Date of Defense

4-24-1998

Department

Chemistry

First Advisor

Tore Ramstad, Pharmacia & Upjohn

Abstract

In this investigation, a method was developed for the determination of purity of the drug PNU-10483 using the technique of capillary zone electrophoresis (CZE). CZE was seen as a potential alternative to the formerly used procedure of ion-pair reversed phase highperformance liquid chromatography (HPLC). Nearly five years earlier, a similar study was performed, in which an attempt was made to develop a method that used CZE to determine the purity of bulk drug and the potency of drug tablets. While this development was mostly successful, sporadically unpredictable results reduced the method's overall viability. These outliers were thought to be caused by inconsistent performance of the vacuum system on the first-generation capillary electrophoresis (CE) instrument used, a PE 270A CE system. As a second-generation instrument had been obtained since that investigation, the procedure that was developed on the original was investigated again and improved.

Separation was carried out by using an untreated fused silica capillary (72 centimeters (cm) total, 50 cm to detector, 50 micrometers inner diameter). Sample injection was via vacuum suction at 5" Hg for 2.5 seconds. The run buffer used was 50 milrimolar (mM) sodium phosphate, monobasic, adjusted to pH 3.30 using 85% phosphoric acid. The sample buffer used was 2.5 mM sodium phosphate, monobasic. For an internal standard, pyridine was chosen, and both it and the PNU-10483 sample were added at a concentration of 0.3 milligrams/millihter. Stabilization of migration times was induced by a rinse cycle of 0.5 molar sodium phosphate, monobasic adjusted to pH 2.5 with 85% phosphoric acid. The voltage applied was 25 kilovolts, and the current observed at this level was about 30 microamps. Detection was on-line at a wavelength of 200 nanometers. Adjusted-area quantitation was used.

The method was selective, as it could separate all the known process and degradation impurities of PNU-10483, as well as its internal standard. When using normal assay concentrations, the procedure was linear over the range 50-120%. For this method, mean recovery was 100.1%, the relative standard deviation (RSD) was 0.97%, and the correlation coefficient for the first-order regression of amount found versus amount added was 0.9982. Furthermore, neither the intercept nor the slope were statistically different from zero or one, respectively, at the 95% confidence level. Residuals were random, not accumulating at any one place. For replicate injections of a single sample solution, the RSD was 0.75%. For single injections of replicate sample solutions at the 100% level, mean recovery was 99.1% and the RSD was 0.82%. At the 80% level, mean recovery was 99.6% and the RSD was 0.87%. Finally, the developed procedure was applied to several research lots of PNU-10483. In this study, the developed method was not compared directly to HPLC, but results found in the earlier study found good agreementbetween the two techniques, suggesting that CZE could serve as a valid alternative to HPLC in the purity determination of PNU-10483.

Access Setting

Honors Thesis-Open Access

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