Biological Matrix Selection

Biomarkers are frequently measured in tissue biopsies, plasma, serum, and/or urine. Establishing the preferred matrix for biomarker analysis is paramount (see Chapter 40). Regardless of species, collection of whole blood for isolation of plasma and serum is semi-invasive and often associated with limited collection volumes (Diehl et al., 2001). Collection of urine is minimally invasive and allows for collection of large sample volumes. The kinetics of safety biomarkers has not been extensively studied therefore, frequent sampling may be informative. However, sampling frequency in the cUnical setting should be balanced with considerations for various factors including but not limited to patient comfort and ease of collection. 

Websites to Regulatory Guidances for Biomarkers in Drug Development 

---

Assay in biologicaf matrix Calibrators and controls in biological matrix Selectivity and specificity Minimal required dilution (MRD)... 

The analytes are typically extracted from the biological matrix using solvent extraction or solid phase extraction (SPE). Most analytes require some form of chemical derivatization prior to analysis by GC-MS techniques, whereas with LC-MS-MS no further treatment of the extract is required. The extracts obtained from urine are relatively dirty because of the many endogenous compounds that are present. It is for this reason that the very selective techniques of GC-MS-MS, GC-HRMS, or LC-MS-MS are required to detect some of the prohibited substances that have low detection levels. 

Selective detectors tend to be employed where the analyte is present in small amounts in a complex matrix such as in bioanalytical procedures where components extracted from the biological matrix along with the analyte can cause interference. Some formulated compounds have only very poor chromophores - these include sugars, lipids, surfactants, amino acids and some classes of drugs, e.g. a number of anticholinergic drugs lack chromophores. In these cases an alternative to UV detection has to be employed. 

For selectivity, there should be evidence that the substance being quantified is the intended analyte. Therefore, analyses of blank samples of the appropriate biological matrix (plasma, urine, or other matrix) should be obtained from at least six sources. Each blank sample should be tested for interference, and selectivity should be ensured at the lower limit of quantification (LLOQ). 

Potential interfering substances in a biological matrix include endogenous matrix components, metabolites, decomposition products, and in the actual study, concomitant medication. Whenever possible, the same biological matrix as the matrix in the intended samples should be used for validation purposes. For tissues of limited availability, such as bone marrow, physiologically appropriate proxy matrices can be substituted. Method selectivity should be evaluated during method development and method validation and can continue during the analysis of actual study samples. 

Selectivity is the ability of the bioanalytical method to measure and differentiate the analytes in the presence of components that may be expected to be present. Specificity is the ability to assess unequivocally the analyte in the presence of components that may be expected to be present. In general, analytical methods are selective, and only in same cases also specific (e.g., an LC-MS/MS bioanalytical method is highly selective but not always also specific because it could be possible to find in the complex biological matrix an interference with the same retention time, molecular weight, and main fragment of the analyte of interest). Even if the 2000 Washington conference focuses only on selectivity, it is up to bioanalytical laboratories to differentiate in their documentation between selectivity and specificity or consider them equivalent and use them interchangeably. 

Limited Quantity of Matrices. For biological matrices that are difficult to obtain, only a single source may be available and is therefore the only possibility for testing selectivity. These matrices include, but are not limited to, human and primate tissue and cerebrospinal fluid. In the absence of the corrected biological matrix, as the 2000 Washington conference final report suggests, a physiologically appropriate proxy matrix can be used. 

It is possible to select a biological matrix and a suitable analytical strategy depending on the aim of the analysis (clinical, medicolegal, epidemiological) and/or on the availability of the sample or on which kind of information is needed. 

While analytical derivatizations are an effective way for extracting compounds, these often require additional steps in the analytical procedure and can introduce side products that may interfere with the analysis. Solid phase extraction has provided an alternative method to this process. The advantage of solid phase extraction is that the reagents, derivatives, and side products are maintained on the solid phase. As needed, these derivatives and side products can be selectively eluted after the desired derivative has been formed on the column. In addition, this method can eliminate potential problems associated with emulsion formulation that may occur with liquid-liquid extraction of compounds from the biological matrix. Finally, solid phase extraction is easily amenable to automation with other analytical detection methods such as gas and liquid chromatography. The phases used in solid phase extraction are the standard ones employed in other extraction methods. ... 

Selectivity in HPLC is obtained by setting optimal chromatographic conditions, such as mobile phase composition, column temperature, and detector wavelength. There are a variety of ways to validate selectivity. One approach is to demonstrate a lack of response in the blank biological matrix. A second approach is to check whether the intercept of the calibration curve is significantly different from zero. 

Figure 1 Example of workflow in natural product isolation from a complex biological matrix using high-performance liquid chromatography for the target compound purification and identification. With successive application of several chromatographic modes of different selectivity (i.e., hydrophobicity/hydrophilicity, charge, molecular size) the chromatographic separation can become multidimensional.

---