Peak area precision

HPLC precision is critical in pharmaceutical analysis.For most pharmaceutical assays under a good manufacturing practice (GMP) environment, retention time and peak area precision of <2.0% RSD must be demonstrated before any samples can be analyzed. This section reviews the fundamental principles of HPLC precision and offers practical guidelines for its enhancement. The reader is referred to Reference 18 for a more detailed treatment of this topic. 

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... 

FIGURE 4 Peak area precision study to evaluate the effect of injection volumes. The resolution of the sampling syringe was about 0.01 jlL as determined by the digital resolution of the stepper motor and the size of the sampling syringe. 

FIGURE 5 Chart plotting the results of a peak area precision study showing the effect of sample dosing speed, bubbles in flush solvent and air-tight vial closure. Reprint from Reference 18 with permission from American Chemical Society. 

Theoretical considerations shown in the above equation also indicate that peak area precision is inversely proportional to the peak sig-nal/noise ratio, and to the number of sampling points across the peak width. For very noisy peaks, the peak area precision is limited by random noise fluctuations (Figure 6). Figure 7 shows that the precision of the peak area degrades rapidly when the signal-to-noise ratio is less than 100. Statistical considerations also stipulate a minimum data sampling... 

FIGURE 6 Peak area precision study to delineate the effect of peak signal-to-noise ratio. 

Recommendations for improving HPLC peak area precision are ... 

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... 

However, a direct interface subjects the exit of the column to vacuum conditions. Tire vacuum may lower the inlet pressure required to obtain the desired mass-flow rate of the carrier gas and also changes its linear-velocity profile across the column. These conditions can cause poor retention-time and peak-area precision and can even make the inlet system stop delivering carrier gas to the column. Thus, analysts should use direct interfaces only with long, narrow-bore columns... 

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... 

Accuracy) Stability Ruggedness Robustness precision Peak area precision Applied voltage Capillary temperature Injection mechanism pressure/vacuum applied Time of applied pressure/ vacuum Height of vial and transition time treatment, capillary temperature, buffer ionic strength, organic modifiers... 

Peak area precision is of great importance in the development of a quantitative assay. The reproducibility of the peak area is dependent upon a number of parameters, all of them instrumentally derived. Chemistry has little impact upon the quantitative reproducibility of an assay except where electrolytic changes in buffer pH can affect the absorption spectra of an analyte or where wall adsorption of the analyte can occur [17]. Most commercial instrumentation allows the use of electroki-netic and pressure injection modes for introduction of sample. 

For unbranched alkyl groups based on ratio of 30 ppm peak area to average of 14, 23 and 32 peak areas. Precision +10%... 

Tropane alkaloids pH, [SDS], [organic modifier] R, T, generated power and current Doehlert design, MLR, RSM hn and peak area precision, linearity, sensitivity, accuracy (63)... 

Tailing factors are a required calculation in most pharmaceutical methods. Tf = 1.0 indicates a perfectly symmetrical peak. Tf > 2 indicates a tailing peak that is typically not acceptable due to difficulty in integrating the peak area precisely. For most peaks (0.5 < Tf < 2.0), the values of As and Tf are fairly similar. For severely tailing peaks, As tends to be somewhat larger than Tf. 

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.

Precision of API peak area Precision of API retention time Asymmetry (USP tailing factor) of API Resolution between API and its isomer Signal-to-noise of precursor... 

Case Study 2 Poor Peak Area Precision Encountered During HPLC System Calibration 258... 

Two real-world examples are used to illustrate how to resolve this common problem encountered during the calibration of an autosampler (Chapter 9). In Table 10.1, the precision of peak area was found to be >1 % RSD, which was above the acceptance criterion of 0.5% RSD. Repeating the experiment showed similar precision results. Since a worn sampling syringe of the autosampler is the most common cause for poor precision, it was replaced. The peak area precision was found to be -0.3% RSD after this replacement. 

Table 10.1. Peak Area Precision Data Before and After Replacing the Sampling Syringe of the Autosampler...

Figure 5.8 (a) Deterioration of peak area precision with increase in spectrum size, (b) Broadening of peak and increase in background scatter with increase in spectrum size... 

Avoidance of statistical errors which are too high for the absolute and relative peak areas (precision). 

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