Oral fluid is an alternative matrix with two primary benefits: It can be collected noninvasively, and positive results indicate acute drug use. This means that specimens can be collected readily in plain sight and that findings reflect an individual’s current drug use status. These benefits make drug testing from oral fluid an attractive option for many physicians and lab clients. Before laboratories add this matrix type to their testing repertoire, however, they must validate their method to demonstrate its reliability in terms of sample handling and testing processes. For laboratories already familiar with developing and validating alternative matrices, this may not be too challenging, but the task may seem daunting for those that currently work only with traditional specimen types.
The basic steps for validating oral fluids essentially are the same as all method validation processes. First, the method must be designed fit-for-purpose. Decisions need to be made regarding scope of analysis, cutoff concentrations, analytical techniques, and results reporting (qualitative versus quantitative). As with other types of tests, necessary validation experiments include verifying the limit of detection, lower limit of quantification, bias, precision, linearity, selectivity, recovery, robustness, and stability. Acceptance criteria should be created prior to the start of validation, and protocols documented.
These commonalities aside, there are some unique considerations when it comes to validating oral fluid tests. One primary issue is that laboratories do not receive pure oral fluid when collection devices are used: Samples will contain both oral fluid and extraction buffer, the latter of which may be proprietary. Lack of knowledge about the ratio of oral fluid to buffer lends itself to qualitative determinations. However, quantitative reporting is possible if a lab discloses that the reported concentration has been derived via testing of this combined fluid mixture.
Another consideration of a more practical nature involves determining the matrix that labs should use to prepare calibration standards and quality control samples. Ideally, a lab will collect oral fluid matrix from volunteers using similar type of collection device type that it expects will be used for the majority of patient samples, then have the combined collected fluid screened by an independent laboratory to ensure negativity. Once the method is up and running, a lab will be able to use its in-house method for this step.
The other option is to utilize matrix-matched calibrators and controls from a non-human source. Labs use matrix matching in analysis to compensate for matrix effects that influence analytical responses. From this perspective, matrix matching works well in demonstrating that the selected calibration and control matrix is appropriate for measurement and as a monitor of ongoing method performance. Commercial vendors offer synthetic saliva and blank extraction buffer; however, since this is not oral fluid mixed with an extraction buffer, labs would do better to perform matrix matching experiments.
Matrix Substitute: Yea or Nay?
During their method validation, labs also need to consider whether they want to use synthetic oral fluid as a matrix substitute for calibration standards and quality control samples. As with traditional specimen types, endogenous compounds or external contaminants may be present that might produce interferences with the measured results. Interferences are non-targeted analytes that can impact the ability to detect, identify, or quantitate a targeted analyte. To ensure that the test excludes interferences that may adversely impact peak shape and/or recovery or produce false positive results, labs absolutely need to test a pre-defined number of pedigreed samples. The same samples also should be spiked with known concentrations of other commonly encountered analytes such as drugs of abuse, prescribed medications, over-the-counter therapeutics, and their major metabolites.
Perhaps the lengthiest experiment that needs to be performed during method validation involves stability, defined as the resistance to chemical change in a matrix under specific storage conditions for given time intervals and temperature conditions. Stability experiments tell the lab the viable time interval and storage conditions under which it can reliably test a sample. With traditional matrices, stability experiments involve adding a drug at varying concentrations and storing the samples for varying lengths of time and under differing temperature conditions.
In testing stability with an oral fluid method, however, the collection device pad adds a complication in that certain drugs may adsorb onto the pad over time, leading to decreased concentrations. While one might argue that this technically does not constitute instability, the potential outcome is the same. One approach to this issue is to add the targeted drug to the collection device with and without the pad in place so that any differences in drug concentration can be assessed as loss due either to stability or to adsorption onto the pad. Experiments of this nature are more extensive than usual simply because of the increased volume of samples that need to be prepared and tested. However, this process provides valuable insight into the kinetics of the substance as it resides in the collection device.
Another approach is to simply perform the stability experiment with the pad in place. While this by itself will not differentiate between adsorption and analyte degradation, it will provide the necessary information to both laboratory and end user.
Labs also should be prepared to perform cross-validation studies, which compare two bioanalytical methods. These studies are necessary when labs use two or more bioanalytical methods to generate data intended for the same study. For example, if an oral fluid test is meant to supplant a test that utilizes a more traditional matrix such as blood, serum, or urine, the lab must compare analytical results obtained from paired samples of oral fluid and of traditional matrix. One study compared and analyzed via validated liquid chromatography/tandem mass spectrometry method results from 21 paired serum and oral fluid samples obtained from 19 patients who had taken fluconazole. Statistical analysis showed that the fluconazole concentration in oral fluid was in good agreement with the serum drug concentration. The authors concluded that oral fluid can be a noninvasive, painless alternative to therapeutic drug monitoring when blood sampling is not possible or desirable (1).
Overall, oral fluid provides reliable toxicology results. Proponents of utilizing this specimen type cite as primary benefits its noninvasive collection and its ability to detect acute drug use. As with any other clinical or forensic laboratory test, oral fluid test results must be proven valid through bioanalytical method validation. Otherwise, any interpretation based upon those results will be compromised. While validating oral fluid can be challenging in comparison to traditional matrices such as blood, serum, or urine, it is readily doable. The key is properly planning experiment design and understanding where some of the routine validation tests may have to be modified to account for testing of oral fluid.
Laura Labay, PhD, F-ABFT, DABCC-TC, is a forensic toxicologist and director of toxicological services at NMS Labs in Willow Grove, Pennsylvania. +Email: [email protected]
CLN's Focus on Mass Spectrometry is supported by Waters Corporation.