Bioanalytical Methods for Sample Cleanup

Preparation of biological samples for chromatographic analyses.
Dec 01, 2012
Volume 25, Issue 12

Roger N. Hayes
High-throughput bioanalytical methods are essential to support the rapid discovery and development of drugs in the pharmaceutical industry. Liquid chromatography coupled with tandem mass spectrometric detection (LC–MS/MS) is considered as the benchmark analytical methodology for quantifying new chemical entities in biological fluids (1–4). Because of the high sensitivity and selectivity of LC–MS/MS, the time required for method development and subsequent sample analysis is dramatically reduced. Rigorous chromatographic resolution of analytes and/or tedious sample extraction protocols are typically not required even when complex biological matrices are used. Most chromatographic techniques have matured and automation is now commonplace (5–7). Nevertheless, with common sample analysis times of less than three minutes, the bottleneck in sample analysis has become the sample preparation step. Sample preparation is still considered to be a slow and labor-intensive process, and it is rare for an analyst to be able to inject samples directly into an LC–MS/MS system with no pretreatment.

The importance of sample preparation stems from three major concerns—removing interferences from the biological sample matrices, concentrating the analyte(s) of interest, and improving analytical system performance (8). An industry survey noted a marked increase in methods requiring limits of quantitation of less than 1 ppb, and the trend toward trace analyses is not diminishing (9). Optimized sample preparation techniques that provide high enrichment factors become crucial for these dilute concentrations.

The choice of sample preparation method should depend on the quality of the data required. It makes little sense to invest weeks of development time attempting to achieve pg/mL sensitivity for a screening assay. However, it may be important to invest such time to develop and validate a method for a lead drug candidate undergoing human safety assessments that are subject to FDA regulatory scrutiny. Indeed, validating analytical procedures is the process of determining a suitable method that is capable of providing useful analytical data. It is important to bear in mind that a method that is valid in one situation could be invalid in another (10, 11).

In the pharmaceutical industry, the most common biological sample matrix is plasma. Moreover, it is common practice to dilute troublesome matrices like urine or cerebrospinal fluid with plasma and apply previously developed plasma extraction protocols. Drugs are most commonly isolated from plasma using one (or occasionally, a combination) of either liquid–liquid extraction, protein precipitation, or solid phase extraction. Other less common choices include column-switching (LC–LC), affinity extraction, and ultrafiltration.

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