Chiral assay procedure

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. 

The guideline states that the objective of validation is to demonstrate that an analytical method is fit for its purpose and summarizes the characteristics required of tests for identification, control of impurities and assay procedures (Table 13-2). As such, it applies to chiral drug substances as to any other active ingredients. Requirements for other analytical procedures may be added in due course. 

The trends among the parameters studied by the UNCA racemization assay have been borne out when other activation methods are used. By a modification of chiral HPLC procedure below, the assay can be used to study any method of activation (for example, uronium or phosphonium methods), keeping in mind that the activation must be rapid and that epimerization by multiple mechanisms becomes possible. These findings also hold for solid-phase synthesis reactions. 

Bonato and Paias [136] developed two sensitive and simple assay procedures based on HPLC and capillary electrophoresis for the enantio-selective analysis of omeprazole in pharmaceutical formulations. Racemic omeprazole and (S)-omeprazole were extracted from commercially available tablets using methanol-sodium hydroxide 2.5 mol/1 (90 10). Chiral HPLC separation of omeprazole was obtained on a ChiralPak AD column using hexane-ethanol (40 60) as the mobile phase and detection at 302 nm. The resolution of omeprazole enantiomers by capillary electrophoresis was carried out using 3% sulfated /1-cyclodextrin in 20 mmol/1 phosphate buffer, pH 4 and detection at 202 nm. 

The purpose of this study is only intended to illustrate and evaluate the decision tree approach for CSP prediction using as attributes the 166 molecular keys publicly available in ISIS. This assay was carried out a CHIRBASE file of 3000 molecular structures corresponding to a list of samples resolved with an a value superior to 1.8. For each solute, we have picked in CHIRBASE the traded CSP providing the highest enantioselectivity. This procedure leads to a total selection of 18 CSPs commercially available under the following names Chiralpak AD [28], Chiral-AGP [40], Chiralpak AS [28], Resolvosil BSA-7 [41], Chiral-CBH [40], CTA-I (microcrystalline cellulose triacetate) [42], Chirobiotic T [43], Crownpak CR(-i-) [28], Cyclobond I [43], DNB-Leucine covalent [29], DNB-Phenylglycine covalent [29], Chiralcel OB [28], Chiralcel OD [28], Chiralcel OJ [28], Chiralpak OT(-i-) [28], Ultron-ES-OVM [44], Whelk-0 1 [29], (/ ,/ )-(3-Gem 1 [29]. 

In November 1997, the Department of Health and Human Services along with the International Conference on Harmonisation (ICH) released a draft guidance for the selection of test procedures, which included chiral drugs. For the development of an enantiopure drug substance, acceptable criteria shall include, if possible, an enan-tioselective assay. This assay should be part of the specification for the identification of an enantiopure drug substance and related enantioenriched impurities [16]. 

Amino acid enantiomers can be separated on a chiral stationary phase after derivatization with chloroformates (Abe et al., 1996). The derivatization procedure is quite simple and rapid, but the derivatizing reagent must be synthesized, which complicates the assay. Another method for the analysis of amino acid enantiomers uses N,0-pentafluoropropionyl isopropyl derivatives and a chiral column with NPD detection (Hashimoto et al., 1992). 

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. 

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... 

Methods have successfully been transferred to various laboratories in inter-company cross-validation exercises for a chiral separation, for an assay of the main component in a formulation and for drug stoichiometry. Revalidation is an alternative to method transfer in case of changes in product composition or analytical procedure (cf. Section I.L). Although a method transfer in CE is not a major difficulty, some aspects have to be considered, especially if a method is transferred to an instrument of another manufacturer. 

S-Bioallethrin. The pyrethroid S-bioallethrin (1R,3R,4 S) and its inactive isomer (1S,3S,4 R) can be readily distinguished by another ELISA procedure, illustrating the assay s ability to determine chirality at the residue level (Figure 1). Antibodies were raised against S-bloallethrln using the allethrin hemisucclnate conjugated to various proteins (11-12). 

Camphorsulfonyl chloride has been widely used as a chiral deriva-tizing agent for the assay of enantiomeric purity of alcohols and amines by NMR techniques. A typical procedure for the preparation of the sulfonate ester or sulfonamide involves mixing a solution of the alcohol or amine in CH2CI2 with camphorsulfonyl chloride in the presence of an amine base (EtsN, py, or DMAP). This reagent has been particularly valuable for determining the enantiomeric purity of secondary alcohols (1, 2) and p-hydroxy esters (3). ... 

The protocol for separating a partial racemic mixture calls for the chromatographic conditions to be modified in such a way that the minor component elutes first and is not lost in the trailing edge of the band for the major component. Errors encountered in the determination of enantiomeric excesses when they are in the range of 98-100%, or close to unique protocol is required for every chiral analyte assayed by chiral-HPLC, requiring considerable development time and constant review of the procedure. 

In the past, pharmacokinetic and pharmacodynamic investigations of chiral drugs have neglected the influences of stereoisomerism. This is primarily a result of the lack of stereospecific analysis procedures. Nonstereospecific assays give pharmacokinetic and pharmacodynamic information which represents a complex combination of the characteristics of the separate stereoisomers. With the advent of stereospecific analysis procedures a better understanding of drug kinetics and action as possible. 

For special cases such as these, and also in some of the earlier CSIRO survey work, laboratory extraction and titration was employed to estimate alkaloid content results were expressed as percentage of dry plant material assuming a mean molecular weight of 3(X) 119, 20. Quantitative determination was also used in the survey of Solarium species for alkaloids, a colorimetric procedure being applied which was based on solasodine as the standard [23]. In addition, a GLC method was developed by CSIRO workers for assaying pyrrolizidine alkaloids [124] vicinal hydroxyls were first converted to the corresponding alkyl boronate derivatives and other hydroxyls protected by trifluoroacetylation, then the derivatised alkaloid mixture was separated on a chiral-phase column. 

Camphorsulfonyl chloride has been widely used as a chiral deriva-tizing agent for the assay of enantiomeric purity of alcohols and amines by NMR techniques. A typical procedure for the... 

Currently employed HPLC methods for pantothenic acid and/or pantothenates have been applied solely to pharmaceuticals and simple matrices such as fortified infant formulas, whereas assays of coenzyme A and its acyl analogs have also been successfully performed on animal tissues. In the last few years, chiral stationary phases have been developed for optical resolution of pantothenic acid and related compounds by HPLC, and also HPLC-MS has become a promising technique. However, the newly developed HPLC procedures still require increased sensitivity and selectivity to make them applicable for the analysis of the total vitamin content in complex matrices such as foods and feeds. 

Several randomly and selectively substituted CD derivatives have been used as chiral selectors in CE. The suppliers of randomly substimted CD derivatives try to provide their products in a reproducible quality and well-characterized form. Nevertheless, the CD derivatives with a known derivatization pattern are recommended to be used for more or less deeply going into mechanistic studies as well as for the development of validated chiral CE assays. Derivatization of a CD in a designed way by selective activation and protection of the hydroxyl groups on the CD rim is a well-established procedure in carbohydrate chemistry. 

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