Accuracy automated methods

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. 

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 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. 

Accuracy. The accuracy of the method expresses the closeness of agreement between the experimental result and the true value. When converting a validated manual method to an automated procedure, it may not be necessary to perform an accuracy study. Instead, it may be sufficient to rely on the comparison to manual data and on additional supporting validation on the automated procedure. When developing and validating the automated method as the first-intent method, it will... 

Equivalency. This test compares the results of the automated procedure with the results of the validated manual method. If accuracy of the automated procedure has been proven, it may not be necessary to perform the equivalency study. However, if the manual method does not exist, then accuracy and reproducibility data should be used to assess the suitability of the automated method. The recommended testing for content uniformity, assays, degradation and impurity methods and dissolution methods are listed in Table 5.4. 

Robustness. Examples of typical possible sources of variation in automated methods are homogenization speed, homogenization time, age of sample, accuracy of solvent dispense, and temperature variation. If all studies described in the method development have been performed, the robustness of the sample preparation has been demonstrated and does not require additional testing. Parameters in relation to the measurement technique may need to be considered and are covered in the relevant chapter. 

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. 

Both manual procedures6,24 and automated methods17,25 have been used to perform thermal chromatography with hydroxyapatite and phosphate buffer. The efficiency and accuracy of automated methods make them desirable for large-scale taxonomic projects. 

Figure 2. Comparison of classification accuracies from an automated system and from visual examination. Accuracies of automated methods are obtained by using SLF16 and a majority-voting ensemble classifier and are presented versus the average accuracy for the same images obtained by visual examination. Each symbol represents a different pattern class. In increasing order of human accuracy these are gppl30, Giantin, LAMP2, TfR, ER, Tubulin, Mitochondria, nucleolin and DNA (both at 100% for human and 99% for computer accuracy), and actin (100% for both). From Murphy (2004).

After amplification, tlie products can be detected by various methods. Simple gel electrophoresis with ethidium bromide staining may suffice. When greater accuracy is required, one of the primers can be fluorescently labeled so that after PCR the fragments are accurately sized on a DNA sequencing device. Alternatively, some form of hybridization assay can be used to verify or analyze the amplified product. Automated methods are always attractive and closed-tube methods are particularly advantageous in the clinical laboratory. Adding a fluorescent dye or probe before amplification allows thermocyclers equipped with optical detection to analyze the reaction as it progresses (real-time PCR) or after the reaction is complete (endpoint measurement) without need to process the sample for a separate analysis step. 

The results of the equipment should be double-checked at regularly scheduled intervals. This actually serves two purposes The precision and accuracy of the automated method may be enhanced as well as corrected, and the analysts keep their ability to perform the referee tests in case of a breakdown of the instrument. 

SEM and TEM are useful techniques for examining colloids (Nomizu et al., 1988) and their application to natural waters has been reviewed by Leppard (1992). Microscopy is the most direct of all methods for sizing particles, but is very time consuming and care must be taken to avoid artifacts. Automated methods have improved the accuracy and efficacy of the technique for producing number-based size distributions (Seaman, 2000). Qualitative chemical information for major elements is provided by energy-dispersive x-ray analysis (EDAX) commonly available with SEM instruments. 

Sucrose quantitation has also been performed by colorimetric methods. However, in recent years, automated enzymatic analyzers and instmmental methods (eg, ion chromatography and hplc) have become increasingly popular, as they provide greater sensitivity and accuracy. Near infrared (nir) spectroscopy is currendy under evaluation as a tool for sucrose quantitation in sugar mills and food processing operations. 

Aperture impedance and most other automated counters measure MCV and RBC independently, in contrast to the manual methods where MCV and MCH accuracies depend on hemocytometer red cell count accuracy. 

So efficient is the automated dideoxy method that sequences up to 1100 nucleotides in length, with a throughput of up to 19,000 bases per hour, can be sequenced with 98% accuracy. After a decade of work, preliminary sequence information for the entire human genome of 2.9 billion base pairs was announced early in 2001- Remarkably, our genome appears to contain only about 30,000 genes, less than one-third the previously predicted number and only twice the number found in the common roundworm. 

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