Method development process

There have been very few method development processes proposed for 2DLC. One study (Schoenmakers et al., 2006) is titled A protocol for designing comprehensive two-dimensional liquid chromatography separation systems. This study advocates that one initially chooses the first-dimension maximum acceptable analysis time, the first-dimension maximum workable pressure drop, and the smallest first-dimension column diameter. The first two variables are then used to construct a Poppe plot (Poppe, 1997)—pronounced Pop-puh (Eksteen, 2007). 

In complex samples, when the range of elution times may not be known beforehand, there is the possibility of wraparound where components from the previous run are still eluting on the next second-dimension elution (Micyus et al., 2005). This situation is of concern and should be eliminated in the method development process for all but the most exploratory of work. This may require collecting fractions and injecting these fractions into the second-dimension column to determine the most retained compound retention time as part of the method development process. 

The method development process with the multisorbent plate consists of three steps. In step 1, the sorbent chemistry and the pH for loading, washing, and elution are optimized. In step 2, optimization of the percentage organic for wash and elution and the pH of the buffer needed is carried out. Step 3 is validation the method developed from the results of the previous two steps is tested for linearity, limits of detection, quantitation of recovery, and matrix effects using a stable isotope-labeled analyte as an IS. 

For PK assays, it is generally believed that most matrix effects are due to the sample matrix (typically plasma). While this is correct in many cases, this assumption has some exceptions (vide infra). One of the most useful tools for avoiding matrix effects is studying the sample matrix and proposed assay by using the post-column infusion technique described by Bonfiglio et al.14S This technique allows visualization of the portion of the chromatographic step affected by ion suppression.161721 Xu et al.101 recommended inclusion of this step in the method development process for drug discovery PK assays. 

There are four different drug products under Part II chemical active substance(s), radiopharmaceutical products, biological medicinal products, and vegetable medicinal products. For example, the GMP production report for biological medicinal products includes description of the genes used, strain of cell line, cell bank system, fermentation and harvesting, purification, characterization, analytical method development, process validation, impurities, and batch analysis (GMP production of biopharmaceuticals is described in Chapter 10). A DMF (Exhibit 8.8) is submitted. 

I I Diagram illustrating the method development process for final... 

In the process we have outlined, method validation is not considered to be the most time-consuming activity in the method development process (only 15% of the development time). 

FIGURE I Schematic overview of an advanced method development process. Method development is a continuous process in which all stakeholders collaborate intensively to design the final method. Reprinted with permission from reference I. 

FIGURE 2 Diagram illustrating the full method development process for late phase methods. Taken with permission from reference I. 

The AMERT is an important part of the analytical method development process. The AMERT allows to verify whether the draft method performs adequately for its intended purpose and complies with the specific country requirements. The concept of performing an AMERT was introduced by Crowther et al. and has been further discussed by Jimidar and De Smet. Detailed description of the approach is presented in reference 1. 

Method validation is only a minor, but important part in the overall method development process. The purpose is to demonstrate by experimentation that the method is suitable for the intended purpose. Method validation in CE is extensively discussed in various chapters of this book. 

The development lab can benefit from valuable objective data to detect method shortcomings and to identify gaps in the method development process. The feedback on method performance should be discussed regularly. During the monitoring a number of key performance indicators are recorded and filled out in feedback sheets by the application labs (stability and operational labs) each time the method is applied. The method feedback sheet is sent together with the method description to the application labs at transfer. An example of such a feedback sheet is shown in Figure 22. 

Analytical potency method development should be performed to the extent that it is sufficient for its intended purpose. It is important to understand and know the molecular structure of the analyte during the method development process, as this will facilitate the identification of potential degradation impurities. For example, an impurity of M + 16 in the mass spectrum of a sample may indicate the probability of a nitrogen oxide formation. Upon successful completion of method development, the potency method will then be validated to show proof that it is suitable for its intended purpose. Finally, the method validated will be transferred to the quality control laboratory in preparation for the launch of the drug substance or drug product. 

We focus on the requirements for registration in Japan. Thus, we provide information that considers the peculiarities of the requirements for the JP heavy metals test at the method development process. The compendial descriptions and relevant ICH guidelines in relation to this chapter are listed in the reference section. The principles for the validation requirements discussed in this chapter can be applied to heavy metal testing in general. 

The overall process from concept to validated method is illustrated on Page 4 (Figure 1). Once an appropriate analytical principle has been selected and the method performance criteria defined, the actual method development process can begin. Usually, this phase is carried out using pure materials and limited samples that are known, or assumed, to be homogeneous. 

When the method development process has been completed, an analytical procedure is subject to validation and transfer to routine use. This process may be called technology transfer which implies migration from the R D environment to routine analytical laboratories. This process needs to be carried out whether the applicability of the procedure is limited to a single laboratory or to many laboratories. 

We have optimized an eight-way MUX coupled to a TOFMS analyzer to carry out eight-channel parallel LC/UV/MS analysis of combinatorial libraries14 in the past 2 years. This system has not only provided the capacity needed for library analysis, but also enabled simultaneous evaluation of experimental parameters to expedite the method development process. In this chapter, we discuss the optimization of this system and present a high-throughput protocol for combinatorial library analysis. We also compare the eight-channel parallel LC/UV/MS system to a conventional single channel LC/UV/MS system in terms of performance and operation. 

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