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Bioanalytical Method Validation: Metabolite Considerations

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Validated bioanalytical methods for the quantitation of drugs in biological matrix (e.g. blood, plasma, serum, or urine) are critical for the successful conduct of nonclinical- and clinical- bioequivalence and pharmacokinetics studies. These validated methods provide critical data to support the safety and effectiveness of drugs. While the analyte required for the analysis in general is the administered drug, active metabolites formed pre-systemically may also be required to be measured.  

Similarly, when a drug is administered as a prodrug that is metabolized rapidly to an active metabolite, levels of the prodrug may be too low to be accurately quantified and bioequivalence may be based on quantitation of the active metabolite. While the focus here is on considerations for metabolites, the same principles apply to prodrugs as well.

Regulatory Expectations

 For a given drug development program, active metabolites will be present in study samples and whether required for analysis or not, regulators do expect that the impact of major metabolites be assessed to support the reliability and reproducibility of the data, including:

  • Method selectivity/specificity: do metabolites interfere with detection of the analyte?
  • What is the quantitative impact of metabolites on the accuracy and precision of analyte measurement?
  • How does sample collection, handling, and long-term sample storage affect the reliability of the data from the bioanalytical method? i.e., what steps need to be followed to preserve the stability of the analyte and the impact from active metabolites from the point of collection, during extraction, in the final extract and during liquid chromatography tandem mass spectrometry (LC-MS/MS) detection

The evaluation of the impact from metabolites for many cases may just be a formality, E.g., metabolites formed by dealkylation, hydroxylation, which are generally quite stable. There are, however, metabolites that will be present in study samples that require special handling conditions to be optimized to preserve the integrity of the samples and minimize any ex-vivo interconversion/back conversion into the analyte of interest, and some examples are list below.

Examples of Bioanalytical methods where metabolites pose a challenge



Special handling Considerations


Atorvastatin lactone

Temperature and pH of plasma at collection, and during extraction to minimize acid-lactone interconversion

Acitretin (trans acitretin)

13-cis Acitretin

Use of anti-oxidants and enzyme inhibitors to minimize ex-vivo isomerization


Regorafenib N-Oxide

Chromatographic separation due to in-source conversion of N-Oxide; use of APCI rather than ESI ionization with less in-source conversion

Acetylsalicylic acid

Salicylic acid

Oxalate fluoride as anticoagulant along with Acidification to minimise ex vivo hydrolysis of acetylsalicylic acid


De-hydro amlodipine

2 amu difference between parent and metabolite, chromatographic separation required

Dabigatran (free)

Dabigatran β-D-glucuronide

Acidified plasma to minimize ex-vivo back-conversion of

glucuronide into Dabigatran

Tenofovir Alafenamide


Careful pH selection and use of Oseltamivir acid to inhibit ex vivo hydrolase activity

Unstable Metabolites: Sources of Instability

Experiments designed in method development, that are later validated, use samples prepared to “simulate” study samples.  In actuality, this is performed using analytical reference standards of available metabolites and sources of instability are not just tested in plasma samples and need to be evaluated from the staring material right through to the final extract that is analyzed by LC-MS/MS:

Unstable Metabolites - Method Validation Considerations for Metabolites blog image.
Unstable Metabolites - Method Validation Considerations for Metabolites blog image 2.

BioPharma’s approach to handling of metabolites during Bioanalytical Method Validation.

At BioPharma Services, our approach to determining the impact from metabolites is customized for each method with intended application in clinical- and non-clinical (GLP) studies.  Major metabolites are sourced, and the following performed:

1. Metabolite Specificity

Specificity is assessed against the analyte lower limit of quantitation (LLOQ) sample by adding the metabolites at the highest expected concentration in clinical samples into a control blank matrix.

  • If the analytical reference standard (RSM) of the metabolite(s) is shown to contain a percentage of the analyte, specificity will be assessed for the internal standard only.

2. Metabolite Quantitative Interference Check

Quantitative impact from metabolites is assessed by adding the metabolites, at the highest expected concentration in clinical samples, into analyte QCs and High and Low levels and accuracy and precision assessed against nominal concentrations

  • If the RSM of the metabolite(s) is shown to have quantifiable levels of the analyte, baseline adjustment of the QCs may be required.

3. Stability in whole blood

Stability of the analyte is assessed with metabolites in whole blood for up to 2 hours.  Freshly collected whole blood is spiked with the analyte at LQC and HQC and metabolite concentrations expected in study samples.

4. Stability in Matrix and in post-extracted samples

Calibration standards and Quality control sample may be prepared only with the analyte of interest. However, clinical study samples will also contain metabolites.  Accordingly, all matrix-based stability assessments (bench-top, freeze-thaw and long-term storage) as well as post extract-stability assessments (processed sample stability, autosampler stability and re-injection reproducibility) are performed with and without metabolites spiked at the highest expected concentration in clinical samples.

Case study: Bioanalytical method for “Free” Edaravone ensuring no impact from its major metabolite, Edaravone sulphate.

Edaravone is a free radical scavenger indicated in the treatment of ischemic stroke, but also received orphan designation in the treatment of amyotrophic lateral sclerosis (ALS).  In vivo, Edaravone is extensively metabolized with the sulfate conjugate being the major moiety in circulating plasma with levels up to 20x higher than that of the parent analyte Edaravone.

In vivo, the sulfate conjugate is prone to deconjugation and therefore ex vivo back-conversion into the parent drug Edaravone is an important consideration in the accurate measurement of “free” or “unconjugated” Edaravone. Accordingly, Edaravone sulfate was included throughout method development and validation and proved to be quite the challenge, as:

    1. The initial batch of reference standard was found to have degraded almost completely to Edararvone. An additional reference standard was acquired, which still contained a small percentage of Edaravone, and baseline adjustments based on the Edaravone levels from the reference material were required.
    2. It was not stabile and was rapidly hydrolysed in methanol
    3. It readily hydrolyzed to Edaravone under mildly acidic and alkaline conditions and when exposed to even moderate heat.
    4. It degraded in the MS Source to Edaravone.
Unstable Metabolites - Method Validation Considerations for Metabolites blog image 3.

Despite the technical challenges, from this unstable metabolite, the final method for “free Edaravone utilized a simple protein precipitation extraction under chilled conditions, avoiding methanol, acid, base and evaporation reconstitution steps. Chromatographic conditions were selected whereby the sulfate conjugate was separated from Edaravone to minimize the impact from in-source conversion.  Under these conditions Edaravone sulfate was found to have no impact on method selectivity, quantitative accuracy, and no impact on all matrix-based stability for “free” Edaravone (Bench-top, Freeze-thaw and long-term frozen storage stability. The method for “free” Edaravone was successfully applied in a pharmacokinetic study with 100% passing batches and 100% incurred sample reproducibility (ISR).

Why Choose BioPharma Services?

Whether required for analysis or not, metabolites will be present in clinical samples.  Inclusion of major metabolites (if commercially available) throughput the development and validation of a bioanalytical method has huge value in simulating clinical samples and ensuring quantitative accuracy of a given method and no/minimal surprises during the application of the method.

This is particularly important for metabolites with known or demonstrated instability. Our approach at BioPharma Services is a major contributing factor to the high quality and success rate of our validated bioanalytical methods applied in clinical and non-clinical (GLP) studies.

Find out why BioPharma might be the right partner for you! Learn more about BioPharma Services and the wide array of bioanalytical services we provide, including bioanalytical testing.

BioPharma Services, Inc., a Think Research Corporation and clinical trial services company, is a full-service Contract Clinical Research Organization (CRO) based in Toronto, Canada, specializing in Phase 1 clinical trials 1/2a and Bioequivalence clinical trials for international pharmaceutical companies worldwide. BioPharma has clinical facilities both in the USA and Canada with access to healthy volunteers and special populations.

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