Quantitative Analytical Methods

Accurate data acquisition is essential for successful metabolic studies. The current trend from HPLC-based to LC/MS/MS-based analytical tools in ADME research, particularly in the pharmaceutical industry, demonstrates this requirement for accurate data acquisition. General aspects of the operation of LC/MS/MS are described below, together with a brief overview of traditional HPLC systems. 

Conversely, the HPLC/UV systems have limited applicability for monitoring aliphatic compounds without conjugated n systems due to the rather poor UV absorbance, and an alternative tool, LC/MS/MS in particular, is recommended if available. 

1 System and Principle LC/MS/MS provides a standard analytic system for DMPK studies in the pharmaceutical industry. This system is versatile, and does not have the prerequisites of LC/UV/FL systems. Instead, MS ionization and ion fragmentation of the molecules of interest become 

HPLC Conditions Columns Reverse phase, and particularly CIS columns such as Waters Symmetry Shield RP18, are most frequently used (Li et ah, 2003 Soars et ah, 2002 Zhang et ah, 2002). 

Mobile phases There are several frequently used combinations of aqueous and organic solvents. The options for the organic mobile phases are rather limited, usually either acetonitrile (AcCN) or methanol (MeOH). The aqueous mobile phases are often lightly acidified and/or buffered (Li et ah, 2003 Zhang et ah, 2002). To limit the potential for ion suppression, the salt concentrations in aqueous mobile phases are usually in the low mM range, much lower than what typically applied in the traditional HPLC/UV methods (Fasco et ah, 1977). 

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An external standardization allows a related series of samples to be analyzed using a single calibration curve. This is an important advantage in laboratories where many samples are to be analyzed or when the need for a rapid throughput of samples is critical. Not surprisingly, many of the most commonly encountered quantitative analytical methods are based on an external standardization. 

The determination of an analyte s concentration based on its absorption of ultraviolet or visible radiation is one of the most frequently encountered quantitative analytical methods. One reason for its popularity is that many organic and inorganic compounds have strong absorption bands in the UV/Vis region of the electromagnetic spectrum. In addition, analytes that do not absorb UV/Vis radiation, or that absorb such radiation only weakly, frequently can be chemically coupled to a species that does. For example, nonabsorbing solutions of Pb + can be reacted with dithizone to form the red Pb-dithizonate complex. An additional advantage to UV/Vis absorption is that in most cases it is relatively easy to adjust experimental and instrumental conditions so that Beer s law is obeyed. 

Quantitative analytical methods using FIA have been developed for cationic, anionic, and molecular pollutants in wastewater, fresh waters, groundwaters, and marine waters, several examples of which were described in the previous section. Table 13.2 provides a partial listing of other analytes that have been determined using FIA, many of which are modifications of conventional standard spectropho-tometric and potentiometric methods. An additional advantage of FIA for environmental analysis is its ability to provide for the continuous, in situ monitoring of pollutants in the field. ... 

Table 1.1 Conspectus of some common quantitative analytical methods...

Thin-layer chromatography (TLC) is used both for characterization of alcohol sulfates and alcohol ether sulfates and for their analysis in mixtures. This technique, combined with the use of scanning densitometers, is a quantitative analytical method. TLC is preferred to HPLC in this case as anionic surfactants do not contain strong chromophores and the refractive index detector is of low sensitivity and not suitable for gradient elution. A recent development in HPLC detector technology, the evaporative light-scattering detector, will probably overcome these sensitivity problems. 

Nuclear magnetic resonance (NMR) spectrometry has seldom been used as a quantitative analytical method but can have some practical importance in the characterization of surfactants [296-298]. 13C-NMR spectrometry has been used for the qualitative and also quantitative analysis of dodecyl, tetradecyl, and cetyl sulfates [299]. H- and, 3C-NMR spectra of sodium dodecyl sulfate are given by Mazumdar [300]. 

In this study, the effect of mobile-phase flow rate, or more accurately, the rate of flow of liquid into the LC-MS interface, was not considered but as has been pointed out earlier in Sections 4.7 and 4.8, this is of great importance. In particular, it determines whether electrospray ionization functions as a concentration-or mass-flow-sensitive detector and may have a significant effect on the overall sensitivity obtained. Both of these are of great importance when considering the development of a quantitative analytical method. 

In preceding chapters we have indicated which tools are nowadays being used routinely or currently are under development. General trends are higher sensitivity, more information, and faster and further automation. Automatic analyses are nice (sample in, report out), but interactive analysis tools are better. It is not realistic to expect the need for more analyses. Some future needs are more reliable quantitation, reference materials and simplification of data management. A particular problem in additive analysis concerns accuracy and traceability. In many cases, extractable rather than total concentration is determined. There are still many quantitative analytical methods waiting to be developed. It is here that the field will advance. Table 10.31 lists some proposed (r)evolutionary developments in polymer/additive analysis. 

Hoffmann, D., Kringle, R. A Total Error Approach for the Validation of Quantitative Analytical Methods. Pharm. Res., 24, 2007, 1157. 

Kumasaka K, Muratsuga M, Fukui T, Kimura M, Takagi Y, Hashizume N (2001) A new quantitative analytical method of serum biotinidase activity using biocytin as a substrate and its clinical significance in Japan. Clin Chim Acta 306 71-77... 

For many years, the economic value of milk was based mainly or totally on its fat content, which is still true in some cases. This practice was satisfactory when milk was used mainly or solely for butter production. Possibly, the origin of paying for milk on the basis of its fat content, apart from its value for butter production, lies in the fact that relatively simple quantitative analytical methods were developed for fat earlier than for protein or lactose. Because of its economic value, there has long been commercial pressure to increase the yield of milk fat per cow by nutritional or genetic means. 

For residue monitoring purposes, it is frequently useful to define MRLs for a particular marker residue. A specific quantitative analytical method for measuring the concentration of the residue with the required sensitivity must be available. The MRL establishes the concentration of the marker residue permitted in the target tissue. Marker residue and target tissue are selected in such a way that total residues in each edible tissue are at or below its safe concentration if the marker residue is at or below the MRLs. For milk or eggs, it may be necessary to select a marker residue different from the marker residue selected for the target tissue representing the edible carcass. 

Quality criteria for quantitative analytical methods, in general, have been proposed or arc lo be proposed by several international organizations including the Association of Official Analytical Chemists, the Food and Drug Administration, the Codex Committee for Residues of Veterinary Drugs in Food, the International Dairy Federation, and the European Union. The European Union, in particular, has laid down minimum quality criteria for quantitative drug residue methods,... 

Dye-coupling/consumption techniques enable quantitation of functional groups on resin. However, this area is at an early stage of refinement more quantitative analytical methods for quantifying a diverse set of organic functional groups are required. 

Ultimately, all quantitative analytical methods rely upon standards, whose composition is determined by the classical techniques of wet chemical quantitative analysis. Obviously, the preferred techniques for analyzing art objects are nondestructive, such as x-ray fluorescence, neutron activation, electron microprobe (both dispersive and nondispersive techniques), and so forth. Emission spectrographic analysis is not suit-... 

Genuine chemical methods were preceded by techniques such as the touchstone,26 and also by the direct investigation of the material or object by the use of the senses. A material could be felt, it could be tasted, its colour could be observed, it could be smelt, and the noise it could be induced to make could be listened to. The use of such methods of organoleptic analysis in the work of the Georgian King, Vakhtang VI (1675-1737) have been described.27 Probably the first demand for a quantitative analytical method arose from a desire to estimate the purity of samples of gold. While... 

The oriental crude drug Mao (Epedrae Herba) is contained in various oriental pharmaceutical preparations (255). Since the content of ephedrine alkaloids in this drug may vary with the Ephedra species used for its preparation and with, e.g., harvest conditions, it has been important to develop quantitative analytical methods in order to evaluate the quality. 

In the absence of suitable reference materials, the procedure should be tested using different sample weights and also measuring recoveries of element added at the beginning of the procedure. It must be remembered, however, that these criteria although necessary, are not sufficient, for the complete demonstration of the validity of the analytical procedure. The application of an independent (different in all respects of sample treatment and analyte quantitation) analytical method to a homogeneous practice sample would provide very useful confirmation of method reliability. 

Studies of interfacial reactions include analysis of both the bulk phases and their interface. Analysis means using not only qualitative and quantitative analytical methods, but also structural studies of the phases and an investigation of the chemical species present by means of thermodynamic calculations and/or experimental techniques. For the interfacial studies on rocks and soils, many different classical and novel methods can be used. In this chapter, the most important methods used for the analysis of solid, liquid, and interface will be presented. 

Dicarboxylic acids react with carbodiimides depending on their structure. From oxalic acid and carbodiimides the corresponding urea, carbon monoxide and carbon dioxide are obtained. This reaction was used by Zetzsche and Friedrich as a quantitative analytical method for the determination of carbodiimides. The carbodiimide content of polymer supported carbodiimides is also determined with oxalic acid." ... 

The Quantitative Evaluation of the Chromatogram Peak Area Measurements Peak Height Measurements Quantitative Analytical Methods for GC and LC Quantitative Analysis by TEC... 

There are many possible permutations for coupling one of the chromatographic or immunoaffinity separations with one or another of the spectrometric detection technologies. HPLC with UV or fluorescence spectrometry/ and HPLC with MS/ are among the most widely used quantitative analytical methods in the pharmaceutical development of new chemical entities because of their general applicability and... 

Surface enhanced resonance Raman scattering (SERRS) is an analytical technique with several advantages over competitive techniques in terms of improved sensitivity and selectivity. We have made great progress in the development of SERRS as a quantitative analytical method, in particular for the detection of DNA. However, one of the main advantages over fluorescence and other optical detection techniques is the ability to multiplex. 

To understand why HPLC is frequently used in quantitative analytical methods, it is useful to assess whether or not an analytical method is suitable for its intended purpose and, in doing so, consider the deficiencies in methods employing classical measurement steps. Analytical method validation is the process of assessing the fitness for purpose of an analytical method in choosing an analytical method issues such as cost, simphcity, operator experience, availability etc. are of secondary importance to the actual validity of the method under consideration. In the validation procedure, tests are typically carried out for the following properties ... 

Wc shall lake up other quantitative analytical methods when we need them nitrogen and sulfur analysis, Sec. 10.12 methoxyl determination. Sec. 17.16 neutralization equivalent, Sec. 18.21 saponification equivalent, Sec. 20.24.)... 

Quantitative analytical methods have been developed to identify metabolites in pathways or classes of compounds. This collective directed approach has been called metabolite profiling or metabolomics. Semiquantitative, NMR-based metabolic fingerprinting has also been applied to high abundance metabolites and has been termed metabonomics [188], Peaks detected in NMR spectra carry information regarding the structure of the metabolites,... 

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