Recent advances in sample preparation techniques to overcome difficulties encountered during quantitative analysis of small molecules from biofluids using LC-MS/MS

Abstract

Liquid chromatography-mass spectrometry analysis of small molecules from biofluids requires sensitive and robust assays. Because of the very complex nature of many biological samples, efficient sample preparation protocols to remove unwanted components and to selectively extract the compounds of interest are an essential part of almost every bioanalytical workflow. This review describes the most common problems encountered during sample preparation, ways to optimize established sample preparation techniques and important recent developments to reduce or eliminate major interferents from biofluids.

The impact of phospholipids and phospholipid removal on bioanalytical method performance

“…The most common solvents used in LLE are mixtures of chloroform, methanol and water (in differing ratios), but chloroform is often replaced with dichloromethane now owing to its lesser toxicity, with similar results (Reis et al, 2013). Modifications of LLE solvent include using additives such as acetic acid, hydrochloric acid, formic acid, citric acid, ethyl acetate and n-butyl chloride, as well as replacing chloroform with MTBE or butanol (Bylda et al, 2014;Jemal et al, 2010;Teo et al, 2015). Most of these modifications are specific to the analyte in question or are for the purpose of combatting matrix effects from a particular species of PL, further supporting the need to individualize the solvent and system used (Michopoulos et al, 2011).…”

“…Most of these modifications are specific to the analyte in question or are for the purpose of combatting matrix effects from a particular species of PL, further supporting the need to individualize the solvent and system used (Michopoulos et al, 2011). For example, with highly hydrophilic compounds that are not easily recovered through LLE, adding high concentrations of salt to increase the polarity of the aqueous phase while using a water-miscible solvent can lead to superior phase separation, aiding in quantification of analytes from biological samples in a process referred to as salt-assisted LLE or SALLE (Bylda et al, 2014). In a 2008 review, Srinivas cautioned against discarding a possible LLE solvent based on its reputation for enhancing matrix effects (Srinivas, 2009).…”

“…Ammonium formate and formic acid solutions (of varying strengths) are also used in many studies that include PL separation or analysis, but these have not been studied individually for PL removal effectiveness. Formic acid is an important solvent for use in solid-phase extraction (SPE) when PLs are a concern (Bylda et al, 2014), so it stands to reason that it could be a useful solvent for clearing columns of adhered PLs. Of course any solvent used will need to be tailored to the individual characteristics of the matrix and analyte being tested, such as hydrophobic or hydrophilic character, pH or polarity, as well as taking into account the specific type of equipment and analysis such as ESI vs APCI, and the type of sample preparation such as protein precipitation (PPT), liquid-liquid extraction (LLE), supported liquid extraction (SLE) or SPE prior to HPLC-MS/MS for example (Brouwers, 2011;Bylda et al, 2014;Little et al, 2006).…”

“…Lytle et al (2000 experimented with using piperidine as a post-column modifier, which promoted deprotonation of the target PLs, thus making them easier to detect in negativemode ESI. Finally, many investigators choose to monitor the MS/MS ion transitions of specific PLs to account for their impact in analyte signal (Bylda et al, 2014;Ismaiel et al, 2010;Little et al, 2006;Schiller et al, 2002;Trufelli et al, 2011).…”

Development and validation of analytical method for the determination of radotinib in human plasma using liquid chromatography-tandem mass spectrometry

“…[7] We applied different protein precipitation/extraction methods using methanol, acetonitrile, and methyl tert-butyl ether (MTBE) as a precipitation/extraction solvent. Methanol and acetonitrile had effectively removed most of plasma proteins.…”