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ADME PK

Plasma stability assay

Use our plasma stability assay to measure the degradation of your compounds in plasma.

The plasma stability assay is one of Cyprotex's in vitro ADME screening services. Cyprotex deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach.

Determination of stability in plasma

  • Determination of the stability of new chemical entities in plasma is important as compounds (with the exception of pro-drugs) which rapidly degrade in plasma generally show poor in vivo efficacy.
  • Instability in plasma can result in misleading in vitro data which can be difficult to interpret (e.g., plasma protein binding data). Storing and analyzing clinical samples from in vivo pharmacokinetic studies may also prove challenging.
  • Compounds with the following functional groups tend to be more susceptible to hydrolysis in plasma: esters, amides, lactones, lactams, carbamides, sulfonamides, and peptic mimetics1.
  • Compounds may exhibit interspecies differences in their stability in plasma.
  • Plasma stability is very useful for screening of prodrugs and antedrugs, where rapid conversion in plasma is desirable.
Plasma stability assay has many applications in drug discovery: to alert teams to labile structural motifs, to prioritize compounds for in vivo studies and to screen prodrugs and antedrugs.

1Di L, Kerns EH, Hong Y and Chen H. (2005) International Journal of Pharmaceutics 297; 110-119

Protocol

Plasma stability assay protocol

Test Article Concentration 1 μM (different concentrations available)
DMSO Concentration 2.5%
Incubation Time 0, 15, 30, 60 and 120 minutes
Test Article Requirements 30 µL of 10 mM DMSO solution
Analysis Method LC-MS/MS
Assay Controls Positive control compound which undergoes degradation in plasma
Data Delivery Percent parent compound remaining at each time point

Follow on metabolite profiling studies

Cyprotex's Plasma Stability assay can be extended to profile the main breakdown product that is formed. Options include a low resolution analysis to identify whether a metabolite is formed, or a cross species comparison to identify potential differences in metabolism which could in turn help to interpret pharmacology and toxicity data. We can also perform ion-transition analysis in order to understand the derivation of metabolites.

Please refer to our Metabolite Profiling and Identification section for further details.

Data

Data from Cyprotex's Plasma Stability assay

4 compounds were incubated with human and rat plasma over 120 min. These compounds show clear differences in stability following incubations with human and rat plasma (Figure 1). These data may be useful in interpreting in vivo efficacy, toxicity and pharmacokinetic studies.

 
Cloe Screen Plasma Stability data for diltiazem

Cloe Screen Plasma Stability data for eucatropine

Cloe Screen Plasma Stability data for enalapril

Cloe Screen Plasma Stability data for procaine

Figure 1
Cyprotex's Plasma Stability data for diltiazem, eucatropine, enalapril and procaine in human and rat plasma (mean ± sd, n = 3).

Q&A

Questions and answers on plasma stability

Please provide an overview of Cyprotex's Plasma Stability assay.

Cyprotex's Plasma Stability identifies compounds which are unstable in plasma.

The test compound (final incubation concentration = 1µM) is incubated with plasma (final DMSO concentration = 2.5%) at five different time points (0, 15, 30, 60 and 120 min). The reaction is terminated by methanol containing internal standard. Following centrifugation, the concentration of test compound in the supernatant is quantified by LC-MS/MS. The percentage of test compound remaining at the individual time points relative to the 0 minute sample is then reported. A control compound known to be metabolized by plasma esterases is also incubated alongside each batch of test compounds.

Stability of eucatropine in human plasma over 120 minutes
Figure 2
Stability of eucatropine in human plasma over 120 minutes (Data represent mean percentage test compound remaining against time with the error bars representing the standard deviation of four separate experiments).

Why is determining plasma stability important?

Typically, unless the compound is a pro-drug, instability in plasma can be detrimental as the concentration of compound in vivo will be reduced which, in turn, influences efficacy. Difficulty may also be experienced in measuring and interpreting the data from in vitro plasma protein binding studies. Storing and analyzing clinical samples from in vivo pharmacokinetic studies may also be challenging.

How do you ensure equivalent recovery from the plasma for all the samples?

If the samples are left in methanol for varying lengths of time post-termination, then the recovery of the compound from the samples can differ. For each time point, the initiation of the reaction is staggered so all time points are terminated with the methanol at the same time. This eliminates the problems experienced with differential recovery across the time points.

How do you know if the compound is degraded by enzymatic processes?

In order to confirm that degradation is caused by enzymatic processes, it is recommended that the compound is also screened through Cyprotex's Chemical Stability assay.

What classes of compounds are metabolized by plasma?

Typically, plasma stability is performed as a secondary screen for particular classes of compounds which feature functional groups more susceptible to hydrolysis in plasma. These include esters, amides, lactones, lactams, carbamides, sulfonamides, and peptic mimetics1.

How are pro-drugs studied in this assay?

If the assay is being used to screen for pro-drug stability and active compound can also be supplied then we can monitor disappearance of pro-drug and appearance of the active compound simultaneously.

If pro-drugs are being screened then the reactions are terminated using acetonitrile rather than methanol. In addition the mobile phases for LC-MS/MS analysis are acetonitrile-based rather than methanol.  This is to try and minimize any transesterifcation of the pro-drug which may occur in the presence of methanol.

Can you investigate stability in matrices other than plasma?

We can investigate stability in other matrices for example tissue homogenates.

References

1 Di L et al. (2005) International Journal of Pharmaceutics 297; 110-119

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