Bioanalysis Zone

Bioanalytical revelation of the month: March

Brian Beato (Laboratory Director, AIT Bioscience):

Brian Beato“When we were recently modifying a method for fentanyl, a well-characterized compound with which we have a lot of experience, we stumbled upon a surprising lack of stability in human plasma that we’d never observed previously. This reminder of the complexities of studying therapeutics in biological matrices from donors with varying disease states, diets, metabolism kinetics, etc., leads to interesting bioanalytical questions.

Over the years in our Indianapolis laboratory, fentanyl has always demonstrated significant stability under ambient storage conditions in K2EDTA human plasma from normal donors. Each assessment of fentanyl-fortified plasma lots from six individual donors demonstrated at least 20 hours of ambient stability, with a recent investigation of a seventh donor easily confirming 47 hours of ambient stability. Of course, we do not store plasma samples at ambient temperature, but when possible, like to establish ambient stability in case of a sample delivery problem.

As part of a more recent validation, however, ambient storage evaluated for fentanyl at approximately 64 hours in an eighth individual “normal” human plasma lot demonstrated biases of -36.7% at the low QC level, and -38.4% at the high QC. This same individual donor lot demonstrated biases of -17.3% at the low QC level, and -17.3% at the high QC level after approximately 24 hours of ambient storage. By the time we could rule out all other potential non-matrix-related causes of these unanticipated results, this individual lot was used up, with no more available from this donor. All matrix lots tested met vendor matrix testing and donor questionnaire specifications regarding negative status for multiple disease states.

As no other K2EDTA human plasma lots from individual donors had ever indicated fentanyl instability, the donor of our eighth plasma lot was thought to potentially be a fast metabolizer for fentanyl, with increased levels of specific plasma enzymes that depleted fentanyl levels in ambient plasma samples. We’ve not evaluated enough individual plasma lots to estimate how common such increased plasma metabolism may be for fentanyl. However, if a pool containing enough plasma from this particular donor type was used to evaluate freeze-thaw stability, or stability in whole blood, perhaps atypical and even irreproducible results could have been generated. What if the analyte in question was not as well characterized as fentanyl, but was instead a newer chemical entity or metabolite? Sometimes the instability of a drug or metabolite in matrix may be more dependent on the chemistry of the individual matrix lot than we think. This possibility could suggest the need for additional stability-confirming experiments involving a wider array of individual donors of matrix.”

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  1. Jim Ferguson on

    With individual difference so large, it seems that PK studies for fentanyl could have resulted in establishing dosing regimens that could either be ineffective, or fatal, if used in outlier patients.

  2. Pingback: Instability of a Stable Drug | AIT Bioscience

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