Thomas Dowling

Enantiomeric separation of dansyl amino acids using MECC with a ligand exchange mechanism

Enantiomeric separation of 11 dansyl amino acids is reported using micellar electrokinetic capillary chromatography. The chiral phase consisted of a copper complex of N, N-di-decyl-D-alanine dissolved in the running buffer. Several factors affecting the separation were studied. The partitioning of the copper complex between the micelles and the buffer interface was investigated, and an attempt to describe the mechanism of separation is presented.

Use of a Quality-by-Design approach to justify removal of the HPLC weight % assay from routine API stability testing protocols

Due to the high method variability (typically > or = 0.5%, based on a literature survey and internal Merck experience) encountered in the HPLC weight percent (%) assays of various active pharmaceutical ingredients (APIs), it is proposed that the routine use of the test in stability studies should be discouraged on the basis that it is frequently not sufficiently precise to yield results that are stability-indicating. The high method variability of HPLC weight % methods is not consistent with the current ICH practice of reporting impurities/degradation products down to the 0.05% level, and it can lead to erroneous out-of-specification (OOS) results that are due to experimental error and are not attributable to API degradation. For the vast majority of cases, the HPLC impurity profile provides much better (earlier and more sensitive) detection of low-level degradation products. Based on these observations, a Quality-by-Design (QbD) approach is proposed to phase out the HPLC weight % assay from routine API stability testing protocols.

Enantiomeric separation of enzymatic hydrolysis products of dihydropyrimidinone methyl ester with cationic cyclodextrin by capillary electrophoresis.

The achiral separation of dihydropyrimidinone (DHP) methyl ester and its corresponding carboxylic acid and the chiral separation of their respective enantiomers were achieved in a single analysis using capillary electrophoresis (CE) with quaternary ammonium-beta-cyclodextrin (QA-beta-CD) as a chiral buffer additive. Separation of the DHP methyl ester from the corresponding carboxylic acid was achieved because the acid was negatively charged at pH 8.3 of the running buffer and the ester is neutral. Upon the addition of QA-beta-CD, the enantiomers of the acid and ester were well resolved before and after the electroosmotic flow, respectively. In addition, the minor DHP methyl ester enantiomer (R isomer) was well separated from several impurities. This CE system was used to monitor the progress of a bioresolution reaction that utilizes an enzyme to convert the R isomer of the ester to its corresponding acid. The quantities of all four enantiomers can be determined using a single set of CE conditions. In addition, it is demonstrated that samples can be directly injected into the capillary without sample pretreatment due to the fact that the coating of the cationic CD on the capillary surface prevents adsorption of the positively charged enzyme. The effects of other experimental parameters such as type of CDs, concentration of CDs, pH, temperature, and the preconditioning of capillary were also studied.

Electrophoretic chiral separation of pharmaceutical compounds with multiple stereogenic centers in charged cyclodextrin media

This paper reports the use of sulfated β-cyclodextrins (S-β-CDs) (degree of substitution (DS) = 7-11 and 15), hepta-6-sulfato-β-CD (HS-β-CD), heptakis (2,3-dimethyl-6-sulfato)-β-CD (HDMS-β-CD), and heptakis (2,3-diacetyl-6-sulfato)-β-CD (HDAS-β-CD) in conjunction with capillary zone electrophoresis (CZE) to separate enantiomers of several commercially available pharmaceutical compounds with multiple stereogenic centers. Compounds studied include eucatropine, fenoterol, nadolol, nafronyl, nylidrin, and pentapiperide. S-β-CD with a relatively high degree of substitution is shown to be effective in separating several of these compounds due to the high selectivity. Resolution of four isomers was achieved for several of the test compounds under counter-electroosmotic flow (EOF) conditions in less than ten minutes. Data illustrating the effects of CD concentration and pH are presented. It is also shown that the migration order of isomers can be manipulated by changing either the CD concentration or buffer pH.

Determination of bis(tributyltin) oxide by GC–MS with on-line hydride derivatization: application to drug substance analysis

We report the determination of residual bis(tributyltin) oxide in a drug substance by GC-MS after extraction and on-line conversion to tributyltin hydride. Gas chromatography was performed using a 15 m x 0.25 mm i.d. DB-5 HT column with a temperature program from 100 to 160 degrees C at 15 degrees C min(-1). A mass range of 165-185 amu was monitored with the MS detector. Hydride generation is performed by placing a small amount of solid sodium borohydride in the injection port of a gas chromatograph and injecting samples and standards through this material. Conversion to tributyltin hydride is shown to be quantitative and linear for levels of bis(tributyltin) oxide between 1 and 100 ppm in the drug substance. The use of GC-MS provides sensitive and selective detection of tin containing species and the tin isotope pattern allows for confirmation of the presence of tin in chromatographic peaks. Recovery at 6 ppm was 89% with an injection precision of 6%. The limit of detection for bis(tributyltin) oxide in drug substance is 1 ppm.

Validation of a Separation of Diastereomers in the Pharmaceutical Industry

Abstract Method validation is an important step in any method development and has important implications in the pharmaceutical industry. Particular efforts should be directed towards the reproducibility, sensitivity and ruggedness of each method developed. In this paper, we report the validation of an HPLC method for the separation of the L-699, 392 and its (S, R) diastereomer. This compound contains two chiral centers and a carboxyl functionality able to participate in a hydrogen bonding process. In order to obtain maximum sensitivity, the elution order of the two diastereomers was adjusted such that the minor diastereomer eluted before the major one. To achieve this elution order a nonpolar mobile phase consisting of methylene chloride and n-propanol containing quinine as a hydrogen bond acceptor was used. In order to optimize the separation, the influence of quinine concentration on the capacity and separation factor of the two diastereomers, influence of the polar modifier in the mobile phase and infl...

Micellar enhanced cyanide ion determination in samples from synthetic organic processes

A method was developed for the recovery and determination of cyanide ion in organic sample matrices. To facilitate the solubilization of cyanide ions, cetyltrimethylammonium bromide (CTAB) was added at concentrations above the critical micelle concentration. Sample cyanation reaction products consisted of solvent mixtures of a hydroxynitrile in DMF-toluene or DMF-isopropylacetate (IPAC). Spectrophotometric determination of cyanide ion at 578 nm by the pyridine-barbituric acid method was automated by flow injection analysis. Recovery of cyanide ion from spiked samples was 93.2% in DMF-IPAC solvent matrix and 93.9% in DMF-toluene. Low alkali concentration was observed to favor solubilization of cyanide ion in the micellar solution.