Bioanalysis Zone

Learn with Lin: part I – basic terms in LC–MS

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In this Learn with Lin article, Zone Leader Lin Wang (Covance, NJ, USA) explores some of the key terms in LC–MS that form the foundations of a good understanding of the bioanalytical applications of this technique. Part I discusses the realm of biological sample preparation, focusing on different methods and possible interferences.

Lin's Wang

Lin Wang received her Bachelor of Science degree in Chemistry from Xiamen University in China and Master of Science in Chemical Engineering from Rutgers University in New Jersey, USA. She is also enrolled in the Business and Science Program at Rutgers University to develop a comprehensive understanding of the interface between science and business and look at the revolution of analytical industry from different perspectives.

She started her career with Envigo (NJ, USA) in the department of Formulation and Inhalation Analysis and is currently the Research Associate II at Covance. She has been working in the CRO industry since 2015 and focusing on method development and validation on both small and large molecules.

Bioanalysis serves an essential role in drug discovery and development. It provides a quantitative measurement of drug concentration in biological matrices like urine, plasma and tissue samples to help gauge important parameters like bioavailability. Bioanalysis also involves metabolism, and ties all of this into the toxicokinetics/pharmacokinetics for a given application [1].  It is estimated by Grand View Research, Inc (CA, USA) that the global bioanalytical testing services market will reach USD 4.83 billion by 2026 [2]. Bioanalysis is a booming industry.

I have spent almost 5 years in dose formulation analysis and recently developed a great interest in bioanalysis. What inspired me to write this article, from a beginner’s point of view, is that when I tried to get to know this field, I couldn’t find a ‘one-stop shop’ to answer all my questions and explain the deep principles behind everything. I hope this article can help more chromatography fans like me who also crave some fun in the bioanalytical world.

Let’s start with a modest selection of basic terms which are fundamental and unique in bioanalysis, with an emphasis on what is new to me, the realm of biological sample preparation.

Sample extraction method [3]

Sample preparation for bioanalysis is critical and often challenging due to the potential difficulty of the selective extraction of the analyte of interest from the matrix and the time involved. Besides, each kind of matrix has its own obstacles. For example, there is a high level of salt in urine. Phospholipids and other components in plasma can cause matrix effects, enhancing or suppressing the analyte response [4].

Here we will discuss four commonly used extraction methods and their pros and cons.

Liquidliquid extraction (LLE)

LLE is based on the differing extents the compounds of the sample mixture partition themselves between two immiscible solvents, following the rule ‘like dissolves like.’ As per the most common application, if the analyte of interest is more favorable in organic solvent than in aqueous solvent, which can be measured as KD (distribution factor), the analyte will be mostly extracted to the organic phase and isolated. Ideally, interferences will be left in the aqueous phase.

liquid-liquid-extractionFigure 1 Schematic of liquid-liquid extraction

 For a typical biological sample extraction, the key steps are as follows when performing LLE:

liquid-liquid-extraction-flow-chartSupported-liquid extraction (SLE)

SLE is a modified LLE method and still follows the partition rule between two immiscible solvents. The major difference is that the aqueous phase in SLE is immobilized on an inert support and deposited as a thin film on the hydrophilic surface. The extraction occurs between this very thin aqueous film and an organic solvent, which is more efficient due to the enlarged interaction surface, compared to the shaking and mixing process utilized in LLE. The traditional support material is diatomaceous earth, a natural silica-based sediment formed from the fossilized remains of diatoms. However, recently synthetic material has been developed as an alternative.

supported-liquid-extraction-diagramFigure 2 Schematic of supported-liquid extraction

Solid-phase extraction (SPE)

SPE is a sample preparation technique that uses selective adsorption, washing and elution to enrich and purify samples through their interaction with a solid medium of specific chemistry, which is akin to the familiar principles of column chromatography.

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