Pharmacokinetic modeling during the drug development process is essential for determining whether an administered drug substance has the necessary characteristics to meet its intended medical use. Typically for small molecules, this is based on time/concentration data generated from biological samples following analysis using LC-MS/MS.
One of the principal challenges for bioanalysts is working with analytes that are unstable in biological fluids and ensuring we report accurate drug concentrations. In many cases, instability can be predictable based on the presence of particular chemical groups within the molecular structure such as lactones, aldehydes, esters or thiols. In addition, the increasing use of pro-drugs designed to release the active moiety post-administration means that the administered drug is inherently unstable but may have to be preserved for measurement in PK studies.
KCAS’ approach is to do a preliminary assessment of analyte stability during method development, particularly if no stability information is available. This is designed to predict any likely issues during later studies. Validation is used to more rigorously monitor analyte stability under typical sampling, storage, extraction and solution conditions. This can determine how samples are treated in the clinic, during extraction in the wet lab or when queued up in the autosampler for LC/MS analysis.
Preserving Compound Stability At Point of Sampling
As analytical experts, we recognize that the best solution for instability may not be practically applicable for our in-life service providers. Generally, the simpler the solution the better, especially for clinical settings. The use of anticoagulants such as sodium fluoride/potassium oxalate or Becton Dickinson’s proprietary anticoagulant tubes may address observed stability issues. This is an ideal approach for clinics because this would not change their sampling routine.
Assessment of room temperature stability in whole blood or in the desired matrix can indicate whether standard clinical routines can be maintained as samples are taken from patients. For slowly degrading molecules, use of in-lab controls during analysis such as ice baths or refrigerated autosamplers may be sufficient to ensure analyte integrity. This can remove the onus of sample treatment from the clinic where the only demands may be reduced transit times from patient sampling to the freezer and sample storage at ultra-low temperatures.
Sample Treatment at Sampling to Counter Analyte Instability
Instability may refer to a compound’s degradation or conversion in situ to more stable chemical forms. In either case it may be feasible to add agents to counter the observed processes in matrix. For example, analytes containing chemical groups that are prone to oxidation such as aldehydes can be preserved by addition of an antioxidant such as sodium metabisulfite to the sample.
If bioanalysis support is needed for multiple species, the enzymatic differences between organisms may have an impact on sample treatment. As an example, ester containing compounds will be susceptible to high esterase levels in rat or mice. Inhibitors such as chlorpyrifos are routinely applied to control enzymatic activity and animal facilities are well versed in this.
In extreme cases where analytes show degradation in biological matrix from time of sampling an alternative approach is to remove the analytes immediately from the matrix. A common approach is to use organic solvents such as chilled acetonitrile to isolate the analytes. This is done to have the compounds present in a solvent system where they are stable enough to allow downstream processing and analysis. Validation should assess stability in the organic solvent system to ensure preservation of the analyte(s).
The basic question to be addressed, when treating samples as soon as they are taken, is whether the clinic has the expertise to do that appropriately. Stabilizing agents such as chlorpyrifos may have toxicity concerns meaning that it’s not optimal for use in a clinical setting so other approaches may be needed.
Additional Strategies to Address Unstable Compounds
Analytes with strained ring systems or other reactive groupsmay be prone to instability in light due to the limited energy needed to drive conversion to a more stable form. Typically, the use of yellow (or other wavelength) lights can be sufficient protection to preserve the analytes during processing. Our wet labs are equipped to allow transition from white to yellow light as needed. It’s important to remember that the injection block should also be protected from light post-reconstitution. If your lab has windows be aware that direct sunlight could affect compound stability even if no degradation effect has been seen under white light.
It is critical during analysis to be aware of what other related compounds may be present throughout the sampling and extraction process. Unstable metabolites, such as glucuronides, can back-convert to the analytes of interest. We have observed an assay where the presence of non-measured metabolites converted to other analytes of interest when reconstituted samples were stored at room temperature leading to an increase in observed concentrations. Using a well-controlled chilled autosampler addressed the issue.
Acyl glucuronides can present additional challenges during analysis. Even if no degradation occurs in plasma, the stability of the glucuronide during the LC/MS procedure should be assessed. The safest approach is to develop chromatographic conditions that separate the glucuronide from the administered drug. If there are potential problems from in-source degradation, chromatographic separation from the drug means there is no effect on quantitation.
A methodical approach to method development from structure review to initial stability testing can provide a sound foundation for validation and sample analysis. Resolving stability issues can involve significant effort particularly as designing readily workable sample collection procedures may be needed for the site(s) while focusing on preserving analytes. Through working with a wide variety of chemical entities, KCAS been able to develop many strategies for dealing with unstable drugs and is always happy to apply that knowledge to future programs.
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