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time demands are significantly reduced. Lastly, SLE in the 96-well plate format is especially amenable to automation, increasing overall sample throughput.
Traditionally, the sorbent used for SLE is highly purified diatomaceous earth (DE). However, as a naturally occurring material, DE consists of irregular fossilised micro- organisms. Consequently, variance in particle-size distribution can generate issues with product manufacturing and batch- to-batch quality control, in turn leading to inconsistent product performance. Figure 2 shows the scanning electron microscope (SEM) image of natural DE sorbent and synthetic SLE sorbent particles, using the same SEM settings. As shown in the Figure 2A, there is noticeable amount of debris in the DE sorbent with particle size inconsistencies. However, for synthetic SLE sorbent (Figure 2B), the particle size is much more uniform, without obvious debris. Further, the inconsistency in DE sorbent particles results in the reduced and inconsistent aqueous phase holding capacity [6,7], leading to a higher risk of sample loss and matrix breakthrough during sample loading and elution. The use of the synthetic SLE substrate allows control of the particle size distribution, in turn leading to improved consistency of method performance.
Fenfluramine (FNN) is an anti-epileptic drug whose mechanism of action is poorly understood. In order to study the distribution of FNN and the accumulation of its major metabolite norfenfluramine (NFNN) in mouse cerebellum, it was necessary to develop a sensitive assay given the
Figure 1: The process and analyte extraction mechanism of supported liquid extraction (SLE).
limitation in tissue mass (ca. 60 mg). Brain homogenate represents a matrix complexity greater than that of traditional plasma owing to significantly higher phospholipid content, and it was therefore necessary to deplete as many of these potential ion suppressors as possible in order to minimise accumulation within the LC/MS system. To this end, the novel synthetic SLE sorbent was evaluated in terms of the efficacy of phospholipid removal, recovery of FNN and NFNN, assay specificity and matrix effect, all benchmarked against traditional diatomaceous earth sorbent.
Experimental Chemicals and Standards
High Performance Liquid Chromatography (HPLC) or Omnisolv grade solvents were sourced from Millipore Sigma, including methanol (MeOH), dichloromethane (DCM), methyl tert-butyl ether (MTBE), 1-chlorobutane, and hexane. Ethyl acetate (EtOAc) and chloroform Optima grade solvents were supplied by Fisher. Other chemicals were purchased from Sigma-Aldrich, including ammonium hydroxide (NH4
OH),
concentrated hydrochloric acid (HCl), and ammonium bicarbonate (NH4
HCO3 ).
Figure 2: SEM images of DE sorbent (A) and synthetic SLE sorbent (B).
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