Automated quantitative analysis standardization

Part—I has three chapters that exclusively deal with General Aspects of pharmaceutical analysis. Chapter 1 focuses on the pharmaceutical chemicals and their respective purity and management. Critical information with regard to description of the finished product, sampling procedures, bioavailability, identification tests, physical constants and miscellaneous characteristics, such as ash values, loss on drying, clarity and color of solution, specific tests, limit tests of metallic and non-metallic impurities, limits of moisture content, volatile and non-volatile matter and lastly residue on ignition have also been dealt with. Each section provides adequate procedural details supported by ample typical examples from the Official Compendia. Chapter 2 embraces the theory and technique of quantitative analysis with specific emphasis on volumetric analysis, volumetric apparatus, their specifications, standardization and utility. It also includes biomedical analytical chemistry, colorimetric assays, theory and assay of biochemicals, such as urea, bilirubin, cholesterol and enzymatic assays, such as alkaline phosphatase, lactate dehydrogenase, salient features of radioimmunoassay and automated methods of chemical analysis. Chapter 3 provides special emphasis on errors in pharmaceutical analysis and their statistical validation. The first aspect is related to errors in pharmaceutical analysis and embodies classification of errors, accuracy, precision and makes... 

Polymerase chain reaction (PCR), on the other hand, has several advantages it can be used to analyze small numbers of tumor cells, DNA from formalin-fixed, paraffin-embedded tumor tissue can be used, and it can be automated and standardized. Quantitative PCR techniques are currently being assessed for their clinical application to HF.R-2 DNA testing (Vona et al., 1999). However, presently the PCR technology is not optimally suited for routine, clinical application (see next section for details of quantitative analysis of HER-2Ineu expression). 

Figure 7.1 An automated quantitation approach that performs all of the necessary tasks required in a quantitative analysis. The user supplies only the molecular ions of the analytes and internal standards. (Cole et al., 1998).

Condition (a) above is not exclusive to headspace analysis in fact, it is a pre-requisite for quantitative analysis of any sample in gas chromatography. Essentially the same is true for condition (b). Condition (d) is primarily a design problem. Finally, constancy of p is assured by proper automation of the system (i.e. by exact repetition of the operational parameters) and by the fact that the calibration standard is carried through the same MHS steps as the sample itself Therefore, the greatest problem is posed by the need to ensure equilibrium between the two phases in the vial. 

Figure 26-18 is a diagram of a dialysis module in which analyte ions or small molecules diffuse from the sample solution through a membrane into a reagent stream, which often contains a species that reacts with the analyte to form a colored product, which can then be determined photometrically. Large molecules, which interfere in the determination, remain in the original stream and are carried to waste. The membrane is supported between two Teflon plates in which complementary channels have been cut to accommodate the two stream flows on opposite sides of the membrane. The transfer of smaller species through the membrane is usually incomplete (often less than 50%). Thus, successful quantitative analysis requires close control of temperature and flow rates for both samples and standards. Such control is easily accomplished in automated flow-injection systems. 

The significant development of chromatography in quantitative analysis is essentially due to its reliability and its use in standardized analyses. Trace and ultratrace analyses by chromatography are used, particularly the EPA methods for environmental analysis, although their costs are rather high. This type of analysis relies mainly on reproducibility of the separation and on the linear relationship between the injected mass of a compound onto the column and the area of the corresponding peak on the resultant chromatogram. This is an excellent comparative method used in many protocols, which, allied with software used for data treatment allow automation of all the calculations associated with these analyses. 

For quantitative analysis applications, these principal components can then be regressed with calibration values of the standards to produce the system model. This model is then used to predict the assay values of a test sample from its spectrum. Together with the assay figures, these models can indicate the errors associated with the prediction and validity of applying the model against the test sample. These are important diagnostics when the approach is a part of an automated analysis system and must be made to failsafe on the assay. 

Densitometric methods. In situ densitometry is an often-used technique for lipid quantitation and has been extensively reviewed by Prosek and Pukl (1996). Lipids are generally sprayed with reagent and their absorption or fluorescence can be measured under UV or visible light by means of a densitometer. The method needs to be standardized and suitable calibration curves need to be constructed to avoid errors. There are several models of densitometer available and some of them are highly automated and coupled to computer systems. Apart from these the use of CCD (charge-coupled device) cameras and colour printers have further improved the densitometric capabilities for accurate quantitations (Prosek and Pukl, 1996). A recent review by Ebel (1996) compares quantitative analysis in TLC with that in HPTLC, including factors that can effect quantitation, the need for careful calibration and errors in quantitative HPTLC analyses. Ebel is of the opinion that as both HPTLC and HPLC are based on the same absorption and fluorescence phenomena they should obtain similar results with respect to quantitation. 

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