HPLC calibration test procedures

Table 9.3. Summary of Typical HPLC Calibration Test Procedures and Acceptance Criteria... 

Significant time is saved in this calibration procedure by minimizing sample preparation and the number of calibrated test apparatus wherein only a balance, a 5-mL volumetric flask, a stopwatch, a thermal probe, two standards, one HPLC column, and one mobile phase are used. The use of MS Word template forms (see Figure 10) saves considerable time by eliminating any notebook entries and also improves the consistency of calibration records. Two important system parameters, dwell volume, and... 

The test procedure can be performed by pumping HPLC grade water at a specified flow rate under a controlled back-pressure, using a commercial back-pressure device connected in-line just after the pump. A f 000 psi back-pressure device is recommended. It is also advised that about f % MeOH be added to the water in order to minimize biological growth. While timing the process with a calibrated stop watch, the pump effluent should then be collected in a volumetric flask. From this, one can calculate the actual solvent flow at each flow rate. 

The mathematical treatment of FMC data can be accomplished by standard procedures via the solution of mass balance equations, on condition that the data were converted to reaction rate data with Eq. (21). As mentioned above, this requires the determination of the transformation parameter a. Two approaches based on calibration were developed and tested. In the first approach, thermometric signals are combined with the absolute activity of IMB, which had been determined by a separate measurement using an independent analytical technique. Figure 5 shows a calibration for the cephalosporin C transformation catalyzed by D-amino acid oxidase. The activity of the IMB was determined by the reaction rate measurement in a stirred-tank batch reactor. The reaction rate was determined as the initial rate of consumption of cephalosporin C monitored by HPLC analysis. The thermometric response was measured for each IMB packed in the FMC column, and plotted against the corresponding reaction rate. From the calibration results shown in Fig. 5 it can be concluded, independently of the type of immobilized biocatalyst, that the data fall to the same line and that there is a linear correlation between the heat response and the activity of the catalyst packed in the column. The transformation parameter a was determined from... 

We might consider the topic of calibration as a good example on which these distinctions can be demonstrated. Calibration of a balance may not involve more than just the placement of the correct calibration weight on the balance and to read off the respective value indicated. Thus, the respective SOP can be kept rather simple. Calibration of an HPLC apparatus for the quantitative determination of test item in a biological matrix will involve, however, more complex and delicate manipulations, so that a much more detailed description of the whole procedure should certainly be advisable. 

After initial qualification, the system is placed in service and is kept in a qualified state using a periodic calibration program. In addition, its performance for the specific application is also verified at the time of use using system suitability testing. This section describes the procedures and documentation used for HPLC hardware qualification. Detailed discussions of this subject can be found in books and articles elsewhere.3,4 The regulations and qualification of computerized data systems and networks with their associated software can be quite elaborate and is described elsewhere.5 7... 

This chapter discusses regulatory issues in HPLC laboratories with a focus on procedures and requirements for system qualification, calibration, method validation, and system suitability testing. Examples in a cGMP pharmaceutical environment are used to illustrate the various tools and systems used to ensure the degree of HPLC data accuracy necessary to achieve the delicate balance of regulatory compliance and laboratory productivity. 

A systematic error was suspected in the application of the HPLC-AAS method which could explain higher values found at the 4 pg level (10000 dilution). This error could not be seen at the higher level (1000 dilution). Indeed, in the case of a small systematic error the calibration slope would be the same and standard additions could not correct for non-additive errors. The laboratory mentioned that a hydride generation procedure was successfully used in their laboratory for lead speciation analysis. No interferences were observed between inorganic Pb, MesPbCl and triethyllead using this method. However, this system is still under development and its ruggedness should be further tested. This method was based on earlier work by Blais and Marshall [122]. 

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