A Three-Pronged Template Approach for Rapid HPLC Method Development

Aug 01, 2013
Volume 26, Issue 8, pg 455–461

High performance liquid chromatography (HPLC) method development is an arduous process requiring considerable experience and scientific judgement. This column instalment 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 liquid chromatography (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 labour-intensive and time-consuming task mostly performed 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 for example, to support the development of new chemical entities (NCE) (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 instalment focuses on the latter part of the process by first summarizing a systematic method development strategy and then proposing a simple three-pronged template approach.

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 below.

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)? Typically, QC methods and testing in a regulated environment 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 analytes such as chemical structure; molecular weight; purity; solubility; LogD value; number of acidic and basic functional groups and chiral centres (pKa); 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.

Initial Method Development: Before initiating method development to obtain the first chromatograms of the sample, some immediate decisions are needed: Selection of HPLC mode, detection technique, column, and mobile phases. In many cases, some default or standard conditions appear to work well for the first "scouting" method; for instance, reversed-phase LC mode with a C18 column, ultraviolet (UV) detection (for chromophoric compounds), mobile-phase A: 0.1% acid in water and mobile-phase B: acetonitrile or methanol. The most common mobile-phase A additives are: Trifluoroacetic acid, formic acid, or phosphoric acid. The actual column dimension and particle size of the packing are dictated by the complexity of the sample and the goals of the method (2,3). For nonchromophoric analytes, refractive index, evaporative light-scattering, charged-aerosol, or mass spectrometry (MS) detection can be used. For isocratic analysis, a process of sequential isocratic steps is generally effective (2,8). This is accomplished by lowering the solvent strength of the mobile phase until all key analytes are retained and resolved. For purity testing or separation of more complex mixtures, a broad linear gradient separation from 5% to 100% mobile-phase B is used to generate the first chromatograms. This approach typically reveals the number of components and overall purity of the sample. The molecular weights and λmax values of the analytes are easily obtained using MS and photodiode-array detection, respectively.

Method Fine-Tuning and Optimization: For purity analysis, test mixtures (cocktails) of process precursors, impurities, degradants, additives, and excipients are used to confirm that the method is able to separate all of these components (that is, to establish that the method is specific). Mother liquors from the final crystallization step and forced degradation samples are used if reference standards of impurities are not available (2,6,7). The use of forced degradation samples is required to establish the stability-indicating nature of a method. Screening of different columns and mobile phases (pH, buffers, organic solvents), and adjustment of operating conditions (temperature, flow rate, gradient time [t G], gradient range — that is, starting and ending %B, and single or multiple-segment gradients) are used to resolve all key analytes. Finally, HPLC conditions are optimized for sensitivity, peak shapes, and analysis time.

The fine-tuning step, often called "selectivity tuning," is the most time-consuming step in the method development process. According to Lloyd Snyder, a pioneer in HPLC and a champion in simulation software, the "trial-and-error" approach of varying one factor at a time (OFAT) still reigns in HPLC method development (9). This often "graduates" to an "enlightened trial-and-error" approach for the experienced scientist with a good understanding of chromatographic principles who can establish the final method conditions in fewer steps. With the numerous factors controlling retention during gradient elution, this OFAT strategy is clearly not a very efficient process, even for the expert. Here, the use of simulation software (such as DryLab [Molnar Institut or ChromSword Group]) and automation systems (for example, a column–mobile phase screening system, Fusion AE QbD [S-Matrix], or AutoChrom [ACD/Labs]) is particularly helpful for the optimization process of methods for difficult samples (2,8–10). For the primary stability-indicating assays used in drug development and QC, a systematic and thorough method development process is becoming increasingly a regulatory expectation if not yet a requirement (11). It is worthwhile to note that HPLC method development during clinical development of new drugs is often an iterative process, repeated many times to accommodate the resolution of unexpected impurities found in new synthetic routes or formulations. To conserve resources, a proactive "stage-appropriate method development and validation approach" is adopted by many pharmaceutical laboratories (2,6).

Method Prequalification: Method validation (or qualification) is needed to ensure data accuracy and "fit for purpose." It is a mandatory requirement for quality control or regulated testing to demonstrate that the method is "suitable for its intended use." Therefore, the last step of method development is often a prequalification step (checking for specificity, linearity, precision, and sensitivity) to demonstrate that the method is "validatable." Method validation is a good manufacturing practice (GMP) process and method development is not a GMP activity. It is thus prudent to make sure that the newly developed method can be validated before the execution of the formal method validation protocol.

Shortcomings and Potential Improvements of the Common Method Development Strategy

The common strategy described above (or other similar process) is generally accepted by most practitioners. Nevertheless, some shortcomings and potential improvements of the process are noted here:

  • The systematic strategy may be too arduous for less-critical samples (such as raw materials or simpler mixtures) or for potency assays.
  • The use of generic methods may be "good enough" for many samples or applications. This approach is fairly common in high-throughput or process chemistry laboratories but is less common in analytical development or QC operation. Benefits of such generic or platform methods include time savings in method development, method validation, and method transfer in addition to efficiency gains from standardization.
  • Developing a stability-indicating method of a complex molecule or sample is the most challenging task and well suited to a systematic method development strategy. Nevertheless, the availability of a method template with useful gradient profiles and operating guidances can facilitate the method fine-tuning step in the development process.

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