Validation of computerized liquid chromatographic systems

Almost all vendors of liquid chromatographs offer dedicated computer-based controller/data-processing systems. These systems are integrated into the operation of the equipment and frequently use proprietary software systems for which complete documentation and program listings are not available to the user. In some instances, data in magnetic media are considered to be "raw data" for regulatory retention requirements. In addition, printed copies of final data used to make decisions and the procedures used to generate these data from raw data must be prepared and retained. In all cases, the systems must be validated to demonstrate that they are suitable for their intended uses. Approaches to validation of these systems are described and discussed. Validation of computerized liquid chromatographic (LC) systems is of interest from scientific and regulatory viewpoints. A scientist wants assurance of reliable data and computed results. A regulator wants tangible evidence of validation, such as written standard operating procedures (SOPs), raw data, guidelines, and policy documents that may be reviewed during inspections of manufacturing facilities. We present and discuss two approaches to such validation -modular and holistic -and describe procedures to satisfy the needs of scientists and regulators. Our remarks are addressed to quantitative analysis of products intended for the marketplace, or components of such products, and exclude qualitative or preliminary investigational work. Modular Validation By modular validation, we mean individual calibration or validation of each distinct, important part of a computerized laboratory system; this general approach was discussed by Tetzlaff in a review of U.S. Food and Drug Administration (FDA) requirements and expectations during inspections of such systems. Our concept of modular validation of computerized LC systems is outlined in Table 1, which is meant to be illustrative, not comprehensive. Although calibration of each module may be useful for troubleshooting purposes, such tests alone cannot guarantee the accuracy and precision of analytical results. Detector Linearity An attempt could be made to verify that the detector’s output voltage is linear with concentration of a standard material. For a UV-VIS LC detector, this test would have to be performed at several wavelengths to determine boundaries of reliability; for example, at which upper and lower wavelengths does stray light start to reduce the concentration range in which the output is linear? Even if these measurements could be made, there would be little assurance they would guarantee linearity when the detector is used for analytical measurements on compounds different from those selected for the detector calibration. Similar arguments apply to other types of LC detectors (fluorometric, electrochemical, etc.). Detector Wavelength Accuracy We have often encountered variable-wavelength UV-VIS LC detectors whose indicated wavelength differs from the actual wavelength by considerable amounts, as much as 20 to 30 nm. Loss of wavelength calibration can occur from mechanical shocks during shipment, and we have observed such errors in detectors newly received from manufacturers. Wavelength inaccuracy is often not apparent to the person operating the detector and can lead to substantial errors in interpretation of data. The operator’s manual usually gives a quick way to adjust the detector to meet the manufacturer’s specification for wavelength accuracy; we urge all laboratories to perform this calibration. Pump Flow Rate An attempt could be made to measure flow rates at several pump settings with various backpressures. Such tests might be repeated over several days to evaluate the reproducibility of the pump’s flow rate. Yet, such measurements are fleeting and of limited value for two reasons: Critical pump parts -valves, especially -deteriorate with time, and pump performance is often influenced by deposits of buffer salts on critical parts. Neither of these effects can be reliably duplicated in standardized tests. 1 Tetzlaff, R. F. (1992) Pharm. Technol. 16 (5), 70, 72, 74-78, 80-83. Injection Volume Similarly, measuring within-day and between-day accuracy and precision of the amount of solution injected automatically or manually might be attempted. And again, such measurements could not be assumed to hold up over useful periods of time due to gradual deterioration in injector parts or due to differences in viscosity or surfactant levels among the solutions used to test the injector and those actually used during analyses. Data Processor; Analog-to-Digital Converter In a rigorous modular validation, an attempt would be made to measure the accuracy of the analog-to-digital converter’s output (numbers) vs. the input signal (detector voltage). However, by connecting the voltage source, electromagnetic interference might be introduced from the test leads, or the load on the input of the analog-to-digital converter might change enough to affect its output accuracy. It is extremely difficult to design electrical test protocols that accurately simulate the conditions present within the instrument when the detector, not the test device, is connected to the analog-to-digital converter. This approach is beyond the capability reasonably to be expected of many laboratories. Data Processor and System Controller; Correctness of Program Code If a laboratory has written its own program code or has purchased a custom-written program code for data processing or system control, the source code should be available for scrutiny and validation. Included here are any "macros" that may be written for an LC system; they must be documented and available for review. Minimum documentation of macros (and other custom-written routines) must include: (1) a statement of what the macro is supposed to do; (2) a written description of how it performs its task, including a clear explanation of each step, the formulas developed for custom-written calculations, and references to the vendor’s user manual for vendor routines utilized; and (3) name(s) of the person(s) who prepared the macro, documented approval of the macro, date of its installation, date(s) of revision, and validation tests. Let us now consider the programs supplied by the vendor of a chromatographic workstation -the programs that process data and control the LC system. Most practicing chemists do not have the ability or inclination to perform systematic software review and validation of the vendor’s programs, which may consist of thousands of lines of code. Further, most chemists use integrators or workstations that contain proprietary algorithms that the manufacturer will not share. The "logic" or the "applicability" of these internal programs cannot be verified because a source listing of the code is not available. Even if the manufacturer supplied the source code, it is unlikely that even the most competent programmer could discern all the faults in all the branches and loops of an unfamiliar program. All equipment manufacturers should have validated their software before releasing it. Some vendors will provide useful information on the behavior and performance of their programs. Inquire about tests the vendor has run on the program code and request copies of the results. Critique of the Modular Approach Most chemists cannot evaluate the proprietary programs running in their computerized LC systems, so they require other procedures for system validation. Individual calibrations of injectors, pumps, detectors, analog-to-digital converters, and the like are admirable but difficult. Although they may be useful for diagnosing problems with LC systems, they alone cannot guarantee reliable analytical results for the reasons given earlier. Holistic Validation In a holistic validation (Table 2), we use a series of tests to measure and evaluate the performance of the entire computerized LC system under the conditions of its intended use: chemical analyses. The minimum holistic validation is a two-step process: initial calibration and running calibration. Each laboratory should develop and include other tests to cover its own special requirements. Initial Calibration--Linearity Before any samples are run, the entire system -injector, pump, column, detector, dataacquisition device, and data-output device -must be calibrated for linearity (or for reproducible non-linearity) by injecting several standard solutions of different concentrations. Use at least four standard solutions to generate this linearity curve; 0 must not be used as a "data point." Initially, the linearity test must be run daily. If the linearity curve is proven reproducible over several days, the test interval may be lengthened: every other day, weekly, or as specified in SOPs. Documented evidence must be obtained to establish that the specified test interval keeps the LC system in control. How shall the span of concentrations be chosen for these standard solutions? For routine work, in which the result is expected to fall within a known range, the standard curve must cover this range plus a bit more for safety. As an example, when analyzing individual tablets by the United States Pharmacopoeia (USP) procedures, results are normally expected to fall between 85 and 115% of the labeled amount. Linearity standards could be set up to cover 50 to 150% of label, for example. Each laboratory must prepare its own written requirements for calibration span of linearity standards and must not report analytical results calculated from data that fall outside this span. In research work the result cannot always be anticipated. Examine the linearity of the system over several concentration decades to assure usable linearity or to set up guidelines for dilutions or concentrations that are within the acceptable range. Initial Calibration--System Precision At first, measure the system precision daily by making at least six replicate injections