In previous columns I have mainly discussed aspects of method validation for product analysis performed in quality control (QC) laboratories, but not for bioanalytical methods. To be specific, a bioanalytical method in the context of this column is the analysis of small drug molecules and any metabolites in biological samples generated from non-clinical and clinical studies. The data generated from these studies are used to evaluate the pharmacokinetics, bioavailability, bioequivalence and toxicokinetics, which are used to support regulatory submissions of new drugs or licensing generic versions of existing drugs.

Typically, the biological fluids analysed are plasma, derived from whole blood by centrifugation, and urine — which needs no further explanation! So let's start from the firm foundation that we use chromatographs for analyses in both QC and bioanalytical methods and we need to have validated methods. These methods are used for the quantitative determination of drugs and their metabolites in biological samples. Therefore, they must generate reproducible and reliable results to allow reliable interpretation of the study samples. It is essential to use fully validated bioanalytical methods that yield reliable results that can be interpreted satisfactorily.

Before discussing the detail of the validation of the methods themselves we will look at the differences in analysis and the HPLC equipment used between QC and bioanalysis. I'm not going to write about reference standards as all analytical methods are predicated on having standards of known purity and stability and these must be documented appropriately.

Methods used in the QC laboratory determine if the manufactured product meets its specifications and a wide variety of analytical techniques can be used, including chromatography, spectroscopy and traditional wet chemistry analysis, such as Karl Fischer titration. The analysis will cover the raw materials used at the start of the manufacturing process, key intermediates and the final product, and will include analysis of known impurities. Usually the amounts to be determined will be known from the product specification and will have a short dynamic range, for example, 50–150% or 75–125% of the nominal amount for the active ingredient in contrast to impurity analysis that will be between 0.05–0.5% of the nominal amount of the active component. Many different analytical methods will be used for product batches in QC analysis, some will be specific to the product and some will be general analytical techniques applicable to all products, for example, loss on drying.

In contrast, there are relatively fewer bioanalytical methods, but they are applied more intensively to samples from both non-clinical and clinical studies. A clinical study can generate up to 5000 samples for analysis depending on the complexity of the study design and its objectives. Instead of a narrow concentration range as found in a product specification, bioanalytical assays can cover 2–4 orders of magnitude. This is especially true if a drug is given intravenously because the assay will follow the time course of the absorption, distribution and elimination of the drug in the body — often until the drug is no longer detected. Thus, the analytical method follows the dynamic analyte concentrations from ingestion of the drug to its final elimination.

HPLC Equipment

The chromatographic pumps used for the bioanalysis are broadly the same as used for QC analysis, however, the autosamplers and detectors used may differ as we will now discuss in more detail.

Autosampler: A traditional autosampler can be used for bioanalysis, but it is important to ensure that the probe can sample just the volume of the sample programmed into the instrument or data system. The older style valve autosamplers where the sample itself was used to flush the value and avoid carryover cannot be used as the bioanalytical sample extracts range from 50–200 μL and there is insufficient volume to flush the value. Thus any autosampler used must have minimal carryover to avoid contaminating the next sample injection.

Many bioanalytical methods use solid-phase extraction to isolate the analytes from the biological sample and the extracts are placed in 96-well plates. Therefore, to avoid sample transfer to vials, the autosampler must also be capable of accepting these plates to inject the sample extracts.

Detector: The typical HPLC detector for the majority of product analysis is a UV detector. This is also used in bioanalysis, but the main detector used is a mass spectrometer, typically a triple quadrupole instrument. This is partly because more potent compounds are produced in pharmaceutical research and development which results in assay concentration ranges in the nanogramme/mL and pictogramme/mL, but is also partly because of the specificity and sensitivity required for the analysis. In this latter context, MS–MS detectors are used along with the associated software to allow the use of multiple ion monitoring, as well as positive and negative ionization modes (with or without chemical ionization). The additional expense for a mass spectrometer detector is offset by the scientific requirements for sensitivity and selectivity as well as the ability to have shortened run times, often less than 5 min per injection.