A collaborative study to investigate the retention reproducibility of barbiturates in HPLC with a view to establishing retention databases for drug identification.

A collaborative study among 10 laboratories has been undertaken to investigate the interlaboratory reproducibility of retention measurements in a high performance liquid chromatographic (HPLC) system for separating barbiturates. The system involved an ODS-Hypersil column with an eluent of methanol:phosphate buffer (40:60, v/v) at pH 8.5; all laboratories used the same batch of packing material. The conventional methods of recording retention properties (retention times and capacity factors) gave poor interlaboratory reproducibility. Better results were obtained by expressing the retentions relative to a standard barbiturate (quinalbarbitone); relative adjusted retention times proved to be more effective than straightforward relative retention times. Retention indices based on the alkylarylketone scale were not as reproducible as the methods based on a single closely related compound. The best reproducibility was obtained using corrected capacity factors based on the retention of four barbiturates in a standard mixture. The results of the study are discussed with a view to selecting the best methods of recording retention in HPLC when considering the establishment of databases for drug identification.

Retention reproducibility of basic drugs in high-performance liquid chromatography on a silica column with a methanol-ammonium nitrate eluent. Batch-to-batch reproducibility of the stationary phase.

The effect of changing the brand and batches of the silica stationary phase used with a methanol-aqueous ammonium nitrate eluent for the high-performance liquid chromatographic separation of basic drugs has been studied. Considerable care had to be taken to obtain a reproducible eluent and the effect of small changes in the concentration of the ammonia solution were examined closely. Large differences in both the capacity factor and relative capacity factors were found for separations on columns packed with four different brands of silica. Significant differences were also observed with columns containing eighteen different batches of Spherisorb S5W, which had been manufactured over a period of several years.

Retention reproducibility of thiazide diuretics and related drugs in reversed-phase high-performance liquid chromatography.

A method has been developed for the separation of thiazide diuretics and a number of related drugs by high-performance liquid chromatography on an ODS-Hypersil column with acetonitrile-1% aqueous acetic acid as the eluent. The effects caused by changes in the separation conditions on the reproducibility and robustness of alternative methods for recording retentions (including capacity factors, retention indices based on the alkyl aryl ketone scale, and relative capacity factors compared to a thiazide standard) have been examined. The results confirm that good interlaboratory reproducibility will only be achieved when operators control the temperature of the column and use the same brand of column packing material. The retentions should be recorded using a relative method, as these were found to be virtually independent of minor variations in the eluent composition.

Retention reproducibility of basic drugs in high-performance liquid chromatography on a silica column with a methanol-ammonium nitrate eluent. The effect of the mobile phase and the operating conditions.

The reproducibility of capacity factors and relative capacity factors are compared as methods for recording retentions for the high-performance liquid chromatography of basic drugs on a silica column with methanol-aqueous ammonium nitrate as the eluent. The effects of changing the column temperature and the ionic strength, pH and proportion of organic modifier in the eluent on the retentions and selectivity have been studied. The results suggest that the mobile phase and operating conditions must be closely defined in order to obtain results of adequate reproducibility to develop a data base of retention values for interlaboratory comparisons and/or for the identification of basic drugs.

Retention reproducibility of basic drugs in high-performance liquid chromatography on a silica column with a methanol-ammonium nitrate eluent. Interlaboratory collaborative study.

The reproducibility of the separation of basic drugs on silica columns has been tested in a collaborative study between nine laboratories. The results from the different laboratories were very similar. Various methods of recording the retention properties of the drugs were compared with reference to their reproducibility and ability to discriminate between different compounds. Relative retention times, relative capacity factors, and corrected capacity factors were much better than retention times and capacity factors, although all the methods suffered from large errors with weakly retained compounds. All the work was carried out using a single batch of silica to avoid variations due to the stationary phase.

Application of retention indices based on the alkylarylketone scale to the separation of the local anaesthetic drugs by high-performance liquid chromatography.

The separation of the local anaesthetic drugs by reversed-phase high-performance liquid chromatography using a Hypersil ODS column has been examined in order to identify critical factors in the establishment of a retention database for interlaboratory comparisons. An eluent of methanol-aqueous orthophosphoric acid (15:85, v/v) (pH 2.5) containing 0.7% hexylamine has been used. The effects of small changes in the eluent composition, pH, temperature and the use of different column packing materials on the retentions of the analytes have been studied. The use of capacity factors, relative capacity factors and retention indices based on the alkylarylketone scale have been compared.