Bioanalytical Services

Instrumentation and Capabilities

Our dedicated, purpose built bioanalytical laboratories incorporate advanced liquid chromatography and tandem mass spectrometry instrumentation and software. Our experienced analysts offer expert bioanalysis capabilities across a variety of matrices, including:

• Method development

• Method validation

• Partial validation to other species

• Analyte stability

• Metabolite quantification

These capabilities incorporate various sample pre-treatment processes, in 96-well format, such as:

• Solid phase extraction

• Protein precipitation

• Liquid-liquid extraction

Toxicokinetic Bioanalysis 

Our bioanalysis team has extensive experience in analysis of samples from toxicokinetic studies supporting non-clinical projects performed in our own facilities and elsewhere.

This specialist expertise ensures a quality analytical service performed according to current best practice standards including requirements for method validation and incurred sample reanalysis. 

Dried Blood Spot 

Historically, toxicokinetic (TK) investigations supporting non-clinical toxicology studies have been performed using plasma samples, which require centrifugation and separation. Hence, relatively large volumes of blood, often up to 0.5 mL, are needed. It is usually not possible to obtain sufficient samples from a single animal for a full TK profile and composite results from different animals have to be used.

Sequani have conducted validation work using the dried blood spot technique, which can be used as an alternative to traditional blood sampling techniques for toxicokinetic purposes. Rather than blood samples being spun and the resultant plasma analysed for API content, the dried blood spot approach requires much smaller volumes of whole blood which are collected onto a blood spot card. Samples of the dried blood spots are then uniformly punched to produce a sample for extraction and analysis.

There are advantages to implementing this technique within development programmes: reduced number of animals, improved quality of TK data and reduced sample shipment costs.

Pharmacokinetic and Toxicokinetic Reporting

Toxicokinetic determinations are an essential component of pivotal toxicity studies and we are able to offer a full service in toxicokinetic data handling and interpretation to support these studies. In addition, we have extensive expertise in design, conduct and data analysis for stand-alone pharmacokinetic and bioavailability studies. All pharmacokinetic and toxicokinetic data are analysed using the WinNonLin® PK/PD modelling and analysis system.

Sequani’s team of toxicokinetic/pharmacokinetic analysts, with extensive training in Good Laboratory Practice (GLP), toxicology and compound development fulfil these functions in every detail. Comprehensive statistical data analysis is provided, using the fully validated networked data handling systems. Data is interpreted and the toxicokinetic/pharmacokinetic report produced, either by itself or as part of a toxicology report ready for regulatory submission.

In Vitro Metabolism 

In vitro metabolism assays can provide valuable information regarding the kinetics and metabolic fate of NCEs in development, giving an indication of metabolic stability and alerting to the formation of major metabolites.  In vitro metabolism data provides justification for the animal species used in pre‑clinical studies and can determine which cytochrome P450 (CYP450) isoenzymes are involved in the metabolism of NCEs and hence indicate the potential for drug-drug interactions.  

Cryopreserved liver microsomes or hepatocytes from common pre-clinical species and man are used in our studies.  Microsomal preparations provide a convenient and inexpensive source of several enzyme functions.  These are mainly associated with Phase I oxidative metabolism but can provide some Phase II functions if appropriate cofactors are included.  Primary hepatocytes provide a more complete in vitro system as they have intact cell membranes and physiological levels of cofactors for complete Phase I and II metabolic processes.  

In Vitro Stability 

• Determination of enzyme kinetics (clearance and half-life, as applicable)

• Systems – cryopreserved liver microsomes or hepatocytes Quantitative LC-MS/MS bioanalysis

• Species available - human, rat, dog, mouse, monkey, mini-pig

Metabolic profiling 

• Major metabolite alert

• Inter-species comparison

• Systems - cryopreserved liver microsomes or hepatocytes

• Qualitative HPLC-UV analysis

• LC-MS and MS/MS characterisation

Cytochrome P450 Inhibition Assays  

Assays to detect inhibition of the major human CYP450s are undergoing in-house validation at Sequani so these studies can be added to our services in the near future.

Plasma Protein Binding 

Determining the extent to which a molecule binds to plasma (or serum) proteins is a critical phase of NCE development since the effective dose is related to the concentration of unbound “free” drug.  The extent of plasma-protein binding of a drug therefore influences compound dosing, efficacy, toxicity, clearance rate and potential for drug-drug interactions.  The extent of binding to proteins can be determined by several methods including ultra-filtration and equilibrium dialysis. 

Ultra-Filtration

• Plasma or serum

• Human, dog and rat (other species on request)

• LC-MS/MS analysis

Equilibrium Dialysis 

Sequani are currently performing in-house validation of assessment of protein binding using equilibrium dialysis methodology.  This may be used in instances where the ultra-filtration method is unsuitable due to non-specific binding.