Stability is an important pre-analytical variable for quantitative LC-MS/MS analysis of drug molecules and/or their metabolites in biological matrices. Instability of an analyte in any stage of the bioanalytical process, including sample collection, processing, storage, extraction and LC-MS/MS analysis, can result in under-/over-estimation if an adequate preventive procedure is not in place. In the current review on practical strategies in quantitative LC-MS/MS bioanalysis of unstable small molecules, the common causes of analyte instability were examined. The instability of some analytes is readily predictable because of the presence of certain chemically or biologically labile moieties in the molecules or because the compounds are in an inter-convertible form, e.g. lactone vs hydroxyl carboxylic acid. However, the instability of many other analytes is not readily predictable. Necessary evaluation needs to be conducted to identify the possible instability issues. The current review highlighted some general considerations and specific approaches for developing a robust LC-MS/MS method. In particular, incurred samples should be used as part of routine short-term stability assessment of any unstable analyte during the early stages of method development and validation. This can help unveil any 'hidden' instability issues that, if left unaddressed, could lead to the invalidation of a 'validated' method.

Strategies in quantitative LC-MS/MS analysis of unstable small molecules in biological matrices.

Salting-out assisted liquid-liquid extraction (SALLE) applies the salting-out effect to separate water-miscible organic solvent such as acetonitrile from plasma or other aqueous biofluids, and can extract a wide range of drug and metabolites, including many hydrophilic compounds. In most cases, the separated organic phase can be directly injected for bioanalysis, or with a simple dilution. SALLE provides similar simplicity to protein precipitation, but cleaner extracts due to a true phase separation. SALLE is also faster, more environmentally friendly and more cost-efficient than conventional liquid-liquid extraction and SPE. Through 96-well automation, SALLE can be easily integrated into the overall high-throughput LC-MS/MS bioanalysis strategy to increase productivity. This article provides a critical overview of the literatures on SALLE and perspectives of the future bioanalytical application of this often overlooked extraction technique. Important parameters impacting SALLE-LC-MS/MS assays are also discussed.

Salting-out assisted liquid–liquid extraction for bioanalysis

https://biblio.ugent.be/publication/4231282/file/4232012.pdf

Development and application of salting-out assisted liquid/liquid extraction for multi-mycotoxin biomarkers analysis in pig urine with high performance liquid chromatography/tandem mass spectrometry.

High-throughput bioanalytical method for analysis of synthetic cannabinoid metabolites in urine using salting-out sample preparation and LC–MS/MS

Where does flow injection analysis mass spectrometry (FIA-MS) stand relative to ambient mass spectrometry (MS) and chromatography-MS? Improvements in FIA-MS methods have resulted in fast-expanding uses of this technique. Key advantages of FIA-MS over chromatography-MS are fast analysis (typical run time <60 s) and method simplicity, and FIA-MS offers high-throughput without compromising sensitivity, precision and accuracy as much as ambient MS techniques. Consequently, FIA-MS is increasingly becoming recognized as a suitable technique for applications where quantitative screening of chemicals needs to be performed rapidly and reliably. The FIA-MS methods discussed herein have demonstrated quantitation of diverse analytes, including pharmaceuticals, pesticides, environmental contaminants, and endogenous compounds, at levels ranging from parts-per-billion (ppb) to parts-per-million (ppm) in very complex matrices (such as blood, urine, and a variety of foods of plant and animal origin), allowing successful applications of the technique in clinical diagnostics, metabolomics, environmental sciences, toxicology, and detection of adulterated/counterfeited goods. The recent boom in applications of FIA-MS for high-throughput quantitative analysis has been driven in part by (1) the continuous improvements in sensitivity and selectivity of MS instrumentation, (2) the introduction of novel sample preparation procedures compatible with standalone mass spectrometric analysis such as salting out assisted liquid–liquid extraction (SALLE) with volatile solutes and NH4
 + QuEChERS, and (3) the need to improve efficiency of laboratories to satisfy increasing analytical demand while lowering operational cost. The advantages and drawbacks of quantitative analysis by FIA-MS are discussed in comparison to chromatography-MS and ambient MS (e.g., DESI, LAESI, DART). Generally, FIA-MS sits ‘in the middle’ between ambient MS and chromatography-MS, offering a balance between analytical capability and sample analysis throughput suitable for broad applications in life sciences, agricultural chemistry, consumer safety, and beyond.

Emerging flow injection mass spectrometry methods for high-throughput quantitative analysis.

High-throughput salting-out assisted liquid/liquid extraction with acetonitrile for the simultaneous determination of simvastatin and simvastatin acid in human plasma with liquid chromatography.

Mathew Hautman

Papers

High-throughput salting-out assisted liquid/liquid extraction with acetonitrile for the simultaneous determination of simvastatin and simvastatin acid in human plasma with liquid chromatography.