Injection precision test

This test uses the entire HPTC system with a specific chromatographic method and validated Cl8 column that should be <10 cm and commercially available. Validated means that the individual column was performance tested before being shipped and that a certified test chromatogram is included with each column. Injection precision testing is typically performed by replicate injections of a test standard (at least six replicates are suggested). One then calculates the peak area %RSD of a stable component in the test standard. Most LC... 

Eor injection precision test details and calculations, refer to the procedure described earlier under Autosampler in section (j) of the OQ guidelines. 

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. 

Experiments 10-27 are designed to check the autosampler injection precision, pump repeatability and detector/system linearity. One programs the system to automatically inject multiple replicate volumes of a certified test standard. One typically injects 6-10 replicates per volume. The standard component s peak areas are used for calculated injection precision (reproducibility) and system linearity whereas, the retention times are used to calculate pump repeatability. 

It is recommended that OQ test the following on an HPLC system flow accuracy, pump compositional accuracy, pressure pulsations, column oven temperature accuracy/stability, detector noise/drift and wavelength accuracy, autosampler injection precision and carryover. 

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. 

The parameters to be tested in OQ/PV for capillary electrophoresis are temperature stability, voltage stability, injection precision, detector performance (noise, drift and wavelength accuracy), and integrator functionality. Additionally, the migration time of a test analyte, using a well-defined method, may be monitored as an indication of the overall function of the instrument. Where the test procedure is performed using a test analyte and method, the usual validation parameters of the... 

Alternatively, the sample injection device can be weighed before and after the injection to determine the amount of sample injected. This test method provides greater precision than the volume delivery method, provided a balance with a precision of 0.01 g is used. 

The peak width increases with injection volume. Therefore this parameter has to be fixed for comparative measurements. It has become the custom to inject low molecular weight test samples in very small volumes at very high concentrations, occasionally even as pure compounds. This extreme is not recommended as it is more important to inject a constant sample amount, reproducibly, in a precisely kept volume. Typical GPC injections are between 50 and 200 /a1. It is better to inject a larger volume of a lower concentration polymer solution. GPC units are often not designed for injection volumes lower... 

The dwell time was 200 msec for the analytes and 100 msec for the IS. At least 500 extracted samples were injected onto each column without any column regeneration. No solvent evaporation and reconstitution steps were involved. Ethyl acetate was preferred over methyl t-butyl ether (MTBE) because MTBE caused pulp-up of the mat. Six blank plasma lots were tested for matrix interference and none was detected in the analyte or IS region. When 100 ng/mL of the analytes were spiked into the blank plasma samples, the relative standard deviations were 1.0 and 1.5% for omeprazole and its metabolite, respectively. Precision and accuracy figures are given in Table 1.9. 

If the precision offered by the external standard technique is not adequate, it is necessary to use an internal standard. In this procedure, a known amount of a reference substance, not originally present, is introduced into the sample. This will result in the appearance of an additional peak in the chromatogram of the modified sample and any variations in the injections volume will equally affect the standard and the test compounds. 

For autosampler precision, 10 consecutive lO-pL injections of an eth-ylparaben solution (20 J,g/mL) are used (Figure 6). A Waters Symmetry column packed with 5- J,m particles is used. The manufacturer s specification for peak area precision at 0.5% RSD is adopted as the acceptance criterion. This stringent precision criterion is required for precise assay testing of drug substances typically specified at 98-102% purity. The linearity test is performed by single injections of 5, 10, 40, and 80 pL of the... 

Finally, Experiment 28 is used to check for any autosampler/ system sample carryover. A blank water sample is injected immediately following the highest injected volume of the preceding precision/repeatability/linearity test. Any carryover is then calculated as in the procedure described earlier under Autosampler in section (j) of the OQ guidelines. 

HPLC methods can usually be transferred without many modifications, since most commercially available HPLC instruments behave similarly. This is certainly true when the columns applied have a similar selectivity. One adaptation, sometimes needed, concerns the gradient profiles, because of different instrumental or pump dead-volumes. However, larger differences exist between CE instruments, e.g., in hydrodynamic injection procedures, in minimum capillary lengths, in capillary distances to the detector, in cooling mechanisms, and in the injected sample volumes. This makes CE method transfers more difficult. Since robustness tests are performed to avoid transfer problems, these tests seem even more important for CE method validation, than for HPLC method validation. However, in the literature, a robustness test only rarely is included in the validation process of a CE method, and usually only linearity, precision, accuracy, specificity, range, and/or limits of detection and quantification are evaluated. Robustness tests are described in references 20 and 59-92. Given the instrumental transfer problems for CE methods, a robustness test guaranteeing to some extent a successful transfer should include besides the instrument on which the method was developed at least one alternative instrument. 

A check of robustness includes preliminary experiments on precision. During robustness testing, a single standard is repetitively analyzed before starting the actual calibration. Without sufficient precision at a single concentration, it is fruitless to calibrate. However, the RSD obtained from repeated injections underestimates the overall error by a factor of up to 3. ... 

Inject the excipient standard solution at the working concentration specified in the method. The relative standard deviation (% RSD) of five or six consecutive injections is NMT 2%. The requirement for % RSD may be modified depending on the properties of the analyte and the test method. In the analysis of Polysorbate 80 in Nasonex, 5% RSD for system precision was acceptable, due to its very low concentration in the formulation. 

The volume precision of the injector can be demonstrated by making at least six replicate injections from a sample. The relative standard deviation (% RSD) of the response of the injections is then calculated to evaluate the precision. A fast and sample analysis should be considered for the test. The run condition for a very simple LC analysis of caffeine is given below as an example. 

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