Important Features of Chiral Derivatization Reactions and Reagents

The presence of a suitable functional group in the molecule of the analyte The prerequisite of the use of any kind of enantioseparation based on covalent, chiral derivatization is the presence of at least one functional group (amino, hydroxyl, carboxyl, epoxy, thiol, etc.) in the molecule of the chiral compound which is capable of reaction with the reactive group of the derivatizing reagent.

Good chromatographic properties of the derivatives

The optimization of the chromatographic parameters of retention time, peak shape and separability of the enantiomers from other components of the complex mixture by proper selection of the column (usually reversed-phase but in some cases normal phase columns), and the composition and pH of the eluent is done in the usual ways for achiral HPLC.

Sufficient separation of the diastereomers formed

This is also influenced by the above-mentioned sol vent composition and column selection, but in this case it is even more important to select a proper derivatizating reagent enabling diastereomers to be formed with sufficiently different molecular fine structures for their chromatographic separation. If the number of the chiral centres in the analyte is more than one, the reagent should enable the separation of all stereoisomers. An example for this is the separation of (S,R), (R,S), (S,S) and (R,R) nadolol in Figure 1.

The structural features which influence the separation of the diastereomers are:

• The distance between the two chiral centres. In the majority of cases this is two to four atoms but there are many exceptions to this rule.

• Conformational rigidity of the diastereomers favours resolution. Bulky groups in the vicinity of one of the chiral centres or the incorporation of one of them into a ring system are especially advantageous. For example, in the case of one of the most widely used chiral derivatizing reagent, GITC (2,3,4,6-tetra-O-acetyl-^-d-glucopyrasonyl iso-thiocyanate, see later) the chiral centres of the reagent are in the pyrane ring. The exchange of the acetyl groups to the bulkier benzoyl groups improves the resolution. The position and bulkiness of the remote functional groups also influences the separation of the diastereomers. In these instances, however, the direction of the influence is difficult to predict. As an example, the investigation leading to the highly efficient derivatizing agent for the amino group ( + )-2-methyl-2^-naphthyl-1,3-ben-zodioxole-4-carboxylic acid chloride is mentioned. It was found that the difference between the bulki-ness of the two substituents at C-2 favourably influences the separation and with the isomeric 5-carboxylic acids only poor separation is achievable.

• The formation of hydrogen bonds. For example, in the case of propranolol the secondary amino group of the molecule is transformed by chiral isocyan-ates or isothiocyanates to diastereomeric urea or thiourea derivatives. The formation of a hydrogen bond between the carbonyl or thiocarbonyl group of the latter and the free hydroxyl group of pro-pranolol plays an important role in the separation: the resolution deteriorates with etherification of the hydroxyl group.

Selection of the elution order This is especially important if the aim of the analysis is the determination of trace level enantiomeric impurity in a drug which is administered as the pure enantiomer. In order to have the impurity peak appear before the main peak the

Figure 1 Resolution of the four diastereomers of the two racemates of nadolol as urea derivatives after reaction with (R)-( - )-1-(naphthyl)ethyl isocyanate and their determination in dog plasma extracts. Column YMC-AM-303 ODS(250 x 4.6 mm, 5-^m); mobile phase, water-acetonitrile (60:40, v/v); flow rate, 1 mL min-1; temperature, 40°C; UV detection at 285 nm. (A) Blank control dog plasma; (B) plasma spiked with 50 ng mT1 of each diastereomer; (C) plasma obtained 2 h after oral administration of 1 mg kg"1 of racemic nadolol. Peaks: 1, (S,R)-nadolol; 2, (RS)-nadolol; 3, (RR)-nadolol; 4 (S,S)-nadolol. Reproduced with permission from Hoshino M, Yajima K, Suzuki Y and Okahira A (1994) Journal of Chromatography B 661: 281, copyright Elsevier.

Figure 1 Resolution of the four diastereomers of the two racemates of nadolol as urea derivatives after reaction with (R)-( - )-1-(naphthyl)ethyl isocyanate and their determination in dog plasma extracts. Column YMC-AM-303 ODS(250 x 4.6 mm, 5-^m); mobile phase, water-acetonitrile (60:40, v/v); flow rate, 1 mL min-1; temperature, 40°C; UV detection at 285 nm. (A) Blank control dog plasma; (B) plasma spiked with 50 ng mT1 of each diastereomer; (C) plasma obtained 2 h after oral administration of 1 mg kg"1 of racemic nadolol. Peaks: 1, (S,R)-nadolol; 2, (RS)-nadolol; 3, (RR)-nadolol; 4 (S,S)-nadolol. Reproduced with permission from Hoshino M, Yajima K, Suzuki Y and Okahira A (1994) Journal of Chromatography B 661: 281, copyright Elsevier.

proper chromatographic system (normal- or reversed-phase) has to be selected; it is even more advantageous to select a derivatizing agent that is available in both the R and S forms. Curves a and b in Figure 2 demonstrate this: the elution order of amino acids changes upon changing from R to S reagent in their derivatization with the o-phthalaldehyde-N-butyryl-cysteine reagent.

Unidirectional derivatization reaction taking place under mild conditions The most widely used reactions are completed at room temperature within 1 h and the reaction mixture can be injected directly into the chromatograph. The necessity of heating or extraction of the reaction mixture does not preclude a reaction being used and nor does the occurrence of side reactions, provided that their products do not interfere with the detection and quantification of the peaks of the main products and the side reactions do not show stereospecificity.

Enantiomeric purity and stability of the derivatizing reagent The enantiomeric purity of the reagent is one of the most important factors determining the success of the determination of the enantiomeric purity of the analyte. It is evident that when using a homochiral reagent containing its antipode as an impurity, the latter also reacts with the main component of the analyte. This results in a diastereomeric derivative which has the same retention time as that originating from the reaction of the main component of the reagent and the impurity of the analyte. It is therefore difficult to estimate if the satellite peak originates from the impurity of the reagent or from that of the analyte. If the aim of the study is the determination of the enantiomeric purity of a drug administered as pure enantiomer and the test limit for the antipode is 0.5%, the enantiomeric purity of the reagent should be at least 99.9%. If the requirement for the enantiomeric purity of the drug is higher, the purity of the reagent should be even higher. If the aim is the determination of commensurable amounts of enantiomers (e.g. in biological samples), 1-2% of the enantiomeric impurity in the reagent is tolerable.

The enantiomeric purity of the reagent can be checked if the enantiomers of the analyte (or at least one of them) are available in enantiomerically pure form. The relative peak area of the diastereomeric impurity after the reaction with the reagent will be characteristic of enantiomeric impurity of the reagent.

The enantiomeric stability of the reagent is also an important prerequisite to obtain reliable results. For example, N-trifluoroacetyl-(S)-( — )-prolyl chloride or anhydride, were found to racemize upon storage. Reagents which are available commercially at the present time fulfil this requirement.

The absence of kinetic resolution and racemization

The absence of kinetic resolution, e.g. difference between the reaction rates of the two enantiomers with the reagent and the enantiomeric stability of the analyte and its diastereomeric derivative are important prerequisites of the applicability of a reagent to a given purpose. These can be checked by comparing the peak areas of the diastereomers during and after the reaction with a racemate. The peak area should be close to unity.

Good chromophoric or fluorophoric properties of the reagent Although the primary aim of derivatiz-ation in chiral chromatography is the formation of easy to separate diastereomeric derivatives it is advantageous if, at the same time, the reagent improves the detectability of the separated enantiomers by introducing chromophoric or fluorophoric groups into their molecules. A typical example for such 'dual-purpose' fluorophoric reagents is ( —)-2-[4-(1-aminoethyl)phenyl]-6-methoxybenzoxazole. The detection limit for the enantiomers of 2-phenyl-propionic acid after derivatization with this reagent is as low as 10 fmol (1.5 pg). Many more examples of this type are presented in the next section.

It is important to note that the UV or fluorescence characteristics of the diastereomers formed are not necessarily equal and therefore have to be checked during the validation of a new method by comparing the spectra and band intensities of the dias-tereomers.

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