomeric esters derived from pairs of h-hydroxy acids and h-blockers are efficiently resolved by normal-phase chromatography, using

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organic solvents such as n-hexane and ethyl acetate as the eluents [206]. Detection wavelengths of 340 nm (excitation) and 420 nm (emission) are typical of the naphthalene structure. Although sub-pmol detection of alcoholic compounds is possible with the derivatization method, the sensitivity might not be adequate for quantitation of enantiomeric alcohols at trace amounts in biological fluids. To obtain high sensitivity, Goto et al. [230] also prepared a similar tagging reagent, (aS)-2V-methoxy-1,1V-binaphthalene-2-carbonyl cyanide (Methoxy-BNCC), which has a methoxy group substituting for the methyl group at the 2V-position. The limit of detection was improved to the 100 fmol level. The enhancement of the sensitivity is due to substitution of the methoxy group, being an electron-donating group on an aromatic nucleus. Many methods for the resolution of enantiomeric h-adrenergic blocking agents involve derivatization with a chiral reagent through a secondary amino functional group. Drugs with a tert-butylamino structure (e.g., bucumolol, carteolol) are less reactive than those with an iso-propylamino structure (e.g., atenolol, propranolol), owing to steric hindrance. Therefore, the quantitative determination of tert-butyl-type compounds by tagging of the amino group is fairly difficult. Whereas the reactive site for Methoxy-BNCC or Methyl-BNCC is the secondary alcoholic group on hblockers, there is equal reactivity with all of the drugs. The method based on MethoxyBNCC was applied to the determination of racemic penbutolol sulfate in dog plasma [207]. 4-(2-Chloroformylpyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole ((S)( )- and (R)(+)-DBD-Pro-COCl) and 4-(2-chloroformylpyrrolidin-1-yl)-7-nitro2,1,3-benzoxadiazole ((S)( )- and (R)(+)-NBD-Pro-COCl) have been developed as chiral reagents for hydroxyl enantiomers [231 – 234] (Fig. 4). These reagents are readily prepared from the corresponding proline derivatives (DBD-Pro and NBD-Pro) by chlorination with PCl5. The acyl halide group exhibits excellent reactivity with hydroxyls in the presence of a HCl scavenger, such as pyridine. Anhydrous organic solvents, such as benzene, are required as the reaction medium, because this type of reagent is sensitive to hydrolysis by moisture. Not only the racemic alcohols (e.g., 2-heptanol and 1-phenylethanol) but also amines (e.g., 1-phenylethylamine and 1-(1-naphtyl)ethylamine) were completely resolved by normal-phase chromatography, while the resolution values in reversed-phase chromatography were relatively small [232]. The reaction with alcohols yields corresponding esters; while diastereomic amides are produced from the reaction with primary and secondary amines. The resolution values in normal-phase chromatography are larger than those in reversed-phase chromatography. When (S)( )-NBD-Pro-COCl is selected as the derivatization reagent, the diastereomers corresponding to the S-configurations are eluted before than those from R-configurations. Opposite elution orders were observed with the use of (R)(+)-NBD-Pro-COCl. 4.4. Thiols, aldehydes, ketones, etc. Numerous chiral reagents have been developed for the labeling of various functional groups. The mercapto group is highly reactive with electrophiles. However, only a few examples are reported for the labelling of thiol compounds. This seems to be due to the low stability of thiols and their derivatives. Possible reagents are of the isothiocyanate type, which are synthesized for the tagging of amines and produce the corresponding dithiocarbamates. The resolution of chiral thiols by HPLC is successsfully carried out with

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GITC which is a familiar reagent for the analysis of primary and secondary amines. In general, the resulting dithiocarbamates are less stable than the thiocarbamoyl derivatives. Other NCS-type reagents, DDITC [31] and PDITC [32], are also applicable to the labeling of SH groups. Separations of the resulting dithiocarbamates are much better than those with GITC. Chiral DBD-PyNCS, reported as the FL reagent for amines and amino acids also reacts with chiral thiols and yields the dithiocarbamate diastereomers [235,236]. The limit of detection in reversed-phase chromatography is sub-pmol level. Recently, the determination of the configuration and stereochemical purity of cysteine residues in peptides has been carried out by utilizing FDNP-Val-NH2 (Val-Marfey’s reagent) [237]. The procedures consist of oxidation of cysteine and cystine residues to cysteic acid, using 30% H2O2 in acetic acid, followed by hydrolysis with TFA/6N HCl, and pre-column derivatization with Val-Marfer’s reagent. The method was applied to the determination of the configuration and epimerization of cysteinyl residues in synthetic and natural peptides, including oxytocin. OPA has been widely used as a label for chiral amino acids in the presence of a chiral mercaptan, such as NAC and NAP. The combination of OPA and a chiral a-amino acids permits the resolution of racemic thiols by HPLC [238]. Of all of the L-amino acids tested as the chiral amine, L-valine gave the best selectivity for racemic thiols, such as cysteine, 3-mercapto-1,2-propanediol, and 1-mercapto-2-propanol. Various types of reactions are possible for the derivatization of carbonyl compounds, aldehydes, and ketones. However, reactions for chromatographic resolution are relatively scarce. Chiral compounds having hydrazino ( –NHNH2) and primary amino (– NH2) groups are usable for the derivatization of aldehydes and ketones and produce the corresponding hydrazones and oximes [239 – 242]. As reagents for chiral ketones, optically active 4-(N,N-dimethylaminosulfonyl)-7-(2-carbazoylpyrrolidin-1-yl)-2,1,3-benzoxadiazole (DBD-ProCZ) and 4-nitro-7-(2-carbazoylpyrrolidin-1-yl)-2,1,3-benzoxadiazole (NBD-ProCZ) were synthesized by the reaction of DBD-Pro-COCl and NBD-ProCOCl with NH2NH2 [243] (Fig. 4). The tagging reactions for aldehydes and ketones proceed under mild conditions at 65 jC for 10 min in the presence trichloroacetic acid (TCA). However, the separation was not adequate for racemic ketones (i.e., 2-phenylcyclohexanone, 2-phenylcycloheptanone, and 1-decalone).

5. Conclusion and further perspective Enantiomeric resolution of numerous chiral compounds has been carried out by direct HPLC, using a CSP column. However, the direct resolution of racemates is usually not adequate for trace analysis in biological specimens, in terms of sensitivity and selectivity. In spite of many problems associated with the indirect method based on diastereomer formation, i.e., optical purity of the reagent, stability of the reagent, possibility of racemization during tagging reaction, and availability of the reagent, the good sensitivity and selectivity of indirect methods, based on fluorescent chiral derivatization reagents, are attractive for the determination of chiral molecules in real-sample analysis. Since the optical purity of chiral derivatization reagents is generally less than 99%, an accurate assay of trace quantities of enantiomers in large amounts of antipodes is relatively difficult with

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indirect methods. Thus, this approach is recommended for such analyses as metabolic studies in biological specimens, because the % CV is usually in the acceptable range of error. The resolution of racemates is largely influenced by the selection of the chiral tagging reagent, derivatization conditions, and chromatographic conditions. The pre-treatment of real samples is another important topic in trace analysis. In the analysis of real samples, such as biological, environmental and food samples, the most significant and major part of the procedure involves the effective sampling of trace analytes from complicated matrices of proteins, fats, and minerals. Sample pre-treatment, i.e., cleanup and concentration of analytes, is inevitable for HPLC measurement with derivatization. Although many chiral derivatization reagents have been developed for various functional groups such as amines, carboxyls, and alcohols, and applied to real-sample analysis, there is no effective reagent for the tagging of tertiary amines, lactones, alkenes, and alkynes. Development of selective and sensitive derivatization reagents for these functional groups is needed. Various modifications of the labelling properties, based upon development of new types of reactions, would provide new reagents for UV – VIS, FL, CL and EC detection. The chiral resolution of important compounds, such as drugs and biogenic amines, has mainly been carried out with conventional HPLC systems. Micro columns of diameters < 1 mm are now on the market. Their use not only increases the sensitivity but also reduces the consumption of harmful solvents. Since the miniaturization of HPLC instruments and analytical columns is in progress, chiral resolution will soon be affected in short run-times and with high sensitivity. HPCE is another attractive means of chiral separation. CE is becoming a popular technique for separating various organic low- and high-molecularmass compounds [244,245]. Capillary electrochromatography (CEC) provides very good separation and detection for chiral compounds more hydrophobic than the compounds suited to CE analysis. Consequently, CEC methods may become powerful techniques in chiral resolution, occupying a middle position between HPLC and HPCE.

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