HPLC system calibration

Calibration, in current Good Manufacturing Practices (GMP) terminology, refers to instrument qualification or performance verification 

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This chapter reviews the principles and strategies used for HPLC system calibration that includes the pump, the detector, the autosampler, and the column oven. A case study is used to illustrate the development of the calibration procedures for all system modules and the rationale of setting up acceptance criteria that balance productivity and compliance. 

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

Summary Sheet for HPLC Since all 10 calibration results are very close to 100% and no trend is apparent, the HPLC system is regarded as being in control. ... 

Valko et al. [37] developed a fast-gradient RP-HPLC method for the determination of a chromatographic hydrophobicity index (CHI). An octadecylsilane (ODS) column and 50 mM aqueous ammonium acetate (pH 7.4) mobile phase with acetonitrile as an organic modifier (0-100%) were used. The system calibration and quality control were performed periodically by measuring retention for 10 standards unionized at pH 7.4. The CHI could then be used as an independent measure of hydrophobicity. In addition, its correlation with linear free-energy parameters explained some molecular descriptors, including H-bond basicity/ acidity and dipolarity/polarizability. It is noted [27] that there are significant differences between CHI values and octanol-water log D values. 

Quantitation is performed by the calibration technique. A new calibration curve with anilide standard solutions is constructed for each set of analyses. The peak area or peak height is plotted against the injected amount of anilide. The injection volume (2 pL) should be kept constant as the peak area or peak height varies with the injection volume. Before each set of measurements, the GC or HPLC system should be calibrated by injection of standard solutions containing about 0.05-2 ng of anilide. Recommendation after constructing the calibration curve in advance, standard solutions and sample solutions are injected alternately for measurement of actual samples. 

Quantitative analysis is performed by the calibration technique. A new calibration curve with a standard solution of each diphenyl ether herbicides is constructed, plotting the peak area against the amount of standard solution injected. Each diphenyl ether herbicide in the sample is measured by using the peak area for each standard. Before each set of measurements, the GC and HPLC system is checked by injecting more than one standard solution containing ca 0.01-2 mg L of each compound. 

Vi = portion of volume of VEnd injected into the GC or HPLC system ( uL) Wa = amount of fluthiacet-methyl for Vi read from calibration curve (ng)... 

The anhydride derivatives obtained as above are dissolved in an appropriate volume of methanol, and a 10-pL aliquot of each solution is injected into the pre-conditioned HPLC system. The peak heights of the fluorescent derivatives of M.A3 and M.A4 are converted to weight using a calibration curve corresponding to each chemical. 

Quantitation is performed by the calibration technique. A standard solution containing 0.1 mgkg of both M.A3 and M.A4 is prepared and 1, 2.5, 5 and 7.5mL of this solution are pipetted into around-bottom flask separately and evaporated. Each sample is converted into the fluorescent anhydride derivative according to the procedures described above. Each sample is dissolved in lOmL of methanol for injection into the HPLC system. The calibration curves are obtained by plotting the peak heights against the amounts of M.A3 and M.A4. The derivatives for preparing the calibration curve should be freshly prepared on a daily basis prior to quantitation. 

To model the amidation and cyclization reactions, an internal standard was weighed into the feed, and the feed placed on a balance for continuous weighing during the reaction. The HPLC system was calibrated using units of mol/kg, so that density effects could be neglected. A series of experiments... 

Blank, calibrator, control, and patient whole-blood samples (50 /iL) were transferred into 1.5 mL conical test tubes, mixed with 100 /xL of the IS, vortexed for 10 sec, and centrifuged at 13,000 g for 5 min. Twenty-five microliters of supernatant were injected onto a Cohesive Technologies Cyclone polymeric turbulent flow column (50 x 1 mm, 50 /flushed with a mixture of methanol and water (10 90 v/v) at a flow of 5 mL/min. Column switching from the TFC to HPLC systems was via a Cohesive Technologies system. The analytical column was a Phenomenex Phenyl-Hexyl-RP (50 x 2.1 mm, 5 /.mi). The mobile phase consisted of methanol and ammonium acetate buffer (97 3 v/v). The buffer was 10mM ammonium acetate containing 0.1% v/v acetic acid. The flow rate was 0.6 mL/min. 

Maintain and calibrate the HPLC system periodically. Preventive maintenance performed by a service engineer annually and system calibrations performed every 6 months are recommended. ... 

Calibration procedures are often similar to those used in the initial operational qualification of each module of the HPLC system. While each company s procedure might differ in the details, most share these common strategies. 

An analyst, a metrologist, or a qualified contractor can perform the calibration, though all must follow the company s prescribed standard operating procedure (SOP) and acceptance criteria. The cost effectiveness of using outside contractors or an internal metrology department is dependent on company size and the number of HPLC systems in the laboratory. 

Most HPLC systems in pharmaceutical laboratories are calibrated every 6-12 months. Periods longer than 12 months are not recommended while periods shorter than 3 months are deemed unnecessary because each HPLC system is also subjected to a daily system suitability check to ensure sufficiency for the application. Ideally, the frequency of calibration should be dictated by the historical data of calibration failures and the manufacturer s recommendation. In practice, 6 months appears to be the norm adopted by most laboratories. Calibration is also required after annual preventive maintenance or major repairs though only the affected modules, and not the entire system, need to be recalibrated. 

In summary, this chapter reviews how to develop expedited calibration procedures and acceptance criteria for all the common modules in an HPLC system. 

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