Chemistry Reference
In-Depth Information
3
HPLC Method
Development and
Optimization with
Validation in Mind
3.1 IntroductIon
Method development and optimization is the foundation of any validated method;
a properly developed and optimized method can help to ensure a method's success
upon implementation. Though the focus of this chapter is on HPLC methods, by
and large, the conceptual steps outlined here for method development and optimiza-
tion will also be applicable to many analytical procedures performed in a regulated
environment, including, but not limited to, gas chromatography (GC), capillary elec-
trophoresis (CE), or mass spectrometry (MS). In this chapter, different approaches
to method development and optimization in a regulatory environment are discussed,
along with suggested HPLC instrument configurations and software tools.
3.2 Hplc metHod development ApproAcHes
An effective analytical method development process involves evaluating and optimiz-
ing various method parameters to satisfy the stated goals of the method or procedure.
There are many literature reports of the experimental design and approach to method
development [1-7], and over the years many different approaches to HPLC method
development have evolved. The selection or development of any new or improved
method often involves tailoring existing approaches and instrumentation to the current
analytes of interest, as well as to the final objectives or requirements of the method.
It often also involves robustness (Chapter 5) or “prevalidation” studies, performed to
ensure that the resulting method is “validatable.” Perhaps the easiest and most straight-
forward method development approach is to survey the existing literature to see if
methods, either exact or related, already exist. In addition to the scientific literature,
many instrument and software vendors offer databases of existing applications, some
with methods that can be directly downloaded into a chromatography data system
(CDS), for example, the D-Library (Dionex Inc., Sunnyvale, California). Another
method development approach involves starting with the structures of the analytes and
developing the method based on information determined from these structures, either
obtained from reference material, or observed or measured. These physical-chemical
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