Autosampler precision

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... 

FIGURE 6 HPLC chromatogram of 10 lL injection of ethylparaben used for the verification of autosampler precision. 

Autosampler precision can be checked by replicate injections of a control sample, with wash injection intervals between every two sample injections. The repeatability of peak areas, mathematically expressed as RSD, is used as a criterion for autosampler precision. For example, when 10 consecutive injections of 10 pL of a solution are performed, the expected RSD for peak area precision ranges from 0.5% to 1.0%. Single injections of different volumes, such as 5, 10, 50, or 80 pL, can also be used, simultaneously checking the linearity of the injector, the detector, and the data system. Another approach to qualify the autosampler involves the gravimetric determination of the average volume of water per injection withdrawn from a tared vial after six 50-pL injections. The procedure... 

Figure 5.6. Diagram illustrating typical autosampler precision vs. injection volumes. CCA, 4 -chloro-3 -sulfamoyl-2-benzophenone carboxylic acid, is a hydrolysis product tor chlorthalidone. Note that peak area precision increases (lower RSD) with larger injection volumes. This was caused by the finite sampling volume precision of the autosampler sampling syringe and stepper motor, which was about 0.01 pL for this particular autosampler. Reprinted with permission from reference 12. See the same reference for additional experimental details.

Autosampler Precision Determine the peak area RSD of six 10-p.L injections of ethylparaben (20pg/mL) RSD <+0.5%... 

For the linearity test the chromatograms of the 100% and diluted benzo-phenone solutions are used. The peak areas of the 2 x 0.1%, 2 x 1%, 2 x 50%, and 2 x 100% chromatograms are integrated and the correlation coefficient of the linear regression is calculated. Note that integration errors of the small peaks may occur which have nothing to do with the autosampler precision. In addition, the ratios of the mean values of peak area 1%-0.1% and 100%- % are calculated. 

Autosampler precision—relative standard deviation of peak areas of 20 consecutive injections must be less that 0.5%. 

This example presents four analytical situations (a) good control, (b) sudden shift in accuracy - perhaps the calibration material has become contaminated, (c) a gradual shift in accuracy-perhaps a reagent has exceeded its expiry date, (d) very poor precision - perhaps an autosampler is working erratically. 

Cool on-column >250 pm column (i.d.) 1 ppm (FID) Reduced thermal degradation and discrimination Wide range of analyte concentrations High sample capacity (LVI) Autosamplers Direct quantification Excellent precision Control of operational conditions (initial oven temperature) Optimisation required Not applicable for polar solvents Column contamination by dirty matrices Poor long term stability... 

The ability of a tPLC system to produce the same values of retention time and peak areas for analytes of interest is determined by evaluating the precision obtained under standardized conditions and analytical methods. The precision (reproducibility) values obtained are functions of the autosampler, cartridge, and detectors employed. Due to the parallel design of the tPLC system described in this chapter, reproducibility evaluations of retention time and peak area involved comparisons of results obtained for these parameters for consecutive runs performed in the same column and across different columns. 

FIGURE 6.28 Precise control of depth of sampling can be achieved with an autosampler employed in a /tPI.C system. The dimensions indicated correspond to those used during the evaluations described. 

Online IS introduction allows loading of samples in the biological matrix without preparation. ISs were introduced online in the quantitation of propranolol and diclofenac in plasma (Alnouti et al. 2006). Plasma samples were loaded into the autosampler without pretreatment. Both the plasma sample (10 /iL) and IS (5 //I. from an IS microreservoir) were aspirated into an injection needle sequentially and injected into the sample loop. After the switching of an injection valve, the mixed solution in the sample loop was loaded into a cartridge containing washing solution for online SPE. The accuracy and precision of the online IS method were comparable (85 to 119% and 2 to 12%, respectively) to values obtained offline (86 to 106% and 2 to 16%, respectively). 

In general, gas chromatography will undoubtedly continue to be the method of choice for characterization of light hydrocarbon materials. New and improved detection devices and techniques, such as chemiluminescence, atomic emission, and mass spectroscopy, will enhance selectivity, detection limits, and analytical productivity. Laboratory automation through autosampling, computer control, and data handling will provide improved precision and productivity, as well as simplified method operation. 

Peak area precision is controlled by the sampling volume precision of the autosampler. In some specific instances, precision can be limited by the signal-to-noise ratio or the sampling rate as described by the equation below. 

Figure 4 shows peak area precision vs. injection volume for a typical autosampler. Note that excellent peak area precision of 0.2% RSD was readily achievable for an injection volume >5 J,L. Precision levels are much poorer (0.5-1% RSD) for sampling volumes <5 J,L, attributable to the finite resolution of the sampling syringe and associated digital stepper motor. To obtain optimum peak area precision, the analyst must avoid potential problem situations such as an overly fast sample... 

Use a precise autosampler (<0.5% RSD) and injection volumes >5 pL. Degas flush solvents used for the autosampler. 

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. 

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

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. 

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. 

HPLC has more or less supplanted GC as a method for quantifying drugs in pharmaceutical preparations. Many of the literature references to quantitative GC assays are thus old and the precision which is reported in these papers is difficult to evaluate based on the measurement of peak heights or manual integration. It is more difficult to achieve good precision in GC analysis than in HPLC analysis and the main sources of imprecision are the mode of sample introduction, which is best controlled by an autosampler, and the small volume of sample injected. However, it is possible to achieve levels of precision similar to those achieved using HPLC methods. For certain compounds that lack chromophores, which are required for detection in commonly used HPLC methods, quantitative GC may be the method of choice, for analysis of many amino acids, fatty acids, and sugars. There are a number... 

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