Liquid chromatography solvent optimization: potential pitfalls when using a black box for developing a quality separation.

ICOS and DIAMOND are two commercially available, semi-automated HPLC solvent optimization software packages. The resultant optimized chromatographic separation is dependent on a combination of the operator's objective, the capability of the software system and the appropriateness of the data input. The latter contains components that represent the match between the requirements of the algorithms used and the information content of the data on which those algorithms operated. Knowledge about the sample content, stability and potential sample-solvent interactions can have a significant effect on the quality of the optimal solvent composition that is calculated. The results generated during the optimization of the separation of a mixture of U-83,757 and a variety of related compounds illustrate the need to consider the significance of the contribution to the calculated optimal separation of each of these potential pitfalls, both individually and in combination with the mode of operation of the relevant algorithms. Our results indicate that the quality of the final result is highly dependent on the intelligence content of the data used.

Automated HPLC optimization--not all systems are the same.

ICOS and DIAMOND are two LC solvent optimization software packages commercially available from Hewlett-Packard and Unicam, respectively. U-83,757 and various related compounds were chosen as the test mixture for the comparison of these systems. Chromatographic data were collected on both systems using the same 10 mobile phase compositions, equally spaced across an iso-eluotropic plane. The comparison focused on determining the performance of both packages with respect to the prediction of the mobile phase composition required to achieve an optimal separation. Both software systems are semi-automatic, with differing amounts of operator involvement required and employing slightly different approaches to interpolating peak movements between the 10 sets of data. The predicted optimal solvent compositions are evaluated in terms of the extent the information collected across the iso-eluotropic plane was used by the various algorithms in the two systems. Our results demonstrate the importance of comprehending the component operations involved in and the limitations of any software package that is used in analytical development. The operator should always remember that both systems are simply tools and design experiments appropriately, since the quality of the final result is highly dependent on a combination of the operator's objective, the capability of the system and the appropriateness of the data input.

High-performance liquid chromatographic method for determining the enantiomeric purity of a benzindene prostaglandin by a diastereomeric separation.

An isocratic high-performance liquid chromatographic (HPLC) method was developed to determine the enantiomeric purity of a benzindene prostaglandin (U-62,840) which is currently being evaluated for pharmaceutical applications. The enantiomers were converted to diastereomeric amide derivatives using optically pure S-(--)-1-phenylethylamine (greater than 99.9%). Separation of the diastereomers was demonstrated on achiral silica-based stationary phases using a hexane-dioxane-water mobile phase and UV detection at 214 nm. Due to the use of an optical derivatizing agent, method validation addressed the issues of optical purity of the reagent and comparative rates of reaction of each enantiomer. Analytical data is reported that demonstrates quantitative derivation, linearity, accuracy, precision, and ruggedness of the optimized assay conditions. Limit of quantitation for the enantiomeric impurity was determined to be 1.1% (signal-to-noise ratio 13). A brief description of the results of a parallel study of an enantiomeric separation using various chiral HPLC columns is included.

Quantitative determination of alprostadil (PGE) in bulk drug and pharmaceutical formulations by high-performance liquid chromatography.

An adsorption high-performance liquid chromatographic method suitable for the quantitative determination of the purity of alprostadil (prostaglandin E1) bulk drug and of the concentration of alprostadil in Prostin V.R. Pediatric Sterile Solution is described. A variety of isomers and related compounds including 8-iso-PGE1, 11-epi-PGE1, 15-epi-PGE1, PGE2, 5,6-trans-PGE2, and 8-iso-PGE2 are separated from alprostadil and are quantifiable. The 2-naphthacyl ester derivatives are employed and derivatization conditions providing maximum response are described.

Quantitative determination of prostaglandins A1 and B1 in alprostadil (PGE1) by high-performance liquid chromatography.

An absorption high-performance liquid chromatography assay for the quantitative determination of prostaglandins A1 and B1 in alprostadil (prostaglandin E1) and in Prostin VR Pediatric Sterile Solution was developed. Prostaglandins A1 and B1 have been shown to be the major degradation products of prostaglandin E1. The adsorption system provided baseline resolution of the 2-naphthacyl esters of prostaglandin A1 from prostaglandin B1. Derivatization conditions providing maximal response for prostaglandins A1 and B1 while minimizing the conversion of prostaglandin E1 to prostaglandins A1 and B1 were established.

Simple GLC determination of ethylene oxide and its reaction products in drugs and formulations.

Convenient rapid GLC methods for the estimation of residual ethylene oxide, ethylene chlorohydrin, and ethylene glycol in ethylene oxide-sterilized bulk drugs and formulations prepared therefrom are described. Ethylene oxide was chromatographed using a porous polymer column; ethylene chlorohydrin and ethylene glycol were chromatographed using either polyethylene glycol 400 or a porous polymer column. All three residuals were determined from the same sample preparation for each type of drug or formulation examined. Recoveries of each of the three residuals were greater than 95% for all samples examined. Detection limits, at moderate electrometer sensitivities, were 2 microgram/g or ml for ethylene oxide and ethylene chlorohydrin and 5 microgram/g or ml for ethylene glycol. The most likely interferences are discussed.

Purity determinations by differential thermal analysis.

Measurement of the melting-point depression of impure samples was compared with a differential method that involved running the impure sample against a pure sample of the same material. Samples of highly purified benzophenone to which had been added known quantities of 4-methylbenzophenone were studied. The area under the curve was proportional to the difference in purity between the sample and reference material over the range studied, 0.3-2.0 mole % with a standard deviation of 0.10 mole %, an uncertainty only half as large as one obtained by the melting-point depression method with the same apparatus.