Assays automatic procedures

In considering the introduction of new test methods, it has been necessary for the USP to recognize the appropriateness of automating compendial assays and tests. The Committee of Revision prefers to adopt automatable procedures, especially for multiple-unit specimens such as those arising from dissolution or content uniformity requirements. Thus, since the publication of the USP XVIII in 1970, the General Notices to the... 

An important aspect is that of data and model stability. The raw data, for example, assay data, need to be updated regularly as new results become available. This process is tedious and time consuming and once a valid protocol for data acquisition and validation has been established, it is suitable for automation. The automatic procedure requires extensive data integrity checks (e.g., do all data points have valid structures and experimental results, how to handle conflicting results from different experiments) and a formalized automated normahzation process. This involves answering questions such as how to handle isomers, do experimental in vivo results have precedence over in vitro results or should all results be shown, should more confidence be put in results from Good Laboratory Practice (GLP)-studies, and so on. The models are then automatically rebuilt using the updated data and auto-updated as was mentioned in the QSAR section. A model may be promoted to use in the production system if it passes some defined validation tests, specific for each model, which was also mentioned earlier. 

Assays for PE. Because PE hydrolyzes pectin to pectic acid and methanol, the enzyme concentration can be assayed by measuring the rate at which free carboxyl groups or methanol is released from the substrate. Kertesz (18) titrated the free carboxyl groups as they were formed by the action of the enzyme on pectin. He used methyl red to indicate the pH (6.2) and added 0.1N NaOH at frequent intervals to maintain the pH relatively constant for 30 min. The pH meter replaced the use of indicators in subsequent modifications (17, 19). Current procedures use automatic pH titrators to titrate alkali at constant pH (25). A blank is used to correct for the consumption of alkali due to its reaction with atmospheric CO2, or the reaction solution is protected from C0 with a blanket of N2. 

Speed of Analysis. The speed with which many immunochemical analyses can be completed illustrates a major advantage of immunochemical procedures. Immunochemical assays are most time and cost effective when the sample load is large. Parker (4) estimated that a single technician could perform 100-5000 radioimmunoassays per day with little or no assay automation in comparison to 20-40 GLC assays (3). Numerous inexpensive systems are available to decrease analysis time. These systems may include solid phase separation techniques, automatic dispensers, test tube racks which will fit directly into a centrifuge and/or scintillation counter, and data handling systems. Alternatively, there are fully automated systems based on RIA or ELISA which require very little operator attention and which handle 25-240 samples/hr. Gochman and Bowie (81) have outlined the basis of operation and summarized the features of automated RIA systems and extensive literature is available from the manufacturers. 

The need to use a discontinuous assay method does not automatically mean that the HPLC method is the procedure of choice. For HPLC to be suitable, it must be possible to separate the components, and some method for detection and quantitation must be available. Next, neither the ingredients in the reaction mixture nor those used to terminate the reaction should produce problems for the separation and detection. Finally, the enzyme itself should be considered. 

Assays according to this procedure are fully automatized for process monitoring and control, both in cell culture and in downstream processing (7,8). Table 5 presents some of the systems available today. 

In a similar way, Fig. 13 shows the effect on the AutoAnalyzer record for the assay of blood glucose of carrying out an initial dilution of the sample in three different ways. In the first procedure, both the blood sample and the diluting fluid were measured by conventional pipets, but in the second, the diluting fluid was added from an automatic dispenser. In the third, sampling and dilution were carried out completely automatically, using the Filamatic diluter. 

A new approach to hydroxyproline assay is a recently introduced enzymatic procedure based on the ability of a multiple-enzyme system in adapted Pseudomonas strains of bacteria to degrade hydroxyproline (R7). Also, a standard colorimetric procedure has been adapted for use in an AutoAnalyzer apparatus (G5). Automatic amino acid analyzers can be easily programmed to isolate hydroxyproline from its more usual place in the shoulder of the aspartic acid peak (see M15). 

In conclusion, in this work we demonstrate the excellent capabilities of using ASE to extract compounds with antioxidant activity from natural sources as rosemary leaves and the microalga Spirulina platemis. ASE shows several advantages compared with traditional extraction processes such as 1) it is faster (IS min vs 2-24h in traditional extraction procedures) 2) it has less solvent volume consumption (13 ml vs 30>S00 mL/lOg) 3) it is more efficient (less dependant on matrix) and 4) it is automatic and allows sequential extraction of samples. The use of in-vitro assays and CE coupled to both, DAD and ESI-MS allows obtaining information about the biological and chemical properties of the ASE extracts. 

The most common method would be off-line or grab samples, which are taken intermittently and transported to the laboratory for analysis (Figure 9.1(a)). The procedure can be slow and the process may be finished before the results are available. This is more suited to quality control samples taken at the end of the process. Increasingly, samples can be taken at-line (Figure 9.1(b)). These types of samples are also taken intermittently but analysed in an instrument that is very close to the process, i.e. in the plant itself. The next type of sample (on-line samples) are taken from the process (usually automatically) and transferred directly into the analytical instrument for analysis without human intervention (Figure 9.1(c)). Pretreatment of the sample may also be carried out automatically as part of the assay. 

Another pressing need fostering the incorporation of computers In clinical chemistry was the growing number, variety and complexity of STAT analyses, performed as fast as possible under conditions of serious risk to the patient s life. STAT assays cannot always be efficiently carried out manually. A special type of automatic analyser has been developed not to meet the need for a large number of analyses, but rather to ensure application of a variety of procedures with the minimum possible set-up. 

A flow chart for the procedures is shown in Figure 6. The main control program for the automated assay was programmed with Visual Basic . The e-sco unit was remotely controlled through its proprietary ActiveX interface. An EXCEL template file contained all relevant information about the sample and the desired automatic processing (number of samples, spectrum file names, peak position and line width). At the end of the assay, the measurement results and signal intensity plots are generated automatically. 

This analytical feature of flow-based methods meets the requirement that more than one method for antioxidant activity characterization should be applied due to the complexity of samples and the absence of a standard antioxidant assay (Huang et ah, 2005). Following the same challenge, an automatic flow procedure based on MSFIA was proposed for the sequential determination of ABTS -scavenging capacity and Folin-Ciocalteu... 

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