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Benzophenone was chosen as the external standard because it is stable, can be weighed out accurately, and is commercially
available at high purity. Benzophenone is also very soluble in compatible solvents, has good chromatographic properties on
open-access gradient HPLC methods, and has a high extinction coefficient at its λmax of 254 nm.
Figure 1: Sample log-on interface of the Yieldaliser software (GlaxoSmithKline, GSK). (ALL FIGURES ARE COURTESY OF BORMAN
ET AL.)
Integrating a standardized approach. To ensure that the HPLC system is functioning satisfactorily, a test mixture and blank solution are automatically injected
early in the day. Immediately afterward, six standard vials of a solution of benzophenone of known concentration (typically
0.35 mg/mL made up in methanol [MeOH]) are injected and the percentage relative standard deviation (i.e., measure of injector
precision) and average response of the benzophenone (i.e., measure of accuracy) are checked against predetermined limits.
If the performance does not fall within these limits, then the system will automatically repeat the standardization. If the
calibration fails again, then the lead user is notified by email and the system is checked for problems such as poor column
performance, solvent leaks, or autosampler issues. The assay functionality is disabled if the standardization criteria are
not met. Figure 1 shows a screenshot of the customized interface on the laboratory computer that is connected to the instrument.
The user selects either the 3- or 8-minute gradient method and then chooses to perform an analysis of impurities or assay
(the assay analysis option is highlighted in red in Figure 1 and only runs on the 8-minute gradient).
Developing sample preparation protocols
Two protocols were developed to enable the analysis of liquid samples (for generating yield in solution data) and solid samples
(assay data). These protocols were created to meet users' expectations. For example, sample preparation had to be quick, easy
to follow, and use vessels that could be disposed of at minimal cost. Chemists needed to be able to to generate data that
were sufficiently precise (within ± 3% w/w of the actual purity) for the majority of their applications. Trained analytical
chemists can perform assays with greater precision (e.g., typically ± 0.5% w/w), however this is far more resource intensive.
