Method precision injection repeatability

For early phase methods, the precision tests only include injection repeatability (also referred to as system repeatability) and method repeatability (also referred to as analysis repeatability). The former is demonstrated by repeating injections of a standard solution and the latter by preparing multiple samples over multiple concentration levels (usually at 80%, 100%, and 120% of the nominal concentration) from the same lot of a composite sample of the dosage form. 

Precision may be considered at three levels repeatability, intermediate precision, and reproducibility (2, 3). Repeatability expresses the precision obtained by repeatedly analyzing, in one laboratory on the same day by one operator using one piece of equipment, aliquots of a homogeneous sample, each of which has been independently prepared according to the method procedure. Repeatability is also termed intra-assay or within-day precision. It is assessed using a minimum of nine determinations. Repeatability can help in determining the sample preparation procedure, the number of replicate samples to be prepared, and the number of injections required for each sample in the final method setting. 

In addition to the ICH levels repeatability and intermediate precision, the system precision, i.e., repeated injections/determinations of a single sample solution (also referred to as injection repeatability or injection precision), provides valuable information. Evaluation of these data will help us to show that the chosen equipment is suitable for its intended use. Injection precision will also become part of the system suitability requirements of the method and an acceptance... 

Alternately, obtain the sample injection device mass before and after injection to determine the amount of sample injected. This method provides greater predsion than the volume delivery method, provided a balance w th a precision of 0.01 mg is used and the syringe is carefully handled to obtain repeatable weighings. 

Several method performance indicators are tracked, monitored, and recorded, including the date of analysis, identification of equipment, identification of the analyst, number and type of samples analyzed, the system precision, the critical resolution or tailing factor, the recovery at the reporting threshold level, the recovery of a second reference weighing, the recovery for the control references (repeated reference injections for evaluation of system drift), the separation quality, blank issues, out of spec issues, carry over issues, and other nonconformances. The quantitative indicators are additionally visualized by plotting on control charts (Figure 23). 

Due to the lower injection precision of CE, it is not the method of first choice for the assay determination because the methods with higher precision are competing (HPLC, titration). However, a few examples in literature can be found and as a rule of thumb a well-developed CE method including internal standards should be able to obtain a repeatability of injections around 1% while values below 0.5% are generally expected for HPLC. 

The study of the precision of a method is often the most time and resource consuming part of a method validation program, particularly for methods that are developed for multiple users. The precision is a measure of the random bias of the method. It has contributions fi om the repeatability of various steps in the analytical method, such as sample preparation and sample injection for HPLC [5-9], and from reproducibility of the whole analytical method fiom analyst to analyst, fiom instrument to instrument and fiom laboratory to laboratory. As a reproducibility study requires a large commitment of time and resources it is reasonable to ensure the overall ruggedness of the method before it is embarked upon. 

The precision of a method is the extent to which the individual test results of multiple injections of a series of standards agree. The measured standard deviation can be subdivided into three categories repeatability, intermediate preci-... 

Adoption of standard methods also allows gathering of extensive knowledge and validation of the methods. The method validation can initially be comprehensive including long-term buffer shelf life, instrument-to-instrument type transfer, analyst-to-analyst repeatability and extensive robustness testing. If the standard method is applied to a new solute then validation is limited and may only need to include assessment of injection precision, linearity, sensitivity, etc., which can be obtained relatively quickly and simply. Internal standards are widely used in CE to improve injection precision and to compensate for solution viscosity differences, which may unduly affect assay results. Standard internal standards can be used for specific methods, which avoids additional work selecting an appropriate choice. 

Because the internal standard method eliminates some of the errors found in the external standard method it does not automatically follow that the internal standard method should always be used. The precision of many LC external standard methods is very good (e.g. <0.4% RSD for a purity determination) given that (i) the repeatability of injection volumes in modern injectors is much better than it used to be, especially if the injection is automated and (b) there are many methods for which sample... 

The internal standard method is less often used in liquid chromatography than in gas chromatography because injection of repeatable volumes has been made easier by the use of precise and reliable injection systems (loop valves). More generally, gradually, the internal standard method is being abandoned. The external standard method is, nowadays, the most common method and the use of an internal standard seems to be restricted to very specific applications for example, when preliminary to the chromatographic analysis, the solute of interest must be extracted by means of a complex protocol. 

The separation performance can be of much higher order of magnitude than in HPLC (up to lO theoretical plates), making CE an extremely valuable method for peptide mapping or DNA sequencing. However, small molecules such as amino acids or inorganic ions can be separated as well. The absolute sample amounts which can be injected are low due to the small volume of the capillaries. A major drawback is the lower repeatability (precision) compared to quantitative HPLC. Preparative separations are not possible. 

Capella-Peiro et al. (28) used a 3 full factorial design to optimize the capillary zone electrophoresis (CZE) separation of a group of seven antihistamines (brompheniramine, chlorpheniramine, cyproheptadine, diphenhydramine, doxylamine, hydroxyzine, and loratadine). In this case, critical parameters such as pH (a concentration of 20 mM phosphate was kept constant in all the experiments) and the applied voltage were studied to evaluate their effect on the resolution and efficiency. Maximum response was achieved at pH 2.0 and an applied voltage of 5 kV. After a repeatability study to check the precision of the electrophoretic method, as well as a suitable calibration, the usefulness of this optimized method was demonstrated through the determination of the listed histamines in pharmaceuticals, urine, and serum samples (recoveries were in agreement with the stated contents). Urine samples were diluted and directly injected in the CE system, while serum samples were previously extracted by means of a solid-phase extraction (SPE) procedure. 

Your instructor will select one experiment for teams to perform validation studies. An example is a gas chromatography experiment such as Experiment 32, but for one analyte. A flow injection analysis (FIA) experiment, such as Experiment 37, would be a good choice as well, since multiple measurements can be made rapidly. The team will determine linearity, accuracy, precision, sensitivity, range, limit of detection, limit of quantitation, and robustness (repeatability) of the method. In addition, a control chart will be prepared over at least one laboratory period. The instructor will have available a reference standard to use for accuracy studies. Plan for two laboratory periods for the completed study. A report of the method will be prepared and documented. Before beginning the experiment, you should review method validation in Chapter 4. 

The attractive features of splitless injection techniques are that they allow the analysis of dilute samples without preconcentration (trace analysis) and the analysis of dirty samples, since the injector is easily dismantled for cleaning. Success with individual samples, however, depends on the selection of experimental variables of which the most important sample size, sample solvent, syringe position, sampling time, initial column temperature, injection temperature and carrier gas flow rate, often must be optimized by trial and error. These conditions, once established, are not necessarily transferable to another splitless injector of a different design. Also, the absolute accuracy of retention times in splitless injection is generally less than that found for split injection. For splitless injection the reproducibility of retention times depends not only on chromatographic interactions but also on the reproducibility of the sampling period and the evaporation time of the solvent in the column inlet, if solvent effects (section 3.5.6.2) are employed. The choice of solvent, volume injected and the constancy of thermal zones will all influence retention time precision beyond those for split injection. For quantitative analysis the precision of repeated sample injections is normally acceptable but the method is subject to numerous systematic errors that may... 

The precision data is generally obtained from triplicate analyses of spiked samples and can be calculated from the accuracy study. The precision of the gas chromatograph and the precision of the method are two different values, and a good chromatographer should calculate both. The precision of the method can be obtained as above by calculating the repeatability of the method. The precision of the instrument can be obtained by multiple injections, usually 10, of one sample solntion. 

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