Method development and quantitation

In carrying out quantitation of an analyte or analytes in a set of samples, it is first necessary to develop a suitable method. The problem must firstly be defined. It is then necessary to collate and examine all the information that is available regarding the problem. Typically this would include  

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An advantage of the mass spectrometer as a detector is that it may allow differentiation of compounds with similar retention characteristics or may allow the identification and/or quantitative determination of components that are only partially resolved chromatographicaUy, or even those that are totally unresolved. This may reduce the time required for method development and is discussed in more detail in Chapter 3. 

Other features of an analytical method that should be borne in mind are its linear range, which should be as large as possible to allow samples containing a wide range of analyte concentrations to be analysed without further manipulation, and its precision and accuracy. Method development and validation require all of these parameters to be studied and assessed quantitatively. 

Within various pharmaceutical laboratories (industrial and academic), the mul-tinuclear technique of solid state NMR has primarily been applied to the study of polymorphism at the qualitative and quantitative levels. Although the technique ideally lends itself to the structure determination of drug compounds in the solid state, it is anticipated that in the future, solid state NMR will become routinely used for method development and problem solving activities in the analytical/materials science/physical pharmacy area of the pharmaceutical sciences. During the past few years, an increasing number of publications have emerged in which solid state NMR has become an invaluable technique. With the continuing development of solid state NMR pulse sequences and hardware improvements (increased sensitivity), solid state NMR will provide a wealth of information for the physical characterization of pharmaceutical solids. 

QuanOptimize from Micromass also allows automated method development for quantitative LC/MS/MS. It automatically identifies the best method for each compound, then runs batches of samples for quantitative analyses and report results in a QuanLynx browser. Thermo recently launched a similar product for automatic MS tuning. Known as QuickQuan, it generates data and stores it in a central Microsoft Access or Oracle database for future access. The infusion-based valve switching auto-tuning device allows individual compounds to be fully and automatically optimized in about 1 min. 

The first step in the method development and validation cycle should be to set minimum requirements, which are essentially acceptance specifications for the method. During method development, a complete list of criteria should be agreed on by the end users so that expectations are clear. Once the validation studies are complete, the method developers should be confident in the ability of the method to provide good quantitation in their own laboratories. The remaining studies should provide greater assurance that the method will work well in other laboratories, where different operators, instruments, and reagents are involved and where the method will be used over much longer periods of time. 

As an identity (ID) test, per ICH guidelines, only selectivity is required in method qualification and validation. Repeatability and intermediate precision are often included to ensure reliability of p7 determinations. Additionally, method robustness should be tested to assure that the assay performance is suitable for QC environment. Quantitative parameters such as LOD/LOQ are not required for an ID assay. If a cIEF method is used for purity determination, then all the purity parameters shown in Section 4 should be qualified. The following sections illustrate an example of method development and qualification procedures for cIEF. 

Quantitative Stmcture-Activity Relationships (QSARs) are estimation methods developed and used in order to predict certain effects or properties of chemical substances, which are primarily based on the structure of the substance. They have been developed on the basis of experimental data on model substances. Quantitative predictions are usually in the form of a regression equation and would thus predict dose-response data as part of a QSAR assessment. QSAR models are available in the open literature for a wide range of endpoints, which are required for a hazard assessment, including several toxicological endpoints. 

If there is no or little information on the method s performance characteristics, it is recommended that the method s suitability for its intended use in initial experiments be proven. These studies should include the approximate precision, working range, and detection limits. If the preliminary validation data appear to be inappropriate, the method itself, the equipment, the analysis technique, or the acceptance limits should be changed. In this way method development and validation is an iterative process. For example, in liquid chromatography selectivity is achieved through selection of mobile-phase composition. For quantitative measurements the resolution factor between two peaks should be 2.5 or higher. If this value is not achieved, the mobile phase composition needs further optimization. 

Meng M, Rohde L, Capka V et al (2010) Fast chiral chromatographic method development and validation for the quantitation of eszopiclone in human plasma using LC/MS/MS. J Pharma... 

An important issue in the method development for quantitative analysis using LC-MS is the possible occurrence of matrix effects. A matrix effect is an (unexpected) suppression or enhancement of the analyte response due to coeluting matrix constituents. It can be easily detected by comparing responses between a standard solution and a spiked pre-treated sample (post-extraction spike). Detailed studies on matrix effects revealed that the ion suppression or enhancement is frequently accompanied by significant deterioration of the precision of the analytical method [115-116]. Therefore, it can be useful to discriminate the two type of matrix effects. The absolute matrix effect indicates the difference in response between the solvent standard and the post-extraction spike, while the relative matrix effect indicates the difference in response between various lots of post-extraction spiked samples [116]. Unless counteraction is taken, an absolute matrix effect will primarily affect the accuracy... 

This section is intended to give a short overview of method development and validation of analytical methods - for qualitative and quantitative analysis of small (Mw < 1000) drugs - from process liquid such as fermentation medium. The same principles can be applied for any low-molecular weight compounds in biotechnological or organic synthesis. 

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