Intermediate precision analytical method

The relatively small mass differences for most of the elements discussed in this volume requires very high-precision analytical methods, and these are reviewed in Chapter 4 by Albarede and Beard (2004), where it is shown that precisions of 0.05 to 0.2 per mil (%o) are attainable for many isotopic systems. Isotopic analysis may be done using a variety of mass spectrometers, including so-called gas source and solid source mass spectrometers (also referred to as isotope ratio and thermal ionization mass spectrometers, respectively), and, importantly, MC-ICP-MS. Future advancements in instrumentation will include improvement in in situ isotopic analyses using ion microprobes (secondary ion mass spectrometry). Even a small increase in precision is likely to be critical for isotopic analysis of the intermediate- to high-mass elements where, for example, an increase in precision from 0.2 to 0.05%o could result in an increase in signal to noise ratio from 10 to 40. 

Analytical procedures are classified as being compendial or non-compendial in character. Compendial methods are considered to be valid, but their suitability should be verified under actual conditions of use. To do so, one verifies several analytical performance parameters, such as the selectivity/specificity of the method, the stability of the sample solutions, and evaluations of intermediate precision. 

For non-compendial procedures, the performance parameters that should be determined in validation studies include specificity/selectivity, linearity, accuracy, precision (repeatability and intermediate precision), detection limit (DL), quantitation limit (QL), range, ruggedness, and robustness [6]. Other method validation information, such as the stability of analytical sample preparations, degradation/ stress studies, legible reproductions of representative instrumental output, identification and characterization of possible impurities, should be included [7], The parameters that are required to be validated depend on the type of analyses, so therefore different test methods require different validation schemes. 

The validation process begun in Phase I is extended during Phase II. In this phase, selectivity is investigated using various batches of drugs, available impurities, excipients, and samples from stability studies. Accuracy should be determined using at least three levels of concentration, and the intermediate precision and the quantitation limit should be tested. For quality assurance evaluation of the analysis results, control charts can be used, such as the Shewart-charts, the R-charts, or the Cusum-charts. In this phase, the analytical method is refined for routine use. 

The authors determined specificity using the known hydrolytic degradation products. The precision of spiked samples of these degradation products were determined and found to be acceptable (99.9 0.4%). Accuracy of the method was determined using spiked recoveries of piroxicam benzoate, and the recoveries were acceptable (99.1-100.5%). Assay precision n = 6, RSD = 0.4%) was in accord with recommended criteria [7]. Within-day precision was performed on two instruments on two separate days, and the overall intermediate precision was 1.0%. The method was linear over the expected analyte concentration range giving a regression line of 1 = 0.999. The detection (DL) and quantification levels (QL) were assessed, and the latter was determined as 0.185 pg/ml (ca. 0.04%). 

An example of the minimum requirement for potency assay of the drug substance and drug product is tabulated in Table 4. Note that the postponement of intermediate precision is aligned with previous discussion that the use of early phase analytical method resides mainly in one laboratory and is used only by a very limited number of analysts. Each individual company s phased method validation procedures and processes will vary, but the overall philosophy is the same. The extent of and expectations from early phase method validation are lower than the requirements in the later stages of development. The validation exercise becomes larger and more detailed and collects a larger body of data to ensure that the method is robust and appropriate for use at the commercial site. 

Calculated repeatability, intermediate precision, and reproducibility values can be compared with those of existing methods. If there are no methods with which to compare the precision parameters, theoretical relative reproducibility and repeatability standard deviations can be calculated from the Horwitz equation and the Horrat value (Table 5). Horwitz RSD values are reported in Table 6. Higher variability is expected as the analyte levels approach the detection limit (see below). Next to the Horwitz equation, the AOAC s Peer Verified Program proposes its own levels of acceptability of %RSD as a function of analyte concentration level [56,72]. 

Depending on the use of the assay, different parameters will have to be measured during the assay validation. Validation of analytical assays is the process of establishing one or more of the following as appropriate to the type of assay accuracy precision (repeatability, intermediate precision), linearity, range, limit of detection, limit of quantification, specificity, and robustness [1]. For physicochemical methods there are accepted defined limits for these test parameters ... 

The extent to which intermediate precision should be established depends on the circumstances under which the procedure is intended to be used. The developing analyst should establish the effects of random events on the precision of the analytical procedure and identify which of the above factors contributes significant variability to the final result. The objective of intermediate precision validation is to verify that in the same laboratory the method will provide the same results once the developmental phase is over. 

The ICH has broadened and redefined these terms to more accurately describe the method s ability to reproducibly generate analytical results. Precision is defined as a combination of repeatability, intermediate precision, and reproducibility. Repeatability is system precision, as defined previously. Intermediate precision includes multiple analyses by multiple analysts on different days using different equipment within a given laboratory. This is only the first step in demonstrating the ruggedness of the method. 

Intermediate precision of the analytical method is known (from method validation). 

Not needed for nonspecific analytical methods (e.g., total organic caibon). All of the compound detected must be attributed to the target analyte unless rationale supporting an alternative has been documented, "in cases where reproducibility has been performed, intermediate precision is not needed. 

Figure 6.5. Experimental design for the assessment of the repeatability, intermediate precision and the reproducibility of an analytical method.

Precision provides an indication of random errors and can be broken down into repeatability and intermediate precision. This procedure should only be performed when the entire analytical method procedure is finalized. 

If automation is utilized, then an intermediate precision test is required to compare results obtained through manual testing versus automated testing (if all solvent composition and analyte concentrations of all actives are identical in both methods). 

Intermediate precision of the method was assessed by analyzing the samples six times on different days, by different chemists, by using different analytical columns of the same manufacture and different HPLC systems. The percentage assay was calculated using the area of the mixed standard preparation. The assay results are shown in Table 2. 

The precision of an analytical method is the closeness of a series of individual measurements of an analyte when the analytical procedure is applied repeatedly to multiple aliquots of a single homogeneous volume of biological matrix [16], The precision is calculated as coefficient of variation (C.V.), i.e., relative standard deviation (RSD). The measured RSD can be subdivided into three categories repeatability (intra-day precision), intermediate precision (inter-day precision) and reproducibility (between laboratories precision) [16, 78, 79, 81],... 

Analytical method transfer should be performed using a validated procedure this transfer data can be useful in determining intermediate precision of the method. The transferring laboratory should ensure that the recipient laboratory(s) is (are) current Good Manufacturing Practice (cGMP) compliant a record of successful audit by QA personnel is essential, especially if the laboratory is a contractor. 

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