Achiral assay

Determination of the drug substance is expected to be enantioselective, and this may be achieved by including a chiral assay in the specification or an achiral assay together with appropriate methods of controlling the enantiomeric impurity. For a drug product where racemization does not occur during manufacture or storage, an achiral assay may suffice. If racemization does happen, then a chiral assay should be used or an achiral method combined with a validated procedure to control the presence of the other enantiomer. 

Where the drug studied is a racemate, the pharmacokinetics, including potential interconversion, of the individual enantiomers should be investigated in Phase I clinical studies. Phase I or II data in the target population should indicate whether an achiral assay, or monitoring of only one optical isomer where a fixed ratio is confirmed, will be adequate for pharmacokinetic evaluation. If the racemate has already been marketed and the sponsor wishes to develop the single enantiomer, additional studies should include determination of any conversion to the other isomer and whether there is any difference in pharmacokinetics between the single enantiomer administered alone or as part of the racemate. 

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... 

For BA studies, measurement of individual enantiomers may be important. For BE studies, this guidance recommends measurement of the racemate using an achiral assay. Measurement of individual enantiomers in BE studies is recommended only when all of the following conditions are met (1) The enantiomers exhibit different pharmacodynamic characteristics. (2) The enantiomers exhibit different pharmacokinetic characteristics. (3) Primary efficacy and safety activity resides with the minor enantiomer. (4) Nonlinear absorption is present (as expressed by a change in the enantiomer concentration ratio with change in the input rate of the drug) for at least one of the enantiomers. In such cases, BE criteria should be applied to the enantiomers separately. 

Drug product. Control of the other enantiomer is necessary if it is a degradation product. Where the enantiomer is not a degradation product, an achiral assay may be sufficient for assay measurement, and an identity test should be established that is capable of verifying the presence of the correct enantiomer or racemate, as appropriate. 

Use of chiral versus achiral analytical assays has been the subject of much discussion in the literature. If the pharmacokinetic profile is the same for both isomers, or a fixed ratio between the plasma levels of enantiomers is demonstrated in the target population, an achiral assay or an assay that monitors one of the stereoisomers may be sufficient. 

If a racemate is being studied, the pharmacokinetics of the two isomers and their potential interconversion should be studied in Phase 1. Based on Phase 1 or 2 pharmacokinetic data in the target population, it should be possible to determine whether an achiral assay or monitoring of just one enantiomer, where a fixed ratio is confirmed, will be sufficient for pharmacokinetic evaluation. 

In general, for PK assessment of chiral compounds, the main difference from other drugs is the decision whether to use an enantioselective or an achiral assay to characterize the pharmacokinetics of each isomer or racemate, respectively. 

Already it is evident that the requirements placed upon validation of a chiral LC-MS method are appreciably more demanding than those for an achiral assay, for... 

Hsu, C.L., Walters, R.R. (1995). Assay of the enantiomers of ibutilide and artilide using solid-phase extraction, derivatization, and achiral-chiral column-switching high-performance liquid chromatography. J. Chromatogr. B 667, 115-128. 

Enantiomeric pnrity assays have also been performed without chromatographic separation being conducted prior to detection, for example, with circular dichroism (CD) and MS. Bertncci et al. [110] developed a chiral assay for pulegone, oxazepam, and warfarin by combining simnltaneons UV, CD, and g factor detection on an achiral separation system with a Hypersil CN colnmn and a mobile phase of hexane 2-PrOH (90 10). The precision (RSD%) of the method ranged from 0.6% to 2.6%, and the LOQs were between 0.1% and 1% (0.2-2.2 j,g). For fnrther information concerning the application of CD and polarometric detection for chiral detection, see the review by Bobbitt and Linder [111]. 

Unnatural (—)-ABA shows one-half to one-third of the activity of (+)-ABA in many bioassays,634 and this small difference in activity between the enantiomers has been explained by the pseudosymmetry of the molecule, which is derived from the 2,6,6-trimethyl-cyclohex-2-en-4-one.635 Figure 26 shows the steric structures of (+)- and (—)-ABAs with the preferable conformation, a half-chair with the pseudoaxial side chain, viewed from the carbonyl group at C-4. In the molecule of (—)-ABA, the C-7 corresponds to the C-9 of (+)-ABA, the C-9 corresponds to the C-7 of (+)-ABA, and the C-8 occupies the space facing the rtf-face of the C-2 in (+)-ABA, whereas a methyl group corresponding to the C-8 of (+)-ABA is absent. This hypothesis has been supported by the achiral analog (38), which shows activity intermediate between (+)- and (—)-ABAs.592 The activity of (—)-ABA is low in the assay of stomatal closure,618 which suggests that the receptor of stomata is more specific to (+)-ABA than to (—)-ABA. 

As noted previously (Section 22.2), derivatization with a chiral derivatizing reagent (CDR) requires the presence of suitable functionality (e.g., —OH, Ar—OH,—SH,—COOH,—CO—NH2,—NRH) within the chiral analyte to serve as a reactive site. Before addressing specific issues with regard to CDR and analyte classes, it may be helpful to review general considerations for achiral derivatization in chromatographic assays. 

The measurement of warfarin enantiomers in serum using coupled achiral/chiral high-performance liquid chromatography" (110), An assay for the serum concentrations of (fi)-warfarin and (S)-warfarin was developed using the BSA CSF coupled to a Pinkerton internal-surface reverse-phase (ISRP) achiral column. The ISRP column was used to separate (R,S)-warfarin from the serum components and warfarin metabolites and to quantitate the total warfarin concentration. The eluent containing the (A,S)-warfarin was then selectively transferred to the BSA CSR where the enantiomers were enantioselectively resolved (a = 1.19) and the enantiomeric composition determined. 

D2 and D3 vitamins (ergocalciferol and cholecalci-ferol) has not been equally successfulJ Vitamin D extracted from natural sources has a single conformational stereochemistry that is one of several isomers produced in synthetic preparations. To certify that the natural form is present in a synthetic product, where it can be accurately assayed in the presence of the other isomers, is a formidable analytical task. Whether direct CD detection can satisfactorily solve it is currently unknown. A prior non-selective derivati-zation reaction might be required on all isomers. The A and E vitamins are achiral and not subject to chiroptical detection unless first derivatized by reaction with a chiral host. 

The experimentally determined purity (purityexp) or activity is the amount of the API present in the material being tested. For chiral drug substances, the achiral purityexp is the amount of both enantiomers in the sample, while the chiral purityexp is the weight percent of the desired enantiomer. The purityexp is used to calculate the assay value of the batch and is used for DP manufacture and for the calculation of dosages used for toxicological testing. 

Counter-ions, usually small polar or ionic compounds, are routinely used to enhance the aqueous solubility and/or stability of the API. Because of their polarity, counter-ions are rarely resolved from the chromatographic solvent front in reversed-phase HPLC and have characteristically poor chromophores which makes detection difficult. The counter-ion can be omitted from the achiral method development sample set with minimal risk when this holds true. Analysis of counter-ions is normally performed using ion chromatography.9,10 This assay is separate from the reversed-phase assay performed to measure organic impurity levels. 

CMPA-based enantiomer separation techniques appear attractive from the viewpoints of conceptual simplicity and flexibility as they operate with relatively inexpensive achiral stationary phases and easy-to-prepare chiral mobile phases. In practice, the development of CMPA-based analytical assays may pose a considerable challenge for a number of reasons. In the course of the development and optimization of CMPA-based enantiomer separations a set of conditions must be identified that favor CMPA-analyte interactions and simultaneously maximize... 

The sample should be as pure as possible, but standards have been used where the major constituent is approximately 85% of the mixture, as long as the remainder is made up of known compounds. Suggestions to use mixtures as reference standards have been made to reduce the number of assays run and the number of samples handled. This is clearly the case for a racemic mixture in an achiral HPLC assay, where both enantiomers have the same retention and detector response. It can also be beneficial to use known mixtures of diastereomers as a standard, provided there is sound reasoning that they give equivalent response to the method of detection. 

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