Components of Method Development

Selection of the appropriate marker residue may be challenging for some antibiotics, particularly those that typically are prepared from fermentation media and may contain multiple active constituents in variable proportions according to the particular manufacturer, such as gentamicin. For these substances, there is batch-to-batch variability, so the ratios of the components are not necessarily consistent, and therefore standards of individual components are preferable for reliable quantification. The rates at which certain components may be more readily metabolized or eliminated from tissues may differ from those of other components. As a result, the residue profile found can vary according to sample collection time since the last treatment, as well as sample storage time and temperature if particular components are subject to degradation on storage prior to analyses. 

Any other factors that may define the analytical requirements should also be considered. For example, when dealing with veterinary drug residue analysis, while the target tissue for domestically produced animals may be liver or kidney, these organ tissues are less commonly available as imported products. The majority of imported meat products are muscle tissue. Therefore, although the method has been validated for analysis of kidney for domestic samples, it is not fit for purpose for use on most import samples until it has also been validated for muscle tissue and possibly even some processed meat products. A method validated for the analysis of an aquaculture drug or natural toxin in oysters from domestic production may also, for example, require validation for shrimp or tilapia for application to imports. In addition, the requirement may include development of a 

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Quality spectroscopy is the second essential component of method development. The technique selected must contain information about... 

In the early stages of method development, when a suitable mobile phase has not yet been identified, the analyst will literally prepare nearly a 100 different mobile phases and plates to obtain the necessary separation. The HPTLC Vario Chamber system allows the user to take one HPTLC plate and cut it into six mini plates and expose them to six mini wells of mobile phase simultaneously. The analyst simply prepares a mixture of the components to be resolved and applies this solution to the six mini plates as a spot. This is a huge time saver since six mobile phases can be evaluated at a time and necessary dilutions can be serially prepared, (see Fig. 13.12). 

The ultimate goal of an assay method is the separation and visualization of all components in a single chromatogram. Proper selection of detection wavelength is a critical part of method development. When choosing a detection wavelength, the following factors need to be taken into consideration ... 

In conclusion, for low-dose dmg products, it is important to be aware of the possibility that adsorption of the dmg from the sample solutions onto surfaces can lead to low or variable assay results. These surfaces include filters, volumetric flasks, and sample vials. Evaluation of the components that come in direct contact with the sample solutions for potential dmg adsorption should be conducted as part of method development. This is especially true for compounds containing active sites such as amino groups, as described in this case study. While, most potency analyses involve the use of some organic solvents in the dissolving solvent, dissolution analyses could be problematic since the compendial media are aqueous buffers. For components such as HPLC vials, it is important to examine not only the type... 

Sample preparation is a critical step of method development that the analyst must investigate. For example, the analyst should investigate if centrifugation (determining the optimal rpm and time) shaking and/or filtration of the sample is needed, especially if there are insoluble components in the sample. This is usually more prevalent with excipient/DS mixtures and with slurry solutions obtained during the synthesis steps of the API. Syringe filters... 

All approaches to method optimization based on multiple experiments have the requirement that all components be detected and that they be tracked between runs. For complex samples, this is typically the most labor-intensive aspect of method development. For unattended method development, the instrument is required to monitor the change in retention of each component automatically. The historical limitations to this technology have been a key stumbling block in the widespread adoption of automated method development. 

Local considerations. There are many other local factors which dictate the path of method development. An increasingly common example is the development of a method which, as well as serving for quantitation, may be directly transferred to HPLC-mass spectrometry (commonly referred to as LC-MS) to allow the identification of minor components in a sample. This is achievable if the desired separation can be obtained using an ammonium acetate buffer instead of the more usual phosphate buffer. On the other hand, if it was intended that the unknown minor components should be isolated in milligram quantities to allow full structural elucidation using techniques such as H NMR as well as mass spectrometry, then conditions would be developed especially for the isolation unless the conditions for quantitative work coincidentally also lent... 

Wu, S.T., Xia, Y.Q., and Jemal, M., High-field asymmetric waveform ion mobility spectrometry coupled with liquid chromatography/electro spray ionization tandem mass spectrometry (LC/ESI-FAIMS-MS/MS) multi-component bioanalytical method development, performance evaluation and demonstration of the constancy of the compensation voltage with change of mobile phase composition or flow rate, Rapid Commun. Mass Spectrom., 21(22), 3667, 2007. 

It can be assumed that with the development and study of new methods, the ability to determine M (S), the method bias component of uncertainty, cannot be done given that it can be evaluated only relative to a true measure of analyte concentration. This can be achieved by analysis of a certified reference material, which is usually uncommon, or by comparison to a well-characterized/accepted method, which is unlikely to exist for veterinary drug residues of recent interest. Given that method bias is typically corrected using matrix-matched calibration standards, internal standard or recovery spikes, it is considered that the use of these approaches provides correction for the systematic component of method bias. The random error would be considered part of the interlaboratory derived components of uncertainty. 

Environmental problems are clearly linked to unbalanced development. This is why EIA, as a component of sound development planning, is particularly important. But these countries face a dilemma. Their need for environmental change is very great. Their resources of trained scientists to participate in environmental surveys and impact assessments are very slender. And a lack of finance, training, and infrastructure may restrict the development modes open to them. The simple transfer of the technologies now employed in the developed nations - including their methods of environmental impact assessment -may not be the best way to alleviate these problems. 

The different types of blanks that can be prepared and how they are used are discussed in this section. Although the focus of this chapter is on method development, it should be stated that blanks are also a fundamental component of method validation and sample analysis, and in fact are critical in terms of supporting the vahdity of the final analysis. 

Selecting a Constant Potential In controlled-potential coulometry, the potential is selected so that the desired oxidation or reduction reaction goes to completion without interference from redox reactions involving other components of the sample matrix. To see how an appropriate potential for the working electrode is selected, let s develop a constant-potential coulometric method for Cu + based on its reduction to copper metal at a Pt cathode working electrode. 

In several important cases, new synthetic strategies have been developed into new production schemes. An outstanding example of this is the production of an entire family of terpene derivatives from a-pinene (29), the major component of most turpentines, via linalool (3) (12). Many of these materials had been produced from P-pinene, a lesser component of turpentine, via pyrolysis to myrcene and further chemical processing. The newer method offers greater manufacturing dexibiUty and better economics, and is environmentally friendly in that catalytic air oxidation is used to introduce functionality. 

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