Several important classes of drugs are inherently unstable in biological fluids. Examples include alkylating agents, cytostatic nucleosides, drugs with ester, aldehyde, thiol, nitroxyl or lactone functional groups, and small molecule compounds with unstable metabolites, such as acylglucuronides.
The rate at which an analyte degrades in blood, other biological fluids, or tissues has a huge impact on how to correctly design the sample collection, processing, extraction, and analysis strategies. Compounds that are very unstable must be stabilized at the point of sample collection, whereas more options are available for compounds with moderate instability. Some drugs (e.g., aspirin) can be stabilized using chilled, conventional blood collection tubes. However, the use of antioxidants, buffers, stabilizing agents and/or derivatizing reagents may be essential to stabilize very unstable analytes.
NITROXYL AND NITRIC OXIDE RELEASING DRUGS
A case in point are drugs that are inherently designed to break down, releasing nitroxyl (HNO) or nitric oxide (NO). There are multiple examples of these unstable compounds, including desthionitrites (also referred to as S-nitrosothiols) which we have worked extensively.
The half-life of desthionitrites in biological samples can be quite short, requiring rapid stabilization of samples upon collection. However, the need for simplicity in sample collection is essential for multi-site Phase II and III clinical studies.
In the case of several desthiontrites we have evaluated, citric acid (0.5 M) added directly to the whole blood samples (1:10) was sufficient to stabilize the compounds from collection to laboratory processing of plasma. However, the addition of formic acid to the plasma prior to freezing was essential to ensure analyte integrity during storage at -70C. This two-step stabilization procedure permitted simple and safe sample collection at the clinic site, leaving the more hazardous addition in a laboratory setting.
Thiols (R-SH) may be the most complex analytes to stabilize, as when in blood they can rapidly form disulfides (R-S-S-R’) with other thiols, most notably glutathione (GSH). There are numerous papers on how best to stabilize thiols, but derivatization has been found to be the most effective. Reagents we have used effectively include 3-methoxyphenacyl bromide (MPBr), pentafluorobenzyl bromide (PFBBr) and N-ethylmaleimide (NEM). In some cases, additional sample stabilization procedures using sodium metabisulfite, phosphoric acid, and/or ascorbic acid are needed to further stabilize the analyte in the plasma fraction.
Of major concern is whether the derivatizing reagent, when added to whole blood, will hemolyze the sample, releasing high levels of reactive GSH. This is particularly problematic in rat blood collections where GSH levels are relatively higher, resulting in poor recoveries without further manipulation. It is essential to find the proper balance in the appropriate level of derivatization reagent needed without incurring significant hemolysis of the red blood cells.
Deciding how to best stabilize an inherently unstable compound can often involve significant thought and effort in developing a bioanalytical method by LC-MS/MS to optimize analyte stability. However, just as important is the attention in designing reasonable sample collection procedures that can be put in place at the site(s) and meet compliance for stabilization. KCAS has had the great fortune to have worked on numerous unstable drug programs throughout our many years, and would be happy to leverage our experience in tackling yours.
Schedule a call today with KCAS’s senior scientific advisor to see if our services are a fit for your immune response characterization needs.