Rapid Optimization of a European Pharmacopoeia Monograph Method

for Analysis of Olsalazine Sodium by HPLC

Introduction
Olsalazine Sodium is the active ingredient of drug formulations used in the treatment of
ulcerative colitis. The European Pharmacopoeia provides a monograph method for the
quantitation of Olsalazine Sodium and known impurities by reversed-phase HPLC. However,
the column specified represents older silica technology that is more prone to stationary phase
degradation at the prescribed pH and the method total assay time of 55 minutes was
considered to be excessive. The goal of this work was therefore to develop a candidate
replacement method that met the following performance requirements:

• incorporated newer, more rugged column technology that was able to resolve and
quantitate Olsalazine Sodium in the presence of all known impurities.
• had an acceptable total assay time.
• performed with the robustness required for a Quality Control method.

This work describes the rapid development and optimization of an HPLC method for
Olsalazine sulphate using the DOE based Fusion AE for Galaxie automated HPLC
experimentation platform. A Quality by Design approach was used, which meant developing
a HPLC method for Olsalazine sodium and known impurities that was designed to meet
performance requirements using sound statistical experimentation principles that accurately
quantified system behavior. Method robustness was considered to be a key method
performance requirement and was also addressed.

Materials and Methods

Instrument – Varian Prostar 500CVM, Agilent 1100 HPLC (Agilent Corp., Palo Alto, CA.).
Composed of: G1311 Quaternary pump, G1313 Autosampler, G1315 Diode Array Detector,
G1316 Column Compartment,.
Columns - 150 x 4.6 mm columns with Phenyl stationary phases.
Mobile Phase - Water/0.1% Phosphoric acid as Solvent A and, Methanol/0.1% Phosphoric
acid as Solvent B
System Parameters Included as Experiment variables - Flow Rate, Gradient Slope,
Gradient Time, and Column Type.
Rapid Method Development Platform - Instrument control, data acquisition and peak
processing was done using the Galaxie chromatography data system (CDS), (Varian Inc.,
Palo Alto, CA.). Statistical experimental design, data analysis, modeling, optimization was
done using the Fusion AE for Galaxie rapid method development software (S-Matrix Corp.,
Eureka, CA.)
Experimental Method – Study factors were varied according to a model-robust screening
design generated by Fusion AE, which also transformed the design into 29 ready-to-run
methods and associated sequence file in the CDS. Experiment was run overnight in walk-
away mode on the HPLC under full control. Results data were imported from the CDS into
Fusion AE via file-less data exchange for automated analysis. Optimization solution searches
were conducted with Fusion AE numerical and graphical optimizers.

Results and Discussion

Fusion AE for Galaxie incorporates a novel and powerful method development approach by
computing and analyzing two “Trend” Responses:

Total Peaks: the total number of integrated peaks in a chromatogram.

Resolved Peaks: the number of peaks in a chromatogram with resolution

≥X (default value of X = 1.5).

The Trend Response results obtained from the experiments were analyzed using the
Automated Analysis mode of Fusion AE. The analysis included multivariate linear
regression, which provided models (equations) relating the experiment variables Pump Flow
Rate, Gradient Time, Final % Methanol, and Column Type to the trend responses.

The Fusion AE numerical and graphical optimizers utilized these models to generate the
prediction results and overlay graphic presented in Figures 1.a and 1.b. below. The Fusion
AE prediction results were graphically and numerically generated based on input goal of
“Maximize” for the two trend responses.

An overlay graph depicts instrument parameter level setting combinations that will not meet
user requirements as shaded regions in the graph. Each color corresponds to a single
response. The overlay graph presented in Figure 1.a, shows that there are only two regions
where the trend response goals can be met. These correspond to low flow rate with high
final % organic, and high flow rate with low final % organic. The “best overall” method as
predicted by the numerical optimizer and shown in Figure 1.b, corresponds to the region of
low flow with high final % organic.
Figures 1.a. & 1.b. Prediction overlay graph and
numerical optimizer prediction table – Trend
Responses.

In addition to the trend responses, Fusion AE for Galaxie also automatically imported and
analyzed the resolution response data sets for all integrated peaks in all chromatograms
and created prediction models for these responses. An overlay graph depicting the
optimum region in terms of resolving all integrated peaks is presented in Figure 2.a
below. Figure 2.b presents the “best overall” method as predicted by the numerical
optimizer.

Figures 2.a. & 2.b. Prediction overlay graph and

numerical optimizer prediction table – Resolution
Responses.
Conclusions
The Phase 1 rapid method development experiment was able to successfully develop a
candidate HPLC method for the separation of Olsalazine and known impurities using the
newer, more robust column technology. The resulting method was able to separate the
API and all known impurities in a substantially reduced run time.

The Phase 1 experiment approach required 29 experiment runs. However, the platform’s
automation enabled the runs to be done overnight in an “inject-and-forget” walk-away
mode.

The statistically rigorous experimental design and subsequent data analysis completed
during this experiment provided statistically defendable Phase 1 data that enabled these
results to be achieved.

It is important to note that the size of the unshaded region in Figure 2.a clearly indicates
that there is opportunity to further optimize the performance of this method. The results
from Phase 1 presented here, including the identification of the correct column, will serve
as the base conditions for the Phase 2 experimental design.

Literature Cited
1) Cornell, John A., Experiments With Mixtures, 2nd Edition, John Wiley and Sons, New
York, New York, 1990.
2)Montgomery, D., Design and Analysis of Experiments, Fourth Edition, John Wiley & Sons,
New York, New York, 1996.
3) Myers, Raymond H. and Montgomery, Douglas C., Response Surface Methodology,
John Wiley and Sons, New York, New York, 1995.
4) Neter, J., Kutner, M., Nachtsheim, C., and Wasserman, W., Applied Linear Statistical
Models, Fourth Edition, McGraw-Hill, Boston, Massachusetts, 1996.
7
Authors
Graham D. Shelver,, Varian Inc, Walnut Creek, CA, USA, Richard Verseput S-Matrix Corp.,
Eureka, CA, USA
Acknowledgements
The Authors would like to thank Chris Butler, UCB Pharmaceuticals and Dr Huqun Liu,
Varian Inc. for their assistance with this project