High performance liquid chromatography (HPLC) method development is an arduous process requiring considerable experience and
scientific judgment. This column installment presents a road map for rapid HPLC method development using a three-pronged approach.
In this approach, three distinct method templates of increasing complexity are defined: fast LC isocratic methods, generic
broad gradient methods, and multi-segment gradient stability-indicating methods. The characteristics, advantages, and limitations
of each template are described, and case studies are used to illustrate their applications. The use of this template approach
is expected to expedite the method development process, particularly during early-phase drug development.
High performance liquid chromatography (HPLC) method development is a labor-intensive and time-consuming task performed mostly
by more-experienced scientists. Paradoxically, the best method development strategy is actually no method development — that
is, if an acceptable method can be found elsewhere. In many cases, method development is unavoidable such as to support the
development of new chemical entities (NCE) (for example, new drugs or chemicals). An HPLC analytical method typically consists
of two major parts: the sample preparation procedure and the HPLC operating conditions. This column installment focuses on
the latter part of the process by first summarizing a systematic method development strategy and by proposing a simple three-pronged
There is no shortage of information on HPLC method development. Useful information can be found in chapters of HPLC books
(1,2), specialized books on method development (3–5) and pharmaceutical analysis (6,7), journal publications, short courses,
and web resources.
An abbreviated synopsis of a "common method development strategy," extracted from references 2 and 3, is included here for
the convenience of our readers. The reader is referred to the original sources for a fuller description of the steps highlighted
Defining Method Goal and Sample Type
The most important question for the analytical method's goal is: Is the method for quantitation of the main component only
(potency methods) or for purity determination (stability-indicating)? Another important question is: Is the method for quality
control (QC)? QC methods and testing in a regulated environment typically have more-stringent method performance and robustness
requirements. Sample type is also important because sample complexity dictates the column length and HPLC operating conditions,
whereas the sample matrix may impose additional requirements in sample preparation and detection.
Gathering Sample and Analyte Information
A thorough literature search can often provide a ready-to-use method or at least some useful starting points for method development.
Knowledge of the analytes such as their chemical structure, molecular weight, purity, solubility, LogD value, number of acidic
and basic functional groups and chiral centers, pK
a, absorbance maximum (λmax), toxicity, degradative pathways, reactivity, and stability are useful. Other valuable resources are material safety data
sheets (MSDS), certificates of analysis (COA), and suppliers' technical packages. Unfortunately, information about the physicochemical
properties of the molecule, while useful to avoid pitfalls, does not necessarily lead to an easier path for method development.