10 February / March 2020
Analysis of Fenfluramine and Norfenfluramine in Mouse Brain and Cerebellum by Liquid Chromatography Tandem Mass Spectrometry using a Novel Solid-Supported Liquid Extraction
by Jeff Plomley and Vinicio Vasquez , Altasciences Inc. , 575 Armand Frappier Boulevard, Laval, Québec, Canada, H7V4B3 Limian Zhao, Agilent Technologies Inc. , 2850 Centerville Road, Wilmington, DE, USA, 19808
This study outlines the application of a new synthetic supported liquid extraction (SLE) sorbent for the quantitative determination of the antiepileptic drug fenfluramine (FNN) and metabolite, norfenfluramine (NFNN), in mouse brain by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Additionally, a comparison of the synthetic SLE sorbent with diatomaceous earth (DE) was conducted, wherein the synthetic SLE sorbent eliminated a greater content of phospholipid while demonstrating higher analyte recovery with improved reproducibility. The validated method supported a two-fold dynamic range with a limit of quantitation (LOQ) of 0.05 µg/g in mouse brain, demonstrated acceptable calibration curve linearity (r2 effect (100 ± 10%).
Introduction
LC-MS/MS has been widely adopted for the high throughput quantitative bioanalysis of small molecules, attributed mainly to the high selectivity, sensitivity, and sampling frequency of the approach. However, even with highly selective analyte monitoring, LC-MS/MS analysis can be deleteriously impacted by changes in ionisation efficiency due to coeluting matrix components such as salts, proteins, lipids (including phospholipids) and other various organic molecules. Such ionisation effects can influence the achievable limit of quantitation (LOQ), method reliability and reproducibility, chromatography and MS source contamination [1]. Therefore, sample preparation is required not only to extract target analytes from matrix, but also to remove unwanted components potentially impacting ionisation efficiency - the latter is often referred to as the matrix effect. The use of appropriate sample preparation techniques is dependent upon the complexity of the matrix, requirements for the detection of target analytes, and the selected instrument detection method. It is understood that sample preparation can
be both time-consuming and costly, but these are the unavoidable factors in order to gain reliable quality analytical results, and to preserve high-value instruments from damage.
Sample preparation products for bioanalysis are often based on a 96-well plate format, which allows automated / semi-automated simultaneous sample processing, thereby supporting the preparation of a large number of samples aligned with high throughput LC-MS/MS analysis. The format also accommodates the relatively small sample sizes associated with biological matrices, typically within several hundreds of microliters. Protein precipitation (PPT), liquid-liquid extraction (LLE) and solid phase extraction (SPE) are the techniques most commonly implemented in the preparation of biological samples for LC-MS/MS analysis, with pros and cons for each approach [2-4].
Although LLE represents a sample preparation process with advantages such as high recovery and extract cleanliness, its mainstream adoption into the modern bioanalytical lab is confounded by the disadvantages associated with automation (time consumption, labour-intensive processes, and the potential for emulsion
> 0.99), intra- and inter-run precision (C.V. < 10%) and accuracy (100 ±10%), and matrix
formation). In contrast, supported liquid extraction (SLE) as a flow-through technique has been increasingly used as an alternative approach to LLE, overcoming many of the disadvantages associated with LLE. [5] The SLE substrate provides a chemically inert but highly hydrophilic surface upon which an aqueous sample adsorbs. When the aqueous sample is loaded onto SLE substrate, a thin layer of aqueous phase is generated and coated onto the SLE sorbent surface. This thin layer of aqueous phase on the sorbent significantly increases the contact surface area during extraction. Following a brief equilibration period, analytes are extracted with a water immiscible solvent either by gravity or through the application of positive or negative pressure while the aqueous phase is retained on the sorbent. The extraction mechanism and workflow process are outlined in Figure 1. Since insignificant mixing of aqueous and organic phases occurs with the SLE workflow, emulsions are eliminated and the intimate contact between phases allows very efficient analyte partitioning, often resulting in high analyte recovery. Due to the simplicity of the SLE workflow (load, soak and elute), labour and
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