Method development peak tracking

A well-known method for peak tracking is based on the phase-system switching idea, " which was developed to solve problems of mobile phase incompatibility in LC/MS target compound analysis. An analytical column is usually connected to a trapping column in tandem mode. A switching valve is placed after the UV detector, and the flow of nonvolatile eluents is directed through the trapping column to waste. When the peak of interest elutes from the analytical column it is... 

Furthermore, in a pharmaceutical development environment (working to GMP), redeveloping a method is not a simple task since peak tracking (by LC-MS) and validation may be required and, in the case of registered methods, results obtained by modified methods may not be acceptable. In such cases, it may be more efficient to use a preparative approach followed by conventional tube NMR where optimum sensitivity can be obtained through the use of cryogenic probes (Figure 6.48) or low-volume 1 mm probes [90]. 

FigurB 25-26 Application of the method development triangle to the separation of seven aromatic compounds by HPLC. Column 0.46 x 25 cm Hypersil ODS (C)e on 5-(j.m silica) at ambient temperature ( 22°C). Elution rate was 1.0 mL/min with the following solvents (A) 30 vol% acetonitrile/70 vol% buffer (B) 40% methanol/60% buffer (C) 32% tetrahydrofuran/68% buffer. The aqueous buffer contained 25 mM KH2P04 plus 0.1 g/L NaN3 adjusted to pH 3.5 with HCI. Points D, E, and F are midway between the vertices (D) 15% acetonitrile/20% methanol/65% buffer (E) 15% acetonitrile/16% tetrahydrofuran/69% buffer (F) 20% methanol/16% tetrahydrofuran/64% buffer. Point G at the center of the triangle is an equal blend of A, B, and C with the composition 10% acetonitrile/13% methanol/11% tetrahydro-furan/66% buffer. The negative dip in C between peaks 3 and 1 is associated with the solvent front. Peak identities were tracked with a photodiode array ultraviolet spectrophotometer (1) benzyl alcohol (2) phenol (3) 3, 4 -dimethoxyacetophenone (4) m-dinitrobenzene (5) p-dinitrobenzene ...

For simple mixtures containing only a few components, prepare each solute at a different concentration. This in combination with diode-array UV detection simplifies peak tracking. For complex mixtures computerized experimental design speeds methods development [6],... 

Recently there has been renewed interest in automated method development in which the optimization software directly interfaces with the instrument in order to run or suggest new experiments based on the prior results that generated the initial resolution maps. In the late 1980s, a number of approaches to this problem were attempted, but none of these tools prevailed, due in part to the challenges of tracking peaks between experiments. 

The selection of the set of samples that best covers the impurity and degradation profiles of the API is critical to successful method development. For a given API there are several ways to design the method development sample set some of which are more advantageous, but incur more risk in terms of the ability to track peaks from column to column and condition to condition. This section will address method development sample set design strategies, experimental concerns, and trade-offs. 

For the degradation conditions outlined in this chapter, all samples generated should be stored at or below 5 °C to preserve kinetic points until HPLC screening analysis can be performed. Key samples can then be used to optimize the analytical methodologies. It is important that these key predictive samples do not continue to react with time, since this would yield nonpre-dictive secondary degradants. These additional variables would make method development (especially peak tracking) extremely difficult in the optimization phase (see Section II.D for more details). 

Where the solute UV spectra are different, peak tracking during method development is conveniently achieved using the spectra. 

The future development of computer simulation will be shaped in part by the limitations of presently available software. The major shortcoming of present computer programs is their limited ability to carry out peak tracking, which is a prerequisite for the use of computer simulation. Peak tracking becomes more difficult when (a) the number of compounds in the sample exceeds about ten and/or (b) trace components are present This tends to complicate the use of computer simulation for many samples e.g., impurity assays, peptide digests, etc. The increasing use of LC-MS should in time both minimize difficulties in peak tracking and further simplify method development... 

The easiest way to unequivocally solve the task of peak assignment and peak tracking is to perform experimental method development using single pure substances, i.e. standards of the compounds to be separated. This procedure is utilized by the software when optimizing the separation of known target substances. However, in most cases, suitable standards are not available for all compounds. For example, in the analysis of the impurities in a product, usually... 

Identifying an unknown by using a standard, as described in the above paragraph, is a quick and easy process. However, what happens when the relative retention time of an unknown does not match that of a standard The next step is to obtain molecular mass and fragmentation data via HPLC/ MS. It is essential to determine the molecular mass of the unknown. Not only does the molecular mass help in the identification of the unknown, but it also enables one to track the correct peak by HPLC if isolation becomes necessary. In order to run LC/MS, a mass spectrometry compatible HPLC method must be available. The mobile phase should contain volatile buffers that are HPLC/MS-compatible. If such a method is not available, then one must be developed, which adds time to the identification time frame. 

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