Chiral purity, determination

In Table 1, the typical validation parameters required for the different types of analytical procedures are listed. For all these analytical procedures CE might be an appropriate analytical technique. In fact numerous validated CE methods for pharmaceutical analysis have been described in literature during the last decade.In Table 2, an overview is listed of the ICH validation parameters included in several reported CE validation studies. Since chiral purity determination is an important application area of CE methods, this test is listed separately as a specific analytical procedure. In addition, the determination of drug counterions has been included as a separate application. This overview illustrates that in general the required validation parameters are addressed in reported CE validation studies. It should be noted, however, that the validation parameters included in Table 2 are not necessarily evaluated exactly according ICH requirements in the reported references. Many pharmaceutical companies apply a phase-related validation approach in which the depth of validation depends on the clinical phase of development of the product involved. 

CE is an important separation technique within the field of pharmaceutical analysis. CE may be an attractive choice as analytical procedure for identification, assay, or (chiral) purity determination. In addition, CE may provide distinct advantages over existing pharmacopoeial... 

Where quantitative chiral purity determinations are required, the minimum level of detection for the minor component and accuracy achieved depend on factors which include the resolution between the resonances from the diastereoisomers formed and the signal-to-noise ratio in the spectrum. 

Shapiro Ml, Archinal AE, Jarema MA. Chiral purity determination by H NMR spectroscopy. Novel use of 1,1 -binaphthyl-2,2 -diylphosphoric acid. J. Org. Chem. 1989 54 5826-5828. 

Ravard A, Crooks PA. Chiral purity determination of tobacco alkaloids and nicotine-like compounds by H NMR spectroscopy in the presence of 1,1 -binaphthyl-2, 2 -diylphosphoric acid. Chirality 1996 8 295-299. 

This is a member of an interesting class of compounds which are chiral, without actually containing a defined chiral centre. They are chiral because their mirror images are non-superimposable. In the case of this molecule, there is no rotation about the bond between the two naphthol rings because of the steric interaction between the two hydroxyl groups, d and T forms can be isolated and are perfectly stable (Optical purity determination by H NMR, D. R Reynolds, J. C. Hollerton and S. A. Richards, in Analytical Applications of Spectroscopy, edited by C. S. Creaser and A. M. C. Davies, 1988, p346). 

The chiral purity of amino acids at large enantiomeric excess can be determined automatically by derivatization with 4-fluoro-7-nitro-2,l,3-benzoxadiazole (127b) followed by CE with cyclodextrin chiral selectors and detection of the LIF excitation at 488 nm. Lod 140 ppm of L-phenylalanine in D-phenylalanine324. 

The use of chiral lanthanide shift reagents (CLSRs) for NMR enantiomeric purity determination has become very popular (6) since the first of these compounds (54a) was reported by Whitesides and Lewis (96). Reagents 54b [Eu(hfbc)3 or Eu(hfc)3] and 54c [Eu(facam)3 or Eu(tfc)3] subsequently independently introduced by Fraser (97) and Goering (98), are most widely used, and are commercially available. 

Dolezalova, M. and Tkaczykova, M., HPLC enantioselective separation of aromatic amino and hydrazino acids on a teicoplanin stationary phase and the enantiomeric purity determination of L-isomers used as drugs. Chirality, 11, 394, 1999. 

Chiral or achiral assay and purity determinations are done according to an external calibration calculation procedure, either with or without internal standardization. The calibration is performed against a 10% w/w (compared to the nominal concentration of the sample solution at 100% w/w) reference standard solution. The sample solution for the purity determination remains at the 100% w/w level, while that of the assay determination is diluted 10 times. The reason for the difference in concentration levels is similar to the purity method. A suggested sample injection sequence can be... 

Capillary electrophoresis (CE) has become a valuable technique in the analytical toolbox for pharmaceutical analysts. CE methods have been successfully applied for identification, assay, purity determination, and chiral separation. ICH guidelines should be followed in meeting regulatory approval if CE methods are used in a registration dossier. Here, the validation parameters required for different analytical procedures are described and a comprehensive overview of CE validation studies presented in literature is given. 

Hammitzsch, M., Rao, R. N., and Scriba, G. K. E. (2006). Development and validation of a robust capillary electrophoresis method for impurity profiling of etomidate including the determination of chiral purity using a dual cyclodextrin system. Electrophoresis 27(21), 4334—4344. 

Substrate/Product/Effector/Ligand Stereospecificity. Assessing the specificity for particular stereoisomers as substrates, products or effectors (surprisingly, effectors such as enzyme activators, rarely have their stereospecificities reported). In such studies, the investigators should clearly state the degree of chiral purity which is present and how it was determined. 

Huang et al. [96] developed a method for the enantiomeric purity determination of (6 )-ornidazole in raw material and injection solution that was used in an preclinical study. In this publication, a mobile phase of n-hexane, MeOH, and 2-PrOH (95 4 1) was used with a Chiralcel OB-H column. No chiral impurity (/ )-ornidazole was detected above the LOD (0.05%) in either the raw material or the injection solution (see Figure 17.4D and E). The separation of the racemate is presented in Figure 17.4A, and the minor peak in Figure 17.4B corresponds to an enantiomeric impurity of 0.5%. 

MIP assays can also be utilized in synthetic organic applications. For example, MIP-based assays have been used to measure the chiral purity of samples in organic solvents. An L-phenylalanine anilide (l-PAA) imprinted polymer was utilized as a recognition element to measure the enantiomeric excess (ee) of PAA samples (Chen and Shimizu 2002). The MIP displays greater capacity for l-PAA versus d-PAA samples of similar concentration, and this difference was used to estimate enantiomeric excess. The enantiomeric excess of an unknown solution was determined by comparing the UV absorbance of the PAA remaining in solution after equilibration against a calibration curve. This MIP assay was demonstrated to be rapid and accurate with a standard error of +5% ee. 

The classical methods for detection and quantitation of racemization require analysis of the chiral purity of the product of a peptide-bond-forming reaction. For example, the Anderson test is used to explore a variety of reaction conditions for the coupling of Z-Gly-Phe-OH to H-Gly-OEt (Scheme 6). 9 The two possible enantiomeric tripeptides are separable by fractional crystallization, so that gravimetric analysis furnishes the racemization data. This procedure has a detection limit of 1-2% of the epimerized tripeptide. A modification by Kemp,1"1 utilizing 14C-labeled carboxy components, extends the detection limit by two to three orders of magnitude by an isotopic dilution procedure. The Young test 11 addresses the coupling of Bz-Leu-OH to H-Gly-OEt, and the extent of epimerization is determined by measurement of the specific rotation of the dipeptide product. 

The precision of enantiomeric purity determinations by gas chromatography is high123 124-1 >s. This statement holds not only for small enantiomeric purities ( 0% ee), e.g., in the differentiation of a true racemate from enantiomerically slightly enriched mixtures (in reactions devoted to the amplification of optical activity under prebiotic conditions), but also for very high enantiomeric purities (— 100% ee), with detection of 1.0 to 0.1% (and less) of enantiomeric impurities (see Section 3.1.5.8). It is always advantageous if the enantiomer present as an impurity is eluted as the first peak from the gas chromatographic column (Section 3.1.5.3.). This is achieved by the proper selection of the chirality of the nonracemic stationary phase147-188 which, unfortunately, is not possible for the cyclodextrin phases. 

Alkylations of lithiated chiral 4,5-dihydrooxa7oles with 2.0 equivalents of a racemic secondary alkyl halide proceed under kinetic resolution10-16. The (S)-alkyl halide is assumed to react preferentially, and, after quenching with water, the excess (7 )-alkyl halide is isolated in 92-99% purity (determined by GC) and in 5 49% optical purity10. Hydrolysis of the alkylated 4,5-di-hydrooxazoles provides the chiral 3-alkylalkanoic acids in 99-99.8% purity (determined by GC) and 13-47% optical purity10. 

For [2.2]paracyclophane-4-carboxylic acid (25) as (—)(R) This result has been mentioned in a footnote in Ref. 1011 but seems never to have been published (see also Ref. 61). The chirality of this acid was correlated via its ( )-aldehyde with a levo-rotatory hexahelicene derivative which, according to the paracyclophane moiety at the terminal, had to adopt (A/)-helicity. Its chiroptical properties are comparable to those of hexahelicene itself101. For the (—)-bromoderivative of the latter the (A/)-helicity was established by the Bijvoet-method 102). In a later study, (—)para-cyclophane-hexahelicene prepared from (—)-l,4-dimethylhexahelicene with known chirality (which in turn was obtained with approximately 12% enantiomeric purity by asymmetric chromatography) confirmed these results. It should be mentioned that [2.2]paracyclophane-4-carboxylic acid (25) was the first planar chiral cyclophane whose chirality was determined 1041 (see also Ref.54 ). The results justmentioned confirmed the assignment (+)( ). 

The idea of two sequential detectors, one conventional the other chiroptical, is the basis of a third strategy for enantiomeric purity determinations using HPLC. It differs from the previous two by not involving a chiral separation. In it the enantiomers co-elute and the total amount is determined from an absorbance measurement. Subsequently a chiroptical... 

Squaric acid 3-Cyclobutene-1,2-dione, 3,4-dihydroxy- (2892-51-5), 76, 190 Stannanes, chiral a-alkoxyallylic, 77, 103 Stereochemical purity, determination of, 76, 52... 

Chiral lanthanide shift reagents represent the most important NMR method for optical purity determination. The potential importance of configuration for chiral drugs has... 

An interesting feature of this study is the enantiomeric purity analysis of the products. By converting the amine functionality of the pyrrolidine to a 3,5-dinitrobenzamide, the substrate can be analyzed by chiral HPLC. To date, the 3,5-dinitrobenzamide of 2-substituted pyrrolidines that the submitters have prepared have beer baseline-resolved by the Pirkle S-N1 N-Naphthylleucine column. This approach obviates the need for MPTA-derivativization which has been previously employed in enantiomeric purity determinations.3 5... 

High-performance liquid chromatography (HPLC) techniques have been used lately for enantiomeric separations of benzazocine having a chiral biaryl axis <1998TA3497> and in analytical methods for purity determination <1996JME669, 1997JME1578, 2000JME2362>. 

The addition of chiral amines to a,/(-unsaturated sulfoximines has been employed for the resolution of racemic sulfoximines 3 utilizing 0.5 equivalents of a chiral amine in chloroform 117. After completion of the reaction, the unreacted starting material is isolated by column chromatography and its optical purity determined by comparison with the reported optical rotation, or by HNMR using a chiral shift reagent. While (—)-(l/f,2.S,)-2-mcthylamino-1-phenyl-l-propanol [(l/ ,2S)-ephedrine] affords material of moderate optical purity, racemic products are isolated from addition reactions with (—)-l-phenyl-2-propanamine [(—)-am-phetamine] or ( + )-( )-l-phenylethylamine. 

---