Precision automated methods

Dl. Dahl-Jorgensen, K, Hanssen, K. F., and Try, K., A fast and precise automated method for the measurement of fructosainine in normal and diabetic individuals. Rev. Bras. Anal. Clin. 16, 138 (1984),... 

Automated methods are more rehable and much more precise than the average manual method dependence on the technique of the individual technologist is eliminated. The relative precision, or repeatabiUty, measured by the consistency of the results of repeated analyses performed on the same sample, ranges between 1% and 5% on automated analy2ers. The accuracy of an assay, defined as the closeness of the result or of the mean of repHcate measurements to the tme or expected value (4), is also of importance in clinical medicine. 

Essential features of an automated method are the specificity, ie, the assay should be free from interference by other semm or urine constituents, and the sensitivity, ie, the detector response for typical sample concentration of the species measured should be large enough compared to the noise level to ensure assay precision. Also important are the speed, ie, the reaction should occur within a convenient time interval (for fast analysis rates), and adequate range, the result for most samples should fall within the allowable range of the assay. 

It has been reported that the differential determination of arsenic [36-41] and also antimony [42,43] is possible by hydride generation-atomic absorption spectrophotometry. The HGA-AS is a simple and sensitive method for the determination of elements which form gaseous hydrides [35,44-47] and mg/1 levels of these elements can be determined with high precision by this method. This technique has also been applied to analyses of various samples, utilising automated methods [48-50] and combining various kinds of detection methods, such as gas chromatography [51], atomic fluorescence spectrometry [52,53], and inductively coupled plasma emission spectrometry [47]. 

Agemian and Chau [55] have described an automated method for the determination of total dissolved mercury in fresh and saline waters by ultraviolet digestion and cold vapour atomic absorption spectroscopy. A flow-through ultraviolet digester is used to carry out photo-oxidation in the automated cold vapour atomic absorption spectrometric system. This removes the chloride interference. Work was carried out to check the ability of the technique to degrade seven particular organomercury compounds. The precision of the method at levels of 0.07 pg/1, 0.28 pg/1, and 0.55 pg/1 Hg was 6.0%, 3.8%, and 1.00%, respectively. The detection limit of the system is 0.02 pg/1. 

This automated procedure was estimated to have a precision of 0.13-0.21mg Hg kg-1 at the lmg Hg kgr1 level with standard deviations varying from 0.011-0.02mg Hg kg-1, i.e. relative standard deviations of 8.4-12% at the 17.2-32.3mg Hg kg-1 level in sediments. Recoveries in methyl mercuric chloride spiking studies were between 85 and 125%. The detection limit for the automated method is dependent upon the weight of sample taken for analysis. It is 0.1 pg Hg L 1 in the aqueous samples. The results for the automated method are routinely reported to a lower limit of O.lmg kg 1 which corresponds to a dry sample weight of 0.25g. 

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... 

The relationship between the concentration of the solute and the peak produced in the chromatogram is, strictly speaking, only valid for peak area measurements, but in most instances it is more convenient to measure peak height. Such peak height measurements should only be used when all the peaks are very narrow or have similar widths. The tedium and lack of precision associated with non-automated methods of peak area measurements may be overcome using electronic integrators, which are features of most modern instruments. 

The USP 24 General Notices state that alternative methods may be used to determine that products comply with the pharmacopoeial standards for the advantages in accuracy, sensitivity, precision, selectivity, adaptability to automation or computerized data reduction, or any other special circumstances. Such alternative or automated methods shall be validated However, when disputed, the compendial method is conclusive as it is the official or referee test. In addition, USP Chapter (61) Microbial Limit Tests states that automated methods may be substituted provided they are validated and give equivalent or better results, whereas USP Chapter (71) Sterility Tests states that alternative procedures may be employed to demonstrate that an article is sterile, provided the results obtained are at least of equivalent reliability. 

One algorithm for blindly approximating physical states has already been proposed [36], although the method requires the number of states to be input. In work to be reported soon, Zhang and Zuckerman developed a simple procedure for approximating physical states that does not require input of the number of states. In several systems, moreover, it was found that sample-size estimation is relatively insensitive to the precise state definitions (providing they are reasonably physical, in terms of the timescale discussion above). The authors are therefore optimistic that a "benchmark" blind, automated method for sample-size characterization will be available before long. 

With this on-line automated method, more than 250 samples have been analyzed unattended in a 24 h period. Good results are obtained over a 10-1000 ng/mL range. The LOQ and LOD are 190pg and 58pg, respectively. Accuracy and precision values are 9.0% or better over the entire range of the assay. 

Automated methods have advantages, in that higher precision is often possible by the elimination of operator bias. Semi-automated techniques are, however, widely used in immunoassays in order to... 

Finally, the automated method should give as good if not better recoveries and precision than the original manual method. Commonly, the automated methods are not as rapid as the manual method. This is because of the serial nature of the automated workstations, whereas manual methods are typically done by vacuum box, which is a batch method. The automated method is superior to manual methods in the area of precision because any repetitive steps of the method are reproducibly delivered and the robot will not make the errors prone to humans. 

Aiach and co-workers have reported on the adoption of five different substrate assays onto an automated discrete analyzer (A2). They observed excellent within-run precision coefficient of variation (CV) values ranging from 1 to 3%, as well as satisfactory day-to-day precision. Ito and Statland similarly evaluated and compared the DuPont aca and Kabi/CentrifiChem automated method for measuring antithrombin III and plasminogen (13). 

As discussed above, the precision of automated methods becomes evident only when tested by double-blind quality control (R4, W3). Although Vestergaard s method appears to be good in this respect, it is interesting and perhaps cautionary that Townsend and James claimed a precision no better than d=1.0 /tg/100 ml for their automated method for plasma cortisol, i.e., coefficient of variation of about 8% of the mean. This was much better (one-third to one-half) when sequential rather than random duplicates were assessed (TIO). 

Until the advent of FIA, all automated methods of analysis emulated not only the sequence, but also the underlying concept of individual operations performed in the course of a manual procedure (Fig. 1.2). A simple chemical assay (Fig. 1.2a) requires mixing of a precisely metered volume of sample solution with a precisely metered volume of reagent. 

Low-temperature heat capacities of the solid coordination compounds Zn(Leu)S04 l/2H20(s) and Zn(His)S04T/2H20(s) (Leu = Leucine and His = Histidine) were measured by a precision automated adiabatic calorimeter over the temperature range between T = 78 K and T = 371 K. Di and coworkers [228,229] determined the initial dehydration temperature of the coordination compounds by analysis of the heat-capacity curve. The experimental values of molar heat capacities were fitted to a polynomial equation with the reduced temperatures (x), [x = f (T)], by a least-squares method. Enthalpies of dissolution of both the complexes were determined by isoperibolic solution-reaction calorimetry. 

The method of choice for serum iron analysis has been recommended by the International Committee for Standardization in Haematology (ICSH) [42]. This reference procedure obviates problems that are inherent in convenient automated methods that are precise but inaccurate [43]. In the recommended reference method, iron is released from transferrin with trichloroacetic acid (TCA), which precipitates apotransferrin and other serum proteins that are removed by centrifugation. Fe in the supernatant fluid is reduced to Fe " with thioglycolic (mercaptoacetic) acid, which is then complexed with a chromogenic iron-chelating reagent whose color is proportional to iron concentration. 

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